Introduction

A hydrogel is a three-dimensional (3D) hydrophilic polymer network [1, 2] with several unique characteristics, including hydrophilicity, swelling, micro-/nanosized pores, and softness. A hydrogel can absorb considerable amounts of water molecules (>90%) from aqueous solutions and swells but does not dissolve in the solution [3]. The maintenance of hydrogels in aqueous solutions is attributed to the chemical or physical crosslinking of its hydrophilic chains [4]. In addition, hydrogels contain internal micro-/nanopores that allow the diffusion of small molecules from and to their surroundings [5,6,7,8]. Hydrogels are also mechanically flexible and soft, and their properties can at least partly mimic those of soft living tissue [9,10,11]. These characteristics of hydrogels make them very useful for various biomedical applications, such as tissue augmentation, drug delivery, and tissue engineering scaffolds.

Hydrogels are typically synthesized with various natural and/or synthetic polymers, and the natural polymers that are commonly used for hydrogel synthesis include hyaluronic acid, alginate, chitosan, collagen, and gelatin. Synthetic polymers include polyethylene glycol (PEG), poly(acryl amide) (PAAm), poly(2-hydroxyethyl methacrylate), and poly(acrylic acid) (PAA). The characteristics of hydrogels, such as toughness, elasticity, and water content, can be tailored by varying the monomer (macromer), crosslinker type(s), and crosslinking densities [12]. Despite the utilities and versatilities of hydrogels, conventional hydrogels still have some limitations in some cases. For example, most pristine hydrogels are mechanically weak [13], whereas hydrogels with high mechanical strength and stability are frequently desired for long-term, load-bearing applications [14]. In addition, novel properties, such as electrical conductivity, have been recently introduced to hydrogels to extend their applicability and/or the realization of new functions while maintaining the original properties (e.g., hydrophilicity and softness) [15,16,17,18,19]. With respect to drug delivery, typical hydrogels often display poor drug-loading and drug-release abilities for some drug molecules (e.g., hydrophobic compounds), which causes some difficulties in the fine tuning of the drug-release profiles [15]. Consequently, many strategies involving the incorporation of double-network, polyampholyte, and nanocomposites have been developed to enhance the mechanical strength and drug-release behaviors of hydrogels and imbue electrical conductivity to hydrogels [20].

Graphene and graphene derivatives (e.g., graphene oxide (GO)) have been widely formulated with hydrophilic polymers to improve the properties of hydrogels, such as mechanical properties, electrical conductivity, and drug loading/release (Fig. 1). Graphene is a two-dimensional single layer of carbon atoms with a hexagonal lattice structure and hybridized sp2 orbitals [21, 22]. In practice, GO is obtained from graphite by oxidative exfoliation using strong oxidants and acids. Due to these harsh synthetic procedures, GO possesses defects on its hybridized sp2 orbitals and diverse functional groups (e.g., epoxide, phenolic hydroxyl, and carboxylic groups) (Fig. 2). These oxygen-containing functional groups of GO aid its dispersion in aqueous solutions and its miscibility with hydrophilic polymer chains in hydrogels [23,24,25]. Consequently, most graphene-based biomaterials, including hybrid hydrogels, have been fabricated using GO. GO generally exhibits hydrophilic edges with oxygenated functional groups (e.g., carboxylic groups and hydroxyl groups) and a hydrophobic basal plane, which enables its multiple molecular interactions with various small and macromolecules, including van der Waals, hydrophobic interactions, and electrostatic interactions [26]. GO can be further chemically reduced to form reduced GO (rGO), which has fewer oxygen-containing functional groups than GO because the chemical reduction of GO leads to the partial restoration of sp2 bonds from the defects in the GO material (Fig. 2). rGO shows improved electrical conductivity and better optical adsorption of light with a wide range of wavelengths. However, rGO is relatively hydrophobic and thus insoluble in water, which result in difficulties in the formulation of composites with hydrophilic polymers. GO and rGO strongly interact with various small and large molecules via various mechanisms, such as π–π interactions, hydrophobic interactions, hydrogen bonding, and electrostatic interactions, which can also result in changes in hydrogel performance [27]. Graphene derivatives (i.e., GO and rGO) can be effective building blocks for generating new chemical and physical properties, such as electrical and thermal conductivity, light adsorption, flexibility and high mechanical strength [6, 28]. GO and rGO can be modified for the inclusion of additional functional groups and their conjugation and immobilization with other micro-/macromolecules, which can extend the application of the resulting hydrogels to drug delivery, biosensors, and tissue scaffolds [29]. It should be noted that a high concentration of GO often results in its assembly to form a gel [30]. However, this gelation occurs under specific conditions, such as low pH, a high GO concentration (>4 mg/mL), and large-sized GO sheets (several microns in lateral sizes). The repulsive forces between GO sheets should be greatly reduced to form self-assembled GO sheets and, in turn, a GO gel. In an acidified solution, the carboxylic groups of GO become protonated and neutral, whereas GO sheets do not form a gel under physiological conditions (neutral pH) due to their excessive repulsive forces. In addition, assemblies of GO in a GO gel slowly change their organization from edge-to-edge binding to basal binding, which eventually results in the formation of GO aggregates and causes instability of the GO gel. This review focuses on GO-incorporated gels or GO gels for potential biomedical applications; hence, GO gels were not included in this review.

