Graphene oxide (GO) holds promise as a versatile biomedical material for drug release within the body and neoplasia treatment, owing to its physicochemical properties and biocompatible nature. Given that carbon is one of the elemental building blocks of our bodies, the utilization of graphene oxide is considered safe for living organisms. In a parallel manner, graffiti, emerging spontaneously through natural processes, seamlessly integrates into various aspects of our lives (Chung et al., 2013). Conversely, graphene stands out as a biocompatible carbon nanomaterial with favorable physicochemical properties, derived from a single layer of graphite (Wang et al., 2010). Graphene is a two-dimensional carbon structure consisting of an sp2 hybrid structure [1]. Graphene is an elastic structure that absorbs very little light and is very strong compared to steel. It is also an extremely resistant material against water and chemical solvents [2]. GO is the single-layer form of the graphite layer separated from each other by oxidation [3,4]. There is partial hydrophobicity on the surface of GO, and there are also hydrophilic parts that can form complexes with hydrogen bonds and metal ions. The oxygen groups in GO make it soluble and stable in water (Ericson et al.). The only obstacle to the molecular use of graphene is that it contains hydrophobic moieties. For this reason, polar and more stable particles can be obtained by coating the GO surface with suitable active substances [5].

Most in vitro studies on graphene have focused on macrophages, which are the first cells to respond to circulating pathogens and foreign substances (Yang et al., 2013; Bianco et al., 2011; Sasidharan et al., 2012). It is believed that macrophages are particularly susceptible to the adverse effects of graphene and its derivatives, triggering an immunological response when graphene oxide (GO) enters the cell through phagocytosis (Yang et al., 2013). In specific investigations, GO was found to elevate the production of cytokines such as interleukin (IL)-(1a,6,10) and tumor necrosis factor α (TNF-α) [6]. Another study noted that 100 μg/mL of graphene induced the production of interferon (IFN)-γ, IL-2, IL-10, and TNF-α cytokines in lipopolysaccharide (LPS)-induced macrophages [6]. A recent study reported that graphene quantum dots negatively impact lipid metabolism in macrophages, leading to a disruption of energy homeostasis [7].

Zinc oxide (ZnO) is a low-cost, chemically stable material that can be used as a biosensor without any toxic properties to the cell [8]. Zinc acts as a second messenger in the immune system. It induces immune activity by activating T cells and cytokines by stimulating pathogen-associated molecular patterns (PAMPs) or pattern recognition receptors (PRRs) via FCE receptors found in zinc signals in the cell. As ZnO has an immunomodulatory effect, it has been used as an adjuvant in some vaccines. There are studies in which ZnO nanoparticles are combined with vaccine antigens and used as an immunomodulator. It has been reported that ZnO is preferred as an adjuvant in various vaccines by using its immunomodulatory property, and it has an immunomodulatory effect by combining with vaccine antigens [[9], [10], [11], [12]]. It has also been reported that ZnO nanowires as tumour antigen carriers enhance cancer immunotherapy [13,14]. ZnO nanoparticles are readily taken up by phagocytic cells such as monocytes and macrophages and show immunomodulatory effects [15]. Due to the dissolution of Zn+2 and the formation of reactive oxygen species (ROS), ZnO has a stimulating effect and leads to the release of inflammatory cytokines and the activation of immune cells [16,17]. ZnO NPs have been found to induce the production of pro-inflammatory cytokines ILs (2-4-5-8-17), TNF-α and IFNs [9]. Reduced graphene oxide (r-GO) has several properties that make it a promising candidate for drug delivery systems in various cancer therapy treatments [[18], [19], [20]]. The combination of large surface area, high drug loading capacity, biocompatibility, surface functionalization, pH sensitivity, photothermal properties and easy functionalization make reduced graphene oxide a promising material for developing drug delivery systems with potential applications in various cancer therapy treatments.

Graphene oxide exhibits immunostimulating properties in macrophage cells, whereas ZnO, recognized as a non-toxic element to cells, plays a crucial role in supporting immune system function. This study focuses on the synthesis of zinc oxide-doped graphene using reduced graphene oxide nanoparticles and varying amounts of a zinc precursor. The investigation specifically targets the production levels of IL-6 and TNF-α cytokines in macrophage cell lines. Notably, no literature was found examining the impact of ZnO-coated graphene oxide on macrophage cells.

The primary aim of this study is to systematically investigate the effects of modified reduced graphene oxide (r-GO) nanoparticles containing graphene oxide (GO) and zinc oxide (ZnO) on J774.2 macrophage cells. The specific focus is on comprehending the potential biomedical applications of these materials. Through the evaluation of the immunostimulatory properties of the synthesized ZnO@r-GO nanoparticles, the study seeks to gain a comprehensive understanding of their interactions at the cellular level and identify potential adjuvant effects. The realization of this objective will contribute to our understanding of the interaction of graphene oxide and zinc oxide combinations with biological systems, paving the way for the development of novel approaches for future biomedical applications.