Graphene is the most outstanding material among the new nanostructured carbonaceous species discovered and produced. Graphene’s astonishing properties (i.e., electronic conductivity, mechanical robustness, large surface area) have led to a deep change in the material science field.
In recent years, nanoscale technologies have become the last frontier in material science and pharmaceutical development [1]. A primary role has been played in this by nanostructured carbonaceous materials [2][3] such as carbon nanotubes and graphene (GF) due to their intrinsic properties and easy functionalization [4].
Nowadays, graphene and related materials represent the most advanced frontier in high-performance carbon materials [5] as witnessed by the European Union research council enforcing a strong action named EU Graphene Flagship [6]. This plan aimed to promote basic investigation on graphene and its related derivatives in order to establish the European Community as a world leader in the field [5]. This was consequent to the top properties of this allotropic one-atom-thick planar sheet of carbon tightly packed into a hexagonal cell structure [7]. Graphene and its related materials’ features can be exploited in a wide range of applications to improve the mechanical robustness and electronic properties of composite materials [8][9][10], both plastics [11][12] and metals [13][14], even at a very limited amount However, its price is not negligible, hindering its way through to the market with respect to cheaper solutions [15][16][17][18][19][20][21][22][23]. Due their high cost, graphene and related materials cannot be used in cheap, large-scale production. However, they can be employed in high-added-cost applications such as those represented by frontier medicine [24].
This field has been boosted up by vicious diseases and the increased concern for human healthiness. Pharmaceutical companies and academic institutions have deeply committed themselves to drive to unreached levels newly designed drugs and procedures [25][26]. Despite the wide numbers of available established protocols, new routes [27][28] are explored to develop new and innovative materials for drug delivery [29], regenerative medicine [30], theragnostic treatment [31], and tissue repairing [32].
In the following section, we report some of the recent advancements in the use of graphene and related materials for drug delivery, tissue engineering, and regenerative medicine.
IGraphen recent years, nanoscale technologies have become-like materials’ viability for biological applications is strongly related to the interactions between graphene and cellular and tissue structures.
Despite the last frontier in material science and pharmaceutical development [1].onishing properties, the use of graphene flakes and rGO as drug carriers without any modification is quite challenging in biological environments due Ato primartheir high hydrophobicity [33]. The use of GO overcole has been played in this by nanostructured carbonaceous materials [2,3] sumes this issue due to its high hydrophilicity. This property leads to a cellular uptake through both endocytotic and macro-pinocytotic mech asnisms carb[34][35].
Nonetheless, nanotubes andthe use of graphene (GF) due to their intrinsic properties and easy functionalizationflakes and rGO in biological environments could be exploited by performing covalent and non-covalent modifications [4][36].
Nowaday Thes,e graphenless invasive and related materials represent the most advanced frontier in high-performancontrolled modifications preserve the high conductivity of graphene flakes and rGO allowing their use in neuronal repairing [37], in vivo cellular cimaging [38], arbon materid stimuli-mediated drug delivery [39][40].
The extended sp2 system of gralphene and rGO represents [5]a strong advas witnessed by the European Union resntage with respect to other platforms used in the biological environment such as inorganic nanoparticles [41] due to their farch councilcile modifications through cycloadditions [42], enf.g., 1,3-diporcinglar cycloadditions a[43], snitrong acene addition [44], and amedide condensation [45]. The intrinsic reactivity EUof Ggraphene Flagship [6].and related materials allows to easily build up compound Thlibraris plan aimed to promote baes filled with plenty of materials with the basic properties of graphene and tuned biological activity.
The mosict investigation oned biological application of graphene and its related derivatives in ordermaterials’ still remains drug delivery [46]. The ability o establish the European Community as a world lf materials to drag and drop chemicals in the human body environment is well exploited by graphene and graphene-related materials [47].
Zhang et al. [48] descr in the field [5].ibed the interactions between Tgraphis was consequent to the top properties of this allotropic one-atom-thick planar sheet of carbon tightly packed into a hexagonal cell structure [7]. Gene and proteins elucidating the ability of graphene-based materials to trigger several protein complexes on the cellular membrane to facilitate uptake. This was due to both electron traphene and its relatedsfer and residual functionalities on graphene-like materials’ features cansurface. Wang et al. [49] bdeeply exploited in a wide range of applications to improve the mechanical robustness and electronic properties of composite materials [8,9,10],red the relationship between graphene’s surface topological defects and its ability of drug delivery, proving a strong effect of topology. The authors described the folding ability of these materials in several shapes ranging from nanoscrolls to polyhedral ones. This protean ability allows different interactions with different bfoth plastics [11,12]lding agents such as membrane proteins or nucleic ancid metals [13,14],s. A further study by Mohammeven at ad et al. [50] vthery limited amount However, its price is not negligible, hindering its way through to the market with respect to cheaper soluoretically clarified the loading ability of pristine and metal-decorated graphene materials. Among them, hydrogel and foam are the most attracting solutions because drug release could be triggered by both chemical and thermal modifications [15,16,17,18,19,20,21,22,23][51].
