CN111848967B - Graphene oxide composite material with multi-arm polyethylene glycol and sulfydryl connected through covalent bonds, preparation method and application - Google Patents
Graphene oxide composite material with multi-arm polyethylene glycol and sulfydryl connected through covalent bonds, preparation method and application Download PDFInfo
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Abstract
The invention discloses a graphene oxide composite material with multi-arm polyethylene glycol and sulfydryl connected by covalent bonds, a preparation method and application. The preparation method comprises the following steps: 1) activating carboxyl on the graphene oxide sheet layer to obtain a graphene oxide dispersion liquid; 2) grafting multi-arm polyethylene glycol and thiol on a sheet of graphene oxide: uniformly mixing the graphene oxide dispersion liquid with multi-arm polyethylene glycol with an amino end group, a condensing agent, water-soluble strong base and cysteine, heating to 50-100 ℃, reacting for 6-24h, and separating to obtain the graphene oxide/amino acid composite material. The graphene oxide composite material has excellent dispersion stability and biocompatibility in water, and the contained sulfydryl can load a water-insoluble anti-cancer drug through a disulfide bond, so that the graphene oxide composite material is a nano drug carrier platform with a controllable release effect.
Description
Technical Field
The invention belongs to the technical field of biocompatible drug carrier materials, and particularly relates to a graphene oxide composite material with covalent bonds connecting multi-arm polyethylene glycol and sulfydryl, a preparation method and application.
Background
Cancer is a disease that seriously harms human life and health, and scientists have made enormous efforts to treat cancer. Although the method for treating the cancer is slightly improved in recent years, the traditional chemotherapeutic drugs have no specific effect on normal cells and cancer cells of a human body, and can also cause great damage to the normal cells in the process of treating the cancer, so that the treatment effect is still unsatisfactory. With the intensive research on cancer tumor cells, people recognize that the physical and chemical environments of cancer tumor parts are greatly different from the environment of normal cells of human bodies, and the target treatment of the tumor cells by using the nanotechnology is an important direction of the current research. The anticancer drug is loaded through the nano material, and the drug is accurately targeted and gathered at a tumor part by combining the high permeability and retention effect (EPR effect) of the solid tumor; the medicine is not released or is released little in the in-vivo delivery process, and is effectively released after reaching the target tumor part, so that the toxic and side effects of the whole body of the medicine can be reduced, and the curative effect of the medicine is improved.
Common drug nano-carriers comprise inorganic nano-particles, polymer nano-particles, dendritic macromolecules, liposomes and the like, but the current drug nano-carriers also have the problems of poor biocompatibility, difficult accurate control of size and appearance, difficult surface modification, low loading rate, ineffective release when reaching tumor parts and the like. In 2004, Geim and Novoselov et al reported a new two-dimensional nanomaterial, graphene, which has attracted much attention from researchers due to its unique physical and chemical properties. Graphene Oxide (GO) is an important derivative in the preparation and research process of graphene materials, has a structure basically the same as that of graphene, and is a quasi-two-dimensional layered structure, but hydroxyl groups and epoxy groups are randomly distributed on a lamellar layer, and carboxyl groups and hydroxyl groups are randomly distributed on the edge of the lamellar layer. Therefore, the graphene oxide has a large specific surface area and a high drug loading capacity as the graphene, and the contained oxygen-containing groups endow the graphene oxide and the derivatives thereof with good hydrophilicity, so that the graphene oxide and the derivatives thereof have good dispersibility in water, and further have good biocompatibility and low cytotoxicity. Meanwhile, the oxygen-containing group contained in the graphene oxide enables the graphene oxide to be easily subjected to chemical modification, and the graphene oxide is connected with drug molecules through covalent bonds, so that the release process of the drug can be accurately controlled. Due to the characteristics, the graphene oxide serving as a drug carrier has great application potential in the field of drug controlled release.
Cancer tumor cells are rich in Glutathione (GSH), which is present at concentrations more than 1000 times higher than in human plasma. The disulfide bond is reported to generate bond breaking reaction under the action of glutathione. If the drug molecules are loaded on the nano-drug carrier through disulfide bonds, when the nano-drug is gathered at a tumor part, the drug molecules loaded on the nano-drug can be released from the drug carrier under the action of glutathione, so that the effective release of the drug is realized.
Disulfide bond is a switch for effectively and controllably releasing drug molecules of a nano-drug carrier, and generation of disulfide bond firstly needs introduction of sulfydryl on the nano-drug carrier. The graphene oxide is used as a targeted anti-tumor nano-drug carrier with good application prospect, and the sulfydryl is introduced into the lamella of the graphene oxide, so that the graphene oxide becomes a nano-drug carrier platform with controllable release effect, and various anti-tumor drug molecules are loaded through disulfide bonds. There have been some attempts to introduce a thiol group on the graphene surface, for example, in 2012, Zhou et al introduces an amino group, a thiol group, and other active groups on the GO sheet layer by a small molecule directly reacting with an epoxy group on the GO surface, and provides suitable reaction sites for stimuli-responsive molecules and targeting molecules (Analyst 2012, 137, 305). However, this method requires a long-term high-temperature reaction, which causes partial thermal reduction of graphene oxide, resulting in aggregation of the modified graphene material in water, and is not suitable for biomedical applications.
That is, the existing method for introducing sulfydryl through surface modification of GO must pass through high temperature and alkaline conditions, and the high temperature and alkaline conditions can cause a certain degree of reduction of GO, and the water dispersion stability and biocompatibility of reduced GO can be deteriorated, which has been reported in other related ways, such as: adv.mater.2008, 20,4490 and ACS nano.2018,12,1390, and for this reason, no study of drug loading was performed in the existing literature with the resulting thiol-modified GO. That is, the introduction of thiol group on graphene oxide lamella as nano drug carrier only stays in the theoretical conclusion, and the practical defect of poor dispersion stability and biocompatibility limits the application of this approach in the biomedical field.