Fig. 1
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Schematic illustration of a graphene-incorporated hydrogel and its novel properties that enable the promotion of new functions

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Fig. 2
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Chemical structures of graphene, graphene oxide (GO), and reduced graphene oxide (rGO)

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Toxicity and biocompatibility of GO-based nanomaterials

GO has been extensively used for various biomedical applications; however, its biocompatibility and toxicity have been debated [31], and a clear conclusion has not been reached. Moreover, its toxicity and biocompatibility appear to be critically associated with the dosage, functionalization, synthetic procedure, and experimental setup. In this review, we attempted to briefly provide information regarding the toxicity of GO and rGO, and then describe the synthesis and application of GO-incorporated hydrogels in the following sections. First, the antibacterial activity of GO has been widely reported, and the antibacterial activities of GO are thought to result from the action of its sharp edges, the induction of oxidative stress, and direct adhesion onto bacterial membranes [32]. Lu et al. observed that GO nanosheets cause physical disruption of the lipid bilayer of Escherichia coli [33]. Similarly, GO can also be toxic to eukaryotic cells depending on the concentrations, exposure time, size, shape, and type of graphene [34] because GO and rGO can destroy cellular membrane functions and induce excessive generation of reactive oxygen species (ROS), which results in cell apoptosis and necrosis [35,36,37]. For example, Dervin et al. observed that the leakage of lactate dehydrogenase from rGO-treated A549 cells is decreased, which implies damage to the cell membrane [38]. Nevertheless, more recent studies have shown that GO and rGO exhibits good biocompatibility with no substantial toxicity at low-to-moderate dosage ranges [39,40,41,42]. De Luna et al. found no significant reduction in macrophage viability after 24 h of incubation at a GO concentration less than 12.5 μg/mL, which is in agreement with the results reported by Mendes et al. and Yue et al. [43,44,45]. Bengston et al. found that GO generates more ROS than the rGO materials; however, after 24 h of incubation, neither GO nor rGO caused cytotoxicity to murine lung epithelial cell viability or genotoxicity [46]. With respect to the in vivo toxicity of GO and rGO, the main concerns include their eventual accumulation in some organs for long periods of time because accumulated GO or rGO can induce inflammation and a granulomatous reaction depending on the dosage [28, 47]. Manjunatha et al. observed that pristine graphene flakes (pG, liquid-phase exfoliation of graphite) cause significant toxicities, including embryonic mortality, delayed hatching, and morphological defects, in the zebrafish embryo model system [48]. In contrast, the administration of GO (0.1 and 0.25 mg) to mice leads to no obvious toxicity [49]. Importantly, the modification of GO critically affects its toxicity and biocompatibility. Xu et al. demonstrated that the biocompatibility and safety of GO can be substantially improved by the surface modification of GO with PAA [50].

