Cerium oxide-based nano material for bone defect repair, preparation method of cerium oxide-based nano material and cerium oxide-based composite bone scaffold

Abstract

The invention discloses a cerium oxide-based nano material for bone defect repair and a preparation method of the cerium oxide-based nano material. The cerium oxide-based nano material for bone defect repair is prepared by performing surface modification on cerium oxide-based nano enzyme, and the surface modification comprises introducing a targeting ligand and / or carbon quantum dots into the cerium oxide-based nano enzyme. The invention also discloses a cerium oxide-based composite bone scaffold. The cerium oxide-based composite bone scaffold is prepared by compounding the cerium oxide-based nano-enzyme and / or the cerium oxide-based nano-material and the bone tissue engineering material.

Description

Technical Field

[0001] The present invention belongs to the field of novel bone tissue engineering materials, and is a preparation process for a novel material for functionally modifying cerium oxide-based nanoenzymes to enable the nanoenzymes to have good bone tissue regeneration promoting function. Background Art

[0002] The bone repair process is usually divided into three stages, including the reaction phase (hematoma and inflammation phase), the repair phase (callus formation phase), and the remodeling phase. Early acute inflammation is an important biological process for eliminating pathogens and maintaining tissue homeostasis. However, if pro-inflammatory stimuli persist, the process may develop into a chronic inflammatory state, which leads to the continuous secretion of cytokines and other factors, continuous tissue destruction, and damage to the internal environment, which is harmful to the body's healing.

[0003] When the body is stimulated by external factors, the innate immune system is activated to reestablish normal homeostasis. Among immune cells, the most important cells are macrophages. Macrophages can be divided into two polarized types according to their phenotype and secreted cytokines: macrophages that mainly secrete pro-inflammatory factors and play a pro-inflammatory function are called M1 macrophages, which mainly play an inflammatory response against microorganisms, play a host immune function, and also cause inflammatory damage to normal tissues of the body. Macrophages that mainly play a role in reducing inflammatory responses and playing a role in tissue repair are called M2 macrophages, especially in the late stage of inflammatory response. They play an anti-inflammatory role and promote wound repair and fibrosis. More importantly, there are abundant folate receptors on the surface of macrophages, which lays the foundation for the distribution and targeted enrichment of materials and drugs in the body.

[0004] Oxidative stress is a physiological state in which the excessive accumulation of oxygen species metabolites caused by the generation of intracellular reactive oxygen species (ROS) or damage to the intracellular antioxidant defense system causes damage to the organism. Nano-cerium oxide has the antioxidant properties of simulating superoxide dismutase (SOD) and catalase (CAT), which can eliminate a variety of ROS (O2 - , O2 - , H2O2, ·OH). Nano-cerium oxide can alleviate the pro-inflammatory M1 polarization by scavenging ROS, while promoting the anti-inflammatory M2 polarization by eliminating the excessive production of ROS triggered by stress stimulation.

[0005] Ideal bone repair materials should be plastic; non-toxic and have good biocompatibility; have an appropriate degradation rate and harmless degradation products; have mechanical strength similar to bone tissue; have evenly distributed and appropriate pores for cell adhesion and growth; and have good bone induction, etc. Functional rare earth oxides are doped into them in a planned way, giving the materials new characteristics: improving the biocompatibility of the materials, promoting osteoblast proliferation and differentiation, inducing the expression of related genes, and improving the local microenvironment, etc.

[0006] Cerium oxide (CeO2) is a common catalytic material. At the nanoscale, due to the generation of surface oxygen defects, some Ce in cerium oxide 4+ Reducing to Ce 3+ To stabilize the defects. At this time, Ce in the material 3+ and Ce 4+ This property enables nano-CeO2 to catalyze and decompose excess free radicals in the body, thereby eliminating superoxide free radicals (O2 ·- ), hydrogen peroxide (H2O2), hydroxyl radicals (·OH), etc. This property helps to slow down the inflammatory response, thereby activating local immune regulation and ultimately providing a favorable microenvironment for new bone regeneration.