Currently, the main approach to improve the biocompatibility and dispersion stability of GO in water is to introduce hydrophilic polymers, such as polyethylene glycol (PEG), to modify the GO lamellae. As the reaction sites on the surface of GO are fewer, more hydrophilic groups can be introduced by connecting multi-arm PEG, so that the biological immune reaction is reduced, and the multi-arm PEG with the total molecular weight of 5000-. In 2008, Dai et al successfully connect multi-arm PEG and graphene through covalent bonds by performing amidation reaction between carboxyl groups of GO and star-shaped polyethylene glycol, and the obtained graphene composite material has good water solubility and biocompatibility, and adsorbs anticancer drugs on the surface of a poly GO sheet layer through physical interaction such as pi-pi stacking, so as to prepare a graphene drug composite (J.Am.chem. Soc.2008,130, 10876). However, the drug-loading mechanism of the document is to physically adsorb drug molecules through the large conjugated surface of graphene, and the drug molecules are not connected through covalent bonds, so that the controllable release of the anticancer drug cannot be realized. Later, the literature shows that the solubility and stability of the GO modified by the polyethylene glycol with star six-arm and single-arm molecular weight larger than 1000 are better in water and biological buffers (Nanomedicine 2011,6, 1327).
In conclusion, a chemical modification method on a graphene oxide lamella needs to be further researched in the field, so that the graphene oxide lamella has the advantages of excellent dispersion stability, biocompatibility, simple preparation process and suitability for large-scale production when a drug-carrying platform is provided.
Disclosure of Invention
The invention aims to overcome the defects that in the prior art, the surface of a graphene oxide sheet layer is easy to aggregate in water when sulfydryl is introduced, and the biocompatibility is poor, and provides a graphene oxide composite material with multi-arm polyethylene glycol and sulfydryl connected through covalent bonds, a preparation method and application thereof. According to the invention, the one-pot method is adopted to simultaneously introduce the sulfydryl and the multi-arm polyethylene glycol on the surface of the graphene sheet layer, the amount of the introduced sulfydryl is easy to control, and the preparation method of the composite material is simple and is suitable for large-scale preparation; the graphene oxide composite material has excellent dispersion stability and biocompatibility in water, and the contained sulfydryl can load a water-insoluble anti-cancer drug through a disulfide bond, so that the graphene oxide composite material is a nano drug carrier platform with a controllable release effect.
The invention provides a preparation method of a graphene oxide composite material (PEG-GO-SH) with covalent bonds connecting multi-arm polyethylene glycol and sulfydryl, which comprises the following steps:
(1) activating carboxyl on the graphene oxide sheet layer to obtain a graphene oxide dispersion liquid;
(2) grafting multi-arm polyethylene glycol and thiol on a sheet of graphene oxide: and (3) uniformly mixing the graphene oxide dispersion liquid with multi-arm polyethylene glycol with an amino end group, a condensing agent, water-soluble strong base and cysteine, heating to 50-100 ℃, reacting for 6-24h, and separating to obtain the graphene oxide dispersion liquid.
The raw materials and process conditions in step (1) are specifically described below:
in step (1), the activation of the carboxyl groups on the graphene oxide sheets is performed by a method conventional in the art, and preferably comprises the following steps: and mixing the graphene oxide aqueous solution with alkali for reaction, and adjusting the pH value to be neutral by using acid to obtain the graphene oxide dispersion liquid.
The graphene oxide aqueous solution is prepared by a conventional method in the field, and is preferably prepared by uniformly dispersing Graphene Oxide (GO) in deionized water. The concentration of the obtained graphene oxide aqueous solution is preferably 0.01mg/mL-0.10mg/mL, more preferably 0.05 mg/mL.
Wherein Graphene Oxide (GO) is represented by the following structural formula:
wherein said uniform dispersion is a routine operation in the art, such as ultrasonic dispersion.
The alkali is preferably a NaOH solution, and the concentration of the NaOH solution is preferably 5mol/L-8mol/L, more preferably 6mol/L-7mol/L, and further more preferably 6 mol/L.
The volume ratio of the graphene oxide aqueous solution to the NaOH solution is preferably 1 (0.01-0.1), more preferably 1 (0.06-0.1).
Wherein, the mixing reaction is preferably performed at room temperature for a period of preferably 1 to 24 hours, more preferably 3 hours.
Wherein, the adjustment of pH to neutral is a routine operation in the field, and generally means the adjustment of pH to neutral by using a pH regulator, and the neutral generally means the pH is between 6.5 and 7.5.
Wherein, the pH regulator is a conventional substance in the field, and is preferably a hydrochloric acid solution. The concentration of the hydrochloric acid solution is preferably 1mol/L to 10mol/L, more preferably 1 mol/L.
The raw materials and process conditions in step (2) are specifically described below:
in the step (2), the multi-arm polyethylene glycol with the amino end group is a commercial raw material in the field, preferably 4-, 6-or 8-arm polyethylene glycol with the amino end group, the number average molecular weight is 6000-10000 g/mol, the molecular weight dispersion degree is less than or equal to 1.3, more preferably six-arm polyethylene glycol with the amino end group, the number average molecular weight is 9000-10000g/mol, and the molecular weight dispersion degree is less than or equal to 1.1.
Wherein, the following structural formula represents multi-arm polyethylene glycol with amino as terminal group:
wherein, the number of arms of the multi-arm polyethylene glycol with the end group of amino is 4, 6 or 8, and the polymerization degree n of the polyethylene glycol on each arm is an integer of 10-20.
In step (2), the condensing agent is an amidation condensing agent conventionally used in the art, preferably 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and/or N, N' -diisopropylcarbodiimide.
