WO2018088978A1

WO2018088978A1 - A method for coating graphene oxide and biocompatible graphene oxide obtained from thereof

Info

Publication number: WO2018088978A1
Application number: PCT/TR2017/000077
Authority: WO
Inventors: Yasemin YUKSEL DURMAZ; Erhan DEMIREL
Original assignee: Istanbul Medipol Universitesi
Current assignee: Istanbul Medipol Universitesi
Priority date: 2016-11-09
Filing date: 2017-07-05
Publication date: 2018-05-17
Anticipated expiration: 2019-05-09

Abstract

The present invention relates to a method (100) for coating graphene oxide and a reduced graphene oxide (2) obtained from thereof. Its aim is to obtain a practical method (100) for coating graphene oxide so as be used in biological applications and a biocompatible reduced graphene oxide (2) platform with no toxic effects. In addition, a method (100) and product which can combine different biomedical applications is realized with the said invention as well.

Description

A METHOD FOR COATING GRAPHENE OXIDE AND

BIOCOMPATIBLE GRAPHENE OXIDE OBTAINED FROM THEREOF

Technical Field

The present invention relates to a method for coating graphene oxide and a reduced graphene oxide obtained from thereof.

Background of the Invention

Graphene known as the thinnest, the most durable and the rigid material in the world is a promising and important material in nanotechnology field due to its characteristics such as electrical conductivity, optical transmittance, mechanical endurance, thermal conductivity and large surface area and it is also much more interesting than other carbon derivative structures. These superior characteristics create a substantial potential for biological applications. Biological applications of graphene as biocompatible interface for drug and gene carrier, biosensor, imaging agent, cells and tissues gradually increase.

The first and most essential point applying to all biomaterials and required for taking graphene applications further and diversifying them is to turn graphene into a biocompatible material. However, it is known that graphene creates toxic effect by many different mechanisms.

Large surface field of graphene may create toxic effect by absorbing cell nutrients within cell and leading the cell to starvation. In addition, increase in lactate dehydrogenase (LDH) amount leads to concentration-based cell deaths by causing generation of active oxygen and creating intracellular stress. Whereas graphene oxide exhibits toxic effect in high doses and its capacity for cell entrance is low. Aromaticity is destroyed at some points on conjugate surface during oxidation and this causes some physical properties to be effected. This situation can be eliminated by using reduced graphene (rGO). Reduced graphene is a single graphene layer which has a more organized honeycomb structure and comprises less functional group in comparison to GO and it has a larger conjugate surface to be used for π-π interaction. However, it does not dissolve in water medium. Therefore, it cannot dissolve in blood and it creates toxic effect upon agglomerating. Again, efficiency of surface modifications carried out by using reduced graphene is at very low levels such as 20-30% for this reason.

In addition, reduction methods of graphene display toxic effect based on concentration and they occur depending on oxidation degrees and carbonyl groups.

In the state of the art, graphene is coated with polyethylene glycol (PEG) (PEGylation) in order that graphene becomes biocompatible. PEG, which is a biocompatible polymer, prevents living organism to be perceived as a foreign substance and to be absorbed by reticuloendothelial system. These PEG chains are attached to graphene surface which is generally oxidized in an uncontrolled way and covalently in a limited number. This functionalizing usually occurs with low efficacy and on the sides of graphene plate. For this reason, free space remains on the plate surface and its functionalizing percentage becomes low and its toxication mechanism cannot be prevented as well. Therefore, agglomeration of graphene in lungs where it indicates accumulation at most cannot be prevented in the long term although it is shown that PEGylation extends blood circulation time of graphene and its accumulation is some organs. In addition, samples for PEGylation of graphene oxide (A) by using pyrene (B) and PEG (C) chains or functionalizing it by other functionalities are available for a single (co)polymer chain in the state of the art. A method for reduction and PEGylation of graphene is disclosed in the article belonging to Jingqin Chen et. al. and titled "One step reduction and PEGylation of graphene oxide for photolhermally controlled drug delivery" in the state of the art. The Chinese patent document no. CN102320599, an application in the state of the art, relates to grafting pyrenyl poly(ethylene glycol) to graphene oxide surface.

Summary of the Invention An objective of the present invention is to obtain a practical method for coating graphene oxide so as be used in biological applications and a biocompatible reduced graphene oxide platform with no toxic effects.

With the said invention, a method and product which can combine different biomedical applications is realized.

Description of the Figures

Figure 1 is molecular view of graft copolymer.

Figure 2 is molecular view of coated graphene oxide. Figure 3 is molecular view of coated graphene oxide in the state of the art. Description of the References in the Figures The components illustrated in the figures are individually numbered, where the numbers refer to the following: 1. Graft copolymer

1 1. PEG

12. Methyl methacrylate

13. Pyrene methacrylate

2. Reduced graphene oxide (rGO)

100. Method

Detailed Description of the Invention

In the invention, a copolymer comprising at least one secondary monomer as at least two branching on an existing polymer chain is defined by graft copolymer (1) or in other words the term of graft copolymer.

In the invention, the term of macromonomer defines a monomer wherein an acrylate group is attached.

