INTRODUCTION

Graphene-based nanomaterials (GBNs), such as graphene, graphene oxide (GO), reduced graphene oxide (rGO), and graphene quantum dots (GQDs) (Fig. 1), attract a significant attention due to their structure and physicochemical properties. Some of the promising applications of GBNs in the field of biomedicine include tissue engineering [1], bioimaging [2, 3] targeted drug delivery [4-9], development of biosensors [10-12] and antiviral [13-16], antibacterial [17-20], and antifungal agents [21, 22], and delivery of biomolecules, such as enzymes [23], proteins [24-26], genes [27-29], RNA [30, 31], and DNA [32, 33] (Fig. 2).

Fig. 1.
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Classification of GBNs.

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Fig. 2.
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Publications on GBN applications.

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GBNs can be modified by covalent [34, 35] and noncovalent [36, 37] functionalization to enhance their electrical [38, 39], optical [40, 41], thermal [42, 43], electronic [44-46], and mechanical [47, 48] properties. Monolayer graphene was first obtained in 2004 by Andre Geim and Konstantin Novoselov [49]. Depending on the method of synthesis, graphene can be produced as mono- or multilayered flakes [50, 51]. It can be synthesized by chemical vapour deposition [52-58], electrochemical exfoliation [59-62], mechanochemical exfoliation [63], and chemical and thermal reduction of GO (synthesis of rGO) [64-70]. rGO is a GO derivative in which almost all oxygen-containing groups are reduced with hydrazine hydrate or biomolecules [71] (see Fig. S1 in the Online Resource 1 for rGO synthesis from GO using l-cysteine [71]).

Graphene consists of sp2-hybridised hexagonal carbon atoms that form two-dimensional nanolayers, while GO additionally has oxygen-containing groups on the surface, e.g., carbonyl, lactol, and carboxyl groups at the edges of GO layers and epoxy and hydroxyl groups on the basal plane (Fig. S2 in the Online Resource 1) [72-74].

GBNs can be functionalized with molecules of various nature due to the presence of functional groups on the GO surface and sp2-hybridised carbon atoms. Reactions that can be carried out on the GO surface (Fig. 3) include amidation, esterification, 1,3-dipolar cycloaddition, and halogenation. Other types of interactions are hydrogen bonding, π–π stacking, and hydrophobic interactions.

Fig. 3.
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Reactions carried out on graphene surface.

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GQDs are graphene nanoparticles less than 100 nm in size. Due to their exceptional properties, such as low toxicity, stable photoluminescence, chemical stability, and pronounced quantum confinement effect, GQDs are considered as new promising materials for biology, optoelectronics, energy industry, and environment [75-78]. GQDs can be prepared using top-down or bottom-up approaches (Fig. 4) [79-81].

Fig. 4.
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Approaches for GQD synthesis: top-down degradation from various carbon sources and bottom-up synthesis from small molecules or polymers.

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GBN CONJUGATES IN BIOMEDICINE

GBNs can be effectively used in the antitumor therapy, e.g., for the development of platforms for the delivery of drugs and genetic constructs, photodynamic therapy (PDT), photothermal therapy (PTT), and theranostics (Fig. 5).

Fig. 5.
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Application of GBNs in cancer treatment.

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To efficacy of GBN-based antitumor nanodrugs can be increased by using specific vectors for their delivery that are developed to recognise tumor-specific receptors, such as HER2, CAIX, and receptors for Tat, LHRH, folate, biotin, and asialoglycoprotein (Fig. 6).

Fig. 6.
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Tumor-specific receptors and ligands used in GBN modification.

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BIOCOMPATIBILITY AND MECHANISMS OF ENDOCYTOSIS

Analysis of publications shows that functionalization of graphene surface decreases hemolysis and, therefore, increases material hemocompatibility. Thus, noncovalent functionalization of GO with chitosan produced a material with no hemolytic activity. Pinto et al. [82] showed that the noncovalent functionalization of graphene surface with polymers [polyvinyl alcohol, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), hydroxyethylcellulose, chondroitin, glucosamine, and hyaluronic acid (HA)] decreased hemolysis to 1.7% for all the resulting materials at concentrations below 500 μg·ml–1. Previously, we have studied the effect of GO enriched (about 85%) with oxygen-containing functional groups (edge-oxidized graphene oxide, EOGO) on the extent of spontaneous hemolysis and found that within the studied concentration range (CGO = 2.5-25 mg·liter–1), this nanomaterial did not affect the level of hemolysis after 1 and 3 h of incubation [83], while, as demonstrated in [84, 85], GO with a lower content of oxygen-containing functional groups (C/O ratio, 2 : 1) caused the rupture of erythrocyte membranes with subsequent release of hemoglobin. Our research group also showed that GO functionalized with L-methionine (GFM) [85], L-cysteine (GFC) [86], glycine (GO-Gly) [87], or folic acid (GO-FA) [88] caused no damage to the erythrocyte membrane at the concentrations up to 25 μg·ml–1.

In comparison with GO, GO functionalized with amino groups caused no activation of platelet aggregation up to C = 2 µg·ml–1. The authors showed that GO-induced aggregation was stronger than the thrombin-induced aggregation [89]. Podolska et al. [90] found that GO, rGO, and rGO-PEG (C = 50 μg mL–1) did not stimulate platelet aggregation in the presence of 2 μmol·ml–1 adenosine diphosphate (ADP). GFC (up to 25 µg·liter–1) caused no ADP-induced stimulation of platelet aggregation, while GFM and EOGO demonstrated the antiplatelet activity at the concentrations up to 25 and 100 µg·liter–1, respectively, in experiments on ADP- and collagen-induced aggregation.

