Monitoring cell endocytosis of liposomes by real-time electrical impedance spectroscopy


Evaluation and understanding the effect of drug delivery in in vitro systems is fundamental in drug discovery. We present an assay based on real-time electrical impedance spectroscopy (EIS) measurements that can be used to follow the internalisation and cytotoxic effect of a matrix metalloproteinase (MMP)–sensitive liposome formulation loaded with oxaliplatin (OxPt) on colorectal cancer cells. The EIS response identified two different cellular processes: (i) a negative peak in the cell index (CI) within the first 5 h, due to onset of liposome endocytosis, followed by (ii) a subsequent CI increase, due to the reattachment of cells until the onset of cytotoxicity with a decrease in CI. Free OxPt or OxPt-loaded Stealth liposomes did not show this two-stage EIS response; the latter can be due to the fact that Stealth cannot be cleaved by MMPs and thus is not taken up by the cells. Real-time bright-field imaging supported the EIS data, showing variations in cell adherence and cell morphology after exposure to the different liposome formulations. A drastic decrease in cell coverage as well as rounding up of cells during the first 5 h of exposure to OxPt-loaded (MMP)-sensitive liposome formulation is reflected by the first negative EIS response, which indicates the onset of liposome endocytosis.

Graphical abstract

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. 1.

    Liu Q, Yu J, Xiao L, Cheuk J, Tang O, Zhang Y, et al. Impedance studies of bio-behavior and chemosensitivity of cancer cells by micro-electrode arrays. Biosens Bioelectron. 2009;24:1305–10.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Minchinton AI, Tannock IF. Drug penetration in solid tumours. Nat Rev Cancer. 2006;6:583–92.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G. Cell death assays for drug discovery. Nat Rev Drug Discov. 2011;10:221–37.

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Zór K, Heiskanen A, Caviglia C, Vergani M, Landini E, Shah F, et al. A compact multifunctional microfluidic platform for exploring cellular dynamics in real-time using electrochemical detection. RSC Adv. 2014;4:63761–71.

    Article  Google Scholar 

  5. 5.

    Garvey CM, Spiller E, Lindsay D, Chiang C-T, Choi NC, Agus DB, et al. A high-content image-based method for quantitatively studying context-dependent cell population dynamics OPEN. Nat Publ Gr. 2016.

  6. 6.

    Radmacher M. Studying the mechanics of cellular processes by atomic force microscopy. Methods Cell Biol. 2007;83:347–72.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Spiller DG, Wood CD, Rand DA, White MRH. Measurement of single-cell dynamics INSIGHT REVIEW. Nature. 2010;465:736–45.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Wakefield ID, Pollard C, Redfern WS, Hammond TG, Valentin J-P. The application of in vitro methods to safety pharmacology. Fundam Clin Pharmacol. 2002;16:209–18.

    CAS  Article  Google Scholar 

  9. 9.

    Rampersad SN, Rampersad NS. Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors. 2012;12:12347–60.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Malich G, Markovic B,Winder C. The sensitivity and specificity of the MTS tetrazolium assay for detecting the in vitro cytotoxicity of 20 chemicals using human cell lines. Toxicology. 1997;124:179–92.

  11. 11.

    Fang Y. Label-free cell-based assays with optical biosensors in drug discovery. Assay Drug Dev Technol. 2006;4:583–95.

  12. 12.

    Xi B, Yu N, Wang X, Xu X, Abassi Y. The application of cell-based label-free technology in drug discovery. Biotechnol J. 2008;3:484–95.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Patching SG. Surface plasmon resonance spectroscopy for characterisation of membrane protein-ligand interactions and its potential for drug discovery. BBA - Biomembr. 2014;1838:43–55.

    CAS  Article  Google Scholar 

  14. 14.

    Giaever I, Keese CR. Monitoring fibroblast behavior in tissue culture with an applied electric field. Proc Natl Acad Sci U S A. 1984;81:3761–4.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Wegener J, Keese CR, Giaever I. Electric cell-substrate impedance sensing (ECIS) as a noninvasive means to monitor the kinetics of cell spreading to artificial surfaces. Exp Cell Res. 2000;259:158–66.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Giaever I, Keese CR. Micromotion ofmammalian cells measured electrically. Proc Natl Acad Sci USA. 1991;88:7896–900.

  17. 17.

