**7. Potential Cytotoxicity and Immunogenicity of Nanocellulose**/**Nanocarbon Composites**

The vast majority of studies dealing with potential biomedical applications of nanocellulose/nanocarbon composites have reported no cytotoxicity or negligible cytotoxicity of these composites, namely of nanocellulose/fullerene composites [35], nanocellulose/graphene composites [68,69,72,87,107], nanocellulose/CNT composites [51,149] and nanocellulose/nanodiamond composites [58,62,97]. Composites containing other carbon nanoparticles, such as activated carbon nanoparticles [30] or carbon quantum dots [28] have also shown no significant cytotoxic effects. In the mentioned studies, cytotoxicity was mainly tested in vitro on various cell types, such as fibroblasts, epithelial and endothelial cells, and mainly on cell lines, including tumor cell lines. The cell viability and proliferation were usually evaluated by tests of the activity of mitochondrial enzymes, such as MTT assay, resazurin (Alamar Blue) assay, or by a direct microscopic examination of the cells. Some composites have also been tested in vivo, e.g., in a rat model (nanocellulose/CNT composites; [106]), a canine model (nanocellulose/CNT composites; [52]), or using a chick chorioallantoic membrane model (nanocellulose/graphene composites, [107]), without adverse effects.

However, the individual components of nanocellulose/nanocarbon composites, particularly carbon nanoparticles, can act as cytotoxic, if they are not bound to any matrix and are free to move. Graphene and graphene-based carbon nanomaterials, such as fullerenes and nanotubes, are hydrophobic in their pristine state, and can enter into hydrophobic interactions with cholesterol in the cell membrane, which can be extracted from the membrane. In this manner, carbon nanoparticles can damage cells even without penetrating them. Another mechanism of cell membrane damage is the generation of reactive oxygen species (ROS) by carbon nanoparticles. In addition, the nanoparticles can penetrate the cell membrane, and can cause oxidative damage to mitochondria, and can also enter the cell nucleus and act as genotoxic agents (for a review, see [10,12,19,20]). Nanodiamonds have been considered to be relatively nontoxic in comparison with other carbon nanoparticles. However, as shown in our earlier studies, hydrophobic, hydrogen-terminated and positively-charged diamond nanoparticles can enter the cells, impair their growth and cause cell death [152,156]. The mechanism of cell damage by nanodiamonds is by generating ROS, and by excessive delivery of sodium ions adsorbed on the nanodiamond surface [168]. Last but not least, carbon nanoparticles can be immunogenic, i.e., they can activate inflammatory reactions, which can be, as has been demonstrated on carbon black, the main pathogenic mechanism of respiratory, cardiovascular and other serious diseases [166,167].

Cellulose nanoparticles, which are generally considered to be biocompatible [34,114] and of a low ecological toxicity [169], can also act as cytotoxic and immunogenic. It has even been speculated that, due to their high aspect ratio and stiffness, CNCs may cause similar pulmonary toxicity as carbon nanotubes and asbestos [170]. In a mouse model, cellulose nanocrystals induced oxidative stress, caused pulmonary inflammation and damage, increased levels of collagen and transforming growth factor-beta (TGF-β) in lungs, and impaired pulmonary functions [170]. In addition, these effects were markedly more pronounced in female mice than in male mice. The immunogenicity of CNCs was also proven in vitro. CNCs and their cationic derivatives CNC-aminoethylmethacrylate and CNC-aminoethylmethacrylamide evoked an inflammatory response in mouse macrophage J774A.1 cells and in peripheral blood mononuclear cells by increasing the level of ROS in mitochondria, the release of ATP from mitochondria and by stimulating the secretion of interleukin-1beta (IL-1β) [171]. The cytotoxicity and immunogenicity of CNCs depend on the preparation conditions and are increased under harsh and caustic conditions, e.g., the so-called mercerization process, i.e., an alkali treatment [149]. CNFs can also cause cytotoxicity and oxidative damage, which can be even more pronounced than in the case of CNCs, and can evoke an inflammatory response (for a review, see [2,172]). The potential cytotoxicity and immunogenicity of nanocellulose, nanocarbon and their composites should therefore be taken into account when they are for use in biomedical applications.