Ding et al. [91] showed that GO (dispersion concentration, C = 100 μg·ml–1) interacted with human serum albumin (HSA) through various types of interactions (covalent and hydrogen bonding, electrostatic forces, hydrophobic interactions, and π–π stacking) that resulted in the HSA dysfunction and its inability to remove toxins due to conformational changes, which indicated a potential toxicity of GO. Functionalization of the GO surface with carboxyl groups (GO-COOH) increased its biocompatibility, as GO-COOH caused no functional changes in HSA. In contrast, Taneva et al. [92] found that interaction of GO (8 mg·ml–1) with HSA did not inactivate HSA in the blood plasma because of the low affinity of GO for HSA. We demonstrated that interaction of modified GO (GFM and GFC) with HSA occurred mainly due to the formation of hydrogen bonds: the dissociation constants for the GFM and GFC complexes with HSA were 185.2 [85] and 1600 [86] μg·ml–1, respectively.

Liu et al. [93] found that GO at the concentrations up to 100 μg ml–1 induced mutagenesis due to its effect on DNA replication and gene expression. Wang et al. [94] reported that GO (up to 100 μg·ml−1) displayed a significant genotoxicity toward human lung fibroblasts because of the DNA damage resulting from the generation of reactive oxygen species (ROS) and surface charge of GO. The authors showed that functionalization of the GO surface with PEG and lactobionic acid (LA) significantly reduced the genotoxicity.

Akhavan et al. [95] showed that the genotoxicity depends on the lateral dimensions of graphene: rGO nanoparticles with an average lateral dimension of 11 ± 4 nm were able to penetrate into the nuclei of human mesenchymal stem cells, leading to DNA fragmentation and chromosomal aberrations even at low rGO concentrations (0.1 and 1.0 mg·ml–1) after 1 h of incubation. At the same time, rGO sheets with an average lateral size of 3.8 ± 0.4 µm did not exhibit genotoxicity at a concentration of 100 mg·ml–1 after 24-h incubation. Our research group showed that GFM and GFC did not display genotoxicity at the concentrations up to 25 μg·ml–1, while EOGO did not exhibit the genotoxic effect up to C = 100 μg·ml–1. We also studied the mechanism of endocytosis of GO conjugates with 1,3,5-triazine-based cytostatic drugs and showed that the transport of these conjugates could occur via two mechanisms – pinocytosis and clathrin-dependent endocytosis [96].

The possibility of selective delivery of the cytostatic drug cytarabine (CYT) was shown in [88]. Using a conjugate of GO with CYT and folic acid (FA) as a vector molecule, our research group demonstrated that the GO-FA-CYT nanoparticles localized in the vicinity of folate receptor-expressing pancreatic carcinoma cells (PANC-1) (Fig. 7).

Fig. 7.
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Fluorescence microscopic images of folate receptor-expressing PANC-1 cells incubated with GO-FA-CYT (a) and GO (b).

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DRUG DELIVERY, PHOTOTHERMAL THERAPY (PTT), AND PHOTODYNAMIC THERAPY (PDT)

Below, we will discuss the use of GBNs in tumor chemotherapy. GBNs can be conjugated with anticancer drugs by noncovalent functionalization of the graphene surface (see Table 1).

Table 1 Cytotoxicity of GBN conjugates with cytostatic drugs in in vitro experiments
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GBNs exhibit a high photothermal conversion efficiency, i.e., they efficiently convert absorbed light into heat. In particular, they can absorb light in the near-infrared (NIR) region, which is a transparency region for biological tissues (750-1700 nm), thus allowing deep tissue heating [118]. Such localized heating can selectively damage or destroy cancer cells in PTT, representing a minimally invasive medical treatment. The size of GBNs promotes their permeability, retention, and selective clustering at the tumor loci [119]. Table 2 summarises information on the use of GBNs in chemotherapy and PTT.

Table 2 Application of GBNs in chemotherapy and PTT
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GO is a highly efficient nanomaterial for PDT, since its irradiation in the NIR region results in the formation of ROS in situ, leading to tumor ablation. The presence of functional groups (epoxy, carbonyl, carboxyl, and hydroxyl) on the GO surface allows to load it with drugs, including photosensitisers, which greatly enhances the efficacy of PDT. GQDs have a significant singlet oxygen quantum yield. Because of their properties, such as suitability for bioimaging, drug loading capacity, and high therapeutic efficacy in PDT, they can be used as a multifunctional nanoplatform in theranostics. These properties also create the possibility of using GBNs in the treatment of cancer. Table 3 summarises the data on the use of GBNs in PDT.

Table 3 The use of GBNs in PDT
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DESIGN OF GBN-BASED THERANOSTIC APPROACHES

Hatamie et al. [161] synthesized GO/cobalt nanocomposites for inducing magnetic fluid hyperthermia (MFH) and as contrast agents in magnetic resonance imaging (MRI) [162]. The composites were obtained by chemical synthesis (using GO as a source material) and assembly of 15-nm cobalt nanoparticles; the concentration of cobalt in the nanocomposites was 80%. The studies of hyperthermia induction showed a superior conversion of electromagnetic energy into heat at a frequency of 350 kHz for the nanocomposite dispersions with the concentrations of 0.01 and 0.005 g/liter. MRI showed that negatively charged GO/cobalt nanocomposites were suitable for T1-weighted imaging.