In step (2), the water-soluble strong base is conventional in the art and preferably comprises sodium hydroxide and/or potassium hydroxide solution. The concentration of the water-soluble strong base is preferably 0.05 to 0.15mol/L, more preferably 0.1 mol/L.
In the step (2), the volume ratio of the graphene oxide dispersion liquid to the water-soluble strong base is preferably 1 (0.001-0.01), more preferably 1 (0.005-0.01).
In the step (2), the mass ratio of the graphene oxide, the multi-arm polyethylene glycol with the end group being an amino group, the condensing agent and the cysteine contained in the graphene oxide dispersion liquid is preferably 1 (2-10): 1-4): 1-3, more preferably 1 (8-10): 2-4): 2-3. The dosage of the multi-arm polyethylene glycol with the amino end group is excessive relative to graphene oxide, so that a hydrophilic polymer is maximally introduced on a graphene oxide lamella; the charging amount of cysteine determines the content of the connecting sulfydryl in the obtained graphene oxide composite material.
In step (2), the mixing is performed according to conventional practice in the art, and preferably, the stirring is performed at room temperature for 2 to 24 hours.
In the step (2), the reaction temperature is preferably 60 to 90 ℃, more preferably 70 to 80 ℃, and still more preferably 80 ℃.
In the step (2), the reaction time is determined by the reaction temperature, preferably when the reaction temperature is 60-90 ℃, the reaction time is 8-14 hours; more preferably, when the reaction temperature is 70-80 ℃, the reaction time is 10-12 hours; still more preferably 80 ℃ for 10 hours.
In a preferred embodiment of the present application, the process conditions are as follows: in the step (2), the volume ratio of the graphene oxide dispersion liquid to the NaOH solution is 1 (0.005-0.01), and the mass ratio of the graphene oxide, the multi-arm polyethylene glycol with the end group as the amino group, the condensing agent and the cysteine is 1 (8-10) to (2-4) to (2-3); the reaction temperature is 80 ℃ and the reaction time is 10-12 hours. Through detection, the content of the multi-arm polyethylene glycol with the amino end group in the obtained graphene oxide composite material is determined by Thermal Gravimetric Analysis (TGA), and the content is 10-30%; the content of sulfydryl is determined by elemental analysis of sulfur element and is 2-6%.
The content of the multi-arm polyethylene glycol with the end group being amino is defined as the mass percentage of the mass of the grafted multi-arm polyethylene glycol with the end group being amino in the total mass of the grafted composite material, and the calculation mode is w ═ 1- (w2/w1)) -100% -w3, wherein w1, w2, w3 and w respectively represent the weight loss rate of PEG-GO-SH at 500 ℃, the weight loss rate of graphene oxide at 500 ℃, the mass fraction of sulfydryl in PEG-GO-SH and the mass fraction of PEG in PEG-GO-SH.
In the step (2), the separation operation is preferably performed, after the reaction is finished, after the reaction solution is cooled to room temperature, the reaction solution is filled into a dialysis bag, and after the reaction solution is dialyzed in deionized water for 1 to 7 days, the filtration separation is performed to remove water-soluble ions.
Wherein, the dialysis bag is preferably permeable to multi-arm polyethylene glycol with amino end group, such as permeable molecular weight of 14,000 g/mol.
Wherein said filtration separation is a routine operation in the art, preferably comprising the steps of: centrifuging, filtering, washing and drying.
The preparation reaction formula of the graphene oxide composite material (PEG-GO-SH) with covalent bonds connecting sulfydryl and multi-arm polyethylene glycol provided by the invention is as follows:
the invention also provides a graphene oxide composite material prepared by the preparation method and connected with the multi-arm polyethylene glycol and the sulfydryl through covalent bonds.
According to a preferred embodiment of the present application:
the average thickness of the sheet layer of the graphene oxide composite material is about 1.5nm, and aggregation does not occur;
the hydration diameter of the sheet layer of the graphene oxide composite material is 123.2nm, the PDI (polydispersity index) is 0.228, and the hydration diameter of the sheet layer after the aqueous dispersion of the graphene oxide composite material is stood for 6 months is 133.6nm, and the PDI is 0.324, which shows that the water dispersion stability of the material is high;
in addition, the content of the hexa-arm polyethylene glycol in the graphene oxide composite material is more than 24%, and the content of sulfydryl is more than 3%.
The invention also provides a graphene oxide composite material (PEG-GO-SH) with covalent bonds connecting the multi-arm polyethylene glycol and sulfydryl, and the structure of the graphene oxide composite material is shown as the following formula:
wherein, the number of arms of the multi-arm polyethylene glycol is 4, 6 or 8, and n is an integer of 10-20.
It should be noted that the edges of the graphene oxide sheets are randomly distributed with carboxyl and hydroxyl groups, which is only schematic in the structural formula, and it is impossible to graft a multi-arm polyethylene glycol with terminal groups removed as amino groups onto each carboxyl group. In addition, it is also schematic for the epoxy groups that it is not possible to substitute each epoxy group with a mercapto group.
The invention also provides application of the graphene oxide composite material with covalent bonds connecting the multi-arm polyethylene glycol and the sulfydryl as a nano-drug carrier platform with targeting and controllable release effects.
The method for loading the anti-cancer drug by the graphene oxide composite material (PEG-GO-SH) with the covalent bond connected with the sulfhydryl group and the multi-arm polyethylene glycol comprises the following steps:
dispersing the graphene oxide composite material with covalent bonds connecting sulfydryl and multi-arm polyethylene glycol in deionized water, mixing with the modified anticancer drug, reacting at room temperature for 6-24 hours, and filtering and separating after the reaction is finished to obtain the graphene oxide composite material loaded with the anticancer drug.
