Indocyanine Green J-aggregate Nanoparticles for Detection of Senescent Cells

Cellular senescence is a response to unrepairable damage and stress characterized by the implementation of a stable cell cycle arrest and an intense pro-inflammatory secretory phenotype (SASP). Upon persistent damage or during aging, senescent cells accumulate in tissues and organs, partially due to an inefficient clearance by the immune system. The presence of senescent cells has been actively implicated in multiple pathological manifestations and chronic disorders including, fibrosis, neurological disorders, diabetes, sarcopenia, inflammatory syndromes and cancer. Evidence during the last decade convincingly demonstrates that the genetic and/or pharmacological removal of senescent cells (senotherapy), in preclinical models, ameliorates a number of aging-associated diseases. In addition, the removal of dysfunctional senescent cells in mice significantly extends not only their health span but also their lifespan (by about 30%). As a consequence of this preclinical success, the field of senotherapies has expanded and the most promising senolytic drugs have been prioritized to early phase clinical trials. Despite treatments showing promising translational results, there is still a lack of tools and techniques to detect, identify and longitudinally monitor senescent cells in *in vivo* settings, thereby emerging as an unmet need to assess the senescent burden pre- and post-senotherapy. Here, within this thesis, we have designed, synthesized and validated the first of its kind J-aggregate nanoparticle, exhibiting fluorescent and photoacoustic imaging properties for longitudinal *in vivo* bioimaging of senescence. First, in this thesis the synthesis of J-aggregates of indocyanine green was explored with the aim to develop a high contrast photoacoustic probe. Reported methods of J-aggregate productions resulted in poorly characterized systems, and the addition of centrifugation steps during the purification resulted in pure J-aggregates, which we named NanoJaggs. These structures are made of a dimer of the clinically approved dye indocyanine green, they were found to be stable in range of conditions, easily sterilized, and could be stored for prolonged periods of time, which makes them well-suited for biomedical applications. The specific chemical composition of NanoJaggs was studied using UV-Vis, cryoTEM, LC-MS and ¹H NMR, resulting in a fully characterized system, exhibiting a strongly enhanced photoacoustic signal (Chapter 4). Following the synthesis optimization and characterization, the potential of NanoJaggs to be used as senescent cell probes was explored *in vitro*. They were found to selectively accumulate in senescent cancer cells and fibroblasts from multiple tissues of origin, and inducers of senescence. NanoJaggs were found to be comparable to established senescence detection methods, such as the lysosomal senescence-associated b-galactosidase (SA-β-gal) activity and to colocalize with the lysosome. As senescent cells possess a higher lysosomal mass and amount, we hypothesized that this was the reason for the selectivity of the NanoJaggs (Chapter 5). The evaluation of the targeting and uptake mechanism using endocytosis inhibitors showed that clathrin-mediated endocytosis as well as macropinocytosis play a role in NanoJagg uptake in senescent cells pointing towards an active mechanism of uptake in addition to lysosomal targeting (Chapter 6). Finally, *in vivo* experiments both exploiting the fluorescence and photoacoustic properties of NanoJaggs were performed. To do so, we employed a mouse model of chemotherapy-induced senescence in tumor xenografts. In both cases a significant accumulation of NanoJaggs in senescent lesions was found, confirming the potential of NanoJaggs to be used as *in vivo* probes to determine the burden of senescent cells in tumor models. Importantly, in this chapter the photoacoustic contrast of NanoJaggs was evaluated to detect cellular senescence and used to map the tumor microenvironment (Chapter 7). Together, these findings demonstrate the significant potential of NanoJaggs for detection of cellular senescence *in vitro*, *ex vivo* and *in vivo* in cancer models. The ability of NanoJaggs to act as a contrast agent for photoacoustic tomography, as well as its simple synthesis and FDA approved building blocks make the NanoJaggs appealing for future clinical applications, and a promising modality for senescence detection and longitudinal bioimaging.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Baker, Andrew

Advisor dc:contributor.advisor

• Fruk, Ljiljana

• Cancer
• Photoacoustic Tomography
• Senescence

Licence

• https://www.repository.cam.ac.uk/bitstreams/12820fea-7735-4724-8a94-04427da58ab8/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.100100

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/354212