Table of Contents

    Topic review

    Graphenic Materials for Biomedical Applications

    View times: 599
    Submitted by: Daniela Plachá


    Graphene nanomaterials have been extensively studied in terms of their properties, modifications and application potential. This entry summarizes the results of research in the fields of graphene, graphene oxide, reduced graphene oxide, which have been obtained in the last two years (2017-2019), in particular those relating to cytotoxicity of graphene nanomaterials, drug delivery systems / genes / proteins and materials. with antimicrobial properties. A brief look at other applications such as biosensors, nanotheranostics, tissue engineering is also included.

    1. Introduction

    Graphene based materials (GBMs) have been very extensively studied in last few decades. Figure 1 shows the number of publications in the period of 2004–2019, dealing with graphene materials, graphene materials in biomedical applications, and graphene materials in drug delivery systems, taking into account review articles as well. According to Web of Sciences/Scopus, more than 195,900 and 144,700 research articles dealing with graphene material have been published in this period, respectively (keyword “graphene”). It can be seen from the Figure 1 that graphene research in general and in biomedical applications started to increase rapidly after 2010. There is an expectation that research will continue to evolve because of the outstanding properties of graphene and derived materials. A growing trend is also evident for research into graphene applications in biomedical areas and, for example, in the application of graphene for drug delivery systems (keywords “graphene, biomedical”; “graphene, biomedical, review”; “graphene, drug delivery and graphene, drug delivery, review”).

    2. Influence and application

    The terms GBMs or graphene family nanomaterial (GFNs) includes graphene (GR) and its derivatives, such as graphene oxide (GO), reduced graphene oxide (rGO), GR/GO dots (GD/GOD), GR nano-onions, and GR nano-ribbons, as well as fullerenes, and carbon nanotubes (CNTs), see Figure 2 .[1][2]

    Nanomaterials 09 01758 g001

    Figure 1. Graphene research: Web of Science (upper part)/Scopus (lower part) review. Data obtained 22 September 2019.

    Nanomaterials 09 01758 g002

    Figure 2. Schematic image of selected graphene-based nanomaterials.

    GR forms a basic structure of GBMs.[3] It is a 2D-planar sheet/crystal of one-atom thickness, consisting of sp2 hybridized carbon atoms. The carbon atoms are arranged into a honeycomb lattice, in which each carbon atom is bound to three adjoining carbon atoms via two single and one double covalent bond.[4][5][6] GR can exist in a single layer, few-layered structure, or forms a graphitic structure. GR is well-known for its exceptional and outstanding mechanical properties, such as an exceptional structural rigidity, accompanied a fracture strength of 130 GPa, an elastic modulus of 32 GPa, and a 1 TPa Young’s modulus. It exhibits high electrical conductivity (104 S/cm), ultra-high thermal conductivity (5300 W/mK), and excellent electron mobility (250,000 cm2/Vs) at room temperature, which is caused by a presence of a delocalized π-electron system in the carbon lattice, where each carbon atom contributes to this system by one unbound electron freely moving through the crystal. The other significant properties are a large specific surface area of 2630 m2/g, the unusual carrier transport velocity which reaches up to 40 GHz, the charge carrier concentration of 1.4 × 1013 cm−2, and high-current carrying capacity of up to 109 A/cm2. Optical transmittance of 97.7% has confirmed these superior optical properties.[5][7][8][9]

    One can imagine GR as a polycyclic aromatic macromolecule which forms a planar structure with a large surface area, allowing the adsorption and anchoring of many compounds, such as biomolecules, metals, and fluorescent species. The compounds can interact with the system of delocalized π-electrons via electrostatic interactions and non-covalent π-π stacking. These kinds of interactions are one of the processes of GR surface functionalization.[3] [10][11]

    There are several procedures for the synthesis of GR, and two major methodological approaches can be distinguished, namely, the ‘bottom-up’ and ‘top-down’ approaches (Figure 3). There are two main considerations for GR fabrication, namely, producing GR of high quality and simultaneously producing said GR in a high quantity.[12][5] Several methods have been properly investigated, such as the mechanical or chemical exfoliation of graphite, the epitaxial growth of GR on carbides of silicon, tantalum, or titanium, or on different metal substrates, such as nickel, copper, cobalt, iridium, platinum, etc. Other methods, such as chemical vapor deposition, solvothermal or organic synthesis could be also named, or synthesis of GR from graphite oxide through oxidation of graphite eventually via un-zipping of CNTs.[5][10][13]

