Photoinitiator- and Radical-free High Resolution Bioprinting

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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Rizzo, Riccardo (ETH )


Introduction. In recent years, the development of novel photo-crosslinking strategies and photoactivatable materials has stimulated a widespread use of light-mediated biofabrication techniques.1 From the high resolution two-photon stereolithography (2P-SL),2 to the novel fast volumetric printing,3 light-based technologies now encompass a powerful set of tools that enables one to mimic multiscale tissue organization with sub-micrometer resolution and centimeter-size scale.
However, despite great improvements towards more efficient and biocompatible photochemical strategies, current photoresins still rely on the presence of photoinitiators (PIs). Upon light exposure, PIs produce radical-initiating species to trigger the crosslinking/polymerization process. In the context of bioprinting where cells are encapsulated in the photoresin, the presence of radicals raises concerns of potential cytotoxicity. On the other hand, near-UV and visible light alone were shown to be biocompatible,4 thus suggesting that the use of a photoinitiator/radical-free photochemistry would be an ideal approach. In this work, we present a universal radical-free crosslinking strategy to be used for light-based technologies.

Methodology. A water-soluble coumarin photocage (DCMAC-OH) was synthesized as previously reported,5 and exploited to mask thiol groups of a thiol-terminated PEG crosslinkers (PEG2SH, PEG4SH). Stability in solutions and dry condition of such crosslinkers were determined by 1H-NMR. Synthetic and natural-derived polymers were modified to bear -ene functionalities (vinyl sulfone, methylsulfone, maleimide). The thiol and -ene component of the photoresin were combined in 1:1 thiol:ene molar ratio and photocrosslinking was monitored with photorheology under oscillation. Potential toxicity of DCMAC-OH was investigated with the MTT assay using primary cells and immortalized cell lines. Photoresins were adopted for high-resolution two-photon stereolithography performed on a LeicaSP8 equipped with MaiTai fs-laser.

Results and conclusions. The radical-free crosslinking method presented in this work relies on base-catalysed thiol-ene chemistry. Due to the caging of thiol groups with the one- and two-photon sensitive photocage DCMAC-OH, crosslinking can be triggered with multiple light-based technologies using 365 nm, 405 nm or two-photon lasers. As demonstrated by photorheology, upon light absorption the photocage is removed and the thiols of the PEG-based crosslinker are free to react with any polymer functionalized with -ene moieties, thus making our method universally applicable. In this work we have adopted three different -ene groups, the widely used maleimides and vinyl sulfone, and a recently discovered phenyl-tetrazole-based methylsulfone.6 Photorheology analysis has shown that reaction with vinyl sulfones is too slow for light-mediated bioprinting, while maleimides and methylsulfones reaction kinetics could be adopted for such methods. The PI-free photoresins were successfully employed for high-resolution 2P-SL. Viability and proliferation assays demonstrated the cytocompatibility of the photocage for stem cells, primary cells and immortalized cell lines. Stability tests of the photocaged crosslinker confirmed excellent stability for up to 6 months, making it a potentially suitable platform for commercialization and off-the-shelf preparations. Our universal PI/radical-free method could represent a paradigm-shift in the light-based technologies, avoiding the need to expose encapsulated cells to harmful radicals.


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