Source: https://pubs.rsc.org/en/content/articlehtml/2019/nh/c8nh00318a?page=search
Timestamp: 2019-04-20 04:14:05+00:00

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Recent years have seen the explosion of biomedical research on graphene-based materials (GBM), owing to the captivating physical and chemical properties of this family of nanomaterials.1 Thousands of scientific papers have been published since 2010, the year Geim and Novoselov received the Nobel Prize for research on graphene; it was pointed out recently that as the number of works continues to grow, “the graphene community could be easily overwhelmed by the collection of this vast knowledge”.2 The nomenclature given to GBM as well as the various methods of synthesis represent a source of confusion since in some papers, what authors describe as graphene/functionalized graphene is another member of this ultrathin carbon family.3 This leads to (sometimes apparent) contradictions in the results ascribed to the same GBM.
In this review, we will shed light on these inconsistencies and sum up the current knowledge about the in vivo bio-interface of freely suspended monolayers of graphene oxide (GO). We focus on GO since the low-cost production and hydrophilic nature of this material still make it preferable to other carbon materials.4 The effects of GO on biological systems are often compared to its reduced form, reduced graphene oxide (rGO). Like GO, rGO can be obtained with several protocols and can be characterized by variable C/O atom ratios. In some works, GO has been compared to GBMs that are different from rGO. In this review, we will stick to the nomenclature used in the original papers, and we will also describe the size data available to facilitate the readers in making comparisons.
The BC of GO is still poorly explored and few works have considered the influence of this layer on in vitro and in vivo effects. Here, we will first discuss the surface features of GO and their links to amino acids and blood protein binding. Then, we will discuss the GO BC composition and how the BC can influence interactions with blood cells. Finally, we will highlight biodistribution and biosafety concerns as well as future challenges related to the development of intravenously injected GO-based pharmaceutical systems. The comprehension of these aspects is involved in and will improve the future design of injectable biocompatible GO.
GO and rGO are often defined as bidimensional materials but only pristine graphene (the freely suspended single-atom-thick sheet of hexagonally arranged, sp2-bonded carbon atoms3) can be considered a true 2D material.9 GO is a chemically modified graphene derived from the oxidation and exfoliation of graphite and rGO is produced from GO with several possible reduction protocols in order to obtain the closest material to pristine graphene.3 rGO can be obtained by methods like thermal reduction at high temperature (>900 °C),4 chemical reduction by reducing agents (like borohydrides, aluminum hydride, hydrohalic acid and sulphur-containing reducing agents),2 hydrothermal, electrochemical and bacteria-mediated reduction.10 Since residual functional groups and defects remain on the basal plane, rGO is not analogous to pristine graphene.
Fig. 1 (a) The Lerf–Klinowski model of the GO structure, adapted11 with permission from The Royal Society of Chemistry. (b) Analysis of protein residue content and the correlation with the protein adsorption capacity on GO, rGO and single-walled carbon nanotubes (SWCNT). (b) The positive correlations between the protein adsorption capacity and protein molecular weight (A), the number of hydrophobic amino acids (B) and the number of Tyr (C), Phe (D), and Trp (E) residues, adapted with permission from ref. 25, American Chemical Society, Copyright (2015). (c) The binding of albumin, fibrinogen and globulin depends on the lateral size and concentration of GO, as shown in this figure adapted6 with permission from The Royal Society of Chemistry.
The groups on the GO surface provide unique opportunities for chemical modification via covalent bonds to obtain functionalized GO. The functionalization of GO can be divided into two categories: edge functionalization (of carboxyl groups) and basal plane functionalization (of hydroxyl and epoxide groups). Reactive intermediate functionalization of rGO to directly functionalize the sp2-hybridized basal plane is also possible; further details can be found in other reviews.11 Often, GO functionalization is used to build GO-based polymer composites to enhance the thermal and mechanical stability of the original polymer. These composites can be produced by covalent modification of GO functional groups or via non-covalent interactions, taking advantage of hydrogen bonding and van der Waals forces between the polymer and GO.11,20 Also, the protein adsorption on GO and rGO can occur via covalent or non-covalent interactions. Covalent binding is based on chemical reactions between the side groups of amino acids and functional groups available on the GO surface.
