Functionalization and optimization-strategy of graphene oxide-based nanomaterials for gene and drug delivery

Abstract

Graphene-family nanomaterials (GFNs) have been widely used in cancer therapy, tissue engineering, antibacterial and biological imaging due to their optical, thermal, and drug absorption properties. When used as drug and gene nanocarrier, the major limitations are aggregation, biocompatibility, and inappropriate release of drugs or genes. To overcome these problems, researchers have developed a variety of functionalization processes. In this review, we grouped the functionalization according to the decoration molecules, putting particular emphasis on the gene delivery. Organic and inorganic materials resulted as the major sets to introduce functional sections onto graphene oxide (GO). We also classified the target molecules used in the GO delivery system, as well as introduced other strategies to increase the delivery efficacy such as controlled release and magnetic targeting.

Introduction

Nanotechnology is one of the most rapidly developing fields. Many different nanomaterials, which possess unique and extraordinary physiological and chemical properties, are currently undergoing preclinical/clinical testing, including dendrimers, liposomes, polymers, metallic nanoparticles (NPs), carbon nanomaterials, and viral NPs. All of them have distinct advantages and disadvantages in terms of functionality, physiochemical properties, biodistribution, pharmacokinetic behavior, immunogenicity, and toxicity [1,2]. Among those, graphene is a type of carbon nanomaterials widely used for in nanomedicine. The theoretical existence of graphene was discussed 60 years ago by Slonczewski and Weiss [3]. Later on (in 2004) single sheets of graphene were isolated through mechanical exfoliation by Novoselov (repeated peeling, scotch-tape technique) [4].

Due to its optical, thermal, mechanical, and electrical properties, graphene is applied for conducting polymers, battery electrodes, printable inks, antibacterial papers [5-7]. To further exploit the pristine graphene, Graphene-family nanomaterials (GFNs) including few-layer-graphene (FLG), ultrathin graphite, graphene quantum dots (GQDs), graphene oxide (GO), reduced graphene oxide (rGO), and graphene nanosheets (GNS) have been developed [6,8,9]. In this way, GFNs are analogous to carbon nanotubes (CNTs), which can vary in wall number, diameter, length, and surface chemistry [6]. Compared to pristine graphene, other GFNs exhibit distinct dispersion/aggregation behaviors, biocompatibility, and other advantages due to their different surface properties [10-12]. In 2008, Sun et al. developed the pegylated GO (PEG-GO) that is soluble in buffers and serum without agglomeration [13]. In 2012, Sasidharan and colleagues revealed that carboxyl functioned graphene has a better hemocompatibility [11]. Moreover, Mendonca et al. found that the toxic effects of rGO are peripheral and transitory in the short-term analysis after systemic administration [14]. A consensus on the toxicity of GFNs impacting the body at different levels such as organs, blood, cells and subcellular structures, has not yet been reached [15]; nonetheless, researchers have reached a standard view on the toxicity of graphene being dependent on their shape, dose, size, time and functionalization [16].

The interaction between GFNs and biological molecules has been addressed by previous studies [6]. In 2008, Liu et al. used PEG and nano-graphene oxide to obtain a delivery material that can absorb the hydrophobic aromatic molecules camptothecin (CPT) analog SN38 [17]. Since then, GFNs have been intensively explored as nanocarriers to be applied in gene delivery drugs, bioimaging, and tissue engineering [18]. Gene therapy mainly depends on ensuring the successful transfer of the therapeutic gene to the targeted cell [19]. The major limitations of gene therapy are poor cellular uptake, degradation by nucleases and rapid renal clearance following systemic administration. The decoration of GFNs prevents target drug or gene aggregation, minimizes its side-effects, controls release at proper time and location in chemotherapy. In this paper, we reviewed the studies on GFNs used in drug and gene delivery published over the recent two years. These functional moieties were summed up into several categories. Furthermore, we presented strategies to ameliorate the delivery efficacy.

Functionalization of graphene used in the delivery

GO that has excellent process ability has become a promising functional nanoreinforcing material for various biomedical applications. Employing the covalent or noncovalent method named “graft” or “load”, GO can be modified with other nanoparticles (NPs) or biomolecules to expand its biomedical applications [20]. Nanohybrids offers several advantages due to the unique properties of each counterpart. In 2017 and 2018, there were nearly 200 papers about GO used as nanocarrier that classified the decoration of GO into certain types (Figure 1). Organic and inorganic are the two major sets. We sorted the organic function into linear polymers, nonlinear polymers, polysaccharides, amino acids-protein-aptamer (APA), and nonpolymers. These categories are listed and censused in Figure 1, except for the nonpolymers that were less frequently used. The representative literature of the first three categories are listed in Table 1. The subgroup was censused in separate categories. Besides, the subgroups, which had passed the in vivo antitumor assay, were red labeled; blue stars indicated that the gene delivery was successful.

