Composite material, preparation method thereof and application of composite material in preparation of bone tissue repair material

Abstract

The invention relates to the field of gene therapy, and discloses a composite material, a preparation method thereof and application of the composite material in preparation of bone tissue repair materials, the composite material comprises a porous scaffold and an active component loaded on the porous scaffold; wherein the porous scaffold comprises a multi-arm polyethylene glycol-polyester block copolymer and an inorganic mineral substance; wherein the active component comprises a nucleic acid drug and a carrier for delivering the nucleic acid drug; wherein in the multi-arm polyethylene glycol-polyester block copolymer, the molar ratio of a monomer structure of a multi-arm polyethylene glycol unit to a monomer structure of a polyester chain segment is 1: (50-10000). The composite material disclosed by the invention can support and fill a tissue part, can slowly release the encapsulated gene nano-carrier, can improve the stability of the gene and the efficiency of entering cells, enables the gene to be continuously expressed in the cells, and plays a long-term effective role in disease treatment and tissue regeneration.

Description

Technical Field

[0001] The present invention relates to the field of gene therapy. Specifically, it relates to a composite material, a preparation method of a composite material, a composite material prepared by the preparation method, and an application of the composite material in the preparation of a bone tissue repair material. Background Art

[0002] Poly(lactic acid) (PLA) porous scaffolds have advantages such as high porosity, large specific surface area, controllable mechanical strength, and adjustable morphology, and can well simulate the extracellular matrix (ECM). Therefore, they are widely used in fields such as drug delivery, tissue engineering, and regenerative medicine. Currently, most porous scaffolds mainly play a supporting and filling role. However, in addition to the supporting and filling roles, in vivo implanted scaffolds also need to have biological functions to promote the repair and regeneration of damaged tissues.

[0003] Adding functional factors to porous scaffolds can endow the materials with partial biological activity and meet the biological function requirements for tissue repair to a certain extent. Commonly used functional factors include two categories: small molecule drugs and proteins. Small molecule drugs usually refer to synthetic drugs with a molecular weight less than 1000 that can affect cell behavior, including antibiotics, antiviral drugs, anticancer drugs, etc. Due to advantages such as low cost, low immunogenicity, high tissue permeability, and easy storage and transportation, small molecule drugs are often added to porous scaffolds for the treatment of various diseases. However, small molecule drugs also generally have problems such as low drug loading and burst release, and obvious side effects will occur if too much drug is used. Proteins are important components in organisms and participate in a large number of biochemical reactions in the body. Therefore, they are often doped in scaffold materials to endow them with biological activity, thereby promoting disease treatment and tissue regeneration. However, proteins are prone to inactivation and denaturation, have relatively strict requirements for temperature, solvents, and processing conditions, and have a short half-life and are easily degraded, and cannot effectively play a role in the body for a long time.

[0004] Gene therapy is a new technology that induces the expression of related proteins by introducing exogenous genes into organisms to achieve the purpose of disease treatment. Compared with small molecule drugs and proteins, genes can be continuously expressed in cells and have long-term effectiveness; at the same time, the newly secreted proteins have high stability and their effects are more obvious. How to apply the technology of gene therapy to the field of tissue engineering to solve the problem that scaffolds in the field of tissue engineering generally lack biological activity is the main research direction in the future. Summary of the Invention

[0005] The object of the present invention is to apply gene therapy to the field of tissue engineering to overcome the problem of the lack of bioactivity in existing porous scaffolds, and to provide a composite material, a preparation method of the composite material, the composite material prepared by the preparation method, and the application of the composite material in the preparation of bone tissue repair materials. While maintaining the performance of the porous scaffold itself, the composite material can slowly release the encapsulated functional gene, regulate the expression of the gene, and effectively promote disease treatment and tissue regeneration.