In blood, non-covalent adsorption occurs through weak van der Waals forces, hydrophobic, electrostatic, and π–π stacking interactions.12,21 The sp2 hybridized honeycomb carbon lattice of rGO and GO is hydrophobic and, therefore, interacts with the hydrophobic regions of proteins, according to the protein geometry.9,22 The basal plane of the GO is also enriched with π electrons, making π–π stacking interactions possible. At the same time the oxygen groups of GO, whose composition is strictly dependent on preparation and storing conditions2, allow further hydrogen bonds and electrostatic bonds.12 These electrostatic bonds are strongly influenced by the charge on the proteins and therefore by the pH and the ionic strength of the buffer. Bonding on GO can also be mediated by van der Waals interactions.23 However, while the electrostatic interactions are more pronounced on GO, both van der Waals and electrostatic interactions play a major role in the adsorption of proteins on rGO due to the increase in the non-functionalized area on the surface.24 In the following sections, we will show how functionalization of the GO surface alters protein adsorption and consequently BC properties.
Chong and colleagues reported that for both GO and rGO, the order of adsorption is fibrinogen > immunoglobulin > transferrin (the iron-binding blood plasma glycoproteins that control the free iron level in biological fluids) > albumin.25 Similar to Chong, in the study of Belling on GO and rGO, the order of protein adsorption is as follows: complement factor H > IgG > albumin.26 These data are in agreement with the Kenry study, since albumin appeared in the supernatant even at very low concentrations (0.5 mg mL−1) followed by globulin and fibrinogen, and was visible in the supernatants at 5 and 10 mg mL−1, respectively (Fig. 1b and ref. 6).
Fig. 2 Milligrams of proteins (bovine serum albumin (BSA), transferrin (Tf), immunoglobulin (Ig) and bovine fibrinogen (BFG)) adsorbed on GO (a) and rGO (b); adapted with permission from the American Chemical Society, Copyright (2015).25 Data for adsorption corrected for available surface indicate a higher capacity of rGO in respect to other materials (c). Comparison between GO and rGO loading of immunoglobulin g (IgG), BSA, human serum albumin (HSA) and factor H; reproduced with permission from ref. 26. (d) Protein adsorbed on three materials, namely carboxylic-functionalized multi-walled CNT (CNT-COOH), 2D graphene nanoplatelets (GNPs) and porous graphene oxide (PGO) illustrated in (e), are reported in (f) albumin, (g) globulin and (h) fibrinogen; adapted with permission from ref. 40.
In summary, the adsorption capacity of GO is influenced by the aromatic residue content, protein size and hydrophobicity. Fibrinogen is better adsorbed than albumin by GO, while GNP, which is less oxygenated, better adsorbs albumin. The hydrophobic interactions between protein amino acids and poorly oxygenated GBM are less-favoured due to the instability of these nanomaterials. Further systematic studies are needed to clarify the influence of surface groups and nanomaterial dispersibility on protein adsorption.
Fig. 3 Main results of GO interaction with blood components are summarized in this illustration of the injection of GO flakes in the bloodstream. The formation of the BC (1) prevents the hemolysis of red blood cells (2a). Thrombosis (2b) and interaction with complement proteins (2c) are ascribed to GO. In (2d) some of the possible fates after macrophage encounters are shown: extracellular blocking or intracellular uptake. The release of cytokines occurs when macrophages uptake GO. Aggregates of GO in macrophage cytoplasm induce the production of pro-inflammatory cytokines. Dendritic cells fail to present antigens to lymphocytes when they uptake GO (2e). Lymphocyte activity is not inhibited, and BC protects lymphocytes from apoptosis (2f).
Direct exfoliation of graphite in human serum is a recently established ultra-sonication protocol to analyze the BC of pristine graphene.46 This method was developed by Castagnola and colleagues to avoid the usage of dispersants that form an adsorbed layer on the graphene surface and affect BC composition. The method consists of 1 to 4 hours of ultrasonication of 10 w/v% of natural flake graphite dispersed in a solution of serum at different concentrations using a bath sonicator.
