Graphene modified with zwitterionic polymer

Abstract

The present invention relates to graphene modified with a zwitterionic polymer, and specifically, in which a zwitterionic polymer, which is a dipolar molecule, is bonded to graphene oxide by a click chemical reaction, and for which the characteristics of exhibiting improved dispersibility and aggregation compared to conventional graphene and having an antioxidant effect have been confirmed, and thus the present invention may be widely used in industrial fields such as semiconductor, battery, materials, bio, and the like.

Description

Graphene modified with zwitterionic polymers

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2024-0092543, filed July 12, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to graphene modified with a zwitter ionic polymer.

[0003]

[0004] Graphene is an allotrope of carbon, a two-dimensional plane of carbon atoms. Oxidized graphene is a material obtained by oxidizing graphene. Graphene is thin and flexible, and possesses excellent mechanical, thermal, electrical, and optical properties, making it a promising candidate for future electronic materials. Due to these properties, graphene holds greater value and promise in practical applications than other carbon nanomaterials, such as fullerenes and carbon nanotubes. However, graphene exhibits poor dispersibility within a matrix due to its insolubility, electrical repulsion between particles, and π-π stacking between graphene layers, leading to irreversible agglomeration and sedimentation.

[0005] Various studies have been conducted to improve graphene aggregate formation, including mechanical exfoliation, surface functionalization, and solvent modification. However, mechanical exfoliation presents limitations, including the difficulty of large-scale production, the high cost of exfoliation equipment, and the risk of defects in graphene during the exfoliation process. Surface functionalization, on the other hand, presents limitations, such as the complexity of chemical reactions and the potential for impurities. Furthermore, changing the specific solvent can affect the electrical and mechanical properties of graphene.

[0006] Poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) is a dipolar polymer that has a stable structure in aqueous solution and can form a denser form than polyethylene glycol (PEG). According to patent document 1 (Korean Patent Publication No. 10-2012-0072404), a core / shell nanocomposite having a PMPC coating layer formed thereon is disclosed to have excellent long-term blood circulation, dispersion stability, and contrast effect, and thus can be used as an MRI contrast agent. According to patent document 2, a hydrogel surface-modified with PMPC is disclosed to have improved antifouling properties by inhibiting the adsorption of proteins and bacteria, and thus a hydrogel lens with excellent antifouling properties can be manufactured.

[0007] Against this backdrop, in the present invention, PMPC was attached to graphene oxide to increase the in vivo usability of graphene, and it was confirmed that the dispersibility and cohesion of graphene oxide were improved in a high salt concentration or low pH environment, thereby developing graphene that can overcome limitations in biological experiments.

[0008]

[0009] [Prior Art Literature]

[0010] [Patent Document]

[0011] (Patent Document 1) Republic of Korea Publication No. 10-2012-0072404 (July 4, 2012)

[0012] (Patent Document 2) Republic of Korea Publication No. 10-2021-0079434 (June 30, 2021)

[0013]

[0014] [Non-patent literature]

[0015] (Non-patent Document 1) Perumal, S.; Atchudan, R.; Cheong, I.W. Recent Studies on Dispersion of Graphene-Polymer Composites. Polymers 2021, 13, 2375.

[0016] (Non-patent Document 2) Jiang, X.; Hong, X.; Jiang, Y.; Shao, Z.; Zhu, D. Recent Developments Concerning the Dispersion Methods and Mechanisms of Graphene. Coatings 2018, 8, 33.

[0017]

[0018] The purpose of the present invention is to provide a graphene variant and a method for producing the same that can solve the problem of graphene agglomeration and low dispersibility in a buffer solution or culture medium.

[0019]

[0020] The present invention provides graphene modified with a zwitter ionic polymer, particularly PMPC (Poly(2-methacryloyloxyethyl phosphorylcholine)).

[0021] The present invention also provides a method for producing graphene modified with a zwitter ionic polymer, comprising the steps of: (a) mixing a RAFT reagent containing a first click chemistry functional group and a zwitter ionic polymer to produce a zwitter ionic polymer having a first click chemistry functional group bonded thereto; (b) producing graphene having a second click chemistry functional group bonded thereto; and (c) combining the zwitter ionic polymer having the first click chemistry functional group bonded thereto and the graphene having the second click chemistry functional group bonded thereto by a click chemistry reaction.

[0022]

[0023] Hereinafter, the present invention will be described in detail.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0025] When the present invention is said to “include” a certain component or a certain step, this does not mean that other components or other steps are excluded, but rather that other components or other steps may be further included, unless specifically stated otherwise.