The inventive method (100) for coating graphene oxide (GO) operates according to the following steps:

- synthesizing the graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality by means of a pre-determined method (101);

- dissolving the obtained graft copolymer (1) within a pre-determined solvent (102); - adding the copolymer (1) solution obtained in a pre-determined ratio into a the graphene oxide (GO) solution having a pre-determined concentration dropwise (103);

- mixing the said copolymer (1) and the graphene oxide solutions at a pre- determined temperature for a pre-determined period of time (104);

- adding a reducing agent to the mixture at a pre-determined ratio (105);

- carrying out both π-π interaction and reduction reaction at a predetermined temperature for a pre-determined period of time (106);

- centrifuging the reaction mixture at a pre-determined rate for a pre- determined period of time (107);

- removing the upper phase and the lower phase comprising the precipitate remains (108);

- dispersing the precipitate in the lower phase within the water again and centrifuging it at a pre-determined rate (109);

- dispersing the precipitate within acetone and centrifuging it at a predetermined rate (1 10);

- drying the obtained sample at a pre-determined temperature (1 11).

In one embodiment, at the 101 step; the graft copolymer (1) is synthesized such that it will comprise at least one PEG (1 1) chain with a group -which has desired functionality- attached to at least one end thereof, and at least one monomer having at least one pyrene. Thus, functionality necessary in that application for a desired application can be given to the PEG (1 1) chain -which is bound to a monomer from one end thereof- by attaching a group having desired characteristics both from this attached end and the other non-attached end thereof. In another embodiment, a desired group can be attached to only one end of the PEG (1 1 ) depending on the application requirement again. In this case, functionality necessary in that application is given to a desired application again. In one embodiment, at the 101 step; the graft copolymer (1) is synthesized such that it will comprise at least one PEG (1 1) chain with one group each -which has desired functionality- attached to both ends thereof, and at least one monomer having at least one pyrene.

In one preferred embodiment, the graft copolymer (1) synthesized at the 101 step comprises at least one macromonomer having at least one PEG (1 1) chain and at least one monomer having at least one pyrene. Preferably, the said macromonomer is a PEG (1 1) chain comprising a methacrylate type of group. In one preferred embodiment, the said copolymer (1) comprises at least one 1- pyrenemethyl methacrylate (13) (PMA), at least one polyethylene glycol methyl ether methacrylate (PEGMA) and at least one methyl methacrylate (12) (MMA). Namely, the macromonomer comprising the PEG (1 1) chain is PEGMA in this embodiment. Molecular weight of PEGMA is 300-4000 g/mol, preferably 500- 2000 g/mol. It is easier to remove PEGMA chains which remain without reacting in the reaction medium after copolymerization in the said molecular weights. In addition, polymers with similar PEG (1 1) percentages can be made by changing molecular weights of PEGMA. In this case, a serial having the same PEG (1 1 ) percentage in terms of coating however different PEG (1 1) chain lengths can be caught and thus a reduced graphene oxide (2) which exhibits similar behaviour in terms of bioavailability but which can be used in different ways in application is created. For example, if a reduced graphene oxide (2) platform comprising PEGMA with 500 and 2000 g/mol molecular weight will be used to deliver drug, both drug loading capacities and drug delivery time and rate of graphene oxides (2) comprising the said PEGMA molecules will be different. This is a preferred status in drug delivery systems.

In the inventive method (100), the graft copolymer (1) is attached to graphene oxide by π-π interaction (stacking) of pyrene groups with graphene oxide surface. This interaction and the area used on the graphene oxide surface are controlled by the percentage of pyrene groups. In addition, molecular weight of PEGMA and its percentage in the copolymer (1 ) and the PEG (1 1 ) density on the graphene oxide surface are controlled. At the 101 step; one of controlled polymerization methods such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT), AGET-ATRP (Activator generated by electron transfer - atom transfer radical polymerization) is preferably used. This is because molecular weights and compositions can be controlled and adjusted in a desired way by means of such methods. Thus, the number of components (monomer and PEG(1 1 ) chains) within a structure can be known.

In one embodiment, ATRP method is used among these methods. Preferably copper (I) bromide (CuBr) is used as catalyst and N, N, N', N", N"-pentamethyl diethylentriamine (PMDETA) is used as a ligand during the graft copolymer (1) synthesis by ATRP method. Ethyl 2-bromo-2-methylpropionate (Ethyl a-bromo isobutyrate (EiBr)) material is an initiator. Ratio of [total mole number of all monomers]/[mole number of PMDETA]/[mole number of CuBr]/[mole number of EiBr] can be respectively 50/1 /1 /1 or 50/1.1/1.1 /1 or 100/1 /1/1 . In one preferred embodiment, this ratio is respectively 50/1/1/1 .

At the 101 step; the reaction starts after at least 3 freeze-pump-thaw (FPT) cycles. Thus, the air oxygen dissolved in the reaction mixture is removed. Thereby, the reaction is performed in a more controlled way. The said polymerization reaction is performed for 6-48 hours and preferably for 24 hours. The reaction temperature is 60- 1 1 0°C, preferably 90°C. The reaction solvent is toluene in one preferred embodiment. This is because toluene increases control when dissolving both polymer and monomer; thus, efficiency increases as well. Tetrahydrofuran or anisole or dimethyl sulphoxide or dimelhyl formamide can be used as reaction solvent in other embodiments. Copper, which is the catalyst material in the reaction, is eliminated by passing the graft copolymer (1) solvent synthesized at the step 101 through the neutral aluminium oxide material. After the catalyst elimination, the graft copolymer (1) is precipitated within diethyl ether or hexane until it reaches full purity. One of diethyl ether or hexane is selected according to impurity type of the graft copolymer (1 ). Then, it is dried in vacuum oven preferably overnight. The graft copolymer (1) can be in viscous liquid or white solid form according to the PEG (11 ) ratio and the molecular weight inside thereof. The graft copolymer (1 ) tends to be viscous liquid upon increase of the PEG (1 1) content.

