CN116942900B

CN116942900B - A method for preparing nanoenzyme-modified orthopedic polymer and its product and application

Info

Publication number: CN116942900B
Application number: CN202310975833.9A
Authority: CN
Inventors: 魏辉; 刘淑杰; 郑力铭
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2023-08-04
Filing date: 2023-08-04
Publication date: 2025-01-28
Anticipated expiration: 2043-08-04
Also published as: CN116942900A

Abstract

The present invention discloses a method for preparing nanozyme-modified orthopedic polymers, and its products and applications, comprising the following steps: obtaining a modified polymer by in-situ growth of nanozymes in a polymer; mechanically processing the modified polymer to form a polymer implant; and using the modified polymer implant for the treatment, relief and/or prevention of orthopedic diseases. The nanozyme-modified orthopedic polymer implant prepared by the present invention can reduce the postoperative medication dosage, reduce adverse drug reactions and economic burden; enhance mechanical properties, reduce complications after implantation; prolong the protective effect on the body; avoid physiological toxicity; and can regulate and design multi-enzyme activities for personalized customization of diseases.

Description

Method for preparing nano enzyme modified orthopedic polymer, product and application thereof

Technical Field

The invention relates to a method for preparing a nano enzyme modified orthopedic polymer, a product and application thereof, belonging to the crossing field of biomedical engineering and functional materials.

Background

Bone tissue provides structural support for the body, and maintaining the health and integrity of bone tissue is critical to the normal life and movement of the human body. However, with the increasing global aging degree and the change of life style of people, the incidence of bone diseases has shown an increasing trend, resulting in impaired bone tissue function of patients. In response to this phenomenon, polymers are widely used in the treatment of orthopedic disorders for their good biocompatibility, processability and mechanical properties, as an implantable medical device to repair, enhance and replace bone tissue function or to act as a support or anchor for artificial cartilage.

Bone tissue maintains the functional stability of bone tissue, i.e., bone metabolic balance, through dynamic balance between osteogenesis and osteoclast. Functional defects in bone tissue are often caused by physical injury or imbalance in bone metabolism that accumulates over time. Orthopedic diseases causing serious damage to bone tissue function include advanced arthritis, osteoporosis, bone tumor, osteomyelitis, large bone defect, cone fracture and the like, and are characterized in that bone metabolism of the body is unbalanced and drug treatment is ineffective, and a focus area needs to be managed by using surgery. In this case, the ideal polymer implant is not only required to exert a supporting effect on the mechanical level, but also to assist the body in restoring bone metabolic balance as much as possible, helping the patient to achieve good postoperative healing and functional recovery. However, conventional orthopedic polymers often fail to achieve good osseointegration and restoration of postoperative bone metabolic balance, the cause of this phenomenon is manifold:

1. Foreign body reaction by the implant.

Although the polymer implant has good biocompatibility and bioinert properties, it acts as a non-host tissue that immediately after implantation causes the immune system of the body to react in that immune cells are activated and accumulate around the implant, releasing reactive oxygen species (Reactive oxygen species, ROS) to attempt phagocytosis and clearance of the implant. However, such phagocytosis is often unsuccessful, which in turn results in the accumulation of excess reactive oxygen species and the release of large amounts of cytokines, ultimately creating excessive inflammatory responses at the host and implant interface and causing damage to surrounding tissues, such as bone tissue resorption and scar tissue formation. Furthermore, in order to isolate and encapsulate the implant, the body forms a fibrous envelope around the implant to limit contact between the implant and the tissue, and the formation of such fibrous envelope may lead to a decrease in the stability of the implant while limiting its osseointegrative effect. Thus, reducing the foreign body response caused by a polymer implant is critical to improving its functional stability in vivo.

2. The micro-environmental regulation capability of the implant is insufficient.

Patients undergoing implantation surgery often suffer from severe bone diseases themselves, with severe imbalance in bone metabolism, often accompanied by complex conditions such as inflammation, autoimmune diseases, tumors, etc., often resulting in increased ROS content in the pathological microenvironment. Implantation surgery can help clear necrotic tissue, but does not thoroughly reverse the progression of the disease, in which case the focal zone microenvironment cannot be effectively modulated by conventional polymer implants alone. Although post-operative drug management can be performed by using drugs such as bisphosphonates, non-steroidal anti-inflammatory drugs, immune antagonists, etc., long-term systemic administration causes serious side effects including kidney damage, dyspepsia, cardiovascular damage, malignant tumor, etc., and aggravates the economic burden of patients. Therefore, modifying the implant to enhance the pathological microenvironment regulation capability is a safe and economical design thought, realizes local immunoregulation of the focus area, and reduces the frequency and dosage of systemic administration. There are studies to improve the pathological microenvironment controlling ability of implants by forming a coating on the surface of the implant and loading and releasing bioactive substances such as cytokines, antioxidants, anti-inflammatory drugs, etc. through the coating. However, the coating is degraded during use, and the carried bioactive substances are gradually dissipated over time, so that the long-term microenvironment regulation effect is difficult to maintain. Therefore, a new modification mode is developed to enable the polymer implant to have long-term physiological microenvironment regulating capability, and has positive effect on the postoperative bone metabolism balance recovery of patients.

