Cu-loaded nanoszyme β-tcp / plga scaffold sustained-release body and preparation method thereof

Abstract

The application provides a beta-TCP / PLGA support sustained-release body loaded with Cu-based nanoscale enzyme and a preparation method, a 3D model of a microstructure of a bone defect is established by measuring bone structure parameters, and a personalized beta-TCP / PLGA support is printed by using beta-TCP / PLGA printing materials through a 3D bioprinter technology. The beta-TCP / PLGA support is immersed in a Cu-HCF nanoscale enzyme solution, so that the beta-TCP / PLGA support is loaded with Cu-HCF nanoscale enzyme, thereby preparing a Cu-HCF nanoscale enzyme beta-TCP / PLGA sustained-release support. In a tumor and inflammatory microenvironment, the enzyme reaction can selectively kill tumor and inflammatory cells, and can protect normal tissues, reduce the possibility of recurrence of bone tumors and inflammation at the bone defect, and avoid possible side effects of drugs on the human body. As a kind of bionic bone support, the beta-TCP / PLGA sustained-release support has good mechanical strength and biodegradability, and also has the effect of keeping a long-acting and stable drug release, can improve the directionality and reduce the toxic side effects. The material cost of the beta-TCP / PLGA sustained-release support is moderate and relatively easy to prepare, and has good mass production prospects and economic applicability.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and specifically to a β-TCP / PLGA scaffold sustained-release formulation loaded with Cu-based nanozymes and its preparation method. Background Technology

[0002] Bone defects refer to the disruption of the structural integrity of bone and are a common clinical condition. Trauma, surgery, tumors, infections, and inflammation can all lead to bone defects. Bone defects typically result in nonunion, delayed healing or nonunion, and local functional impairment. Treatment for bone defects is lengthy, requires advanced techniques, and is prone to complications such as nonunion fractures during the treatment process.

[0003] Current treatments for bone defects primarily focus on repairing the defects and treating inflammation at the site of the lesion. Among the treatment methods for repairing bone defects, PLGA scaffolds have received considerable attention. PLGA, or polylactic acid-glycolic acid copolymer, is a polymer material with excellent biocompatibility and biodegradability. The degradation products of PLGA are lactic acid and glycolic acid, which are also byproducts of human metabolism. Therefore, its application in pharmaceuticals and biomaterials does not cause toxic side effects, and its degradation rate is controllable. It has wide applications in biomedical engineering and has been used to fabricate artificial catheters, tissue engineering scaffold materials, etc. Furthermore, its use as a carrier for protein and enzyme drugs is currently a hot research topic.

[0004] In the treatment of inflammation at bone defect sites, reactive oxygen species (ROS) imbalance is associated with inflammatory diseases including rheumatoid arthritis, cardiovascular disease, and cancer. Highly efficient anti-inflammatory enzymes capable of scavenging ROS can protect tissues from inflammation-induced damage. However, natural ROS-scavenging enzymes are highly sensitive to the environment and difficult to mass-produce. Since the report of HRP nanozymes in 2007, nanozyme research has rapidly emerged and its scope has broadened. Studies have shown that metal ion nanozymes, as catalytic nanomaterials mimicking enzyme activity, can serve as ROS-scavenging enzymes. Nanozymes are a class of enzyme mimics that possess both the unique properties of nanomaterials and catalytic functions. They are characterized by high catalytic efficiency, stability, economy, and large-scale preparation, and have been widely applied in medicine, chemical engineering, food, agriculture, and environmental fields.

[0005] The basic principles guiding the further clinical translation of nanozymes include: (1) the nanozyme components must be biocompatible, completely free of toxic elements, or biodegradable into non-toxic substances; (2) the size and shape of the nanozyme should have a sufficiently small hydrodynamic diameter to be excreted by the kidneys and avoid retention in the reticuloendothelial system; (3) the surface charge of the nanozyme should be zwitterionic or neutral surface coating to minimize non-specific tissue / organ uptake; (4) the nanozyme should have high chemical stability in aqueous solvents and serum; (5) the nanozyme should be conducive to drug metabolism kinetics and targeted accumulation, effectively targeting the disease while being completely eliminated from the body within a reasonable time; (6) the nanozyme preparation process should be simple and easy to implement, easy to scale up and synthesize, and highly reproducible. Therefore, based on meeting the above basic principles, combining PLGA scaffolds with nanozymes will have significant implications for the treatment of bone defects. However, no relevant technical solutions have been documented in current research regarding how to obtain personalized PLGA scaffolds with high matching degree to bone defects, mineralize and modify the PLGA scaffolds, and combine nanozymes with PLGA scaffolds to prepare nanozyme scaffold sustained-release bodies. Therefore, this invention provides a method for preparing β-TCP / PLGA scaffold sustained-release bodies loaded with Cu-based nanozymes. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides a β-TCP / PLGA scaffold sustained-release formulation loaded with Cu-based nanozymes and its preparation method.