    GO is derivative of GR. It is a flake-like material that is characterized by the occurrence of polar oxygen-containing functional groups. The GO flakes consist of a typical graphenic planar structure, composed of sp2 carbon atoms that is disrupted by the presence of carbon atoms in the sp3 hybridization state. While on the planar surface, there are mainly hydroxyl and epoxy functional groups, while at the edges of the flakes there are carboxylic groups.[9][14][15] This composition is a reason for the aforementioned hydrophilicity, the good aqueous dispersity, and the excellent ability to easily cross cell membranes. The functional groups also allow for the capture, anchoring, or immobilization of polymers or biomolecules, such as nucleic acids, as well as drugs, NPs, etc., [16] and, for that reason, GO is a suitable carrier for delivering proteins, peptides, RNA, DNA, and various molecules of biologically active substances with antiviral, antibacterial, or anticancer activities into cells. [17]

    Nanomaterials 09 01758 g003

    Figure 3. Selected methods of graphene synthesis.

    For applications in the biomedical area, especially for delivering systems or antimicrobial materials, GO is usually synthetized from graphite using various modifications of Hummers’ method. One possibilities for a modified Hummers’ method is schematically shown in Figure 4. The effective delamination of graphite can be achieved in at a high yield by selecting suitable reagents and conditions for the manufacturing process. Thus, it can be produced in large quantities at affordable costs. The fully delaminated material may have a thickness of about 1 nm. [7][10][13] The polar groups are usually further modified by functionalization with the biocompatible and hydrophilic polymers, such as polyethylene glycole (PEG) or polyvinylpyrrolidone (PVP), then different drugs are loaded on the GR plane. Among GBMs, GO is an excellent material for drug delivery systems, precisely because the presence of functional groups makes it easy to modify its surface.[3][18]

    The interactions of GO flakes are strongly influenced by their size. Functionalized GO flakes can be easily broken down to a smaller size by mild sonication and then are able to enter into cells. It is known that GOs with lateral dimensions higher than 100 nm do not easily penetrate into cells, however GO flakes of approximately 100 nm and less can readily cross the cell membrane. If the diameter reaches below 40 nm, their penetration in the cells may be further increased. [19]

    The reduction of GO functional groups results in rGO. The structure of rGO is similar to that of GR, and it is associated with its higher electrical conductivity compared to GO. [12][14][20] Simultaneously, due to the lower content of oxygen-containing functional groups, it is more hydrophobic. Large-scale rGO production is usually performed in two steps. First, the graphite is chemically oxidized to form GO, which is then reduced to rGO. Hydrazine is the best-known reductant here, however, it is poisonous and hazardous for the environment. Due to this fact, alternatives have been studied, such as organic acids, amino acids, proteins, sugars, microorganisms, plant extracts, and antioxidants, which are called “green reducing agents”. [21] Another method of reduction is thermal, solothermal, electrochemical, microwave, and photoreduction. [2][10][15] It was found in some instances that not all functional groups were eliminated during the reduction, and some of them remained preserved on the rGO basal plane, e.g., –OH, –C–O–C–, –CO, and –COOH. The resulting properties of rGO are influenced by the reduction method used and the conditions under which it is performed.

    Nanomaterials 09 01758 g004

    Figure 4. Scheme of graphene oxide (GO) preparation using Hummers’ method.

    Other representatives of GBMs include fullerenes and CNTs. Fullerene is produced by spherically wrapping 2D GR sheets into 0D closed formations . [22][23] CNTs are formed by graphenic layers containing carbon atoms in sp2-hybridized state, which are rolled up into a hollow, cylindrical arrangement. [24][25][26] Their important feature is their very high aspect ratio, which is given by their diameter, which is in the range of several nanometers, and length, which can even reach up to millimeters. According to chirality and possible adatoms, they have a metallic or semiconductive character. They can effectively bind biomolecules by hydrophobic interactions, π-π stacking, or van der Waals forces, similarly to GR. [22] Their properties and resulting interactions with biological systems are influenced by many factors, such as the number of layers, the lateral dimensions, shape, purity, and density of defects. [27] A huge number of scientific articles have been devoted to these materials and are therefore not included in this review.

