Graphene derivatives such as GO, rGO, and GQDs have been considered in a wide range of medical applications due to the mutable surface chemistry and excellent physical properties of these materials.

The intriguing applications of graphene-based materials in the medical field are biosensors, Bioimaging applications, tissue engineering, drug and gene delivery, novel anticancer treatments, graphene coatings on implants, and medical devices.

Introduction

The “wonder material” graphene has concurred the medical world much like many other application areas. The application areas of graphene-based materials are vast including energy storage, environmental protection, sensors, material sciences, supercapacitors, solar cells, and medical applications. The use of graphene-based materials has especially gained popularity due to their biocompatible nature, flexible surface chemistry, excellent mechanical properties such as high Young’s modulus (~1 TPa), mechanical strength (~40 N N m⁻¹), strong electrical conductivity (10⁶ S cm⁻¹), strong thermal conductivity (5000 W m⁻¹ k⁻¹), optical transmittance (~97.7%) and large specific surface areas (~2600 m² g⁻¹). Researchers have worked on characterizing, developing, and producing new medical application methods based on these excellent properties of graphene-based materials. Different medical applications benefit from different graphene derivatives thanks to the mutable nature of graphene through surface functionalization. The most prominent graphene derivatives in medical fields are found to be graphene oxide (GO), reduced graphene oxide (rGO), and graphene quantum dots (GQDs). Most of the medical applications of graphene-based materials take on the tedious tasks of the field such as cancer treatment, novel therapy and diagnostic methods, and antimicrobial protection of the clinical environment. Today, we will take look at the most prominent medical applications of graphene-based materials including biosensor and Bioimaging applications, tissue engineering, drug and gene delivery, graphene coatings on implants, and medical devices, and finally, tumor therapy applications.

Medical Applications of Graphene

Biosensor Applications

Biosensors are important devices in the medical field for diagnostic and therapeutic applications. Biosensor systems are based on the detection of biomarkers such as tissues, enzymes, nucleic acids, antibodies, and microorganisms. As technology evolves and the understanding of biological systems improves the need for accurate, sensitive, and selective biosensors also gains importance. Graphene-based materials have shown great potential for the development of next-generation biosensors due to their excellent electrical and thermal conductivity, high surface area, two-dimensional structure, and durability. In biosensor applications, graphene is utilized as a transducing agent. GO, rGO, and GQDs are commonly utilized graphene materials in biosensor applications. Three different approaches are utilized in graphene-based biosensor applications. One of these approaches uses a probe molecule on the graphene sheet that interacts with the analyte while another approach is label-free and based on the measurement of the electrical changes on graphene upon the interaction with an analyte. The third approach is based on the fluorescent properties of GQDs. The functional groups on GO and rGO present excellent binding sites to the biomarker molecules. GO is especially preferred due to its high biocompatibility, biodegradability, water solubility, and high specific surface area. The water solubility of GO provides ease of biosensor production while the high surface area of GO allows the detection of small molecules such as estrogen, nicotinamide, adenine dinucleotide (NAD) and adenine triphosphate. Furthermore, the oxygen-containing compounds on the GO surface such as hydroxy, carbonyl, carboxyl, and epoxide as well as the surface charge of the GO surface promote immobilization of biomolecules to the sensor. GO can be applied as a thin film to various sensor surfaces to not only capture biomolecules but also to support cell growth suitable for live-cell biosensing. Fluorescent biosensors based on the optical changes of the substrate upon interaction with the analyte have been greatly utilized in diagnostics. However, traditional fluorescent materials such as CdS, ZnS, InP, and PbSe quantum dots (SQDs) show non-negligible cytotoxicity. GQDs have the advantage of biocompatibility over SQDs and offer sensing properties. GQD based biosensors are utilized in cancer diagnosing, analysis of glucose, DNA, and protein biomarkers, as well as the immunological assay studies.

