Functionalized graphene oxide nanodrug carrier and preparation method thereof

Abstract

The application discloses a functionalized graphene oxide nano drug carrier and a preparation method. The application covalently grafts phospholipid and hyaluronic acid with a certain structure on two sides of graphene oxide, and obtains a nano drug carrier with stable positive charge on one side and stable negative charge on the other side in an arthritic inflammatory environment through design of the grafted structure. The nano drug carrier can be targetedly adsorbed on the negatively charged cartilage surface in a sliding joint interface, and self-sequencing nano drug carrier layers with stable layered gradient structures are formed on the cartilage surface along with shear stress. The stable nano drug carrier layer integrates drug loading, fluid lubrication of hyaluronic acid, boundary lubrication of graphene oxide and hydration super-lubrication of phospholipid, and can stabilize drug delivery while fully guaranteeing good lubrication effect of the joint in different movement states.

Description

TECHNICAL FIELD

[0001] The application relates to a joint nanomedicine carrier, in particular to a preparation method of a functionalized graphene oxide nanomedicine carrier. BACKGROUND

[0002] Osteoarthritis (OA) is a common degenerative joint disease, mainly characterized by joint cartilage damage, extracellular matrix degradation and bone remodeling. Under normal physiological conditions, the joint cartilage and synovial fluid interact to maintain the lubrication properties of the joint. Synovial fluid is mainly composed of water, hyaluronic acid, phospholipid and lubricin. Among them, hyaluronic acid mainly plays a fluid lubrication role, and phospholipid and lubricin mainly play a boundary lubrication role. However, under the condition of OA, the secretion of hyaluronic acid and phospholipid is insufficient, which leads to the decrease of the lubrication performance of the joint cartilage, the wear of the cartilage, and further accelerates the development of OA. The main treatment method in the clinic is to inject hyaluronic acid into the joint, which can supplement the free hyaluronic acid in the synovial fluid to a certain extent. However, free hyaluronic acid is usually negatively charged and cannot be stably adsorbed on the surface of the cartilage which is also negatively charged, resulting in poor lubrication performance under boundary conditions and difficulty in treating OA.

[0003] The phospholipid in the joint mainly relies on hydration lubrication to play a lubricating role under boundary conditions. The hydration lubrication of phospholipid utilizes the charge characteristics of phospholipid molecules to adsorb water molecules around the phosphocholine group to form a hydration layer. In the process of shearing, water molecules will quickly exchange to reduce the stress of the cartilage. Shi et al. used phosphatidylcholine to wrap methacrylated chondroitin sulfate to play a lubricating and sustained-release role in the joint.

[0004] Graphene oxide is an inorganic two-dimensional carbon nanomaterial, which has attracted widespread attention in biological medicine due to its unique physical and chemical properties. Studies have shown that graphene oxide has adsorption capacity for friction pairs under boundary lubrication conditions, and can form a stable lubricating protective film on the friction interface. At the same time, the graphene oxide sheet contains a large number of oxygen-containing groups, which has the ability to load drugs. For example, CN111558047A prepared a carrier that can load antitumor drugs by using graphene oxide, polyethylene glycol diamine and oxidized sodium alginate. Therefore, graphene oxide also has good development prospects in joint lubrication and drug carriers. SUMMARY

[0005] In view of this, the application discloses a preparation method of a functionalized graphene oxide nanometer drug carrier. The nanometer drug carrier can be targeted to be adsorbed on the negatively charged cartilage surface in the sliding joint interface, and a stable self-ordered layered gradient structure nanometer drug carrier layer is formed on the cartilage surface along with the shear stress. The stable nanometer drug carrier layer integrates the drug loading, the fluid lubrication of hyaluronic acid, the boundary lubrication of graphene oxide and the hydrated superlubrication of phospholipid, and can ensure good lubrication of the joint in different movement states while stably delivering drugs.

[0006] To achieve the above object, the application provides the following technical scheme.

