During this project, we have established new synthetic routes for the synthesis of cyclic peptide / polymer conjugates (WP 1.1) by exploring different polymerisation techniques, namely RAFT polymerisation, different conjugation protocols, including isocyanate chemistry and strained alkyne – azide coupling (copper free click chemistry). We have also studied the self-assembly of the structures by neutron scattering and electron microscopy (WP1.2) and have elucidated the mechanism of self-assembly.
We have established the stability in physiological conditions (WP2.1) and toxicity profiles (WP2.2) of a library of materials, based on peptide conjugated to PEG, Pox, PHPMA and also asymmetric systems based on poly(PEG) and PolyBuA). We explored the cell internalisation mechanisms of these materials, and established that most enter cells through endocytosis (WP2.3).
We also established the properties of self-assembly of our tubular supramolecular brushes in biological media (WP2), including testing their self-assembly properties using Asymetric Field Flow Flow Fractionation (AF4) (WP1.2). We engineered supramolecular brushes based on asymmetric conjugates, in which one polymeric chains is hydrophobic and the other is hydrophilic, to form amphiphilic tubular suprastructures, which we coined tubisomes. We have completed the toxicity profile of our lead candidates (WP2.2) and studied the cell uptake pathways on model cell line sand model cancer cell lines. We have further studied the systems in vitro by exploring their interactions with tumour models (spheroids) (WP2.3). We have engineered and tested nanotubes carrying anticancer drugs, either via encapsulation (tubisomes carrying doxorubicin) or by direct attachment to the peptide core structure (camptothecin) (WP2.4). After much delay sin obtaining licences and ethical clearance, due to the impact of the world pandemic, we also were able to test our systems in vivo on small animals. We have established the pharmacokinetic parameters of the nanotubes and showed that their circulation time is much enhanced by comparison to simpler polymers, but they can also be excreted after 12-24 h, thus avoiding the usual issues of accumulation typically observed for nanoparticles (WP2.5).
Our results show that the SPBs have unique properties in biodistribution, with extended circulation time in the animals, thus enabling an enhanced activity, yet their supramolecular structure enable their complete clearance. This feature is unique to our system and is a major finding in the nanomedicine field, making our SPBs the candidates of choice for future drug delivery systems. Indeed, a typical problem encountered by nanoparticles in this field is their accumulation in organs, which lead to toxicity. Our materials are new, invented and designed through the TUSUPO project, and their in vivo behaviour opens up opportunities for new projects centred around these materials. These results are the culmination of the project, as we now have designed and engineered anticancer drug delivery systems that can not only deliver the drugs to a tumour, but also have optimal circulation time whilst avoiding accumulation in kidney, liver or spleen.
