Crosslinking reactions occurring at physiological conditions based on the bioinspired cyanobenzothiazole (CBT)/cysteine ligation

• Applicant: Dr. Julieta Paez
• Subject Area: Polymer Materials ; Biomaterials
• Term: from 2019 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 422041745

• Final Report Year: 2023

Final Report Abstract

Crosslinked polymeric materials represent an enormous global market encompassing coatings, adhesives and hydrogels. Due to the climate crisis, there is currently an increasing interest in environmentally friendly conditions for polymer curing and processing via diverse technologies. Thus, the need for novel effective crosslinking reactions in water is urgent. Such crosslinking reactions should fulfill several criteria: i) develop under mild conditions: in water as solvent, at moderate temperature, without catalysts; efficiently and selectively; ii) be chemically orthogonal to conventional polymerizations for uncomplicated integration to existing processing technologies; and iii) offer the possibility of on-demand and temporal control. In this project, a novel bioinspired crosslinking reaction that meets above requirements has been identified and developed into soft crosslinked networks for application in healthcare. Inspired by the biochemical cycle of luciferin inside of the firefly body, we selected the covalent coupling reaction between cyanobenzotiazole (CBT) and cysteine (Cys) groups, known as “CBT-Cys ligation” as crosslinking reaction. This reaction works effectively under physiological conditions, it is derived from inexpensive reagents, and its onset can be triggered by simple environmental changes. By providing a new chemistry for crosslinking that presents low environmental impact and cost-effectiveness, this project utilizes the CBT-Cys ligation for the obtention of crosslinked networks and demonstrates its applicability in the biomedical field. This project was divided in 5 stages. First, efficient synthesis pathways for CBT and Cys bearing building blocks were developed, thus delivering reactive monomers and macromers. Secondly, those building blocks were reacted under mild aqueous conditions to form 3D crosslinked networks (hydrogels). Reaction conditions to make the CBT-Cys ligation chemistry technologically attractive for hydrogel production were explored, and the stability of derived networks under physiological conditions was assessed. Third, the CBT-Cys hydrogels were characterized at the micro/macroscopic levels. Physical chemical and mechanical methods were applied to study how the crosslinking kinetics dictates network properties, which are relevant for the envisioned biomedical applications. In a fourth stage, the chemical compatibility of the CBT-Cys reaction with industrially-relevant polymerizations was investigated, and protocols for their combined use were produced. Moreover, chemical tools to trigger and control ondemand the formation of CBT-Cys hydrogels were developed. In the final stage, the good biocompatibility of CBT-Cys hydrogels was revealed, prompting their usage in the production of soft biomaterials. The excellent performance of CBT-Cys biomaterials for cell biology and tissue engineering applications was demonstrated. The findings of this project have direct impact in advancing the biomaterials chemistry and tissue engineering fields. The outcomes pave the way for a broader use of the CBT-Cys chemistry and derived materials in healthcare applications.

Publications

• “Chemistry of Tunable Gels for 3D Cell Culture”. Mechanobiology of Cancer Summer School 2019, Mechano-Control EU-Project. Prulláns, ES. 17-21.09.19.
  Paez JI.
  
• “Filling the kinetic gap: new thiol-mediated crosslinking chemistry for 3D cell culture hydrogels”. FBPS 2019. Tenerife, Canarias, ES. 19-23.05.19.
  Paez JI.
  
• “Thiol-mediated chemistries for bioconjugation and hydrogelation: towards versatile (bio)materials”. Universidad Nacional de Córdoba, AR; and at INTEC Seminar. INTEC, AR. Dec19.
  Paez JI.
  
• Thiol-Methylsulfone-Based Hydrogels for 3D Cell Encapsulation. ACS Applied Materials & Interfaces, 12(7), 8062-8072.
  Paez, Julieta I.; Farrukh, Aleeza; Valbuena-Mendoza, Rocío; Włodarczyk-Biegun, Małgorzata K. & del, Campo Aránzazu
  
• “Designing soft materials for ‘the living’: hydrogels based on the luciferin ligation for cell encapsulation”. 1st Engineered Living Materials Conf. Saarbrücken, DE. 12-13.02.2020.
  Jin, M.
  
