Graphene derivates: a general overview and possible ways to detox

Formation of self-assembling gel of glutathione in the presence of graphene oxide

Legend:

G = graphene

GO = graphene oxide

rGO = reduced graphene oxide

In the first part of this post I present you a brief summary of the excellent paper with the title “Synthesis and Toxicity of Graphene Oxide Nanoparticles: A Literature Review of In Vitro and In Vivo Studies” (source: https://doi.org/10.1155/2021/5518999)

The second part is more focused on potential ways to detox from graphene derivates.

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1. What is graphene? A short overview.

Graphene is carbon-based nanomaterial which was first discovered in 2004 by the working group of Andre Geim. Due to its unique properties and diverse properties such as transparency (depending on number of layers), thermal property, electrical conductivity and mechanical strength make it a versatile material for various fields. These include for example electronics, car industry, medical field (especially DNA sequencing), development of biosensors as well as cell differentiation and growth.

Graphene is insoluble in water and therefore the use is limited to passive platforms for detection and cell work. GO on the other hand can be used to administer anticancer drugs in biological cells and gene delivery. Due to the large surface area, the stability of the drug is maintained as its not altering the biological activity. Self-healing hydrogels for wound healing which can be injected include quaternized chitosan (QCS), polydopaminecoated reduced graphene oxide (rGO-PDA), and poly(N-isopropylacrylamide) (PNIPAm).

The toxicity is limiting it’s use and mainly depends on the route of administration, administered dose, method of synthesis of GO and its physicochemical properties.

2. How is it synthesized

A standard method for the synthesis of GO does not exist, because each method creates a different type of GO. The distinct physicochemical properties are structure and reactivity vary depending on the method and carbon source.

3. Structure of GO

Several structures have been suggested.

4. Characterization of GO

Atomic force spectroscopy (AFM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy, solid state nuclear magnetic resonance (Ss-NMR), Fourier transform infrared spectroscopy (FT-IR), X-ray induced photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and thermogravimetric analysis (TGA).

Raman spectroscopy is the most specific method to characterize GO:

2 peaks only: G and D band.

G band (sp²) is at about 1580 cm^-1 and is attributed to the ordered crystal structure’s phase vibrations, while the D band(∼1350 cm^-1, sp³) is attributed to the disorder’s crystal structure.

2D band is a harmonic of D band at around 2700 cm^-1 and provides information on the graphene planes’ stacking order.

Monolayer graphene: intense Lorentian peak (~ 4 times intensity of G band). Shape of 2D strip changes based on the number of planes. From 5 planes of thickness is identical to hexagonal graphite.

5. Properties of GO

Main characteristic: behaves like hydrophilic material. Hydroxyl groups, epoxides, ketones and carboxylic acids allow biochemical and bioconjugation reactions to occur at the basal plane and the edges of GO.

Functionalization with proteins, antibodies and DNA are possible. High adsorption capacity for proteins and antibodies. Polypeptide can be adsorbed on the GO’s surface by hydrophobic interaction, van der Waals forces, electrostatic interactions and hydrogen bonds. Protein is protected against proteolysis. Stacking interactions π- π may occure due to the fact of abundant π electrons.

6. Toxicity in cell models

Factors: dose, lateral size, and surface charge.

GO with a dose less than 20 μg/mL did not exhibit toxicity to human fibroblast cells, and the dose of more than 50 μg/mL exhibits cytotoxicity such as decreasing cell adhesion, inducing cell apoptosis, and entering into lysosomes, mitochondrion, endoplasm, and the cell nucleus.

Cytotoxicity depends on cell type. Neuroblastoma cell link shows no cytotoxicity up to 80 µg/ml.

Lateral size affects toxicity. Thermally reduced GO more toxic than chemically reduced GO.

GO with smaller size cause more oxidative stress.

Surface charge: Interaction of the negative charged oxygen groups with the positive charged phosphatidylcholine on the outer membrane of red blood cells. Negative charge induced platelet activation and aggregation. Moreover, GO induces cardiotoxicity, mitochondrial disruption, generation of ROS and DNA interactions.

=> degree of toxicity is a function of physicochemical properties of GO and the experimental conditions.

7. Toxicity of GO In Vivo

Natural pathways into an organism: inhalation, ingestion and dermal

For biomedical application: injections.

8. Toxicity Mechanisms

Direct interaction of GO nanosheets causes physical damage to cell membranes. Once GO penetrates the cell, it can destroy high amounts of lipid membrane phospholipids and inducing cell membrane degradation. ROS leads to apoptosis and is the primary mechanism for GO toxicity.

Graphene derivates

Here I present you a very quick summary of the difference between graphene and its derivates.