Figure 1.

Functionalization of GO. Organic and inorganic were the two major sets. Organic set was further divided into linear polymers, nonlinear polymers, polysaccharides, amino acids-protein-aptamer (APA), and nonpolymers (not show). The subgroups which had passed the in vivo antitumor assay were red labeled. Blue stars indicated the gene delivery have been realized. Inorganic and APA have not undergone these screen cause of its complexity.

Table 1.

The representative literature of first three categories of organic subset were listed

Classification	Modified materials	Nanocomposites	Loaded drugs	In vitro/in vivo model	Results	references
Linear polymers	PEG, polyethylene glycol	FA/PEGylated GO nanosheet	HDAC1/K-Ras siRNAs	MIA PaCa-2 tumor bearing mice	Irradiation prompt the siRNA release; FA/GO/(HDAC1+K-Ras) siRNA showed 52% reduction of tumor volume	[24]
	PLGA, poly (D, L-lactic-co-glycolic acid)	GO/PP-SS-DOX, GO/PEG-PLGA-doxorubicin	PEG-PLGA-SS-DOX	B16 tumor bearing mice	Redox-sensitive PP-SS-DOX specifically release DOX in cancer cells, result in higher inhibition rate than free DOX	[29]
	PDA, polydopamine	rGO-PDA	Ara, cytarabine hydrochloride	HeLa cells tumor bearing mice	Greater inhibition of cancer cell and tumor growth than free Ara when combine the chemotherapy and phototherapy	[34]
	PVP, polyvinylpyrrolidone	PVP-rGO/Bi2S3	DOX	BEL-7402 tumor bearing mice	Greater inhibition of cancer cell and tumor growth than free DOX when combine the chemotherapy and phototherapy	[37]
Nonlinear polymers	PEI, polyethylenimine	GO-PEG-PEI	Cas9/sgRNA (EGFP and CXCR-4)	AGS.EGFP cells	Transfection efficacy is about 39%	[179]
	PAMAM, polyamidoamine	GPD, GO-PEG-PAMAM	EPAC1 siRNA tagged with Cy5	HUVEC cells MDA-MB-231	Higher transfection capability than Lipofectamine 2000; A maximum transfection efficiency of 56.5%; lower cell invasion	[57]
	hPG, hyperbranched polyglycerol	nanographene sheets-hPG	DOX	HeLa cells tumor bearing mice	Efficiently inhibit tumor growth	[59]
Polysaccharide	CHI, chitosan	GC-GO, galactosylated chitosan-GO	DOX	HepG2 tumor bearing mice	GC-GO-DOX shown higher inhibition rate than free DOX	[64]
	CMC, carboxymethyl cellulose	CMC-GO nanocomposite hydrogel beads	DOX	SW480 cells	GO-CMC/DOX complexes shown lower cytotoxicity under the same conditions than free DOX	[65]
	CD, cyclodextrins	GO-HP-β-CD, GO-hydrophilic hydroxypropyl-β-CD	DEX, dexamethasone		No anticancer assay	[71]
	ALG, alginate	GO-ALG	5-FU, 5-fluorouracil	HT-29 tumor bearing mice	Mice treated with GO-ALG/5-FU showed prolonged survival time and inhibited tumor growth	[73]

Organic decoration

Linear polymers

Polyethylene glycol (PEG) is the most frequently used linear polymer. Biocompatible neutrally charged PEG leads to high aqueous solubility and stability in physiological solutions including serum [21,22]. In 2008, Liu et al. used PEG and GO and obtain a delivery material that can absorb the hydrophobic aromatic molecules camptothecin (CPT) analog SN38 [17]. Ribonuclease A (RNase A) and protein kinase A (PKA) were also successfully loaded on PEGylated GO [23]. Moreover, Yin et al. used the PEGylated GO as a vehicle to co-deliver HDAC1 and K-Ras siRNAs into MIA PaCa-2 cells in BALB/c mice [24].

PLGA (poly (D, L-lactic-co-glycolic acid)) was initially used as clinical suture material due to its excellent biocompatibility and tunable rate of in vivo biodegradation. PLGA-based micro/nanoparticles can be used for the delivery of macromolecules, such as protein or various types of nucleic acid [25]. GO/PLGA nanofibers are formed by electrospinning technique, where human embryonic kidney 293 cells or mesenchymal stem cells (MSCs) can be successfully transfected by pGFP-GO/PLGA [26]. Besides, 5-iodo-2-deoxyuridine (IUdR) or 5-fluorouracil can be loaded on PLGA functionalized GO, which can further improve the properties of the particles (fits function, magnetic targeting property and MRI ability) [27,28]. DOX is released from GO/PP-SS-DOX (conjugat mPEG-PLGA (PP) with DOX via disulfide bond) nanohybrids in cancerous cells due to the reductive environment [29], while bone morphogenetic protein-2 (BMP-2) is delivered using GO-PLGA as microcarrier in bone tissue engineering [30].

