Near-infrared excitable probes and nanoprobes for the intracellular detection and quantification of nitric oxide

Arnau Del Valle, Carla (2022) Near-infrared excitable probes and nanoprobes for the intracellular detection and quantification of nitric oxide. Doctoral thesis, University of East Anglia.

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Abstract

The main objective of this thesis was to develop novel near-infrared (NIR) excitable fluorescent systems for the intracellular detection of nitric oxide (NO). NO is a double-edged sword in biology depending on its concentration and intracellular location. Therefore, the detection and quantification of NO is crucial to understand its role in biology. The systems were designed to be NIR excitable to achieve high photostability, high biological tissue penetration and minimal photodamage upon long-term irradiation. Additionally, the use of nanoparticles was chosen to gain water solubility and improved stability, and to achieve tuneability that allowed for further functionalisation to incorporate ratiometric pairs for the quantification of intracellular NO.

A probe for the detection of NO was synthesised consisting on a NIR excitable fluorophore (1,8- naphthalimide) modified with a o-phenylenediamine moiety. The reaction of the probe with NO yielded an enhancement of the fluorescence emission intensity via the cancellation of the photoinduced electron transfer (PET) mechanism between the o-phenylenediamine and the fluorescent core. The sensitivity, selectivity, pH stability and the ability to detect NO in acidic pHs were examined in solution. The probe was used to detect endogenous and exogenous NO in a variety of cellular environments including mouse and human macrophages and endothelial cells. Confocal laser scanning microscopy (CLSM), images and fluorescence emission spectra, multiphoton microscopy and flow cytometry were used to confirm the intracellular NO detection by the probe.

Following the excellent performance of the molecular probe, a novel NIR excitable nanoprobe was synthesised using gold nanoparticles (AuNPs). A thiolated derivative ligand of the NO sensitive probe was synthesised and used for the functionalisation of AuNPs. Upon optimisation and full characterisation of the nanoprobe in solution, its capability to detect intracellular NO levels was confirmed in mouse and human macrophages, endothelial cells and breast cancer cells. The NO detection was confirmed using CLSM recording images and fluorescence emission spectra. Multiphoton microscopy and flow cytometry were also used to further confirm the ability of the nanoprobe to detect intracellular NO. The potential of the nanoprobe to detect different concentrations of exogenous NO in a breast cancer cell line was also confirmed.

To quantify NO within the cells, a ratiometric nanoprobe was developed functionalising AuNPs with two fluorescence ligands, a NO sensitive and a reference. As for previous systems, the fluorescence emission of the NO sensitive ligand was quenched in the absence of NO and enhanced upon addition of NO. Simultaneously, the fluorescence emission intensity of the reference ligand remained constant in the presence of NO allowing for ratiometric measurements. Following the study of its sensitivity, selectivity and pH stability, the ratiometric nanoprobe was used to detect intracellular NO in breast cancer cells. Additionally, a calibration curve in the CLSM was calculated between the ratio of intensities of both ligands and the concentration of NO within mouse macrophages using a NO donor and the quantification of endogenous NO was achieved in stimulated mouse macrophages.

Other strategies for the development of ratiometric nanoprobes for the quantification of NO were studied using 1) AuNPs functionalised with two NO sensitive ligands and 2) quantum dots (QDs) as reference signal functionalised with a NO sensitive ligand. Although promising results were obtained with these nanosystems, further optimisation is required prior to be used intracellularly.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Chemistry
Depositing User:	Nicola Veasy
Date Deposited:	30 Nov 2022 14:08
Last Modified:	30 Nov 2022 14:08
URI:	https://ueaeprints.uea.ac.uk/id/eprint/89974
DOI:

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