[0007] The technical solution of the present invention is as follows:

[0008] A method for preparing a β-TCP / PLGA scaffold sustained-release formulation loaded with Cu-based nanozymes includes the following steps:

[0009] Step 1: Use a Micro-CT scanner to collect bone structure parameters of the bone defect site, and based on the bone structure parameter data, use the computer's built-in 3D modeling software to establish a microstructure model of the bone defect.

[0010] Step 2: Weigh out a certain amount of PLGA solid and β-TCP solid, add them sequentially to an organic solvent, and dissolve and mix them under stirring conditions to prepare a paste-like β-TCP / PLGA printing material;

[0011] Step 3: Use a 3D bioprinter to read the microstructure model data of the bone defect established in Step 1, use the β-TCP / PLGA printing material prepared in Step 2 to print the β-TCP / PLGA scaffold for bone defect in a low-temperature molding chamber, and perform low-temperature shaping and sterilization treatment on the β-TCP / PLGA scaffold.

[0012] Step four, preparation of Cu-HCF nanozyme, includes the following steps: 1. Weigh a certain amount of K4[Fe(CN)6] solid and disperse it in deionized water to prepare a K4[Fe(CN)6] solution; 2. Weigh a certain amount of CuSO4·5H2O solid and disperse it in deionized water to prepare a CuSO4 solution; 3. Under vigorous stirring, slowly add the CuSO4 solution dropwise to the K4[Fe(CN)6] solution and maintain the reaction until a Cu2[Fe(CN)6] suspension is obtained; 4. Weigh an excess of PEG-SH solid and add it to the Cu2[Fe(CN)6] suspension under stirring and maintain the reaction until a Cu-HCF nanozyme precipitate is obtained; 5. Collect the Cu-HCF nanozyme precipitate by centrifugation and wash it with water to obtain the Cu-HCF nanozyme.

[0013] Step 5 involves preparing the β-TCP / PLGA scaffold sustained-release and freeze-drying it, including the following steps: 1. Weigh a quantitative amount of Cu-HCF nanozyme and add it to a 3% gelatin solution, mix well, and dilute to prepare a Cu-HCF nanozyme solution with a total mass concentration of 3 g / L; 2. Immerse the β-TCP / PLGA scaffold in the Cu-HCF nanozyme solution and maintain it for a certain period of time to obtain the β-TCP / PLGA scaffold sustained-release; 3. Freeze the β-TCP / PLGA scaffold sustained-release in a low-temperature environment and then perform low-temperature vacuum drying.

[0014] Furthermore, in step one, the bone structure parameters include the three-dimensional structure of the trabeculae, the number of trabeculae, the thickness of the trabeculae, the trabecular separation, the structural model index, bone density, bone pore size, and bone volume fraction.

[0015] Furthermore, in step one, the 3D modeling software includes Mimics software or Geomagic Studio software; the spatial resolution of the Micro-CT scanner is 10μm×10μm×10μm; and the scanning coverage area of ​​the Micro-CT scanner is ≥1.5 times the bone defect area.

[0016] Furthermore, in step two, the organic solvent is 1,4-dioxane, the ratio of the total solid weight of PLGA and β-TCP to the liquid volume of 1,4-dioxane is 18:100; the mass ratio of PLGA to β-TCP is 1:3; the mass values ​​of PLGA, β-TCP and the liquid volume of 1,4-dioxane can be increased or decreased proportionally.