3. The mechanical function of the implant is degraded.

Depending on the role that the orthopaedic polymer plays in bone tissue and the mechanical properties required, it can be divided into load-bearing polymer materials and non-load-bearing polymer materials. Load-bearing polymeric materials are used to carry loads and are commonly used in bone repair and replacement procedures, such as artificial joint shims, bone fusion devices, and bone plates, etc., which are generally high in strength, rigidity, and wear resistance. However, load-bearing polymer implants still suffer from deterioration of mechanical properties under prolonged loading, manifested by reduced strength, reduced toughness, particle fall-off, and even cracking and fracture. Not only does the degradation of mechanical properties result in the implant not functioning properly, but it may even present serious health risks to the patient. For example, after joint replacement surgery, about 10% of patients need revision surgery 10-20 years after surgery because of aseptic loosening, and particles of the polymer liner that fall under long-term load are the main cause of aseptic loosening. The dropped particles activate the host's immune system, resulting in a massive accumulation of ROS, resulting in excessive activation of osteoclasts and peri-implant osteolysis, ultimately resulting in loosening of the prosthesis and even surrounding fractures. It follows that maintaining the long-term mechanical properties of the polymer implant intact is an important means of ensuring its long-term functioning.

In summary, the defects of the current polymer implant for orthopaedics can be found that the ideal modification direction of the polymer implant for orthopaedics is as follows, (1) reducing foreign body reaction caused by the implant, (2) long-term stable pathological microenvironment regulating capability and (3) long-term stable mechanical property.

ROS are oxygen metabolites involved in cell signaling and regulation of cell function, but in pathological conditions of inflammation, autoimmune diseases, tumors, foreign body implantation, infection, etc., excessive ROS can lead to oxidative stress, which in turn exacerbates the disease. Extensive research has been conducted to eliminate excess ROS as a means of regulating inflammatory resolution and restoring bone metabolic balance. Conventional antioxidants, however, eliminate ROS in a sacrificial manner, and their effectiveness decreases over time. Although natural enzymes can efficiently scavenge ROS in a non-chemical dose-dependent form, their instability limits practical use.

The doping of the nano-filler is an effective method for enhancing the mechanical properties of the polymer, and the nano-material can obviously improve the mechanical properties, the thermal stability, the wear resistance and the like of the polymer through a high specific surface area effect, an interface enhancement effect and a nano-size effect. However, the reinforcing effect is limited by the dispersion condition of the nano-filler in the polymer matrix, and the agglomeration of the nano-filler is often caused by directly mechanically blending the nano-filler and the polymer and then molding, so that the mechanical property of the polymer is reduced. Thus, nanoparticles grown in situ in the polymer generally have better dispersibility and better mechanical reinforcement compared to conventional physical mixing.

The nano enzyme is a nano material with enzyme-like catalytic property, and has both the active center of the enzyme-like and the physicochemical property of the nano material. Nanoenzymes have various enzyme activities including superoxide dismutase (SOD) and Catalase (CAT), and have been widely used for antioxidant therapy in various disease models. Meanwhile, the nano enzyme has high stability and controllability, and can be suitable for severe working environments which cannot be tolerated by various natural enzymes.

The patent (application number: 2021104391305) discloses a bone implant material based on nano enzyme modification, and a preparation method and application thereof, wherein the bone implant material based on nano enzyme modification is obtained by modifying a bone implant material by nano enzyme or a precursor of nano enzyme (nano enzyme can be prepared in situ). However, the modification of polyethylene stays on the raw material level, and the processing and mechanical property test of the product are not performed, so that whether the modification mode of the polyethylene can affect the processing, forming and mechanical properties of the implant cannot be determined, the proposed modification mode of the polyethylene is suitable for polyethylene with the molecular weight of 200 ten thousand, and the modification mode of the polyethylene cannot meet the requirement of clinically practical ultra-high molecular weight polyethylene (the molecular weight is more than or equal to 500 ten thousand). The single yield of the modification mode proposed in the patent is in the milligram level, and the raw material consumption required by the actual product preparation is difficult to meet. In addition, in the modification method of the patent, the obtained modified polyethylene is subjected to liquid nitrogen cooling and crushing, and the molecular weight of the polyethylene is reduced in this way, so that the mechanical properties of the finished product are further reduced. Moreover, the nano-enzyme modified titanium alloy implant modification proposed in this patent is to perform nano-enzyme modification on the surface of the titanium alloy to form a coating. In view of the fact that titanium alloy prostheses are commonly used in prosthetic joint implants for the preparation of articular surfaces, femoral head balls, and the like, their anti-friction properties and mechanical aging properties are particularly important for the long-term stability of the prostheses, however, the patent does not address the relevant aspects of testing modified titanium alloy prostheses. In addition, in practical applications, peeling and degradation of the nano-enzyme coating may occur with long-term reciprocating rubbing movement, so that long-term stability of the nano-enzyme surface modification may lag behind in-situ modification.

In summary, aiming at the defects existing in the current orthopedic polymer implant, the use of nano enzyme as a modification medium is a method capable of simultaneously improving the mechanical property and the physiological microenvironment regulating capability of the implant.

Disclosure of Invention

The invention aims to solve the defects of the existing polymer implant for orthopaedics, and provides a method for improving the performance of the polymer implant by modifying nano enzyme, which specifically comprises the steps of ① reducing foreign body reaction caused by the implant, ② prolonging the maintenance time of the physiological microenvironment regulating capability of the implant, ③ improving the mechanical performance of the implant, delaying the generation of wear particles and ④ reducing the immune reaction caused by the wear particles.

The first object of the invention is to provide a method for synthesizing nano enzyme in situ in polymer, the second object of the invention is to provide a method for processing polymer after modification of nano enzyme, and the third object of the invention is to provide the application of nano enzyme modified polymer implant in improving orthopedic diseases.

The invention provides a method for preparing a nano enzyme modified polymer for orthopaedics, which aims to solve the technical problems and comprises the following steps:

(1) Dissolving polymer powder in solvent A for assisting polymer swelling and/or dissolution, swelling and/or dissolution under heating,

(2) Dispersing or dissolving one or more nano-enzyme or nano-enzyme precursor in its good solvent B solvent, and adding the B solvent into the swelled and/or dissolved polymer,

(3) The resulting nanoenzyme-modified polymer was subjected to solvent substitution using a C solvent (dielectric constant difference of 30 or more) which was not miscible with the a solvent, followed by washing and drying.