    Caviglia C, Zór KZ, Montini L, Tilli V, Canepa S, Melander F, et al. Impedimetric toxicity assay in microfluidics using free and liposome-encapsulated anticancer drugs. Anal Chem. 2015;87:2204–12.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Caviglia C, Zór K, Canepa S, Carminati M, Larsen LB, Raiteri R, et al. Interdependence of initial cell density, drug concentration and exposure time revealed by real-time impedance spectroscopic cytotoxicity assay. Analyst. 2015;140:3623.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Ceriotti L, Ponti J, Broggi F, Kob A, Drechsler S, Thedinga E, et al. Real-time assessment of cytotoxicity by impedance measurement on a 96-well plate. Sensors Actuators B. 2007;123:769–78.

    CAS  Article  Google Scholar 

  20. 20.

    Lundstrom K. Cell-impedance-based label-free technology for the identification of new drugs. Expert Opin Drug Discov. 2017;12:335–43.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Starkuviene V, Pepperkok R. The potential of high-content high-throughput microscopy in drug discovery. Br J Pharmacol. 2007;152:62–71.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Liu Z, Lavis LD, Betzig E. Imaging live-cell dynamics and structure at the single-molecule level. Mol Cell. 2015;58:644–59.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Isherwood B, Timpson P, McGhee EJ, Anderson KI, Canel M, Serrels A, et al. Live cell in vitro and in vivo imaging applications: accelerating drug discovery. Pharmaceutics. 2011;3:141–70.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Fischer LM, Tenje M, Heiskanen AR, Masuda N, Castillo J, Bentien A, et al. Gold cleaning methods for electrochemical detection applications. Microelectron Eng. 2008;86(86):1282–5.

    CAS  Article  Google Scholar 

  25. 25.

    Dimaki M, Vergani M, Heiskanen A, Kwasny D, Sasso L, Carminati M, et al. A compact microelectrode array chip with multiple measuring sites for electrochemical applications. Sensors. 2014;14:9505–21.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Vergani M, Carminati M, Ferrari G, Landini E, Caviglia C, Heiskanen A, et al. Multichannel bipotentiostat integrated with a microfluidic platform for electrochemical real-time monitoring of cell cultures. IEEE Trans Biomed Circuits Syst. 2012;6:498–507.

    Article  PubMed  Google Scholar 

  27. 27.

    Sasso L, Heiskanen A, Diazzi F, Dimaki M, Le’on JC, Vergani M, et al. Doped overoxidized polypyrrole microelectrodes as sensors for the detection of dopamine released from cell populations. Analyst. 2013;138:3651–9.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Caviglia C, Carminati M, Heiskanen A, Vergani M, Ferrari G, Sampietro M, et al. Quantitative label-free cell proliferation tracking with a versatile electrochemical impedance detection platform. J Phys Conf Ser. 2012;407:012029.

    CAS  Article  Google Scholar 

  29. 29.

    Hoelder S, Clarke PA, Workman P. Discovery of small molecule cancer drugs: successes, challenges and opportunities. Mol Oncol. 2012;6:155–76.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Hengel SR, Spies MA, Spies M. Small-molecule inhibitors targeting DNA repair and DNA repair deficiency in research and cancer therapy. Cell Chem Biol. 2017;24:1101–19.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Belizário JE, Sangiuliano BA, Perez-Sosa M, Neyra JM, Moreira DF. Using pharmacogenomic databases for discovering patient-target genes and small molecule candidates to cancer therapy. Front Pharmacol. 2016;7:312.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Rani K, Paliwal S. A review on targeted drug delivery: its entire focus on advanced therapeutics and diagnostics. Sch J Appl Med Sci. 2014;2:328–31.

    CAS  Article  Google Scholar 

  33. 33.

    Mishra N, Pant P, Jaiswal J. Targeted drug delivery : a review targeted drug delivery : a review. Am J PharmTechnol Res. 2016;6:1–24.

    CAS  Google Scholar 

  34. 34.

    Sharma G, Anabousi S, Ehrhardt C, Kumar MNVR. Liposomes as targeted drug delivery systems in the treatment of breast cancer. J Drug Target. 2006;14:301–10.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Samad A, Sultana Y, Aqil M. Liposomal drug delivery systems: an update review. Curr Drug Deliv. 2007;4:297–305.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Sercombe L, Veerati T, Moheimani F, Wu SY, Sood AK, Hua S. Advances and challenges of liposome assisted drug delivery. Front Pharmacol. 2015;6:286.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Verhoef JJF, Anchordoquy TJ. Questioning the use of PEGylation for drug delivery. Drug Deliv Transl Res. 2013;3:499–503.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Paliwal SR, Paliwal R, Vyas SP. Drug delivery a review of mechanistic insight and application of pH-sensitive liposomes in drug delivery a review of mechanistic insight and application of pH-sensitive liposomes in drug delivery. Drug Deliv. 2015;22:231–42.

