Impact of the Graphene Production Methods Sonication\nand Microfluidization on <i>In Vitro</i> and <i>In\nVivo</i> Toxicity, Macrophage Response, and Complement Activation

Graphene, a material composed of a two-dimensional lattice of carbon\natoms, has due to its many unique properties a wide array of potential\napplications in the biomedical field. One of the most common production\nmethods is exfoliation through sonication, which is simple but has\nlow yields. Another approach, using microfluidization, has shown promise\nthrough its scalability for commercial production. Regardless of their\nproduction method, materials made for biomedical applications need\nto be tested for biocompatibility. Here, we investigated the differences\nin toxicity, macrophage response, and complement activation of similar-sized\ngraphene flakes produced through sonication and microfluidization,\nusing <i>in vitro</i> cell assays and <i>in vivo</i> assays on zebrafish larvae. <i>In vitro</i> toxicity testing\nshowed that sonicated graphene had a high toxicity, with an EC<sub>50</sub> of 100 μg mL<sup>–1</sup> for endothelial cells\nand 60 μg mL<sup>–1</sup> for carcinoma cells. In contrast,\nmicrofluidized graphene did not reach EC<sub>50</sub> at any of the\ntested concentrations. The potency to activate the complement system\nin whole blood was 10-fold higher for sonicated than for microfluidized\ngraphene. In zebrafish larvae, graphene of either production method\nwas found to mainly agglomerate in the caudal hematopoietic tissue;\nhowever, no acute toxic effects were found. Sonicated graphene led\nto an increase in macrophage count and a macrophage migration to the\nventral tail area, while microfluidized graphene led to a transient\nreduction in macrophage count and fewer cells in the ventral trail\narea. The observed reduction in macrophages and change in macrophage\ndistribution following exposure to microfluidized graphene was less\npronounced compared to sonicated graphene and contributed to masking\nof the fluorescent signal rather than cytotoxic effects. Summarized,\nwe observed higher toxicity, macrophage response, and complement activation\nwith graphene produced through sonication, which could be due to oxygen-containing\nfunctional groups introduced to the edge of the carbon lattice by\nthis production method. These findings indicate that microfluidization\nproduces graphene more suitable for biomedical applications.