AbstractExtracellular vesicles (EVs) are phospholipid bilayer enclosing vesicles with sizes ranging from 30–2000 nm, and are secreted by most cells types. They are increasingly gaining attention for their role in cell-cell communication, disease progression and therapeutic effect, as well as for their potential in diagnostics as disease biomarkers and as therapeutic drug delivery systems. Integrating imagingbased EV tracking techniques at the whole-body level into the development of EVs will enable monitoring their biodistribution in vivo, and also facilitate their potential clinical translation. Among the different medical imaging modalities available, positron emission tomography (PET) imaging stands out as it allows short- and long-term whole-body tracking of radiolabelled compounds in both animals and humans, and with excellent sensitivity and quantification properties. In this work it was hypothesised that the PET radiotracer [89Zr]Zr(oxinate)4, recently developed as a cell and liposome radiolabelling agent, should allow intraluminal radiolabelling of EVs. Therefore, the work presented in this thesis is aimed at developing a simple, efficient and direct EV radiolabelling method using [89Zr]Zr(oxinate)4, and to test it both in vitro and in vivo using PET imaging.
Firstly, two milder, simpler and improved [89Zr]Zr(oxinate)4 syntheses were developed and optimised for EV radiolabelling. Compared to the standard chloroform extraction method, previously used for [89Zr]Zr(oxinate)4 synthesis, these new radiosynthetic methods ensured that excess reagents do not damage the EVs during the process, and that sufficient EV radiolabelling can be achieved for in vivo PET imaging. Following conversion of [89Zr]Zr(oxalate)4 to [89Zr]ZrCl4, either oxine in ethanol (Method 1), or an oxine kit formulation (Method 2) was added. For both methods, 40 μg oxine was sufficient for the formation of [89Zr]Zr(oxinate)4 complex with a radiochemical yield of >90%. Method 2, however, yielded highly concentrated [89Zr]Zr(oxinate)4 in a small volume, more suitable for in vivo imaging of EVs.
Secondly, small EVs (sEVs or exosomes) were isolated and characterised using several techniques (nanoparticle tracking analysis, dot blot and flow cytometry). All five types of sEVs (MDA-MB-231, B16-F10.GFP, MDA-MB-231.CD63-GFP, PANC1 and 4T1) were of 50% stable in PBS over 24 h.
In vitro cell uptake studies demonstrated that both 89Zr-labelled PANC1 and 4T1 sEVs were sufficiently stable to enter cells. However, macrophage uptake of 4T1 sEVs could not be inhibited by fucoidan (a known macrophage scavenger receptor blocker). Nevertheless, PET-CT imaging of 89Zr-PANC1 sEVs in healthy mice over 24 h demonstrated rapid uptake of these vesicles in liver, spleen, brain and several lymph nodes. In vivo imaging also revealed differential biodistribution of intact PANC1 sEVs compared to intentionally heat-damaged ones, with significantly reduced spleen uptake for the latter. Since 89Zr is a bone-tropic radionuclide, the spleen:bone uptake ratio highlighted the difference between intact (8.1 ± 2.6) and heat-damaged (2.6 ± 1.7) sEV uptake. This led to the proposal of using this ratio as an imaging biomarker for PANC1 EVs’ stability in vivo when using this radiolabelling method.
Finally, following the development of intra-articularly administered neutrophilderived EVs as a potential nanomedicine for rheumatoid arthritis (RA) and based on the results described above, the biodistribution of 89Zr-labelled neutrophil EVs in a RA mouse model was evaluated. It was hypothesised that intravenously injected neutrophil EVs will preferentially accumulate in the inflamed joints. Neutrophil EVs were isolated from healthy human blood and a RLY of 7.8 ± 0.6% was achieved after incubation with [89Zr]Zr(oxinate)4. Using PET imaging, significantly higher accumulation of 89Zr-neutrophil EVs in liver and lymph nodes at the peak of inflammation was observed compared to at an earlier stage. However, no significant difference in inflamed joint uptake was observed.
To summarise, in this thesis, it has been demonstrated that EVs can successfully and efficiently be radiolabelled with [89Zr]Zr(oxinate)4, without substantial effect on their characteristics, and its limitations and advantages were identified. 89Zrlabelled EVs, derived from both cancer cell lines and primary cells can be imaged in vivo for up to 24 h using PET imaging. It is hoped this method will facilitate further studies aiming at understanding the biodistribution of EVs in vivo.
|Date of Award||1 Nov 2022|
|Supervisor||Rafael T. M. de Rosales (Supervisor) & Khuloud Al-Jamal (Supervisor)|