[0026] In the present invention, the term "graphene" is one of the allotropes of carbon, in which carbon atoms are present at the vertices of a hexagon (sp 2 (Combination) It refers to a material that forms a two-dimensional planar crystal structure in the shape of a widely spread hexagonal honeycomb. Graphene is a film made of one atom thick and can exist as a stable structure.

[0027] In the present invention, the term "graphene oxide (GO)" refers to a material in which various oxidized functional groups (-OH, -COOH, -C=O, -CHO, etc.) are attached to the edges of graphene and form several to several tens of layers.

[0028] The above graphene oxide may be obtained by a method known to those skilled in the art. The above graphene oxide may be produced from graphite by physical exfoliation, chemical vapor deposition, chemical exfoliation, or epitaxial growth, but is not limited thereto.

[0029] In the present invention, the graphene oxide variant may mean a complex in which an additional substance / material is combined with the graphene oxide to improve the dispersibility and cohesion of the graphene oxide, and specifically, it may be a complex in which an additional substance / material such as a functional group, a metal particle, a magnetic particle, a nanoparticle, a polymer, a peptide, and / or a nucleic acid is combined / attached to the nano-sized graphene oxide through a physical van der Waals force bond, an ionic bond, a hydrogen bond, a covalent bond, and / or a non-covalent bond.

[0030] The above graphene oxide may have the form of a nanofilm, nanosheet, nanowire, nanorod, nanotube, pulverized nanowire, nanotetrapod, tripod, bipod, nanocrystal, nanodot, quantum wire, or nanoparticle.

[0031] The above graphene oxide may include graphene quantum dots (GQDs). In addition, it may include graphene particles having a width, length, and height of several nm manufactured through appropriate processing, but is not limited thereto.

[0032] The structural and chemical properties of the above graphene can be verified by X-ray diffraction, X-ray photoelectron spectroscopy, Raman analysis, and Fourier transform infrared spectroscopy. X-ray diffraction is a technique that can confirm the structural information of a material, such as chemical composition, crystal structure, crystal size, strain, preferred orientation, and layer thickness, in a laboratory, and X-ray photoelectron spectroscopy is a sensitive quantitative spectroscopy technique based on the photoelectric effect that can identify not only elements existing within a material or covering the surface, but also the overall electronic structure and density within a material. In addition, Raman analysis is a technique that can distinguish the number of layers of graphene or confirm the degree of structural defects, and Fourier transform infrared spectroscopy is a technique used to obtain an infrared spectrum absorbed or emitted from a solid, liquid, or gas.

[0033]

[0034] Graphene has the disadvantage of being difficult to disperse in solvents and tending to aggregate due to its large surface area, van der Waals forces and ð-ð interactions, electrical and mechanical properties impaired by functional groups present on the surface, and hydrophobic characteristics. Consequently, when applied in the body, there has been a problem that the unique characteristics of existing graphene are lost due to aggregation and toxicity may occur. In the present invention, to solve the above problems, PMPC, which is expected to maximize the hydrophilic effect compared to PEGylated graphene using a dipolar polymer, was combined with graphene, thereby confirming the effect of improving the dispersibility and aggregation of graphene.

[0035]

[0036] Accordingly, the present invention relates to graphene modified with a zwitter ionic polymer.

[0037] The zwitterionic polymer may be at least one selected from the group consisting of PMPC (Poly(2-methacryloyloxyethyl phosphorylcholine)), PVA (Polyvinyl alcohol), PAMPS (Poly(acrylamidomethylpropane sulfonic acid)), PDMAEMA (Poly(N,N-dimethylaminoethyl methacrylate)), PEGMA (Poly(ethylene glycol) methyl ether methacrylate), PHEMA (Poly(2-hydroxyethyl methacrylate)), and PEI (Poly(ethylene imine)). Preferably, the zwitterionic polymer may be PMPC (Poly(2-methacryloyloxyethyl phosphorylcholine)).

[0038] The above graphene may be any one selected from the group consisting of graphene oxide, graphene oxide variant, and graphene quantum dots, and may preferably be graphene oxide or graphene oxide variant, but is not limited thereto.

[0039] The above graphene oxide may be nano graphene oxide.

[0040] The above nano-graphene oxide may have an average of 1 to 3 layers and a diameter of 10 to 20 mm, but is not limited thereto.

[0041] The above graphene may have improved dispersibility and cohesion.

[0042] The above dispersibility may refer to the ability to uniformly distribute graphene particles within a solvent or matrix.

[0043] The above cohesion may refer to a phenomenon in which graphene particles clump together to form lumps.

[0044] The above graphene may have an antioxidant effect.

