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Authors: Han, Shen
Title: Visualisation of nanoparticle-cell interactions by correlative microscopy
Online publication date: 5-Oct-2021
Language: english
Abstract: Nanoparticles (NPs) have been a part of our daily life and have emerged to be ideal drug carriers mainly due to their small size and controllable functionalisation for potential cell targeting. Since the 1990s, NPs have been used for clinical purposes, and they have been continuously showing their immense capacity in drug delivery such as the recent RNA vaccine. On one hand, engineered NPs provide a relatively stable carrier system for fragile but active agents like proteins and RNAs. On the other hand, NPs inevitably interact with components in biological milieu and the interaction is define and affect the efficacy of these NPs. Therefore, it is very crucial to understand and to visualise the interactions between the engineered NPs and cells in order to facilitate the smarter design of more effective nanomedecine. The aim of this thesis is to extract useful information from the intracellular journey of NPs by the leverage of various microscopic techniques. With the elemental mapping by energy-dispersive X-ray (EDX) spectroscopy in transmission electron microscopy (TEM), inorganic silica nanocapsules (SiNCs) were clearly identified from the organic cellular environment. It was demonstrated that different stages of SiNCs in HeLa cells including the initiate uptake and the distribution in late endosomes were pinpointed. The possible degradation of SiNCs was further confirmed by EDX elements mapping. It was proven that SiNCs were degradable in HeLa cells and could be a potential candidate for drug carrier despite their inorganic composition. In order to achieve an equally efficient identification of organic NPs in cells, light microscopy (LM) and electron microscopy (EM) were combined to image carbon-based fluorescent nanodiamonds (fNDs) with inherent fluorescence. The exploitation of in-dish and on-section correlative light and electron microcopy (CLEM) proposed general strategies to balance the preservation of fluorescence and cellular structures after EM preparations. Further applications of several imaging techniques in TEM revealed even single fND upon uptake and in a mitochondrion. By employing these precise imaging techniques to visualise fNDs in cells, single particle events like subcellular targeting in mitochondria or endosomal escape are able to be captured. The application of core-shell NCs for medical purposes have gained so much attention especially the NCs made from biocompatible materials such as proteins. In order to achieve the precise localisation of such NCs, on-section CLEM was performed to identify bovine serum albumin (BSA) NCs, marked with nanoplatelets (NPLs) in cells. With the help of the fluorescence from NPLs, the distribution of intracellular NCs were tracked at different time points. It was shown that the internalised BSA NCs followed the endolysosomal pathway and ended up in endolysosomes and late endosomes for degradation. This work shows how to trace the intracellular fate of protein NCs with the help of particles exhibiting inherent fluorescence. The ultimate goal to visualise intracellular polymeric NPs by on-section CLEM is to resolve the fluorescence from organic dyes which are used for the labelling. In order to achieve this goal, an optimised on-section CLEM workflow was developed for organic dye-labelled NPs and NCs. It was demonstrated that organic dyes BODIPY and Cy5 were preserved within polystyrene NPs (PS NPs) and hydroxyethyl starch (HES) NCs after EM preparation with osmium tetroxide (OsO4) and EPON resin embedding. This work presented for the first time how CLEM could be applied to localise intracellular polymeric NPs which are labelled with organic dyes. The most troublesome problem when developing polymeric NPs for medical applications is the formation of the protein corona. It represents the protein coat which absorbs onto the NPs once they are introduced into biological fluid and will eventually alter the biological behaviour of NPs. We took advantages of the optimised on-section CLEM to further explore the intracellular fate of protein corona. By performing volume CLEM with array tomography (AT) using a Cy5 labelled protein corona and BODIPY labelled PS NPs, the unique endosomal distribution of protein corona and NPs was illustrated within a 3-dimensional model. It was shown that the protein corona was gradually separated from the NPs. This happens during the maturation from early endosomes to late endosomes and multivesicular bodies (MVBs).
DDC: 570 Biowissenschaften
570 Life sciences
Institution: Johannes Gutenberg-Universität Mainz
Department: FB 10 Biologie
Sonderforschungsbereiche (SFB)
Place: Mainz
Version: Original work
Publication type: Dissertation
License: in Copyright
Information on rights of use:
Extent: III, 148 Seiten
Appears in collections:JGU-Publikationen

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