[0007] A functionalized graphene oxide nanometer drug carrier, which is prepared from phospholipid, hyaluronic acid and graphene oxide.

[0008] A preparation method of a functionalized graphene oxide nanometer drug carrier, which comprises the following steps.

[0009] Disperse graphene oxide in distilled water to obtain a graphene oxide dispersion liquid;

[0010] Dissolve hyaluronic acid in distilled water, add a carboxyl activating agent, then add a crosslinking agent, and place the mixture to react to obtain solution A; after dialysis, the solution A is dried to obtain crosslinking agent modified hyaluronic acid;

[0011] Dissolve phospholipid in a nonpolar solvent to obtain a phospholipid solution;

[0012] Dissolve the crosslinking agent modified hyaluronic acid in distilled water to obtain a crosslinking agent modified hyaluronic acid solution;

[0013] Mix the graphene oxide dispersion liquid and the phospholipid solution to form emulsion A under the action of mechanical force;

[0014] Add the carboxyl activating agent to the emulsion A, inject the crosslinking agent modified hyaluronic acid solution into the emulsion A to obtain emulsion B; place the emulsion B at room temperature to react for a certain period of time, take the middle emulsion layer, and wash and dry the middle emulsion layer with a nonpolar solvent and distilled water to obtain the functionalized graphene oxide nanometer drug carrier;

[0015] Preferably, the molecular weight of the hyaluronic acid is 8000-2500000 Dalton;

[0016] Preferably, the carboxyl activating agent is one or more of 1-ethyl-(3-dimethylaminopropyl) carbodiimide, diisopropyl carbodiimide, dicyclohexyl carbodiimide and N-hydroxy succinimide;

[0017] Preferably, the cross-linking agent is adipic acid dihydrazide or ethylenediamine.

[0018] Preferably, the non-polar solvent includes chloroform, diethyl ether, n-hexane, dichloromethane, petroleum ether.

[0019] Further, the concentration of the graphene oxide dispersion solution is 0.1-2 mg / mL, and / or the concentration of the phospholipid solution is 1-5 mg / mL, and / or the concentration of the cross-linking agent modified hyaluronic acid solution is 0.5-5 mg / mL.

[0020] Further, the mass ratio of the hyaluronic acid to the cross-linking agent is 1:0.2-1.

[0021] Further, the C / O of the graphene oxide is (20-40) / 100, and the size of the graphene oxide is 5-0.5 μm.

[0022] Further, the phospholipid is one or more of phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine.

[0023] Further, the volume ratio of the graphene oxide dispersion solution to the phospholipid solution is 1:0.2-1.

[0024] Further, the mechanical force is 100-600 W ultrasonic oscillation or 6000-30000 rpm mechanical stirring.

[0025] Further, the volume ratio of the emulsion A to the cross-linking agent modified hyaluronic acid solution is 40:1-10.

[0026] Further, the hyaluronic acid is dissolved in distilled water, a certain amount of carboxyl activating agent is added, a certain amount of cross-linking agent is added, and then the solution A is obtained by placing the solution at a predetermined temperature for a certain period of time.

[0027] The application also provides a preparation method of the functionalized graphene oxide nanomedicine carrier.

[0028] The hyaluronic acid is dissolved in distilled water, a certain amount of carboxyl activating agent is added, a certain amount of cross-linking agent is added, and then the solution A is obtained by placing the solution at a predetermined temperature for a certain period of time.

[0029] In one preferred embodiment, the molecular weight of the hyaluronic acid is 8000-2500000 Dalton.

[0030] In a preferred embodiment, the carboxyl activation reagent is one or a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0031] In a preferred embodiment, the cross-linking agent is adipic acid dihydrazide; and the mass ratio of the hyaluronic acid to the cross-linking agent is 1:0.3-0.8.