Most GO-related toxicity has been tested using dispersed GO, and thus, most studies have focused on the direct interactions between the material and biological components (e.g., cells). In contrast, the entrapment of GO in hydrogel matrices can minimize the prompt and direct effects of GO on cells, such as cell membrane disruption. Therefore, GO-incorporated hydrogels can reduce the potential acute toxicity of GO. GO- or rGO-incorporated PAAm hydrogels support myoblast growth and differentiation without inducing cytotoxicity. In addition, the tissue reactions of these hydrogels are similar to those of GO-free PAAm hydrogel controls [51, 52]. GO- and rGO-incorporated hydrogels possess beneficial biological activities, including antioxidizing activity and promotion of the growth and differentiation of some types of cells [53,54,55,56,57,58,59]. For example, GO and rGO enhance the osteogenesis of mesenchymal stem cells (MSCs), which is considered to result from the adsorption of osteogenic inducers (e.g., dexamethasone) and extracellular matrix (ECM) on graphene and GO [40]. In addition, GO-incorporated chitosan substrata promote the adhesion and osteogenic and neurogenic differentiation of human MSCs (hMSCs) [60]. Hence, GO-embedded hydrogels can not only reduce the potential toxicity of GO but also provide special biological activity.

Applications of graphene-incorporated hydrogels

In this review, the roles of graphene derivatives in hydrogels are discussed with a focus on their biomedical applications. When GO and hydrophilic polymers are composited, the GO components can increase the mechanical properties and stabilities of hydrogels compared with those of GO-free hydrogels. The incorporation of GO into hydrogels can also enhance the molecular adsorption and electrical conductivity of hydrogels. In the subsequent sections, the basic roles of graphene components in hydrogels are illustrated with various examples. Note that numerous composite hydrogels containing various nanomaterials (e.g., metal nanoparticles and organic nanoparticles) have been fabricated to obtain hydrogens with improved properties [48, 61,62,63,64,65]. Compared with such conventional nanomaterials, GO presents an extremely high surface-to-volume ratio with a large lateral dimension, flexibility, electrical conductivity, biological activity, and multiple strong molecular interactions with various molecules. These unique characteristics of GO-incorporated hydrogels are beneficial for various biomedical applications.

Improvement of mechanical properties and stabilities

As mentioned in the previous section, conventional hydrogels are inherently fragile and weak. In certain cases, tougher and stronger hydrogels are required [10]. The mechanical properties of hydrogels (e.g., elasticity and toughness) are typically engineered by adjusting the concentration of the monomer(s) and crosslinker(s) [66]. Stiffness and toughness are tightly correlated with each other; hence, simple control over the crosslinking density of a certain hydrogel is often insufficient to achieve a hydrogel with suitable mechanical strength [67]. As an alternative, the hybridization of various polymers with various nanomaterials (e.g., organic and inorganic nanomaterials) has been performed to create composite hydrogels [48, 61,62,63,64,65, 68]. The introduction of nanomaterials to hydrogels can stabilize the structures of the hydrogels by forming diverse physicochemical interactions with polymeric chains and thereby improve the mechanical properties of the bulk hydrogels [69]. In this sense, extensive studies have attempted to produce tough and/or rigid hydrogels composed of GO-based nanomaterials [6, 52, 66, 69,70,71,72,73,74,75,76,77,78,79]. GO and rGO strongly interact with macromolecules, including polymer chains, via various interaction modes (e.g., hydrogen bonding, electrostatic interactions, hydrophobic interactions), which in turn critically determines the overall hydrogel structure and the mechanical properties of the composite hydrogels, such as elastic modulus, toughness, and tensile strength [66]. The molecular interactions of polymer chains and GO flakes are greatly affected by the chemistry, purity, and structures of both components [80]. Zhang et al. obtained high-strength GO/PVA composite hydrogels using a freeze/thaw method. These researchers reported that the addition of 0.8 w% GO increases the tensile strength and compressive strength of the gels by 132% and 36%, respectively, compared with those of GO-free PVA gels [74]. Jang et al. investigated the effects of GO incorporation into PAAm hydrogels on their mechanical properties and found that an increase in the GO content up to 4 mg/mL resulted in sevenfold and fourfold increases in rigidity and toughness, respectively [81]. Similarly, Shin et al. formulated GO and gelatin methacrylate (GelMA) and photopolymerized them by UV exposure to form GO/GelMA composite hydrogels for cell encapsulation [82]. In their studies, the GO concentrations (0–2 mg/mL) and UV exposure times (10–360 s) in the presence of 5% GelMA solution were varied to tune the mechanical properties of the composite GO/GelMA gels from 4 to 24 kPa (Fig. 3a). These researchers found that the cells in GO-incorporated GelMA (2 mg/mL GO) exhibited higher metabolic activities compared with those in GO-free GelMA. Zhou et al. prepared GO-incorporated collagen hydrogels for 3D culture of bone marrow-derived hMSCs. These GO-containing collagen gels showed an elastic modulus that was approximately fourfold higher than that of GO-free collagen gels [83]. Interestingly, these researchers also found that the osteogenesis of hMSCs was promoted by GO/collagen hydrogels, and this finding implies that GO might contribute to hydrogel stiffness, which can induce osteogenesis via mechanotransduction, and osteogenic activity, which can induce ECM deposition in hydrogels. Cha et al. covalently incorporated GO into gelatin using methacrylate-grafted GO (MeGO) and GelMA to obtain improved mechanical properties and cell encapsulation [66]. Interestingly, the covalently incorporated MeGO–GelMA hydrogels displayed substantially enhanced structural integrity and fracture resistance without any effect on rigidity (Fig. 3b). Choe et al. took advantage of the improved mechanical stability of GO-incorporated hydrogels and produced alginate/GO (alg/GO) composites that could be used as bioinks for 3D printing [84], and the printed alg/GO scaffold exhibited enhanced printability and structural stability. After 10 days of incubation in culture medium, the printed alg/GO scaffolds maintained their original shapes and fine features, whereas the scaffolds composed of alginate along (GO free) showed substantial impairment of their macro- and microstructures (Fig. 3c).