Surface modification is a commonly adopted approach to improve the biocompatibility of GO and to control the formation of the BC.
Tan and colleagues compared the interactions of serum proteins with GO and nGO-PEG, a nanometric GO functionalized by a 10 kDa amine-terminated six-arm-branched polyethylene glycol (PEG) via amide formation.55 Unlike GO, which adsorbs a significant amount of serum proteins without specificity, nGO-PEG exhibits reduced protein binding and selectivity toward six proteins: four immune-related factors (C3a/C3a (des-Arg), clusterin, histidine-rich glycoprotein, vitronectin) and two coagulation factors (contained platelet factor 4 and thrombin). However, the association of thrombin and platelet factor 4 might be a pseudo effect, since their circulation levels are extremely low, but increase by more than 3 orders of magnitude during the clotting protocol in serum preparation.55 Further, given the size difference between nGO-PEG (nanometric flakes) and GO (micrometric flakes), the potential size effect on the above interactions should be investigated.
Xu and colleagues measured the effect of GO surface modification on the composition of GO-BC in mouse serum (Fig. 4). GO was chemically modified to obtain aminated GO (GO-NH2), prepared with GO dispersion in ammonia with hydrazine hydrate as a reducing agent, GO-polyacrylamide (GO-PAM), GO-polyacrylic acid (GO-PAA) and GO-PEG.
Fig. 4 Protein corona analysis of GO, GO-NH2, GO-PAM, GO-PAA and GO-PEG formed in mouse serum at 37 °C for 1 h. (a) SDS-PAGE analysis of protein corona. (b) Normalized OD values of each protein corona, indicating the amount of protein adsorbed on GO. Asterisks (*) denote p < 0.05 compared to pristine GO. (c) Eleven highly abundant components identified by mass spectrometry. Reproduced with permission from ref. 56.
In summary, the composition of G-BC is affected both by the nanomaterial state and the protein source. We can speculate that the work of Castagnola and colleagues46 defines, to some extent, the configuration of the BC of injected pristine graphene and that the enrichment in apolipoproteins might be useful for targeted delivery applications (as discussed in the conclusions). However, unfunctionalized pristine graphene instability remains the Achilles’ heel of this nanomaterial. As we will see in Section 6, when GO is injected in blood, aggregates form, but in a size and dose-dependent fashion.58 This means that GO is more stable and is suitable for an injected delivery system and consequently, its BC should be precisely controlled. GO adsorbs a large amount of proteins thanks to the highly available surface,49 and this is generally looked at as a disadvantageous feature in vivo, since the more proteins “mark” the foreign nanomaterial, the better it is attacked by our immune system.59 Many functionalization strategies are therefore exploited to improve GO stealth properties. However, this protein enrichment gives GO some advantages over other nanomaterials in diagnostics and pharmaceutical applications. Indeed, the higher protein adsorption in the BC can be exploited to select and enrich poorly concentrated biomarkers in patients’ blood52 and develop diagnostic tools based on the BC.7 Secondly, the list of proteins found in GO-BC includes ApoE, vitronectin and clusterin, which are important blood–brain barrier (BBB)-directing molecules (ApoE) as well as targets of therapies (Table 2).60–62 rGO, whose BC, to the best of our knowledge, still has to be fully characterized, can enter the brain thanks to a transitory decrease in the BBB paracellular tightness.63 Future studies could be focused on delivery applications based on GO/rGO selectively adsorbed proteins.
As explained above, in vivo injection implies the formation of the BC around GO and rGO (Fig. 3-1) and regardless of the BC composition, several groups have demonstrated the physical hindrance of BC between GO and eukaryotic cells (Fig. 3-2a). The presence of a BC around GO flakes can completely inhibit the interaction and therefore RBC hemolysis.67 GO hemolysis is also prevented by chitosan, dextran and curcumin coating or by functionalization of the GO surface.66,69,70 An interesting study proposed the use of mussel-inspired dopamine (DA) for both the GO reduction and functionalization.71 DA has many properties: (i) it adheres to solid surfaces in water solution without surface pretreatment; (ii) once on the surface, DA can anchor a secondary functional biopolymer via the thiol, imino and amine groups; (iii) the catechol groups of DA can convert GO into chemically reduced rGO. Cheng and colleagues exploited these features to obtain heparin-grafted polyDA-rGO (Hep-gpRGO) and BSA-grafted polyDA-rGO (BSA-g-pRGO) that greatly suppressed hemolysis ratios (lower than 1.8%, even with a high concentration of 200 μg mL−1).