Polydopamine (PDA) was used for surface modification or to steady the nanocarrier due to its excellent attachment property [31]. PDA doped graphene nanohybrids are used in bioimaging when absorbing DNA [32], and in drug delivery, while enwrapping the mesoporous silica nanoparticles [33]. For example, antitumor assay in vivo was conducted in HeLa bearing mice treated with combined chemotherapy and photothermal therapy [34]. Yet, so far, no gene delivery has been reported in the PDA-GO delivery system.

Most of the other linear polymers were the derivatives of polyethylene (DPEs), among which polyvinylpyrrolidone (PVP) was most frequently used. PVP is a nonionic, nontoxic and biocompatible polymer surfactant that could also serve as a biocompatible stabilizer of GO in the physiological environment [35]. PVP functionalized GO has been identified as nanocarriers for SN-38 [36]. PVP-rGO/Bi₂S₃ nanocomposite has a high storage capacity for DOX and simultaneously displays perfect photothermal conversion efficiency in the NIR region [37]. The DPEs also include polyethylene glycosylated [38], poly acrylic acid (PAA) [39,40], poly methacrylic acid (PMAA) [41]. In addition, other polymers like poly lactic acid (PLA) [42] and polypyrrole (PPy) [43].

Nonlinear polymers

Nonlinear polymers such as hyperbranched polyethylenimine (PEI), polyamidoamine (PAMAM), polyglycerol (PG) and cyclodextrins (CDs) have prominent spatial structure and are treated as self-carrier. Cationic polymers such as PEI can condense plasmid DNA and RNA into stable complexes via electrostatic interactions [44]. Wang et al. compared the intracellular delivery efficiency of GO-PEI and GO-PEG in Raw264.7 cells and found that GO-PEI has better aggregating features in the cytoplasm [45]. Thus, PEG and PEI dual-functionalized GO are more suitable to be used as a gene nanocarrier. Moreover, GO-PEG-PEI (also named as RGPP) exhibited various delivery abilities for both siRNA and large size plasmids in 11 cell lines including human, mouse, cancer and normal [46]. Furthermore, GO-PEG-PEI loaded with plasmid-based Stat3 siRNA can suppress malignant melanoma growth in mice [47,48]. When loaded with miR-7b overexpression plasmid or siRNA-targeting Ckip-1 increases the bone formation [49,50]. Moreover, Yue and his team have constructed the GO-PEG-PEI nanocarrier for the delivery of high-molecular-weight Cas9/single-guide RNA (sgRNA) complexes and efficiently executed the function of gene editing in human AGS cells [51]. Most of the studies on the GO-PEI gene delivery system have focused on the cell level, except for an earlier report on cardiac repair in vivo [24,52]. Although relatively fewer studies have addressed the drug delivery; in these, the antitumor assays were performed in vivo via GO-PEI drug delivery system [53].

Dendrimers are highly branched nearly spherical and symmetrical macromolecules. Poly (amidoamine) (PAMAM) are the most commonly used dendrimers, in which interior cavities are used to encapsulate hydrophobic or hydrophilic drugs, while their terminal functionalized outside surface change the physicochemical, reactivity, dynamics, and biological properties [54]. In 2012, graphene-oleate-PAMAM or GO-PAMAM were used to deliver the plasmid DNA of EGFP into HeLa and MG-63 cells [55]. DOX and MMP-9 shRNA plasmid co-delivery can also be performed by GO-PAMAM leading to higher cytotoxicity of MCF-7 cells [56]. Yadav et al. have developed a PEG and PAMAM modified GO (GPD), which, when coated with EPAC1 siRNA inhibited cell migration and decreased invasion of MDA-MB-231 cells [57]. Moreover, PAMAM functioned graphene nanostars loaded with metalloproteinase 9 (MMP-9) overexpression plasmid reduced hepatic fibrosis of artificial liver cirrhosis mice [58]. Besides, graphene nanostars, which are new kind of GFNs, after being linked to PAMAM-G5 and loaded with the plasmid encoding for metalloproteinase 9, could significantly reduce hepatic injury and improve liver restoration in mice with liver cirrhosis [58]. However, no in vivo antitumor assays have been tested using GO-PAMAM nanocarrier.