Su et al. [163] engineered a noncovalent based mitomicine C–graphene–BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)–mPEG (MGBP) nanoconjugate that ensured extensive ROS production and high photothermal conversion efficiency (48%) and demonstrated an excellent therapeutic efficacy in vitro (decreased HeLa cell viability to 17%). Apart from the synergistic photo/chemo therapy, MGBP can be used in fluorescence and photothermal dual-mode imaging, as BODIPY emits fluorescence when exposed to laser irradiation (see Fig. S3 in the Online Resource 1 for the use of MGBP in theranostics).

Taratula et al. [164] reported a novel cancer-targeting nanoplatform for imaging and treatment of unresected ovarian cancer tumors by intraoperative multimodal phototherapy. To develop this theranostic system, low-oxygen-containing graphene nanosheets were chemically modified with polypropylenimine dendrimers loaded with phthalocyanine (Pc) as a photosensitiser. Such molecular design prevented the quenching of Pc fluorescence by graphene nanosheets, providing the possibility of fluorescence imaging. Furthermore, the developed nanoplatform was conjugated with PEG to improve its biocompatibility and with luteinising hormone-releasing hormone (LHRH) peptide for the tumor-targeted delivery (Fig. S4 in the Online Resource 1). Notably, a low-power NIR irradiation at a single wavelength was used for both heat generation by the graphene nanosheets (PTT) and ROS production by Pc (PDT). Such combinatorial phototherapy resulted in an enhanced destruction of ovarian cancer cells, with a killing efficacy of 90-95% at low doses of Pc and low-oxygen-containing graphene, presumably, due to the synergistic cytotoxic effect of generated ROS and mild hyperthermia. In vivo studies confirmed that Pc loaded into this nanoplatform can be employed as a NIR fluorescence agent for the imaging-guided drug delivery. Hence, the developed Pc-graphene nanoplatform has a significant potential as an efficient NIR theranostic probe for imaging and combinatorial phototherapy.

Lamb et al. [165] multifunctionalized graphene nanoflakes (GNFs) with (i) peptide-based Glu–NH–C(O)–NH–Lys ligand capable of binding prostate-specific membrane antigen (PSMA), (ii) potent antimitotic drug (R)-Ispinesib, (iii) chelator desferrioxamine B (DFO), and (iv) albumin-binding tag used to extend the half-life of the developed agent in vivo. 68Ga-labelled conjugates were used in in vitro and in vivo experiments to evaluate the performance of GNFs as a theranostic agent (Fig. S5 in the Online Resource 1).

Using the dose-response curves and flow cytometry analysis, it was shown that GNFs loaded with (R)-Ispinesib inhibited the kinesin spindle protein (KSP) and induced cell cycle arrest at the G2/M checkpoint. Experiments on the cellular uptake and blocking demonstrated that GNFs functionalized with the Glu–NH–C(O)–NH–Lys ligand showed a specificity toward PSMA-expressing cells (LNCaP cell line). The distribution profile and the excretion rates of 68Ga-labelled GNFs in athymic nude mice were evaluated using the time–activity curves derived by dynamic positron-emission tomography (PET). Imaging experiments showed that GNFs demonstrated low accumulation and retention in background tissues and had a rapid renal clearance.

Tomasella et al. [166] used GO and reduced thiolated GO (rGOSH) as 2D substrates to fabricate nanocomposites with gold nanospheres (AuNSps) or nanorods (AuNRs) via in situ reduction of the metal salt precursor and seed-mediated growth processes. The plasmonic sensing capability of the gold-decorated nanosheets was evaluated by UV-visible spectroscopy. In vitro experiments on the toxicity of the obtained nanocomposites in human neuroblastoma SH-SY5Y cell indicated a high potential of these hybrids as a plasmonic theranostic platform.

Usman et al. [167] synthesized a bimodal GO-based theranostic nanodelivery system using CA as an anticancer agent, while Gd and AuNPs were used as contrast agents for MRI. CA and Gd were simultaneously loaded on the GO nanolayers via hydrogen bonding and π–π noncovalent interactions to form the GOGCA nanocomposite. Subsequently, AuNPs were doped on the GOGCA surface by means of electrostatic interactions (Fig. S6 in the Online Resource 1). The efficacy (cytotoxicity) of the resulting conjugate was demonstrated in HepG2 hepatocellular carcinoma cells (IC50 = 25 μg/ml). At the same time, the conjugate displayed no toxicity toward normal 3T3 fibroblasts. The T1-weighted images of the conjugate obtained by MRI demonstrated contrast enhancement in comparison with the conventional MRI contrast agent Gd(NO3)3.

Chawda et al. [168] engineered rGO nanoparticles decorated with Gd3+ ions. The resulting Gd-containing rGO nanosheets (Gd-rGONSs) were found to enhance the loading of 5-FU (loading capacity, 34%) (Fig. S7 in the Online Resource 1). The drug release was sustained and reached ~92% within 72 h. Gd–rGONSs provided a strong contrast in comparison to the optically responsive bare GO in the swept source optical coherence tomography. The longitudinal relaxivity rate (r1) for Gd–rGONSs at a magnetic field strength of 1.5 T was 16.85 mM−1·s−1, which was four times higher than that of the commercial contrast agent Magnevist (4 mM−1·s−1).

Samadian et al. [169] developed a drug delivery nanosystem based on AuNPs, decorated PEG, and FA-conjugated GO. Initially, the graphite powder was oxidised to GO and then functionalized with chloroacetic acid to produce carboxylated graphene oxide (GO–COOH). The obtained GO–COOH was functionalized with the amine end-caped PEG, FA, and 3-amino-1-propanethiol to produce GO–PEG–FA–SH. AuNPs were synthesized through a citrate-mediated reduction and then decorated onto/into GO–PEG–FA–SH through the formation of the Au–S bond to produce the GO–PEG–FA/AuNP nanosystem (Fig. S8 in the Online Resource 1).

