FIREFLY-INSPIRED BIOMATERIALS AS TUNABLE, TRIGGERABLE, AND CELL-INSTRUCTIVE MATRICES FOR 3D CELL ENCAPSULATION

Jun 29, 2022, 2:10 PM
10m
Room: S3 B

Room: S3 B

Speaker

Paez, Julieta (University of Twente)

Description

"Introduction. One challenge of developing biomaterials for tissue engineering is the capability to precisely engineer desired properties, while keeping robustness and versatility in the system. Cell-encapsulating hydrogels are used as extracellular matrix mimics for basic study of cell function, high-throughput drug screening, and therapeutic delivery. In their molecular design, the crosslinking chemistry plays a vital role in regulating important properties, such as gelation rate, mechanical strength, and bioactivity [1]. Despite the many covalent crosslinking strategies reported so far, they are often not economic, user-friendly, or tunable enough to facilitate the adaptability of the encapsulating system to a variety of biomedical scenarios. To overcome this challenge, and inspired by the biochemistry of fireflies, we present a bioinspired covalent chemistry for the fabrication of precisely tunable, inexpensive, and versatile polyethylene glycol (PEG) hydrogels for 3D cell culture [2]. It is based on the condensation reaction between cyanobenzothiazole and cysteine groups, known as “luciferin click ligation”.

Methodology. 4-arm, 20-kDa PEG macromers bearing cyanobenzothiazole or cysteine functional groups were synthesized. Hydrogels were prepared under physiological conditions (37°C, HEPES buffer, pH 7-8). Crosslinking process, biofunctionalization with enzymatically cleavable and cell-adhesive peptides, and encapsulation of human mesenchymal stem cells (hMSCs) took place one-pot. The resulting hydrogels were cultured for 1-3 days. Cell viability, cell behavior and cell-materials interactions were evaluated by live/dead assay, F-actin cytoskeletal and morphological characteristics of cell analyses, respectively. Cell proliferation ability was assessed by Ki67+ nuclei staining. Mechanical strength and gelation kinetics of hydrogels were characterized by shear rheology.

Results. PEG hydrogels showed efficient and pH-regulable gelation rate, adjustable mechanical strength within physiologically relevant values, and high materials homogeneity at the microscale. By incorporating biochemical cues (i.e., cell-adhesive and cell-degradable ligands) to the hydrogel network, cell behavior and cell-materials interactions were modulated. Our gels supported the culture of hMSCs: encapsulated cells showed high cell viability (demonstrating the good cytocompatibility of these gels) and maintained their proliferation capability. 3D cell spreading (volume expansion), accompanied by high degree of cell protrusion and F-actin stress fiber formation, was observed in the presence of both cell-adhesive and cell-degradable cues in the gels.
To further develop the firefly-inspired hydrogel system as an injectable platform, novel redox-triggerable hydrogel precursors were introduced to the molecular design. The cysteine-based precursor was modified with a protecting group at the thiol residue, thus blocking gel crosslinking. Upon addition of a biocompatible reductant, the cysteine group was deprotected and the crosslinking reaction was triggered with exquisite control of the reaction rate. The storage stability of precursors was also improved, which is convenient for future upscaling and translation [3].

Conclusions. Firefly-inspired gels are robust and provide versatility for easy adaptation to diverse biomedical situations. Molecular engineering confers higher user control for the fabrication of injectable biomaterials. These biomaterials are expected to become valuable platforms for tissue engineering.

References:
[1] Paez, J.I. et al. Biomacromolecules 22, 7, 2874 (2021).
[2] Jin, M. et al. ACS Appl. Mater. Interfaces 14, 4, 5017 (2022).
[3] Jin, M., Paez, J.I.
, unpublished results."

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