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Fouladi F, Steffen KJ, Mallik S. Enzyme-responsive liposomes for the delivery of anticancer drugs. Bioconjug Chem. 2017;28:857–68.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Deshpande PP, Biswas S, Torchilin VP. Current trends in the use of liposomes for tumor targeting. Nanomedicine (Lond). 2013;8:1509–28.

    CAS  Article  Google Scholar 

  41. 41.

    Gialeli C, Theocharis AD, Karamanos NK. Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting. FEBS J. 2011;278:16–27.

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Jabłońska-Trypuć A, Matejczyk M, Rosochacki S, Jabłon AJ, Trypuć J-T. Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM) enzymes in collagen degradation, as a target for anticancer drugs Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM) enzymes in collagen degradation, as a target for anticancer drugs. J Enzym Inhib Med Chem. 2016;31:177–83.

    CAS  Article  Google Scholar 

  43. 43.

    Gjetting T, Jølck RI, Andresen TL. Effective nanoparticle-based gene delivery by a protease triggered charge switch. Adv Healthc Mater. 2014;3:1107–18.

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Kelland L. The resurgence of platinum-based cancer chemotherapy. Nat Rev Cancer. 2007;7:573–84.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Raymond E, Faivre S, Woynarowski JM, Chaney SG. Oxaliplatin: mechanism of action and antineoplastic activity. Semin Oncol. 1998;25:4–12.

    CAS  PubMed  Google Scholar 

  46. 46.

    Adams RA, Meade AM, Seymour MT, Wilson RH, Madi A, Fisher D, et al. Intermittent versus continuous oxaliplatin and fluoropyrimidine combination chemotherapy for first-line treatment of advanced colorectal cancer: results of the randomised phase 3 MRC COIN trial. Lancet Oncol. 2011;12:642–53.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Alcindor T, Beauger N. Oxaliplatin: a review in the era of molecularly targeted therapy. Curr Oncol. 2011;18:18–25.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Seeland S, Kettiger H, Murphy M, Treiber A, Giller J, Kiss A, et al. ATP-induced cellular stress and mitochondrial toxicity in cells expressing purinergic P2X7 receptor. Pharmacol Res Perspect. 2015;3:1–13.

    Article  Google Scholar 

  49. 49.

    Meissner R, Eker B, Kasi H, Bertsch A, Renaud P. Distinguishing drug-induced minor morphological changes from major cellular damage via label-free impedimetric toxicity screening. Lab Chip. 2011;11:2352–61.

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Kobayashil H, Takemura Y, Ohnuma T. Relationship between tumor cell density and drug concentration and the cytotoxic effects of doxorubicin or vincristine: mechanism of inoculum effects*. Cancer Chemother Pharmacol. 1992;31:6–10.

    Article  Google Scholar 

  51. 51.

    Straubinger RM, Hong K. Endocytosis of liposomes and intracellular fate of encapsulated molecules : encounter with a low pH compartment after internalization in coated vesicles. Cell. 1983;32:1069–79

  52. 52.

    Yoshida A, Sakai N, Uekusa Y, Imaoka Y, Itagaki Y, Suzuki Y, et al. Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis. PLoS Biol. 2018:16.

Download references


We acknowledge Rasmus Eliasen for the help with liposome de-PEGylation.


The authors received funding from the EU for the FP7 project EXCELL (NMP4-SL-2008-214706) and the Horizon 2020 MSCA-ITN project Training4CRM (H2020-MSCA-ITN-2016). Kinga Zór received financial support during the preparation of the manuscript from the Danish National Research Foundation (DNRF122) and Villum Fonden (Grant No. 9301) for Intelligent Drug Delivery and Sensing Using Microcontainer and Nanomechanics (IDUN).

Author information



Corresponding authors

Correspondence to Claudia Caviglia or Kinga Zór or Jenny Emnéus.

Ethics declarations

Conflict of interest

The authors declare that there are no conflicts of interest.

Human participants and/or animals

The studies presented in this article comprise neither human participants nor animals.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Published in the topical collection featuring Female Role Models in Analytical Chemistry.

Electronic supplementary material


(PDF 5 kb)


(AVI 8045 kb)


(AVI 7987 kb)


(AVI 8160 kb)


(AVI 8421 kb)


(AVI 6879 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Caviglia, C., Garbarino, F., Canali, C. et al. Monitoring cell endocytosis of liposomes by real-time electrical impedance spectroscopy. Anal Bioanal Chem 412, 6371–6380 (2020).

Download citation


  • Real-time monitoring
  • Electrical impedance spectroscopy
  • Cell morphology
  • Matrix metalloproteinase
  • Cytotoxicity
  • Liposome endocytosis