Development and application of oxygen nanosensors for the assessment of molecular oxygen in 2D and 3D cell culture
Nanoparticle science is a rapidly developing field within a variety of scientific disciplines. Developing nanosensor-based nanoparticles for measuring intracellular oxygen is of great interest as a diagnostic tool. It helps to understand and investigate biological responses that affect health, the s...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English English |
| Published: |
2023
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| Subjects: | |
| Online Access: | https://eprints.nottingham.ac.uk/72292/ |
| _version_ | 1848800730531495936 |
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| author | Abohujel, Reem |
| author_facet | Abohujel, Reem |
| author_sort | Abohujel, Reem |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Nanoparticle science is a rapidly developing field within a variety of scientific disciplines. Developing nanosensor-based nanoparticles for measuring intracellular oxygen is of great interest as a diagnostic tool. It helps to understand and investigate biological responses that affect health, the status of the disease, and the mechanisms of curative therapies. The measurements are based on changes in luminescence intensity quenched by the increase in oxygen concentration. This approach provides information on cellular spatial and temporal measurement and the assessment of kinetics in multiple biological samples for different applications.
This study describes the development and characterisation of ratiometric oxygen nanosensors to improve the monitoring of oxygen in a biological system. Moreover, in order to obtain biological insights that would help to expand the knowledge of nanosensor trafficking, pH nanosensors were used to investigate intracellular pH responses. Thus, oxygen and pH nanosensors were tracked in situ to provide the intracellular fate that correlates with physiological relevant mechanisms. The ratiometric approaches were used in both oxygen and pH nanosensors. Oxygen nanosensors designed by conjugating a platinum porphyrin to the surface of the polyacrylamide matrix and entrapping 5-(and-6) - carboxytetramethylrhodamine (TAMRA) in polyacrylamide nanoparticles. Ratiometric nanosensors for pH sensing were prepared by entrapping the OG (Oregon green) and (FAM) Fluorophores 5- and 6-carboxyfluorescein sensitive fluorophores to pH and TAMRA insensitive fluorophore to pH in a polyacrylamide matrix.
Nanosensors were developed and fluorophores conjugating to polyacrylamide nanoparticles optimised and calibrated using fluorescence microscopy imaging. Both oxygen and pH nanosensors were investigated in terms of their co-localisation with lysosome and mitochondria in the cells. This was done to overcome the limitations of oxygen monitoring problems. Intracellular monitoring of oxygen by nanosensors brings hope for cellular responses to micro-environmental change especially responses that report cellular vital signs activity. Oxygen nanosensors have been applied to the biological systems of immune cells (THP-1 differentiated macrophage) and hepatic cells (HepG2 cells), resulting in five major findings:
1) Nanosensors were successfully delivered to macrophage and HepG2 cells without affecting cell viability and metabolic activity. In addition, calibration of sensors was conducted in fixed cells within a controllable oxygen chamber connected to a fluorescence microscope.
2) In studying nanosensors application, oxygen nanosensors were delivered to macrophages in order to control phenotype modulation by monitoring oxygen. However further investigation has revealed that Pt porphyrin modulates macrophage phenotype with continuous imaging. This is caused by enhancing ROS generation which leads to unreliable metabolic endpoint measurements. Therefore, application of nanosensors in biological system in this thesis focuses on studying oxygen monitoring in hepatic cells.
3) Nanosensors location were determined in hepatic cells with respect to lysosomes and mitochondria in order to help correlate oxygen consumption with cellular metabolic activity. It has been found that oxygen nanosensors are most likely to be distributed in the cytoplasm.
4) Endpoint measurement of oxygen has been assessed in hepatic cells exposed to different insults using imaging microscopy. Reported oxygen concentrations were supported with an apoptotic assay to understand the effect of cellular functionality.