(source: https://nanografi.com/blog/what-is-the-difference-between-graphene-oxide-and-reduced-graphene-oxide/)

Main difference relies on the following properties:

Potential solutions for detoxification

An article named "Endoperoxides Revealed as Origin of the Toxicity of Graphene Oxide" stated that EDTA does not influence the endoperoxides induced by GO, but an oligonucleotide linked with a fluorescein derivative significantly decreased the reactivity. Enter the following quote into Sci-Hub: “DOI: 10.1002/anie.201507070”

Humic acids alleviate the toxicity of reduced graphene oxide modified by nanosized palladium in microalgae

https://doi.org/10.1016/j.ecoenv.2022.113794

2 papers that mention kaolin to alleviate the toxicity of GO:

DOI: 10.1021/acs.estlett.8b00135

DOI: 10.1039/C8MD00633D

Regarding glutathione to „detox“ from GO

One-step reduction of graphene oxide with L-glutathione

(source: DOI:10.1016/j.colsurfa.2011.05.019 )

Finally, similar to the case of reduction of GO by vitamin C, the mechanism for reduction of GO via GSH under the employed conditions are currently unclear. However, the stabilization mechanism of the graphene nanosheets suspension may originate from the oxidized product of GSH. In the reduced state, each GSH
is able to release a proton and then reacts with another reactive glutathione to form glutathione disulfide (GSSG) as shown in Fig. 8a. It is well-known that GO contain mainly two types of reactive oxygen species, including hydroxyl and epoxy functional groups on the basal plane. The protons have commonly high binding to these oxygen-containing groups, yielding water molecules and the yellow–brown GO aqueous dispersion has been changed into black homogenous solution after reduction as shown in Fig. 8b”

The conclusion is that reduction of GO by non-toxic agents such as GSH to graphene or rGO increases its biocompatibility. Thus, its rendered to be less toxic, but see the next paper.

Self-assembly of natural tripeptide glutathione triggered by graphene oxide†

(source: DOI: 10.1039/c2sm25938a)

Just by accident, I’ve found this paper that GSH and GO can form a self-assembling gel with unique structures depending on the concentration of GO in the GSH solution. This adds another layer of complexity in regards to the reduction of GO with GSH.

Glutathione (g-glu-cys-gly, GSH) is an important tripeptide in the human body because of its well-characterized functions, including the detoxification of exogenous electrophiles and reactive oxygen species, maintenance of cellular thiol status, and serving as a cofactor in the biosynthesis of endogenous compounds. It has been reported that the oxidized disulfide form (GSSG) of GSH and its derivatives can self-assemble into transparent, thermoreversible gels. These gels were a fibrous network of filaments approximately 75 nm wide, and the structure appeared to consist of straight fibers with multiple branching ‘‘nodes’’, which intersected and tangled to form a three-dimensional web-like fiber network. Additionally, complexes of GSSG with both lipids and surfactants could form fibers with lengths of at least a few microns. A GSH–pyrene conjugate was reported to form a fluorescent hydrogel in water with a branched fibrous network. The trigonal conjugate of GSH was shown to spontaneously self-assemble into nanospheres, a process driven by hydrogen bonding and the electrostatic interactions between GSH units. These results suggest that GSH or its derivatives could act as building blocks for further supramolecular assembly.

Herein, we develop a facile and straightforward approach for producing GSH/GO gels or GSH/reduced graphene oxide (RGO) gels. Unlike the initial fiber structure of GSH gel, the GSH/GO gels exhibit different shapes, for example straight fibers, bead-like, or globular structures, by adjusting the content of GO. The change in morphology may be ascribed to the rearrangement of the hydrogen bonding pattern in the b-sheet-like structures. It may be useful for understanding the formation of GSH gels and may additionally provide a new route to design engineered, self-assembled peptides. The GSH/GO gels exhibit good adsorption and controlled releasability of Rhodamine B, which was used a model drug, and may be exploited for the controlled release of drugs in the future.

I want to emphasize that the picture above was taken with a TEM. A proper darkfield microscope will not get to the nm level. Hence, you’ll not see the same structures.

For GSH gels, GSH (80 mM) in DMSO was gently heated to 60

°C, and then left to gelate. GSH gels with different concentrations were prepared by diluting the 80 mM of GSH solution and
then left to gelation.

I doubt that this will happen inside the human body if you take DMSO, because a 60°C environment is very unlikely. This whole experiment was done in vitro, but my question is that if glutathione can be triggered to form self-assembling structures inside the human body?

However, the formation of these specific GSH/GO gels or GSH/reduced graphene oxide (RGO) gels need to be confirmed first in vax damaged patients via Raman spectroscopy, TXRD and/or FT-IR before some of us jump into conclusions. Nethertheless it’s an interesting finding that might be useful for the bigger picture. Who knows.