Wherein the polymer powder in the step (1) comprises one or more of ultra-high molecular weight polyethylene, low molecular weight polyethylene, polypropylene, polyvinyl chloride, polytetrafluoroethylene, polystyrene, polyamide, polyether ether ketone, polylactic acid-co-glycolic acid, polylactic glycolic acid, ethylene-vinyl acetate copolymer, polycarbonate, polyurethane, polycaprolactone, polylactic acid-co-caprolactone, polymethyl methacrylate or polyethylene terephthalate.

Preferably, the polymer powder is ultra-high molecular weight polyethylene (molecular weight not less than 500 ten thousand).

Wherein the solvent A in the step (1) comprises one or more of isopentane, n-pentane, decalin, petroleum ether, hexane, cyclohexane, isooctane, trifluoroacetic acid, trimethylpentane, cyclopentane, heptane, trichloroethylene, toluene, paraxylene, chlorobenzene, orthodichlorobenzene, benzene, isobutanol, dichloromethane, butanol, n-propanol, tetrahydrofuran, ethanol, ethyl acetate, petroleum ether, cyclohexane, chloroform, benzene, toluene, dichloromethane, diethyl ether, ethyl acetate, isopropanol, chloroform, pyridine, acetone, acetic acid, acetonitrile, aniline, dimethylformamide, methanol, ethylene glycol, dimethyl sulfoxide or water.

Preferably, the solvent A is decalin.

Wherein the nanoenzyme in step (2) comprises one or more combinations of carbon-based nanoenzyme, nitrogen-based compound nanoenzyme, oxygen-based compound nanoenzyme, metal organic framework-based nanoenzyme, covalent organic framework-based nanoenzyme, metal coordination nanoenzyme or composite nanoenzyme.

Further, the carbon-based nanoenzyme includes one or more of a fullerene-based nanoenzyme, a carbon fiber-based nanoenzyme, a carbon nanotube-based nanoenzyme, a graphene-based nanoenzyme, or other carbon-based nanoenzyme.

The nitrogen group compound nanoenzyme includes one or more combinations of nitride-based nanoenzymes or phosphide-based nanoezymes.

The oxy-nanoenzyme comprises one or more combinations of an oxide-based nanoenzyme, a sulfide-based nanoenzyme, a selenide-based nanoenzyme, or a telluride-based nanoenzyme.

The metal nano enzyme comprises one or a combination of more of gold nano enzyme, copper nano enzyme, silver nano enzyme, platinum nano enzyme, palladium nano enzyme, rhodium nano enzyme, ruthenium nano enzyme or alloy nano enzyme.

The metal organic frame based nano-enzyme comprises one or more of iron-based metal organic frame nano-enzyme, zinc-based metal organic frame nano-enzyme, copper-based metal organic frame nano-enzyme, zirconium-based metal organic frame nano-enzyme, hafnium-based metal organic frame nano-enzyme, vanadium-based metal organic frame nano-enzyme, metal doped metal organic frame nano-enzyme or carbon-based nano-enzyme.

The covalent organic framework nano-enzyme comprises one or more combinations of boron covalent organic framework nano-enzyme, imine covalent organic framework nano-enzyme, triazine covalent organic framework nano-enzyme or other types of covalent organic framework nano-enzyme.

Preferably, the nanoenzyme is an oxygen group compound nanoenzyme, the oxygen group compound nanoenzyme is an oxide group nanoenzyme, and the oxide group nanoenzyme is cerium oxide.

The nano enzyme contains one or more of a lithium element, a magnesium element, an aluminum element, a potassium element, a calcium element, a scandium element, a titanium element, a vanadium element, a chromium element, a manganese element, an iron element, a cobalt element, a nickel element, a copper element, a zinc element, a gallium element, a germanium element, a rubidium element, a strontium element, an iridium element, a zirconium element, a niobium element, a molybdenum element, a ruthenium element, a rhodium element, a palladium element, a silver element, a tin element, an antimony element, a cerium element, a hafnium element, an iridium element, a platinum element, a gold element or a bismuth element.

Preferably, the nano-enzyme contains cerium element.

Wherein the nano-enzyme precursor comprises one or more of organic metal salt, metal chloride salt, metal bromide salt, metal nitrate salt, metal acetate salt, metal sulfate salt or metal cyanide complex.

Preferably, the nano-enzyme precursor is an organometallic salt.

Wherein the organic metal salt comprises one or more of metal acetylacetonate, metal alkoxide, metal acid salt, metal ether acid salt, metal ether alkoxide, metal amide salt, metal alkoxide, metal carboxylic acid amide salt, metal ether amide salt, metal imide salt, metal phosphorus complex or metal nitrogen complex.

Preferably, the organometallic salt is a metal acetylacetonate.

Wherein the metal acetylacetonate contains a metal element including one or more of lithium element, magnesium element, aluminum element, potassium element, calcium element, scandium element, titanium element, vanadium element, chromium element, manganese element, iron element, cobalt element, nickel element, copper element, zinc element, gallium element, germanium element, rubidium element, strontium element, iridium element, zirconium element, niobium element, molybdenum element, ruthenium element, rhodium element, palladium element, silver element, tin element, antimony element, cerium element, hafnium element, iridium element, platinum element, gold element, or bismuth element.

Preferably, the metal acetylacetonate is cerium acetylacetonate.

Wherein, the good solvent B solvent for dispersing the nano enzyme or nano enzyme precursor in the step (2) comprises one or a combination of more of ethanol, ethyl acetate, isopropanol, chloroform, pyridine, acetone, acetic acid, acetonitrile, aniline, dimethylformamide, methanol, glycol, dimethyl sulfoxide or water.