[0006] To achieve the above object, in the first aspect of the present invention, a composite material is provided, which comprises a porous scaffold and an active ingredient loaded on the porous scaffold;

[0007] Wherein, the porous scaffold comprises a multi-arm polyethylene glycol-polyester block copolymer and an inorganic mineral;

[0008] Wherein, the active ingredient comprises a nucleic acid drug and a carrier for delivering the nucleic acid drug;

[0009] Wherein, in the multi-arm polyethylene glycol-polyester block copolymer, the molar ratio of the multi-arm polyethylene glycol unit to the monomer structure of the polyester segment is 1:50-10000.

[0010] Preferably, the number-average molecular weight of the multi-arm polyethylene glycol-polyester block copolymer is 12000-1200000 g / mol; and / or

[0011] Wherein, the porosity of the porous scaffold is more than 40%; and / or

[0012] Wherein, the melt index of the porous scaffold at 190 °C and a load of 2.16 kg is 1-50; and / or

[0013] Wherein, the compression modulus of the porous scaffold is 50-500 kPa; and / or

[0014] Wherein, the melt strength of the porous scaffold is 3-30 mN; and / or

[0015] Wherein, the average pore diameter of the porous scaffold is 200-800 μm.

[0016] In the second aspect of the present invention, a preparation method of a composite material is provided, and the method comprises the following steps:

[0017] S1. In the presence of a purified catalyst, bring the purified multi-arm polyethylene glycol into contact with a polyester monomer and carry out a polymerization reaction to obtain a multi-arm polyethylene glycol-polyester block copolymer;

[0018] S2. Mix the multi-arm polyethylene glycol-polyester block copolymer with an inorganic mineral and perform a shaping treatment to obtain a porous scaffold;

[0019] S3. Load the active ingredient onto the porous scaffold to obtain the composite material;

[0020] Among them, the active ingredient includes a nucleic acid drug and a carrier for delivering the nucleic acid drug;

[0021] Among them, the molar ratio of the multi-arm polyethylene glycol to the polyester monomer is 1:50 - 10,000.

[0022] The third aspect of the present invention provides a composite material prepared by the method as described above.

[0023] The fourth aspect of the present invention provides the application of the composite material as described above in the preparation of a bone tissue repair material.

[0024] In the present invention, gene therapy technology and tissue engineering technology are combined. By loading a carrier containing a nucleic acid drug into a porous scaffold, a porous scaffold with gene activity, that is, the composite material described in the present invention, is prepared. While supporting and filling the tissue site, this composite material can slowly release the encapsulated gene nanocarrier, which can improve the stability of the gene and the efficiency of entering cells, enabling it to continuously express in cells and play a long-term and effective role in disease treatment and tissue regeneration.

[0025] The composite material prepared by the method described in the present invention has high cell compatibility, enabling the survival rate of cells on the material to reach more than 90%, and even up to 99%. Moreover, the in vitro protein expression of DNA encapsulated in vesicles is 4.8 times higher than that of naked DNA. This composite material has potential application prospects in the regeneration and repair of bone tissue, skin, nervous system and other fields. Description of the Drawings

[0026] Figure 1 is the release curve of pT7T3D-PacI plasmid (naked and vesicle-encapsulated) in the composite material of Example 1;

[0027] Figure 2 is the release curve of pT7T3D-PacI plasmid (naked and vesicle-encapsulated) in the composite material of Comparative Example 1. Detailed Embodiments

[0028] In the ranges disclosed herein, the endpoints and any values are not limited to the exact range or value. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0029] In a first aspect of the present invention, a composite material is provided, which composite material comprises a porous scaffold and an active ingredient loaded on the porous scaffold;

[0030] Wherein, the porous scaffold comprises a multi-arm polyethylene glycol-polyester block copolymer and an inorganic mineral;

[0031] Wherein, the active ingredient comprises a nucleic acid drug and a carrier for delivering the nucleic acid drug;

[0032] Wherein, in the multi-arm polyethylene glycol-polyester block copolymer, the molar ratio of the multi-arm polyethylene glycol unit to the monomer structure of the polyester segment is 1:50 - 10000.