[0007] Surface modification of nanoparticles is a common modification method. In general, physical and chemical methods are used to treat the surface of ions to purposefully change the physical and chemical properties of the particle surface, such as the surface atomic layer structure and functional groups, surface hydrophilicity, electrical properties and reaction characteristics, so that researchers can control the surface of nanoparticles. Doping is another common method to improve material properties. Purposefully adding a small amount of other elements or compounds to a certain material or matrix can make the material or matrix produce specific physical, electrical, magnetic and optical properties, thereby giving it specific value or use.

[0008] Based on this, the cerium oxide-based bone tissue engineering material involved in the present invention will be based on the unique biological effects of nano-cerium oxide, and a new type of material with excellent osteogenic differentiation promoting effect will be invented, in order to guide and reference the development of bone repair materials. Summary of the invention

[0009] The purpose of the present invention is to provide a cerium oxide-based nanomaterial for bone defect repair and a preparation method thereof in view of the above technical analysis.

[0010] The cerium oxide-based nanomaterial for bone defect repair of the present invention is prepared by surface modification of the cerium oxide-based nanozyme, wherein the surface modification comprises introducing targeting ligands and / or carbon quantum dots into the cerium oxide-based nanozyme.

[0011] Furthermore, the size distribution range of the cerium oxide-based nanomaterial for bone defect repair of the present invention may be 2 nm to 100 nm, preferably 5 nm.

[0012] The cerium oxide-based nanozyme involved in the cerium oxide-based nanomaterial for bone defect repair of the present invention has a narrow distribution of grain size of 5 nm. Smaller size means larger specific surface area and more efficient Ce 3+ With Ce 4+ The conversion of cerium ions into 3+ and 4+ valences can make cerium act as both an oxidation catalyst and a reduction catalyst, thereby showing a variety of enzyme-mimicking activities, such as the enzyme activities of superoxide dismutase (SOD) and catalase (CAT).

[0013] Further, the targeting ligands used for surface modification include folic acid, mannose, phosphatidylserine or lipopolysaccharide. For example, folic acid ligands can be captured by folic acid receptors on the surface of macrophages, thereby enriching the cerium oxide-based nanomaterials on the surface of macrophages.

[0014] Macrophages are important immune cells in the human body. According to their phenotype and secreted cytokines, they can be divided into classically activated M1 and alternatively activated M2 macrophages. As targeted cerium oxide-based nanomaterials are internalized by cells, they can remove excessive reactive oxygen species (ROS) in macrophages by simulating the antioxidant properties of superoxide dismutase and catalase, thereby reducing the number of M1 phenotype macrophages that play a pro-inflammatory role and increasing the number of M2 phenotype macrophages that play an anti-inflammatory role.

[0015] Furthermore, the carbon quantum dots used for surface modification include carbon quantum dots containing amino groups, hydroxyl groups, carboxyl groups or double bonds, and the cerium oxide-based nanozyme surface-modified with amino-containing carbon quantum dots emits yellow or red fluorescence under visible light or near-infrared light excitation.

[0016] Furthermore, the cerium oxide-based nanozyme is prepared by the following methods: solvothermal method, thermal decomposition method or sol-gel method.

[0017] The present invention also discloses a cerium oxide-based composite bone scaffold, which is prepared by subjecting cerium oxide-based nanoenzymes and / or cerium oxide-based nanomaterials to a composite step with bone tissue engineering materials.

[0018] Furthermore, the above-mentioned composite step includes ball milling, covalent grafting or physical adsorption.

[0019] Furthermore, the above-mentioned bone tissue engineering material includes one or more of bioglass, hydroxyapatite, and polycaprolactone.

[0020] Furthermore, the mass fraction of the above-mentioned cerium oxide-based nanozymes and / or cerium oxide-based nanomaterials in the cerium oxide-based composite bone scaffold is 1wt.% to 20wt.%.

[0021] The cerium oxide-based nanozyme and / or cerium oxide-based nanomaterial for bone defect repair is doped in the bone scaffold material and is released to the bone defect site as the scaffold degrades.

