Among all the nanomaterials, carbon nanomaterials exhibit the most unique properties. Many prominent applications involving materials synthesized with carbon nanomaterials have gained popularity in recent years. Owing to their unique geometry, and properties closely related to single-walled carbon nanotubes (SWCNTs), carbon nanohorns (CNHs) have attracted significant attention. However, CNHs have different profiles of dispersion, structural geometry, surface chemistry, and synthesis methods and hence have a different properties. The toxicity of carbon nanomaterials is also a significant limitation for bio-related applications. Many metals are used as catalysts for the synthesis of carbon nanotubes (CNT) and other carbon nanomaterials (1). Owing to the inter-wall attraction force (van der Waals) of the CNTs, the dispersion of CNTs is a cumbersome process, and harsh chemical treatments using acids, and surfactants are necessary (2, 3).
CNHs may be an effective alternative to CNTs because they exhibit excellent purity and does not require post-synthesis treatment. CNHs have horn-shaped capped ends of approximately 2–3 nm, the wider ends aggregate to other CNHs, resulting in a Dahlia-like spherical structure (4). The void between the tips of adjacent CNHs allows the CNH aggregates to have an active dispersion profile compared to CNTs. Although functionalization improved CNHs dispersion, the CNH aggregates could not be separated into single CNHs by chemical functionalization (5). The three-dimensional arrangement of the CNHs aggregates allows electron transport and influences the bonding within the polymer along the surface, enhancing the mechanical properties of the composite, compared to the one-dimensional confinement of CNTs. Research on oxidized CNHs to entrap anticancer agents to treat lung cancer was reported as early as 2005 (6). Polymer composites comprising poly-(vinyl alcohol), graphene, and CNHs have also been reported recently (7).
On the other hand, cellulose is an abundantly available natural polymer (8). In particular, Hydroxyethylcellulose (HEC) is a derived cellulose that is used as a thickening agent, which are widely used materials in industries. HEC is also known for its application in the pharmaceutical industry for capsule formulations to improve hydrophilization of drugs (9). Research on cellulose composites with carbon nanomaterials has been increasingly, with multiple reports on the numerous applications of cellulose-carbon nanomaterials. CNTs and cellulose composites have recently been studied as aerogels for vapor sensing (10) and, water sensors (11). In addition, graphene oxide coated with cellulose nanofibers have been used for designing transparent conductive paper (12). A few other have reports included the doping of conductive nanomaterials such as Ag nanowires, for electromagnetic interference shielding (13) and, PEDOT: PSS/MWCNT for supercapacitor electrodes (14).
In the area of smart, bio-compatible, wearable devices carbon nanomaterials are being widely researched. To accommodate a device in close contact with human body/skin the compatibility of the material is a very crucial aspect. To evaluate allergic reactions and safety of different materials to human skin, will help in further exploring different applications of new materials. Such as, allergic dermatitis caused by skin sensitizers is one of the prominent items safety compliance considerations. To evaluate the skin sensitization potential of the prepared Cnh-cel sheets, h-CLAT, an in vitro skin sensitization test [including cytotoxicity test] was carried out. The cytotoxicity of CNH is expected to be low because of its spherical aggregates, as no metal catalyst is used during the synthesis of CNHs. CNHs and their composite materials are applicable to living bodies, especially in the medical field, safety evaluations, such as those evaluating toxicity and allergy, are strictly tested and regulated for safety.
For the safety evaluation of substances, allergic dermatitis is one of the essential items. After skin contact, substances causing allergic reactions, such as rash, are termed skin sensitizers, and the processes causing allergic reactions are termed skin sensitization (GHS 2017). The mechanisms of skin sensitization have been summarized in the form of an adverse outcome pathway (AOP) ranging from early events at the molecular level to adverse events through intermediate events, including the following four events (OECD 2014) (16). The first event is the formation of covalent bonds between the electrophile; in other words, skin sensitizers covalently bind to the nucleophilic center of the proteins present in the skin. The second event includes inflammatory reactions, particularly in the keratinocytes in the skin, activation of the antioxidants/electrophilic substance responsive element (AREs) dependent signal transduction pathways. The third event is the activation of antigen-presenting cells called dendritic cells in the immune system, which is assessed by the expression of specific cell surface markers, chemokines, and cytokines. The fourth event is the proliferation of T cells, which play a central role in the host immune response. The evaluation of skin sensitization using animal (in vivo) and non-animal (in vitro) experiments aims to reproduce all or part of the AOP. In vivo animal experiments include guinea pig maximization (OECD 1992) (17) and the mouse regional lymph node test (OECD 2010) (18).
From the viewpoint of animal protection, regulations on animal experiments have become stricter in recent years. Particularly in the cosmetic industry in the EU, animal experiments for the production and evaluation of raw materials, processed products, and final products, have been banned (Ban on animal testing 2019). Additionally, sales and imports of such materials and products have also been banned; therefore, manufacturers exporting these to the EU are forced to comply. Thus, non-animal tests for the evaluation of skin sensitization are necessary. Currently, peptide binding tests (the first event of AOP) (DPRA 2019, OECD 2019; Gerberick et al. 2004, 2007), keratinocyte reporter assay (the second event of AOP) (24–28), and human cell line activation test h-CLAT (the third event of AOP) are available and have been summarized in the OECD test guidelines as alternative evaluation methods (OECD, 2015a(22); OECD, 2015b(28); OECD, 2016 (29)).
The h-CLAT is a test method to evaluate the third event of the AOP by measuring the expression level of the cell surface marker (OCED, 2016) (30). It is known that upon the activation of dendritic cells, the expression levels of cell surface markers such as CD86 and CD54 increase also (31). In h-CLAT, the human monocytic leukemia cell line THP-1 cells were used as the dendritic cell model to evaluate the ability of test substances to activate dendritic cells.
In this study, we aimed to evaluate skin sensitization and cytotoxicity of novel composites of CNH and cellulose sheets using h-CLAT. These Cnh-cel sheets enabled the application of the novel composites in the field of restricted animal experiments.