    GBMs have also received great attention in the field of biomedicine due to their exceptional features and possible applications (Figure 5).

    The proper characterization of materials used to explore possibilities outside of biomedical technologies is one of the most important steps. The main reason for these proper characterizations is that the materials are prepared in scientific laboratories as well as by different manufacturers, using different procedures, which differ in their quality. This is further reflected in the quality of the resulting material. Materials should be described as single-layer GR, multi-layer GR, ultra-thin graphite, GR nanosheets, and GR nanomaterials. According to Wick et al. [27], a few-layer GR is composed of 2–10 GR layers composed of flake-like stacks, while ultrathin graphite consists of more than 10 layers, where the thickness is below 100 nm. This should be applied also for GO.[27][6][28]

    Nanomaterials 09 01758 g005

    Figure 5. Application of graphene-based materials (GBMs) in the field of biomedicine.

    GR- and GO-based materials can be characterized according to the procedure proposed by Wick et al. [27]. The basic qualities which should be carefully observed are the number of layers (thickness), lateral size distribution, and C/O ratio, as is represented in Figure 6. [7][23][27] The lateral size of GR flakes is one of the major factors influencing GR properties, because differences in size and geometry encourage a change in the ratio between the edge and the bulky structures, resulting in limited space in specific dimensions and a consequent change in mechanical and electrical properties. [29] This characterization is very important for biomedical applications, because it is obvious that 2D GBMs with rough surfaces, a small size, and sharp edges more readily penetrate cells as opposed to smoother GBMs with larger dimensions. Because each carbon atom lies on the surface, these materials, especially in the case of monolayered GR, have a theoretical maximum surface area. For that reason, they have an exceptionally large capacity for the adsorption of molecules and biomolecules. Their capacity corresponds to the specific surface area and bending stiffness, and is associated with the number of layers. Obviously, more 2D GR layer material has a lower adsorption capacity.[30]

    Analytical methods, such as scanning and transmission electron microscopy (SEM and TEM), atomic force microscopy (AFM), infrared and/or Raman spectroscopy, X-ray photoelectron spectroscopy, and inductively coupled plasma mass spectrometry (ICP/MS), are the most useful tools for characterization. [31][32][33][34] TEM or Raman spectroscopy and AFM allow the determination of the number of layers and lateral dimensions. An accurate and real view of sheet quality, the number of defects, and delamination achieved can be obtained from AFM, which allows the determination of lateral dimensions by analyzing the shape and height of the GR/GO flakes. [6][28][35][36][37]

    There are many other methods for characterization for different purposes, for which the prepared GR is used. Among them, dynamic light scattering, zeta potential and optical absorbance measurements, such as UV/VIS spectroscopy, can be listed.[38] For example, UV/VIS spectroscopy can be used to observe changes in GO structure after the functionalization of its surface. GO has a strong absorption of UV/VIS radiation at 230 nm. Characteristic absorbance at 300 nm is done by π→π* transitions of C=C bond and the n→π* transitions of C=O bond, respectively. Upon the binding of the functional groups to C=C bonds, a shift from 230 to 266 nm occurs, and the shoulder at 300 nm is reduced. [19][34][39]

    Nanomaterials 09 01758 g006

    Figure 6. Grid for the classification of GBMs, based on their atomic C/O ratio, lateral dimension, and number of layers. GBMs shown in the corners of the grid correspond to the ideal cases. The grid is in nanoscale, but one can use it for the microscale. Reprinted from[27] with permission from John Wiley and Sons, copyright 2019.