• “Introducing external triggers to bioinspired hydrogels design for flexible cell encapsulation”. ESB 2021, Porto, PT. 5-9.09.2021.
  Jin, M.
  
• “Luciferin-Bioinspired Hydrogel Scaffolds with Tunable Properties for 3D Cell Encapsulation”. NBTE 2021, Lunteren, NL. 4-5.04.2022.
  Jin. M.
  
• Current strategies for ligand bioconjugation to poly(acrylamide) gels for 2D cell culture: Balancing chemo-selectivity, biofunctionality, and user-friendliness. Frontiers in Chemistry, 10.
  Wolfel, Alexis; Jin, Minye & Paez, Julieta I.
  
• Gelation Kinetics and Mechanical Properties of Thiol‐Tetrazole Methylsulfone Hydrogels Designed for Cell Encapsulation. Macromolecular Bioscience, 23(2).
  de Miguel‐Jiménez, Adrián; Ebeling, Bastian; Paez, Julieta I.; Fink‐Straube, Claudia; Pearson, Samuel & del Campo, Aránzazu
  
• Luciferin-Bioinspired Click Ligation Enables Hydrogel Platforms with Fine-Tunable Properties for 3D Cell Culture. ACS Applied Materials & Interfaces, 14(4), 5017-5032.
  Jin, Minye; Koçer, Gülistan & Paez, Julieta I.
  
• Redox-triggerable firefly luciferin-bioinspired hydrogels as injectable and cell-encapsulating matrices. Polymer Chemistry, 13(35), 5116-5126.
  Jin, Minye; Gläser, Alisa & Paez, Julieta I.
  
• “A bioinspired strategy for controlled bio-functionalization of polyacrylamide hydrogels with bioligands”. NBTE 2021, Lunteren, NL, 4-5.04.2022.
  Wolfel, A.
  
• “Bio-inspired redox-responsive hydrogel as injectable matrices”. NWO Chains Conference 2022. Veldhoven, NL 21-22.09.2022.
  Jin, M.
  
• “Bioinspired chemical approaches to engineer crosslinked networks with tailorable properties for cell encapsulation”. MESA+ day, University of Twente, NL 13.06.2022.
  Jin. M.
  
• “Bioinspired chemical tools to molecularly engineer smart matrices for tissue engineering”. 1st Symposium Engineering matrices: from cells to tissues. Lyon, FR. 21.11.2022.
  Paez, JI.
  
• “Bioinspired injectable hydrogel with redox-responsiveness for cell-instructive matrix cues”. 1st MSEB, Heidelberg, DE. 19-21.10.2022.
  Jin, M.
  
• “Chemo-selective biofunctionalization of hydrogels with cell-adhesive ligands”. TechMed Research Day 2022, University of Twente, NL. 16.06.2022. ‘Best Poster Award’.
  Wolfel, A.
  
• “Firefly-bioinspired hydrogels with redox-responsiveness as cell-encapsulating injectable matrices”. ESB 2022, Bordeaux, FR. 4-8. 09.2022.
  Jin, M.
  
• “Firefly-Inspired Biomaterials as Tunable, Triggerable, and Cell-Instructive Matrices for 3D Cell Encapsulation“. 2022 TERMIS-EU. Krakow, PL. 28.06-01.07.2022.
  Paez JI.
  
• “Firefly-inspired hydrogel as tunable soft matrices for cell encapsulation”. TechMed Research Day 2022, University of Twente, NL. 16.06.2022.
  Jin, M.
  
• “Hydrogels with Engineered Crosslinks: Towards Tunable, Processable, and Adaptable Biomaterials for Cell Encapsulation”. NBTE 2021. Lunteren, NL. 04-05.04.2022.
  Paez JI.
  
• “Molecularly engineering smart hydrogels using firefly luciferin-bioinspired chemistry”. 1st MSEB. Heidelberg, DE. 19-20.10.2022.
  Paez, JI