Bioimaging Applications

Another playing field for graphene in the medical industry is the bioimaging applications. Bioimaging is utilized in the visualization of biological processes under the optical system and to get the three-dimensional structure or image of the fixed specimen. Recently, bioimaging applications have especially focused on tumor cell observation. The observation of cells, tissues, or multicellular organisms offers valuable insight into metabolites and interactions. Graphene has been used for optical bioimaging methods due to its excellent electrical and optical properties, biocompatibility, and durability. The common bioimaging methods that utilize graphene-based materials are fluorescence microscopy, two-photon fluorescence, positron emission tomography, magnetic resonance imaging, Raman imaging, multimodal imaging, and photoacoustic imaging and computed tomography. Graphene oxide and graphene quantum dots are favored in bioimaging applications. GO nanosheets are characterized by visible and near-infrared fluorescence which allows bioimaging with lower background. The in vivo imaging of cells is successively achieved with the use of GO nanosheets. Biosensors utilizing GO nanosheets are found to be effective for the imaging of anti-adenosine triphosphate aptamer. Additionally, GQDs are strong contestants in bioimaging applications because of their broad absorption and narrow emission spectra, high photostability, the strong quantum confinement, and bright fluorescence. Near-infrared reflectance of GQDs is appropriate for deeper tissue imaging. The fluorescence wavelength of GQDs is commonly between 270-680 nm and varies based on the synthesis method, functional groups, and excitation wavelengths. In bioimaging, variations of fluorescence color are widely utilized. The most common fluorescence wavelengths present the colors green and blue. Functionalization of GQDs is frequently considered to decrease the GQD concentration needed for quality imaging since it is found that high concentrations of GQD showed cytotoxicity. Functionalization is achieved through the inclusion of folic acids (FA) or heteroatoms. Graphene-based bioimaging applications hold an important place in diagnostics and therapeutic practices. These imaging materials are especially utilized in the diagnosis of tumor cells, Alzheimer’s disease, and AIDS.

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Tissue Engineering

Tissue engineering is an up-and-coming interdisciplinary field that requires the input of biological engineering and material sciences. Tissue engineering applications focus on the development of biological substitutes that can repair, maintain, or improve tissue’s function. The practice of tissue engineering is mainly based on the development of degradable scaffolds with biocompatible chemistry to provide cellular attachment, proliferation, differentiation, and support new tissue formation as well as the mechanical strength to support and mimic the tissue. Furthermore, these scaffolds must be biodegradable to avoid the need for surgery for removal, highly porous to facilitate cell adhesion and diffusion, have compatible pore size and pore interconnectivity to favor tissue integration and vascularisation. Tissue engineering scaffolds are often composites of polymers and bioactive materials. Biocompatible polymers that can be natural or synthetic are used to obtain biodegradable scaffolds. However, more often than not polymer networks alone lack mechanical strength and fall short on the requirements of tissue engineering. Graphene-based materials, GO and rGO, are great candidates to improve the physical properties and bioactivity of tissue engineering scaffolds. The inclusion of graphene-based materials into scaffolds significantly increases the tensile strength and elasticity modulus which is particularly important for natural polymer-based scaffolds since natural polymers tend to lack mechanical strength and show high degradation rates. Graphene improves the mechanical strength of natural polymers while providing control over the degradation rate. Graphene improves cell adhesion, viability, proliferation, and differentiation due to its surface chemistry and high surface area. Improved bioactivity of graphene containing scaffolds is utilized in stem cell differentiation. Furthermore, the high electrical conductivity of graphene is a very valuable asset in neural tissue engineering since it influences the behavior of electro-active neural cells. The inclusion of graphene into the scaffolds is also an important topic to consider. Graphene can be coated or attached directly onto the polymer network or included as a composite material with other bioactive materials such as HAp and bioactive glass. In the composite structure, graphene improves the bioactivity as well as the mechanical strength of other bioactive materials.