Tu et al. have obtained graphene derivatives with the property of pH-triggered surface charge conversion, and near-infrared irradiation (NIR) controlled DOX release by using hyperbranched polyglycerolamine and 2,3-dimethylmaleic anhydride (DA) to modify graphene. This nanohybrid showed high antitumor efficacy in tumor bearing-mice [59]. The ring form polymer cyclodextrins (CDs) will be presented in the polysaccharide subgroup.

Polysaccharide

Polysaccharides are a class of commonly used biomacromolecules, most of which are naturally derived [60]. Chitosan, derived from a deacetylated form of chitin, is extensively used in the fabrication of drug carriers or other biomedical materials due to its biocompatible, antibacterial, and biodegradable properties [61]. In 2011, Chitosan functionalized GO was first obtained via a facile amidation process and was used to successfully deliver camptothecin (CPT) or pDNA (pRL-CMV) into human cancer cell lines [62]. MDR1 siRNA could be transfected by chitosan functionalized GO and could efficiently knockdown the MDR1 mRNA and its translation product P-gp expression levels in MCF-7/Dox cells [63]. Wang et al. have measured the in vivo antitumor efficacy of galactosylated chitosan decorate GO [64].

Cellulose, which is another polymer of glucose, is also used in drug delivery systems. Carboxymethyl cellulose (CMC) and hydroxyethyl cellulose (HEC), for example, are frequently used to decorate GO. DOX loaded CMC/graphene quantum dot (GQD) nanocomposite hydrogel film or CMC/GO has a pH-dependent release profile and strong cytotoxicity to K562 or SW480 cells [65,66]. Drug loading and release behavior of folic acid (FA) on rGO-HEC nanohybrids have been also investigated [67].

Cyclodextrins (CDs) belongs to a family of macrocyclic compounds consisting of several glucose subunits with a relatively hydrophobic central cavity and a hydrophilic outer surface [68]. Thus, CDs are widely used in drug delivery by hosting the poorly soluble drugs and forming a biocompatible complex. CDs functionalized GO as a scaffold with or without further decoration could deliver DOX, CPT, Dexamethasone (DEX) or SN38 (7-ethyl-10-hydroxycamptothecin) to alter the cell growth [69-72]. Yet, so far, the antitumor efficacy of those CDs has not been tested in vivo. As far as other polysaccharides are concerned, GO-alginate (ALG) possesses pH-controlled 5-fluorouracil (5-FU) release and high antitumor efficacy [73]. The cytotoxicity of Dextran (DEX) conjugated GO loaded with curcumin (CUR) to 4T1 and MCF-7 nucleolin over-expressed cancer cells have been detected [74].

Hydrogel-based drug delivery systems facilitate controlled and adjustable drug release [75]. The most commonly used hydrogels are polysaccharide-based materials. These include CMC [65], Sterculia gum and carbopol polymers [76], sterculia gum-polyacrylamide hydrogel [77], gum tragacanth [78], poly (ethylene glycol) dimethacrylate [79], polyacrylamide hydrogels [80], chitosan [81], chitosan-g-poly (N-isopropylacrylamide) (CPN) [82]. Also, tripeptide hydrogels (not polysaccharide) [83] have been studied to modified GO. Among them, CMC [65], chitosan [81] and CPN [82] have been testing for cancer therapy. Especially, GOFA-DOX/HACPN have been tested in nude mice implanted with MCF-7/LUC cell [82].

Compared to hydrogels, nanogels can be used as a more feasible delivery system due to their small size [84]. So far, the in vitro anticancer assay of alginate [85], isopropylacrylamide [86], salep, and isopropylacrylamide [87] modified GO nanogels loaded with DOX have been performed. Aerogels based on GO and polyvinyl alcohol (PVA) can be potentially used for dermal delivery and treatment of trauma bleeding [88]. The release of 5-FU from the hybrid aerogels of chitosan, CMC and GO has also been addressed by previous studies [89]. Polyacrylamide/graphene oxide/gelatin/sodium alginate (PAM/GO/Gel/SA) composite hydrogel and accelerate peripheral nerve regeneration [90]. These hydrogel-graphene nanocarriers have a near-infrared (NIR) or pH-dependent release. Nevertheless, there are no reports on the delivery of RNA or DNA being reported via hydrogel-graphene nanocarrier.

Amino acid-protein-aptamer

Protein and amino acid functionalization are a commonly used tools for GO. Ginsenoside Rh2 (Rh2) can be loaded on the Arg or Lys functionalized pristine graphene [91]. Collagen-coated 3D graphene foams itself without additional load, stimulating the differentiation of DA neurons from MSC [92]. FA-BSA decorated GO load with DOX secure an increased effective drug concentration to MCF-7 and A549 cells [93]. Gelatin [90] and tripeptide [83] hydrogels decorated GO are used for nerve regeneration or drug delivery. Moreover, the Caspase-3 specific peptide probe is tagged with FAM and further anchored to GO. Thus, acquired a caspase-3 activated imaging agent can detect cell apoptosis [94]. Moreover, some peptides or antibodies (such as octaarginine (R8) and anti-HER2 antibody [95]) that are treated as target molecules are addressed in the latter part.