Fig. 3
figure 3

GO-incorporated hydrogels exhibit enhanced mechanical properties. a Compressive moduli of GO–GelMA hydrogels produced with various GO concentrations and UV exposure times. Reproduced from [82] with permission from Wiley. b Photographs of MeGO–GelMA and GO–GelMA hydrogels after uniaxial compression. Reproduced from [66] with permission from Wiley. c Photographs of 3D-printed alginate/GO scaffolds containing various GO concentrations after 10 days of incubation in cell culture medium. Reproduced from [84] with permission from the Royal Society of Chemistry. d Photographs of original and stretched PAA/rGO hydrogels (left), strain–stress curves of PAA and PAA/rGO (top) and a cyclic stretching test with PAA/rGO (bottom). Reproduced from [89] with permission from Elsevier

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As described above, rGO contains fewer oxygenated functional groups than GO and thus exhibits a higher capability of π–π and hydrophobic interactions than GO [85]. However, due to its hydrophobicity, rGO readily aggregates in aqueous solutions and/or highly hydrophilic environments. By adjusting the degree of reduction or chemical modification, rGO can be miscible in various hydrophilic polymers and exhibit substantial molecular interactions between the polymeric chains [86, 87]. Sayyar et al. added 3 wt% rGO into a matrix composed of chitosan and lactic acid to create a strong and conductive hydrogel, in which the rGO sheets were well dispersed in the composite matrix, and this addition of a small amount (3 wt%) of rGO resulted in increases in the tensile strength (>200%) and modulus (~130%) [88]. PAA/rGO hydrogels exhibit substantially improved tensile strength compared with pure PAA hydrogels [89] (Fig. 3d). Table 1 shows examples of hydrogels prepared with graphene derivatives to achieve enhanced mechanical properties. The GO- or rGO-containing hydrogels can be further designed to exhibit long-term structural stability, and the resulting hydrogels can serve as a promising platform for sustained drug delivery and tissue engineering scaffold applications.

