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Printable polypeptide hydrogels with antimicrobial properties

Periodic Reporting for period 1 - GelPrint (Printable polypeptide hydrogels with antimicrobial properties)

Reporting period: 2020-04-01 to 2022-03-31

Biomaterials with antimicrobial properties which can be used for wound healing and tissue engineering applications offer high application potential due to the global increase of antimicrobial resistance. While polypeptides own this potential, their integration into a materials platform has not been realised to date. The overall objective of this project is to develop 3D printable antimicrobial or bacteriostatic polypeptide hydrogel materials, which can be employed in tissue regeneration applications to prevent bacterial growth. In particular, the goals include synthesis of sets of cross-linked polypeptide hydrogels based on lysine (Lys) and investigation of their hydrogel properties and 3D printability. The project is broadly interdisciplinary, as disciplines of polymer chemistry, biomaterials science and engineering, microbiology and in vitro assessment techniques will be combined. The project objectives were met by the synthesis of a set of novel hydrogel forming star polypeptides by ring-opening polymerisation containing different amnio acid compositions. Through evaluation of their rheological properties and cell compatibility, a lead materials was identified which combined excellent antimicrobial properties against E. Coli, was extrudable and printable while displaying good cell compatibility. This proof of concept demonstration confirms their feasibility in tissue regeneration, specifically in wound dressing applications. The results demonstrate for the first time that through compositional and structural optimisation of synthetic polypeptides their biomaterial properties can be tuned to address biomedical challenge.
A family of 8 arm star polypeptides based on lysine, tyrosine and cysteine amino acids was prepared. 10 different polypeptides were synthesized with a variation of the sequence of the blocks and the number of the monomeric units. This resulted in a set of polymers with different molecular characteristics that affect the rheological properties of the hydrogels. The structures and molecular weights of all polypeptides was confirmed by IR spectroscopy, Gel Permeation Chromatography and NMR spectroscopy. Rheology measurements revealed the rheological profile of the different hydrogels revealing the strength of the hydrogels as well as their shear thinning properties.
Due to the presence of lysine monomeric units the hydrogels provide antimicrobial properties. 3 out of the ten synthesized polymers were selected for this test based on the rheological applications. Hydrogels were tested with bacterial culture, either E. coli (Gram negative) or S. aureus (Gram positive). The results showed that all hydrogels were more effective against S. Aureus than E. Coli. The highest log reduction against S. Aureus, one of the leading pathogens for deaths associated with antimicrobial resistance, was 3.7.
The lead hydrogel (according to rheological properties) was used to investigate their printability. An object in the shape of pyramid was successfully printed. In vitro toxicity was carried out on rat mesenchymal stem cells (rMSCs) to evaluate the potential leachable cytotoxicity of the hydrogels. This was carried out by measuring the metabolic activity of the cells at days 1, 3, and 7 compared to untreated cell as per ISO guidelines (ISO standard 10993-5). Generally the hydrogels demonstrated good biocompatibility and the absence of cytotoxic leachables, although two hydrogels caused some reduction in metabolic activity at day 7. Additionally, Live/Dead imaging of the rMSCs was taken after 7 days exposure to the hydrogels. Visually, the results corroborate the trend demonstrated by the metabolic activity, whereby there are no significant differences in live cells visualised in the lead material compared to the untreated cells alone group.

The commercial exploitation of the results was discussed with the RCSI TTO. While it was agreed that the materials are innovative it was decided that IP filing would be premature and required additional data including in vivo data. The results will inform future projects with a clinical focus and are currently reviewed by experts in tissue engineering as well as clinical microbiology groups for exploitation within their technologies.

While some project data were recently accepted for publication in Macromolecular Bioscience (open access), the main data set is currently written up for publication. Some data were disseminated as conference posters.
It was known from the literature and particularly from work carried out in the host group at RCSI that lysine containing polypeptides have antimicrobial properties. However, all samples produced previously also showed significant cytotoxicity, which rendered them unsuitable for applications in tissue regeneration (e.g. wound healing). It was also knows that polypeptide hydrogels can be 3D printed. This project progressed beyond the state-of-the-art in that for the first time a systematic study was conducted with the aim to balance hydrogel properties like printability, antimicrobial properties and biocompatibility by controlling the polypeptide structure and composition. The results clearly demonstrated that by small changes in the polymer chemistry, bulk hydrogel properties can be fine-tuned. For example, some investigated hydrogels displayed superior rheological properties, which allowed the 3D printing of defined objects. By changing the polymer composition, improved antimicrobial properties were obtained while reading biocompatibility. The results thus inform about design characteristics on molecular level and their translation into hydrogel properties. This is crucially important for developing next generation biomedical hydrogels. While in vivo studies have to be carried out to complement the preliminary data, these results suggest applicability of the hydrogel as antimicrobial would healing materials. Considering the increasing issues around antimicrobial resistance, these materials could, in the long term, be alternatives for traditional wound dressings containing antibiotic agents. The socio-economic as well as clinical impact can be expressed in improved patient treatment, improved health and reduced healthcare costs due to a reduction in hospitalisation.
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