    The publication can be found here:


    1. Kasturi Muthoosamy; Ibrahim Babangida Abubakar; Renu Geetha Bai; Hwei-San Loh; Sivakumar Manickam; Exceedingly Higher co-loading of Curcumin and Paclitaxel onto Polymer-functionalized Reduced Graphene Oxide for Highly Potent Synergistic Anticancer Treatment. Scientific Reports 2016, 6, 32808, 10.1038/srep32808.
    2. Tahriri, M.; Del Monico, M.; Moghanian, A.; Tavakkoli Yaraki, M.; Torres, R.; Yadegari, A.; Tayebi, L.; Graphene and its derivatives: Opportunities and challenges in dentistry.. Materials Science and Engineering: C 2019, 102, 171-185, 10.1016/j.msec.2019.04.051..
    3. Nishtha Panwar; Alana Mauluidy Soehartono; Kok Ken Chan; Shuwen Zeng; Gaixia Xu; Junle Qu; Philippe Coquet; Ken-Tye Yong; Xiaoyuan Chen*; Nanocarbons for biology and medicine: Sensing, imaging, and drug delivery.. Chem. Rev. 2019, 119, 9559–9656, 10.1021/acs.chemrev.9b00099.
    4. Mariana Ioniță; George Mihail Vlăsceanu; Aiza Andreea Watzlawek; Stefan Ioan Voicu; Jorge Burns; Horia Iovu; Graphene and functionalized graphene: Extraordinary prospects for nanobiocomposite materials. Composites Part B: Engineering 2017, 121, 34-57, 10.1016/j.compositesb.2017.03.031.
    5. Josphat Phiri; Patrick Gane; Thad C. Maloney; General overview of graphene: Production, properties and application in polymer composites. Materials Science and Engineering: B 2017, 215, 9-28, 10.1016/j.mseb.2016.10.004.
    6. Gao Yang; Lihua Li; Wing Bun Lee; Man Cheung Ng; Structure of graphene and its disorders: a review. Science and Technology of Advanced Materials 2018, 19, 613-648, 10.1080/14686996.2018.1494493.
    7. Johannes Walter; Thomas J. Nacken; Cornelia Damm; Thaseem Thajudeen; Siegfried Eigler; Wolfgang Peukert; Determination of the Lateral Dimension of Graphene Oxide Nanosheets Using Analytical Ultracentrifugation. Small 2014, 11, 814-825, 10.1002/smll.201401940.
    8. Garima Mittal; Vivek Dhand; Kyong Yop Rhee; Soo-Jin Park; Wi Ro Lee; A review on carbon nanotubes and graphene as fillers in reinforced polymer nanocomposites. Journal of Industrial and Engineering Chemistry 2015, 21, 11-25, 10.1016/j.jiec.2014.03.022.
    9. Abdulazeez T. Lawal; Graphene-based nano composites and their applications. A review. Biosensors and Bioelectronics 2019, 141, 111384, 10.1016/j.bios.2019.111384.
    10. Xingying Zhang; Ying Wang; Gaoxing Luo; Malcolm Xing; Two-Dimensional Graphene Family Material: Assembly, Biocompatibility and Sensors Applications.. Sensors 2019, 19, 2966, 10.3390/s19132966.
    11. André F. Girão; Maria Concepcion Serrano; António Completo; Paula A. A. P. Marques; Do biomedical engineers dream of graphene sheets?. Biomaterials Science 2019, 7, 1228-1239, 10.1039/c8bm01636d.
    12. Monisha Chakraborty; M. Saleem J. Hashmi; Wonder material graphene: properties, synthesis and practical applications. Advances in Materials and Processing Technologies 2018, 4, 1-30, 10.1080/2374068x.2018.1484998.
    13. Kristina E. Kitko; Qi Zhang; Graphene-Based Nanomaterials: From Production to Integration With Modern Tools in Neuroscience.. Frontiers in Systems Neuroscience 2019, 13, 26, 10.3389/fnsys.2019.00026.
    14. Meng-Ying Xia; Yu Xie; Chen-Hao Yu; Ge-Yun Chen; Yuan-Hong Li; Ting Zhang; Qiang Peng; Graphene-based nanomaterials: the promising active agents for antibiotics-independent antibacterial applications.. Journal of Controlled Release 2019, 307, 16-31, 10.1016/j.jconrel.2019.06.011.