The resulting nanosystem was loaded with DOX·HCl (76 wt. %), and its drug-loading capacity and pH-dependent drug release were investigated. The anticancer activity of the developed theranostic agent against MCF-7 cells was evaluated using the MTT assay (IC50 = 20 µg/ml after 24 h). This nanomaterial can also be used in the chemotherapy/PTT therapy of solid tumors due to the presence of AuNPs.

Yang et al. [170] developed a biocompatible HA–glutathione (GSH) conjugate (HG) with stabilised gold nanoclusters (AuNCs) combined with GO and loaded with 5-FU (25.3 wt. %) as a novel theranostic platform (HG–AuNC/GO–5-FU) [170]. This multifunctional nanomaterial possessed an excellent fluorescence, photosensitivity, and ability to specifically target cancer cell. Moreover, in the presence of lysosomal hyaluronidase (HAdase) and laser illumination, the recovery of fluorescence and 1O2 and complete release of 5-FU could be achieved, which allows the use HG–AuNC/GO–5-FU in imaging, tumor chemotherapy, hyperthermia treatment, and PDT. This multifunctional complex holds a great potential as a versatile theranostic platform for application in bioimaging-assisted cancer therapy.

Guo et al. [171] double-functionalized GO with FA and Ce6 for combined targeted PTT/PDT against MCF-7 cells and RAW 264.7 macrophages (Fig. S9 in the Online Resource 1). GO–FA/Ce6 exhibited good photothermal properties and high ROS-generating capacity.

This nanomaterial penetrated rapidly into cancer cells via folate receptor-mediated endocytosis, as well as into macrophages. A combination of PTT and PDT allowed to increase the therapeutic efficiency against MCF-7 cancer cells (cell death, up to 65%) compared to individual treatment. GO–FA/Ce6 also efficiently eliminated RAW 264.7 macrophages due to the effect of PTT/PDT (cell death, up to 94%).

Baktash et al. [172] designed and optimized a hybrid theranostic nanosystem by combining Fe3O4 magnetic nanoparticles (MNPs) for imaging and chitosan-grafted GO as a pH-sensitive smart nanocarrier (chitosans with different molecular weights and at different concentrations were used) and investigated the drug (DOX) loading and release properties, biocompatibility, and magnetic characteristics of the developed Fe3O4/GO/chitosan nanosystem. It was determined that grafting of the concentrated high-molecular-weight chitosan on MNPs/GO provided efficient drug release and improved DOX loading. Studying the effects of GO and chitosan on the magnetic behavior of the Fe3O4/GO system showed that GO decreased the contrast efficiency of the MNPs, while grafting of MNP/GO with hydrophobic chitosan enhanced the contrast, as was seen from a sharp decrease in the r1 relaxivity, which is very desirable for MRI applications (the r2/r1 value for this composite was 28.95, while the r2/r1 values for Fe3O4/GO and Fe3O4 were 6.37 and 14.66, correspondingly). The cytotoxicity assay using L929 cells (normal mouse adipose fibroblasts) revealed a high biocompatibility of the MNP/GO/chitosan nanosystem. Further assays carried out using MNP/GO/chitosan loaded with DOX demonstrated an improved performance of MNP/GO grafted with-low-molecular weight chitosan against MCF-7 cells (cell viability was 39% at 4 μg/ml DOX vs. 53% in the presence of DOX only).

Pan et al. synthesized a covalent conjugate based on GO and silicon phthalocyanine (SiPc) (Fig. S10 in the Online Resource 1) [173].

In vitro studies of the GO–SiPc conjugate in cells showed that this nanomaterial synchronously caused the photothermal effect, intracellular fluorescence, and ROS generation. Efficient photoablation of cancer cells could be triggered by either 671- or 808-nm lasers due to the synergistic PTT/PDT or NIR photothermal effect, respectively. When systemically administered to MCF-7 xenograft mice, GO–SiPc efficiently accumulated at the tumor loci and strongly inhibited tumor growth after laser irradiation.

Chen et al. [174] reported a novel approach to a one-step fabrication of magnetic graphene hybrid nanocomposites GO–PEG–γ-Fe2O3 (GPFs) using pulsed laser ablation in liquid method [174]. Due to their good magnetic and photothermal performance, GPFs were employed as nanotheranostic agents for the multimodal imaging-guided chemo/photothermal synergistic therapy. The results of multifunctional in vivo imaging confirmed the GPF uptake by the tumors after intravenous injection. Moreover, using the GPF–DOX conjugate allowed to achieve a superior synergistic antitumor effect via combined chemotherapy/PTT. Figure S11 in the Online Resource 1 presents a photograph of hepatocellular carcinoma (H22)-bearing nude mice under different treatments (Fig. S12 in the Online Resource 1 demonstrates the difference in the relative tumor volume after the treatment).