Preferably, the B solvent is ethanol.

Wherein, the solvent C used for solvent replacement in the step (3) is not mutually soluble with the solvent A, and the dielectric constants of the solvent C and the solvent A are different by more than 30.

Wherein the C solvent comprises one or more of ethanol, ethyl acetate, isopropanol, chloroform, pyridine, acetone, acetic acid, acetonitrile, aniline, dimethylformamide, methanol, ethylene glycol, dimethyl sulfoxide or water.

Preferably, the C solvent is methanol.

Wherein, in the step (3), the nano enzyme modified polymer powder after solution replacement is subjected to ethanol filtration and washing for at least 10 times, and is heated in a vacuum oven at 80 ℃ for 7 days to volatilize the residual solvent.

The invention also provides an orthopaedics polymer prepared by the method.

The invention also provides a method for preparing a modified polymer implant by using the orthopedic polymer, which comprises the following steps:

(1) Performing mechanical molding and morphology processing on the modified polymer to obtain a crude modified polymer implant;

(2) And (3) carrying out post-processing treatment on the crude modified polymer implant to obtain the modified polymer implant.

Wherein the mechanical molding step of the nano enzyme modified polymer in the step (1) comprises one or more of hot press sintering, hot press molding, injection molding, blow molding, extrusion molding, calendaring molding, 3D printing molding, vacuum molding or spray molding.

Preferably, the molding mode is hot press molding.

Wherein the 3D printing forming comprises one or more of a fused deposition method, a photo-curing method, a laser sintering method, a powder melting method or an electron beam melting method.

Preferably, the 3D printing forming mode is a fused deposition method.

Wherein the topography processing method in step (1) comprises one or more of cutting, grinding, drilling, milling or cutting.

Preferably, the machining is milling.

Wherein the post-processing treatment step in step (2) comprises one or more combinations of cleaning, polishing, sandblasting, acid washing, plasma treatment, solvent treatment, silane coupling agent treatment, chemical coating treatment, or ion implantation treatment.

Preferably, the post-processing treatment is cleaning.

The invention also provides a modified polymer implant prepared by the method.

Wherein the nano enzyme modified polymer product for orthopaedics comprises an artificial joint gasket, a bone plate, bone nails, bone cement, a bone bracket, a bone fusion device, a bone filler, a bone fixing belt, an artificial bone or periosteum.

Preferably, the molded device is an artificial joint spacer.

The invention also provides application of the orthopedic polymer in preparing an orthopedic implant for treating, relieving and/or preventing orthopedic diseases.

Wherein the orthopedic disease comprises advanced arthritis, aseptic loosening, bacterial loosening, osteoporosis, bone tumor, repair of large bone defects, osteomyelitis or fracture.

The principle of the invention is as follows:

1. According to the invention, the polymer is swelled and then is injected into the nano enzyme precursor to prepare the modified polymer, and the intermolecular gap is increased after the polymer is swelled, so that the growth and dispersion of the nano enzyme in the polymer are facilitated. Compared with the traditional physical mixing, the nano-enzyme obtained by in-situ growth can realize good dispersion in a polymer matrix and improve mechanical properties. In addition, the melt environment formed after the polymer swells delays the oxidation process in the cerium oxide crystallization process, which is conducive to the formation of smaller-sized nano-enzymes. Compared with the preparation of the nano-enzyme by the hydrothermal method proposed in patent (application number: 2021104391305), the nano-enzyme grown in situ in polyethylene at the same time has smaller particle size, and the smaller particle size helps to realize more excellent catalytic performance.

2. Considering that the polymer implant needs to exert oxidation resistance and bear mechanical load in the application process, the invention simultaneously utilizes the mechanical enhancement property and enzyme-like catalytic property of the nano enzyme as the nano material, thereby realizing the improvement of polymer properties in multiple aspects. Compared with the patent (application number: 2021104391305), the invention has more comprehensive modification on the polymer and is more close to the clinical practical application situation.

3. In the preparation process, the modified polymer is subjected to post-synthesis treatment in modes of swelling, solvent replacement, cooling crystallization and the like, so that the modified polymer is converted from a molten state to a state which can be used for mechanical processing. Compared with the patent (application number: 2021104391305), the method does not use the steps of intense stirring, liquid nitrogen crushing and the like in the process of collecting and treating the modified polymer, does not reduce the molecular weight of the polymer, and is beneficial to the maintenance of the mechanical properties of subsequent products.

4. According to the invention, the polymer is swelled by using the nonpolar solvent decalin in the preparation process, so that the molecular weight range of the modified polyethylene is widened, and the yield of the single modified polymer is improved. Compared with the patent (application number: 2021104391305), the modified polyethylene can meet the modification requirement of high molecular weight polyethylene with molecular weight of more than 500 ten thousand, and in addition, the mass preparation is beneficial to practical processing application.

5. The invention carries out mechanical molding and morphology treatment on the obtained modified polymer raw material, can prepare implants with different morphologies, and meets the treatment requirements under different pathological conditions. Compared with the patent (application number: 2021104391305), the artificial joint spacer product is prepared by the patent, and the feasibility of the polymer modification and subsequent processing method is proved.