At the 102 step; the graft copolymer (1) is dissolved in a pre-determined concentration within a pre-determined solvent, preferably dimethylformamide (DMF).

At the 103 step; the graft copolymer (1) is added into graphene oxide solution (GO dispersion in water) having 0.1-10 mg/mL, preferably 4 mg/mL concentration. The particle size of at least %95 part of the said GO solution is 800 nm at most. Volumetric ratio of the solvent in the copolymer (1) solution to the water in the graphene oxide solution is less than 1 , preferably 0.5. Thus, it is ensured that the graft copolymer (1 ) prefers the graphene oxide surface not the solution; in other words, it is obliged to be coated to the graphene oxide surface. While doing this, polymers which do not completely dissolve in water are prevented from precipitating during the reaction. The weight ratio of the GO to the graft copolymer (1) is in the range of 2-50, preferably in the range of 5-10 and more preferably in the range of 5 or 10. This ratio is preferably 10 while being coated with the graft copolymers (1) comprising 2 to 3 pyrenes whereas it is 5 with the graft copolymers (1) comprising more pyrenes. At the 104 step; the graft copolymer (1) and the graphene oxide solution is mixed for 2 hours at 20-25°C temperature with a rate such that the reaction mixture will remain homogeneously. These are pre-determined periods and temperatures. At the 105 step; hydrazine monohydrate (N2H4) which is easy to remove, compatible with the solvent and does not require severe reaction conditions is added into the mixture as reducing agent. Preferably, the hydrazine monohydrate has at least 98% purity by mass. The reaction can be carried out at approximately 80°C in the next step by using the said hydrazine monohydrate. Thus, there is no risk of protein degradation when a protein bonded copolymer (1) is used. Preferably, the said agent comprises 64-65%) hydrazine by mass and water in a complementary ratio. The ratio of the GO to the said agent is 0.2-5 w/v (g/mL), preferably 1 w/v (g/mL). In alternative embodiments, at the 105 step; benzyl alcohol or borohydride or aluminium hydride or A1/HC1 is used as reducing agent.

At the 106 step; the reducing reaction of the GO and coating the graft copolymer (1) to graphene oxide are realized for 2-24 hours, preferably for 20 hours and at 80°C. These are pre-determined periods and temperatures. In one preferred embodiment, the colour turns from dark brown into black during this reducing reaction and gas bubbles are observed in the meantime.

At the 107 step; the pre-determined centrifuging period of the reaction mixture is 5-20 minutes, preferably 15 minutes and its rate is 5000-15000 rpm, preferably 12000 rpm.

At the 109 step; the precipitate is dispersed within water in an amount enough to disperse the solid with preferably ultrasonication and centrifuged with 5000- 15000 rpm rate, preferably 1 1000-12000 rpm rate. These are pre-determined rates in the said step.

At the 110 step; the precipitate is dispersed within pure acetone in an amount enough to disperse the solid and centrifuged with 5000-15000 rpm rate, preferably 1 1000-12000 rpm rate. These are pre-determined rates in the said step.

At the 1 1 1 step; the sample is dried at a temperature in the range of 20-25°C as a pre-determined temperature and preferably in a vacuum oven.

With the inventive method (100), since the PEG (1 1) chains are dissolved more homogeneously and such that they are emitted to the surface, toxic effects occurring by agglomeration of graphene in some organs in time are prevented and its circulation time in blood is increased.

The said method (100) eliminates the toxic effect generated because of large surface field of graphene oxide by absorbing cell nutrients inside cell and leading the cell to starvation, due to the fact that it covers the graphene oxide surface by the graft copolymers (1) comprising pyrene.

The reduced graphene obtained by reduction of the graphene oxide during the π-π interaction reduces intracellular stress and toxicity caused by functional groups thereof. Another advantage of using graphene in a reduced form is that characteristics of graphene such as optical, thermal and conductivity are kept in a more superior way by keeping its π conjugate structure. However, the reduced graphene oxide (2) does not dissolve in water medium. Therefore, the method is initiated by the graphene oxide (GO).

The homogeneous reaction medium and the repeated pyrene units in the inventive method (100) increase reaction yield. Due to the fact that the content of the synthesized graft copolymer (1 ) can be known very well, it is also possible to functionalize the graphene oxide surface by changing monomer percentages or including new monomers as requested. For example, in the event that the graft copolymer (1) comprises fluorophore or targeting agent in one embodiment, graphene may have ability to tend to a certain cell or it can create a multi-functional structure for different embodiments upon being synthesized such that it will comprise fluorophore or peptide. In addition, the graft copolymer (1) can comprise azide groups (e.g., 3-azido propyl methacrylate) in another embodiment. Upon binding of azide groups, reactivity level increases to a large extent. The said groups creating different functionalizing can also be bound to the PEG (1 1) end.

In addition, protein can be included to the graft copolymer (1) in the inventive reduced graphene oxide (2) by using widely-known binding methods (such as "click" reactions) or a new treatment can be provided by adding a polymer that can carry gene or the graphene oxide (2) can be used as a dual drug which is both therapeutic and genetic carrier. Imaging can be performed by fluorescent method in the event that the reduced graphene oxide (2) is functionalized by NIR (Near- infrared) dye. Even, anti-cancer drug can be loaded. For example, since the inventive reduced graphene oxide (2) is negatively charged, it can be used for drug delivery to brain.

