Implementation of a photo-cleavable crosslinker in proteinosomes, protocellular materials and hydrogels

Abstract

Proteinosome technology, which seeks to develop the uses, life-like nature and complexity of this protocell type has advanced rapidly over the last decade. This has included the recent development of protocellular materials (PCMs), covalently interconnected, macroscopic consortia of proteinosomes. As a further part of progress in proteinosome technology, significant advancements have been made by researchers in the area of proteinosome communication, e.g.,
through the use of encapsulated cascade enzymes that allow transmission of small molecule "signals" (such as H2O2) between individual proteinosomes. However, despite this progress, there is currently no method to achieve controllable release of diverse molecular cargoes from within
proteinosomes - a highly necessary step-forward to increase the real-world utility of proteinosomes (in drug delivery, for example). This work aims to address this gap by using a photo-cleavable poly(ethylene glycol)-based crosslinker, functionalised with photo-cleaving ortho-nitrobenzyl moieties. This thesis also aims to increase and explore the ways in which the photo-cleaving
crosslinker can be used - namely, in the development of a novel cargo release from proteinosomes system and in the synthesis of polymeric networks to include within proteinosomes. Chapters 1 and 2 cover a discussion of the background literature and outline the various experimental methods used to support this project, respectively. Chapter 3 covers work which investigates the synthesis of the crosslinker. A series of optimisation experiments are described
and a new synthetic route is attained. Chapter 4 presents attempts to synthesise photo-cleaving proteinosomes and the associated challenges that were (unexpectedly) faced. After these initial chapters, Chapter 5 discusses work surrounding attempts to develop a new method of light-induced proteinosome communication. This work centres around the use of the photo-cleaving crosslinker to connect high molecular weight protein "anchors" to low
molecular weight diffusible signals, forming "anchor-signal conjugates". These conjugates could be entrapped within proteinosomes by virtue of their molecular weight being above the intrinsic molecular weight cut-off of the proteinosome membrane. On irradiation with UV the conjugate breaks apart and releases the signal to diffuse out throught the proteinosome membrane.
Aside from light-induced proteinosome communication, proteinosomes (and protocellular materials) have relatively poor mechanical integrity. Chapter 6 covers work intended to address this challenge by incorporating a polymeric network within the proteinosome lumen. Moreover, this polymer network is constructed with the photo-cleavable polymer and so provides excellent
spatio-temporal control over the mechanical properties of the proteinosome and any resultant PCMs. Also in Chapter 6, we take the concept of photo-degradable polymer networks further and synthesise photo-weakening hydrogels, capable of being photo-patterned. In summary, this work demonstrates the variety of applications of a photo-cleavable crosslinker,
where ultraviolet light can be used to selectively destroy the membranes of individual proteinosomes or precisely modify the mechanical properties of hydrogels (either in bulk or encapsulated within proteinosome lumina within a PCM).

Date of Award	10 Dec 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Sebastien Rochat (Supervisor) & Pierangelo Gobbo (Supervisor)