In the method for loading anticancer drugs, the filtration and separation are conventional operations in the art, and preferably comprise the following steps: centrifuging, filtering, washing and drying.
In the method for loading the anticancer drug, the anticancer drug is a conventional substance in the field, and preferably paclitaxel, oridonin or camptothecin.
In the method for loading the anticancer drug, the mass ratio of the graphene oxide composite material with the covalent bond connecting the sulfydryl and the multi-arm polyethylene glycol to the modified anticancer drug is preferably 1 (1-2), wherein the modified anticancer drug is preferably paclitaxel modified by a disulfide bond. Preferably, the modified anticancer drug is dissolved in a solvent such as methanol to form a solution and then mixed with the aqueous dispersion of the graphene oxide composite. The graphene oxide composite material is dispersed in deionized water, and the concentration of the formed aqueous dispersion is preferably 0.05 mg/mL. The anti-cancer drug is excessive relative to the graphene oxide composite material, so that the anti-cancer drug can fully react with the sulfhydryl group contained in the graphene oxide composite material.
According to a preferred embodiment of the application, the paclitaxel modified by the disulfide bond is taken as an anticancer drug, the paclitaxel modified by the disulfide bond is added into the graphene oxide composite material aqueous dispersion, and after stirring for 24 hours at room temperature, the graphene oxide composite material loaded with the paclitaxel by the disulfide bond is obtained through filtration and separation. Through testing ultraviolet-visible spectrum (UV/vis) of the graphene oxide composite material loaded with paclitaxel and drug release behavior in a simulated solid tumor environment, the graphene oxide composite material loaded with the anti-cancer drug can controllably and effectively release the anti-cancer drug in a Glutathione (GSH) environment, and can more effectively release the anti-cancer drug in a specific acidic environment of a solid tumor.
In the invention, the room temperature has the conventional meaning in the field, and generally means 5-35 ℃.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: the method is simple and easy to operate, the multi-arm polyethylene glycol and the sulfydryl are introduced to the surface of the graphene oxide lamella by a one-pot method, the content of the sulfydryl introduced by covalent bonds on the surface of the graphene oxide lamella is easy to control, the repeatability is good, and the method is suitable for large-scale preparation; the obtained graphene oxide composite material with covalent bonds connecting sulfydryl and multi-arm polyethylene glycol has good water dispersion stability and biocompatibility, the contained sulfydryl provides active sites for controllable and targeted loading of anticancer drugs, and the graphene oxide composite material is a nano drug carrier platform with the functions of targeting and controllable release.
Drawings
Fig. 1(a) and 1(b) are Atomic Force Microscope (AFM) images and height distribution diagrams of Graphene Oxide (GO) (left panel) and graphene oxide composite material (PEG-GO-SH) in which thiol and multi-arm polyethylene glycol are covalently linked (right panel), respectively;
FIG. 2 is a graphene oxide composite (PEG-GO-SH) aqueous dispersion with covalent bonds linking thiol groups and multi-arm polyethylene glycol and a Dynamic Light Scattering (DLS) curve for the dispersion standing for 6 months;
FIG. 3 is an infrared spectrum (FT-IR) of a graphene oxide composite (PEG-GO-SH) with covalent bonds to thiol and multi-arm polyethylene glycol;
FIG. 4 is a Thermogravimetric (TGA) curve of Graphene Oxide (GO), six-arm polyethylene glycol (PEG), and graphene oxide composite (PEG-GO-SH) with covalent bonds linking thiol and multi-arm polyethylene glycol;
FIG. 5 is an ultraviolet-visible spectrum (UV/vis) of a graphene oxide composite (PEG-GO-SH) covalently linked to a thiol group and a multi-arm polyethylene glycol;
FIG. 6 is an X-ray photoelectron spectroscopy (XPS) of a graphene oxide composite (PEG-GO-SH) covalently linked to a thiol group and a multi-arm polyethylene glycol;
fig. 7 is an ultraviolet-visible spectrum (UV/vis) of Paclitaxel (PTX) loaded graphene oxide composite with covalent bonds linking thiol groups and multi-arm polyethylene glycol;
fig. 8 is a drug release profile of paclitaxel loaded graphene oxide composite with covalent bonds linking sulfhydryl groups and multi-arm polyethylene glycol in Glutathione (GSH) buffer monitored by high performance liquid chromatography;
wherein, fig. 8(a) is the release profile in 10mmol/L GSH and 0mmol/L GSH buffer (pH 7.4), respectively, and fig. 8(b) is the release profile in 10mmol/L GSH buffer at pH 7.4 and pH 5.0, respectively.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1: activation of graphene oxide
Uniformly dispersing Graphene Oxide (GO) in deionized water to obtain 0.05mg/mL graphene oxide aqueous solution, adding 6mol/L NaOH aqueous solution to react for 3 hours at room temperature, and then adjusting the pH value to be neutral by using 1mol/L hydrochloric acid solution to obtain activated graphene oxide dispersion liquid. The volume ratio of the graphene oxide aqueous solution to the sodium hydroxide aqueous solution is 1: 0.1.
Example 2 (a): preparation of graphene oxide composite material (PEG-GO-SH) with sulfhydryl and multi-arm polyethylene glycol connected by covalent bond through one-pot method
The general preparation method of the graphene oxide composite material (PEG-GO-SH) with the covalent bond connecting the sulfydryl and the multi-arm polyethylene glycol by the one-pot method comprises the following steps: adding multi-arm polyethylene glycol with an amino end group, amidation condensing agent, 0.1mol/L NaOH solution and cysteine into the activated graphene oxide dispersion liquid, stirring at room temperature for 2-24 hours, and heating to 50-100 ℃ for reaction for 6-24 hours; and after the reaction is finished, cooling the reaction liquid to room temperature, filling the reaction liquid into a dialysis bag, dialyzing in deionized water for 1-7 days to remove water-soluble ions, and filtering and separating to obtain the graphene oxide composite material (PEG-GO-SH) with the covalent bond connecting the sulfydryl and the multi-arm polyethylene glycol.