A multifunctional theranostic nanoplatform based on GO and MnWO4 was developed by in situ growth of MnWO4 nanoparticles onto GO surfaces in a PEG-containing hyperthermia polyol medium [175]. In comparison with GO and MnWO4/PEG, the NIR absorbance of the GO/MnWO4/PEG nanocomposite was significantly improved, resulting in an enhanced photothermal conversion capability and good photoacoustic (PA) imaging performance. In addition, the longitudinal relaxivity r1 of GO/MnWO4/PEG reached 11.34 mM−1·s−1 in a 0.5-T magnetic field, which was significantly higher than for ordinary Mn(II)-based T1 agents. In vivo MRI and PA imaging studies demonstrated that GO/MnWO4/PEG could be used as an efficient bimodal contrast agent to guide cancer treatment. GO/MnWO4/PEG showed a high loading capacity for DOX (550 mg/g); the resulting conjugate demonstrated a pronounced cytotoxic activity towards 4T1 (human breast carcinoma) and HUVEC (human umbilical vein endothelial cells) cell lines. For example, cells incubated with 100 µg/ml GO/MnWO4/PEG/DOX (containing 5 µg/ml DOX) and then exposed to laser irradiation showed the highest mortality rate (about 90%) vs. 50% in the case of DOX (C = 5 µg/ml) or GO/MnWO4/PEG.

Prasad et al. [176] reported the results of in vivo photo-triggered tumor regression induced by application of a biodegradable red emissive nanotheranostic composite based on liposomes fortified with GO flakes and functionalized with FA (GO–Lipo–FA) and loaded with DOX (Fig. S13 in the Online Resource 1) [176].

The synthesized nanocomposite has a good aqueous dispersibility, quick photothermal response (54°C in 5 min), high biocompatibility, deep intracellular localization, feasibility for 4T1 visualisation, and long-term tumor-binding ability of the injected emissive nanohybrid. GO enhanced the stability of the drug-loaded liposomes in the extracellular environment, which prevented premature release of the loaded anticancer drug from the liposomal cavity. In addition, the authors demonstrated the developed nanocomposite caused tumor regression (~300 to 25 mm3) in 4T1 Balb/c mice.

Foroushani et al. [177] developed a theranostic system based on GO integrated with PDA, BSA, diethylenetriaminepentaacetic acid (DTPA)–Mn(II) contrast agent, FA, and 5-FU for targeting CT-26 colon cancer cells via folate receptors overexpressed on cancer cells. According to the results of biodistribution assessment, the conjugate was observed mainly in the tumors and, therefore, provided highly efficient drug delivery to CT-26 cells. In vitro and in vivo MRI and therapy examination confirmed the ability of the conjugate to enhance the contrast in tumor imaging (diagnostics) and to inhibit the growth of cancer cells (therapy).

Luo et al. [178] proposed an easy method for the synthesis of a theranostic agent based on superparamagnetic iron oxide nanoparticles loaded onto GO nanosheets (SPIONs@GO) and cis-aconitic anhydride-DOX prodrug (CAD) attached to the carboxylic groups of GO through the 2-poly(amidoamine) dendrimer (G2.NH2) linker (Fig. S14 in the Online Resource 1).

The release of DOX from the conjugate was pH-sensitive: 66.91 ± 3.16% at pH 5.5 and 47.51 ± 1.87% at pH 6.5 within 12 h. The viability of 4T1 cells after treatment with CAD–SPIONs@GO for 24 h decreased cell viability from 93.8% to 38.3% at the DOX concentration of 1.3-20 µM (similar to the treatment with free DOX). According to the results of biodistribution experiments, 4 h after injection, CAD–SPIONs@GO mainly localized to the spleen and liver. The total Fe amount in all major organs decreased greatly 12 h after injection, suggesting that CAD–SPIONs@GO was cleared out of the body. The authors proposed that the interface effect between GO and in situ growth of SPIONs contributed to the significant increase in the r1 value and decrease in the r2 value. In vivo studies results confirmed a possibility of conjugate application in high-resolution T1-weighted MRI.

Shi et al. [179] synthesized a theranostic agent based on rGO conjugated to the anti-CD105 antibody (TRC105) and a complex of 64Cu (PET label; half-life, 12.7 h) with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA, chelator). In vivo experiments on the blockade of the agent uptake by 4T1 cells with an excess of TRC105, as well as flow cytometry and histology data, confirmed the stability of 64Cu–NOTA–rGO–TRC105 and its specificity for CD105 of the tumor vasculature. Noteworthy, 64Cu–NOTA–RGO–TRC105 exhibited little extravasation in 4T1 cells, indicating that targeting tumor vasculature (instead of tumor cell) can be a valid and preferred approach for the application of nanomaterials. Since rGO can be used for PTT, the tumor-specific rGO conjugate may serve as a promising theranostic agent that integrates imaging and therapeutic components.

Cheng et al. [180] developed a mild thermal annealing procedure to induce blue fluorescence in GO suspensions (Fig. S15 in the Online Resource 1) [180]. The procedure preserved the oxygen functional groups, which enabled conjugation of a cancer drug and resulted in nontoxic and harmless nanomaterial. The authors demonstrated the capability of GO to simultaneously act as a cellular imaging agent and a drug delivery agent in CT26 cancer cells without the need for additional fluorescent protein labelling. The authors also covalently annealed GO with CP (elemental content of Pt in the conjugate, ~3 wt. %) and determined that the annealed GO boosted the therapeutic performance of CP in killing CT26 cancer cells.

Hu et al. [181] synthesized a new conjugate based on rGO, PDA, and ICG for amplifying the PA imaging and PTT effects for cancer phototheranostic (Fig. S16 in the Online Resource 1). The procedure for the ICG–PDA–rGO preparation included the following steps: (i) dopamine monomers were loaded on the GO surface and spontaneously self-polymerised via the Michael addition/Schiff reaction to form a PDA coating on the rGO surface, (ii) free ICG dye was absorbed on the PDA–rGO surface via hydrogen bonds and π–π stacking interactions.

ICG–PDA–rGO exhibited stronger PTT effect and higher PA contrast than pure GO and PDA–rGO. After PA imaging-guided PTT treatment, the tumors in 4T1 breast subcutaneous and orthotopic mice models were suppressed completely; no treatment-induced toxicity was observed.