[0022] The method for preparing a cerium oxide-based nanomaterial for bone defect repair disclosed in the present invention comprises the following steps:

[0023] 1) Preparation of cerium oxide-based nanozymes;

[0024] 2) Introducing targeting ligands and / or carbon quantum dots into the cerium oxide-based nanozyme.

[0025] The steps of preparing the cerium oxide-based nanozyme include:

[0026] Step 1: dissolve ammonium cerium nitrate and crystalline sodium acetate in water, add acetic acid for acidification, stir to dissolve and transfer to an autoclave lined with polytetrafluoroethylene, and obtain a milky white suspension by solvent thermal reaction in a blast oven.

[0027] Step 2: Wash and centrifuge the suspension obtained in step 1, and finally freeze-dry it in vacuum to obtain cerium oxide-based nanozyme.

[0028] Furthermore, in step 1, the molar ratio of ammonium cerium nitrate to crystalline sodium acetate is 1:12 to 1:16, preferably 1:15; the temperature of the solvent thermal reaction is 220° C., and the duration is 12 hours.

[0029] In the present invention, the cerium oxide-based nanozyme prepared by the above method is generally a milky white fluffy powder.

[0030] In the present invention, the step of introducing the targeting ligand and / or carbon quantum dots into the cerium oxide-based nanozyme comprises:

[0031] Step 1: Add the cerium oxide-based nanozyme to a mixed solution of polyacrylic acid and diethylene glycol, heat and stir under an inert gas environment, then cool to room temperature and add anhydrous ethanol to produce a brown precipitate. After washing, centrifugation, and vacuum freeze-drying, a carboxyl-modified cerium oxide-based nanozyme is obtained.

[0032] Step 2: Disperse the carboxyl-modified cerium oxide-based nanozyme in a 2-(N-morpholine)ethanesulfonic acid solution, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to activate the carboxyl group, and obtain a dispersion containing the carboxyl-activated cerium oxide-based nanozyme.

[0033] Step 3: Mix and stir the dispersion containing carboxyl-activated cerium oxide-based nanozyme with amino-containing carbon quantum dots, wash, centrifuge, and vacuum freeze-dry to obtain carbon quantum dot composite cerium oxide-based nanozyme.

[0034] Step 4: Mix and stir the dispersion containing the carboxyl-activated cerium oxide-based nanozyme with the polyethyleneimine aqueous solution, wash, centrifuge, and vacuum freeze-dry to obtain the amino-modified cerium oxide-based nanozyme.

[0035] Step 5: Disperse the amino-modified cerium oxide-based nanozyme into water, mix and stir with the folic acid ligand whose carboxyl group is activated in advance with EDC and NHS, wash, centrifuge and freeze-dry in vacuum to obtain the folic acid-modified cerium oxide-based nanozyme, i.e., the targeted cerium oxide-based nanozyme.

[0036] Furthermore, in step 1, the molar ratio of polyacrylic acid to the cerium oxide-based nanozyme is 1:6, the heating temperature is 240° C., the duration is 1.5 hours, and the reaction environment is an inert gas.

[0037] Furthermore, in step 2, the pH of the 2-(N-morpholine)ethanesulfonic acid solution is 5.5.

[0038] Furthermore, in step 2 and step 4, the molar ratio of EDC to NHS is 2:5, and the time for activating the carboxyl group is 0.5 to 2 hours.

[0039] Furthermore, in step three, the carbon quantum dot composite cerium oxide-based nanozyme emits yellow or red fluorescence under the excitation of visible light or near-infrared light. Beneficial Effects

[0040] The present invention has at least the following technical effects or advantages.

[0041] Cerium oxide-based nanomaterials have excellent functions of scavenging reactive oxygen species, thereby alleviating inflammation and promoting the regeneration of bone tissue. They can regulate the local microenvironment, reduce the inflammatory response at the bone defect site, and achieve good bone immune regulation.