Example 2(b)
The following is a specific preparation process of the graphene oxide composite material with covalent bonds connecting sulfydryl and six-arm PEG:
to 800mL of the activated graphene oxide dispersion prepared in example 1 (the addition volume of the aqueous sodium hydroxide solution and the aqueous hydrochloric acid solution was small compared to that of the aqueous graphene oxide solution, and therefore, the mass of graphene oxide was calculated to be negligible, that is, 40mg was calculated by multiplying the mass of graphene oxide contained in this example by 800 mL), 400mg of hexa-armed polyethylene glycol having a number average molecular weight of 9,500g/mol and a molecular weight dispersity of 1.05 was added, and the mixture was ultrasonically dispersed for 5 minutes. Then, 160mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 200mg, 0.1mol/L sodium hydroxide and 40mg of cysteine (the mass ratio of the graphene oxide contained in the graphene oxide dispersion liquid, the multi-arm polyethylene glycol with the end group being the amino group, the condensing agent and the cysteine is 1:10:4:1) were added to the mixed solution, and the reaction was carried out at room temperature for 6 hours, and then the reaction system was heated to 80 ℃ for 10 hours. After the reaction is finished, cooling the reaction system to room temperature, filling the reaction solution into a dialysis bag (with a permeable molecular weight of 14,000g/mol), placing the dialysis bag in deionized water for dialysis for 5 days, and then centrifuging, filtering, washing and vacuum drying the reaction solution to obtain the graphene oxide composite material with the mercapto group and the six-arm polyethylene glycol connected by the covalent bond, wherein the yield is 95% (relative to the mass of the fed graphene oxide).
Effect example 1
The graphene oxide composite material covalently connecting the thiol group and the six-arm polyethylene glycol obtained in example 2(b) was characterized by Elemental Analysis (EA), Atomic Force Microscopy (AFM), infrared spectroscopy (FT-IR), ultraviolet spectroscopy (UV/vis), thermogravimetric analysis (TGA), Dynamic Light Scattering (DLS), and X-ray photoelectron spectroscopy (XPS).
Elemental Analysis (EA) 3.20% (S), 2.62% (N), 46.73% (C), 7.56% (H), 40.32% (O).
Fig. 1(a) is an AFM image and a height distribution diagram of a raw material Graphene Oxide (GO), and fig. 1(b) is an AFM image and a height distribution diagram of a graphene oxide composite material. According to the AFM image, the size and the appearance of the obtained graphene oxide composite material are basically consistent with those of the raw material graphene oxide, and the obtained graphene oxide composite material is not aggregated; from the height distribution graph, the average thickness of the graphene oxide sheet layer as the raw material is about 1nm, and the average thickness of the graphene oxide composite sheet layer is about 1.5nm, which indicates that the thickness of the graphene oxide composite sheet layer is increased after the six-arm PEG is connected to the surface of the graphene oxide composite sheet layer.
Fig. 2 is a Dynamic Light Scattering (DLS) curve of the graphene oxide composite aqueous dispersion and the composite after standing for 6 months, according to the detection result given by a dynamic light scattering instrument, the hydration diameter of the graphene oxide composite lamella is about 123.2nm, the PDI (polydispersity index) is 0.228, and the graphene oxide composite lamellar can be used as a nano carrier of a targeted anticancer drug within the size requirement range required by the retention effect (EPR effect) of a solid tumor. According to the detection result given by a dynamic light scattering instrument, the hydration diameter of the graphene oxide composite sheet layer of the graphene oxide composite aqueous dispersion liquid after being placed for 6 months is about 133.6nm, and the PDI is 0.324, which indicates that the water dispersion stability of the material is high.
The results of fig. 3 are as follows: FT-IR (cm)-1):3400(νO-H，νN-H),2960(νC-H),1725(νC＝O), 1363(νC-O)。
Fig. 4 is a thermal weight loss curve of the graphene oxide composite material, and it can be seen from the thermal weight loss curve that the residual mass fraction of the six-arm PEG at 500 ℃ is only 1.3%, and it can be considered that the organic matter is completely decomposed at this temperature; the residual mass fraction of the raw material graphene oxide at 500 ℃ was 53.2%, indicating that the raw material contained about 46.8% of organic components; the residual mass fraction of the graphene oxide composite material at 500 ℃ is 34.5%, which indicates that the graphene oxide composite material contains about 65.5% of organic components, which are higher than the organic components in the raw materials by about 19%, and the mass fraction of the organic components introduced by covalently linking the six-arm polyethylene glycol and the sulfydryl is 28.6%.
The mass content of the introduced organic component in the graphene oxide composite material is calculated in a mode of w ═ (1- (w2/w 1)). 100%, wherein w1, w2 and w respectively represent the weight loss rate of PEG-GO-SH at 500 ℃, the weight loss rate of graphene oxide at 500 ℃ and the mass fraction of the introduced organic component in the PEG-GO-SH.
Fig. 5 is an ultraviolet-visible spectrum (UV/vis) curve of the graphene oxide composite material, and it can be seen from the ultraviolet-visible spectrum curve that the graphene oxide composite material has an obvious absorption peak at 255nm, which is different from the unique absorption peaks at 234nm and 300nm of graphene oxide, and polyethylene glycol has no obvious characteristic absorption peak, which fully indicates that graphene oxide has been converted into a graphene oxide composite material under covalent modification of multi-arm polyethylene glycol and thiol. Further, the mass content of the thiol in the composite material is subtracted from the mass content of the introduced organic component, namely 28.6% -3.2%, and the mass content of the polyethylene glycol in the graphene oxide composite material can be calculated to be 25.4%.