These contrasting results are not unique in the literature on GO and cells, but they are explainable by the experimental conditions. In most of the literature, the cytotoxicity of GO and rGO against many cell lines has been demonstrated to be caused by cellular membrane penetration and/or oxidative stress induction.6,72 However as for RBC, the physical damage to the cell membrane is largely attenuated when GO is incubated with BSA or FBS, due to the extremely high protein adsorption ability of GO.14,25,39,73,74 In cell culture medium supplemented with FBS, GO is enriched with a BC of albumin and IgG, irrespective of the lateral size GO.75 In summary, the presence of proteins in the cell culture medium influences the results on cytotoxicity and we could consider that GO and rGO are not hemolytic in vivo where abundant protective BC form on their surfaces (Fig. 3-2a).
Hemostasis cascade prevents blood loss from injured tissue and maintains blood fluidity. The final hemostasis is driven by platelets, which form the clot, a mixture of red blood cells, aggregated platelets, fibrin and other cellular elements (Fig. 3-2b). If the clot forms abnormally, it can induce thrombosis.
The group of proteins of the complement system (Fig. 3-2c) that promotes antigen phagocytosis and recruitment of neutrophils and macrophages to the site of inflammation is highly abundant in the GO corona.49 The in vitro GO complement-activation test provided evidence that this material significantly triggers complement activation by the increase in the concentration of fragments C3a (proportional to GO concentration) and C5a.
Authors have claimed that this activation might be driven by both hydroxyl groups and the hydrophobic surface of the GO skeleton.64 A detailed study of the relation between GO surface oxidation and complement activation confirmed this hypothesis.79 In this work, GO was synthesized through the modified Hummers’ method and was mildly reduced to obtain three nanomaterials having different oxygen content (i.e. 36%, 29%, 24% atomic percentage) and size of a few μm (from AFM characterization). It was found that the decrease in oxygen content reduced complement activation, as reflected in the lower levels of both C5a and SC5b-9. This could be explained by the instability of the flakes and the diminished exposure of the GO surface in less oxidized GO, which is more prone to forming irreversible flocculates in solution. On the other hand, this phenomenon may be attributed to a combined effect of oxygen-type functionality and topological change arising from a wrinkling of the GO surface that may modulate the affinity for recognized molecules or interaction with complement regulators.79 Since C5a can substantially potentiate IL-6 production in lipopolysaccharide (LPS)-stimulated peripheral human blood leukocytes, the effect of GO on IL-6 release in human whole blood was tested in the same study. At concentrations below the GO-mediated complement activation, the LPS-induced responses were inhibited by GO, probably through a direct interaction of GO with LPS or GO and LPS binding protein. This effect disappeared when the GO concentration was above the complement activation threshold and the IL-6 cytokine release was induced.79 Pre-coating of GO or rGO with a corona of albumin or complement H factor, obtained after incubation of the nanomaterials at 37 °C for 2 hours with these proteins, can reduce complement activation by 40% and 90%, respectively.26 This interesting effect is mediated by both the steric blocking of the interaction with complement components and, for the complement H factor corona, by regulation of the complement cascade.26 In summary, GO activates the complement system but the reduction of oxygen content and the precoating with BC might prevent this effect.
A direct comparison of the effect of materials with similar dispersibility but different oxidation states (GO and rGO nanoplatelets with size <100 nm) was conducted for monocytes and macrophage precursors (Fig. 5). GO nanoparticles have been obtained with the modified Hummers’ method and sonication, while rGO was obtained via UV photoreduction of the former.88 rGO is better ingested in respect to GO but induces differential expression patterns of antioxidative enzymes. The effects of exposed THP-1 cells could also pass to THP-1a as shown in Fig. 5, reproduced from ref. 88 GO nanoparticles (GONPs) demonstrated a stronger inhibition of THP-1a phagocytosis towards E. coli as compared to rGO nanoparticles (rGONPs) and both GONPs and rGONPs impaired the phagocytosis and endocytosis abilities of THP-1a.