[0042] Under the stimulation of the cerium oxide nanozymes contained in the cerium oxide nanomaterials, macrophages will secrete biological factors related to osteogenesis and angiogenesis, stimulating downstream osteoblasts and vascular epithelial cells to transform into new bone tissue and blood vessels respectively. At the same time, the two cells can also achieve cross-stimulation of biological factors, promoting bone formation and angiogenesis at the same time, and ultimately achieving bone integration in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a roadmap for the preparation process of the targeted cerium oxide-based nanomaterial of the present invention.

[0044] Figure 2 This is a transmission electron microscope photograph of the targeted cerium oxide-based nanomaterial of the present invention.

[0045] Figure 3 It is an infrared spectrum of the targeted cerium oxide-based nanomaterial of the present invention. Implementation

[0046] The advantages and various effects of the present invention will be specifically described below in conjunction with specific embodiments and examples. Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. If there is a conflict, this specification takes precedence.

[0047] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0048] The cerium oxide-based nanomaterial for bone defect repair and its preparation method of the present application will be described in detail below in combination with examples and experimental data.

[0049] Embodiment 1:

[0050] The method for preparing cerium oxide-based nanozyme for bone defect repair comprises the following steps.

[0051] Step 1: Dissolve 2.74 g of ammonium cerium nitrate and 10.00 g of crystalline sodium acetate in 56 mL of deionized water, then add 10 mL of acetic acid to the solution and stir at room temperature for 1 hour to dissolve.

[0052] Step 2: The mixed solution was transferred to a 100 mL polytetrafluoroethylene-lined autoclave, and the solvent thermal reaction was carried out at 220 °C for 20 h, and then naturally cooled to room temperature.

[0053] Step 3: The suspension in the kettle is washed several times and centrifuged (10,000 rpm) to separate the cerium oxide-based nanozyme suspension containing a small amount of water. The suspension is pre-frozen and placed in a vacuum freeze dryer, and dried at -80°C for 24 hours under vacuum to obtain the cerium oxide-based nanozyme.

[0054] Embodiment 2:

[0055] The invention discloses a method for preparing a targeted cerium oxide-based nanomaterial for repairing bone defects, comprising the following steps.

[0056] Step 1: Disperse 100 mg of the cerium oxide-based nanozyme described in Example 1 in 2 mL toluene for standby use. 300 mg of polyacrylic acid and 30 mL of dihexanediol were placed in a 150 mL three-necked flask and stirred evenly. Vacuum was evacuated several times, nitrogen was introduced, and the system was slowly heated to 110 ° C for 30 min. 50 mg / mL of cerium oxide-toluene dispersion was slowly injected into the three-necked flask with a syringe and continued to stir at 110 ° C for 1.5 hours. The system was then heated to 240 ° C and continued to stir for 1.5 hours. After the reaction was completed, it was naturally cooled to room temperature, 30 mL of ethanol was added to precipitate the product, and centrifuged at 6000 rpm. After centrifugation, the supernatant was discarded, deionized water was added to the product, and the above washing and centrifugation operations were repeated several times to obtain a carboxyl-modified cerium oxide nanozyme suspension containing a small amount of water. The suspension was pre-frozen and placed in a vacuum freeze dryer, and dried at -80°C for 24 hours under vacuum to obtain a carboxyl-modified cerium oxide-based nanozyme.

[0057] Step 2: Disperse 100 mg of carboxyl-modified ceria nanozyme into 50 mL of 2-(N-morpholino)ethanesulfonic acid solution (10 mM, pH = 5.5), then add 40 mM EDC and incubate at room temperature with shaking for 15 minutes. Then add 100 mM NHS to the mixed solution and continue stirring for 2 hours.

[0058] Step 3: Slowly add 50 mL of PEI aqueous solution (1 mg / mL) to the mixed solution and stir at room temperature for 24 hours. The crude product was washed several times and centrifuged (8000 rpm) to separate a suspension containing a small amount of water. The suspension was pre-frozen and placed in a vacuum freeze dryer, and dried at -80 °C under vacuum for 24 hours to obtain amino-modified cerium oxide-based nanozymes.