[0045] The graphene may be combined with a zwitterionic polymer by one or more methods selected from the group consisting of click chemistry, reverse atom transfer radical polymerization (RATRP), activators generated by electron transfer for atom transfer radical polymerization (AGENT ATRP), and in situ polymerization, but is not limited thereto.

[0046] The click chemistry reaction described above is a chemical reaction that allows for rapid and efficient compound synthesis. It is characterized by high yields, simple reaction conditions, and the absence of a catalyst. This method is useful for synthesizing functional groups on graphene, effectively introducing various functional groups onto the graphene surface. Click chemistry is particularly widely used for binding to biomolecules, developing drug delivery systems, and synthesizing functional nanomaterials. This reaction offers high selectivity and reproducibility, significantly expanding the range of graphene applications.

[0047] The above-mentioned RATRP is a type of radical polymerization that utilizes a catalyst and initiator to control the growth and termination of molecules. To enhance the controllability of radical polymerization, a metal catalyst is used to carry out the reaction. During the polymerization process, reaction conditions can vary, playing a crucial role in controlling the physical and functional properties of the final polymer. Because RATRP allows for precise control over the composition and structure of molecules, it is useful for the synthesis of polymer materials and biocompatible materials.

[0048] The above-mentioned AGENT ATRP is a method for conducting a radical polymerization reaction using an activator generated through electron transfer. It uses an electron transfer agent to initiate and control the polymerization reaction, allowing for precise control of the molecular weight and molecular weight distribution of the polymer. AGENT ATRP is useful for the synthesis of environmentally friendly polymers and the development of biocompatible materials.

[0049] The above in situ polymerization refers to a method of polymerizing functional groups in situ on the surface of graphene in the presence of graphene. Ultrasound or plasma may be used during the polymerization process, which promotes reaction efficiency and uniform distribution of nanoparticles.

[0050] The above graphene may have a diameter of 0.1 to 100 nm and a thickness of 0.1 to 100 nm, preferably a diameter of 1 to 50 nm and a thickness of 0.5 to 50 nm, but is not limited thereto.

[0051] In the present invention, the diameter refers to the average diameter in the lateral size of graphene having a certain thickness, and the lateral size can be interpreted as the relatively longer length among the horizontal and vertical lengths when the length of graphene having a certain thickness is measured based on a rectangular frame, or the longest distance among the distances connecting any two ends of graphene having a certain thickness.

[0052] The above graphene may be a graphene to zwitterionic polymer combined in a ratio of 1.0:0.01 to 1.0:100.0, preferably a graphene to zwitterionic polymer combined in a ratio of 1.0:0.01 to 1.0:20.0, and most preferably a graphene to zwitterionic polymer combined in a ratio of 1.0:0.1 to 1.0:15.0, but is not limited thereto.

[0053] When the ratio of the above-mentioned zwitterionic polymer is 1.0:100.0 or more, there may be a limit at which the effect of the graphene according to the present invention is limited, and when it is 1.0:0.01 or less, there may be a limit at which the hydrophilic effect disappears.

[0054]

[0055] The present invention also relates to a method for producing graphene modified with a zwitter ionic polymer, comprising the steps of: (a) mixing a RAFT reagent containing a first click chemistry functional group and a zwitter ionic polymer to produce a zwitter ionic polymer having a first click chemistry functional group bonded thereto; (b) producing graphene having a second click chemistry functional group bonded thereto; and (c) combining the zwitter ionic polymer having the first click chemistry functional group bonded thereto and the graphene having the second click chemistry functional group bonded thereto by a click chemistry reaction.

[0056] The zwitterionic polymer may be at least one selected from the group consisting of PMPC (Poly(2-methacryloyloxyethyl phosphorylcholine)), PVA (Polyvinyl alcohol), PAMPS (Poly(acrylamidomethylpropane sulfonic acid)), PDMAEMA (Poly(N,N-dimethylaminoethyl methacrylate)), PEGMA (Poly(ethylene glycol) methyl ether methacrylate), PHEMA (Poly(2-hydroxyethyl methacrylate)), and PEI (Poly(ethylene imine)). Preferably, the zwitterionic polymer may be PMPC (Poly(2-methacryloyloxyethyl phosphorylcholine)).

[0057] The combination of the first click chemical functional group and the second click chemical functional group may be at least one selected from the group consisting of an azide group-alkyne group, a thiol group-alkyne group, an amine group-epoxy group, a thiol group-epoxy group, an amine group-acroyl group, a thiol group-acroyl group, and a cyclooctene-tetrazine, but is not limited thereto.