[0017] Furthermore, in step three, the low-temperature molding and sterilization treatment of the β-TCP / PLGA scaffold includes the following steps: a. Using a low-temperature freezer, the β-TCP / PLGA scaffold is frozen and solidified at -37°C; b. After freezing, the β-TCP / PLGA scaffold is placed in a -80°C freezer for ≥30 minutes, then quickly transferred to a pre-cooled freeze dryer rack for freeze-drying until all organic solvents in the β-TCP / PLGA scaffold are completely removed; c. The β-TCP / PLGA scaffold is soaked in a 95% ethanol solution for ≥4 hours, and then irradiated with ultraviolet light for ≥24 hours before being naturally dried and sealed for storage.

[0018] Furthermore, in step three, the printing design parameters of the 3D bioprinter are as follows: nozzle diameter 200μm, nozzle temperature 20-40℃, printing aperture 1.0mm; nozzle reverse rotation 3.0mm×0.1mm, forward rotation 3.0mm×0.1mm, delay 0s, and travel speed 20mm / min.

[0019] Furthermore, in step three, the temperature of the low-temperature molding chamber is -30℃; the low-temperature molding chamber is a space of 290mm×300mm×210mm, with a silicone film placed on top of it; the low-temperature molding chamber is also equipped with an openable operating door, and the operating door is equipped with an observation hole.

[0020] Furthermore, in step four, the molar mass ratio of K4[Fe(CN)6] to CuSO4·5H2O is 1:2; the mass values ​​of K4[Fe(CN)6], CuSO4·5H2O and PEG-SH can be increased or decreased proportionally.

[0021] Furthermore, in step five, the β-TCP / PLGA scaffold is immersed in the Cu-HCF nanozyme solution for ≥12 hours; the low-temperature freezing temperature of the β-TCP / PLGA scaffold sustained-release body is -37°C, the freezing time is ≥2 hours, and the low-temperature vacuum drying time is ≥72 hours.

[0022] The β-TCP / PLGA scaffold sustained-release formulation prepared by the above preparation method comprises a β-TCP / PLGA scaffold and a Cu-based nanozyme loaded on the β-TCP / PLGA scaffold, wherein the β-TCP / PLGA scaffold is loaded with 20 μg-100 μg of Cu-based nanozyme per gram weight.

[0023] The beneficial effects of this invention are:

[0024] 1) The smaller the size of a single nanozyme particle, the larger its surface area on a macroscopic scale. This increases the likelihood of contact between the nanozyme particle and the substrate, resulting in higher catalytic activity. Therefore, Cu-HCF nanozymes exhibit superior catalytic activity compared to ordinary enzymes. Furthermore, Cu-HCF nanozymes can exhibit superoxide dismutase activity under neutral conditions, eliminating harmful reactive oxygen species (ROS) groups; under acidic conditions, they can exhibit oxidase activity, catalyzing the production of reactive oxygen species. Since normal human tissues are mostly in a neutral environment, while tumors and inflamed tissues are mostly in a slightly acidic environment, Cu-HCF nanozymes, as a sustained-release medium, can selectively kill tumor and inflammatory cells through enzymatic reactions in the tumor and inflammatory microenvironment, while protecting normal tissues. This reduces the possibility of bone tumor and inflammation recurrence at bone defects, avoiding potential side effects of drugs on the human body.

[0025] 2) The β-TCP / PLGA scaffold is a biomimetic bone scaffold with good mechanical strength and degradability. When applied to human tissues, it has extremely low immunogenicity and high tissue compatibility. At the same time, the β-TCP / PLGA scaffold sustained-release formulation can maintain a local long-term and stable drug release effect, which can improve the directionality and reduce toxic side effects. Furthermore, the β-TCP / PLGA scaffold is easy to prepare, and the material and manufacturing costs are moderate, which makes it a promising candidate for mass production and economically viable. Attached Figure Description

[0026] Figure 1 : A schematic flowchart of the method described in this invention;

[0027] Figure 2 Schematic diagram of the low-temperature molding and sterilization process for β-TCP / PLGA stents;

[0028] Figure 3 : A schematic diagram of the printed β-TCP / PLGA scaffold sustained-release formulation;

[0029] Figure 4 A schematic diagram of a 3D skeletal model created using Mimics software. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and by way of embodiments.

[0031] A method for preparing a β-TCP / PLGA scaffold sustained-release formulation loaded with Cu-based nanozymes includes the following steps:

[0032] Step 1: Collect bone structure parameters at the bone defect site of the patient and establish a microscopic structural model of the bone defect.

