Detection of PLGA-based nanoparticles at a single-cell level by synchrotron radiation FTIR spectromicroscopy and correlation with X-ray fluorescence microscopy

Lorella Pascolo,¹ Barbara Bortot,¹ Nuria Benseny-Cases,² Alessandra Gianoncelli,³ Giovanni Tosi,⁴ Barbara Ruozi,⁴ Clara Rizzardi,⁵ Eleonora De Martino,¹ Maria Angela Vandelli,⁴ Giovanni Maria Severini<sup>1

1</sup>Institute for Maternal and Child Health, Istituto di Ricovero e Cura a Carattere Scientifico Burlo Garofolo, Trieste, Italy; ²European Synchrotron Radiation Facility, Polygone Scientifique Louis Néel, Grenoble, France; ³Elettra-Sincrotrone Trieste, Area Science Park, Basovizza, Trieste, Italy; ⁴Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; ⁵Department of Anatomical Pathology, Department of Pathology and Forensic Medicine, University of Trieste, Trieste, Italy

Abstract: Poly-lactide-co-glycolide (PLGA) is one of the few polymers approved by the US Food and Drug Administration as a carrier for drug administration in humans; therefore, it is one of the most used materials in the formulation of polymeric nanoparticles (NPs) for therapeutic purposes. Because the cellular uptake of polymeric NPs is a hot topic in the nanomedicine field, the development of techniques able to ensure incontrovertible evidence of the presence of NPs in the cells plays a key role in gaining understanding of their therapeutic potential. On the strength of this premise, this article aims to evaluate the application of synchrotron radiation-based Fourier transform infrared spectroscopy (SR-FTIR) spectromicroscopy and SR X-ray fluorescence (SR-XRF) microscopy in the study of the in vitro interaction of PLGA NPs with cells. To reach this goal, we used PLGA NPs, sized around 200 nm and loaded with superparamagnetic iron oxide NPs (PLGA-IO-NPs; Fe₃O₄; size, 10–15 nm). After exposing human mesothelial (MeT5A) cells to PLGA-IO-NPs (0.1 mg/mL), the cells were analyzed after fixation both by SR-FTIR spectromicroscopy and SR-XRF microscopy setups. SR-FTIR-SM enabled the detection of PLGA NPs at single-cell level, allowing polymer detection inside the biological matrix by the characteristic band in the 1,700–2,000 cm^-1 region. The precise PLGA IR-signature (1,750 cm^-1 centered pick) also was clearly evident within an area of high amide density. SR-XRF microscopy performed on the same cells investigated under SR-FTIR microscopy allowed us to put in evidence the Fe presence in the cells and to emphasize the intracellular localization of the PLGA-IO-NPs. These findings suggest that SR-FTIR and SR-XRF techniques could be two valuable tools to follow the PLGA NPs’ fate in in vitro studies on cell cultures.

Keywords: PLGA-NPs, cell targeting, SR-FTIR, SR-XRF, imaging

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