Determination of nanoparticle localisation within subcellular organelles in vitro using Raman spectroscopy

Abstract

Ease of sample preparation, narrow spectral bandwidth and minimal influence from water are features of Raman spectroscopy which make it a powerful, label-free way to study a wide range of biological structures and phenomena. In this context, given the concerns over their toxicology arising from their increased production and use, evaluation of nanoparticle uptake and localisation in biological systems and determination of the mechanisms of subcellular interaction and trafficking can provide long-term solutions for nanotoxicology, and potential strategies for nanomedicine. In this study, Raman spectroscopy is explored to monitor the sequential trafficking of nanoparticles through subcellular organelles in-vitro and to establish the spectroscopic signatures of those organelles. A549 human lung carcinoma cells were exposed to 40 nm carboxylate-modified and fluorescently-labelled polystyrene nanoparticles for 4, 12 and 24hrs. Raman spectroscopy was applied to nanoparticle exposed cells to determine the localisation within cellular compartments. Confocal laser scanning fluorescence microscopy (CLSM) with different organelle staining kits confirmed the localisation of the nanoparticles in organelles at the chosen exposure periods and colocalization was quantified using ImageJ with the JACoP colocalisation plugin. To confirm nanoparticle localisation and elucidate the spectroscopic signatures of the different subcellular organelles, a combination of K-means clustering (KMCA) and Principal components analysis (PCA) was applied to the Raman spectroscopic maps. The study showed the applicability of the techniques for elucidation of the localisation of polystyrene nanoparticles within the cell as well as determination of their local environment, differentiating the spectral signatures of intracellular compartments such as endosomes, lysosomes and endoplasmic reticulum, in a completely label free manner.

In vitro monitoring of time and dose dependent cytotoxicity of aminated nanoparticles using Raman spectroscopy

“…Previous studies have confirmed that the polystyrene nanoparticles (PS-COOH) are taken up into endosomes and carried to lysosomes 15,26,[51][52][53] . Most of the nanoparticles are observed in lysosomes after 8 hr particle exposure.…”

“…24 . Also, the ability of the Raman spectroscopy to differentiate different cellular compartments, such as endosomes and lysosomes, as well as localisation of the nanoparticles in these compartments has been demonstrated 15 . For the case of Carbon Nanotubes, Knief et al 25 probed the correlation of Raman biospectroscopic markers with conventional cytotoxicity assays, indicating that Raman spectroscopy can potentially be employed as a single, label free assay to localise nanoparticles, identify trafficking mechanisms and evaluate nanoparticle toxicity.…”

“…Basically, particles are taken up across the cell membrane by the formation of early endosomes which carry the nanoparticles to the lysosomes. After particles are engulfed by lysosomes, they are carried to the endoplasmic reticulum 15,33,34 . In the case of PS-NH 2 , exposure of cells to the nanoparticles can induce toxicity due to the formation of Reactive oxygen species (ROS).…”

“…Aminated polystyrene nanoparticles (PS-NH 2 ) are chosen as model nanoparticles due to their welldocumented cytotoxic mechanisms. Human lung adenocarcinoma (A549) cells were chosen for consistency with other studies 15,26 , which show common modes of action in a number of cell lines, and as they act as models for human exposure by inhalation. Cells were exposed to PS-NH 2 in different doses and for different exposure times.…”

Determination of spectral markers of cytotoxicity and genotoxicity using in vitro Raman microspectroscopy: cellular responses to polyamidoamine dendrimer exposure

“…[7][8][9][10] Moreover, Raman microspectroscopy has been used to localise nanoparticles in cellular compartments. [11][12][13] The dose and time dependent effects of aminated polystyrene nanoparticles (PS-NH 2 ) on different cell lines has been spectroscopically characterised 14 and the mechanisms leading to cell death have been differentiated between cancerous and non-cancerous cell lines with the aid of multivariate analysis techniques. 15 In the current study, the applicability of Raman microspectroscopy as a label-free and in vitro high content screening technique to identify spectral markers of cytotoxicity and genotoxicity is demonstrated.…”