Aptamers are oligonucleotide molecules that can bind to targets with similar affinities and specificities to antibodies. This affinity is applied in biosensor and biomedicine [96]. Most of the fluorophore-tagged aptamers are attached to GO via π-π stacking interactions where they display a weak fluorescence signal due to efficient quenching; the fluorescence is activated when nanocomposites entry the cells and aptamer bind to higher affinity target molecules. This tactic has been used in Cytochrome c (Cyt c) [94], cancerrelated microRNA (miRNA) [97] and tumor exosomes detection [98]. Thrombin binding aptamer (TBA) undergoes a conformational change when potassium (K+) is added, which thus far has been used in potassium detection when combined with FRET-based sensors [99]. The affinity of aptamer molecules applied in targeted delivery is discussed later on.

Nonpolymers

Researchers have used the nonpolymeric organic materials to improve the functional properties of GO. Ma et al. have used soy phosphatidylcholine (SPC) membrane to encapsulate DOX-loaded NGO (NGO/DOX) and to increase its stability and biocompatibility. Administration of DOX-loaded NGO in mice, can inhibit tumor growth and prolong the survival time [100]. Moreover, fluorinated graphene, which possesses better properties in molecular sensing, was used to deliver DOX and CPT to Hela and oral epithelial cells [101]; while adipic dihydrazide (ADH) and heparin (Hep) modified GO were used to deliver DOX to HepG2 and MCF-7 cells, and have a decreased DOX release in heart, lung and kidney [102]. GO-DOPA-maleimide-c (RGDfC) synthesized via Thiol-Maleimide ‘Click’ Chemistry was previously applied to the targeted DOX chemotherapy and photothermal to MDA-MB-231 and HeLa cells [103]. Sedghi et al. have used 3-aminopropyltriethoxysilane (APTES, Si(OEt)3) as a crosslink between electrospun nanofibers and GO [104]. Also, pegylated phospholipids could be used as a link to anchor the functional molecules to GO [105].

Inorganic NPs decoration

Inorganic NPs, which possess high surface to volume ratio, controllable shape and size, facile surface modification, stability and unique optical and magnetic properties [106] include metal NPs (Au, Ag, Cu, Pt and Ti), metal oxide NPs (Fe₃O₄, ZnO and TiO₂), Rare-Earth NPs (Gd, Ce, Eu), as well as other inorganic materials (SiO₂ and so on) [107-109]. Organic-inorganic nanohybrid offers numerous advantages by combining the unique properties of organic and inorganic counterparts. In 2012, Huang et al. established a grapheneinorganic nanohybrid, which has been currently applied in electronics, optics, electrochemical energy conversion and storage, solar energy harvesting, and so on [8]. This paper reviewed graphene-inorganic nanohybrids used as nanocarriers.

Metal NPs

In 2013, PEI-functionalized GO-encapsulating gold NPs (GOPEI-AuNPs) were applied to deliver DNA into HeLa cells as a novel pDNA gene vector and its transfection efficiency was higher than PEI-functionalized GO [110]. GO sheet attached to Au@PANI (polyaniline) core-shell nanoparticles exhibited NIR/pH-responsive DOX release property and excellent NIR photothermal transduction efficiency, leading to synergistic therapeutic efficacy in tumor-bearing mice [111]. Moreover, Usman et al. have doped GO with rare earth metal Gd (Gadolinium) and protocatechuic acid via hydrogen bonding and π-π interactions, then coated the nanomaterial with AuNPs through electrostatic interactions, thus fabricating the GAGPAu that can be used as both PA nanocarrier and magnetic resonance imaging (MRI) contrast agent [112]. Graphene-gold composites could also deliver miRNA-101 or miRNA-122 into cancer cells [113,114]. Most of the reports used gold as MRI or SERS (Surface-Enhanced Raman Spectroscopy) contrast agent, while GO as a load moiety. In graphene-silver composites, the silver section can also provide antibacterial activity [115], while Habiba et al. revealed that the photodynamic therapy of Ag-GQDs is enhanced by Ag compared to GQDs [116]. Cu-crosslinked carboxymethylcellulose/naproxen/graphene quantum dot nanocomposite has been reported as an oral drug delivery nanocarrier [117]. GO coated titanium as a bone implant, was always used to deliver drug resulting in cell differentiation [118]. Rare earth paramagnetic metal Gd based NPs are used as MRI contrast agents [119].