[0033] The patient's bone defect was scanned in vitro using a Micro-CT scanner to obtain data on bone structure parameters, including but not limited to the three-dimensional structure of trabeculae, the number of trabeculae (Tb.N), the thickness of trabeculae (Tb.Th), the separation of trabeculae (Tb.Sp), the structural model index (SMI), bone mineral density (BMD), bone porosity, and bone volume fraction (BV / TV).

[0034] The bone structure parameters were converted to Dicom format using a Micro-CT scanner.

[0035] The micro-CT scanner connects to the computer via a data cable to transmit bone structure parameters in DIcom format to the computer.

[0036] The computer is equipped with 3D modeling software, such as Mimics or Geomagic Studio, which can read bone structure parameter data in Dicom format and build a microscopic structural model of bone defects.

[0037] Mimics software is a widely used medical image processing software in the medical device field, enabling medical professionals to accurately process patient medical images. Mimics can perform 3D reconstruction from tomographic images of CT scanners, MRI machines, and even 3D color ultrasound. It is widely used in clinical, biomedical engineering, and materials engineering fields. It can import DICOM, JPEG, TIFF, BMP, X-ray, or initial image data to quickly build accurate 3D solid models and output files in CAD, FEA, RP, and other formats.

[0038] Geomagic Studio is a reverse engineering and 3D inspection software produced by Raindrop Systems, Inc. It can scan raster models to create high-quality polygonal or mesh models and convert them into NURBS surfaces. In the medical field, it can read DICOM format 3D images, process them accordingly, and generate 3D models. Geomagic Studio can perfectly complement CAD, CAE, and CAM tools, and can output industry-standard formats, including STL, IGES, STEP, and CAD, among many others. This software has a wide range of applications and mature technology. For example, the report "3D Human Body Modeling Technology Based on Geomagic" by Li Yan et al. from Wuhan University of Science and Technology was recorded in the May 2008 issue of the *Journal of Textile Research*, Volume 29, Issue 5.

[0039] Therefore, this step will not elaborate on the 3D modeling principles and operation process of Mimics or Geomagic Studio software.

[0040] In this step,

[0041] 1. The preferred Micro-CT scanner is the Skyscan1076 scanner manufactured by Bruker micro-CT in Belgium.

[0042] 2. The spatial resolution range of the Micro-CT scanner is 2.8-81.8μm, and the preferred spatial resolution set in this step is 10μm×10μm×10μm;

[0043] 3. When performing in vitro scanning of bone defects in personnel, since the Skyscan 1076 scanner requires the tissue to have a diameter of less than 8cm and a length of less than 30cm, the scanning coverage area should be ≥1.5 times the bone defect area when the bone defect area is small, in order to obtain high-accuracy modeling data, while meeting the sample size restrictions.

[0044] Step 2: Prepare β-TCP / PLGA printing material.

[0045] Accurately weigh 10 grams of analytical grade PLGA solid and 30 grams of analytical grade β-TCP solid, and grind them into powder;

[0046] Measure an appropriate amount of 1,4-dioxane liquid into a beaker, turn on the magnetic stirrer and stir continuously and evenly for 4±0.5h. To obtain better stirring effect, set the stirring speed of the magnetic stirrer to 800rpm.

[0047] While stirring, add PLGA powder to the 1,4-dioxane liquid in batches until completely dissolved to obtain PLGA solution A;

[0048] While maintaining stirring, add β-TCP powder to PLGA solution A in batches. After stirring for ≥30 minutes, turn off the magnetic stirrer to obtain a paste-like mixture. The paste-like mixture is the β-TCP / PLGA printing material and should be stored in a light-proof environment at room temperature.

[0049] In this step,

[0050] 1. The amount of 1,4-dioxane liquid should be sufficient to fully dissolve and mix the PLGA solid and β-TCP solid, and to make the finished printing material appear as a paste. In this step, it is preferable to use a powder weight (i.e., the total weight of PLGA solid and β-TCP solid, in g) to solution volume (i.e., the volume of 1,4-dioxane liquid, in ml) ratio of 18:100 for experimental operation. Alternatively, it can be adjusted according to the requirements of the printing job for the form of the printing material.

[0051] 2. In addition to 1,4-dioxane, the organic solvent used in this step can also be chloroform.

[0052] 3. The PLGA to β-TCP quality ratio ranges from 1:2 to 1:5, with a preferred quality ratio of 1:3.