[0058] When the above combination is an alkyne-azide group, it may be a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or a strain-promoted alkyne-azide cycloaddition (SPAAC) that does not utilize a copper catalyst.

[0059] The click chemistry reaction described above is a chemical reaction that allows for rapid and efficient compound synthesis. It is characterized by high yields, simple reaction conditions, and the absence of a catalyst. This method is useful for synthesizing functional groups on graphene, effectively introducing various functional groups onto the graphene surface. Click chemistry is particularly widely used for binding to biomolecules, developing drug delivery systems, and synthesizing functional nanomaterials. This reaction offers high selectivity and reproducibility, significantly expanding the range of graphene applications.

[0060] The above graphene may be any one selected from the group consisting of graphene oxide, graphene oxide variant, and graphene quantum dots, and may preferably be graphene oxide or graphene oxide variant, but is not limited thereto.

[0061] The above graphene oxide may be nano graphene oxide.

[0062] The above nano-graphene oxide may have an average of 1 to 3 layers and a diameter of 10 to 20 mm, but is not limited thereto.

[0063] The above graphene may have improved dispersibility and cohesion.

[0064] The above dispersibility may refer to the ability to uniformly distribute graphene particles within a solvent or matrix.

[0065] The above cohesion may refer to a phenomenon in which graphene particles clump together to form lumps.

[0066] The above graphene may have an antioxidant effect.

[0067] The above graphene may have a diameter of 0.1 to 100 nm and a thickness of 0.1 to 100 nm, preferably a diameter of 1 to 50 nm and a thickness of 0.5 to 50 nm, but is not limited thereto.

[0068] In the present invention, the diameter refers to the average diameter in the lateral size of graphene having a certain thickness, and the lateral size can be interpreted as the relatively longer length among the horizontal and vertical lengths when the length of graphene having a certain thickness is measured based on a rectangular frame, or the longest distance among the distances connecting any two ends of graphene having a certain thickness.

[0069] The above graphene may be a graphene to zwitterionic polymer combined in a ratio of 1.0:0.01 to 1.0:100.0, preferably a graphene to zwitterionic polymer combined in a ratio of 1.0:0.01 to 1.0:20.0, and most preferably a graphene to zwitterionic polymer combined in a ratio of 1.0:0.1 to 1.0:15.0, but is not limited thereto.

[0070] When the ratio of the above-mentioned zwitterionic polymer is 1.0:100.0 or more, there may be a limit at which the effect of the graphene according to the present invention is limited, and when it is 1.0:0.01 or less, there may be a limit at which the hydrophilic effect disappears.

[0071]

[0072] Graphene modified with a zwitter ionic polymer (PMPC) according to the present invention can be utilized as graphene with high potential for in vivo applications by exhibiting effects of reduced aggregation and improved dispersibility even in environments where aggregates are formed and dispersibility of existing graphene is low.

[0073]

[0074] Figure 1 illustrates the synthesis process of a RAFT reagent having an azide group.

[0075] Figure 2 shows the PMPC synthesis process using the above RAFT reagent.

[0076] Figure 3 illustrates the propargyl NGO synthesis process.

[0077] Figure 4 shows the results of confirming the amide bond of propargyl NGO generated after the EDC coupling reaction between NGO and propargyl amine using FT-IR.

[0078] Figure 5 shows the synthesis process of NGO-PMPC combined by click chemistry reaction.

[0079] Figure 6 shows the results of confirming the covalent bond of NGO-PMPC using FT-IR.

[0080] Figure 7 shows an image of the shape of PMPC-ized graphene confirmed by transmission electron microscopy (TEM).

[0081] Figure 8 shows an enlarged image of the above image.

[0082] Figure 9(a) shows the absorbance analysis results of NGO according to wavelength, Figure 9(b) shows the absorbance analysis results of NGO according to concentration, Figure 9(c) shows the absorbance analysis results of PMPC-N3 according to wavelength, and Figure 9(d) shows the absorbance analysis results of NGO-PMPC according to wavelength and concentration.

[0083] Figure 10(a) shows the results of the analysis of aggregate formation according to the salt concentration of NGO and NGO-PMPC, and Figure 10(b) shows the Z-average according to the salt concentration of NGO and NGO-PMPC.

[0084] Figure 11(a) shows the results of the analysis of aggregate formation according to pH of NGO and NGO-PMPC, and Figure 11(b) shows the Z average according to pH of NGO and NGO-PMPC.

[0085] Figure 12(a) shows the results of the aggregate formation analysis in DMEM and triple-distilled water of NGO and NGO-PMPC, and Figure 12(b) shows the Z-average in DMEM and triple-distilled water of NGO and NGO-PMPC.