[0032] The graphene oxide is dispersed in distilled water to obtain a graphene oxide dispersion liquid with a concentration of 0.1-2 mg / mL. The phospholipid and the cross-linking agent-modified hyaluronic acid are respectively dissolved in a non-polar solvent and distilled water to obtain a phospholipid solution with a concentration of 1-5 mg / mL and a cross-linking agent-modified hyaluronic acid solution with a concentration of 0.5-5 mg / mL. The graphene oxide dispersion liquid and the phosphatidylethanolamine solution are mixed to form an emulsion A under the action of a mechanical force.

[0033] In a preferred embodiment, the C / O of the graphene oxide is 25-40%, and the size of the graphene oxide is 5-0.5 μm.

[0034] In a preferred embodiment, the phospholipid is phosphatidylethanolamine, phosphatidylserine or phosphatidylcholine.

[0035] In a preferred embodiment, the non-polar solvent includes dichloromethane and petroleum ether.

[0036] In a preferred embodiment, the volume ratio of the graphene oxide dispersion liquid to the phospholipid solution is 1:0.3-0.7.

[0037] In a preferred embodiment, the mechanical force is 300-500 W ultrasonic oscillation or 12000-20000 rpm mechanical stirring.

[0038] A certain amount of carboxyl activation reagent is added to the emulsion A, and the cross-linking agent-modified hyaluronic acid solution is injected into the water layer in the emulsion A to obtain an emulsion B. The emulsion B is placed at room temperature for a certain period of time, and then the middle emulsion layer is taken out and washed with a non-polar solvent and distilled water and dried to obtain a functionalized graphene oxide nanomedicine carrier.

[0039] In a preferred embodiment, the carboxyl activation reagent is one or a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0040] In a preferred embodiment, the non-polar solvent is dichloromethane or petroleum ether.

[0041] In a preferred embodiment, the volume ratio of the emulsion A to the cross-linking agent-modified hyaluronic acid solution is 20:1-5.

[0042] In a preferred embodiment, the predetermined temperature is 25-40 DEG C, and the predetermined time is 20-30 hours.

[0043] Advantages

[0044] Technical effects and advantages of the present application:

[0045] First, the present application uses phospholipid and hyaluronic acid to covalently graft graphene oxide on both sides, to prepare an active targeting joint cartilage interface functionalized graphene oxide nanomedicine carrier; in an inflammatory environment, the synovial fluid will gradually acidify, and in an acidic environment, the amino site of the phospholipid will be positively charged, which can target and adsorb the negatively charged cartilage; this nanomedicine carrier has the performance of actively targeting the cartilage interface and self-sequencing to form a stable nanomedicine carrier layer in the OA environment.

[0046] Second, the nanomedicine carrier prepared by the present application takes into account the fluid dynamic lubrication of hyaluronic acid, the boundary lubrication of graphene oxide, and the hydration superlubrication performance of phospholipid, so that the cartilage can have excellent lubrication ability in different sliding states, making up for the defect that the existing hyaluronic acid lubricant in clinical practice does not have boundary lubrication.

[0047] Third, the stable nanomedicine carrier layer formed by the nanomedicine carrier prepared by the present application on the cartilage inflammation interface provides a high-efficiency nanomedicine carrier for targeted joint cartilage delivery of drugs. BRIEF DESCRIPTION OF DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0049] Figure 1 It is a structure schematic diagram of the preparation process of the nanomedicine carrier in Embodiment 1 of the present application;

[0050] Figure 2 It is a structure schematic diagram of the nanomedicine carrier in Embodiment 1 of the present application;

[0051] Figure 3 It is a schematic diagram of the nanomedicine carrier and cartilage targeting adsorption in Embodiment 1 of the present application;

[0052] Figure 4 It is a Fourier transform infrared spectrum of the nanomedicine carrier in Embodiment 2 of the present application;

[0053] Figure 5Figure for lubrication performance of the nano-drug carrier in Example 2 of the present application at the interface of natural cartilage;

[0054] Figure 6 Figure for characterization of the cross section of the cartilage after shearing in Example 2 of the present application;

[0055] Figure 7 Figure for the results of biocompatibility detection of the nano-drug carrier in Example 2 of the present application;

