Integrated multiplatform method for in vitro quantitative assessment of cellular uptake for fluorescent polymer nanoparticles

Raffaele Ferrari, Monica Lupi, Francesca Falcetta, Paolo Bigini, Katia Paolella, Fabio Fiordaliso, Cinzia Bisighini, Mario Salmona, Maurizio D'Incalci, Massimo Morbidelli, Davide Moscatelli, Paolo Ubezio

Research output: Contribution to journalArticlepeer-review

Abstract

Studies of cellular internalization of nanoparticles (NPs) play a paramount role for the design of efficient drug delivery systems, but so far they lack a robust experimental technique able to quantify the NP uptake in terms of number of NPs internalized in each cell. In this work we propose a novel method which provides a quantitative evaluation of fluorescent NP uptake by combining flow cytometry and plate fluorimetry with measurements of number of cells. Single cell fluorescence signals measured by flow cytometry were associated with the number of internalized NPs, exploiting the observed linearity between average flow cytometric fluorescence and overall plate fluorimeter measures, and previous calibration of the microplate reader with serial dilutions of NPs. This precise calibration has been made possible by using biocompatible fluorescent NPs in the range of 20-300 nm with a narrow particle size distribution, functionalized with a covalently bonded dye, Rhodamine B, and synthesized via emulsion free-radical polymerization. We report the absolute number of NPs internalized in mouse mammary tumor cells (4T1) as a function of time for different NP dimensions and surface charges and at several exposure concentrations. The obtained results indicate that 4T1 cells incorporated 103-104 polymer NPs in a short time, reaching an intracellular concentration 15 times higher than the external one.

Original languageEnglish
Article number045102
JournalNanotechnology
Volume25
Issue number4
DOIs
Publication statusPublished - Jan 31 2014

Keywords

  • cellular uptake
  • endocytosis
  • flow cytometry
  • imaging
  • nanoparticles

ASJC Scopus subject areas

  • Bioengineering
  • Chemistry(all)
  • Electrical and Electronic Engineering
  • Mechanical Engineering
  • Mechanics of Materials
  • Materials Science(all)

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