Fig. 5 Illustration of the short-term effects of GONPs and rGONPs on THP-1 cells, and the long-term effects on THP-1a differentiation from THP-1 cells. GONPs and rGONPs could have induced ROS formation and activated the NF-κB pathway in THP-1 cells. rGONPs could not fully transcript proinflammatory genes due to lack of additional transcription factors. Reproduced with permission from ref. 72.
Other important phagocytic cells are dendritic cells (DC) that activate antigen-specific T cells, after (i) antigen capture, (ii) intracellular processing and (iii) the presentation of the antigen within the MHC complex on the cell surface.
The focus of this review is the GO interaction with blood components and BC in light of the future design of GO pharmaceutical delivery systems. Intravenously injected drug delivery systems (DDS) developed so far include PEGylated nanographene sheets for tumor passive targeting,93 rGO functionalized with chitosan and iron oxide magnetic nanoparticles for the delivery of doxorubicin94 and epidermal growth factor receptor antibody-conjugated PEGylated nanographene oxide for epirubicin delivery in tumors55 (for a comprehensive outlook of DDS based on graphene see ref. 95). Nanoparticles intended for drug delivery applications are being engineered to reduce their clearance and extend systemic circulation times and thus increase the opportunity for targeted delivery. However, the disadvantage of prolonged circulation times is the greater chance of interaction with blood components and activation of adverse effects.
Before any nanomaterial translation into clinical therapy, there are biosafety concerns that need to be addressed. We have seen how GO interacts with blood system components and how BC can influence these interactions, but what is the biodistribution and the toxicity when GO is administered intravenously (i.v.)?
The early study of Zhang and colleagues determined the distribution and biocompatibility of i.v. injected GO in mice.99 The half-life of GO in blood is much longer than in other carbon nanomaterials (∼5 hours). Within 48 hours after i.v. injection, GO is cleared from the bloodstream and distributed throughout various organs with preferred accumulation in the lungs, liver, and spleen. The lack of pathological changes was reported after 14 days of treatment at a low dose (1 mg kg−1), but at a higher dose (10 mg kg−1), granulomatous lesions, pulmonary edema, inflammatory cell infiltration, and fibrosis throughout the lung were observed.99 Many studies confirmed that the primary site of GO accumulation and toxicity in vivo is the lungs.97 It seems that the pathological effects on the lungs are proportional to the degree of dispersion and oxidation of GO. When directly injected into the lungs, GO induces severe long-term (21 days) lung injury, while graphene flakes, either in a dispersed or aggregated state, do not increase apoptosis in lung macrophages.100 The biodistribution of GO is size-dependent. In the report by Zhang, the size of GO ranged from 10 to 800 nm and that this caused a distinctive clearance behavior: particles with small size were quickly eliminated through the renal route within 12 h post injection, while large particles were intercepted by the lungs.99 A systematic study on GO size, dose and dosing frequency was conducted by Liu and colleagues.58 Liu intravenously administered two types of GO: small GO flakes (s-GO, average hydrodynamic diameter of ∼250 nm) and large GO flakes (l-GO, average hydrodynamic diameter of ∼900 nm) at a single high dose (2.1 mg kg−1) or seven repeated low doses (0.3 mg kg−1); irrespective of size, the single high-dose administration of GO induced lung damage and infiltration of inflammatory cells. In the lungs, GO accumulated in the macrophages but not in the lymphocytes, which were recruited but were not able to trap GO. In this study, the authors claimed that although oxidative stress is a widely existent phenomenon in cells exposed in vitro to GO, the protective effect of proteins forming a BC around GO should be considered in vivo.
Interesting size-dependent results were reported for multiple-dose exposure. The s-GO did not induce renal damage or accumulate in the kidneys since it was quickly eliminated through the glomeruli. Conversely, l-GO failed to be cleared through kidneys and induced damage. The lungs were damaged only after multiple doses of l-GO. This effect depends on the aggregation of GO with proteins that induce the blockage of large GO-complexes in the lungs. The hypothesis relies on the formation of multiple complexes of l-GO and proteins that enter the capillaries and create multiple injury points and inflammatory cell recruitment. s-GO could instead pass through lungs capillaries after each low-dose administration. The kidneys and lungs were more damaged by l-GO, while the s-GO preferentially accumulated in the liver with toxic effects.