[0056] Figure 8 Figure for the dispersion results of the nano-drug carrier with low oxidation degree and high phospholipid content in Example 3 of the present application. DETAILED DESCRIPTION

[0057] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0058] Embodiment 1

[0059] The exemplary embodiment 1 of the present application provides a preparation method of a functionalized graphene oxide nano-drug carrier, and the specific steps are as follows:

[0060] Step S1, 1 g of hyaluronic acid with a molecular weight of 18000000 Dalton is dissolved in 300 mL of distilled water, 80 mg of 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride is added, then 500 mg of adipic acid dihydrazide is added, and the solution is placed at 25℃ for 10 h to obtain solution A. After dialysis, the solution A is dried to obtain adipic acid dihydrazide modified hyaluronic acid.

[0061] Step S2, 20 mg of graphene oxide with a carbon-oxygen ratio of 25% is dispersed in distilled water to obtain a graphene oxide dispersion solution with a concentration of 0.1 mg / mL. Phosphatidylethanolamine and adipic acid dihydrazide modified hyaluronic acid are respectively dissolved in petroleum ether and distilled water to obtain a phosphatidylethanolamine solution with a concentration of 1.5 mg / mL and an adipic acid dihydrazide modified hyaluronic acid solution with a concentration of 0.7 mg / mL. 200 mL of the graphene oxide dispersion solution is mixed with 60 mL of the phosphatidylethanolamine solution to form an emulsion A under the action of ultrasonic.

[0062] Step S3, 30 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 100 mg of N-hydroxysuccinimide were added to the emulsion A, and 40 mL of adipic acid dihydrazide modified hyaluronic acid solution was injected into the water layer of the emulsion A to obtain emulsion B. The emulsion B was placed at 25℃ for 20 h, and the middle emulsion layer was taken and washed with petroleum ether and distilled water, and dried to obtain the functionalized graphene oxide nanomedicine carrier. The preparation process, structural schematic diagram and cartilage targeting adsorption schematic diagram are shown in Figures 1-3 .

[0063] Step S4, the functionalized graphene oxide nanomedicine carrier powder, graphene oxide powder, phosphatidyl ethanolamine and adipic acid dihydrazide modified hyaluronic acid were added to potassium bromide at an addition amount of 1%, and then pressed into a tablet for infrared characterization. Figure 4 Compared with graphene oxide, the functionalized graphene oxide nanomedicine carrier has an additional characteristic peak related to methylene at 2852 cm -1 , 2923 cm -1 , which indicates that the phosphatidyl ethanolamine is successfully grafted onto the graphene oxide. Meanwhile, there is an additional new absorption peak at 1645 cm -1 , which belongs to the carbonyl stretching vibration of amide, indicating that the graphene oxide is connected with the hyaluronic acid through amide reaction.

[0064] Example 2

[0065] The example embodiment 2 of the present application provides a preparation method of the functionalized graphene oxide nanomedicine carrier for actively targeting the articular cartilage interface, and the specific steps are as follows:

[0066] Step S1, 1 g of hyaluronic acid with a molecular weight of 12000 daltons was dissolved in 300 mL of distilled water, 80 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride was added, and then 500 mg of adipic acid dihydrazide was added. After being placed at 30℃ for 30 h, solution A was obtained. After dialysis, the solution A was dried to obtain adipic acid dihydrazide modified hyaluronic acid.

[0067] Step S2, graphene oxide was dispersed in distilled water to obtain graphene oxide dispersion with a concentration of 1 mg / mL and a carbon-oxygen ratio of 40%. Phosphatidyl ethanolamine and adipic acid dihydrazide modified hyaluronic acid were respectively dissolved in petroleum ether and distilled water to obtain a phosphatidyl ethanolamine solution with a concentration of 3 mg / mL and an adipic acid dihydrazide modified hyaluronic acid solution with a concentration of 3 mg / mL. 80 mL of graphene oxide dispersion was mixed with 60 mL of phosphatidyl ethanolamine solution to form emulsion A under the action of ultrasonic.