[0086] Figure 13 shows the results of sediment formation after dispersing NGO and NGO-PMPC in DMEM.

[0087] Figure 14 shows the results of an antioxidant evaluation of PMPC-modified graphene according to the present invention. M24001 represents PMPC-modified graphene manufactured at a ratio of 1:3.8 (NGO to PMPC), and M24002 to M24004 represent PMPC-modified graphene manufactured at a ratio of 1:0.6 (NGO to PMPC).

[0088] Figure 15 shows the antioxidant evaluation results of the above M24001 and M24002.

[0089]

[0090] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0091]

[0092] [Example 1]

[0093] Fabrication of nano-graphene oxide

[0094] <1-1> Production of graphene oxide

[0095] Graphite (Qingdao Kropfmuehl, China) was used as a carbon structure, and nano-graphene oxide was synthesized through the Couette-Tayler flow method, which generates a vortex in a container containing a graphite mixture to allow sulfuric acid to penetrate the graphite layers and promote a chemical reaction. Sulfuric acid (H2SO4), sodium nitrate (NaNO3), and potassium permanganate (KMnO4) below were all purchased from Samchun Pure Chemicals. Graphite (graphite) and sulfuric acid (H2SO4) were mixed in a ratio of 1:40 to 1:70, and sodium nitrate (NaNO3), which lowers the viscosity of the mixed solution and facilitates stirring, was mixed in a ratio of 1:0.2 to 1:0.7 to graphite (graphite). At this time, sulfuric acid (H2SO4) is inserted between the interlayer structures of the graphite, expanding the interlayer spacing.

[0096] The above mixture is mixed at 150 rpm to 500 rpm for 1 to 3 hours. Thereafter, the mixture and potassium permanganate (KMnO4) are mixed at a ratio of 1:5 to 1:15, and a rotational force of 1,200 rpm to 5,000 rpm is applied for 1 to 72 hours to promote interlayer expansion and separation of graphite.

[0097] At this time, appropriately managing the reaction time and rotational force through oxidation reaction control is crucial for controlling the interlayer spacing and producing single-layer graphene oxide. The graphene oxide produced through the above process is nanosized by applying ultrasonic waves of at least 10 W to 900 W. Nano-ization conditions vary depending on the size of the graphene oxide being nanosized.

[0098]

[0099] <1-2> Nano-fication of graphene oxide

[0100] To enhance the frictional strength of the obtained graphene oxide, 50 to 100 times the amount of deionized water (DI water) or PBS solvent is added relative to the solid content of graphene oxide. A certain amount of graphene oxide mixed in the solvent is fed into a nano-processing device that uses a physical method to physically reduce the particle size of the graphene oxide. In addition, the ratio of each material applied when producing graphene oxide can act as a ratio that promotes the nano-processing of graphene oxide. In this experiment, nano-processed graphene oxide can be separated through a physical method in the nano-processing device. In addition, it can be separated by size through a centrifugal process. At this time, the centrifuge can be used by applying a rotation speed of 8,000 to 20,000 rpm depending on the size.

[0101]

[0102] [Example 2]

[0103] RAFT polymer synthesis and polymer synthesis

[0104] Reversible addition-fragmentation chain-transfer polymerization (RAFT) is a method of controlling radical polymerization using an addition-fragmentation chain transfer agent, and has the advantage of being able to obtain polymers of uniform size due to a narrow distribution of polymer molecular weight.

[0105] In the present invention, a RAFT reagent having an azide group was synthesized to prepare PMPC having an azide group (-N3) at the end that can be combined with NGO by referring to previous studies.

[0106] 3-Azidopropanol, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, 4-dimethylaminopyridine (DMAP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC hydrochloride), triethylamine (TEA), and dichloromethane (DCM) were used.

[0107] Under anhydrous conditions and a nitrogen atmosphere, slowly add anhydrous DCM containing 3-azidopropanol, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, and EDC hydrochloride dissolved in DMAP. Anhydrous TEA is also added to the solution. Stir at room temperature for 6 hours, and the solvent is removed. The residue is purified by column chromatography using a 1:1 ratio of hexane to ethyl acetate.

[0108] PMPC was synthesized by radical polymerization using the synthesized RAFT polymer. The reagents used were 2-Methacryloyloxyethyl phosphorylcholine (MPC), RAFT polymer, 4,4-Azobis(4-cyanovaleric acid) (ACVA), and MeOH (1 M).

[0109] MPC, RAFT polymer, and ACVA were dissolved in MeOH, dissolved oxygen was removed, and the mixture was stirred at room temperature in a nitrogen atmosphere. The reactant was purified by precipitation with acetone, and the solvent was removed.