Turcheniuk et al. [182] produced a theranostic agent based on AuNRs coated with pegylated rGO (AuNRs@rGO–PEG) and modified with sulfo-cyanine7 fluorescent dye (Cy7) and Tat protein (see Fig. S17 in the Online Resource 1).

Selective targeting of tumors was ensured by specific interaction between the Tat protein and human glioblastoma astrocytoma cells (U87MG). Due to the presence of NIR fluorescent dye integrated onto the rGO shell, the conjugate acted as fluorescent cellular marker. In vivo experiments in mice implanted with U87MG cells showed that irradiation at 800 nm (0.7 W/cm2, 10 min) suppressed tumor growth after 5 days. Histological analysis of tumor tissues revealed an active uptake of the nanoparticles by the tumor stromal cells and selective damage of tumor vessels.

Wang et al. [183] synthesized a novel nanomaterial for the PTT/immunotherapy of cancer by the self-assembly of oleate-capped Fe3O4 nanoparticles (FNPs) and rGO through electrostatic interaction, followed by modification with PEG–NH2 [182]. FNP/rGO–PEG nanocomposites can be used for the MRI-guided cancer PTT/immunotherapy due to their excellent magnetic properties. Under laser irradiation (805 nm), FNP/rGO–PEG improved the PTT efficacy by increasing the temperature up to 60°C and killing 80% of 4T1 orthotopic mouse breast tumor cells. In addition, FNP/rGO–PEG nanocomposites could be used to stimulate immune response by triggering the maturation of dendritic cells (CD11c+ CD86+) and secretion of cytokines (IL-12p70, IL-6). Intratumoral injection of FNP/rGO–PEG nanocomposites in combination with NIR laser irradiation significantly increased the median survival time of tumor-bearing animals.

Bansal et al. [184] developed a theranostic agent based on GQDs conjugated with a biosurfactant isolated from Candida parapsilosis through the amine-carboxyl coupling reaction and noncovalent modification with FA. The obtained conjugate had a homogenous dispersion and showed the photoluminescence properties and demonstrated enhanced uptake by cancerous cells in comparison with non-modified GQDs. In the MTT assay, the conjugate decreased the viability of MCF-7 cells by more than 60% after 24 h of incubation and by 75% after 48 h [184].

Ko et al. [185] synthesized GQDs for the diagnostics and therapy of breast cancer via conjugation with two precursors. DOX-disulfide-GQDs provided chemotherapy and PEG-disulfide-herceptin enhanced the half-life and ensured the targeting of HER2 (Fig. S18 in the Online Resource 1) [184]. The cleavage of disulfide links at a physiologically relevant glutathione concentration in cancer cells provided controlled drug release. The authors demonstrated an enhanced cellular uptake of the conjugate by SK-BR-3 cells (HER2-positive) in comparison with MDA-MB-231 cells (HER2-negative). As a result, the viability of SK-BR-3 cells was significantly decreased (to <50%) at the conjugate concentration of 50 mg/ml, whereas the viability of MDA-MB-231 cells was reduced to >85%.

Iannazzo et al. [9] developed a novel conjugate based on GQDs covalently modified with the tumor targeting module biotin (BTN) and noncovalently modified with DOX (GQD-BTN-DOX, Fig. S19 in the Online Resource 1), as well as the GQD-DOX conjugate [9]. The DOX content in GQD-BTN-DOX and GQD-DOX was 16.6 and 17.8 wt. %, respectively. GQD-DOX nanoparticles were preferentially accumulated in the cytoplasm, while DOX localized to the nuclei. At the same time, GQD-BTN-DOX nanoparticles concentrated in the endosomal compartment after endocytosis-mediated internalisation. The cytotoxicity of GQD-BTN-DOX towards A549 cells strongly depended on the uptake by the cells, which was more pronounced and delayed for GQD-BTN-DOX in comparison with GQD-DOX and DOX only.

Li et al. [186] synthesized a covalent GQD-FA conjugate and loaded it with IR780 iodide (33.19 wt. %) via π–π stacking interactions (see Fig. S20 in the Online Resource 1). In vivo NIR fluorescence imaging and biodistribution analysis demonstrated that in BALB/c nude mice xenografted with HeLa cells, the conjugate preferentially accumulated in the tumors. When irradiated with an 808-nm laser, IR780/GQDs-FA caused hyperthermia (photothermal conversion efficiency, 87.9%) and induced apoptosis of cancer cells and tumor necrosis, resulting in complete tumor disappearance without relapse.

Ding et al. [187] developed a novel type of GQD-based theranostic agent with a superior therapeutic performance against 4T1 cancer cells both in in vitro [IC50 (theranostic agent) = 1.5 g/ml, IC50 (DOX) = 4 g/ml] and in vivo (the conjugate reduced the tumor volume 2.7 times more than DOX alone) due to the improved tissue penetration and cellular uptake [187]. GQDs were synthesized via facile chemical oxidation and exfoliation technique using polyacrylonitrile carbon fibres as a raw material. The NIR fluorescent molecule Cy5.5 was covalently attached to GQDs via the cathepsin D-responsive peptide (Phe-Ala-Ala-Phe-Phe-Val-Leu-Cys, P); functionalized GQDs were then loaded with DOX via π–π interactions. The synthesized construct allowed to track the delivery and release of the anticancer drug, as well as to monitor drug-induced apoptosis of cancer cells through GQD, DOX, and Cy5.5 characteristic fluorescence.