[0033] In the present invention, the multi-arm polyethylene glycol-polyester block copolymer has a multi-arm polyethylene glycol segment as the core and covalently connects the polyester segments, and is a star copolymer.

[0034] Preferably, in the multi-arm polyethylene glycol-polyester block copolymer, the molar ratio of the multi-arm polyethylene glycol unit to the polyester monomer unit is 1:200 - 2000, such as 1:200, 1:500, 1:800, 1:1000, 1:1200, 1:1500, 1:1800, 1:2000 and any range formed between any two values.

[0035] Preferably, based on the weight of the porous scaffold, the content of the multi-arm polyethylene glycol-polyester block copolymer is 30 - 95 wt%, such as 30, 40, 50, 60, 70, 80, 90, 95 wt% and any range formed between any two values, more preferably 50 - 90 wt%, and the content of the inorganic mineral is 5 - 70 wt%, such as 5, 10, 20, 30, 40, 50, 60, 70 wt% and any range formed between any two values, more preferably 10 - 50 wt%.

[0036] Preferably, the number-average molecular weight of the multi-arm polyethylene glycol-polyester block copolymer is 12000 - 1200000 g / mol, such as 12000, 50000, 100000, 200000, 400000, 600000, 800000, 1000000, 1200000 g / mol and any range formed between any two values, more preferably 100000 - 300000 g / mol.

[0037] Preferably, by weight, in the multi-arm polyethylene glycol-polyester block copolymer, the residue amount of the monomers used to prepare the polymer is 1 wt% or less, the catalyst residue amount is 150 ppm or less, and the organic solvent residue amount is 1000 ppm or less. Among them, for the specific types of each component, refer to the second aspect. The organic solvent residue amount is the total content of various organic solvents after the copolymer is prepared in step S1.

[0038] Preferably, based on the weight of the porous scaffold, the loading amount of the active ingredient is 5 wt% or less, for example, it can be 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5 wt% or less, and any range composed of any two values.

[0039] Preferably, in the active ingredient, the weight ratio of the nucleic acid drug to the carrier is 1:1 - 20, for example, it can be 1:1, 1:5, 1:10, 1:15, 1:20, and any range composed of any two values, and more preferably 1:3 - 12.

[0040] Preferably, the porosity of the porous scaffold is 40% or more, for example, it can be 40, 50, 60, 70, 80, 90% or more, and any range composed of any two values, and more preferably 50 - 80%.

[0041] Preferably, the melt index of the porous scaffold at 190 °C under a load of 2.16 kg is 1 - 50, for example, it can be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, and any range composed of any two values, and more preferably 5 - 30.

[0042] Preferably, the compression modulus of the porous scaffold is 50 - 500 kPa, for example, it can be 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 kPa, and any range composed of any two values, and more preferably 200 - 350 kPa.

[0043] Preferably, the melt strength of the porous scaffold is 3 - 30 mN, for example, it can be 3, 5, 10, 15, 20, 25, 30 mN, and any range composed of any two values, and more preferably 15 - 25 mN.

[0044] Preferably, the average pore size of the porous scaffold is 200 - 800 μm, for example, it can be 200, 300, 400, 500, 600, 700, 800 μm, and any range composed of any two values.

[0045] Preferably, the multi-arm polyethylene glycol unit is derived from multi-arm polyethylene glycol. In the present invention, multi-arm polyethylene glycol refers to polyethylene glycol with at least three arms. Preferably, the number of arms of the multi-arm polyethylene glycol is 3-12, such as 3, 4, 6, 8, 10, 12, and any range formed between any two values, preferably 4-8.

[0046] Preferably, the number-average molecular weight of the multi-arm polyethylene glycol is 2000-200000 g / mol, such as 2000, 5000, 10000, 20000, 40000, 60000, 80000, 100000, 200000 g / mol, and any range formed between any two values, more preferably 10000-50000 g / mol.

[0047] Preferably, the monomer unit of the polyester segment is derived from a polyester monomer, and the polyester monomer is lactide.

[0048] In the present invention, the lactide is L-lactide.