[0110]

[0111] [Example 3]

[0112] Propargyl NGO synthesis

[0113] To prepare a precursor of NGO capable of covalent bonding with PMPC, an alkyne group was introduced into NGO through an amide coupling reaction between the carboxylic acid group of NGO and propargylamine, thereby enabling a click reaction with PMPC-N3. The reagents used were NGO (1.0 mg / mL), propargylamine (0.156 mmol), sulfo-NHS, EDC hydrochloride, and DW.

[0114] EDC·HCl and Sulfo-NHS were each dissolved in water and added to the NGO dispersion, followed by the addition of propargylamine. The pH was adjusted to 8 with 1 M NaOH solution and stirred at room temperature for one day. The mixture was then purified using a dialysis membrane, a fixed volume was freeze-dried, and the concentration was determined. FT-IR analysis was performed. The amide bond formed after the EDC coupling reaction between NGO and propargylamine was confirmed by FT-IR (Fig. 4).

[0115]

[0116] [Example 4]

[0117] NGO-PMPC synthesis using click reactions

[0118] To conjugate super-hydrophilic PMPC using the click reaction between NGO-Propargyl and PMPC-N3, reagents including NGO-Propargyl (5.7 mg / mL), PMPC-N3, copper sulfate pentahydrate, sodium ascorbate, and DW were used.

[0119] NGO-Propargyl and PMPC-N3 were dissolved in DW, and then copper sulfate pentahydrate and sodium ascorbate were added to the reaction solution. The mixture was stirred at room temperature for one day. The mixture was purified by dialysis using a dialysis membrane against a 0.1% EDTA solution and triple-distilled water. After purification, a portion was lyophilized to determine the concentration and subjected to FT-IR analysis. FT-IR spectral analysis confirmed that NGO and PMPC were covalently bonded, as a triazole group peak was generated as a product of the Click reaction.

[0120]

[0121] [Example 5]

[0122] TEM

[0123] The morphology of the synthesized PMPC-modified graphene particles was analyzed by Cs-corrected HRTEM (JEM-ARM200F, Cold FEG, JEOL Ltd, Japan) after loading them onto a 400-mesh carbon-coated copper grid (Fig. 7).

[0124]

[0125] [Example 6]

[0126] UV-Vis absorption analysis of synthesized NGO-PMPC

[0127] To determine the optimal NGO:PMPC ratio within the NGO-PMPC, absorption spectrum analysis was performed. The NGO prepared in Example 1 at a concentration of 1 mg / mL was sequentially diluted two-fold with distilled water, and UV-Vis absorbance was measured. PMPC was also dissolved at 1 mg / mL and 3 mg / mL, and absorbance was measured (Fig. 9).

[0128] Comparing the absorption regions of NGO (a) and PMPC (c), we can see that NGO has a much stronger absorption, and in the region around 400 nm, PMPC shows almost no absorption, while NGO shows strong absorption. In addition, looking at the graph of absorption according to NGO concentration in the 400 nm region (b), we can see that the concentration and absorbance are linearly related.

[0129] Therefore, measuring the absorbance at 400 nm can quantify the amount of NGO in NGO-PMPC. The absorbance was observed while diluting the NGO-PMPC solution (1.39 mg / mL) (d). When the NGO-PMPC solution was diluted threefold, a spectrum nearly identical to the 400 nm wavelength absorbance of 0.1 mg / mL NGO was obtained.

[0130] That is, since NGO-PMPC 0.4 mg / mL contains about 0.1 mg / mL of NGO, it can be seen that the optimized ratio of NGO: PMPC is about 1:3.

[0131]

[0132] [Example 7]

[0133] Dispersibility analysis in brine

[0134] DLS analysis can be used to compare the aggregation rates of the two substances to determine their aggregation tendencies. To determine the effect of salt concentration on aggregate formation, NGO dispersions and NGO-PMPC dispersions were mixed and then diluted in 50, 100, 150, and 300 mM NaCl solutions to a final concentration of 0.1 mg / mL based on NGO. Aggregate formation was analyzed using dynamic light scattering (DLS) (Figs. 10a and 10b).

[0135] At salt concentrations ranging from 50 to 300 mM, particle size was compared between NGO and NGO-PMPC by comparing overall dispersibility and Z-average. Overall, increasing salt concentration tended to decrease dispersibility and increase aggregate formation.