[0053] 4. The PLGA used is composed of 75% lactic acid and 25% glycolic acid.

[0054] 5. The mass values ​​of the substances in this step can be increased or decreased proportionally according to the required amount during actual production;

[0055] 6. In the laboratory operating environment, the preferred temperature is 25℃ and the preferred humidity is 30%. Since 1,4-dioxane is a Group 2 carcinogen, the operation should be carried out in a closed system with full ventilation. Operators should wear half-face masks and protective glasses, and the laboratory should use an explosion-proof ventilation system.

[0056] Step 3: 3D printing to fabricate β-TCP / PLGA scaffolds for bone defects.

[0057] The β-TCP / PLGA scaffold was printed using a 3D bioprinter to read the microstructural model data of the bone defect established in step one and a low-temperature rapid prototyping method.

[0058] The preferred 3D bioprinter is the German-made Tissue Form II, with the following printing design parameters:

[0059] The nozzle diameter is 200μm, the nozzle temperature is 20-40℃, and the printing aperture is 1.0mm. The nozzle rotates 3.0mm×0.1mm in reverse and 3.0mm×0.1mm in forward direction, with a delay of 0s and a travel speed of 20mm / min to begin shaping. A β-TCP / PLGA scaffold with the spatial morphology of the bone defect is printed in layers in an orderly manner. The β-TCP / PLGA scaffold has a diameter of 7.5mm and a height of 10.0mm, and the porosity of the porous structure in the scaffold is approximately 80%.

[0060] The printing steps are as follows:

[0061] The β-TCP / PLGA printing material prepared in step two is poured into the molding hopper. Driven by the 3D model, in a -30°C low-temperature molding chamber environment, the nozzle of the 3D bioprinter extrudes and freezes the printing material onto the molding platform. The 3D bioprinter stacks the material layer by layer along the scanning path until a 3D reconstructed model is printed. The scanning motion module of the 3D bioprinter controls the nozzle module's scanning within the XY horizontal plane; controls the molding platform's downward layer-by-layer movement along the Z-axis; and ensures precise stacking of the printing material after it is extruded from the nozzle.

[0062] The cryogenic forming chamber is a 290mm × 300mm × 210mm space equipped with an openable operating door. The door has an observation window, lined with glass on both the inside and outside, used primarily to monitor the forming process of the β-TCP / PLGA bracket. A silicone-pressed film is installed directly above the forming chamber. This film serves two purposes: firstly, it isolates the forming chamber from the outside environment, preventing hot air from affecting the chamber temperature through convection; secondly, it does not interfere with the scanning motion, ensuring the accuracy of the β-TCP / PLGA bracket printing; and thirdly, it also provides dust protection.

[0063] The printed β-TCP / PLGA scaffold undergoes low-temperature molding and sterilization, as follows:

[0064] a. Use a low-temperature freezer to cure and shape the β-TCP / PLGA bracket at -37°C;

[0065] b. After freezing the β-TCP / PLGA holder, place it in a -80℃ freezer for ≥30 minutes and then quickly transfer the β-TCP / PLGA holder to the pre-cooled freeze dryer material rack for freeze drying for 24-48 hours until all the 1,4-dioxane solvent in the β-TCP / PLGA holder is removed.

[0066] c. Soak the β-TCP / PLGA stent in 95% ethanol solution for ≥4 hours, then sterilize it by irradiating it with ultraviolet light for ≥24 hours. After sterilization, allow it to air dry and store it in a sealed container.

[0067] In this step,

[0068] 1. Low-temperature rapid prototyping (LT-RP) is a manufacturing technology used to create 3D models and prototypes. It utilizes frozen or cryogenic materials, combined with heating or solidification techniques, to rapidly manufacture objects with complex geometries in a short time. The advantages of LT-RP include high manufacturing speed, low cost, and wide applicability. It has important applications in model making, prototyping, medical devices, and tissue engineering.

[0069] 2. This step enables the β-TCP / PLGA bracket to have a microporous structure. The microporous structure is formed by thermal phase separation caused by the slurry extruded from the nozzle and solidified in a -30°C molding chamber environment, as well as by the sublimation of the solvent 1,4-dioxane during the subsequent freeze-drying process.

[0070] Step 4: Prepare Cu-HCF nanozymes.