[0049] Preferably, the number-average molecular weight of the polyester segment is 10000-1000000 g / mol, such as 2000, 5000, 10000, 20000, 40000, 60000, 80000, 100000, 200000 g / mol, and any range formed between any two values, more preferably 90000-250000 g / mol.

[0050] In a preferred technical solution of the present invention, the multi-arm polyethylene glycol-polyester block copolymer is a four-arm polyethylene glycol-polylactic acid block copolymer.

[0051] Preferably, the inorganic mineral is selected from at least one of calcium phosphate, hydroxyapatite, bioactive glass, and calcium carbonate.

[0052] Preferably, the nucleic acid drug is DNA and / or RNA.

[0053] Preferably, the DNA is a plasmid encoding an active factor, and its length is within 1 million bp, more preferably 2000-100000 bp.

[0054] Preferably, the RNA is small interfering RNA, and its length is within 100 bp, more preferably 15-35 bp.

[0055] The DNA and RNA can be obtained by conventional means in the art. For example, reference can be made to S. El-Ashram, I. A. Nasr, X. Suo, Nucleic acid protocols: Extraction and optimization. 2016, 12, 33. Those skilled in the art can select appropriate DNA or RNA molecules according to needs. For example, it can be a DNA sequence encoding bone morphogenetic protein BMP-2 (which can exist in the form of a plasmid, such as pT7T3D-PacI plasmid, a plasmid expressing the gene encoding keratinocyte growth factor KGF in vector gWiz TM in which the plasmid expressing the gene encoding keratinocyte growth factor KGF), matrix metalloproteinase MMP-2 siRNA sequence.

[0056] Preferably, the carrier is a lipid nanoparticle carrier or an amphiphilic block copolymer carrier. The carrier can exist in the form of vesicles. Preferably, the diameter of the carrier is less than 1000 nm, more preferably 100 - 400 nm. In the preferred case, the quality of the composite material can be further improved, and the release rate of the nucleic acid drug is slower and more uniform.

[0057] The raw materials for preparing the lipid nanoparticle carrier can include lipid compounds and cholesterol. Preferably, the lipid is phospholipid, preferably selected from at least one of EPC, SPC, DOTAP, DSPC, DOPC, DPPC, DMPC, POPC, DSPG, DPPG, and DMPG.

[0058] In the present invention, the preparation method of the lipid nanoparticle carrier can be referred to A. Akbarzadeh, et al. Liposome: classification, preparation, and applications. 2013, 8, 102. For example, phospholipid and cholesterol can be sonicated in the presence of water to obtain liposomes, wherein the molar amount of phospholipid to cholesterol is preferably 1:0.5 - 2.

[0059] In a preferred embodiment of the present invention, RNA (such as the sense strand and antisense strand of siRNA) is mixed at a molar ratio of 1:0.9 - 1.1, and it is mixed with sodium hyaluronate (weight ratio of 1:0.5 - 2), dissolved in deionized water, then protamine is added (the content of protamine is 1:1 - 1.5 compared to 1 weight part of the total amount of RNA), incubated at room temperature to obtain an aqueous phase material, and a liposome solution is added thereto (the volume ratio of the liposome solution to the aqueous phase material is 1:1 - 2), and incubated at room temperature to form RNA-loaded liposomes.

[0060] Preferably, the amphiphilic block copolymer contains a hydrophilic segment and a hydrophobic segment covalently linked.

[0061] Preferably, the hydrophobic segment is selected from at least one of polylactic acid, polycaprolactone, polyglycolic acid, and polysiloxane.

[0062] Preferably, the hydrophilic segment includes at least one of polyethylene glycol, polyvinyl alcohol, polyacrylic acid, and polyethyleneimine.

[0063] Preferably, the molar ratio of the hydrophobic segment to the hydrophilic segment is 1:0.01 - 1:100, such as 1:0.01, 1:0.1, 1:1, 1:10, 1:20, 1:50, 1:100, and any range formed by any two values.