Metal oxide NPs

Metal oxide NPs include iron oxide, titanium oxide, and zinc oxide. Mostly used magnetic nanoparticles include iron oxide and its derivatives (such as CoFe₂O₄ [115]) [120]. Iron oxide nanoparticles (IONP) have been extensively studied in biomedical applications, mainly as magnetic guided cell signaling due to their high magnetic properties [121]. When combined with GO or GQDs, IONP has been applied for MRI imaging [27,122], magnetic hyperthermia therapy [123,124], and magnetic-guided behavior [27,125]. Moreover, magnetic hyperthermia of IONP facilitated the temperature-dependent drug release of GO [124]. TiO₂-based nanohybrid has drawn significant attention in the photocatalysis field [126]. Hybrid nanocomposite Pr-TiO₂/NGO, which consists of graphene oxide nanosheets and TiO₂ nanocrystals doped with rare earth metal praseodymium (Pr), combined the photocatalytic activity therapy, photothermal therapy and anticancer drug DOX, thus leading to significant inhibition of HeLa cell growth [127]. Mesoporous zinc oxide (ZnO) scaffolds coated with drop-cast graphene oxide (GO) forming a delayed-release biolayer system could be used in bone tissue engineering [128].

Other inorganic NPs

Mesoporous silica nanoparticles (MSNs) possess high surface area available for drug loading [129]. GO or GQDs coated drug-loaded MSNs could break up because of NIR-induced thermal effect, thus achieving controlled drug release [43,130]. There are different modes between silica and GO, such as GQDs that can be incorporated into the cavity of MSNs hollow. This controlled release drug delivery platform has been proved in tumor-bearing mice [131].

Except for those inorganic NPs mentioned above, ZnS QD is a diagnostic agent candidate due to its optical characteristics. Zeng et al. obtained a theranostic nanoparticle by combining InP/ZnS (core/shell) QDs to GO loaded with miR-122, which can be used for targeted imaging and induction of apoptosis of drug-resistant hepatoma cells in tumor-bearing mice [132]. Similarly, Diaz-Diestra et al. combined ZnS:Mn to DOX leaded GO obtaining a theranostic platform [133].

Hydroxyapatite (HAP) had biocompatibility, bioactivity, and osteoconductivity, which have been extensively used in the medical field, especially for bone repair or regeneration and drug delivery applications. GO-HAP composites, apart from bone regeneration, could deliver absorbing ibuprofen (IBU) [134], 5-FU [135]. Cheang et al. have used GO-HAP nanocomposites to deliver a plasmid that expressed suicide gene herpes simplex virus thymidine kinase (HSV-TK) to inhibit the proliferation of cancer cells [136].

Optimization strategies for improving the delivery efficacy

Although dozens of new drugs appear each year, almost all of them face the challenge of effectively targeting the diseased tissue and cells. To increase the delivery efficacy and reduce the side-effects, there appear some efficient strategies. Among them, target agent modification is a feasible strategy for NPs-based drugs [137]. Various target agents have been used to improve GO-based nanocarrier [138]. Trigger controlled delivery, which dependent on tumor microenvironment, specifically gives the drug efficacy to designated cells [139]. Here we presented several targeting agents and trigger controlled systems that have been used in optimizing the GO-based platform (Figure 2). The representative literatures which have compare the target-effect are listed in Table 2.

Figure 2.

Optimizing strategies to improve the delivery efficacy. Photothermal and acidic condition were the commonly used controlled release stimuli. Target molecules include ligand, protein, peptide and aptamer. Magnetic targeting was a powerful way for inorganic NPs and GO nanohybrid.

Table 2.

The representative target molecules used in GO-based nanoplatform

Target molecules	Targets	Nanocomposites	Loaded drugs	In vitro/in vivo model	Results	references
FA	Folate binding protein on the cell membrane	GO/PP-SS-DOX, GO/PEG-PLGA-DOX	PEG-PLGA-SS-DOX	B16 tumor bearing mice	The tumor growth inhibition rate of non-targeting and the targeting nanohybrids was 60.70% and 81.78% respectively.	[29]
HA	CD44 on the cell membrane	HA-rGO	indocyanine green	KB cells tumor bearing mice	The targeting nanohybrids almost completely inhibited the tumor growth, while poorer effect using non-targeting nanohybrids.	[153]
GA	Protein Kinase C of mitochondrion	GA-GO	DOX	HepG2 tumor bearing mice	The average tumor weights of GA-GO@DOX, GO@DOX, and control groups were 0.59, 0.70, and 1.29 g after 21 d treatment, respectively.	[155]
antibodies of VEGF	VEGF	PTX-GHP-VEGF	PTX, paclitaxel	SW-13 tumor bearing mice	The survival rate of mice in the PTX-GHP-VEGF+NIR-treated group was 100%, and dramatically higher than nontarget group.	[161]
P-L-Arg	glycosaminoglycan receptors on the cell membrane	P-L-Arg-Au@GO	miRNA-101	MCF7, MDA and HU-02	Au@GO-PEG-(P-L-Arg) displayed more toxicity on MCF7 cell line in comparison to Au@GO-PEG.	[113]
AS1411 aptamer	nucleolin on the cell membrane	GO-DEX-Apt-CUR	CUR, curcumin	4T1, MCF-7 and CHO	GO-DEX-Apt-CUR are significantly more cytotoxic compared with GO-DEX-CUR in MCF-7 or 4T1 cells.	[74]