With the inventive method (100), the graphene oxide surface is coated with well- defined graft copolymer (1 ) such that it density and distribution can be controlled by the π-π interaction and the graphene oxide is reduced at the same time.

Efficiency of the π-π interaction has increased to approximately 65-66% due to the fact that it is initiated by the GO and the said GO is reduced in the method (100). Because commercially easily-accessible materials are used in the said method (100), this method (100) is an easy, practical and efficient method which can be practiced by everyone.

The inventive reduced graphene oxide (2) comprises at least one graft copolymer

(I) comprising at least one group comprising at least one type of monomer and at least one type of macromonomer or having at least two types of monomers and desired functionality.

In one embodiment, the graft copolymer (1) comprised by the rGO (2) comprises at least one PEG (11) chain with a group -which has desired functionality- attached to at least one end thereof , and at least one monomer having at least one pyrene.

In one embodiment, the graft copolymer (1) comprised by the rGO (2) comprises at least one PEG (1 1) chain with one group each -which has desired functionality- attached to both ends thereof, and at least one monomer having at least one pyrene.

In one preferred embodiment, the inventive rGO (2) comprises the graft copolymer (1) comprising at least one macromonomer having at least one PEG

(I I) chain and at least one monomer having at least one pyrene. Preferably, the said macromonomer is a PEG (1 1) chain comprising a methacrylate type of group. More preferably, this macromonomer is a methoxy PEG (1 1) chain comprising methacrylate on one end thereof. In one preferred embodiment, the said copolymer (1) comprises at least one 1 -pyrenemethyl methacrylate (13) (PMA), at least one polyethylene glycol methyl ether methacrylate (PEGMA) and at least one methyl methacrylate (12) (M A). In other words, the macromonomer comprising the PEG (1 1) chain is PEGMA in this embodiment. In one preferred embodiment, the inventive biocompatible reduced graphene oxide (2) has the graft copolymer (1) comprising 2 to 10 PMA (13), 4 to 19 PEGMA and 10 to 37 MMA (12). More preferably, the said copolymer (1) comprises 3 to 7 PMA (13). In the event of working by longer chains (increase of molecular weight), these numbers can be extended.

The graft copolymer (1) comprised by the biocompatible reduced graphene oxide (2) contains 6-90% PEGMA, 15-85% MMA (12) and 7-15% PMA (13) by mole. The said copolymer (1) comprises 40-90% PEG (1 1) by weight. Molecular weight of the graft copolymer (1) is 5000-50000 g/mol. Polymerization degree is 20-100. Preferably, polymerization degree is about 50, thus molecular weight of the graft copolymer (1) is kept under 50000 g/mol value. The inventive reduced graphene oxide (2) is in nano order and its size is 500 nm at maximum and preferably less than 350 nm. Thus, it is suitable for biological applications.

The reduced graphene oxide (2) obtained in the invention can provide functionality necessary in that application is provided for a desired application by attaching a group having desired characteristics to the PEG (1 1) chain -which is bound to a monomer from one end thereof due to its structure- both from this bound end and the other non-bound end thereof. In another embodiment, again depending on application requirements, a desired group can be attached to only one end of the PEG (1 1) (for example, to the end bound or non-bound to the monomer). In this case, functionality necessary in that application is provided for a desired application. Thus, the reduced graphene oxide (2) obtained in the invention can be used as a starting material for many biomedical applications. For example, considering drug delivery systems, a controllable drug delivery profile can be obtained by means of density and distribution of the controllable PEG (11). Faster and slower drug delivery can be provided according to characteristic of the PEG (1 1) selected in the copolymer (1). In addition, it can also be used as implant material. With the inventive method (100) and the reduced graphene oxide (2), entrance of graphene derivatives to living cell can be facilitated.

Thcrmogravimetric Analysis Thermogravimetric analysis was carried out for the purpose of comparing a coating method starting by the rGO (2) with different embodiments of the inventive method (100). Consequently, it is seen that efficiency of coating is significantly high in the inventive method (100). The method (100) is initiated by the GO in the first four examples in the Table 1 whereas it is started by the rGO (2) in the last example. In addition, the related results are shown in the Graphic 1 below.

Table 1 : Results of thermogravimetric analysis.

According to the above-stated TGA analysis, the graft copolymer (1) used in this method (100) exhibits a significant mass loss about 400°C. On the other hand, the reduced graphene oxide (2) (rGO) exhibits only a mass loss of 20% even when it is heated to (rGO) 800°C. Whereas the GO used as starting material maintains the first mass loss occurring via separation of some groups from about 100°C to about 200°C by a sudden loss. Whereas the graphene oxides (2) coated by the graft copolymer (1) and reduced at the same time exhibit mass loss similar to the copolymer (1) loss however at a higher temperature. The coating carried out with a pyrene number of 3.6 and a graft copolymer (1) ratio (w/w) of 5 was realized with 58.87% efficiency whereas the coating carried out with a pyrene number of 6.5 and by using the same ratio was realized with 62.77% efficiency. It is seen that increasing number of pyrene affects the coating positively even a little. In the event that the ratio of graft copolymer(l)/GO (w/w) is taken into account as 10, it is calculated that the copolymer (1) having 6.5 pyrene number is coated by 65.37%. Increasing the graft copolymer (l)/GO ratio (w/w) contributed increase of the efficiency even a little. The graft copolymer (l)/GO ratio (w/w) can be applied as 10 in order to increase the efficiency in coating the graft copolymers (1) comprising less pyrene. The coating carried out by using graft copolymer (1) (graft copolymer (l)/GO (w/w): 5) having reduced graphene oxide (2) (rGO) (the reduction process is carried out previously not at the same time) and 6.5 pyrene number as the starting material, was realized with only 33.77% efficiency. This exhibits efficiency of the developed method (100) once again. The present invention is not limited to the above-mentioned applications and a person skilled in the state of the art can exhibit different embodiments of the invention easily. These can be evaluated within the scope of protection demanded by the claims of the invention. Extreme values in the said range values mentioned in the claims should be regarded as being included in the range. For example, the expressions mentioned in the claims such as "in the range of 1-10" or "1 to 10" includes the values of 1 and 10 as well.

