Exploring Pretargeted PET Imaging Strategies for Liposomal Nanomedicines

Student thesis: Doctoral ThesisDoctor of Philosophy


Liposomal nanomedicines have emerged as efficient drug carriers. These nanomedicines can passively accumulate in tumours/inflammation through the enhanced permeation and retention (EPR) phenomenon while reducing off-site drug toxicity. However, tumoral targeting can be variable, leading to heterogeneous uptake among tumour types and fluctuating clinical efficacies in patients. While nuclear imaging (e.g., PET and SPECT) provides valuable spatiotemporal (whole-body biodistribution) information and informed clinical management, it requires long-lived radionuclides and produces high radiation doses. Therefore, pretargeted nuclear imaging could improve our ability to study nanomedicine with the advantage of lower radiation doses and allow the use of short-lived radionuclides that would not otherwise be compatible with the long half-life of PEGylated liposomes.

In this thesis we have explored two strategies for pretargeted imaging of liposomal nanomedicines. Chapter 2 reports a novel metal chelation pretargeting system based on tris(hydroxypyridinone)(THP). The THP chelator can be radiolabelled at low concentrations with the generator-produced radionuclide gallium-68 (t1/2 = 68 min) at room temperature and physiological conditions with high efficiency, and it could be used to create a novel pretargeted nuclear imaging system for PEGylated liposomes. The bifunctional chelator THP-NCS was conjugated to a DSPE-PEG2000 amine phospholipid and incorporated into the bilayer of the PEGylated liposomes to give THP-PL-liposomes. Pretargeting experiments were performed in vitro under high dilution conditions in serum, showing high THP-PL-liposomes radiolabelling. In vivo imaging experiments were performed in healthy BalB/c mice at multiple time points. Pretargeting group (administration of THP-PL-liposomes, followed either after 5 h or 27 h by neutralised 68Ga3+(68Ga-acetate)) was compared with positive control (administration of directly radiolabelled PEGylated liposomes (67Ga-THP-PL-liposomes)) and negative control (administration of 68Ga-only). At earlier time point, when the liposomes were majorly circulating in the blood, pretargeting was observed, showing radioactive accumulation in the blood pool, heart, liver, and spleen. No difference between the in vivo biodistribution of directly labelled liposomes (positive control) and the pretargeting group was observed, indicating an effective pretargeting. However, lower pretargeting was observed at the later time point, with a slight difference between the pretargeting group and the negative control. These findings suggests that pretargeting via metal chelation is not feasible at the later time point possible due to the inability of the 68Ga to reach the THP-PL-liposomes accumulated in the liver and spleen.

This metal chelation pretargeting was also examined for the bone targeting bisphosphonate THP-Pam, to examine the ability of pretargeting in targets other than the liver and spleen. Imaging experiments were performed in healthy BalB/c mice at multiple time points (5 and 27 hours). The pretargeting group (administration of THP-Pam followed either after 5 h or 27 h by neutralised 68Ga3+ (68Ga-acetate)) was compared with both a directly radiolabelled THP-Pam (68Ga-THP-Pam) and a 68Ga-only (negative control). Moderate pretargeting was observed at both time points showing higher observed uptake in the target tissues of bones compared to the negative control (p<0.05), but lower than directly labelled THP-Pam. While this metal chelation pretargeting method did not work for the liver and spleen localised agents, it worked effectively for tracking agents in the blood, heart, and bones.

In chapter 3, a biorthogonal pretargeted nuclear imaging system that has shown promise with antibodies was examined for PEGylated liposomes. We hypothesised that IEDDA biorthogonal reaction between tetrazine and transcyclooctene known to take place in physiological conditions with high reaction rates, could be used to create a pretargeted nuclear imaging system for PEGylated liposomes. To this end, we made two components of this pretargeting system: TCO-PL-liposomes (PEGylated liposomes with a transcycloctene containing phospholipid embedded into the bilayer) and THP-tetrazine (TCO reactive THP bioconjugate with high affinity for 68Ga). In vitro pretargeting experiments performed in high dilution conditions in PBS and serum showed high biorthogonal reaction rates between TCO-PL-liposomes and 68Ga-THP-tetrazine. Imaging experiments were performed in healthy BalB/c mice at 4 h and 26 h post-injection. Pretargeting TCO-PL-liposomes followed either by neutralised 68Ga-THP-tetrazine with both a directly radiolabelled PEGylated liposomes (67Ga-TCO-PL-liposomes) and a 68Ga-THP-tetrazine (negative control) were compared. At both time points, pretargeting was observed, showing a higher accumulation of radioactivity in the blood pool, heart, liver and spleen (target organs for PEGylated liposomes) compared to the negative control (p<0.05). These results were also confirmed in a fibrosarcoma tumour model in mice (p<0.05). However, no pretargeting was observed within the tumours. The radioactivity uptake observed in tumours in pretargeting group showed no significant difference to uptake observed in negative control and was low compared to directly labelled TCO-PL-liposomes.

In Chapter 4, we aim to tackle another significant disadvantage faced by liposomal nanomedicines, which is their inability to cross the blood brain barrier (BBB) and reach the brain, as well as the areas of interest (e.g., inflammation, tumours, and neurodegeneration). However, new methods to disrupt the BBB can make brain targets accessible to liposomes. Therefore, we examined whether rapid short pulse (RaSP) can deliver liposomes to the murine brain in vivo. Fluorescent DiD-PEGylated liposomes were synthesised and injected intravenously alongside microbubbles and applied focussed ultrasound at different pressures. The delivery and biodistribution were assessed by fluorescence imaging of the brain sections, and the safety profile of the sonicated brains was evaluated by histological staining. RaSP was shown to deliver liposomes locally across the BBB at 0.53 MPa with a more diffused and safer profile compared to the long pulse ultrasound sequence. Cellular uptake of liposomes was observed in neurons and microglia, while no uptake within astrocytes was observed in both RaSP and long pulse-treated brains.
Date of Award1 Jan 2023
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorRafael T. M. de Rosales (Supervisor) & James Choi (Supervisor)

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