[0071] Accurately weigh 0.1 mmol (36.8 mg) of K4[Fe(CN)6] and 0.3 g of CA, disperse them in 20 mL of deionized water to prepare K4[Fe(CN)6] solution B;

[0072] Accurately weigh 0.2 mmol (50 mg) of CuSO4·5H2O and disperse it in 10 mL of deionized water to prepare CuSO4 solution C;

[0073] Using a magnetic stirrer, CuSO4 solution C was slowly added dropwise to K4[Fe(CN)6] solution B under vigorous stirring conditions. Immediately after addition, a wine-red precipitate, namely Cu2[Fe(CN)6] precipitate, was formed. After maintaining the reaction of the mixture for ≥24 hours, Cu2[Fe(CN)6] suspension D was obtained.

[0074] Weigh 100 mg of PEG-SH and add it to Cu2[Fe(CN)6] suspension D under stirring conditions, and maintain the reaction for 24 hours to obtain Cu-HCF nanozyme precipitate;

[0075] The Cu-HCF nanozyme precipitate was collected by centrifugation and washed with water at least three times to obtain Cu-HCF nanozyme.

[0076] In this step,

[0077] 1. The operation should be performed under aseptic conditions, in a biosafety cabinet in the laboratory.

[0078] 2. The mass values ​​of the substances in this step can be increased or decreased proportionally according to the required amount during actual production;

[0079] 3. An excess of PEG-SH solid powder can be added. PEG-SH is easily soluble in water. Through repeated water washing, the unreacted PEG-SH solid can be removed from the Cu-HCF nanozyme precipitate.

[0080] Step 5: Preparation of β-TCP / PLGA scaffold sustained-release formulation:

[0081] Prepare a 3% gelatin solution and add 1% acetic acid to obtain gelatin solution E;

[0082] Weigh a certain amount of Cu-HCF nanozyme and add it to an appropriate amount of gelatin solution E. Mix it with ultrasound to prepare Cu-HCF nanozyme solution F with a total mass concentration of 6 g / L. Further dilute to prepare Cu-HCF nanozyme solution G with a total mass concentration of 3 g / L.

[0083] Place Cu-HCF nanozyme solution G in a beaker, immerse the β-TCP / PLGA scaffold in Cu-HCF nanozyme solution G for ≥12 hours and then remove it to obtain the β-TCP / PLGA scaffold sustained-release body.

[0084] Freeze-drying treatment of β-TCP / PLGA scaffold sustained release: Freeze the β-TCP / PLGA scaffold sustained release at -37°C, for example, in a low-temperature freezer for ≥2 hours; then perform low-temperature vacuum drying in a freeze dryer for ≥72 hours.

[0085] In this step,

[0086] 1. The mass of Cu-HCF nanozyme is 10% to 15% of the mass of PLGA, preferably 12%;

[0087] 2. Cu-HCF nanozymes can be uniformly dispersed in gelatin solution by ultrasonic mixing. To obtain better dispersion, the concentration of Cu-HCF nanozyme solution G should be controlled within the range of 1 g / L-5 g / L. Too high a concentration will result in too many Cu-HCF nanozyme particles loaded in the micropores of the β-TCP / PLGA scaffold, causing multiple Cu-HCF nanozyme particles to compete in the micropores of the bone scaffold, resulting in unstable linkage. In this step, the preferred concentration of Cu-HCF nanozyme solution G is 3 g / L.

[0088] Since the water absorption capacity of the β-TCP / PLGA scaffold is 20 μl / g, the prepared β-TCP / PLGA scaffold sustained-release body has a Cu-HCF nanozyme loaded per unit weight ranging from 20 μg to 100 μg. Preferably, the Cu-HCF nanozyme loaded per unit weight is 60 μg.

[0089] 3. Cu-HCF nanozymes are covalently loaded onto β-TCP / PLGA scaffolds, exhibiting good binding force;

[0090] 4. The operation should be performed under aseptic conditions, in a biosafety cabinet in the laboratory.