Compared with the prior art, the invention has the following remarkable advantages:

1. The nano enzyme is selected as a modification medium of the implant, has the catalytic capability of various enzymes, can efficiently catalyze and eliminate various ROS, realizes the alleviation of inflammation, and further improves the treatment effect of the anti-inflammatory drug. In addition, the reduced foreign body response intensity caused by the nano-enzyme modified implant also helps to reduce the dosage of post-operative drug. The polymer implant modified by the nano enzyme can help reduce the dosage of the medicine after operation and avoid adverse reaction of long-term medicine and economic burden of patients by partially compensating the medicine and weakening the stimulation to organisms;

2. The invention enhances the mechanical performance and reduces the complications after implantation by growing the nano-enzyme in situ in the polymer matrix and utilizing the physical and chemical properties of the nano-enzyme as the nano-material, thereby mechanically enhancing various polymers and being beneficial to the mechanical stability of the polymer implant. In addition, by reducing the falling of implant particles, the generation of postoperative complications can be effectively inhibited, and the service life of the prosthesis is prolonged;

3. The invention can realize modification of polymer materials on macroscopic and integral level by growing nano enzyme in situ in polymer matrix, and the nano enzyme in polymer matrix can maintain stability of structure and function under long-term mechanical action. In addition, even if the modified polymer device falls off the particle fragments, nano enzyme with catalytic activity still exists in the fallen particles, so that the up-regulation of ROS secretion caused by the particles can be relieved, and the long-term protection of the physiological environment of the organism is realized;

4. The invention can ensure that the nano enzyme can play a role in the implantation part for a long time by in-situ growth of the nano enzyme in the polymer, endowing the nano enzyme activity of the polymer matrix, and simultaneously protecting and fixing the nano enzyme by the polymer matrix. The risks of falling off and absorption of the nano enzyme particles are avoided, the nano toxicity is reduced, and the safety of the nano material applied to the physiological environment is improved;

5. The nano enzyme has various enzyme activities including ROS scavenging activity, ROS generating activity, hydrolase activity and the like. The nano enzyme itself can be used as nano material to realize the expression and reduction of the target enzyme activity through various synthesis means, so that ideal nano enzyme can be designed according to the pathological environment and treatment requirements of different diseases, and then the ideal nano enzyme is applied to the modification of polymers to realize the personalized customization of the implant;

6. The modification mode is simple, is suitable for various polymer matrixes, and is convenient for mass preparation and industrial production.

Drawings

FIG. 1 is a joint prosthesis pad prepared from a nanoenzyme modified polymer;

FIG. 2 is a Transmission Electron Microscope (TEM) image at various magnifications of cerium oxide grown in situ in ultra high molecular weight polyethylene, low magnification on the left and high magnification on the right;

FIG. 3 is an X-ray diffraction (XRD) result of the cerium oxide nano enzyme modified ultra high molecular weight polyethylene before and after hot pressing;

FIG. 4 is a graph showing the oxidation aging resistance of a cerium oxide nanoenzyme modified ultra high molecular weight polyethylene;

FIG. 5 is an elongation at break result of cerium oxide nanoenzyme modified ultra high molecular weight polyethylene;

FIG. 6 is a drawing showing the tensile properties of the cerium oxide nanoenzyme modified ultra high molecular weight polyethylene;

FIG. 7 is an impact performance result of cerium oxide nanoenzyme modified ultra high molecular weight polyethylene;

FIG. 8 is a graph showing the results of the coefficient of friction of the cerium oxide nanoenzyme modified ultra high molecular weight polyethylene;

FIG. 9 is a graph showing the abrasion rate results for cerium oxide nanoenzyme modified ultra high molecular weight polyethylene;

FIG. 10 shows the results of superoxide anion elimination (SOD-like enzymatic activity) of cerium oxide nanoenzyme modified ultra-high molecular weight polyethylene;

FIG. 11 is a graph showing the hydrogen peroxide scavenging effect (CAT-like enzyme activity) of cerium oxide nanoenzyme modified ultra-high molecular weight polyethylene;

FIG. 12 is a graph showing the hydroxyl radical scavenging effect of cerium oxide nanoenzyme modified ultra high molecular weight polyethylene;

FIG. 13 is a graph showing the results of cerium oxide nanoenzyme modified ultra high molecular weight polyethylene particles slowing down macrophage ROS production;

FIG. 14 is a graph showing the results of cerium oxide nanoenzyme modified ultra high molecular weight polyethylene particles reducing bone resorption in a skull bone dissolution model;

FIG. 15 is a graph showing the results of cerium oxide nano-enzyme modified ultra-high molecular weight polyethylene particles promoting osteogenesis in a skull bone dissolution model;

FIG. 16 is a graph showing the results of cerium oxide nano enzyme modified ultra high molecular weight polyethylene particles inhibiting inflammation in a skull bone dissolution model;

FIG. 17 is a graph showing the results of cerium oxide nanoenzyme modified ultra high molecular weight polyethylene particles reducing bone resorption in a femoral implant model;

FIG. 18 is a graph showing the results of inhibition of foreign body reaction by cerium oxide nano-enzyme modified ultra-high molecular weight polyethylene particles in a femoral implant model;

fig. 19 is a graph showing the results of inhibition of inflammation by cerium oxide nano-enzyme modified ultra-high molecular weight polyethylene particles in a femoral implant model.

Detailed Description

The technical scheme of the invention is further described below with reference to the accompanying drawings.

EXAMPLE 1 Synthesis preparation of cerium oxide nanoenzyme modified ultra high molecular weight polyethylene

1. 10G of ultra-high molecular weight polyethylene (molecular weight is 550 ten thousand) is dispersed in 800mL of decalin solvent to obtain a mixed system;

2. heating the mixed system obtained in the step 1 to 120 ℃ in a nitrogen environment, and continuously stirring until the ultra-high molecular weight polyethylene is completely swelled;

3. 1347.56mg of cerium acetylacetonate is dissolved in 20mL of absolute ethanol to obtain a precursor solution;

4. slowly dripping the precursor solution obtained in the step 3 into the system obtained in the step 2, and converting the atmosphere into air atmosphere;

5. stirring and heating the system obtained in the step 4 for 24 hours;

6. pouring the mixture obtained in the step 5 into methanol at the temperature of 0 ℃ for solvent replacement, and pouring out redundant solvent after the mixture is cooled;

7. Filtering, washing and drying the mixture obtained in the step 6 for 10 times by using ethanol;

8. and (3) placing the modified polymer obtained in the step (7) into a vacuum oven at 80 ℃ to volatilize the residual solvent.

