Graphene’s interactions with biological systems have been extensively studied due to its potential applications in biomedical fields, ranging from biosensing to tissue engineering. The material’s unique physicochemical properties, including high surface area, mechanical strength, and electrical conductivity, make it an attractive candidate for interfacing with biological components. However, its biocompatibility and cytotoxicity depend on multiple factors, including lateral size, surface chemistry, and degradation behavior. Understanding these interactions is critical for safe and effective medical applications.

Protein adsorption is one of the first events that occur when graphene is introduced into a biological environment. The high surface area of graphene allows for significant protein binding, which can influence cellular responses. Studies have shown that graphene oxide (GO) and reduced graphene oxide (rGO) exhibit different protein adsorption profiles due to variations in surface oxygen content. Hydrophobic graphene surfaces tend to adsorb more proteins non-specifically, while functionalized graphene with hydrophilic groups can exhibit selective binding. The adsorbed protein layer, known as the corona, affects cellular uptake, immune responses, and overall biocompatibility. For instance, fibrinogen adsorption on pristine graphene has been linked to platelet activation, increasing thrombogenic risks. In contrast, albumin-coated graphene surfaces show reduced immune recognition, improving biocompatibility.

Cell viability assays are essential for evaluating graphene’s cytotoxicity. Numerous studies using in vitro models have demonstrated that graphene’s biological impact is highly size-dependent. Larger graphene sheets (micrometer-scale) tend to induce physical damage to cell membranes due to their sharp edges, leading to necrosis or impaired proliferation. In contrast, smaller graphene flakes (nanometer-scale) are more readily internalized by cells, potentially causing oxidative stress and inflammatory responses. Quantitative assessments reveal that concentrations above 50 µg/mL of graphene oxide often reduce cell viability in various cell lines, including fibroblasts and epithelial cells. However, surface modifications can mitigate these effects. For example, PEGylation of graphene oxide significantly decreases cytotoxicity by reducing direct membrane disruption and oxidative stress.

Degradation pathways of graphene in biological systems remain an area of active research. Unlike some biodegradable polymers, graphene is highly resistant to enzymatic breakdown. However, oxidative degradation can occur under certain conditions. Myeloperoxidase (MPO), an enzyme secreted by neutrophils and macrophages, has been shown to degrade graphene oxide through oxidative mechanisms, producing smaller fragments that can be cleared more easily. The rate of degradation depends on the degree of oxidation; highly oxidized GO degrades faster than rGO or pristine graphene. Long-term accumulation of non-degraded graphene may lead to chronic inflammation or granuloma formation, emphasizing the need for controlled degradation strategies in medical applications.

Surface functionalization plays a crucial role in enhancing graphene’s biocompatibility. Covalent and non-covalent modifications can tailor graphene’s interactions with biological systems. Common functionalization strategies include carboxylation, amination, and PEGylation, which improve hydrophilicity and reduce nonspecific protein binding. For instance, amine-functionalized graphene exhibits enhanced compatibility with neuronal cells, making it suitable for neural interface applications. Non-covalent modifications using biocompatible polymers or biomolecules (e.g., chitosan, hyaluronic acid) further improve stability and reduce immune recognition. The choice of functional groups also influences cellular uptake mechanisms; positively charged surfaces promote higher internalization rates via endocytosis, while negatively charged surfaces may reduce uptake but improve circulation time in vivo.

Standardization of graphene-based materials for medical use is a pressing need. Variability in synthesis methods, purity levels, and physicochemical properties leads to inconsistent biological outcomes. Key parameters requiring standardization include lateral size distribution, layer number, oxygen content (for GO/rGO), and residual metal impurities from synthesis. Regulatory agencies must establish clear guidelines for characterization protocols, such as Raman spectroscopy for defect analysis and dynamic light scattering for size distribution. Batch-to-batch reproducibility is critical for clinical translation, as even minor variations can alter toxicity profiles. Additionally, standardized in vitro and in vivo testing models are necessary to compare results across studies reliably.

In summary, graphene’s interactions with biological systems are complex and influenced by material properties such as size, surface chemistry, and degradation behavior. Protein adsorption dictates initial biological responses, while cytotoxicity is highly dependent on physical and chemical characteristics. Surface functionalization offers a pathway to enhance biocompatibility, but long-term degradation and clearance mechanisms require further investigation. Standardization efforts are essential to ensure reproducibility and safety in medical applications. Addressing these challenges will pave the way for graphene’s successful integration into biomedical technologies without overlapping with drug delivery-specific applications. Future research should focus on establishing universal benchmarks for material characterization and biological evaluation to facilitate regulatory approval and clinical adoption.