1. Integrated multiplatform method forin vitroquantitative assessment of cellular uptake for fluorescent polymer nanoparticles
- Author
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Massimo Morbidelli, Francesca Falcetta, Davide Moscatelli, Cinzia Bisighini, Maurizio D'Incalci, Paolo Bigini, Raffaele Ferrari, Fabio Fiordaliso, Katia Paolella, Mario Salmona, Paolo Ubezio, and Monica Lupi
- Subjects
Materials science ,Free Radicals ,Polymers ,education ,Nanoparticle ,Biocompatible Materials ,Mammary Neoplasms, Animal ,Bioengineering ,Nanotechnology ,Fluorescence spectroscopy ,Flow cytometry ,Mice ,chemistry.chemical_compound ,Cell Line, Tumor ,Fluorometer ,Rhodamine B ,medicine ,Animals ,General Materials Science ,Particle Size ,Electrical and Electronic Engineering ,health care economics and organizations ,Fluorescent Dyes ,chemistry.chemical_classification ,Drug Carriers ,Microscopy, Confocal ,medicine.diagnostic_test ,Rhodamines ,Mechanical Engineering ,technology, industry, and agriculture ,General Chemistry ,Polymer ,Flow Cytometry ,Fluorescence ,Kinetics ,Spectrometry, Fluorescence ,chemistry ,Mechanics of Materials ,Drug delivery ,Biophysics ,Nanoparticles ,Female - 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 10(3)-10(4) polymer NPs in a short time, reaching an intracellular concentration 15 times higher than the external one.
- Published
- 2014
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