[0068] Step S3: 80 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 160 mg of N-hydroxysuccinimide were added to emulsion A, and 20 mL of adipic acid dihydrazide-modified hyaluronic acid solution was injected into the aqueous layer of emulsion A to obtain emulsion B. Emulsion B was reacted at 35°C for 15 h. The middle emulsion layer was collected and washed with petroleum ether and distilled water, and dried to obtain a functionalized graphene oxide nanodrug carrier.

[0069] Step S4: To explore the cartilage lubrication and targeted adsorption properties of the functionalized graphene oxide nanodrug carrier, natural bovine cartilage was used as a friction pair to compare the lubrication properties of the functionalized graphene oxide nanodrug with those of clinically injected HA at the same concentration.

[0070] The experimental settings were: load 0.5-2.5 N, rotation speed 60-180 rpm / min, test time 0.5-3 h, and reciprocating stroke 5 mm. The functionalized graphene oxide nanoparticle drug carrier was found to have good tribological properties. The friction coefficient of the lubricated sustained-release nanoparticles at all rotation speeds was lower than that of clinically used hyaluronic acid, such as Figure 5 As shown by Figure 5 It can be seen that the maximum friction coefficient of this nano drug carrier is reduced by 51.7% compared with the clinically used hyaluronic acid. The cross section of the cartilage after shearing was observed using a scanning electron microscope. The results are as follows Figure 6 As shown by Figure 6 It can be seen that the nano drug carriers are targetedly adsorbed and formed into a film at the cartilage interface.

[0071] Step S5: Biocompatibility test of the drug carrier of the present invention and the drug of the present invention. The CCK-8 method was used to determine the effect of different concentrations of the drug carrier of the present invention on the proliferation activity of macrophage RAW264.7. RAW264.7 cells were prepared into uniform 2×10 5 / mL of cell solution was transferred to a 96-well plate at a density of 2×104 cells / well and cultured in a 37°C incubator (5% CO2) for 24 h. 100 μL of 1, 5, 10, 25, 50, 100, and 200 μg / mL modified graphene oxide dispersion of Example 1 was added to the experimental group. 100 μL of culture medium was added to the blank group and the control group, and 6 replicate wells were set up in each group and incubated for 24 h. 10 μL of CCK-8 reagent was added to each well, and after continuing to culture for 1.5 h, the OD value was measured using a microplate reader with the wavelength controlled at 450 nm to calculate the cell survival rate. The results are shown in Figure 2. Figure 7 As shown, it can be seen that the nanomedicine has almost no effect on the activity of RAW264.7 cells at a concentration of 200 mg / mL.

[0072] Example 3

[0073] The example embodiment 3 of the present application provides a method for preparing a functionalized graphene oxide nanodrug carrier, and the specific steps are as follows:

[0074] Step S1, 1 g of hyaluronic acid with a molecular weight of 18000000 Dalton is dissolved in 300 mL of distilled water, 80 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is added, and then 500 mg of adipic acid dihydrazide is added, and the solution is placed at 40℃ for 40 h to obtain solution A. After dialysis, the solution A is dried to obtain adipic acid dihydrazide modified hyaluronic acid.

[0075] Step S2, 20 mg of graphene oxide with a carbon-oxygen ratio of 20% is dispersed in distilled water to obtain a graphene oxide dispersion solution with a concentration of 0.1 mg / mL. Phosphatidylethanolamine and adipic acid dihydrazide modified hyaluronic acid are respectively dissolved in petroleum ether and distilled water to obtain a phosphatidylethanolamine solution with a concentration of 1.5 mg / mL and an adipic acid dihydrazide modified hyaluronic acid solution with a concentration of 0.7 mg / mL. 100 mL of the graphene oxide dispersion solution is mixed with 100 mL of the phosphatidylethanolamine solution to form an emulsion A under ultrasonic action.