FIG. 6 is an X-ray photoelectron spectroscopy (XPS) curve of a graphene oxide composite material, wherein a characteristic peak of 180eV on the curve is a 2p peak of elemental sulfur, which indicates that a mercapto group is introduced on a sheet layer of the graphene oxide composite material; the elemental analysis data of elemental sulfur can be approximated to a mercapto group content of 3.2% by mass.
In the embodiment, the graphene oxide composite material is characterized by Elemental Analysis (EA), an Atomic Force Microscope (AFM), an infrared spectrum (FT-IR), an ultraviolet-visible spectrum (UV/vis), thermal weight loss analysis (TGA), Dynamic Light Scattering (DLS), and X-ray photoelectron spectroscopy (XPS). By controlling the reaction temperature and the reaction time, the graphene oxide composite material has high preparation efficiency while ensuring good dispersion stability in water; meanwhile, the mass fraction of the thiol introduced on the surface of the graphene sheet layer can be controlled to a certain extent by controlling the dosage of cysteine. The method for preparing the graphene oxide composite material is not reported, has the remarkable advantages of simple and easy operation, good controllability, high repeatability and the like, and is suitable for large-scale preparation.
Example 3(a)
The procedure of covalently bonding 4-and 8-arm polyethylene glycol in the graphene oxide composite material was the same as that of example 2(b) for bonding 6-arm polyethylene glycol, and only the polyethylene glycol was replaced with 4-or 8-arm polyethylene glycol during the reaction. The content of polyethylene glycol and the mass fraction of thiol group in the graphene oxide composite material prepared using 4-, 6-and 8-arm polyethylene glycol, respectively, under the same conditions as described above are shown in table 1.
TABLE 1 polyethylene glycol and mass fractions in graphene oxide composites prepared using multi-arm polyethylene glycols of different arm numbers
Example 3(b)
This example further demonstrates that: by controlling the dosage of cysteine, the mass content of the introduced sulfydryl in the graphene oxide composite material can be adjusted. In the step of preparing the graphene oxide composite material in which the thiol group and the six-arm polyethylene glycol are covalently bonded in example 2(b), graphene oxide composite materials with different mass fractions of thiol groups can be obtained by changing the amount of cysteine, and the data are shown in table 2.
TABLE 2 Mass fractions of six-arm polyethylene glycol and thiol groups in graphene oxide composites with covalently-bonded thiol groups and six-arm PEG prepared with different amounts of cysteine
Example 3 (c):
control test: influence of reaction temperature on preparation of graphene oxide composite material (PEG-GO-SH) with sulfhydryl and multi-arm PEG connected by covalent bond in one-pot method
The procedure of covalently bonding six-armed polyethylene glycol in the graphene oxide composite material was the same as that of the raw materials and the charge ratio selected in example 2(b), and only the reaction temperature and the reaction time were changed during the reaction. The mass fractions of polyethylene glycol and thiol in the graphene oxide composite materials prepared at different reaction times and reaction temperatures are shown in table 3.
TABLE 3 Mass fractions of six-arm polyethylene glycol and thiol groups in graphene oxide composites with covalently bonded thiol groups and six-arm PEG prepared at different reaction times and reaction temperatures
As can be seen from the data in the table, in the present invention, increasing the thiol content requires increasing the temperature and prolonging the reaction time, but increasing the reaction time causes the PEG content to decrease and the reduction degree of the graphene oxide to increase, resulting in the deterioration of the water dispersion stability, i.e. it is difficult to increase both the thiol content and the PEG content. Experimental data indicate that some aggregation occurs with a hexa-armed polyethylene glycol content of less than about 15%. Therefore, it is considered that the content of the hexa-arm polyethylene glycol is more than 16%, the content of the mercapto group is more than 2%, the content of the hexa-arm polyethylene glycol is more than 24%, and the content of the mercapto group is more preferably more than 3%.
By controlling the reaction time and temperature, the contents of the six-arm polyethylene glycol and the sulfydryl in the graphene oxide composite material with the sulfydryl and the six-arm polyethylene glycol connected by the covalent bond can be adjusted, the content of the sulfydryl can be increased along with the adjustment of the reaction temperature, but the content of the six-arm polyethylene glycol can be reduced when the temperature is higher than 80 ℃, so that the graphene oxide composite material with the sulfydryl and the six-arm polyethylene glycol connected by the covalent bond is poor in dispersion stability in water and aggregation phenomenon is easily caused; at lower temperatures, as shown by the data at 50 ℃ in the table, the mercapto content decreases and cannot be increased even with prolonged reaction times (14 h).
Experiments show that the temperature of the temperature rise reaction in the step (2) has a key influence on the dispersion stability and preparation efficiency of the graphene oxide composite material (PEG-GO-SH) in water: when the reaction temperature is low, the obtained graphene oxide composite material has good dispersion stability in water, but the reaction speed is slow, and the mercapto content is low; when the reaction temperature is high, the reaction speed is high, but the content of the multi-arm polyethylene glycol in the obtained graphene oxide composite material is reduced, the dispersion stability in water is poor, and the aggregation is easy.
Experiments show that: the preparation of the graphene oxide composite material with the sulfhydryl and the six-arm polyethylene glycol connected by the covalent bond can be realized at 50-100 ℃, but the preferable reaction temperature is 80 ℃. After temperature optimization, high grafting content can be obtained in a relatively short time, i.e. the reaction is carried out at a suitable temperature, and high grafting content can be obtained in a short time; the temperature is high or low, the reaction time is prolonged, and the grafting content cannot be increased.
