Impact of the Graphene Production Methods Sonication and Microfluidization on In Vitro and In Vivo Toxicity, Macrophage Response, and Complement Activation

Jan-Lukas Førde; Abdelnour Alhourani; Tian Carey; Adrees Arbab; Kari E Fladmark; Silje Skrede; Tom Eirik Mollnes; Lars Herfindal; Hanne R Hagland

doi:10.1021/acsomega.4c03189

Impact of the Graphene Production Methods Sonication and Microfluidization on In Vitro and In Vivo Toxicity, Macrophage Response, and Complement Activation

ACS Omega. 2024 Sep 18;9(39):40468-40476. doi: 10.1021/acsomega.4c03189. eCollection 2024 Oct 1.

Authors

Jan-Lukas Førde^{1

2}, Abdelnour Alhourani³, Tian Carey^{4

5}, Adrees Arbab⁴, Kari E Fladmark⁶, Silje Skrede^{7

8}, Tom Eirik Mollnes^{9

10}, Lars Herfindal², Hanne R Hagland³

Affiliations

¹ Department of Internal Medicine, Haukeland University Hospital, 5009 Bergen, Norway.
² Centre for Pharmacy, Department of Clinical Science, University of Bergen, 5020 Bergen, Norway.
³ Department of Chemistry, Bioscience and Environmental Engineering, University of Stavanger, 4021 Stavanger, Norway.
⁴ Textile Two Dimensional Ltd., London WC2H 9JQ, England.
⁵ School of Physics, CRANN & AMBER Research Centre, Trinity College, Dublin, 2, Ireland.
⁶ Department of Biological Sciences, University of Bergen, 5020 Bergen, Norway.
⁷ Section of Clinical Pharmacology, Department of Medical Biochemistry and Pharmacology, Haukeland University Hospital, 5021 Bergen, Norway.
⁸ Department of Clinical Science, University of Bergen, 5020 Bergen, Norway.
⁹ Research Laboratory, Nordland Hospital Trust, 8092 Bodø, Norway.
¹⁰ Department of Immunology, Oslo University Hospital and University of Oslo, 0372 Oslo, Norway.

Abstract

Graphene, a material composed of a two-dimensional lattice of carbon atoms, has due to its many unique properties a wide array of potential applications in the biomedical field. One of the most common production methods is exfoliation through sonication, which is simple but has low yields. Another approach, using microfluidization, has shown promise through its scalability for commercial production. Regardless of their production method, materials made for biomedical applications need to be tested for biocompatibility. Here, we investigated the differences in toxicity, macrophage response, and complement activation of similar-sized graphene flakes produced through sonication and microfluidization, using in vitro cell assays and in vivo assays on zebrafish larvae. In vitro toxicity testing showed that sonicated graphene had a high toxicity, with an EC₅₀ of 100 μg mL^-1 for endothelial cells and 60 μg mL^-1 for carcinoma cells. In contrast, microfluidized graphene did not reach EC₅₀ at any of the tested concentrations. The potency to activate the complement system in whole blood was 10-fold higher for sonicated than for microfluidized graphene. In zebrafish larvae, graphene of either production method was found to mainly agglomerate in the caudal hematopoietic tissue; however, no acute toxic effects were found. Sonicated graphene led to an increase in macrophage count and a macrophage migration to the ventral tail area, while microfluidized graphene led to a transient reduction in macrophage count and fewer cells in the ventral trail area. The observed reduction in macrophages and change in macrophage distribution following exposure to microfluidized graphene was less pronounced compared to sonicated graphene and contributed to masking of the fluorescent signal rather than cytotoxic effects. Summarized, we observed higher toxicity, macrophage response, and complement activation with graphene produced through sonication, which could be due to oxygen-containing functional groups introduced to the edge of the carbon lattice by this production method. These findings indicate that microfluidization produces graphene more suitable for biomedical applications.