[0091] The β-TCP / PLGA scaffold sustained-release formulation loaded with Cu-based nanozymes prepared by the above-described method comprises two parts: a β-TCP / PLGA scaffold and a Cu-based nanozyme. The Cu-based nanozyme nanoparticles are dispersed and loaded onto the surface and within the micropores of the β-TCP / PLGA scaffold, with each gram of β-TCP / PLGA scaffold loading 20 μg-100 μg of Cu-based nanozyme. During use, as the β-TCP / PLGA scaffold gradually degrades in the human tissue environment, the Cu-HCF nanozyme loaded on the β-TCP / PLGA scaffold is gradually released, selectively killing tumor and inflammatory cells through enzymatic reactions while protecting normal tissues.

[0092] The explanations of the terms and English abbreviations used in the above steps are as follows:

[0093] 1. Cu-HCF nanozymes refer to self-assembled Cu2[Fe(CN)6]-PEG nanozymes. Self-assembly refers to a technology in which basic structural units, such as molecules, nanomaterials, or substances at the micrometer or larger scale, spontaneously form an ordered structure. During self-assembly, basic structural units spontaneously organize or aggregate into a stable structure with a certain regular geometric appearance through non-covalent interactions. Self-assembly technology is simple and easy to implement, requires no special equipment, usually uses water as a solvent, and has the advantages of molecular-level control over the deposition process and membrane structure.

[0094] 2. PLGA is a polylactic acid-glycolic acid copolymer, which is formed by the random polymerization of two monomers - lactic acid and glycolic acid. Different monomer ratios can prepare different types of PLGA. The PLGA used in this specification is 75:25, that is, the polymer is composed of 75% lactic acid and 25% glycolic acid.

[0095] 3. β-TCP is type II calcium phosphate. Calcium phosphate (TCP) is divided into high-temperature α-TCP and low-temperature β-TCP. The transition temperature between the α and β phases is 1120-1180℃. Compared with α-TCP, β-TCP has better degradation performance and good cell compatibility. Animal or human cells can grow, differentiate and reproduce normally on the material.

[0096] 4. CA stands for citric acid.

[0097] 5. PEG-SH is polyethylene glycol mercaptoethanol, a white solid powder that is easily soluble in water.

[0098] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for preparing a β-TCP / PLGA scaffold sustained-release formulation loaded with Cu-based nanozymes, characterized in that, Includes the following steps: Step 1: Use a Micro-CT scanner to collect bone structure parameters of the bone defect site, and based on the bone structure parameter data, use the computer's built-in 3D modeling software to establish a microstructure model of the bone defect. Step 2: Weigh out a certain amount of PLGA solid and β-TCP solid, add them sequentially to an organic solvent, and dissolve and mix them under stirring conditions to prepare a paste-like β-TCP / PLGA printing material; Step 3: Use a 3D bioprinter to read the microstructure model data of the bone defect established in Step 1, use the β-TCP / PLGA printing material prepared in Step 2 to print the β-TCP / PLGA scaffold for bone defect in a low-temperature molding chamber, and perform low-temperature shaping and sterilization treatment on the β-TCP / PLGA scaffold. Step four, preparation of Cu-HCF nanozyme, includes the following steps:

1. Weigh a certain amount of K4[Fe(CN)6] solid and disperse it in deionized water to prepare K4[Fe(CN)6] solution; 2. Weigh a certain amount of CuSO4·5H2O solid and disperse it in deionized water to prepare CuSO4 solution; 3. Under vigorous stirring, slowly add the CuSO4 solution dropwise to the K4[Fe(CN)6] solution, maintaining the reaction until a Cu2[Fe(CN)6] suspension is obtained; 4. Weigh an excess of PEG-SH solid and add it to the Cu2[Fe(CN)6] suspension under stirring, maintaining the reaction until a Cu-HCF nanozyme precipitate is obtained; 5. Collect the Cu-HCF nanozyme precipitate by centrifugation and wash it with water to obtain the Cu-HCF nanozyme. Step 5 involves preparing the β-TCP / PLGA scaffold sustained-release and freeze-drying it, including the following steps:

1. Weigh a quantitative amount of Cu-HCF nanozyme and add it to a 3% gelatin solution, mix well, and dilute to prepare a Cu-HCF nanozyme solution with a total mass concentration of 3 g / L; 2. Immerse the β-TCP / PLGA scaffold in the Cu-HCF nanozyme solution and maintain it for a certain period of time to obtain the β-TCP / PLGA scaffold sustained-release; 3. Freeze the β-TCP / PLGA scaffold sustained-release in a low-temperature environment and then perform low-temperature vacuum drying.

