Graphene derivatives and various graphene-based nanomaterials hold great potential in drug delivery due to their high loading capacity and stimuli-responsive behavior for numerous drug molecules. These properties are attributed to their large surface area, chemical and mechanical stability, and excellent thermal, electrical, and optical characteristics.
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The exceptional functionalization potential of these nanomaterials allows for their integration with various macromolecules, polymers, and nanoparticles, enabling the creation of novel nanocarriers with enhanced trigger sensitivity and biocompatibility.
In 2008, graphene was first used as a drug delivery system for cancer treatment, establishing its suitability for this application. However, challenges remain with its application in drug delivery, particularly its cytotoxicity in biological solutions, often caused by its tendency to aggregate.1,2
Challenges of Graphene Toxicity
Graphene-based nanomaterials have been extensively studied for their ability to cross cell membranes and alter physiological environments. For example, graphene derivatives and nanoparticles can penetrate the plasma membrane, triggering an inflammatory response due to the leakage of cytoplasmic contents.
In the cytosol, graphene-based nanomaterials interact with various cellular organelles and the cytoskeleton, producing toxic effects through the generation of reactive oxygen species (ROS). These ROS can lead to mitochondrial disorders by damaging the cell membrane, causing lactate dehydrogenase release and reducing mitochondrial membrane potential.
For example, a study examining the immunotoxicity of graphene oxide and vanillin-functionalized graphene oxide in THP-1 cells (a human acute monocytic leukemia cell line) found evidence of cytotoxicity, including mitochondrial membrane potential loss, decreased ATP levels, elevated lactate dehydrogenase, and cell death.
Vanillin-functionalized graphene oxide and graphene oxide triggered strong inflammatory responses in THP-1 cells by stimulating the secretion of various chemokines and cytokines. Cells treated with vanillin-functionalized graphene oxide exhibited higher oxidative stress and toxicity compared to those treated with graphene oxide alone.
Graphene-based nanomaterials can also cause genotoxic effects upon reaching the nucleus, with particle size playing a critical role in determining their toxicity and biocompatibility. For example, graphene oxide nanowells are significantly more cytotoxic in vitro than graphene oxide nanoplates. Larger graphene nanoparticles with a higher degree of oxidation typically exhibit greater cytotoxic effects.
Recent studies have also shown that graphene oxide and graphene induce distinct toxicities in vivo and in vitro based on their physical characteristics. These nanomaterials interact with red blood and dendritic cells, leading to various toxicological effects.
Aggregated graphene sheets show lower hemolytic activity, while smaller graphene oxide particles exhibit higher hemolytic activity. Similarly, both multilayer and monolayer graphene oxide promote ROS generation in dendritic cells, causing cell disruption and immunotoxicity.2,3
Approaches to Mitigating Graphene Toxicity
Reducing the toxicity of graphene derivatives and graphene-based nanomaterials is critical for their safe and effective use in drug delivery. This can be achieved through surface modifications, controlling particle size, and using biocompatible coatings.
Many studies have shown that graphene with a small particle size and a biocompatible coating does not exhibit toxicity in animals when administered within a reasonable dose range. Functionalizing graphene derivatives with suitable polymers enhances their stability, solubility, and biocompatibility.
Polyethylene glycol (PEG) has been extensively studied as a biocompatible polymer for graphene modification. PEG functionalization reduces graphene’s toxicity, improves physiological stability, and minimizes its uptake by the reticuloendothelial system.1
For example, one study developed a ROS-responsive nanofiber membrane using reduced graphene oxide as a nanocarrier and poly (ethylene glycol) diacrylate-1,2-ethanedithiol copolymer as a ROS-sensitive motif for fucoxanthin delivery. In a hydrogen peroxide environment, this membrane exhibited sustained fucoxanthin release with low toxicity.
Another study reduced graphene’s cytotoxicity by functionalizing graphene oxide nanocolloids with bovine serum albumin protein, demonstrating its effectiveness as an anticancer drug carrier.