    15. Duarte De Melo-Diogo; Rita Lima-Sousa; Cátia G. Alves; Ilídio J. Correia; Llidio Correia; Graphene family nanomaterials for application in cancer combination photothermal therapy.. Biomaterials Science 2019, 7, 3534-3551, 10.1039/c9bm00577c.
    16. Saifullah Bullo; Kalaivani Buskaran; Rabia Baby; Dena Dorniani; Sharida Fakurazi; Mohd Zobir Hussein; Dual Drugs Anticancer Nanoformulation using Graphene Oxide-PEG as Nanocarrier for Protocatechuic Acid and Chlorogenic Acid.. Pharmaceutical Research 2019, 36, 91, 10.1007/s11095-019-2621-8.
    17. Himani Tiwari; Neha Karki; Mintu Pal; Souvik Basak; Ravindra Kumar Verma; Rajaram Bal; Narain Datt Kandpal; Ganga Bisht; Nanda Gopal Sahoo; Functionalized graphene oxide as a nanocarrier for dual drug delivery applications: The synergistic effect of quercetin and gefitinib against ovarian cancer cells.. Colloids and Surfaces B: Biointerfaces 2019, 178, 452-459, 10.1016/j.colsurfb.2019.03.037.
    18. Jalil Charmi; Hamed Nosrati; Jafar Mostafavi Amjad; Ramin Mohammadkhani; Hosein Danafar; Polyethylene glycol (PEG) decorated graphene oxide nanosheets for controlled release curcumin delivery.. Heliyon 2019, 5, e01466, 10.1016/j.heliyon.2019.e01466.
    19. Akram Assali; Omid Akhavan; Fatemeh Mottaghitalab; Mohsen Adeli; Rassoul Dinarvand; Shayan Razzazan; Ehsan Arefian; Masoud Soleimani; Fatemeh Atyabi; Cationic graphene oxide nanoplatform mediates miR-101 delivery to promote apoptosis by regulating autophagy and stress. International Journal of Nanomedicine 2018, 13, 5865-5886, 10.2147/IJN.S162647.
    20. S. Taniselass; M.K. Md Arshad; Subash C.B. Gopinath; Graphene-based electrochemical biosensors for monitoring noncommunicable disease biomarkers. Biosensors and Bioelectronics 2019, 130, 276-292, 10.1016/j.bios.2019.01.047.
    21. K.K.H. De Silva; H.-H. Huang; R.K. Joshi; M. Yoshimura; Chemical reduction of graphene oxide using green reductants. Carbon 2017, 119, 190-199, 10.1016/j.carbon.2017.04.025.
    22. Ozlem Erol; Idil Uyan; Meryem Hatip; Canelif Yilmaz; Ayse B. Tekinay; Mustafa O. Guler; Recent advances in bioactive 1D and 2D carbon nanomaterials for biomedical applications.. Nanomedicine: Nanotechnology, Biology and Medicine 2017, 14, 2433-2454, 10.1016/j.nano.2017.03.021.
    23. Rasoul Madannejad; Nahid Shoaie; Fatemeh Jahanpeyma; Mohammad Hasan Darvishi; Mostafa Azimzadeh; Hamidreza Javadi; Toxicity of carbon-based nanomaterials: Reviewing recent reports in medical and biological systems.. Chemico-Biological Interactions 2019, 307, 206-222, 10.1016/j.cbi.2019.04.036.
    24. Reza Eivazzadeh-Keihan; Ali Maleki; Miguel De La Guardia; Milad Salimi Bani; Karim Khanmohammadi Chenab; Paria Pashazadeh-Panahi; Behzad Baradaran; Ahad Mokhtarzadeh; Michael R. Hamblin; Carbon based nanomaterials for tissue engineering of bone: Building new bone on small black scaffolds: A review.. Journal of Advanced Research 2019, 18, 185-201, 10.1016/j.jare.2019.03.011.
    25. Zihao Li; Ling Wang; Yu Li; Yiyu Feng; Wei Feng; Carbon-based functional nanomaterials: Preparation, properties and applications. Composites Science and Technology 2019, 179, 10-40, 10.1016/j.compscitech.2019.04.028.
    26. Antonio Setaro; Advanced carbon nanotubes functionalization. Journal of Physics: Condensed Matter 2017, 29, 423003, 10.1088/1361-648x/aa8248.