Example 4: paclitaxel loaded graphene oxide composite material with covalent bond connecting sulfydryl and six-arm polyethylene glycol
The graphene oxide composite material obtained in example 2(b) and covalently bonded to a thiol group and a six-arm PEG was uniformly dispersed in deionized water to obtain a dispersion of 0.05mg/mL of the graphene oxide composite material. 20mL of the dispersion of the graphene oxide composite material was mixed with 2mg of a methanol solution of disulfide-modified paclitaxel (2mg means the mass of disulfide-modified paclitaxel), and stirred at room temperature for 24 hours. After the reaction is finished, removing unreacted paclitaxel molecules through multiple times of centrifugation, and obtaining the paclitaxel-loaded nano targeted anticancer drug through filtration, washing and vacuum drying. The obtained paclitaxel-loaded nano-targeted anticancer drug is characterized by ultraviolet-visible spectrum (UV/vis), and the drug release behavior is tested in Glutathione (GSH) environment.
Fig. 7 is an ultraviolet-visible spectrum curve of the paclitaxel-loaded nano-targeted anticancer drug, and on the ultraviolet-visible spectrum curve, a peak at 230nm is a characteristic absorption peak of paclitaxel, which illustrates that paclitaxel is loaded on a graphene oxide composite sheet layer through a covalent bond.
The prepared taxol-loaded nano-targeted anticancer drug is dispersed into 0.2 mg/mL of uniform dispersion liquid in deionized water, 10mL of nano-targeted anticancer drug dispersion liquid is added into 90mL of buffer solution simulating an entity tumor environment, the concentration of the taxol in the buffer solution is monitored by High Performance Liquid Chromatography (HPLC), and the medicine release behavior of the taxol-loaded nano-targeted anticancer drug is tested. The influence of Glutathione (GSH) on the drug release behavior of the nano-targeted anticancer drug is firstly compared in a neutral environment (pH 7.4). Fig. 8(a) is a graph showing the relationship between the amount of the nano-targeted anticancer drug released in the buffer solution without GSH and the buffer solution containing 10mmol/L GSH and the time, and it can be seen from the graph that the drug release rate is less than 15% in the environment without GSH, and the drug release rate can reach 60% in the environment containing GSH. Then, the influence of the specific acidic environment of the solid tumor on the drug release behavior of the nano-targeting anticancer drug is researched. Fig. 8(b) is a graph showing the relationship between the drug release amount of the nano-targeted anticancer drug in 10mmol/L GSH buffer solution with pH 7.4 and pH 5.0 (which simulates the acidic environment of solid tumors) and time, and it can be seen from the graph that the drug release rate can reach 60% in the neutral environment where GSH exists, and the drug release rate can be higher than 85% in the acidic environment specific to solid tumors. The nano-targeting anticancer drug prepared by loading the anticancer drug on the graphene oxide composite material through the disulfide bond can be accurately and controllably and effectively released in the environment of solid tumors, and further embodies that the graphene oxide composite material (PEG-GO-SH) with the covalent bond connecting the sulfydryl and the multi-arm polyethylene glycol provided by the invention is a nano-drug carrier platform with controllable release effect.
Comparative example 1
To 800mL of the activated graphene oxide dispersion liquid prepared in example 1, 400mg of six-arm polyethylene glycol having a number average molecular weight of 9,500g/mol and a molecular weight dispersion degree of 1.05 was added, and the mixture was ultrasonically dispersed for 5 minutes. Then, 40mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was added to the mixture, ultrasonic dispersion was continued for 30 minutes, and 120mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was further added, and the mixture was reacted at room temperature for 12 hours. After the reaction, the reaction solution was filled in a dialysis bag (permeable molecular weight: 14,000g/mol) and dialyzed in deionized water for 5 days. The yield obtained in this step was 90%.
5mL of 0.1mol/L sodium hydroxide and 40mg of cysteine were added to the dispersion obtained by dialysis, and the reaction system was heated to 80 ℃ to react for 10 hours. After the reaction is finished, cooling the reaction system to room temperature, filling the reaction solution into a dialysis bag (with a permeable molecular weight of 14,000g/mol), placing the dialysis bag in deionized water for dialysis for 5 days, and then centrifuging, filtering, washing and vacuum drying the reaction solution to obtain the graphene oxide composite material with the mercapto group and the six-arm polyethylene glycol connected by the covalent bond, wherein the yield of the graphene oxide composite material is 85%.
The result shows that the multi-arm PEG is firstly modified on the graphene oxide lamella to improve the dispersion stability of the graphene oxide in water, and then the sulfydryl is introduced on the graphene oxide lamella, so that the sulfydryl-containing nano-drug carrier with good dispersibility in water can be obtained. However, each step of the method needs to separate and purify the modified graphene oxide, the operation is complicated, multiple dialysis operations are easy to cause artificial product loss, the efficiency is low, and large-scale preparation is difficult to perform.
Claims (19)
1. A preparation method of a graphene oxide composite material with covalent bonds connecting multi-arm polyethylene glycol and sulfydryl is characterized by comprising the following steps:
(1) activating carboxyl on the graphene oxide sheet layer to obtain a graphene oxide dispersion liquid;
(2) grafting multi-arm polyethylene glycol and thiol on a sheet of graphene oxide: uniformly mixing the graphene oxide dispersion liquid with multi-arm polyethylene glycol with an amino end group, a condensing agent, water-soluble strong base and cysteine, heating to 70-80 ℃, reacting for 10-12 hours, and separating to obtain the graphene oxide dispersion liquid;
in the step (1), the activation of the carboxyl groups on the graphene oxide sheet layer comprises the following steps: mixing the graphene oxide aqueous solution with alkali for reaction, and adjusting the pH to be neutral by using acid to obtain a graphene oxide dispersion liquid; the concentration of the graphene oxide aqueous solution is 0.01mg/mL-0.10 mg/mL;
in the step (1), the alkali is NaOH solution, the concentration of the NaOH solution is 5-8 mol/L,
the volume ratio of the graphene oxide aqueous solution to the NaOH solution is 1 (0.01-0.1);
in the step (2), the mass ratio of graphene oxide, multi-arm polyethylene glycol with amino as an end group, a condensing agent and cysteine in the graphene oxide dispersion liquid is 1 (8-10) to (2-4) to (2-3);
the condensing agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and/or N, N' -diisopropylcarbodiimide;
in the step (2), the water-soluble strong base comprises sodium hydroxide and/or potassium hydroxide solution,
the concentration of the water-soluble strong base is 0.05-0.15mol/L,
the volume ratio of the graphene oxide dispersion liquid to the water-soluble strong base is 1 (0.001-0.01); the multi-arm polyethylene glycol with the end group of amino is the polyethylene glycol with the end group of 4, 6 or 8 arms of amino, the number average molecular weight of 6000-10000 g/mol and the molecular weight dispersion degree of less than or equal to 1.3,
or the multi-arm polyethylene glycol with the end group as the amino group is the polyethylene glycol with 4, 6 or 8 arms and the polymerization degree n of the polyethylene glycol on each arm is an integer of 10-20.
