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The Role of Graphene Oxide Nanocarriers in Treating Gliomas

Gliomas are the most common primary malignant tumors of the central nervous system, and their conventional treatment involves maximal safe surgical resection combined with radiotherapy and temozolomide chemotherapy; however, this treatment does not meet the requirements of patients in terms of survival and quality of life. Graphene oxide (GO) has excellent physical and chemical properties and plays an important role in the treatment of gliomas mainly through four applications, viz. direct killing, drug delivery, immunotherapy, and phototherapy. This article reviews research on GO nanocarriers in the treatment of gliomas in recent years and also highlights new ideas for the treatment of these tumors.


INTRODUCTION
Glioma, originating from neuroectodermal cells, is the most common primary malignant tumor of the central nervous system and is aggressive and lethal. Gliomas account for 75% of intracranial primary malignant tumors and have a high morbidity and mortality (1,2). Glioblastoma multiforme (GBM) is the most common malignant type, accounting for approximately 57% of all gliomas (3). At present, the standard treatment for GBM is simultaneous maximal safety surgical resection and chemoradiotherapy with subsequent adjuvant chemotherapy for 6 months (4). Owing to the invasive and temozolomide-resistant nature of GBM, the 1-year survival rate of patients with the disease is approximately 41.4% whereas the 5-year survival rate is less than 5% (5). In recent years, much progress has been made in the diagnosis and treatment of gliomas (6,7). The proposal of molecular pathology allows for a more precise diagnosis, which will enable more effective therapeutic approaches (8). The optimization of traditional therapy includes easing the intraoperative delineation and improving spatial and time radio exposure (9,10). Tumor treating fields (TTF) is a newly approved therapeutic approach in high-grade gliomas patients, which is both effective and safe (11). In addition, new therapies such as molecular targeting and immunotherapy have also accumulated more and more experience, but few achievements can change clinical practice protocols or outcomes (12)(13)(14). Nanocarriers are pharmaceutically-relevant colloidal systems with sizes in the range of 1-1000 nm. These colloidal systems are capable of treating tumors through direct action or their ability to as drug delivery systems (15,16). The applicability of these nanocarriers in tumor targeting and treatment is accomplished through the following: 1) loading anti-tumor drugs through their ability to act as drug delivery systems, 2) functional modification of the drug-loaded nanoparticles by adding targeting ligands, and 3) using specific physics, chemistry, or biological methods to release the drug in the appropriate amount to exert the desired therapeutic effect (17,18). Graphene oxide (GO) nanocarriers have the characteristics of easy modification and good dispersibility and are thus postulated to become a new nanocarrier-based therapeutic option for the management of gliomas. Consequently, research on GO and GO nanocarriers has gradually become a hot topic in recent years (19).

Graphene Oxide
Graphene is a 2-dimensional sheet of sp 2 -hybridized carbon with a honeycomb-like structure (20). Graphene has a relatively complete structure, high stability, and weak interaction with other media, which is not conducive to its use as a drug carrier. GO and reduced GO (rGO) are derivatives of graphene. The surfaces of GO and rGO contain a large number of oxygencontaining functional groups such as carboxyl, epoxy, and hydroxyl groups, which can be chemically modified as active sites (21). The existence of a large number of oxygen-containing groups leads to good water solubility and dispersibility of GO and rGO (22). The skeleton of GO and rGO is an aromatic ring with a large specific surface area and abundant functional groups, which can increase drug loading via p-p interactions and bind biological macromolecules, such as proteins, nucleotide acid fragments, and aptamers to facilitate functional modification and target recognition (22,23) (Table 1).