Ligand

The most commonly used target agent is folate acid (FA). The overexpression of the folate acid receptor has been found in many the cancer cells [140]. Targeting efficacies of FA that modifies GO NPs have been proven in vitro and in vivo. So far, different particles coated with FA have been tested, including GO carrying DOX, colchicine (COLC) [141], paclitaxel [142], curcumin [143], camptothecin [144], copper complex (regard as a substitution cis-dichlorodiamineplatinum) [145], and siRNA [24]. Mice bearing HELA cells [100], HepG2 cells [141], PaCa-2 cells [24], B16 cells [29], MCF-7 cells [82] or EAT (Ehrlich Ascites Tumor) cells [146] have been used as models in previous studies. Among these in vivo assays, two showed a significant inhibitory effect of tumor growth [29,100].

Hyaluronic acid (HA) is extensively used as a major ligand that binds to the CD44 (hyaluronan) receptor with high affinity. Similar to FA receptor, CD44 receptor is overexpressed in many cancer tissue [147]. HA modified GO or GQDs have been used to deliver DOX [148], Histamine dihydrochloride (HDC) [149], 5-Fluorouracil (5-FU) [150], SNX-2112 [151], miR-21 peptide nucleic acid (PNA) probes [152] in vitro or in vivo. Anti-tumor assays have been identified in HELA cells [148], Panc-1 cells [150], MDA-MB-231 bearing mice [152]. However, there is still no chemotherapy research comparing the differences of in vivo anti-tumor efficacy of GO-based material with or without HA; nonetheless, Miao et al. obtained remarkable results in photothermal therapy in 2015 [153].

In GO-based nanocarrier, Triphenylphosphonium (TPP) [59,154], Glycyrrhetinic acid (GA) [155], Hypericin [156] are used to target mitochondria. The alteration of mitochondria functions is associated with malignant transformation, and this provide targets for novel cancer therapeutics [157]. Among them, GA is believed to target the hepatocellular carcinoma (HCC) cells and exhibit a certain degree of targeting property to the SMMC-7721 [158]. Other drug delivery systems use these mitochondria target molecules, offering enhanced drug efficacy via preferential mitochondria accumulation to push the cells to apoptosis stage.

Protein

The antibody of marker molecules which overexpressed on the cancer cells (CMMs) can direct the nanohybrid to cancer cells [159]. P-glycoprotein (P-gp) is overexpressed in cancer cells when they acquire multi-drug resistance (MDR) [160]. Zeng et al. have synthesized GPMQNs (graphene-P-gp loaded with miR-122-InP@ZnS quantum dots nanocomposites) which utilize P-gp antibody to target the tumor in nude mice [132]. The monoclonal antibodies have been used against vascular endothelial growth factor (VEGF) to modify GO and to increase the delivery efficacy to VEGF overexpressed cancer cells [161]. Epidermal growth factor receptor (EGFR) is highly expressed on the surface of tumor cells. Cetuximab (CET, an EGFR monoclonal antibody) modified GO may inhibit the growth of xenograft tumors with implanted CT-26 cells [162]. Moreover, the antibody of heparin sulfate proteoglycan glypican-3 (GPC3), a potential molecular target for hepatocellular carcinoma (HCC), is used to decorate rGO [163]. Anti-HER2, an antibody of a transmembrane tyrosine kinase receptor overexpressed in 25%-30% of human breast cancer, is used in S180 cells bearing mice [95]. Proteins, such as transferrin (TF) or lactoferrin (LF), could guide the DOX-loaded GO NPs to melanoma cells or glioma cells, respectively through their receptors [164,165].

Peptide and aptamer

Cell-penetrating peptides (CPPs) and cell-targeting peptides (CTPs) facilitate the selective delivery of chemotherapeutic drugs [166,167]. These peptides, including poly-L-arginine (P-L-Arg) [113], Arg-Gly-Asp (RGD) [168], c (RGDfC) [103], c (RGDyK) [169], octaarginine (R8) [95] are used to enhance the selectivity of GO nanohybrid. CPPs and CTPs decorated GO loaded with miRNA or drug have been already tested in vitro and in vivo. The chimeric peptide (MPG-2H1) has been designed to facilitate the endocytosis of pDNA loaded GQDs [170].