Drug Delivery

Medical drugs have been the backbone of medical practices for a very long time. However, conventional drugs have some crucial drawbacks including non-specific targeting, short blood circulation time, and burst release, resulting in low availability, poor therapeutic efficiency, and side effects. Hence, the new generation of medical applications has focused on targeted drug delivery systems to overcome these drawbacks of conventional drugs. Targeted drug delivery systems offer selectivity, controlled release of drugs, easier, accurate and less frequent dosing, decrease in required drug concentration, and toxic effects on the human body. Amongst the various different vehicles for targeted drug delivery applications, graphene-based materials, especially GO and GQDs, have attracted attention. GO is considered due to its unique 2D structure, ultra-large surface area, excellent stability, facile-modification surface characteristics, good biocompatibility, water solubility, and potential for mass production. The single-atom-layer structure benefits the adsorption and anchoring of drug molecules on both sides of the GO surfaces, contributing to higher drug loading performance. Furthermore, the oxygen-containing compounds on the GO surface provide control over the surface chemistry, binding sites to drug molecules, targeting, and stimuli-response properties to the drug delivery vehicles. The drug loading capability of GO can be altered by adjusting the pH and concentration. Even though most of the studies have focused on GO-based drug delivery applications, GQDs have also present great potential in this area. The fluorescent behavior of GQDs allows the tracking of movement in the cells and understanding the cellular drug uptake mechanisms. Targeted drug delivery vehicles based on graphene structures either employ GO and GQDs solely or in combination with biocompatible polymer networks such as chitosan. Graphene-based drug delivery systems have been especially useful in anticancer treatments since targeted drug delivery reduces the notorious side-effects of cancer treatments while enhancing the anticancer performance greatly. In addition to facilitating the delivery of anticancer agents, graphene-based drug delivery systems can be utilized for the delivery of peptides and biomolecules as well.

Gene Delivery

Apart from the drug delivery applications, the surface chemistry, and high adsorption properties of graphene oxide are also used in gene delivery and gene therapy applications. Gene therapy is based on repairing the gene damage which requires selective delivery of the gene into damaged cells. Gene delivery refers to the transportation of DNA and RNA strands anchored onto the vehicle molecule. Even though GO is considered to be a promising agent for gene delivery the surface modification of GO is required to eliminate the effect of hindrance caused by electrostatic repulsion of DNA molecules. Low-molecular-weight branched polyethyleneimine (PEI) is frequently used for the surface modification of GO which facilitates the immobilization of DNA and RNA plasmids. In addition to polyethyleneimine, chitosan-functionalized GO which is frequently used in drug delivery is also applicable to gene delivery systems. Another GO composite for gene delivery is cationic lipids modified GO nanoparticles (GOCL NPs) which have good biocompatibility and potential for clinic applications. Gene delivery systems are mostly exploited for cancer treatment applications to offer an effective method for this tedious problem. Another advantage of GO-based vehicles in cancer treatment applications is the capability to load both gene and anticancer drugs simultaneously onto the GO surface enhancing the anticancer effect significantly.

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Graphene Coating on Implants

For the past few decades, biomedical implants have been excessively used in medical applications. These implants are mostly used in coronary/cardiovascular stents, cranial fixation, orthopedic stents, and dental implants. The most commonly used materials for biomedical implants are novel alloys such as stainless steel and nitinol due to their excellent mechanical strength. The challenges with alloy based implants are acquiring biocompatibility, corrosion, and wear resistance. Graphene with its atomically smooth surface, chemically inert nature, and high durability is a good candidate for overcoming the drawbacks of alloy implants. The studies have shown that the chemical inertness of graphene coatings on implants improves the biocompatibility of the material while the durability of graphene provides high wear resistance to the material. The corrosion resistance of graphene-based coatings is attributed to the impermeable nature of graphene to all gasses as well as the chemical inertness. The most commonly used method to obtain graphene coating on implants is chemical vapor deposition since it is a simple well-known method and allows the deposition of thin homogenous layers of graphene.

Graphene Coatings on Medical Devices

Sterility is crucial in clinical environments to decrease hospital-acquired infections. Major sources of hospital-acquired infections are medical devices such as catheters, cardiac pacemakers, implants, joint prostheses, prosthetic heart valves, and dentures. Bacteria can attach to medical devices and form biofilms resistant to antibiotics and the immune system. This is why the prevention of bacterial attachment to the surface of medical devices hold an important place in reducing hospital-acquired infections. Antimicrobial coatings on medical devices are considered as an appropriate method to prevent bacteria colonization. These coatings shouldn’t interfere with the bulk mechanical properties, offer long-term durability, resistance to bacteria attachment, and development. Antimicrobial properties and excellent mechanical strength of GO and rGO have particularly attracted attention as an antimicrobial coating material on medical devices. Furthermore, the solubility of GO in water allows mixing with polymer solutions to form hydrogel structures while the functional surface groups allow the attachment of antibacterial particles such as silver. The antibacterial property of GO is based on three different mechanisms.