[0059] Step 4: Take 20 mg of folic acid powder and dissolve it in 50 mL of dimethyl sulfoxide, then add 100 mM EDC and incubate it at room temperature with shaking for 15 minutes. Then add 250 mM NHS to the mixed solution and continue stirring for 2 hours. Slowly add 50 mL of amino-modified cerium oxide-based nanozyme solution (2 mg / mL) to the above mixed solution and stir it at room temperature for 24 hours. The crude product can be separated into a suspension containing a small amount of water after multiple washings and centrifugation (8000 rpm). The above suspension is pre-frozen and placed in a vacuum freeze dryer, and dried at -80 °C under vacuum for 24 hours to obtain the desired folic acid-modified cerium dioxide nanomaterial, that is, a targeted cerium oxide-based nanomaterial for bone defect repair.

[0060] Embodiment 3:

[0061] The method for preparing carbon quantum dot composite cerium oxide-based nanozyme for bone defect repair comprises the following steps.

[0062] Step 1: Disperse 100 mg of the cerium oxide-based nanozyme described in Example 1 in 2 mL of toluene for standby use. 300 mg of polyacrylic acid and 30 mL of dihexanediol were placed in a 150 mL three-necked flask and stirred evenly. Vacuum was evacuated several times, nitrogen was introduced, and the system was slowly heated to 110 ° C for 30 min. 50 mg / mL of cerium oxide-toluene dispersion was slowly injected into the three-necked flask with a syringe and continued to stir at 110 ° C for 1.5 hours. The system was then heated to 240 ° C and continued to stir for 1.5 hours. After the reaction was completed, it was naturally cooled to room temperature, 30 mL of ethanol was added to precipitate the product, and centrifuged at 6000 rpm. After centrifugation, the supernatant was discarded, deionized water was added to the product, and the above washing and centrifugation operations were repeated several times to obtain a carboxyl-modified cerium oxide nanozyme suspension containing a small amount of water. The suspension was pre-frozen and placed in a vacuum freeze dryer, and dried at -80°C for 24 hours under vacuum to obtain the desired carboxyl-modified cerium oxide-based nanozyme.

[0063] Step 2: Disperse 100 mg of carboxyl-modified ceria nanozyme into 50 mL of 2-(N-morpholino)ethanesulfonic acid solution (10 mM, pH = 5.5), then add 40 mM EDC and incubate at room temperature with shaking for 15 minutes. Then add 100 mM NHS to the mixed solution and continue stirring for 2 hours.

[0064] Step 3: Mix the dispersion containing carboxyl-activated cerium oxide-based nanozyme with 1 mg / mL amino-containing carbon quantum dots, stir at room temperature for 4 hours, wash, centrifuge, and vacuum freeze-dry to obtain carbon quantum dot composite cerium oxide-based nanozyme.

[0065] Embodiment 4:

[0066] The method for preparing a cerium oxide-based composite bone scaffold for repairing bone defects comprises the following steps.

[0067] Step: 1: Add 26.8 g of ethyl orthosilicate, 5.6 g of calcium nitrate, 3.0 g of triethyl phosphate and 4 g of 1 mol / L hydrochloric acid solution into 300 mL of anhydrous ethanol, stir at room temperature for 24 hours, then expose to the air, wait for the solvent to evaporate completely, grind with an agate mortar to obtain white powdered bioglass.

[0068] Step 2: Mix the cerium oxide-based nanozyme of Example 1 with 2 g of bioglass powder at a mass fraction of 1%, and ball mill it for 5 hours to obtain a bone tissue engineering scaffold doped with cerium oxide for implantation in bone defect sites.

[0069] Experimental test analysis results:

[0070] Reference Figure 1 , which shows the synthetic route of folate-modified cerium oxide-based nanozymes, i.e., cerium oxide-based nanozymes were functionalized with polyacrylic acid through ligand exchange reaction. Subsequently, polyethyleneimine was used as a bridge, and the folate ligand was modified on the surface of the nanozyme through a two-step EDC / NHS coupling reaction. After purification, the desired folate-modified cerium dioxide nanomaterial was obtained, i.e., the targeted cerium oxide-based nanozyme for bone defect repair.

