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Remote-Activated Delivery of Therapeutic Exosomes (RADoTE) via an Injectable PEG Hydrogel Carrier

Periodic Reporting for period 1 - RADoTE (Remote-Activated Delivery of Therapeutic Exosomes (RADoTE) via an Injectable PEG Hydrogel Carrier)

Reporting period: 2016-11-01 to 2018-10-31

The overall objective of the RADoTE project was to develop a hydrogel system that would improve extracellular vesicle (EV)-based therapies. EVs are cell-derived particles that contain unique biological content derived from their parent cells, and they can be internalized by target cells. Several research studies have suggested that EVs derived from stem cells might help to heal diseased tissues, such as damaged cardiac tissue following a heart attack. These cell-free biological particles could be useful for both tissue regeneration and drug delivery applications. However, because EVs are very small (about 120 nm in size), when they are injected into the blood stream they are rapidly cleared away, minimizing the intended therapeutic effects. For this project, an injectable hydrogel-based delivery system was used to control where EVs were administered, while keeping them stabilized until release from the hydrogel. The hydrogels designed for this project were degraded by tissue-penetrating near-infrared (NIR) light, meaning hydrogel degradation and EV release could be triggered on-demand by applying NIR light from outside of the body. To achieve this, the hydrogels contained UV-degradable crosslinks and embedded upconversion nanoparticles (UCNPs) that locally convert NIR light to UV. UCNPs represent an exciting new type of nanomaterial that can be used as light transducers for both imaging and photochemistry applications. During this project, a NIR-degradable hydrogel system was developed and validated, a model bioluminescent EV system enabled quantification and verification of bioactivity, and EV stability in degradable hydrogels and controlled release were assessed. Overall, these results provided proof-of-concept demonstration that this type of technology could be used in the future for controlled EV delivery. As a MSCA fellow, I co-authored publications in Nature Communications, Advanced Materials, ACS Nano, and Chemical Science, all of which are open access articles.

This ambitious project involved a collaboration with the National University of Singapore (NUS) to provide UCNPs as well as expertise and insight, but the majority of the work was possible due to the placement within the world-renowned Stevens Group at Imperial College London. Because of the interdisciplinary nature of the research plan, this diverse research group was the ideal host for this project. I benefited greatly from working alongside experts in chemistry, materials science, cell biology, and EV technologies.
The following summarizes the main research tasks and results from this fellowship:

1. The components required in order to fabricate photodegradable multi-arm PEG hydrogels were synthesized and characterized. A new thiol-functionalized o-nitrobenzyl group was designed and synthesized, which provides a highly useful new synthetic strategy to make thiolated photodegradable crosslinkers. Thiol-ene crosslinking chemistry is widely used for many types of biomaterials, so this new synthetic strategy will make photodegradable chemistry more accessible for many researchers.
2. UCNPs were incorporated into photodegradable hydrogels, and degradation in the presence of NIR was confirmed. It was demonstrated that hydrogel degradation was directly correlated with exposure to NIR. However, exposing cells to the continuous wave NIR laser resulted in cell death due to heating effects. To address this challenge and to improve the biocompatibility of the process, a controlled shutter system was introduced. This permitted high peak energy pulses to activate UCNPs, with improvements in cell viability.
3. Bioluminescent EVs were harvested and purified, and long-term stability in physiological conditions was verified. After screening several different labelling methods and cell types, the best signal was achieved with EVs expressing a bioluminescent protein. The bioluminescence signal emitted from EVs following exposure to an appropriate substrate molecule enabled EV quantification as well as assessment of their stability over time. It was found that EVs could be maintained for 1 week (with best results up to 4 days) in physiological conditions.
4. The rate of degradation for photodegradable hydrogels was controlled by varying the hydrogel formulation and NIR exposure time and intensity. The degradation rate of the photodegradable hydrogels was directly related to the release of entrapped cargo. The results from this work package verified that they hydrogel formulation and light exposure could be tuned in order to achieve a variety of desired controlled release characteristics.
5. It was verified that EVs could be entrapped in hydrogels and released on demand, and their cellular uptake characteristics remained intact. This work serves as preparation for the next stage of in vivo experiments to be conducted in mice, after the completion of the Marie Curie fellowship.

The results and key findings from this project were disseminated by presenting at international research conferences including the Gordon conference on Biomaterials in 2017, TERMIS-EU 2017 in Switzerland, and TERMIS-World Congress 2018 in Japan. I co-authored publications in Advanced Materials, Nature Communications, ACS Nano, and Chemical Science, which are all available open access and acknowledge MSCA funding. Two further publications are currently in preparation for submission to high-quality journals for novel biomaterials. During this funding period, I participated in several outreach activities as a way to disseminate my research interests and findings to broader audiences. I participated in the ‘Meet a Scientist’ event at the London Science Museum, presented my work to female secondary school students at the Women@Imperial outreach event, and conducted hydrogel demonstrations for the London International Youth Science Forum.
The results from this MSCA-funded project have progressed the state of the art in several research areas. First, a new synthetic strategy was developed to thiol-functionalize photodegradable linkers using simple solid-phase peptide synthesis techniques. This development should be very useful in enabling more researchers to synthesize and use these types of crosslinkers in their work. Typically, this type of chemistry is only implemented by research groups that have strong expertise in chemical synthesis. Second, a new type of bioluminescence labelling technique for EVs was implemented and its usefulness for tracking EVs and assessing bioactivity and stability was demonstrated. Most labelling techniques used for EVs within the scientific literature are highly unstable, meaning EVs typically cannot be tracked reliably for more than 1-2 days. With bioluminescence labelling, EVs can be tracked up to 1 week. Bioluminescent EVs should be hugely useful for the growing field of EV-based therapeutics. Additionally, we showed that EVs can be entrapped in degradable hydrogels and then released following degradation by a variety of mechanisms including enzymatic activity, UV light, or NIR. The proof-of-concept validation of this delivery strategy should enhance the therapeutic efficacy of EV-based treatments in the future. This is expected to have several positive impacts on society, as these types of degradable hydrogels could be used for controlled release of a variety of therapeutics even beyond EVs.
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