Example 2 detection of the Synthesis of nanoenzyme in nanoenzyme modified polyethylene

1. The nano enzyme modified ultra high molecular weight polyethylene prepared in example 1 was tested using XRD, and the results are shown in figure 3. According to the characteristic peak card comparison of the polyethylene and the cerium oxide at the lower side of the picture, the modified polyethylene has characteristic peaks of polyethylene and also has obvious characteristic peaks of cerium oxide compared with the polyethylene. Indicating successful in situ growth of cerium oxide in the polyethylene matrix and furthermore no effect on the structure of the polyethylene itself.

2. 10Mg of nano enzyme modified ultra-high molecular weight polyethylene is dispersed in 10mL of decalin, and stirred and heated at 120 ℃ until the polyethylene swells;

3. Taking 50 mu L of the mixed solution in the step 2, dripping the mixed solution into 1mL of ethanol, and shaking and dispersing the mixed solution;

4. The mixed solution obtained in step 3 was dropped on a copper wire mesh and observed by TEM, and the result is shown in fig. 2. The cerium oxide particles were seen from the left low-magnification TEM image, with a size of about 5nm, and the 0.31nm lattice distance, which is a typical lattice distance of the cerium oxide (111) crystal plane, was measured from the right high-magnification TEM image, indicating that the in-situ grown cerium oxide in polyethylene also has good crystallization.

EXAMPLE 3 Nano enzyme modified polyethylene Molding Process

1. Placing the nano enzyme modified polymer obtained in the example 1 in a hot-pressing die, and compacting the die under the pressure of 10Mpa at normal temperature, and repeating for a plurality of times until the polymer is compacted and fills the die cavity;

2. Placing the die after the loading in a hot press, heating to 210 ℃, and preserving heat and pressure for 1h;

3. naturally cooling to room temperature under the condition of unchanged pressure after the heat preservation and pressure maintaining are finished, and then taking out the polymer block;

4. Slicing the block after hot pressing to obtain slices with the thickness of about 100 mu m, and then soaking the slices in squalene and heating at high temperature to perform oxidation aging treatment. The oxidation index of the polyethylene sheet and the modified polyethylene sheet after aging was monitored by a fourier infrared spectrometer, and the results are shown in fig. 4. After the same oxidative ageing procedure, the modified polyethylene shows a lower oxidation index than the common polyethylene, which indicates that the modified polyethylene has stronger antioxidation.

5. The hot-pressed blocks are cut according to national standard test standards and tested for relevant mechanical properties, and the tensile properties (fig. 5 and 6), impact properties (fig. 7), friction properties (fig. 8) and abrasion properties (fig. 9) of the blocks are tested, and the elongation at break results (fig. 5) show that the elongation at break of the polyethylene is not obviously reduced compared with that of common polyethylene, and the in-situ growth of cerium oxide in the polyethylene can not damage the tensile properties of the polyethylene. Furthermore, the ultimate tensile strength (fig. 6) and impact strength (fig. 7) of the polyethylene are improved by growing cerium oxide in situ, which is particularly important for the clinical application of the modified polyethylene. The friction performance (figure 8) and the wear performance (figure 9) of the common polyethylene and the modified polyethylene are tested by a friction and wear tester by taking the titanium alloy ball as a friction pair, the friction coefficient of the modified polyethylene is remarkably reduced, and the volume of particles dropped under the same wear time is smaller, which shows that the wear resistance is enhanced by in-situ growth of cerium oxide in the polyethylene.

6. And (3) carrying out numerical control processing on the polymer block obtained in the step (3) to prepare an artificial joint gasket model shown in figure 1.

Example 4 measurement of the enzyme-like Activity of nanoenzyme modified polyethylene

1. Cooling the ultra-high molecular weight polyethylene modified by the nano enzyme by using liquid nitrogen;

2. Crushing the cooled polyethylene obtained in the step 1 by using a planetary ball mill, wherein the oscillation frequency is 50Hz, 30s each time and 10 times are a period, and the total period is 10;

3. washing and drying the crushed polymer particles for multiple times, and dispersing the polymer particles in PBS containing 0.05% polyoxypropylene ethylene oxide glycerol ether for later use;

4. The superoxide anion scavenging activity of the polymer particles was measured using the nitrotetrazolium chloride method and the results are shown in FIG. 10. Compared with common polyethylene particles, the polyethylene particles modified by the nano enzyme show higher inhibition rate of superoxide anions;

5. the hydrogen peroxide scavenging activity of the polymer particles was tested using an oxygen electrode. The nano-enzyme modified polymer particles realize the removal of hydrogen peroxide by catalytically decomposing hydrogen peroxide into oxygen and water, and the oxygen electrode measures the removal capacity of different particles to hydrogen peroxide by monitoring the concentration of dissolved oxygen in the liquid environment, and the result is shown in figure 11. Compared with common polyethylene particles, the polyethylene particles modified by the nano enzyme show the generation amount of dissolved oxygen which increases with time, which shows that the polyethylene particles have remarkable hydrogen peroxide scavenging capability;

6. The hydroxyl radical scavenging activity of the polymer particles was measured using electron paramagnetic resonance spectroscopy, and the amount of hydroxyl radicals was measured by the intensity of the electron paramagnetic resonance spectroscopy signal using 5, 5-dimethyl-1-pyrroline-N-oxide as a hydroxyl radical scavenger, and the results are shown in fig. 12. Compared with common polyethylene particles, the polyethylene particles modified by the nano enzyme have obvious effect of scavenging hydroxyl free radicals, and the characteristic peak of the hydroxyl free radicals is obviously reduced.