Claims

A reduced graphene oxide (2) characterized in that it comprises at least one graft copolymer (1) which has at least one type of monomer and at least one type of macromonomer.

A reduced graphene oxide (2) according to Claim 1 , having the graft copolymer (1 ) which comprises macromonomer having at least one PEG (1 1) chain and monomer having at least one pyrene.

A reduced graphene oxide (2) according to Claim 1 or 2, comprising the graft copolymer (1) which has macromonomer with a PEG (1 1) chain comprising an acrylate type of group.

A reduced graphene oxide (2) according to Claim 3, comprising the graft copolymer (1) which has macromonomer with a PEG (1 1) chain comprising methacrylate on one end thereof.

A reduced graphene oxide (2) according to any of the preceding claims, comprising the graft copolymer (1) which comprises at least one macromonomer having a PEG (11) chain, at least one PMA (13) and at least one MMA(12).

A reduced graphene oxide (2) according to Claim 5, having the graft copolymer (1) which comprises at least one PMA (13), at least one PEGMA (polyethylene glycol methyl ether methacrylate) and at least one MMA (12).

A reduced graphene oxide (2) according to any of the preceding claims, having the graft copolymer (1) which comprises 6-90% PEGMA by moles.

8. A reduced graphene oxide (2) according to any of the preceding claims, having the graft copolymer (1) which comprises PEGMA with 300-4000 g/mol molecular weight.

9. A reduced graphene oxide (2) according to any of the preceding claims, having the graft copolymer (1) which comprises PEGMA with 500-2000 g/mol molecular weight.

10. A reduced graphene oxide (2) according to any of Claim 5 to 9, having the graft copolymer (1) which comprises 2 to 10 PMA (13), 4 to 19 PEGMA and 10 to 37 MMA (12).

11. A reduced graphene oxide (2) characterized in that it comprises at least one graft copolymer (1) having at least two group with at least one two types of monomer and desired functionality.

12. A reduced graphene oxide (2) according to Claim 1 1, comprising at least one PEG (1 1) (poly(ethylene glycol)) chain with a group -which has desired functionality- attached to at least one end thereof, and at least one monomer having at least one pyrene.

13. A reduced graphene oxide (2) according to Claim 1 1 or 12, having the graft copolymer (1) which comprises at least one PEG (1 1 ) chain with one group each -which has desired functionality- attached to both ends thereof, and at least one monomer having at least one pyrene.

14. A reduced graphene oxide (2) according to any of Claim 1 1 to 13, having the graft copolymer (1) which comprises at least one PEG (1 1 ) chain with a group -which has desired functionality- attached to at least one end thereof, at least one PMA (13) (1-pyrenemethyl methacrylate) and at least one MMA(12) (methyl methacrylate).

15. A reduced graphene oxide (2) according to any Claim 5 to 15 or 14, having the graft copolymer (1) which comprises 15-85% MMA (12), 7- 15% PMA (13) by mole and 40-90% PEG (1 1) by weight.

16. A reduced graphene oxide (2) according to Claim 15, having the graft copolymer (1) which comprises 3 to 7 PMA (13).

17. A reduced graphene oxide (2) according to any of the preceding claims, having the graft copolymer (1) which has a molecular weight of 5000- 50000 g/mol.

18. A reduced graphene oxide (2) according to any of the preceding claims, having the graft copolymer (1) which has a polymerization degree of 20- 100.

19. A reduced graphene oxide (2) according to any of the preceding claims, having the graft copolymer (1) which has a polymerization degree of about 50 in order to keep the molecular weight of the copolymer (1) under 50000 g/mol.

20. A reduced graphene oxide (2) according to any of the preceding claims, which has a size of 500 nm at most.

21. A reduced graphene oxide (2) according to any of the preceding claims, which has a size less than 350 nm such that it will adapt to biological applications.

22. A graphene oxide coating method (100); characterized in that the graphene oxide is coated with a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer.

23. A graphene oxide coating method (100); characterized in that the graphene oxide is coated with a graft copolymer (1) comprising at least two types of monomer and a type of group having the desired functionality.

24. A graphene oxide coating method (100) according to Claim 23, wherein the graphene oxide is coated with a graft copolymer (1) comprising at least one PEG (1 1) chain with a group -which has desired functionality- attached to at least one end thereof, and at least one monomer having at least one pyrene.

25. A graphene oxide coating method (100) according to Claim 22, wherein the graphene oxide is coated with at least one macromonomer having a PEG (1 1) chain and at least one monomer having at least one pyrene.