5) Oxygen nanosensors were applied on 3D cellular model of spheroids to evaluate oxygen nanosensors in tumour model. Spheroid is a multicellular aggregate which is a complex architecture model of tumour. These characteristics limit the detection capability of oxygen sensing probe and failure of oxygen detection. With these challenges, oxygen sensors are becoming extremely useful for screening drugs by detecting oxygen gradients in spheroids. The kinetic of oxygen concentration was monitored to assess the effect of environmental reactions. |
| first_indexed | 2025-11-14T20:56:12Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-72292 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English English |
| last_indexed | 2025-11-14T20:56:12Z |
| publishDate | 2023 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-722922025-02-28T15:17:10Z https://eprints.nottingham.ac.uk/72292/ Development and application of oxygen nanosensors for the assessment of molecular oxygen in 2D and 3D cell culture Abohujel, Reem Nanoparticle science is a rapidly developing field within a variety of scientific disciplines. Developing nanosensor-based nanoparticles for measuring intracellular oxygen is of great interest as a diagnostic tool. It helps to understand and investigate biological responses that affect health, the status of the disease, and the mechanisms of curative therapies. The measurements are based on changes in luminescence intensity quenched by the increase in oxygen concentration. This approach provides information on cellular spatial and temporal measurement and the assessment of kinetics in multiple biological samples for different applications. This study describes the development and characterisation of ratiometric oxygen nanosensors to improve the monitoring of oxygen in a biological system. Moreover, in order to obtain biological insights that would help to expand the knowledge of nanosensor trafficking, pH nanosensors were used to investigate intracellular pH responses. Thus, oxygen and pH nanosensors were tracked in situ to provide the intracellular fate that correlates with physiological relevant mechanisms. The ratiometric approaches were used in both oxygen and pH nanosensors. Oxygen nanosensors designed by conjugating a platinum porphyrin to the surface of the polyacrylamide matrix and entrapping 5-(and-6) - carboxytetramethylrhodamine (TAMRA) in polyacrylamide nanoparticles. Ratiometric nanosensors for pH sensing were prepared by entrapping the OG (Oregon green) and (FAM) Fluorophores 5- and 6-carboxyfluorescein sensitive fluorophores to pH and TAMRA insensitive fluorophore to pH in a polyacrylamide matrix. Nanosensors were developed and fluorophores conjugating to polyacrylamide nanoparticles optimised and calibrated using fluorescence microscopy imaging. Both oxygen and pH nanosensors were investigated in terms of their co-localisation with lysosome and mitochondria in the cells. This was done to overcome the limitations of oxygen monitoring problems. Intracellular monitoring of oxygen by nanosensors brings hope for cellular responses to micro-environmental change especially responses that report cellular vital signs activity. Oxygen nanosensors have been applied to the biological systems of immune cells (THP-1 differentiated macrophage) and hepatic cells (HepG2 cells), resulting in five major findings: 1) Nanosensors were successfully delivered to macrophage and HepG2 cells without affecting cell viability and metabolic activity. In addition, calibration of sensors was conducted in fixed cells within a controllable oxygen chamber connected to a fluorescence microscope. 2) In studying nanosensors application, oxygen nanosensors were delivered to macrophages in order to control phenotype modulation by monitoring oxygen. However further investigation has revealed that Pt porphyrin modulates macrophage phenotype with continuous imaging. This is caused by enhancing ROS generation which leads to unreliable metabolic endpoint measurements. Therefore, application of nanosensors in biological system in this thesis focuses on studying oxygen monitoring in hepatic cells. 3) Nanosensors location were determined in hepatic cells with respect to lysosomes and mitochondria in order to help correlate oxygen consumption with cellular metabolic activity. It has been found that oxygen nanosensors are most likely to be distributed in the cytoplasm. 4) Endpoint measurement of oxygen has been assessed in hepatic cells exposed to different insults using imaging microscopy. Reported oxygen concentrations were supported with an apoptotic assay to understand the effect of cellular functionality. 5) Oxygen nanosensors were applied on 3D cellular model of spheroids to evaluate oxygen nanosensors in tumour model. Spheroid is a multicellular aggregate which is a complex architecture model of tumour. These characteristics limit the detection capability of oxygen sensing probe and failure of oxygen detection. With these challenges, oxygen sensors are becoming extremely useful for screening drugs by detecting oxygen gradients in spheroids. The kinetic of oxygen concentration was monitored to assess the effect of environmental reactions. 2023-07-22 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en cc_by https://eprints.nottingham.ac.uk/72292/1/PhD%20thesis_Reem%20Abohujel_%20Updated_1.docx.pdf application/pdf en cc_by https://eprints.nottingham.ac.uk/72292/2/End%20of%20PhD%20form%2014%20Jan.pdf Abohujel, Reem (2023) Development and application of oxygen nanosensors for the assessment of molecular oxygen in 2D and 3D cell culture. PhD thesis, University of Nottingham. Nanosensors |
| spellingShingle | Nanosensors Abohujel, Reem Development and application of oxygen nanosensors for the assessment of molecular oxygen in 2D and 3D cell culture |
| title | Development and application of oxygen nanosensors for the assessment of molecular oxygen in 2D and 3D cell culture |
| title_full | Development and application of oxygen nanosensors for the assessment of molecular oxygen in 2D and 3D cell culture |
| title_fullStr | Development and application of oxygen nanosensors for the assessment of molecular oxygen in 2D and 3D cell culture |
| title_full_unstemmed | Development and application of oxygen nanosensors for the assessment of molecular oxygen in 2D and 3D cell culture |
| title_short | Development and application of oxygen nanosensors for the assessment of molecular oxygen in 2D and 3D cell culture |
| title_sort | development and application of oxygen nanosensors for the assessment of molecular oxygen in 2d and 3d cell culture |
| topic | Nanosensors |
| url | https://eprints.nottingham.ac.uk/72292/ |