title: Integrated multiplatform method for in vitro quantitative assessment of cellular uptake for fluorescent polymer nanoparticles.
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.
authors.forename: Raffaele
authors.initials: R
authors.lastname: Ferrari
authors.forename: Monica
authors.initials: M
authors.lastname: Lupi
authors.forename: Francesca
authors.initials: F
authors.lastname: Falcetta
authors.forename: Paolo
authors.initials: P
authors.lastname: Bigini
authors.forename: Katia
authors.initials: K
authors.lastname: Paolella
authors.forename: Fabio
authors.initials: F
authors.lastname: Fiordaliso
authors.forename: Cinzia
authors.initials: C
authors.lastname: Bisighini
authors.forename: Mario
authors.initials: M
authors.lastname: Salmona
authors.forename: Maurizio
authors.initials: M
authors.lastname: D'Incalci
authors.forename: Massimo
authors.initials: M
authors.lastname: Morbidelli
authors.forename: Davide
authors.initials: D
authors.lastname: Moscatelli
authors.forename: Paolo
authors.initials: P
authors.lastname: Ubezio
mesh_terms: D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry; D045744:Cell Line, Tumor; D004337:Drug Carriers / Q000737:chemistry; D005260:Female; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry*; D005609:Free Radicals; D007700:Kinetics; D015674:Mammary Neoplasms, Animal / Q000473:pathology; D051379:Mice; D018613:Microscopy, Confocal; D053758:Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D011108:Polymers / Q000737:chemistry; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D001672:Biocompatible Materials; D004337:Drug Carriers; D005456:Fluorescent Dyes; D005609:Free Radicals; D011108:Polymers; D012235:Rhodamines; C029773:rhodamine B