Table 1 Mechanical properties of various graphene-incorporated hydrogels
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Electrical conductivity

Electrically conductive biomaterials have been widely studied in the fields of tissue engineering, bioelectrodes, and biosensors because these can efficiently mediate electrical signals with biological systems [51, 90, 91]. Electrically conductive biomaterials can permit electrical stimulation of various types of cells to induce improvements in their growth, differentiation, and migration [52, 92, 93]. In particular, a conductive hydrogel has garnered great attention as a new type of biomaterial that can mimic the biological and electrical properties of soft tissues (e.g., muscles and nerve tissues) possessing electroactive properties [51, 92]. Conductive hydrogels that exhibit sufficient electrical conductivity for transmitting an electrical signal and mechanical properties similar to those of a target tissue in the body are necessary and highly desired for implanted bioelectrode and tissue engineering scaffold applications [90, 91]. In general, conductive hydrogels have been fabricated by the blending of polymeric hydrogel materials and conductive materials (e.g., gold, polypyrrole, polyaniline, and poly(3,4-ethylenedioxythiophene)) [63, 94, 95]. However, a trade-off between the electrical and mechanical properties of conductive hydrogels has been frequently observed. For example, increases in the conductive components in hydrogels lead to increases in both electrical conductivity and rigidity/brittleness. As a result, conductive hydrogels are normally weak and brittle and do not exhibit sufficient flexibility [96, 97], whereas soft hydrogels present insufficient electrical properties. Consequently, the production of soft and conductive hydrogels is a major challenge. The use of GO as an electrically conductive component can address this issue because GO (or rGO) is electrically conductive and flexible, has a high surface-to-volume ratio and exhibits relatively good electrical conductivity. In addition to electrical conductivity, GO and rGO can strongly interact with hydrogel polymer chains via hydrogen bonds, ionic bonds, and hydrophobic interactions, which provides mechanical ductility and structural stability [98, 99]. The Fan Group blended only GO into the PCL scaffold to produce conductive hydrogels for use as a nerve guidance conduit [100]. The generated hydrogel showed a relatively high conductivity (4.55 × 10−4 S/cm) but very high rigidity (48.32 MPa). The GO concentration in the hydrogel needed to this high conductivity was very high (2%), and thus, the hydrogel was very rigid with a high Young’s modulus compared with GO-free hydrogels.

Although GO typically shows good dispersion in aqueous solutions and/or hydrophilic polar environments, GO inherently exhibits insufficient electrical conductivity due to its structural defects (e.g., oxygenated functional groups) [101]. The use of rGO can improve the electrical properties of conductive composite hydrogels because rGO has restored sp2 carbon bonds and π–electron clouds on the flake sheets [87]. However, rGO is hydrophobic and readily aggregates in aqueous solutions, which causes difficulties in its preparation and mixing with hydrophilic polymers for the synthesis of electrically conductive rGO/hydrogel composites. Hence, the production of conductive hydrogels using rGO remains an unaddressed issue. In other words, the conductivity of rGO-containing hydrogels can be realized by well-dispersed percolating networks of rGO components within the hydrogel matrix without substantial aggregation of rGO flakes. Our group previously developed a simple and effective method for producing conductive hydrogels containing rGO [51]. Specifically, a GO-and-PAAm composite hydrogel was first polymerized, and then chemically reduced to covert the GO entrapped in the hydrogel to rGO (Fig. 4a). This approach could maintain the dispersion of the rGO components without severe aggregation within the hydrogel. Mild chemical reduction of GO/PAAm with ascorbic acid at 37 °C did not affect the hydrogel networks, as evidenced by the similar mechanical properties (reduced GO/PAAm) to unreduced GO/PAAm hydrogels. The resultant reduced GO/PAAm hydrogel exhibited elastic and conductive characteristics with a Young’s modulus of ~50 kPa and a conductivity of 1.4 ± 0.4 × 10−4 S/cm. In another study, Jing et al. produced a self-healable conductive hydrogel using GO and dopamine-conjugated chitosan and found that this hydrogel has a conductivity of 1.22 × 10−3 S/cm and an excellent ability to recover post cyclic compression [102]. Interestingly, these researchers reduced GO and simultaneously formed rGO-containing hydrogels through the in situ reduction of GO in a hydrogel network by dopamine; in this network, dopamine is spontaneously oxidized and accordingly causes the reduction of GO. Shin et al. directly reduced GO to rGO using ascorbic acid and further coated rGO with GelMA to increase the dispersion of rGO (Fig. 4b). These researchers polymerized rGO and GelMA to produce conductive gelatin hydrogels [103], and these conductive hydrogels showed repeated contraction and relaxation in response to external electrical field application and sufficient elasticity and conductivity suitable for the construction of engineered cardiac tissues. Table 2 shows other examples of conductive hydrogels with GO and rGO that have great potential to be widely used in electroactive tissue engineering, bioelectrodes, and biosensors because they resolve the incompatible relationship between the electrical and mechanical properties of conventional conductive hydrogels.