Kim et al. have fabricated a fibroblast activation protein (FAP) activated promelittin decorated rGO nanosheet DOX delivery system, which displayed a better antitumor efficacy via tumor microenvironment targeted therapy in vivo. Moreover, Melittin used in this system is a nonspecific cytolytic peptide that attacks all lipid membranes. FAP is a serine protease that uniquely cleaves the Pro-Xxx amino acid bond; FAP has been reported to be selectively overexpressed on the membranes of cancer-associated fibroblasts (CAFs) [105].

AS1411 aptamer, which is an ssDNA aptamer that can be recognized by nucleolin and can improve the intracellular uptake, assists CUR-loaded GO to target 4T1 and MCF-7 cells [74]. ST-3 aptamer facilitates entry of GO into the biofilm, making the antibacterial activity of ST-3-GO superior to that of its component molecules [171].

Trigger controlled delivery system

Trigger controlled drug release, and the applications have been developed in a new drug delivery system to provide enhanced efficiency or more beneficial therapy [172]. GO-based hydrogels were discussed in previous section. These delivery platforms could prolong and sustain drug release. Dynamic bonding interactions between GO and its cargo have shown the controlled response to external stimuli such as changes in pH or temperature. The photothermal effect has been suggested for cancer therapy and controlled drug release due to the induced heating of graphene via absorption of NIR light [173,174]. Many drugs, such as DOX [133], curcumin (Cur) [143], colchicine (COLC) [141], 5-FU [73] have shown much faster release in acidic conditions (just as tumors) than in neutral conditions. NIR or pH is the capital stimulus in the controlled drug release of GO-based nanocarriers. There was a different example with alginate (ALG) dissolved in the alkaline environment of the colon, where 5-FU loaded on GO-ALG had a specific release into the colon via oral administration, thus significantly inhibiting the tumor growth [73].

Glutathione (GSH)-triggered release has shown to be successful using GPMQNs (graphene oxide InP@ZnS QDs) as nanocarrier [132]. Fibroblast activation protein (FAP) has been used to activate a cytolytic peptide (detail in previous section) [105]. The particular target microenvironment offered an opportunity to design stimuli-responsive drug delivery systems for controlled release. IONP and GO nanohybrids can be guided by using an external magnetic field [175]. Lu et al. have developed cetuximab (CET) and magnetic dual-target delivery system and combined chemotherapy with photothermal therapy [162].

Conclusion and perspective

GFNs have been widely used for cancer and bacteria treatment, tissue engineering, and biological imaging. Most of these applications rely on the excellent absorption property of GO-based materials. Many efforts have been made to improve GO-based materials as gene and drug nanocarriers via various decorations. In the present study, we grouped the functionalization according to the decoration molecules, placing a higher emphasis on the used nucleic acid deliveries (Figure 1). Organic and inorganic materials are the major sets used to introduce functional sections onto GO. The representative literature of the first three categories of the organic subgroup is listed in Table 1. In terms of cancer treatment efficiency, there is no significant difference in antitumor inhibition rate when using GO delivery nanoplatforms and free drugs. However, it will be different if combined with other strategies such as: phototherapy, target delivery, controlled release and so on.

Controlled and targeted release restricts the nanocarrier delivery efficacy and clinical application. GO-based nanoplatform offer some feasible strategies to improve the drug efficacy. Photothermal and pH-controlled release are excellent characteristics of GO nanocarrier [59,73]. The most common target molecules used in the GO delivery system are shown in Figure 2. Most of the target molecules specifically have an affinity to targets of cancer cells. Cleavable chemical bonds, under the catalytic of a particular enzyme or other molecules in cancer microenvironment, could explicitly give an accumulation or activation of drugs [29,105]. Besides, macrophages or monocytes, due to their tumor-tropic migratory property and strong phagocytic capacity, can serve as a cellular carrier to enhance the delivery efficacy, also known as biomimetic delivery systems (BDS). GO-based nanomaterials combine to macrophage-mediated delivery display appreciable tumor inhibition rate [176].

Though chemotherapy is successfully conducted in tumor-bearing mice, most of the gene therapy remained at the cell level except in Paul’s work on cardiac repair [52], and Yin’s work on antitumor [24]. Both plasmid DNA and small RNA can be delivered via GO-based nanocarrier. In vivo gene delivery was, in most cases, carried out for tissue engineering and bioimaging [177,178]. In conclusion, GO-based nanomaterials still have a long way to go to be applied in clinical work, especially in gene delivery. We believe that target delivery and controlled release, as optimization strategies, are certain fields to be further explored.

Disclosure of conflict of interest

None.