Badrigilan et al. [188] produced a theranostic agent based on superparamagnetic iron oxide and bismuth (III) oxide (Bi2O3) with GQDs for in vitro computed tomography (CT)/MR dual-mode bioimaging and PTT (Fig. S21 in the Online Resource 1).

The GQD-Fe/Bi nanocomposite had the following advantages: (i) the photothermal conversion efficacy was 31.8% with a high photostability upon irradiation with a NIR 808-nm laser; (ii) photothermal ablation of HeLa and MCF-7 cells in vitro resulted in a significant decrease in cell viability (~50% at 100 µg/ml) in comparison with laser treatment only (3.0%); (iii) obtained nanoparticles exhibited a superior X-ray attenuation capability (175%) in comparison with Dotarem (macrocyclic gadolinium-based contrast agent), as well as showed a strong T2-relaxation shortening capability (r2 = 62.34 mM−1·s−1) as a contrast agent for CT/MRI.

The same authors synthesized GQD-coated bismuth nanoparticles and assessed the possibility of their application for CT imaging and PTT [189].

Lee et al. [190] developed rGQDs derived by rGO top-down oxidation and HA-GQDs (HGQDs) that were hydrothermally synthesized by the bottom-up method [190]. The obtained nanomaterials possessed substantial NIR absorption and fluorescence throughout the visible and NIR regions, which is beneficial for in vivo imaging. Aqueous dispersions of rGQDs and HGQDs added to HeLa cells and irradiated with NIR laser (λ = 808 nm, 0.9 W/cm2, 10 min) facilitated an increase in temperature up to 54.5°C, leading to the decrease in the HeLa cell viability from 80% for RGQDs (C = 1.5 mg/ml) and 60% for HGQDs (C = 1.7 mg/ml) without irradiation down to ~40% (RGQDs) and ~20% (HGQDs) after irradiation.

Sung et al. [191] synthesized a unique conjugate composed of porous carbon/silica nanosponge encapsulated with GQDs loaded with docetaxel (DTX) via π–π interactions; then, the particles were capped with the red blood cell (RBC) membrane and cetuximab via fusion (see Fig. S22 in the Online Resource 1).

The obtained conjugate has the following advantages: (i) the stability of the RBC lipids and proteins on porous particles was higher than that of lipids of liposomal particles due to a high adhesion energy; (ii) the porous surface of the particles exhibited an excellent lateral bilayer fluidity, thus improving the targeting efficacy; (iii) RBC-coated nanoparticles had a considerably longer circulation time than PEGylated nanoparticles due to the presence of transmembrane protein CD47 that induces signalling through the phagocyte receptor CD172a, inhibits immune response, and suppresses particle recognition by the immune system (see Fig. S23 in the Online Resource 1 for the mechanism of conjugate action).

Due to the synergistic effect of biomimetic targeting and penetration of DTX/GQD nanoparticles followed by irradiation (1.5 W/cm2, 10 min), it was able to achieve a significant reduction in the size of A549 tumor during the first 10 days of treatment.

Xuan et al. [192] synthesized nanoparticles for bioimaging and combined chemotherapy/PTT based on AuNSp clusters (diameter of 50 nm) coated with GQDs covalently modified by FA using carbodiimide method and noncovalently modified with DOX (94.39 ± 0.39%) (see Fig. S24 in the Online Resource 1 for the scheme of conjugate synthesis).

The obtained nanoparticles formed stable aqueous dispersions and demonstrated an excellent PA and CT imaging performance, low cytotoxicity, and PTT conversion efficiency up to 51.31%. In addition, the authors showed a significant decrease in the relative tumor volume in BALB/c nude mice (SPF males, 4-week-old) inoculated with HeLa cells (Fig. S25 in the Online Resource 1).

Wu et al. [193] developed a new type of theranostic agent named PC@GCpD(Gd) [192]. First, the authors synthesized GQDs covalently modified with the Ce6 photosensitiser (GCpD) and coated with PDA layers, yielding water-compatible and biocompatible nanoparticles with a substantial photothermal/photochemical effect. Then, the Cy3-labelled nonmethylated CpG oligodeoxynucleotide (5′-TCC ATG ACG TTC CTG ACG TT-3′-Cy3) was condensed with the biodegradable cationic poly(l-lysine) (PLL) polypeptide to obtain immunoactive nanoparticles (PCs). GCpD nanocomposites easily self-assembled on the surface of PC nanoimmunocores and then were chelated with Gd3+ (see Fig. S26 in the Online Resource 1).

The obtained photo/immunoactive hybrid PC@GCpD(Gd) nanostructures decreased the viability of cancer cells, released endogenous cancer cell antigens, and contemporaneously regulated tumor microenvironment to facilitate the immunostimulatory effect. The authors characterised the cellular uptake, MRI/fluorescence imaging, and phototherapeutic and immunostimulatory activity towards the murine mammary cancer EMT6 model, as well as the biosafety of PC@GCpD(Gd) nanoparticles. It was shown that laser irradiation (660 nm, 1 W/cm2, 10 min) simulated the PTT and PDT effects, leading to a significant decrease in the EMT6 cell viability in mice, secretion of proinflammatory cytokines, maturation of dendritic cells, and recruitment of CD4+ and CD8+ T cells into the tumor, resulting in a higher therapeutic efficacy. MRI/fluorescence imaging traced specific accumulation and retention of PC@GCpD(Gd) in the tumor-draining lymph nodes.