In a related study, graphene quantum dot-iron complexes were synthesized by co-encapsulating graphene quantum dots and iron within engineered ferritin nanocages obtained from the archaeon Archaeoglobus fulgidus.
These nanocages exhibited strong fluorescence at low pH, high relaxivity in MRI, and a high doxorubicin loading capacity, with minimal cytotoxicity. This combination of properties suggests their potential use as MRI agents, drug carriers, and pH-responsive fluorophores for cancer diagnosis and therapy.
Functionalized graphene quantum dots have also demonstrated low toxicity. For instance, a recent study synthesized a smart folic acid-PEG-graphene quantum dot-carboxylic acid drug vehicle/nanodrug delivery system, which exhibited low systemic toxicity and strong antitumor efficiency.
Additionally, doxorubicin-loaded oxidized graphene nanoribbons, modified with phospholipid-PEG, showed no in vivo toxicity and were excreted from the body through urine, indicating that this nanocarrier could enhance therapeutic efficacy while reducing the risk of side effects within the body.1,4
Recent Developments
A recent study published in ACS Nano used two-dimensional graphene oxide nanosheets with varying serum protein binding profiles—high and low hard-bound protein corona (HChigh/low)—as screening tools to assess the roles of protein corona in nanomaterial toxicities in vivo. The researchers investigated the hypothesis that the in vivo nanotoxicity and biocompatibility of graphene oxide are dependent on both the protein corona and host immunity.
The results showed that HClow graphene oxide induced more severe lung injury compared to the HChigh graphene oxide in both immunodeficient and immunocompetent mice. Additionally, HClow graphene oxide caused more significant liver injury in both groups, with immunodeficient mice being more susceptible to its hepatotoxic effects.
Moreover, administration of HClow graphene oxide administration led to increased serum pro-inflammatory cytokines and heightened hematological toxicity in both immunocompetent and immunocompromised mice.
Despite the promising biomedical applications of graphene derivatives and nanomaterials, their biological responses have been limited by inconsistent data and an incomplete understanding of their potential impacts on physiological systems. More research is necessary to develop safe and effective graphene-based drug delivery systems.5
In conclusion, recent advancements in surface modifications and biocompatible coatings have shown promising results in reducing graphene toxicity while preserving the valuable properties of graphene derivatives and nanomaterials. Future research must focus on developing novel strategies for its safe and effective use in drug delivery and other biomedical applications.
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References and Further Reading
- Khakpour, E., Salehi, S., Naghib, SM., Ghorbanzadeh, S., Zhang, W. (2023). Graphene-based nanomaterials for stimuli-sensitive controlled delivery of therapeutic molecules. Frontiers in Bioengineering and Biotechnology. DOI: 10.3389/fbioe.2023.1129768, https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1129768/full
- Magne, TM. et al. (2021). Factors affecting the biological response of Graphene. Colloids and Surfaces B: Biointerfaces. DOI: 10.1016/j.colsurfb.2021.111767, https://www.sciencedirect.com/science/article/abs/pii/S0927776521002113
- Ghulam, AN., Hazeem, L., Backx, BP., Bououdina, M., Bellucci, S. (2022). Graphene Oxide (GO) Materials—Applications and Toxicity on Living Organisms and Environment. Journal of Functional Biomaterials. DOI: 10.3390/jfb13020077, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9224660/
- Jampilek, J., Kralova, K. (2021). Advances in Drug Delivery Nanosystems Using Graphene-Based Materials and Carbon Nanotubes. Materials. DOI: 10.3390/ma14051059, https://www.mdpi.com/1996-1944/14/5/1059
- Li, YT. et al. (2024). Graphene Oxide Nanosheets Toxicity in Mice Is Dependent on Protein Corona Composition and Host Immunity. ACS Nano. DOI: 10.1021/acsnano.4c08561, https://pubs.acs.org/doi/10.1021/acsnano.4c08561