2. The method for preparing the graphene oxide composite material according to claim 1, wherein the graphene oxide aqueous solution is obtained by uniformly dispersing graphene oxide in deionized water.
3. The method for preparing the graphene oxide composite material according to claim 2, wherein the graphene oxide aqueous solution is obtained by uniformly dispersing graphene oxide in deionized water, and the uniform dispersion is ultrasonic dispersion;
and/or the concentration of the graphene oxide aqueous solution is 0.05 mg/mL.
4. The method for preparing a graphene oxide composite material according to claim 1, wherein the mixing reaction is performed at room temperature for 1 to 24 hours;
and/or the pH regulator used for regulating the pH to be neutral is hydrochloric acid solution.
5. The method for preparing a graphene oxide composite material according to claim 4, wherein the mixing reaction is performed at room temperature for 3 hours;
and/or the pH regulator used for regulating the pH to be neutral is a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is 1-10 mol/L.
6. The method for preparing the graphene oxide composite material according to claim 1, wherein in the step (1), the concentration of the NaOH solution is 6mol/L to 7mol/L,
the volume ratio of the graphene oxide aqueous solution to the NaOH solution is 1 (0.06-0.1);
and/or the pH regulator used for regulating the pH to be neutral is a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is 1 mol/L.
7. The method for preparing the graphene oxide composite material according to claim 6, wherein the concentration of the NaOH solution is 6 mol/L.
8. The method for preparing the graphene oxide composite material as claimed in claim 1, wherein in the step (2), the multi-arm polyethylene glycol with the end group of amino is six-arm polyethylene glycol with amino, the number average molecular weight is 9000-10000g/mol, and the molecular weight dispersity is less than or equal to 1.1.
9. The method for preparing the graphene oxide composite material according to claim 1, wherein in the step (2), the concentration of the water-soluble strong base is 0.1 mol/L;
and/or the volume ratio of the graphene oxide dispersion liquid to the water-soluble strong base is 1 (0.005-0.01).
10. The method for preparing a graphene oxide composite material according to claim 1, wherein in the step (2), the uniformly mixing is performed by stirring at room temperature for 2 to 24 hours;
and/or, the separating operation is: after the reaction is finished, after the reaction liquid is cooled to room temperature, the reaction liquid is filled into a dialysis bag, and after the reaction liquid is dialyzed in deionized water for 1 to 7 days, the filtration and the separation are carried out to remove water-soluble ions.
11. The method for preparing a graphene oxide composite material according to claim 10,
the dialysis bag is a multi-arm polyethylene glycol dialysis bag with amino groups at the end groups;
the filtration and separation comprises the following steps: centrifuging, filtering, washing and drying.
12. The method for preparing a graphene oxide composite material according to claim 1, wherein the temperature of the reaction in the step (2) is 80 ℃.
13. The method for preparing a graphene oxide composite according to claim 12, wherein the reaction time is 10 hours when the reaction temperature is 80 ℃.
14. A graphene oxide composite covalently linking a multi-arm polyethylene glycol and a mercapto group prepared by the preparation method according to any one of claims 1 to 13.
15. Use of the covalently linked multi-arm polyethylene glycol and thiol-group graphene oxide composite material of claim 14 as a nano-drug carrier platform with targeting and controlled release effects.
16. The use of claim 15, wherein the method for loading the anticancer drug by covalently linking the thiol group and the multi-arm polyethylene glycol graphene oxide composite material comprises the following steps:
dispersing the graphene oxide composite material with covalent bonds connecting sulfydryl and multi-arm polyethylene glycol in deionized water, mixing with the modified anticancer drug, reacting at room temperature for 6-24 hours, and filtering and separating after the reaction is finished to obtain the graphene oxide composite material loaded with the anticancer drug.
17. The use of claim 16, wherein in said method of loading an anticancer drug, said filtering step comprises the steps of: centrifuging, filtering, washing and drying;
and/or the anticancer drug is paclitaxel, oridonin or camptothecin;
and/or the mass ratio of the graphene oxide composite material with the covalent bond connecting the sulfydryl and the multi-arm polyethylene glycol to the modified anticancer drug is 1 (1-2);
and/or, dissolving the modified anticancer drug in a solvent to form a solution, and mixing the solution with the graphene oxide composite material for reaction;
and/or the graphene oxide composite material is dispersed in deionized water, and the concentration of the formed aqueous dispersion is 0.05 mg/mL.
18. The use of claim 16, wherein the modified anticancer agent is paclitaxel modified with disulfide bonds.
19. The use of claim 17 or 18, wherein the modified anticancer agent is dissolved in a solvent to form a solution, and then mixed with the graphene oxide composite material for reaction, wherein the solvent is methanol.
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