EXAMPLE 5 stimulation of macrophages by nano-enzyme modified Polymer particles

1. RAW264.7 macrophages were grown in 12 well plates at a concentration of 3×10 ⁵ cells/well for 24h;

2. Dispersing nano enzyme modified polyethylene particles and common polyethylene particles in a cell culture solution respectively at the concentration of 0.2mg/mL, 0.4mg/mL and 0.8 mg/mL;

3. driving the mixed culture medium obtained in the step 2 into the 12-hole plate in the step 1, sealing the hole by using a sealing film after filling the hole, and then turning the hole plate to enable polyethylene particles and modified polyethylene particles to float upwards respectively and contact macrophages, and incubating for 24 hours;

4. Pouring out the culture solution, washing cells by using PBS buffer solution in a shaking way, adding PBS containing 10 mu M of 2, 7-dichlorofluorescein diacetate probe, and incubating in a cell incubator for 40 minutes in a dark place;

5. sucking out PBS containing the probe after 40 minutes, flushing the cells for 3 times by shaking the PBS, flushing the cells, collecting the cells by a centrifuge tube and preserving the cells in a dark place;

6. Macrophage ROS content was monitored using a flow cytometer and the results are shown in fig. 13. The relative fluorescence intensity of the active oxygen probe caused by the polyethylene particles to stimulate macrophages increases with the increase of the concentration of the particles, and is far higher than that caused by the nano enzyme modified polyethylene particles, which indicates that the active oxygen secretion of the macrophages caused by the nano enzyme modified polyethylene particles is less.

Example 6 physiological Properties of nanoenzyme modified Polymer particles in a skull dissolution model

1. ICR mice (victims biotechnology limited) were grouped into control, polyethylene and modified polyethylene groups. Wherein the control group is a healthy mouse, and the polyethylene group and the modified polyethylene group are respectively injected into different particles. Respectively injecting 50mg of polyethylene particles and modified polyethylene particles into the central seam of the skull of the ICR mouse, suturing, and taking materials after 14 days;

2. detecting tissues, wherein the detection content comprises Micro-CT, maoson staining and interleukin 1-beta staining of skull;

3. Statistical analysis was performed on the results of the test, including relative bone volume (fig. 14), relative bone formation face (fig. 15), and relative interleukin 1-beta fluorescence intensity (fig. 16). Polyethylene particle stimulation resulted in a significant decrease in relative bone volume fraction (fig. 14), relative bone formation surface (fig. 15) in mice compared to control mice, indicating that polyethylene stimulation aggravates osteoclastic resorption and inhibits new bone formation, which was alleviated in the modified polyethylene group. From interleukin 1 beta fluorescence statistics (fig. 16), it was found that mice of the polyethylene group exhibited the highest interleukin 1 beta secretion, and the inflammatory level was enhanced compared to the control group and the modified polyethylene group.

Example 7 physiological Properties of nanoenzyme modified Polymer particles in femoral implant model

1. Incision was made at left knee joint of ICR mice (victims biotechnology limited) and a k-wire with a diameter of 0.8mm and a length of 5mm was implanted at distal femur;

2. Mice were grouped into control, polyethylene and modified polyethylene groups. Control mice were no longer injected with particles and were implanted with only k-wire. Respectively injecting 50mg of common polyethylene particles and modified polymer particles into a polyethylene group and a modified polyethylene group mouse at the knee joint at the distal end of femur, suturing, and taking materials after 42 days;

3. Detecting tissues, wherein the detection content comprises Micro-CT, maoson staining and interleukin 1-beta staining of skull;

4. Statistical analysis of the results of the assay was performed, including relative bone volume (FIG. 17), fibrous layer thickness (FIG. 18), and relative interleukin 1-beta fluorescence intensity (FIG. 19). The decrease in relative bone volume in mice stimulated with polyethylene particles compared to control mice implanted with only kirschner wires (fig. 17) suggests that the reduction in the osteogenic capacity of mice after polyethylene particle stimulation is improved in the modified polyethylene particle group. It was found from the fiber layer thickness statistics that the fiber layer around the metal implant was thicker in the mice of the polyethylene particle group compared to the mice of the control group and the mice of the modified polyethylene group (fig. 18), indicating that the polyethylene particles aggravate the foreign body reaction caused by the metal implant, which is detrimental to the long-term stability of the implant. Interleukin 1 beta fluorescence intensity statistics found that the mice stimulated with polyethylene particles had an elevated interleukin 1 beta secretion content in the bone tissue region (fig. 19), indicating the presence of a severe inflammatory response.

In summary, the polymer obtained by the method for modifying the polymer by using the nano-enzyme provided by the invention can be used for preparing the implant for orthopaedics, and has enhanced mechanical property and antioxidant enzyme activity. The prepared implant can realize the weakening of foreign body reaction after implantation, the elimination of inflammation of focus areas and the promotion of osseointegration by eliminating ROS. In addition, the nano-enzyme shows long-term structural and functional stability in the polymer matrix, so that the long-term maintenance of the function of the prosthesis can be ensured, and the nano toxicity of the nano-material can be avoided. By the novel nano enzyme modified polymer synthesis mode, the implant with bone microenvironment regulation and mechanical property enhancement can be prepared, and has the potential of batch preparation and industrial production.

Claims

1. A method for preparing a nanozyme-modified orthopedic polymer, comprising the following steps:

(1) Dissolve ultra-high molecular weight polyethylene with a molecular weight of 5.5 million in decalin and swell it under heating;

(2) dispersing or dissolving one or more nanozymes or nanozyme precursors in a good solvent B solvent, and injecting the B solvent into the swollen and/or dissolved polymer;

(3) using a C solvent that is immiscible with decahydronaphthalene to perform solvent replacement on the obtained nanozyme-modified polymer, and then washing and drying to obtain the nanozyme-modified orthopedic polymer; the dielectric constants of the decahydronaphthalene and the C solvent differ by more than 30.