Ezzati et al. [52] Dprodue their high cost, grapheneced a biocompatible graphene foam surface tailored with alanine, cysteine, and glycine for cisplatin drug and related ease. The authors tested the materials cannot be used in cheap, large-scale production. However, they can be employ with a high load of cisplatin on MCF-7 and HepG2 human cancer cell lines with good results. Additionally, the graphene-based carrier underwent biodegradation inside the body according to the mechanism reported in Figure 1.
Graphigh-ene added-cost applications such as those represennd related materials also represent a very promising platform for drug release upon biological stimuli, as reported by frontier medTrusek et al. [53] icine [24].
T this field has been boosted up by vici case of doxorubicin.
Doxorus diseases and the increased concern for humanbicin is a widely diffused anticancer drug acting on the S phase of the cellular metabolic pathway [54]. hSevealthiness. Pharmaceutical companies and academic instiral studies reveal the efficacy of graphene-based materials as carriers for doxorubicin as reported by Shen et al. [55]. Contrary to polymer-based formutions have deeply committed themselves to drive to unreached levels newly designed drugs and procedures [25,26].lations, graphene and graphene-like materials could act not merely as delivery systems but also as smart platforms to target and release doxorubicin upon several stimuli on a large time window and to monitor the cell Despvitalitey [56]. Furthe wide numbers of available established protocols, new routes [27,28]rmore, graphene-like materials could be also coupled with polymers to enhance classical formulation effects. The authore explored to develop new and innovativs proved the effectiveness of chitosan-tailored graphene materials for drug delivery [29],as carriers through molecular dynamic calculation. Doxorubicin interacts with both the π orbital system of gregenerative medicine [30],aphene and the polar functionalities of the chitosan. After reaching the taragnostic treatment [31],get, doxorubicin interacts with protein amine and tcarboxylissue repairing [32].
c residues losing the chitosan interactions. In this case following section, we report some of the recent adv, the weak interactions with the graphene surface are not sufficient, and it was efficiently released.
This effect was magncements in the uified considering that the base of graphene and related mathe drug delivery systems is the pH release mechanism [57].
GO-based systems werials for druge also used for the delivery of ketamine [58], tissuebuprofen [59], and mengineerdicine for hormone therapy [60]. In all cases, the high cong, and regenerative medicinetrol on drug release prevented the sudden increment of drugs, leading to a time-prolonged release.
GThe combination of graphene and its grelatedaphene derivatives lack of some properties such as bandgap or opticalwith plenty of different matrices has represented a game changing event in the production of biomaterials [61][62]. Fixing or reprlacing boperties. This reduces the possible applications,th tissues and organs has been boosted by the graphene-based composites by combining biocompatible scaffolds due to a variety of surface interactions [63].
Tissue aend an engineered material could offset these limitations. In recentgineering is one of the frontiers of medicine, aiming to overcome the drawbacks of the classical transplant procedure through the production of hybrid materials able to perform the same activity as the tissues replaced times,[64]. gGraphene quantum dots (GQDs) have received great attention for their possible uniqueand its related materials have shown very attractive results related to the production of polymeric composites with great mechanical performances together with high biocompatibility for several applications [75]ranging from asbone a solid alteto soft tissue repair [65].
Bahrnatmi et al. [66] combivne to classicald polyurethane with graphene, GO, and rGO.
Grap for thene quantum dots consist of graphene sheets, which could be single-sheet or multi-layered. They are nanoproduction of conducting-polymer-based-material tissue regeneration. The authors aimed to grow L929 fibroblast and blood vessel endothelial cells on a membrane containing graphene flakes. This was particles with dimensions smaller than 100 nm, commonly under 20 nm, and their shapes are usually circulularly interesting in the case of endothelial cells. Indeed, they could be grown on the inner surface of tubular scaffolds by mimicking the native blood vessel structure [67]. The author or elliptical. However, other shapes are produced with different synthetic routes [76].
Ths proved the ability of composites to support the attachment, spreading, and proliferation of cell lines. A very close approach was used synthesis methby Pant et al. [68] fodsr tohe produce GQDs are both ttion of coated stents by using graphene oxide mixed with polyurethane.