[0071] Reference Figure 2 , which shows that the targeted cerium oxide-based nanomaterials for bone defect repair are nanoscale after multi-step surface modification, with a size distribution of 5 to 10 nm.

[0072] Reference Figure 3 , the figure shows that through Figure 1 The steps shown successfully prepared targeted cerium oxide-based nanomaterials with surface modified with folic acid ligands for bone defect repair.

Claims

1. A cerium oxide-based nanomaterial for bone defect repair, characterized in that: The cerium oxide-based nanozyme is prepared by surface modification of the cerium oxide-based nanozyme, wherein the surface modification includes introducing a targeting ligand such as mannose, phosphatidylserine, lipopolysaccharide, triphenylphosphine, a cell targeting peptide (including but not limited to RGD, RGDM, CRGDYK or RGDFC) and / or carbon quantum dots into the cerium oxide-based nanozyme; and compounding the nanozyme into a scaffold by means of ball milling, covalent grafting or physical adsorption to prepare a cerium oxide-based composite bone scaffold, characterized in that the scaffold includes the following steps: 1) Preparation of cerium oxide-based nanozymes; 2) introducing targeting ligands and / or carbon quantum dots into the cerium oxide-based nanozyme; 3) Compounding the above-mentioned cerium oxide-based nanozyme into the bone scaffold.

2. The cerium oxide-based composite bone scaffold simulates the properties of biological enzymes including but not limited to superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione peroxidase (GSH-Px), glucose oxidase (GOx), lipoxygenase (LOX), polyphenol oxidase (PPO), cytochrome reductase, and various oxygenases, hydrogenases, etc., to adjust the different phenotypes of macrophages, alleviate pro-inflammatory M1 polarization, and promote anti-inflammatory M2 polarization, thereby achieving anti-inflammatory effects. Under the stimulation of the cerium oxide-based nanozymes contained in the cerium oxide-based nanomaterials, macrophages will secrete biological factors related to osteogenesis and angiogenesis, stimulating downstream osteoblasts and vascular epithelial cells to transform into new bone tissue and blood vessels, respectively. At the same time, the two cells can also achieve cross-stimulation of biological factors, while promoting bone formation and angiogenesis, and ultimately achieving bone integration in vivo. In addition, the combination of cerium oxide-based nanozymes and bone scaffolds not only increases the density of the composite scaffold, but also plays the role of cerium oxide as a nucleating agent in the glass-ceramic formation process, significantly improving the mechanical strength of the original bone scaffold, making the composite scaffold have broader application prospects in the field of bone tissue engineering.

3. The cerium oxide-based nanomaterial for bone defect repair according to claim 1, characterized in that: The size distribution ranges from 2 nm to 200 nm.

4. The cerium oxide-based nanomaterial for bone defect repair according to claim 1, characterized in that: The carbon quantum dots include carbon quantum dots containing amino groups, hydroxyl groups, carboxyl groups and / or double bonds, and the cerium oxide-based nanozyme surface-modified with the amino-containing carbon quantum dots emits yellow or red fluorescence under the excitation of visible light or near-infrared light.

5. The cerium oxide-based nanomaterial for bone defect repair according to claim 1, characterized in that: The cerium oxide-based nanozyme is prepared by the following methods: solvothermal method, thermal decomposition method or sol-gel method.

6. A cerium oxide-based composite bone scaffold, characterized in that: The cerium oxide-based nanozyme and / or cerium oxide-based nanomaterial described in claim 1 is prepared by subjecting the material to ball milling, covalent grafting or physical mixing.

7. The cerium oxide-based bone scaffold according to claim 5, characterized in that: The bone tissue engineering material includes one or more of bioactive glass, hydroxyapatite, tricalcium phosphate, polylactic acid-co-glycolic acid, polycaprolactone, gelatin, sodium alginate, and collagen.

8. The cerium oxide-based bone scaffold according to claim 5, characterized in that: The mass fraction of the ceria-based nanozyme and / or the ceria-based nanomaterial in the ceria-based bone scaffold is 1 wt.% to 20 wt.%.

Images