2. The method for preparing a β-TCP / PLGA scaffold sustained-release formulation loaded with Cu-based nanozymes according to claim 1, characterized in that, In step one, the bone structure parameters include the three-dimensional structure of the trabeculae, the number of trabeculae, the thickness of the trabeculae, the trabecular separation, the structural model index, bone mineral density, bone pore size, and bone volume fraction.

3. The method for preparing a β-TCP / PLGA scaffold sustained-release formulation loaded with Cu-based nanozymes according to claim 1, characterized in that, In step one, the 3D modeling software includes Mimics software or Geomagic Studio software; the spatial resolution of the Micro-CT scanner is 10μm×10μm×10μm; and the scanning coverage area of ​​the Micro-CT scanner is ≥1.5 times the bone defect area.

4. The method for preparing a β-TCP / PLGA scaffold sustained-release formulation loaded with Cu-based nanozymes according to claim 1, characterized in that, In step two, the organic solvent is 1,4-dioxane, and the ratio of the total solid weight of PLGA and β-TCP to the liquid volume of 1,4-dioxane is 18:100 g / mL; the mass ratio of PLGA to β-TCP is 1:3; the mass values ​​of PLGA, β-TCP, and the liquid volume of 1,4-dioxane can be increased or decreased proportionally.

5. The method for preparing a β-TCP / PLGA scaffold sustained-release formulation loaded with Cu-based nanozymes according to claim 1, characterized in that, Step three, the low-temperature shaping and sterilization treatment of the β-TCP / PLGA scaffold includes the following steps: a. The β-TCP / PLGA bracket is freeze-cured at -37°C. b. After freezing, the β-TCP / PLGA scaffold is transferred to an environment of -80°C and frozen for ≥30 minutes. Then, it is quickly transferred to the pre-cooled material rack of the freeze dryer and freeze-dried until the organic solvent in the β-TCP / PLGA scaffold is completely removed. c. Soak the β-TCP / PLGA scaffold in a 95% ethanol solution for ≥4 hours, then irradiate the β-TCP / PLGA scaffold with ultraviolet light for ≥24 hours, and then allow it to air dry and store it in a sealed container.

6. The method for preparing a β-TCP / PLGA scaffold sustained-release formulation loaded with Cu-based nanozymes according to claim 5, characterized in that, In step three, the printing design parameters of the 3D bioprinter are as follows: nozzle diameter 200μm, nozzle temperature 20-40℃, printing aperture 1.0mm, nozzle rotation 3.0mm×0.1mm in reverse and 3.0mm×0.1mm in forward, delay 0s, and travel speed 20mm / min.

7. The method for preparing a β-TCP / PLGA scaffold sustained-release formulation loaded with Cu-based nanozymes according to claim 5, characterized in that, In step three, the temperature of the low-temperature molding chamber is -30℃; the low-temperature molding chamber is a space of 290mm×300mm×210mm, and a silicone film is placed on top of it; the low-temperature molding chamber is also equipped with an openable operating door, and an observation hole is provided on the operating door.

8. The method for preparing a β-TCP / PLGA scaffold sustained-release formulation loaded with Cu-based nanozymes according to claim 1, characterized in that, In step four, the molar mass ratio of K4[Fe(CN)6] to CuSO4·5H2O is 1:2; the mass values ​​of K4[Fe(CN)6], CuSO4·5H2O and PEG-SH can be increased or decreased proportionally.

9. The method for preparing a β-TCP / PLGA scaffold sustained-release formulation loaded with Cu-based nanozymes according to claim 1, characterized in that, In step five, the β-TCP / PLGA scaffold is immersed in the Cu-HCF nanozyme solution for ≥12 hours; the low-temperature freezing temperature of the β-TCP / PLGA scaffold sustained-release body is -37℃, the freezing time is ≥2 hours, and the low-temperature vacuum drying time is ≥72 hours.

10. A β-TCP / PLGA scaffold sustained-release formulation loaded with Cu-based nanozymes, characterized in that, The β-TCP / PLGA scaffold sustained-release formulation comprises a β-TCP / PLGA scaffold and a Cu-based nanozyme loaded on the β-TCP / PLGA scaffold, wherein the β-TCP / PLGA scaffold is loaded with 20 μg-100 μg of Cu-based nanozyme per gram.

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