• Mechanical destruction of the cell membrane by the sharp edges of GO sheets. This mechanism is also called the nano-blade effect which causes the leakage of cytoplasmic constituents and the death of microorganisms.
• Chemical destruction of cells through cell oxidation. GO induces oxidative stress on bacteria interfering with cellular metabolism and leading to cell necrosis/apoptosis.
• The third mechanism is based on the isolation of microorganisms from the environment preventing the nutrition and proliferation of the cell. However; this method is mainly observed in the solution phase rather than coatings.

Antimicrobial graphene coatings can be based solely on the antimicrobial properties of GO and rGO or the combined effect of GO and other antibiotic agents (such as lysozyme, silver, and chitosan). In the second case, GO is used to enhance the mechanical and antibacterial properties of the composite material. GO-based antimicrobial coatings are prepared by several different methods such as spin coating and electrospinning, vacuum filtration, electroless plating, phase inversion, electrophoretic deposition, hydrogel self-assembly/crosslinking, wet chemical reduction, solvent evaporation, and solution-casting. Each of these methods presents different advantages and disadvantages. In particular, hydrogels offer an increase in biocompatibility while deposition methods such as electrophoretic deposition increase the sharp edges of GO sheets and consequently the antimicrobial behavior. The important parameters to consider for GO coatings are biocompatibility, GO lateral size, concentration, and functionalization.

Tumor Therapy

So far we have discussed the use of graphene-based cancer treatment methods that are mostly focused on cellular interference. However, there are other tumor therapy methods utilizing the promising properties of graphene and focus on the cancerous tissue altogether. Such methods include photothermal therapy and radiotherapy. Photothermal therapy is a non-invasive method which is based on the exogenous NIR irradiation. Compared to other methods such as chemotherapy, surgery, and radiotherapy, photothermal therapy shows fewer side-effects and systemic toxicity, negligible drug-resistance, and controllable application. Photothermal therapy involves planting photosensitizing agents into the cancerous tissue and exposing this area to NIR laser to generate considerable heat to kill cancerous cells due to hyperthermia. Graphene-based materials fill the role of photosensitizing agents in this therapy method. It is found that rGO shows a better photosensitizing effect compared to GO and graphene. Furthermore, rGO also shows drug loading properties enabling the combination of targeted drug delivery based chemotherapy and phototherapy. The delivery of rGO to the cancerous tissue is often facilitated with the use of polymers such as PEG and hyaluronic acid (HA). Radiotherapy is a well-known clinical tumor therapy method and a widely used practice all over the world. This method is based on the irradiation of high-energy rays which initiate ionizing radiation producing significant damages to DNA and leading to efficient anticancer effect. However, conventional radiotherapy applications lack targeted application, face radiation resistance, and causes considerable side-effects. The use of graphene-based materials in radiotherapy applications shows promising effects on reducing these drawbacks. Go and rGO are used as radionucleotide carriers to obtain targeted radiotherapy effects. Furthermore, the active reaction of GO with oxygen-free radicals, GO can effectively protect normal cells. The use of rGO also allows combining radiotherapy and photothermal therapy creating an effective anticancer treatment method. 

Conclusion

From the time of its discovery, graphene and graphene-based materials have attracted a lot of attention in various different fields. Medical applications were no exception to these areas and jumped onto the graphene bandwagon quite rapidly. The intrinsic properties of graphene and graphene-based materials such as mechanical strength, electrical and thermal conductivity, optical properties, and high surface area are exploited in medical applications to create novel solutions for the challenging problems of the medical field. Furthermore, the mutable surface chemistry of graphene is utilized greatly. The graphene-derivatives such as GO, rGO, and GQDs themselves are the product of the modified graphene surface. These graphene derivatives attracted the most attention in medical applications leaving the infamous graphene behind. Graphene-based materials are most prominently used in sensors, Bioimaging applications, tissue engineering, gene and drug delivery, graphene coatings on implants and medical devices, and novel tumor therapy applications.

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