In summary, s-GO is safer than l-GO, but at a single high dose the aggregation with blood proteins, and therefore the BC, induces the formation of toxic large complexes. These results are in accordance with the study by Qu and colleagues, in which GO (30–1000 nm) aggregation was suppressed with the addition of Tween-80 surfactant.101 While GO aggregated and accumulated in the lung macrophages, Tween-80 GO was more stable and accumulated in the liver inside Kuppfer cells (macrophages).101 Interestingly, rGO seems to have reduced thrombogenicity in vivo, improved biocompatibility and brain tissue targeting.63,77,102 The intravenous administration of rGO (with a lateral size of 342 nm administered at 7 mg kg−1 in a single dose) led to minor signs of toxicity in the blood, liver and kidneys and a lack of inflammation after 7 days.
The addition of polymer coatings such as PEG to the nanomaterial surface is a tool for avoiding recognition by the immune cells. PEG is known to prolong particle circulation in the blood and significantly decrease uptake by the spleen and liver-resident phagocytes. It has been hypothesized that PEG creates a steric shield around the coated particle, effectively preventing plasma proteins from adhering to the particle surface and thus avoiding subsequent uptake by mononuclear phagocytes. PEGylated GO sheets (10–30 nm) were reported to be mainly distributed in the liver and spleen and did not exhibit toxicity at high doses (20 mg kg−1) within 90 days.74,104 This was likely due to both the small size and PEGylation, which stabilized the nanomaterial and prevented the aggregation as well as reduced the BC formation.
Luo and colleagues demonstrated that PEGylation of GO does not prevent macrophage activation.105 nGO-PEG, a functionalized GO with a lateral size of ∼200 nm, prevents macrophages uptake, but through physical contact with their cell membranes, boosts the release of cytokines, potentially leading to further immunological responses downstream.
Finally, the degradation of injected GO is an important biosafety concern. Long-term interaction (14 days) of GO with plasma causes reduction and biodegradation with hole formation caused by the action of hydroxyl radicals.54 Once internalized by the immune cells, biodegradable particles are digested and cleared from the body, while non-biodegradable particles accumulate in cells for extended periods.
Despite the great scientific advances, future studies for in vivo application should focus on some weaknesses in graphene research. First, graphene materials should be designed to have more than just a stable small size for rapid excretion, and degradable composition to limit toxicity. Detailed physicochemical characterization, including size, surface area, charge, purity, oxygen content, stability in body fluids and the composition of the BC, should be described in papers aimed at the pharmacological administration of graphene.
– perform biocompatibility tests, cytotoxicity, genotoxicity, biodegradation, distribution and accumulation into organs, metabolism.
In the last decade, the design of injectable nanoparticles has affected the control of the BC formation, the interface seen by cells and tissues, in vivo. Combining all the experimental results presented in this review, we can conclude that the BC significantly affects several interactions of GO. The BC inhibits the hemolytic effects of GO, regulates complement activation, and mediates immune response activity and biodistribution.
Given the difficulty of precisely controlling the in vivo interaction with proteins, most of the strategies designed to modulate the BC are based on functionalization with anti-fouling polymeric residues that suppress protein adsorption, altogether lowering the targeting efficiency.43 In the case of GO, pre-coating with chitosan to reduce non-specific protein adsorption, functionalization with dextran or PEG to improve elimination from the body or modification of surface functional groups by amination and carboxylation are examples of approaches to modulating the BC.
Based on the great advances in GO research, we can foresee that new opportunities in nanomedical applications will be available. We hope that the arguments presented in this review will help researchers in moving forward and pushing the limits of GO toward clinical applications.
This work was supported by Fondazione Umberto Veronesi (Postdoctoral Fellowships Grant 2018 to V. Palmieri). We are thankful to Giulia Peragallo for her advice for Fig. 3 design.
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