26. A graphene oxide coating method (100) according to any of Claim 22 to 25, wherein the reducing reaction of the graphene oxide is carried out.

27. A graphene oxide coating method (100) operating according to the steps of:

- dissolving the obtained graft copolymer (1) within a pre-determined solvent (102);

- adding the copolymer (1) solution obtained in a pre-determined ratio into a the graphene oxide (GO) solution having a pre-determined concentration dropwise (103);

- mixing the said copolymer (1) and the graphene oxide solutions at a predetermined temperature for a pre-determined period of time (104); - adding a reducing agent to the mixture at a pre-determined ratio (105);

- drying the obtained sample at a pre-determined temperature (1 1 1).

28. A graphene oxide coating method (100) according to Claim 27, wherein the graft copolymer (1 ) comprising macromonomer having at least one PEG (11) chain and monomer having at least one pyrene is synthesized at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

29. A graphene oxide coating method (100) according to Claim 27, wherein the graft copolymer (1) is synthesized at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)" such that it will comprise at least one PEG (1 1) chain with a group -which has desired functionality- attached to at least one end thereof, and at least one monomer having at least one pyrene.

30. A graphene oxide coating method (100) according to Claim 27 or 29, wherein the graft copolymer (1) comprising at least one PEG (1 1) chain with one group each -which has desired functionality- attached to both ends thereof, and at least one monomer having at least one pyrene is synthesized at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

31. A graphene oxide coating method (100) according to Claim 27 or 28, wherein the graft copolymer (1) having macromonomer which is a PEG (1 1) chain comprising a methacrylate type of group is synthesized at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

32. A graphene oxide coating method (100) according to Claim 31 , wherein the graft copolymer (1) having macromonomer which is a PEG (1 1) chain comprising methyl ether methacrylate on one end thereof is synthesized at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

33. A graphene oxide coating method (100) according to Claim 27 or 29 or 30, wherein the graft copolymer (1 ) comprising at least one PEG (1 1 ) chain with a group -which has desired functionality- attached to at least one end thereof, at least one PMA (13) (1 -pyrenemethyl methacrylate) and at least one MMA (12) (methyl methacrylate) is synthesized at the step of

"synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

34. A graphene oxide coating method (100) according to any of Claim 27 or

28 or 31 or 32, wherein the graft copolymer (1) comprising at least one macromonomer having at least one PEG (1 1) chain, at least one PMA (13) and at least one MMA (12) is synthesized at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

35. A graphene oxide coating method (100) according to Claim 34, wherein the graft copolymer (1) comprising at least one 1-pyrenemethyl methacrylate (13), at least one polyethylene glycol methyl ether methacrylate and at least one methyl methacrylate (12) is synthesized at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

36. A graphene oxide coating method (100) according to any of Claim 27 to 35, wherein the graft copolymer (1) comprising PEGMA with molecular weight of 300-4000 g/mol is synthesized at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

37. A graphene oxide coating method (100) according to any of Claim 27 to 36, wherein the graft copolymer (1) comprising PEGMA with molecular weight of 500-2000 g/mol is synthesized at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

38. A graphene oxide coating method (100) according to any of Claim 27 to

36, wherein the graft copolymer (1) is synthesized by one of controlled polymerization methods such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT), AGET- ATRP (Activator generated by electron transfer - atom transfer radical polymerization) at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

39. A graphene oxide coating method (100) according to Claim 38, wherein copper (I) bromide (CuBr) is used as catalyst during synthesis of the graft copolymer (1) by ATRP method at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

40. A graphene oxide coating method (100) according to Claim 38 or 39, wherein N, N, N', N", N"-pentamethyl diethylentriamine (PMDETA) is used as ligand at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

41. A graphene oxide coating method (100) according to any of Claim 38 to 40, wherein ethyl 2-bromo-2-methylpropionate material is used as an initiator at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

42. A graphene oxide coating method (100) according to Claim 39 and 40 and

41, wherein the ratio of [total mole number of all monomers]/[mole number of PMDETA]/[mole number of CuBr]/[mole number of EiBr] is 50/1/1/1 or 50/1.1/1.1/1 or 100/1/1 /1 at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

43. A graphene oxide coating method (100) according to any of Claim 27 to

42, wherein the reaction starts after at least 3 freeze-pump-thaw (FPT) cycles in order to remove the air oxygen dissolved from the reaction mixture and thus to perform the reaction in a more controlled way at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

44. A graphene oxide coating method (100) according to any of Claim 27 to

43, wherein the polymerization reaction is performed for 6-48 hours at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

45. A graphene oxide coating method (100) according to any of Claim 27 to 44, wherein the polymerization reaction is performed for 24 hours at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

46. A graphene oxide coating method (100) according to any of Claim 27 to

45, wherein the reaction temperature is 60-1 10°C at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

47. A graphene oxide coating method (100) according to any of Claim 27 to

46, wherein the reaction temperature is 90°C at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

48. A graphene oxide coating method (100) according to any of Claim 27 to

47, wherein the reaction solvent used is toluene or tetrahydrofuran or anisole or dimethyl sulphoxide or dimethyl formamide at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

49. A graphene oxide coating method (100) according to any of Claim 39 and

27 to 48, wherein the copper -which is the catalyst material in the reaction mixture- is eliminated by passing the copolymer (1) solution through the neutral aluminium oxide material, the graft copolymer (1) is precipitated within diethyl ether or hexane until it reaches full purity and then dried overnight at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

50. A graphene oxide coating method (100) according to any of Claim 27 to 49, wherein the graft copolymer (1) in viscous liquid or white solid form is synthesized at the step of "synthesizing a graft copolymer (1) comprising at least one type of monomer and at least one type of macromonomer or comprising at least two types of monomers and at least one type of group with a desired functionality (101)".