Ruiyi et al. [194] synthesized histidine (His)- and octadecylamine (OA)-functionalized GQDs (His/OA-GQDs). The obtained nanoparticles were used for the fabrication of His/OA-GQD-NaYF4:Yb,Tm nanocages that exhibited a 140.2-fold enhancement of upconversion fluorescence, stability in aqueous solutions, and high DOX-loading capacity (461.2% within 30 min) (see Fig. S27 in the Online Resource 1) [194]. The authors also developed a drug delivery system (GYAuDOX) which included His/OA-GQD-NaYF4:Yb,Tm gold nanoparticles as a core, and MGC-803 cell membrane as a shell. The obtained material exhibited a high biocompatibility, selective targeting of homotypic tumor cells, pH- and light-stimulated DOX release, and capacity for chemotherapy/PTT. The data on the efficacy of the obtained theranostic agent are presented in Fig. S28 in the Online Resource 1.

Liu et al. [195] synthesized GQDs with a strong absorption (1070 nm) in the NIR-II region (1000-1700 nm) by a one-step solvothermal treatment using phenol (carbon precursor) and hydrogen peroxide (oxidising agent) in the magnetic field with an intensity of 9 T (see Fig. S29 in the Online Resource 1) [195].

The synthesized nanoparticles possessed a uniform size (3.6 nm), tunable fluorescence (quantum yield, 16.67%), and high photothermal conversion efficacy (33.45%). The obtained nanomaterial ablated tumor cells, inhibited tumor growth upon NIR-II irradiation, and, at the same time, provided an enhanced NIR imaging of tumors in mice.

Zhang et al. [196] developed a nanomaterial (named R-NCNP) by coating a mesoporous carbon nitride (C3N4) layer on a core–shell nitrogen-doped GQD (N-GQD)@ HMSNs and decorated it with a P-PEG-RGD polymer consisting of a purified hematoporphyrin derivative photofrin (P) and the tumor-homing peptide RGD (Arg-Gly-Asp) connected by PEG as a linker, to achieve the targeted delivery (see Fig. S30 in the Online Resource 1).

The obtained material has the following advantages for biomedicine applications: (i) R-NCNPs catalyzed water decomposition in the tumor microenvironment with the generation of oxygen, thus decreasing local hypoxia; (ii) the generated oxygen bubbles enhanced generation of an echogenic signal, making them laser-activatable ultrasound imaging agents; (iii) activation of the encapsulated photosensitisers and C3N4-layered photosensitiser at λ = 630 nm stimulated ROS formation; (iv) combination of PTT with PDT for tumor eradication; (v) P-PEG-RGD promoted efficient accumulation of particles in the tumor; (vi) R-NCNPs acted as multimodal real-time monitoring agent.

Prasad et al. [197] synthesized a theranostic agent based on GQD-embedded mesoporous silica which displayed a high penetration and retention ability in solid tumors (see Fig. S31 in the Online Resource 1). The obtained material had a uniform particle size distribution, improved stability, high surface area (850 m2/g), DOX loading capacity of 31%, and high photothermal response. It was shown that administration of carbanosilica in 4T1 female Balb/c mice led to a temperature rise (to ~55°C after 5 min of exposure to NIR light), fluorescence intensity of 108 p/s/cm2/sr, and as a result, provided 68.75% tumor shrinking compared to 34.48% without NIR irradiation.

Yang et al. [198] developed a self-assembly approach to the theranostic agent synthesis based on the acidity-activated GQD nanotransformers (GQD NTs) by mixing (i) GQDs (loading module) that provided large surface area for the loading of photosensitiser [tetrakis(4-carboxylphenyl) porphyrin, TCPP] and MRI contrast agent (Mn-TCPP), (ii) RGD peptide as a targeting module due to its affinity to αVβ3 integrin, and (iii) linking module that connected the first two modules through the host–guest interactions between β-CD and adamantine [198]. As seen from Fig. S32 in the Online Resource 1, the acidity of tumor microenvironment triggered GQD NT transformation and drugs release.

The synthesized theranostic agent provided an efficient targeting and long-term retention in the tumor (over 96 h), possibility of MRI/fluorescence imaging, and photothermal effect, which enhanced cell membrane permeability, as well as an efficient photosensitiser uptake and repeated PDT at a photosensitiser content 10-30 times lower than in previously published papers. As seen from Fig. S33 in the Online Resource 1 (survival and tumor growth curves of A549 tumor-bearing mice after different treatments), the developed nanomaterial significantly inhibited tumor growth and increased mouse survival.

CONCLUSION

Since their discovery in 2004, graphene and its derivatives have become some of the most promising materials due to a broad range of potential applications in various fields of science and technology, such as biotechnology, biomedicine, tissue engineering, bioanalysis, etc. (Fig. 8).

Fig. 8.
figure 8

Directions of GBN scientific applications.

Full size image

Graphene has a unique two-dimensional flat structure, unique physical and chemical properties, and high biocompatibility, which promotes its application in the creation of high-tech materials for biomedical purposes. The use of graphene and its derivatives for the treatment of solid tumors is one of the promising areas of modern oncology. Along with the advantages of GBNs, there are also some limitations that need to be considered. One of the main problems is the lack of information about metabolic pathways and toxicokinetics of graphene materials used in biomedical applications. This limits the ability to fully evaluate the safety and efficacy of these materials in living organisms. Another important problem is poor reproducibility of the synthesis of graphene-based materials and common lack of comprehensive studies on their structure and composition. Both these factors lead to a poor reproducibility of biological effects of graphene-based materials in living systems. Also, water dispersions of GBNs are prone to aggregation, which affects their biological activity and mechanism of biological action. In this regard, it is necessary to conduct a comprehensive physico-chemical investigation of their stability, including the studies of optimal stabilizers. Let us hope that these problems will be solved in the XXI century – the century of nanotechnology.