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Assessing the Stability of Fluorescently Encoded Nanoparticles in Lysosomes by Using Complementary Methods.

ABSTRACT: Nanoparticles (NPs) are promising tools in biomedical research. In vitro testing is still the first method for initial evaluation; however, NP colloidal behavior and integrity, in particular inside cells (that is, in lysosomes), are largely unknown and difficult to evaluate because of the complexity of the environment. Furthermore, while the majority of NPs are usually labeled with fluorescent dyes for tracking purposes, the effect of the lysosomal environment on the fluorophore properties, as well as the ensuing effects on data interpretation, is often only sparsely addressed. In this work, we have employed several complementary analytical methods to better understand the fate of fluorescently encoded NPs and identify potential pitfalls that may arise from focusing primary analysis on a single attribute, for example, fluorophore detection. Our study shows that in a lysosomal environment NPs can undergo significant changes resulting in dye quenching and distorted fluorescence signals.

SUBMITTER: Milosevic AM

PROVIDER: S-EPMC5659134 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Assessing the Stability of Fluorescently Encoded Nanoparticles in Lysosomes by Using Complementary Methods.

Milosevic Ana M AM Rodriguez-Lorenzo Laura L Balog Sandor S Balog Sandor S Monnier Christophe A CA Petri-Fink Alke A Rothen-Rutishauser Barbara B

Angewandte Chemie (International ed. in English) 20170922 43

Nanoparticles (NPs) are promising tools in biomedical research. In vitro testing is still the first method for initial evaluation; however, NP colloidal behavior and integrity, in particular inside cells (that is, in lysosomes), are largely unknown and difficult to evaluate because of the complexity of the environment. Furthermore, while the majority of NPs are usually labeled with fluorescent dyes for tracking purposes, the effect of the lysosomal environment on the fluorophore properties, as w ...[more]

PMID: 28767191

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Project description:BackgroundIn the field of nanoscience there is an increasing interest to follow dynamics of nanoparticles (NP) in cells with an emphasis on endo-lysosomal pathways and long-term NP fate. During our research on this topic, we encountered several pitfalls, which can bias the experimental outcome. We address some of these pitfalls and suggest possible solutions. The accuracy of fluorescence microscopy methods has an important role in obtaining insights into NP interactions with lysosomes at the single cell level including quantification of NP uptake in a specific cell type.MethodsHere we use J774A.1 cells as a model for professional phagocytes. We expose them to fluorescently-labelled amorphous silica NP with different sizes and quantify the colocalization of fluorescently-labelled NP with lysosomes over time. We focus on confocal laser scanning microscopy (CLSM) to obtain 3D spatial information and follow live cell imaging to study NP colocalization with lysosomes.ResultsWe evaluate different experimental parameters that can bias the colocalization coefficients (i.e., Pearson's and Manders'), such as the interference of phenol red in the cell culture medium with the fluorescence intensity and image post-processing (effect of spatial resolution, optical slice thickness, pixel saturation and bit depth). Additionally, we determine the correlation coefficients for NP entering the lysosomes under four different experimental set-ups. First, we found out that not only Pearson's, but also Manders' correlation coefficient should be considered in lysosome-NP colocalization studies; second, there is a difference in NP colocalization when using NP of different sizes and fluorescence dyes and last, the correlation coefficients might change depending on live-cell and fixed-cell imaging set-up.ConclusionsThe results summarize detailed steps and recommendations for the experimental design, staining, sample preparation and imaging to improve the reproducibility of colocalization studies between the NP and lysosomes.

Dataset Information

Assessing the Stability of Fluorescently Encoded Nanoparticles in Lysosomes by Using Complementary Methods.

Publications

Assessing the Stability of Fluorescently Encoded Nanoparticles in Lysosomes by Using Complementary Methods.

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