51. A graphene oxide coating method (100) according to any of Claim 27 to

50, wherein the copolymer (1) is dissolved within dimethylformamide at the step of "dissolving the obtained graft copolymer (1) within a predetermined solvent (102)".

52. A graphene oxide coating method (100) according to any of Claim 27 to

51 , wherein the dissolved copolymer (1) is added into water-graphene oxide solution having 0.1-10 mg/mL concentration at the step of "adding the copolymer (1) solution obtained in a pre-determined ratio into a the graphene oxide (GO) solution having a pre-determined concentration dropwise (103)".

53. A graphene oxide coating method (100) according to any of Claim 27 to

52, wherein the dissolved copolymer (1) is added into water-graphene oxide (2) solution having 4 mg/mL concentration at the step of "adding the copolymer (1) solution obtained in a pre-determined ratio into a the graphene oxide (GO) solution having a pre-determined concentration drop wise (103)".

54. A graphene oxide coating method (100) according to any of Claim 27 to

53, wherein the particle size of the GO solution is 800 nm at most at the step of "adding the copolymer (1) solution obtained in a pre-determined ratio into a the graphene oxide (GO) solution having a pre-determined concentration drop wise (103)".

55. A graphene oxide coating method (100) according to any of Claim 27 to

54, wherein at least 95% of the GO solution has particle size of 800 nm at most at the step of "adding the copolymer (1) solution obtained in a predetermined ratio into a the graphene oxide (GO) solution having a predetermined concentration dropwise (103)".

56. A graphene oxide coating method (100) according to any of Claim 27 to

55, wherein the volumetric ratio of the solvent in the copolymer (1 ) solution to the water in the graphene oxide solution is less than 1 at the step of "adding the copolymer (1) solution obtained in a pre-determined ratio into a the graphene oxide (GO) solution having a pre-determined concentration dropwise (103)".

57. A graphene oxide coating method (100) according to Claim 56, wherein the volumetric ratio of the solvent in the copolymer (1) solution to the water in the graphene oxide solution is 0.5 in order to ensure that the graft copolymer (1) prefers the graphene oxide surface at the step of "adding the copolymer (1) solution obtained in a pre-determined ratio into a the graphene oxide (GO) solution having a pre-determined concentration dropwise (103)".

58. A graphene oxide coating method (100) according to any of Claim 27 to 57, wherein the weight ratio of the GO to the graft copolymer (1) is between 2-50 at the step of "adding the copolymer (1) solution obtained in a pre-determined ratio into a the graphene oxide (GO) solution having a pre-determined concentration dropwise (103)".

59. A graphene oxide coating method (100) according to any of Claim 27 to

58, wherein the weight ratio of the GO to the graft copolymer (1 ) is between 5 to 10 at the step of "adding the copolymer (1) solution obtained in a pre-determined ratio into a the graphene oxide (GO) solution having a pre-determined concentration dropwise (103)".

60. A graphene oxide coating method (100) according to any of Claim 27 to

59, wherein the weight ratio of the GO to the graft copolymer (1) is 5 or 10 at the step of "adding the copolymer (1 ) solution obtained in a pre- determined ratio into a the graphene oxide (GO) solution having a predetermined concentration dropwise (103)".

61. A graphene oxide coating method (100) according to Claim 60, wherein the weight ratio of the GO to the graft copolymer (1) comprising 2 to 3 pyrenes is 10 whereas its ratio to the copolymers (1 ) comprising more pyrenes is 5 at the step of "adding the copolymer (1) solution obtained in a pre-determined ratio into a the graphene oxide (GO) solution having a predetermined concentration dropwise (103)".

62. A graphene oxide coating method (100) according to any of Claim 27 to

61 , wherein the copolymer (1) and the graphene oxide solutions are mixed for 2 hours at the step of "mixing the said copolymer (1) and the graphene oxide solutions at a pre-determined temperature for a pre-determined period of time (104)".

63. A graphene oxide coating method (100) according to any of Claim 27 to

62, wherein the copolymer (1) and the graphene oxide solutions are mixed at a temperature in the range of 20-25°C at the step of "mixing the said copolymer (1) and the graphene oxide solutions at a pre-determined temperature for a pre-determined period of time ( 104)".

64. A graphene oxide coating method (100) according to any of Claim 27 to

63, wherein the copolymer (1) and the graphene oxide solution is mixed at a rate such that it will remain homogeneously at the step of "mixing the said copolymer (1) and the graphene oxide solutions at a pre-determined temperature for a pre-determined period of time (104)".

65. A graphene oxide coating method (100) according to any of Claim 27 to

64, wherein hydrazine monohydrate which is easy to remove, compatible with the solvent and does not require severe reaction conditions is added into the mixture as reducing agent at the step of "adding a reducing agent to the mixture at a pre-determined ratio (105)".

66. A graphene oxide coating method (100) according to Claim 65, wherein hydrazine monohydrate with at least 98% purity by mass is added at the step of "adding a reducing agent to the mixture at a pre-determined ratio (105)".

67. A graphene oxide coating method (100) according to any of Claim 27 to

66, wherein the said agent comprises 64-65% hydrazine monohydrate by mass and water in a complementary ratio at the step of "adding a reducing agent to the mixture at a pre-determined ratio (105)".