    27. Peter Wick; Anna E. Louw-Gaume; Melanie Kucki; Harald F. Krug; Kostas Kostarelos; Bengt Fadeel; Kenneth A. Dawson; Anna Salvati; Ester Vázquez; Laura Ballerini; et al. Classification Framework for Graphene-Based Materials. Angewandte Chemie International Edition 2014, 53, 7714-7718, 10.1002/anie.201403335.
    28. Giovanni Bottari; Maria Angeles Herranz; Leonie Wibmer; Michel Volland; Laura Rodríguez-Pérez; Dirk M. Guldi; Andreas Hirsch; Nazario Martín; Francis D'souza; Tomás Torres; et al. Chemical functionalization and characterization of graphene-based materials. Chemical Society Reviews 2017, 46, 4464-4500, 10.1039/C7CS00229G.
    29. Li-Shang Lin; Wei Bin-Tay; Zabeada Aslam; A.V.K. Westwood; R. Brydson; Determination of the lateral size and thickness of solution-processed graphene flakes. Journal of Physics: Conference Series 2017, 902, 12026, 10.1088/1742-6596/902/1/012026.
    30. Xuezhong Gong; Guozhen Liu; Yingshun Li; Denis Yau Wai Yu; Wey Yang Teoh; Functionalized-Graphene Composites: Fabrication and Applications in Sustainable Energy and Environment. Chemistry of Materials 2016, 28, 8082-8118, 10.1021/acs.chemmater.6b01447.
    31. Namdev Dhas; Khushali Parekh; Abhijeet Pandey; Ritu Kudarha; Srinivas Mutalik; Tejal Mehta; Shrinivas Mutalik; Two dimensional carbon based nanocomposites as multimodal therapeutic and diagnostic platform: A biomedical and toxicological perspective. Journal of Controlled Release 2019, 308, 130-161, 10.1016/j.jconrel.2019.07.016.
    32. Piaopiao Wei; Jian Shen; Kangbing Wu; Nianjun Yang; Defect-dependent electrochemistry of exfoliated graphene layers. Carbon 2019, 154, 125-131, 10.1016/j.carbon.2019.07.100.
    33. Manonmani Mohandoss; Soujit Sen Gupta; Ramesh Kumar; Rabiul Islam; Anirban Som; T. Pradeep; Azhardin Ganayee Mohd.; Shihabudheen M Maliyekkal; Self-propagated combustion synthesis of few-layered graphene: an optical properties perspective. Nanoscale 2018, 10, 7581-7588, 10.1039/c7nr09156g.
    34. Elvin Aliyev; Volkan Filiz; Muntazim M. Khan; Young Joo Lee; Clarissa Abetz; Volker Abetz; Structural Characterization of Graphene Oxide: Surface Functional Groups and Fractionated Oxidative Debris.. Nanomaterials 2019, 9, 1180, 10.3390/nano9081180.
    35. Joon Hyong Cho; Seung Ryul Na; Saungeun Park; Deji Akinwande; Kenneth M Liechti; Michael A Cullinan; Controlling the number of layers in graphene using the growth pressure.. Nanotechnology 2019, 30, 235602, 10.1088/1361-6528/ab0847.
    36. Jiang-Bin Wu; Miao-Ling Lin; Xin Cong; He-Nan Liu; Ping-Heng Tan; Raman spectroscopy of graphene-based materials and its applications in related devices. Chemical Society Reviews 2018, 47, 1822-1873, 10.1039/c6cs00915h.
    37. Cameron J Shearer; Ashley D Slattery; Andrew J Stapleton; Joseph G Shapter; Christopher T Gibson; Accurate thickness measurement of graphene. Nanotechnology 2016, 27, 125704, 10.1088/0957-4484/27/12/125704.
    38. C. Mellado; T. Figueroa; R. Baez; M. Meléndrez; K. Fernández; Effects of probe and bath ultrasonic treatments on graphene oxide structure. Materials Today Chemistry 2019, 13, 1-7, 10.1016/j.mtchem.2019.04.006.
    39. Juan Amaro-Gahete; Almudena Benítez; Rocío Otero; Dolores Esquivel; César Jiménez-Sanchidrián; Julián Morales; Álvaro Caballero; Francisco J. Romero-Salguero; A Comparative Study of Particle Size Distribution of Graphene Nanosheets Synthesized by an Ultrasound-Assisted Method. Nanomaterials 2019, 9, 152, 10.3390/nano9020152.
    1. Please check and comment entries here.