[0136] Furthermore, at all salt concentrations, NGO-PMPC exhibited a tendency toward reduced cohesion compared to NGO. Up to a salt concentration of approximately 150 mM, equivalent to the concentration of physiological saline, NGO-PMPC exhibited significantly increased dispersibility and reduced cohesion compared to NGO.

[0137]

[0138] [Example 8]

[0139] Dispersibility analysis in buffer solution

[0140] To determine the effect of pH and buffer solution salt type on the dispersibility of NGO, acetate (pH 5.5), phosphate (pH 7.4), tris (pH 9.0) 10 mM, and ionic strength 150 mM were used. NGO dispersion and NGO-PMPC dispersion were mixed and then diluted to a final concentration of 0.1 mg / mL based on NGO. Aggregate formation was analyzed by dynamic light scattering (DLS) (Figs. 11a and 11b).

[0141] The analysis results show that NGO-PMPC has higher dispersibility than NGO across all pH ranges. NGO is found to form large aggregates at slightly acidic pH (pH 5.5). This is a unique characteristic that occurs when the carboxyl residues on the NGO surface become protonated at acidic pH. On the other hand, NGO-PMPC also exhibits increased aggregation at pH 5.5, but its dispersibility is significantly higher than that of NGO.

[0142]

[0143] [Example 9]

[0144] Dispersibility analysis in cell culture medium

[0145] The dispersibility in DMEM containing 4500 mg / L D-glucose and L-glutamine and tertiary distilled water was compared (Figs. 12a and 12b). NGO dispersions and NGO-PMPC dispersions were mixed in DMEM and tertiary distilled water, respectively, to a final concentration of 0.1 mg / mL based on NGO, and then diluted. Aggregate formation was analyzed using dynamic light scattering (DLS). Additionally, aggregation was observed after 12 hours of storage at 37°C, similar to cell culture conditions.

[0146] As shown in [Figure 13], the measurement results show that the dispersibility of NGO and NGO-PMPC in triple-distilled water is almost similar. However, when exposed to DMEM, the cohesiveness of NGO increases significantly, while the cohesiveness of NGO-PMPC is hardly observed.

[0147] After dispersing NGO and NGO-PMPC samples in DMEM and storing them at 37°C for 12 hours, visual observation revealed that precipitates were observed only in the NGO / DMEM sample, while no precipitates were observed in the NGO-PMPC sample.

[0148] Through the above example, it can be seen that when PMPC is bonded to NGO, the characteristics of NGO that exhibit serious aggregation phenomenon under high salt concentration or low pH conditions are changed, thereby showing the effect of improving dispersibility.

[0149]

[0150] [Example 10]

[0151] Antioxidant evaluation

[0152] Antioxidant assessment evaluates the ability of a substance to remove reactive oxygen species (ROS) and reactive nitrogen species (RNS) that occur in vivo. There are various chemical methods to evaluate the antioxidant effect. For example, radical / ROS-based scavenging tests (Trolox-equivalent antioxidant capacity (TEAC / ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC) test, chemiluminescence method, total radical trapping antioxidant parameter (TRAP), total oxygen radical scavenging capacity (TOSC), beta-carotene decolorization test, etc.), non-radical integrated reduction potential-based tests (iron reducing antioxidant power (FRAP), copper reducing antioxidant capacity (CUPRAC), nanoparticle-based methods, and electrochemical methods), metal chelation capacity, and total phenol content tests.

[0153] In order to evaluate the antioxidant effect of PMPC-modified graphene in the present invention, PMPC-modified graphene was prepared according to the ratio as shown in [Table 1] below, and the scavenging ability to remove reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated in a living body was evaluated (Figs. 14 and 15).

[0154] RAW264.7 cells in culture were washed with PBS. The washed cells were separated from the culture vessel using culture medium (DMEM High Glucose) and centrifuged to create a cell pellet. This was mixed again with 1 ml of culture medium to determine the cell count. At this time, the cells and trypan blue reagent were mixed and placed in a hemocytometer, and the cell count was calculated by observing under a light microscope. After that, 2.5 x 10 were seeded into a 24-well cell culture plate. 5After seeding cells at a concentration of 10 cells / well, they were cultured in a CO2 incubator for 24 hours. LPS (1 mg / ml) was added to the culture medium containing the cells to generate reactive oxygen species (ROS) and reactive nitrogen species (RNS). Simultaneously, the graphene of the present invention was added to the PMPC-modified graphene experimental group. After another 24 hours of culture, 50 μL of conditioned medium from each experimental group's well was transferred to a 96-well plate. To create a nitrite baseline prior to evaluation, 1 μL of the nitrite standard from the Griess reagent system was added to 999 μL of the culture medium and diluted 1 / 1000-fold. To measure the nitrite concentration, add 50 μL of reagent A (sulfanilamide solution) included in the Griess reagent system to the wells containing the sample and standard solution, and incubate for 5 minutes at room temperature. Add 50 μL of Griess reagent B (NED Solution) to each well, and incubate for 5 minutes at room temperature. Finally, the absorbance was measured at 535 nm, and the absorbance of the experimental group was measured using the y value of the standard curve formula to calculate the concentration of X (Nitric Oxide).