2. The method according to claim 1, characterized in that the nanozyme in step (2) includes one or more of oxide-based nanozymes, sulfide-based nanozymes, selenide-based nanozymes, telluride-based nanozymes, nitride-based nanozymes, phosphide-based nanozymes, gold nanozymes, copper nanozymes, silver nanozymes, platinum nanozymes, palladium nanozymes, rhodium nanozymes, ruthenium nanozymes, alloy nanozymes, iron-based metal organic framework nanozymes, zinc-based metal organic framework nanozymes, copper-based metal organic framework nanozymes, zirconium-based metal organic framework nanozymes, hafnium-based metal organic framework nanozymes, vanadium-based metal organic framework nanozymes, metal-doped metal organic framework nanozymes, boron-based covalent organic framework nanozymes, imine-based covalent organic framework nanozymes, triazine-based covalent organic framework nanozymes, fullerene-based nanozymes, carbon fiber-based nanozymes, carbon nanotube-based nanozymes, graphene-based nanozymes or other nanozymes.

3. The method according to claim 2 is characterized in that the oxide-based nanozyme contains metal elements, and the metal elements include lithium, magnesium, aluminum, potassium, calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, rubidium, strontium, iridium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, tin, antimony, cerium, hafnium, platinum, gold or bismuth. One or more elements.

4. The method according to claim 1 is characterized in that the nanozyme precursor in step (2) comprises one or more of an organic metal salt, a metal chloride salt, a metal bromide salt, a metal nitrate salt, a metal acetate salt, a metal sulfate salt or a metal cyanide complex.

5. The method according to claim 4, characterized in that the organic metal salt comprises one or more of metal acetylacetonate, metal alkoxide, metal acid salt, metal ether salt, metal ether acid salt, metal ether alkoxide, metal amide salt, metal alcoholate, metal carboxylic acid amide salt, metal ether amide salt, metal imine salt, metal phosphorus complex or metal nitrogen complex.

6. The method according to claim 5, characterized in that the metal acetylacetonate contains metal elements including one or more of lithium, magnesium, aluminum, potassium, calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, rubidium, strontium, iridium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, tin, antimony, cerium, hafnium, platinum, gold or bismuth.

7. The method according to claim 1 is characterized in that the good solvent B solvent for the nanozyme and nanozyme precursor in step (2) includes one or more of ethanol, ethyl acetate, isopropanol, chloroform, pyridine, acetone, acetic acid, acetonitrile, aniline, dimethylformamide, methanol, ethylene glycol, dimethyl sulfoxide or water.

8. The method according to claim 1, characterized in that the C solvent incompatible with decalin in step (3) comprises one or more of ethanol, ethyl acetate, isopropanol, chloroform, pyridine, acetone, acetic acid, acetonitrile, aniline, dimethylformamide, methanol, ethylene glycol, dimethyl sulfoxide or water; the cleaning and drying method comprises filtering and washing the nanozyme-modified polymer after solution replacement with ethanol multiple times, and volatilizing the residual solvent in a vacuum oven.

9. A method for preparing a nanozyme-modified orthopedic polymer, comprising the following steps:

(1) dissolving ultra-high molecular weight polyethylene with a molecular weight of 5.5 million in decalin and swelling it under heating to obtain a mixed system;

(2) dissolving cerium acetylacetonate in anhydrous ethanol, adding the solution dropwise to the mixed system in step (1), converting the atmosphere to air, stirring and heating to obtain a nanozyme-modified polymer;

(3) Using methanol to replace the solvent of the nanozyme-modified polymer in step (2), and then washing and drying to obtain the nanozyme-modified orthopedic polymer.

10. An orthopedic polymer prepared by the method according to any one of claims 1 to 9.

11. A method for preparing a modified polymer implant using the orthopedic polymer according to claim 10, characterized in that it comprises the following steps:

(1) Mechanically shaping and morphologically processing the modified polymer to obtain a crude modified polymer implant;

(2) post-processing the crude modified polymer implant to obtain a modified polymer implant; the mechanical forming method in step (1) includes one or more combinations of hot pressing sintering, hot pressing molding, injection molding, blow molding, extrusion molding, calendering molding, 3D printing molding, vacuum molding or spray molding.

12. The method according to claim 11, characterized in that the 3D printing molding includes one or more of a fused deposition method, a photo-curing method, a laser sintering method, a powder melting method or an electron beam melting method.

13. The method according to claim 11, characterized in that the topography processing method in step (1) comprises one or more of cutting, grinding, drilling, milling or cutting.

14. The method according to claim 11, characterized in that the post-processing step in step (2) includes one or more of cleaning, polishing, sandblasting, pickling, plasma treatment, solvent treatment, silane coupling agent treatment, chemical coating treatment or ion implantation treatment.

15. A modified polymer implant prepared by the method according to any one of claims 11 to 14.

16. The modified polymer implant according to claim 15, characterized in that the modified polymer implant comprises an artificial joint spacer, a bone plate, a bone nail, a bone cement, a bone scaffold, a bone fusion cage, a bone filler, a bone fixation band, an artificial bone or a periosteum.

17. Use of the orthopedic polymer according to claim 10 in the preparation of an orthopedic implant for treating, alleviating and/or preventing orthopedic diseases, characterized in that the orthopedic diseases include advanced arthritis, aseptic loosening, bacterial loosening, osteoporosis, bone tumors, repair of large bone defects, osteomyelitis or fractures.