Fig. 4
figure 4

GO-incorporated electrically conductive hydrogels. a rGO-incorporated PAAm hydrogels (i.e., r(GO/PAAm)) produced by mild chemical reduction of GO/PAAm hydrogels. The Young’s moduli, conductivities, and impedances of GO/PAAm and reduced GO/PAAm (r(GO/PAAm)) obtained after different reduction times are compared with those of bare PAAm hydrogel. Reproduced from [52] with permission from Elsevier. b Schematic illustration of conductive rGO/GelMA hydrogels. rGO reduced with ascorbic acid was primarily coated with GelMA and polymerized with GelMA to obtained improved dispersion and conductivity of the rGO components. Reproduced from [103] with permission from Wiley

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Table 2 Various electrically conductive rGO-incorporated hydrogels
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Molecular adsorption (drug loading/delivery)

Conventional drug administration methods usually require a high dose or repeated administration, which is associated with low efficiency, poor targeting, and short residence time. To address these problems, various controlled drug delivery systems using nanoparticles, liposomes and hydrogels have been studied [104]. Hydrogels are attractive materials for drug carriers due to their excellent biocompatibility, possible biodegradability, and abilities to protect drugs from external degradation factors (chemical or enzymatic) and easily encapsulate hydrophilic drugs. In addition, it is possible to modulate the release profiles of drugs from hydrogels by controlling their porosity or degradability through modification of the polymer backbone or controlling the degree of crosslinking [104,105,106]. However, hydrogels generally exhibit a limitation in the loading and release of hydrophobic drug molecules due to discrepancies in hydrophilicity/hydrophobicity between hydrogels and hydrophobic molecules. In addition, the low mechanical strength of typical hydrogels hinders stable residence at target local sites after their administration. The release of drug molecules from a hydrogel is generally achieved through diffusion and/or gradual decomposition of the hydrogel; hence, it is generally difficult to finely control the release of the drugs from the hydrogel matrix in a predictable manner. In many cases, the weak interactions of the drug with the polymer matrix cause the burst release of the drugs from the hydrogels [107, 108]. Thus, to engineer the interactions between drug vehicles and drugs of interest, composite hydrogels have been fabricated by blending or grafting with other polymers or nanomaterials [109, 110]. This approach can provide not only improved release kinetics of various drug molecules but also smart drug delivery (e.g., stimuli-responsiveness) [111].

The incorporation of GO into a hydrogel can increase the drug-loading capacity and allow sustained drug-release because GO provides various additional functional groups capable of interacting with drug molecules within the hydrogel and much larger surface areas. Various examples of hydrogels containing graphene derivatives for drug delivery systems are shown in Table 3. For example, Wang et al. introduced GO to Konjac glucomannan/sodium alginate (KGM/SA) hydrogels to enhance the mechanical properties of the hydrogels and their ability to adsorb drugs. These researchers found that the addition of 3.0 mg/mL GO increased the loading capacity of a colorectal cancer drug, 5-fluorouracil (5-FU), from 17.49 to 22.73%, and a decreased 5-FU release rate was observed from GO-incorporated KGM/SA [112].

