Magnetic Fe3O4@graphene oxide improves the therapeutic effects of embryonic stem cells on acute liver damage

Abstract Objective Acute liver failure is usually associated with inflammation and oxidation of hepatocytes and has high mortality and resource costs. Mesenchymal stem cell (MSCs) has occasionally been reported to have no beneficial effect due to poor transplantation and the survival of implanted cells. Recent studies showed that embryonic stem cell (ESC)‐derived MSCs are an alternative for regenerative medicine. On the other hand, graphene‐based nanostructures have proven useful in biomedicine. In this study, we investigated whether magnetic graphene oxide (MGO) improved the effects of ESC‐MSC conditioned medium (CM) on protecting hepatocytes and stimulating the regeneration of damaged liver cells. Materials and methods To provide a rat model of acute liver failure, male rats were injected intraperitoneally with carbon tetrachloride (CCl4). The rats were randomly divided into six groups, namely control, sham, CCl4, ESC‐MSC‐CM, MGO and ESC‐MSC‐CM + MGO. In the experimental groups, the rats received, depending on the group, 2 ml/kg body weight CCl4 and either ESC‐MSC‐CM with 5 × 106 MSCs or 300 μg/kg body weight MGO or both. Symptoms of acute liver failure appeared 4 days after the injection. All groups were compared and analysed both histologically and biochemically 4 days after the injection. Finally, the results of ESC‐MSC‐CM and MSC‐CM were compared. Results The results indicated that the use of MGO enhanced the effect of ESC‐MSC‐CM on reducing necrosis, inflammation, aspartate transaminase, alanine aminotransferase and alkaline phosphatase in the CCl4‐induced liver failure of the rat model. Also, the expression of vascular endothelial growth factor and matrix metalloproteinase‐9 (MMP‐9) was significantly upregulated after treatment with MGO. Also, the results showed that the ESC‐MSC‐CM has more efficient effective compared to MSC‐CM. Conclusion Magnetic graphene oxide improved the hepatoprotective effects of ESC‐MSC‐CM on acute liver damage, probably by suppressing necrosis, apoptosis and inflammation of hepatocytes.


| INTRODUC TI ON
Acute liver failure (ALF) is a life-threatening clinical syndrome characterized by rapid hepatocellular necrosis due to hepatotoxicity, viral infection and immune-mediated attacks. 1,2 It has high mortality and resource costs. 3 Liver transplantation has some limitations such as the lack of donors, costs and immunosuppressive complications. 4 Mesenchymal stem cells (MSCs) have also been used to treat ALF.
They are easy to obtain and do not pose an ethical problem. 5 MSCs have functions such as secretion of growth factors, angiogenesis and immunosuppression, as well as properties such as anti-inflammatory and anti-apoptosis effects. They can also prevent hepatocyte cell death and stimulate the regeneration of liver cells by paracrine mechanism or direct differentiation. 2,6 In view of the ALF treatment with stem cells, the available data are somewhat contradictory. Although MSC injection has some benefits, [7][8][9][10][11] it has occasionally been reported to have no beneficial effect due to poor transplantation and the survival of implanted cells. [12][13][14] In addition, the failure of MSCs to adhere to the target tissue leads to apoptosis of MSCs. 14 However, limitations to harvesting MSCs from adult tissue include the invasive procedures in most cases, availability of suitable donors, limited number of cells obtained during the harvesting process, and restricted in vitro expansion capacity. 15 Recently, it has been shown that MSCs could be derived from ESC, with similar phenotypic characteristics, along with favourable immunomodulatory and antiinflammatory properties that make them attractive candidates for regenerative medicine 15,16 and they have been used to treat various animal models disease such as experimental autoimmune encephalitis. [17][18][19] On the other hand, administration of MSC-conditioned medium (ESC-MSC-CM) has been shown to improve tissue injury. 20,21 The use of modern biotechnologies such as stem cells, laser and nanomaterial has attracted a great deal of attention in medical applications such as regenerative medicine. 22−30 Graphene and its derivatives have been shown to improve the proliferation and differentiation of stem cells. 12,13 Due to their physicochemical properties and biocompatibility, sometimes they act as natural extracellular matrices (ECM) and are able to regulate the differentiation of stem cells. 31 It has already been shown that the degree of cell differentiation and proliferation under different graphene oxide derivatives are different. For example, although in the absence of any osteogenic inducers the graphene nanogrids showed slight patterned osteogenic differentiations, the graphene sheets could not present any differentiation. On the other hands, the highly improvement of differentiation on the reduced graphene oxide nanoribbon grid was assigned to both its excellent effects on adsorption of the chemical osteogenic inducers and physical properties such as stresses oxidative. 32,33 Graphene oxide (GO) as a new class of carbon-based nanomaterials is a derivative of graphene with a two-dimensional honeycomb structure. The main difference between graphene and GO is the controllable hydrophilic nature of GO, which makes it welldispersible in water. 34 Because of its small size, ease of use and large specific surface area, 35 GO has been recommended for biomedical applications such as biosensors, [36][37][38] drug/gene delivery, [39][40][41][42] and antibacterial effects. [43][44][45] In addition, GO can absorb surface factors including proteins and small molecules, which are the essential components for the differentiation of MSCs. 46,47 However, it is difficult to separate GO from the solution by conventional centrifugation and filtration methods as a result of its hydrophilicity, high dispersibility and small size. 34 Fe 3 O 4 superparamagnetic nanoparticles are used in magnetically assisted drug delivery. 48 They can easily be separated by a magnetic field. 49−53 Nanocomposites based on magnetic graphene oxide (MGO) possess unique properties; they have high specific surface area, surface-active sites, excellent magnetic characteristics, high chemical stability, an adjustable size and shape, and can be simply functionalized or modified. 34 The biocompatibility and anticancer effects of Fe 3 O 4 in combination with graphene oxide have already been shown. 54 In this study, we investigated whether the synthesized MGO increases the protective effects of ESC-MSC-CM in the treatment of liver damage in animal models. We studied the possible in vivo hepatoprotective effects of MGO mixed with ESC-MSC-CM on the rat model of acute liver failure induced by carbon tetrachloride (CCl 4 ).
CCl 4 is a well-known hepatotoxin and is often used to induce acute liver failure. 21 The ability of ESC-MSC-CM mixed with MGO in the treatment of acute liver failure in rats was analysed by evaluating the serum level of enzymes and histopathological parameters.