As shop–down in Figure 2, graphend bottom–up as summarized ine-oxide-based composites create a stable coating Figure 4on bythe using different precursors and synthetic strategiessurface of the stent without any tearing or ablation phenomenon.
The mosdificat common approach is the top–down one that usually consists of physical processes that reduce the dimensions of carbon materials as carbon fibres [78]ion of interphase properties due to the presence of graphene clearly increased the durability of the stent due to the radical-scavenging effect, which preserves the por coal [79], lymeric malong with acid oxirix from degradation [80] and hydpromothermal treatment [81]ed by extracellular fluids.
Soimilvothermal route [82],ar results microwave [83], ould be and sonication [84] arhieved also promising alternative approaches.
Aby using titanium-based materials [69], top–down physical process is the acid exfoliation, followed by the oxidation of thhich however show the critical drawback of cellular morphology alteration.
Rege nano-obtained product. Li et al. [85]erative medicine is another field where suggested an electrochemical synthesis routeraphene-based materials have released their full potential to[70][71]. producThe a variety of GQDs, although the products differ in size, emission colours, and other intrinsicdherence of a cell on graphene is a critical properties. Hydrothermaly as shown by Morçimen et al. [72]. By studynthesis can be done just on precise precursors, such as GO, with a functionalization with oxidizing agents to introduce functional groups on the product surface [86].
The bottom–ing the adherence and proliferation of the SH-SY5Y neuron cell line on graphene foams, aup method provides for the generation of aromatic molecules as precursors ars proved the viability of this approach in more complex procedures.
Fend can lead to high yield. Li et al. et al. [73] [87] produceveloped a protocol to fabricate GQDs through direct pyrolysis on citric acid.
The d an iron-oxide-based, magnetic, electrospun, synthesis routes mentioned above lead to a differentiatiort nanofibre-wrapped GO for a guiding cellular behaviour as shown in Figure 3.
As shown in ofFigure 3, the GQDauthors produced and their different physicochemical properties which depend on procedure, precursors, defects, and functional groups [88,89] short GO magnetic fibres by electrospinning techniques achieving materials with diameters of up to 300 nm and a length of up to 80 μm and a magnetization saturation of up to 50.33 emu/g. The GQDs’ properties of interest are absorbance, photoluminescence (PL), electrochemical photoluminescence among the optical ones and of course their biocompatibility; all these properties make them great candidates for bioimaging and theragnostic applications.
Thee fibres were able to induce an extremely tight adhesion with cells due to the functionalized GO. The observations were particularly promising for tissues manipulation absorption characteristics of GQDs are shown in the short-wavelength region because of C=C bonds and around 270–390 nm they show peaks for the C=O bonds. The passivy using external magnetic fields to selectively move specific cellular groups by tailoring the GO surface. Furthermore, the creation of the surface with a functional group can alter the disposition of the peaks; it strongcontrolled cellular aggregates could be a step forward in specific site repairing.
Agarwaly deet al. [74] producends on the type of dot.
The prd a highly elastincipal and most fascinating property of the carbon dots is their photoluminescence. The unclear photoluminescence mechanism is one of the impediments to understand completely the physical mechanism behind these promising materials. The tuneable property of photoluminescence is size-dependent since it is related to quantum confinement. Moreover, other factors such as shape, defect, and doping also influence the effect. In detail, Zhu et al. [90] r and electroconductive graphene/collagen cryogel for spinal cord regeneration. Firstly, the authors tailored the graphene surface with amine residues promoting an efficient cross-linking with collagen showing a conductivity of up to 3.8 ± 0.2 mS/cm and a Young modulus of up to 347 kPa. The authors teporsted that PL is related to the quantum confinement effect of the electrons in sis composite for stem cell transplantation and neural tissue regeneration through the cryogelation app2 carbons [91].ach by Ausinother structure-dependent characteristic of GQDs is the electron transfer [92]. Bg the BM-MSCs cell line. They reported a good stem cell growth and expression enhancompatibilityement of CD90 and almostCD73 absgenent toxicity make them eligible for in vivo applications. In vitro studies have been made by Shang et al. [93] upon electric stimulation of up to 100 mV/mm without the loss of stemness. Additionally, and they studied the GQDs’ effects on the cellular life finding out how the nanomaterial did not affect either the viability or the reproduction capacity. In vivo studies have been made on mice by exposing them to authors observed an ATP secretion increment that could be helpful to favour neuronal regeneration and immunomodulation. Graphene-based cryogel promoted the neuronal different nanomaterials. The results showed no abnormalities in organs or habits and brought to awareness their excellent biocompatibility, raising interest in the possible bio-applications.