Graphene Oxide Nanocarriers
The tumor cells are growing disorderly and dense, so conventional drug delivery systems work only on the surface of the tumor, making it difficult to penetrate deep into the tumor tissue (44). Molecules and nanomaterials with sizes ranging between 10 and 100 nm can be retained in tumors because of the abnormal blood vessels and the defective lymphatic circulation in tumor tissues. This is known as the enhanced permeability and retention effect (EPR) (45)(46)(47). EPR provides an option to achieve spontaneous accumulation of GO for the passive delivery of anti-tumor drugs to tumors. However, EPR provides less than a 2-fold concentration increase in tumor tissues compared with non-target tissues and organs, and sometimes the efficiency of EPR is too low to reach the drug concentrations sufficient for curing cancer (48). There is difficulty in translating the EPR effect from bench to bedside because of intra-and inter-tumoral heterogeneity, heterogeneity in nanoparticle accumulation in the different tumors, and EPR effects differences between rodent tumors and human tumors (49)(50)(51)(52). The GO nanocarrier system has more advantages than the traditional anti-tumor drug delivery system, like liposomes. These include ① good blood compatibility and optimal dispersibility in the liquid environment of the human body (53); ② large specific surface area facilitating multi-functional modification by biomolecules and small molecules, such as proteins and single-stranded DNA bases (54); ③ single atomic layer of thickness, and an ultra-high drug loading capacity compared with other nanomaterials (55); ④ sustained release and prolonged drug half-life (56); ⑤ relatively good biological safety and acceptable toxicity (57)(58)(59)(60)(61). Various modification methods have been employed to improve the solubility, stability, and cytotoxicity of GO, while there is still a need to further evaluate the biocompatibility and toxicity of GO nanocarriers into the human body (57, 62).

Functional Modification of Graphene Oxide Nanocarriers
Graphene is too stable to react with other materials, which limits its application as a drug nanocarrier; however, GO can easily be functionally modified (63,64). Functional modification of the structure and properties of GO is an effective way to improve the utilization of graphene carriers. At present, functional modification methods for GO are mainly divided into two Poly (acrylic acid) (PAA) Improving biocompatibility of GO (43) types: covalent and non-covalent modifications. Covalent modification refers to the use of chemical reactions to modify the oxygen-containing functional groups on the surface of GO. The application of non-covalent bond modification is ingenious. It uses p-p conjugation, ionic bonds, and hydrogen bonds to modify GO. Functional modification can provide new properties to GO and thus enhance the dissolution of GO in polar solvents to improve its dispersibility (65).

Covalent Modification of Graphene Oxide Nanocarriers
The surface of GO contains a large number of active oxygen groups, such as hydroxyl, carboxyl, and carbonyl groups, which can chemically react with other groups through covalent bonds to optimize its performance by forming interactions such as ester and amide bonds. Marcelo et al. used D-mannose to covalently modify GO (man-GO) using mannosylated ethylenediamine, which was found to improve the RBC toxicity and protein corona formation of GO (66). Covalent modification of GO is important in therapeutic pathways. Ouyang et al. covalently modified GO with arginine-glycine-aspartic acid (RGD) and silicon phthalocyanine (SiPc), which is a fluorescence imaging-guided photothermal and photodynamic therapy for cancer (67). Chen et al. further bound GO to anti-HER2 antibodies loaded with dual-drug DOX and 9-aminoacridine, which increased cytotoxicity against cancer cells (27). Both RGD and anti-HER2 antibodies are one of the motifs helping improve active tumor-targeting properties. FA also acts as a molecular recognition motif for folate-receptor-positive cancer cells (68 (72). The modification of the GO surface via a covalent route is strong and not susceptible to the inconsistent external environment, which makes the GO nanocarriers stable with biological systems in vitro or in vivo.

Non-Covalent Modification of Graphene Oxide Nanocarriers
The surface of GO has many charges, which can be functionally modified by DNA, some small molecule drugs, or small molecules through non-covalent bonds, such as p-p conjugation, ionic bonds, and hydrogen bonds, making GO an ideal nanocarrier for drug delivery. Pan et al. combined 5-FU with Tau-GO through a non-covalent bonding mechanism to form 5-FU-Tau-GO, which was reported to continuously release 5-FU in the acidic environment of the tumor, thus prolonging the duration of action of 5-FU more effectively than 5-FU alone (73). Wang  , and DOX showed in pH-dependent sustained-release manner (35). However, noncovalently modified GO may load fewer aromatic drugs than covalently modified GO.