[0155] NXM Draft M24001 M24002 M24004 Note Input (NGO:PMPC) 1:0.6 1:3.8 1:0.6 1:0.6 Actual measured weight Output (NGO:PMPC) 1:3 1:0.6 8 1:0.17 1:0.25 Absorbance conversion value

[0156]

[0157] The PMPC-modified graphene according to the present invention exhibits the effect of reducing aggregation and improving dispersibility even in an environment where aggregates of existing graphene are formed and dispersibility is low, so it can be utilized as graphene with high potential for in vivo application, and thus has industrial applicability.

Claims

1. Graphene modified with zwitter ionic polymer.

2. Graphene according to claim 1, characterized in that the zwitterionic polymer is at least one selected from the group consisting of PMPC (Poly(2-methacryloyloxyethyl phosphorylcholine)), PVA (Polyvinyl alcohol), PAMPS (Poly(acrylamidomethylpropane sulfonic acid)), PDMAEMA (Poly(N,N-dimethylaminoethyl methacrylate)), PEGMA (Poly(ethylene glycol) methyl ether methacrylate), PHEMA (Poly(2-hydroxyethyl methacrylate)), and PEI (Poly(ethylene imine)).

3. In the first paragraph, the graphene is at least one selected from the group consisting of graphene oxide, graphene oxide variants, and graphene quantum dots.

4. In the third paragraph, the graphene is nano-oxidized graphene.

5. In the first paragraph, the graphene has improved dispersibility and cohesion.

6. In the first paragraph, the graphene has an antioxidant effect.

7. In the first paragraph, the graphene is a graphene in which the zwitterionic polymer and the graphene are combined through at least one method selected from the group consisting of click chemistry, reverse atom transfer radical polymerization (RATRP), atom transfer radical polymerization using an activator generated by electron transfer for atom transfer radical polymerization (AGENT ATRP), and in situ polymerization.

8. In the first paragraph, the graphene has a diameter of 0.1 to 100 nm and a thickness of 0.1 to 100 nm.

9. In the first paragraph, the graphene is graphene in which graphene and a zwitterionic polymer are combined in a ratio of 1.0:0.01 to 1.0:100.

0. 10.(a) A step of mixing a RAFT reagent containing a first click chemical functional group and a zwitter ionic polymer to prepare a zwitter ionic polymer having a first click chemical functional group attached thereto; (b) a step of manufacturing graphene having a second click chemical functional group attached thereto; and (c) a step of combining the zwitterionic polymer having the first click chemical functional group and the graphene having the second click chemical functional group by a click chemical reaction; A method for producing graphene modified with a zwitter ionic polymer, comprising:

11. A manufacturing method according to claim 10, wherein the zwitterionic polymer is at least one selected from the group consisting of PMPC (Poly(2-methacryloyloxyethyl phosphorylcholine)), PVA (Polyvinyl alcohol), PAMPS (Poly(acrylamidomethylpropane sulfonic acid)), PDMAEMA (Poly(N,N-dimethylaminoethyl methacrylate)), PEGMA (Poly(ethylene glycol) methyl ether methacrylate), PHEMA (Poly(2-hydroxyethyl methacrylate)), and PEI (Poly(ethylene imine)).

12. A manufacturing method in claim 10, wherein the combination of the first click chemical functional group and the second click chemical functional group is at least one selected from the group consisting of an azide group-alkyne group, a thiol group-alkyne group, an amine group-epoxy group, a thiol group-epoxy group, an amine group-acroyl group, a thiol group-acroyl group, and a cyclooctene-tetrazine.

13. A manufacturing method according to claim 10, wherein the graphene is at least one selected from the group consisting of graphene oxide, graphene oxide variants, and graphene quantum dots.

14. A manufacturing method according to claim 13, wherein the graphene oxide is nano graphene oxide.

15. A manufacturing method according to claim 10, wherein the graphene has a diameter of 0.1 to 100 nm and a thickness of 0.1 to 100 nm.

16. A manufacturing method in claim 10, wherein the graphene is a combination of graphene and a zwitterionic polymer in a ratio of 1.0:0.01 to 1.0:100.0.

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