Tiation of BM-MSCs with enhanced expression of MAP-2 kinase and β-tubulin III. In vitro observation proved a high indoleamine 2,3 dioxygenase activity under inflammatory conditions together possible bio-applications of GQDs are cellular imaging [94],with the proliferation of macrophages, which could aid in tissue repal-timir.
Te ichan vivo biosensing [95], iyom et and drug delivery. [74] [96].
Tshowe main advantages of GQDs upon traditional imaging solution is red that osteoblast differentiation and gene expression could also be modulated to the extremely high biocompatibility together with facile tailoring properties. Furthermore, the metabolic excretion after the procedure promotes a rapid removal of toxic formulation elements, e.g., heavy metals such as gadolinium or europium used in imaging procedures [97]by GO coating of titanium materials due to an upregulation of the expression of the bone matrix protein genes during late-stage osteoblast differentiation.
Graphene and its related derivatives lack of some properties such as bandgap or optical properties. This reduces the possible applications, and an engineered material could offset these limitations. In recent times, graphene quantum dots (GQDs) have received great attention for their possible unique applications [75] as a solid alternative to classical graphene, GO, and rGO.
Graphene quantum dots consist of graphene sheets, which could be single-sheet or multi-layered. They are nanoparticles with dimensions smaller than 100 nm, commonly under 20 nm, and their shapes are usually circular or elliptical. However, other shapes are produced with different synthetic routes [76].
The synthesis methods to produce GQDs are both top–down and bottom–up as summarized in Figure 4 by using different precursors and synthetic strategies.
Figure 4. Scheme of graphene quantum dots (GQDs) top–down and bottom–up approaches as reported by [77] (under CC license).
The most common approach is the top–down one that usually consists of physical processes that reduce the dimensions of carbon materials as carbon fibres [78] or coal [79], along with acid oxidation [80] and hydrothermal treatment [81]. Solvothermal route [82], microwave [83], and sonication [84] are also promising alternative approaches.
A top–down physical process is the acid exfoliation, followed by the oxidation of the nano-obtained product. Li et al. [85] suggested an electrochemical synthesis route to produce a variety of GQDs, although the products differ in size, emission colours, and other intrinsic properties. Hydrothermal synthesis can be done just on precise precursors, such as GO, with a functionalization with oxidizing agents to introduce functional groups on the product surface [86].
The bottom–up method provides for the generation of aromatic molecules as precursors and can lead to high yield. Li et al. [87] developed a protocol to fabricate GQDs through direct pyrolysis on citric acid.
The synthesis routes mentioned above lead to a differentiation of the GQDs produced and their different physicochemical properties which depend on procedure, precursors, defects, and functional groups [88][89]. The GQDs’ properties of interest are absorbance, photoluminescence (PL), electrochemical photoluminescence among the optical ones and of course their biocompatibility; all these properties make them great candidates for bioimaging and theragnostic applications.
The absorption characteristics of GQDs are shown in the short-wavelength region because of C=C bonds and around 270–390 nm they show peaks for the C=O bonds. The passivation of the surface with a functional group can alter the disposition of the peaks; it strongly depends on the type of dot.
The principal and most fascinating property of the carbon dots is their photoluminescence. The unclear photoluminescence mechanism is one of the impediments to understand completely the physical mechanism behind these promising materials. The tuneable property of photoluminescence is size-dependent since it is related to quantum confinement. Moreover, other factors such as shape, defect, and doping also influence the effect. In detail, Zhu et al. [90] reported that PL is related to the quantum confinement effect of the electrons in sp2 carbons [91]. Another structure-dependent characteristic of GQDs is the electron transfer [92]. Biocompatibility and almost absent toxicity make them eligible for in vivo applications. In vitro studies have been made by Shang et al. [93], and they studied the GQDs’ effects on the cellular life finding out how the nanomaterial did not affect either the viability or the reproduction capacity. In vivo studies have been made on mice by exposing them to different nanomaterials. The results showed no abnormalities in organs or habits and brought to awareness their excellent biocompatibility, raising interest in the possible bio-applications.
The possible bio-applications of GQDs are cellular imaging [94], real-time in vivo biosensing [95], and drug delivery [96].
The main advantages of GQDs upon traditional imaging solution is related to the extremely high biocompatibility together with facile tailoring properties. Furthermore, the metabolic excretion after the procedure promotes a rapid removal of toxic formulation elements, e.g., heavy metals such as gadolinium or europium used in imaging procedures [97].