68. A graphene oxide coating method (100) according to any of Claim 27 to

67, wherein the ratio of the GO to the said agent is 0.2-5 w/v at the step of "adding a reducing agent to the mixture at a pre-determined ratio (105)".

69. A graphene oxide coating method (100) according to Claim 68, wherein the ratio of the GO (2) to the said agent is 1 w/v at the step of "adding a reducing agent to the mixture at a pre-determined ratio (105)".

70. A graphene oxide coating method (100) according to any of Claim 27 to 69 except for Claim 65 to 67, wherein benzyl alcohol or borohydride or aluminium hydride or A1/HC1 is added as reducing agent at the step of "adding a reducing agent to the mixture at a pre-determined ratio (105)".

71. A graphene oxide coating method (100) according to any of Claim 27 to 70, wherein the reducing reaction of the GO and coating the graft copolymer (1) to graphene oxide are realized for 2-24 hours at the step of "carrying out both π-π interaction and reduction reaction at a predetermined temperature for a pre-determined period of time (106)".

72. A graphene oxide coating method (100) according to any of Claim 27 to

71, wherein the reducing reaction of the GO and coating the graft copolymer (1) to graphene oxide are realized for 20 hours at the step of "carrying out both π-π interaction and reduction reaction at a predetermined temperature for a pre-determined period of time (106)".

73. A graphene oxide coating method (100) according to any of Claim 27 to

72, wherein the reducing reaction of the GO and coating the graft copolymer (1) to graphene oxide are realized at 80°C at the step of "carrying out both π-π interaction and reduction reaction at a pre- determined temperature for a pre-determined period of time (106)".

74. A graphene oxide coating method (100) according to any of Claim 27 to

73, wherein the reaction mixture is centrifuged for 5-20 minutes at the step of "centrifuging the reaction mixture at a pre-determined rate for a pre- determined period of time (107)".

75. A graphene oxide coating method (100) according to any of Claim 27 to

74, wherein the reaction mixture is centrifuged for 15 minutes at the step of "centrifuging the reaction mixture at a pre-determined rate for a pre- determined period of time (107)".

76. A graphene oxide coating method (100) according to any of Claim 27 to 75, wherein the reaction mixture is centrifuged at 5000-15000 rpm rate at the step of "centrifuging the reaction mixture at a pre-determined rate for a pre-determined period of time (107)".

77. A graphene oxide coating method (100) according to Claim 76, wherein the reaction mixture is centrifuged at 12000 rpm rate at the step of "centrifuging the reaction mixture at a pre-determined rate for a predetermined period of time (107)".

78. A graphene oxide coating method (100) according to any of Claim 27 to

77, wherein the precipitate is dispersed within water with ultrasonication at the step of "dispersing the precipitate in the lower phase within the water again and centrifuging it at a pre-determined rate (109)".

79. A graphene oxide coating method (100) according to any of Claim 27 to

78, wherein the precipitate is dispersed within water in an amount enough to disperse the solid at the step of "dispersing the precipitate in the lower phase within the water again and centrifuging it at a pre-determined rate (109)".

80. A graphene oxide coating method (100) according to any of Claim 27 to

79, wherein the precipitate is centrifuged with 5000-15000 rpm rate at the step of "dispersing the precipitate in the lower phase within the water again and centrifuging it at a pre-determined rate (109)".

81. A graphene oxide coating method (100) according to Claim 80, wherein the precipitate is centrifuged with 1 1000-12000 rpm rate at the step of "dispersing the precipitate in the lower phase within the water again and centrifuging it at a pre-determined rate ( 109)".

82. A graphene oxide coating method (100) according to any of Claim 27 to 81, wherein the precipitate is centrifuged with 5000-15000 rpm rate at the step of "dispersing the precipitate within acetone and centrifuging it at a pre-determined rate (1 10)".

83. A graphene oxide coating method (100) according to Claim 82, wherein the precipitate is centrifuged with 1 1000-12000 rpm rate at the step of "dispersing the precipitate within acetone and centrifuging it at a predetermined rate (1 10)".

84. A graphene oxide coating method (100) according to any of Claim 27 to

83, wherein the precipitate is dispersed within pure acetone in an amount enough to disperse the solid at the step of "dispersing the precipitate within acetone and centrifuging it at a pre-determined rate (1 10)".

85. A graphene oxide coating method (100) according to any of Claim 27 to

84, wherein the sample is dried at a temperature in the range of 20-25°C at the step of "drying the obtained sample at a pre-determined temperature (11 1)".

86. A graphene oxide coating method (100) according to any of Claim 27 to

85, wherein the sample is dried in a vacuum oven at the step of "drying the obtained sample at a pre-determined temperature (11 1)".

87. A reduced graphene oxide (2) obtained from a method (100) according to any of Claim 27 to 86.

88. A reduced graphene oxide (2) according to any of Claim 1 to 21 or 87 having the graft copolymer (1) which comprises fluorophore and/or targeting agent and/or peptide and/or azide groups.

89. A reduced graphene oxide (2) according to any of Claim 1 to 21 or 87 or 88; which is used for gene delivery or drug delivery and release or as an implant material.

90. A reduced graphene oxide (2) according to any of Claim 1 to 21 or 87 to

89; which is used as a dual drug that is both therapeutic and genetic carrier, or by loading an anti-cancer drug, or for drug delivery to brain.