[0076] Step S3, 30 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 100 mg of N-hydroxysuccinimide are added to the emulsion A, and 40 mL of the adipic acid dihydrazide modified hyaluronic acid solution is injected into the water layer in the emulsion A to obtain an emulsion B. The emulsion B is placed at 25℃ for 20 h, and the middle emulsion layer is taken and washed with petroleum ether and distilled water, and then dried to obtain a functionalized graphene oxide nanodrug carrier with high phospholipid content. The dispersion result of the material in water is shown in Figure 8 Due to the reduction of oxygen content in graphene oxide and the excessive content of phospholipid, the dispersion performance of the material in aqueous solution is reduced, and agglomeration occurs.

[0077] The steps S1-S4 in the embodiments 1-3 are only for displaying the specific embodiments of the present application, and do not limit the method steps of the present application. The steps can be changed and the order can be changed according to actual needs.

Claims

1. A functionalized graphene oxide nano drug carrier, characterized in that: The raw materials for its preparation include phospholipids, hyaluronic acid and graphene oxide, and the drug carrier is covalently grafted with phospholipids and hyaluronic acid on both sides of the graphene oxide; The C / O ratio of the graphene oxide is (25-40) / 100; The phospholipid is one or more of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine; The preparation method of the functionalized graphene oxide nano drug carrier comprises the following steps: dispersing graphene oxide in distilled water to obtain a graphene oxide dispersion; Dissolve hyaluronic acid in distilled water, add a carboxyl activation reagent, then add a cross-linking agent, and allow to react to obtain solution A. Then, dialyze solution A and dry it to obtain cross-linker-modified hyaluronic acid. dissolving the phospholipid in a non-polar solvent to obtain a phospholipid solution; dissolving the cross-linker-modified hyaluronic acid in distilled water to obtain a cross-linker-modified hyaluronic acid solution; The graphene oxide dispersion is mixed with the phospholipid solution to form emulsion A under the action of mechanical force; A carboxyl activation reagent is added to emulsion A, and a cross-linker-modified hyaluronic acid solution is injected into emulsion A to obtain emulsion B; emulsion B is allowed to react at room temperature, and the middle emulsion layer is taken and washed with a non-polar solvent and distilled water, and dried to obtain a functionalized graphene oxide nano-drug carrier; The molecular weight of hyaluronic acid is 8,000 to 2,500,000 Daltons; The carboxyl activation reagent is one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, diisopropylcarbodiimide, dicyclohexylcarbodiimide and N-hydroxysuccinimide; The cross-linking agent is adipic acid dihydrazide or ethylenediamine; The non-polar solvent includes chloroform, ether, n-hexane, dichloromethane, and petroleum ether; The volume ratio of the graphene oxide dispersion to the phospholipid solution is 1:0.3-0.

7.

2. The functionalized graphene oxide nano drug carrier according to claim 1, characterized in that The concentration of the graphene oxide dispersion is 0.1-2 mg / mL, and / or the concentration of the phospholipid solution is 1-5 mg / mL, and / or the concentration of the cross-linker-modified hyaluronic acid solution is 0.5-5 mg / mL.

3. The functionalized graphene oxide nano drug carrier according to claim 1, characterized in that The mass ratio of the hyaluronic acid to the cross-linking agent is 1:0.2~1.

4. The functionalized graphene oxide nano drug carrier according to claim 1, characterized in that The sheet diameter of the graphene oxide is 5-0.5 μm.

5. The functionalized graphene oxide nano drug carrier according to claim 1, characterized in that The mechanical force is 100-600 W ultrasonic vibration or 6000-30000 rpm mechanical stirring.

6. The functionalized graphene oxide nano drug carrier according to claim 1, characterized in that The volume ratio of the emulsion A to the cross-linker-modified hyaluronic acid solution is 40:1-10.

7. The functionalized graphene oxide nano drug carrier according to claim 1, characterized in that Dissolve hyaluronic acid in distilled water, add a certain amount of carboxyl activation reagent, and then add a certain amount of cross-linking agent, and place it at 25-40°C to react for 10-40 hours to obtain solution A.

Images