Biocompatibility of Graphene Oxide Nanocarriers
The potential toxic effect of GO on living cells and organs limits its use in cancer therapy. Clinical use of GO requires extensive studies on its potential short-and long-term toxicity in vivo and in vitro, mainly through cell experiments and animal models to assess its biosafety. GO is much less toxic than graphene since the surface of GO is rich in a large number of carboxyl, hydroxyl, epoxy, and other active functional groups, which induces good water-solubility and biocompatibility of GO (76,77 (58). Inhalation, ingestion, and dermal are the major routes of entry of GO, and intravenous GO is distributed mainly in the lungs, liver, and spleen (57). GO and rGO exhibit potential toxicity in non-target organs in laboratory mammals, including liver, lung, spleen, kidney, and reproductive organs, which need further investigation (62). revealed that rGO can promote the apoptosis of gliomas through caspase-and mitochondrion-dependent apoptotic pathways and has a direct killing effect on gliomas in glioblastoma U87 cell line (96). In another study, Jaroslaw found that rGO could reduce the expression of voltage-dependent ion channel genes and extracellular receptors in U87 cells, and induce the damage of cell membrane and the changes the of cell membrane potential (97 , which significantly prolonged the survival of mice with glioma, and the treatment strategy included chemotherapy and photothermal therapy (109). Nucleolin is one of the major nuclear proteins, but it is overexpressed on the membranes of glioma cells and vascular endothelial cells, which makes it a promising target (110)(111)(112)(113). AS1411 is a DNA aptamer rich in guanine, which can specifically bind to Nucleolin, and has good tumor targeting to anti-glioma (114). Du et al. coated GO with AS1411 to load B3, a berberine derivative, and proved the nanocarrier a promising treatment for tumors (115). GO can also achieve targeted therapy of tumors through appropriate modification, including molecular targeting, external magnetic field targeting, and other targeting forms. Lactoferrin (Lf) is an iron-transporting serum glycoprotein that binds to receptors overexpressed on the surface of glioma cells and vascular endothelial cells of the BBB. Song et al. established the GO-Fe3O4-DOX delivery system and systematically evaluated its anti-glioma effect. The system was reported to prolong the action of DOX and increase uptake of the molecule by glioma cells, which displayed stronger cytotoxicity against C6 glioma cells (116). Angiopep-2 (ANG-2), a polypeptide and a specific ligand for the low-density lipoprotein receptor-related protein-1 (LRP-1), has a strong ability to pass through the BBB and has a high transcytosis capacity; it can act as a target to increase the ability to deliver drugs to the brain (117). Zhao et al. modified GO with ANG-2 and then attached DOX to the carrier system. The resulting nanocarriers more significantly enhanced tumor growth inhibition than DOX or DOX-GO both in vitro and in vivo (118). The glioma cell line U87-MG with high expression levels of EGFR on the surface is an optimized model for targeting by cetuximab (CET), an EGFR antibody. Lu et al. attached irinotecan (CPT-11) onto GO-CET and then added chitosang-poly(N-isopropylacrylamide) (CPN), a thermosensitive gel, and subsequently entrapped stomatin-like protein 2 (SLP2) short hairpin RNA (shRNA). The final GO-CET-CPT11-shRNA-CPN nanocarrier system targeted gliomas with a high expression level of EGFR, formed a hydrogel in the tumor area, prolonged the duration of action of CPT11, and induced cells apoptosis both in vitro and in vivo (119). This system was found to perform dual functions in chemotherapy and gene therapy, thus broadening the treatment options of glioma. The glioma surface is rich in folic acid (FA) receptors. Therefore, Wang et al. added FA and TMZ to GO and the system showed a pHdependent response as well as sustained release properties after reaching the glioma area, effectively reducing the drug dose and prolonging its duration of action in vitro (120). Transferrin (Tf)mediated transport has been proven to cross the blood-brain barrier, which makes Tf a potential molecular therapeutic target in the treatment in the treatment of glioma (121). In addition, Tf receptors are overexpressed on the surface of glioma cells (122). Hence, Liu et al. used Tf as a target functional group and DOX as a chemotherapeutic drug to construct a new type of gliomatargeted Tf-PEG-GO-DOX nanocarrier delivery system. In vivo experiments showed that the Tf-PEG-GO-DOX system effectively increased the intratumoral concentration of DOX and significantly prolonged the survival period of tumorbearing rats, indicating that the system could effectively improve the efficacy of glioma treatment (123). CTX, a peptide of scorpion venom, has high selectivity for gliomas by binding to matrix metalloproteinase-2 (124). Consequently, Wang et al. modified GO with CTX and loaded DOX to form CTX-GO-DOX nanocarriers, which significantly increased the concentration of DOX in the tumor cells and ultimately enhanced the killing effect of the drug against C6 glioma cells in vitro (35). CTX-GO is, therefore, a promising drug delivery system for the targeting of gliomas. GO-Fe3O4 can be used for targeted delivery through a magnetic field; it can release the loaded drug in this field to induce the desired therapeutic effect. Wang et al. used GO-Fe3O4 to load TMZ and yielded the GO-Fe3O4-TMZ system, which was controlled by an external magnetic field and had a strong drug-loading capacity for TMZ with a strong killing effect on C6 glioma cells in vitro (125). External magnetic field targeting with GO can play a role not only in targeted drug delivery but also in auxiliary imaging. Sakine et al. modified GO with superparamagnetic iron oxide nanoparticles (SPION) and poly(lactic-co-glycolic acid) (PLGA) and then attached the radiosensitizing drug IUdR on it to form IUdR-GO-SPION-PLGA. The latter system was enriched in gliomas with the use of an external magnetic field during in vivo experiments and reported to significantly strengthen the apoptosis-inducing effect of IUdR during glioma radiotherapy when monitored under MRI (126,127). This system can increase the therapeutic effect of IUdR, effectively reduce its dosage, and in turn reduce its toxicity to healthy biological tissues. There are a variety of other modified groups that are used to target gliomas, and the effectiveness of these groups is worthy of further exploration.

