Periodic Reporting for period 4 - TUSUPO (Tubular Supramolecular Polymers: A new class of therapeutic polymers) Période du rapport: 2020-01-01 au 2021-06-30 Résumé du contexte et des objectifs généraux du projet This research programme has established a new class of materials and developed them into functional devices for biomedical applications. We have designed tubular supramolecular polymers (supramolecular polymer brushes, SPBs), based on the self-assembly of cyclic peptide – polymer conjugates. The synergy between the cyclic peptide, which directs the formation of the SPBs and the polymer conjugate, which provides functionality, opens the route to a wealth of new functional structures. We have generated new synthetic routes for the ligation of polymers to peptides, developed new protocols for the characterisation of the materials, and established the mechanism of supramolecular polymerisation.The functionality and versatility of the SPBs obtained in this work open the route to a wealth of applications, and we have focused on one specific target: the fabrication of drug delivery vectors. We exploited the unique combination of features presented by this new class of polymer therapeutics, such as multiple attachment points for one or more drug(s) / targeting ligands / markers, the ability to self-disassemble into smaller and easy-to-excrete components, and an elongated shape that enables diffusion and interaction with cells more efficiently than traditional globular delivery systems. We studied the pharmacology properties of the SPBs, including their stability, toxicity, mode of cell penetration and ability to deliver a single or a combination of bioactive agent(s) (in the case of concerted mechanisms).The program also provided significant training opportunities for postgraduate and postdoctoral researchers in a range of skills encompassing organic synthesis, polymer synthesis, compound characterisation and physical measurements (using techniques which are required for employment in Europe’s growing bio- and nanotechnology industry) and biology. Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent 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. Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent) - We have established an approach to tune the length of the nanotubes by using charged polymers. Catrouillet et al. ACS Macro Lett., 2016, 5 (10), 1119–1123 DOI: 10.1021/acsmacrolett.6b00586- We established the properties of the supramolecular assemblies, published in Nature communication and chem Rev: Rho et al. Nat Commun, 2019, 10, 4708 DOI:10.1038/s41467-019-12586-8 and Song et al. Chem. Rev. 2021, 10.1021/acs.chemrev.0c01291- In particular, we have shown the structures assemble in a dynamic process thus suggesting their ability to self-disassemble in site, to enable rapid clearance when used in vivo. Rho et al. Adv. Funct. Mater. 2017, 1704569 DOI: 10.1002/adfm.201704569- We have also establish the fundamental understanding of polymer conjugates structure on their ability to be uptaken by cells. Ellacott et al. Biomacromolecules 2021, 22, 2, 710–722 - We have shown the SPBs are effective drug delivery vectors for Iridium based anticancer drugs. Larnaudie et al. Biomacromolecules, 2018, 19 (1), pp 239–247 AND Larnaudie et al. Biomaterials, 2018, DOI:https://doi.org/10.1016/j.biomaterials.2018.03.047- Finally we have developed hierarchical tertiary structures which were used as drug delivery vectors. Yang et al. Angewandte Chemie 2020, DOI: 10.1002/anie.201916111 AND Brendel et al. Angewandte Chemie 2018, DOI: 10.1002/anie.201808543 Artistic impression of Supramolecular Polymer Brushes