Table 3 Drug delivery with various GO-incorporated hydrogels
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Importantly, hydrophobic drug loading is greatly enhanced by the use of GO-containing hydrogels due to possible hydrophobic interactions and π–π interactions between the hydrophobic compounds and the hydrophobic portion of GO flakes. The hydrophobic interactions of GO in hydrogels can be further increased by using rGO. However, unlike GO, rGO displays poor dispersion in water and readily undergoes severe aggregation; hence, the incorporation of rGO into a hydrogel should be carefully designed if the hydrogel will be used as an effective drug delivery vehicle. Chen et al. chemically reduced GO to rGO with chitosan derivatives (CSDs) and obtained CSD/rGO-incorporated alginate hydrogels with relatively good dispersion of rGO in aqueous solution. The fluorescein sodium (FL) loading efficiency could be significantly increased by the use of CSD/rGO/alginate hydrogels. Specifically, the FL loading efficiencies obtained for CSD/rGO/alginate, CSD/GO/alginate, and alginate hydrogels are 82.8%, 31.9%, and 18.1%, respectively. In addition, CSD/rGO/alginate display excellent sustained release of FL for up to 150 h [113]. Our group developed an interesting strategy for the generation of rGO-incorporated alginate hydrogels that minimizes rGO aggregation. A GO/alginate hydrogel is first prepared, and then chemically reduced to lessen the aggregation of GO (or rGO) and maintain a high effective surface area; these reduced GO/alginate hydrogels are more highly absorbent to rhodamine B (RB) than unreduced GO hydrogels or GO-free hydrogels. The RB adsorption capacities of rGO/alginate, GO/alginate, and GO-free alginate hydrogels are 730, 361, and 320 mg/g, respectively (Fig. 5a). The adsorption characteristics of reduced GO/alginate and GO/alginate hydrogels were further examined using various compounds in the rhodamine family, and we found that rGO/alginate can efficiently adsorb hydrophobic compounds containing hydrophobic chains with zwitterionic characteristics [114].

Fig. 5
figure 5

GO-incorporated hydrogels for drug delivery. a Various GO (or rGO)-containing alginate hydrogels for rhodamine B (RB) adsorption. GO-containing hydrogels (GO/Alg) were chemically reduced to r(GO/Alg) for different times (3 and 12 h). In addition, rGO was directly composited with alginate to form rGO/Alg. Photographs (top) of various samples were acquired after incubation in RB solution (100 mg/L) at room temperature for 72 h. Reproduced from [114] with permission from IOPscience. b DOX release profiles from GO-hybrid supramolecular hydrogel (HSH) at different pH values. The HSH hydrogel was polymerized with GO nanosheets, N-isopropylacrylamide (NIPAM), and ureido-pyrimidinone (UPyMA). After incubation of the hydrogel in DOX solution (200 µg/mL) for 24 h at 4 °C with shaking, the amounts of DOX released from the hydrogels in PBS solutions at different pH values were measured. Reproduced from [115] with permission from Wiley. c NIR and pH responsive release of DOX from CMC-rGO/CHO-PEG hydrogels. The release of DOX from the hydrogels was investigated under different conditions, such as acidified solution (pH 6.5) and/or NIR laser radiation (left). The sample, marked as pH, was incubated in PBS (pH 6.5). The sample, marked as NIR, was incubated in PBS solution and exposed to an NIR laser (808, 1.0 W cm−2). Illustrations show the pH responsive release mechanism of DOX from the CMC-rGO/CHO-PEG hydrogel (right). Reproduced from [118] with permission from Elsevier

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pH-sensitive releases of small molecules from GO can be achieved if the primary interaction of the drug molecules with GO is ionic and originates from the carboxyl groups of the GO. This ionic interaction between GO and the drug is sensitively affected by the pH of the environment. For example, the release of doxorubicin (DOX) from GO can be triggered by a shift in the pH. When carboxyl groups of GO are deprotonated at neutral pH values (pH 7.4), strong electrostatic interactions with the amine groups of DOX are formed. Hence, at neutral pH, the release of DOX from GO is slow, whereas under acidic conditions, most of the carboxyl groups of GO are protonated and present minimal negative charges, leading to a relatively rapid release of DOX due to the absence of electrostatic interactions between GO and DOX [115] (Fig. 5b). Bai et al. reported that poly(vinyl alcohol) (PVA)/GO composite hydrogels allow pH control and selective drug release. Vitamin B12 is released from PVA/GO hydrogels more rapidly in neutral solution than in acidic solution, which suggests that these drug delivery systems can potentially be used for pH-neutral organs, such as the intestine [116].