Immunotherapy of Graphene Oxide Nanocarriers
Tumor immunotherapy is mainly based on the relationship between the immune function of the body and the status of tumors, and it is important to find optimized intervention measures to regulate the body's immune response to tumors to achieve anti-tumor effects (128). Dendritic cells (DCs) are the most important antigen-presenting cells known in current research to date. Under normal circumstances, there are very small amounts of DCs in the human body. Only once DCs can normally perform the antigen-presenting function can the body effectively recognize the pathogen, induce an immune response, and produce a normal immune response (129). DCs in cancer patients are defective and cannot present tumor antigens properly. Reactivating DCs to initiate anti-tumor immune responses has become an important hot spot in tumor immunotherapy in recent years. For central nervous system tumors, activated DCs can simultaneously promote the infiltration of lymphocytes in the tumor microenvironment and monitor the entire central nervous system accurately and specifically (130). GO excels in loading and delivering antigen (Ag). Wang et al. used the glioma peptide Ag from T98G, a human glioma cell line, to modify GO. GO-Ag has been reported to activate DCs in vitro, effectively inducing a specific anti-glioma immune response, promoting the arrival of lymphocytes and significantly upregulating the secretion of interferon-g (IFN-g) (131). GO-Ag provides a new idea for glioma immunotherapy; it is, therefore, important to find a safe form of Ag and also a safe form of the composite GO-Ag for use in clinical settings. Lu et al. developed Fe3O4 nanoparticles (FNPs)-rGO-PEG for used for MRI-guided photothermal-immunotherapy, which could activate DCs after laser irradiation in vivo (132). Indoleamine-2,3dioxygenase (IDO) is an immunosuppressive enzyme capable of inhibiting T cells, and IDO inhibitors (IDOi) boost the efficiency of immune-based cancer therapies (133). Yang