| MATERIAL S AND ME THODS
In this study, the GO and magnetic GO were produced according

| Quantification of serum biochemical
The blood samples were kept at room temperature for 1 h and then centrifuged at 1500 g for 12 min at 4°C. The serum was separated and kept at 20°C until analysis. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were measured using an automated analyser (Hitachi) and commercially available kits (Pars Azmun) according to the manufacturer's instructions.

| Liver histology and immunohistology
The liver tissues were stained with haematoxylin-eosin (H&E) and

| Statistical analysis
The data were analysed using one-way analysis of variance for multiple comparisons. The significance level was set at p < 0.05 and p < 0.01. All data are expressed as means ± SE. Kolmogorov-Smirnov test was used to study normal distribution. Parametric continuous data with normal distribution between different groups were compared by one-way analysis of variance (ANOVA) followed by post hoc, Tukey test.

| Synthesis of MGO nanohybrid
Scanning electron microscopy (SEM) image of typical morphological features is depicted in Figure 1. As seen, the as-prepared GO confirmed the synthesis of GO nanosheets with quite smooth surface, where the distinctive layered structure of the sample was evident. A sharp diffraction peak for the pristine graphite (2θ = 30.93°, index of 002, θ the Bragg angle, JCPDS No. 00-012-0212) is corresponding to a d-spacing of 0.337 nm. The observation is in consistent with the previous study 58 The as-prepared GO showed a characteristic peak at 2θ = 10.58° which resulted from the diffraction on its 002 layer together with a broad diffraction peak at 22.38°. Similar results have been reported in the previous study. 59 The results for DLS and Zeta potential for pristine GO nanosheets and other nanoparticles analyses are given in Table 1.   64 The EDX analyses are given in Table 2.

| MGO inhibits the release of liver enzymes and improves survival rate
Our results indicated that treatment with MGO significantly improved the survival rate of CCl 4 -induced liver failure in rats that

| MGO injection improves histopathologic appearance and reduces neutrophil and Kupffer cell infiltration
We also examined the histopathologic changes in H&E and immu-

| MGO lowers the level of proinflammatory cytokines and increases the expression of vascular endothelial growth factor and MMP-9 in damaged livers
In

| DISCUSS ION
The ideal treatment for the CCl 4 -induced liver damage is to reduce symptoms such as apoptosis and inflammation and to promote immediate liver regeneration. In this study, we have shown that in- They secrete more anti-inflammation and antiapoptosis factors, attenuating the inflammation and also cardiomyopathy symptoms. 70 These results confirm the findings of Jiang et al. 63 They showed that transplantation of ESC-MSCs has a stronger effect in organ regeneration than that of adult-MSCs. 71 However, we observed in our study that ESC-MSCs mixed with  Note: −, +, ++, and +++ were used to assess hyperemia and accumulation of inflammatory cells and indicate no effect, slight, mild, and intensive, respectively. although the condition medium improved the survival and increased secretory proteins with trophic and immunomodulatory effects, its effects were less than those of MGO mixed with the medium. This could be due to the effects of the nanodeposition on the surface of trophic factors in serum followed by ECM protein absorption, which is essential for the adhesion, differentiation and growth of hepatocytes.

TA B L E 3 Microscopic evaluation
Nevertheless, it could also be due to the capacity of MGO with condition medium to alter the migration of immune cell into the damaged liver. 21 In addition to its superior mechanical properties, internalization and intracellular fate, GO has a large surface area that makes it suitable for drug delivery and regenerative medicine. 47,[73][74][75][76][77][78][79][80] The spontaneous differentiation induction of MSCs without extrinsic biochemical manipulation is another property of GO. 81,82 Therefore, in addition to the anti-apoptotic and proliferative ef- In this work, we synthesized MGO and examined its role in increasing proliferation, angiogenesis and decreasing apoptosis in the damaged liver cells through the ability to bind and release growth factors. We proposed MGO nanoparticles as a new multifunctional target platform for effective delivery of biomolecules capable of treating damaged liver in vivo. We showed that GO in combination with Fe 3 O 4 nanoparticles as biocompatible magnetic material accelerates the delivery of growth factors to the damaged tissue. One of the involved mechanisms is that graphene derivatives strongly adhered to macromolecules such as growth factors, which act as cell-adhesion substrate and growth factor-delivery carrier, thereby helps in the regeneration and improving damaged organs. 47 On the other hand, ESC-MSCs induce a higher rate of hepatocyte proliferation and have stronger antiinflammatory properties than bone marrow MSCs. Although MSC-CM and ESC-MSC improve liver function, they do not increase survival. 16 our results showed that MGO can improve the survival from CCl 4induced liver failure in rats received ESC-MSC-CM.

ACK N OWLED G EM ENTS
We thank Dr. Elaheh Motamedi for technical assistance and data analysis.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available within the manuscript or available from the authors upon request.