Other external stimuli have been used to trigger drug release from GO-incorporated hydrogels. Near-infrared (NIR) irradiation can photothermally induce the release of drug molecules bound on GO flakes. Wu et al. designed peptide-GO hybrid hydrogels using peptides functionalized with a GO-binding motif (pyrene) and a photocrosslinking motif. These researchers found that the release of the encapsulated DOX could be triggered by NIR irradiation, which could be explained by the decreases in π–π interactions between GO and the drugs observed at elevated temperature [117]. Liu et al. fabricated composite hydrogels consisting of carboxymethyl chitosan (CMC)-functionalized rGO and aldehyde-functionalized PEG (CHO-PEG) for DOX delivery and found that the release of encapsulated DOX could be regulated through external NIR irradiation and pH changes [118] (Fig. 5c). Moreover, electrical stimulation can lead to controlled drug release from GO-incorporated hydrogels. Liu et al. demonstrated that an rGO/PVA hydrogel can serve as an electrically responsive drug-release system. The application of periodic electrical stimulation to the hydrogel resulted in highly controllable and repeatedly pulsatile lidocaine release [119]. Kenna et al. prepared rGO/PEG-diglycidyl ether (PEGDGE)/Jeffamine EDR148 hydrogels and found that electrical stimulation led to the induction of drug release due to the repulsion between rGO and the negatively charged drug. These researchers also reported that the drug-release rate could be further controlled by the concentration and polarity of GO and the amplitude of the applied electric potential [120].

Importantly, the drug adsorption and release characteristics of GO-incorporated hydrogels can be further engineered by GO functionalization. For example, GO functionalization with cationic polymers forms positively charged GO flakes and in turn increases the interaction with negatively charged drugs and genes [121, 122]. For example, Paul et al. modified GO with polyethyleneimine (PEI) and prepared a GO/GelMA hydrogel for vascular endothermal growth factor (VEGF) gene delivery. The GO/GelMA hydrogels exhibited strong binding affinity with VEGF DNA, which allowed sustained local gene delivery [123]. Chengnan et al. prepared carboxylic acid-enriched rGO (rGO-COOH)/metformin hydrogels and found that NIR irradiation induced dissolution of the hydrogel and active metformin release [124]. Altogether, the above-described findings demonstrate that GO and rGO are excellent building blocks for the preparation of hydrogels that enable high loading, sustained release, and smart delivery.

Conclusions

In this review, we focused on the properties and functions of hydrogels incorporating graphene derivatives. Based on the unique abilities of GO and rGO to interact with various molecules, structurally stable and mechanically strong hydrogels can be fabricated with these materials. For the preparation of GO-incorporated hydrogels, the characteristics of both the hydrogel polymers and GO (or rGO) need to be carefully considered to achieve the desired functions. In particular, GO hydrogels can serve as interesting platforms for mediating electrical signals with biological systems because the GO components can create electrically conductive networks within the hydrogels, and these hydrogels can thus serve as electrically conductive and mechanically soft interfaces for bioelectrodes and tissue scaffold applications. Because previous studies on conductive hydrogels have mainly focused on soft properties, their electrical conductivities are often insufficient. Hence, future studies should attempt to improve their electrical properties and stability by increasing the GO concentrations and modulating their assembly. With respect to drug delivery, various modes of molecular interactions are possible with GO, which makes it possible to efficiently deliver hydrophilic or hydrophobic drug molecules through the use of GO-incorporated hydrogels. The smart and controlled release of a drug from GO-containing hydrogels has recently gained great attention due to the ability to use hydrogels to precisely deliver target drugs that respond to pH changes, NIR irradiation, or electrical stimulation. As a prerequisite, GO and rGO can be used as biologically compatible components because small to moderate amounts of these materials do not cause severe toxicity, and their direct and acute responses to biological systems can be attenuated through their modification and/or their incorporation/entrapment in hydrogels. However, the biocompatibility of the GO-incorporated hydrogels should be carefully evaluated based on their applications prior to clinical use. It should be noted that GO and rGO can exhibit various properties depending on their sources and production methods, which results in variations in their functions and/or possible toxicities. In-depth studies on the molecular interactions between GO and polymeric chains will allow us to tailor the properties of GO-incorporated hydrogels for specific applications. Altogether, the above-described research demonstrates that GO-incorporated hydrogels will be beneficial for various biomedical applications, such as tissue augmentation, drug delivery, and use in bioelectrodes.