Phototherapy of Graphene Oxide Nanocarriers
Phototherapy includes photothermal therapy (PTT) and photodynamic therapy (PDT). Photothermal therapy refers to injecting materials with strong light-to-heat conversion properties into the human body, targeting them to the tumor tissues, and irradiating them with a light source to kill tumor cells by converting light energy into heat (135,136). GO is a potential photosensitizer. It can convert light energy into heat energy under 808 nm near-infrared laser irradiation, with most biological systems not being sensitive to light in this region. Therefore, the use of GO for photothermal treatment can kill tumor cells contactless and non-invasive. Furthermore, it is non-invasive and safer than conventional treatment methods such as surgery, chemotherapy, and radiotherapy (137,138). Samira et al. found that IUdR-GO-SPION-PLGA has radiosensitizing and photothermal treatment effects on gliomas in the near-infrared (NIR)region, significantly increasing the glioma killing effect of IUdR in vitro (139). Li et al. coupled the targeting molecule Tf on the surface of GO to prepare functionalized GO-Tf-FITC microparticles, which were irradiated with infrared rays at 808 nm. Flow cytometry detected the killing effect on glioma U251 cells with GO-Tf-FITC, with the apoptosis and death indices of the GO-Tf-FITC group being significantly higher than those of the GO-FITC and blank control groups. Furthermore, functionalized GO-Tf-FITC were found to have a significant targeted photothermal killing effect on glioma U251 cells when viewed using fluorescence imaging following fluorescence labeling (140). Dong et al. further established GO-PEG-Tf as a drug nanocarrier and loaded it with DOX to perform dual functions in chemotherapy and photothermal therapy. The system showed a great killing effect on gliomas in in vivo and in vitro experiments (141). Omid et al. transformed rGO into reduced graphene oxide nanomeshs (rGONMs) using TiO 2 , and then used RGD to modify GO targeting of U87-MG to establish rGONM-PEG-Cy7-RGD (142). Owing to the strong NIR absorption ability of rGONMs, this system can significantly improve the absorption capacity of NIR, which enhances the photothermal therapy ability on gliomas. Joshua et al. also established a similar model and further proved that the system is efficient and could be used for effective photothermal therapy using a mouse glioma model with low-dose GO at a low laser power (143). The improvement of photothermal therapy capabilities with GO is also an important direction for future research.
Photodynamic therapy uses photo-biochemical processes to convert light energy into free radicals, which then play a role in inducing programmed cell death (136). U87-MG has been reported to cause an overexpression of integrin avb3 protein receptors (144). Pyropheophorbide-alpha (PPa) is a promising second-generation photosensitizer (145). Wei et al. prepared a GO carrier, PPa-GO-mAb, coupled with an integrin avb3 monoclonal antibody as the targeting ligand. The experimental results showed that this nanocarrier system could actively target U87-MG, through gathering around the mitochondria of glioma cells and producing free radicals to exert a killing effect under near-infrared laser irradiation. A CCK8 cytotoxicity test confirmed that the nanocarriers could effectively kill U87-MG cells, and the killing effect was concentration-dependent (146) ( Table 2).

CONCLUSIONS AND PERSPECTIVES
In general, GO nanocarriers are widely studied in the treatment of gliomas because of their excellent physical and chemical properties and have shown broad application prospects in medicine, such as in chemotherapy, immunotherapy, and phototherapy (151,152 (155). After further exploration, the potential of GO nanocarriers in the clinical applications of gliomas will be limitless. At present, the use of GO as a nanocarrier is still in the preliminary stages of research; there are still many problems to be solved, such as the effectiveness and safety of functionally modified GO nanocarriers in the body and their distribution in tumor tissues. GO is more water-soluble than graphene, but it tends to aggregate under physiological conditions, even leading death to mice (156). The modification GO surface can purposefully change its physical and chemical properties and biocompatibility to improve the drug carrier efficiency (53). Covalent modification of GO often requires multi-step chemical reactions, which may affect the activity of biomolecules, while non-covalent modification can avoid this deficiency, which is only limited to chemical or biological molecules with a specific structure (157). There is still room for improvement in the preparation and modification of GOs, which is worthy of further optimization research. The relationship between GO functionalization and biological system and the mechanism of biological clearance is yet to be completely understood. Key theories for controlling drug release of GO nanocarrier are still immature. The current GO nanocarriers are mainly used for drug loading via non-covalent interactions, which makes the drug loading amount unstable, and the drug loading stability of the GO nanocarriers is even more debatable (158). GO could passively accumulate to glioma tissue due to the EPR effect (24). Besides passive targeting such as EPR, practicing active targeting will improve clinical practicability. Hence, its targeting still needs to be improved and optimized. In addition, multifunctional and multi-target modifications of GO nanocarriers to improve the specificity of targeting gliomas and prolonging the retention time in tumors should also be the focus of future research, such as a combination of chemotherapy, immunotherapy, and phototherapy (152). After the release of loaded drugs, the elimination of GO in the body is also an issue that must be paid attention to in cancer therapy (159). GO is mainly used to load small molecule drugs, but not biomacromolecules such as DNA and protein, and a number of studies on GO are carried out in vitro for the potentially harmful effects, lacking in vivo experimental data (119,160). At present, many studies of GO nanocarriers focus on classical chemotherapy drugs, anti-tumor traditional Chinese medicine components, and gene drugs. Further research is needed to improve the diversity of loaded substances and explore in vivo processes (161). In conclusion, there are still many problems in the study of GO, which requires scientific research teams to strengthen disciplinary integration and jointly solve the difficulties.

AUTHOR CONTRIBUTIONS
BW carried out the primary literature search, drafted and revised the manuscript, and participated in discussions. HG, HY, YC, HX, and GZ helped modify the manuscript. All authors read and approved the final manuscript.