[0064] In a preferred embodiment of the present invention, an aqueous solution of a plasmid containing a DNA molecule (the concentration can be 1 - 20 mg / mL) is mixed with an organic solution of the amphiphilic block copolymer (the organic solvent can be chloroform, dichloromethane, etc.) to obtain a water / oil emulsion, and then it can be dropped into deionized water containing, for example, PVA (the concentration can be 0.1 - 1 wt%), and the organic solvent is removed to obtain polymer vesicles loaded with the plasmid.

[0065] For the preparation method of the polymer vesicles loaded with the plasmid, reference can also be made to R.Ghasmi.et al.mPEG - PLA and PLA - PEG - PLA nanoparticles as new carriers for delivery of recombinant human Growth Hormone(rhGH).2018,8,9854.

[0066] In the present invention, the melt index is tested by a melt index instrument, the molecular weight is tested by GPC, the compression modulus is tested by dynamic thermomechanical analysis, and the melt strength is tested by a melt strength tester.

[0067] The second aspect of the present invention provides a preparation method of a composite material, and the method includes the following steps:

[0068] S1. In the presence of the purified catalyst, the purified multi - arm polyethylene glycol is contacted with the polyester monomer and subjected to a polymerization reaction to obtain a multi - arm polyethylene glycol - polyester block copolymer;

[0069] S2. The multi - arm polyethylene glycol - polyester block copolymer is mixed with an inorganic mineral and subjected to a shaping process to obtain a porous scaffold;

[0070] S3. The active ingredient is loaded onto the porous scaffold to obtain the composite material;

[0071] Among them, the active ingredient includes a nucleic acid drug and a carrier for delivering the nucleic acid drug;

[0072] Among them, the molar ratio of the multi-arm polyethylene glycol to the polyester monomer is 1:50 - 10,000.

[0073] Preferably, the molar ratio of the multi-arm polyethylene glycol to the polyester monomer is 1:20 - 2,000, such as 1:200, 1:500, 1:800, 1:1,000, 1:1,200, 1:1,500, 1:1,800, 1:2,000 and any range formed between any two values.

[0074] Preferably, the dosages of each component are such that based on the weight of the porous scaffold, the content of the multi-arm polyethylene glycol-polyester block copolymer is 30 - 95 wt%, such as 30, 40, 50, 60, 70, 80, 90, 95 wt% and any range formed between any two values, preferably 50 - 90 wt%, and the content of the inorganic mineral is 5 - 70 wt%, such as 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70 wt% and any range formed between any two values, preferably 10 - 50 wt%.

[0075] Preferably, the dosages of each component are such that based on the weight of the porous scaffold, the loading amount of the active ingredient is 5 wt% or less, such as 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5 wt% or less and any range formed between any two values.

[0076] Preferably, in the active ingredient, the weight ratio of the nucleic acid drug to the carrier is 1:1 - 20, such as 1:1, 1:5, 1:10, 1:15, 20 or less and any range formed between any two values, preferably 1:3 - 12.

[0077] Preferably, the catalyst is selected from at least one of stannous octoate, stannous caprylate, tributylaluminum, and diethylzinc.

[0078] Preferably, the dosage of the catalyst is 0.1 - 1 wt% of the multi-arm polyethylene glycol, such as 0.1, 0.2, 0.4, 0.6, 0.8, 1 wt% and any range formed between any two values, preferably 0.2 - 0.6 wt%.

[0079] For the descriptions of the multi-arm polyethylene glycol, polyester monomer, inorganic mineral, nucleic acid drug, and carrier, please refer to the first aspect and will not be elaborated here.

[0080] Preferably, in step S1, the manner of contacting and carrying out the polymerization reaction includes: performing first impurity removal on the multi-arm polyethylene glycol, then adding a catalyst for second impurity removal, and then adding a polyester monomer and carrying out the polymerization reaction to obtain a multi-arm polyethylene glycol-polyester block copolymer.

[0081] Preferably, the manner of the first impurity removal is to maintain at 60-140 °C for 1-4 h under vacuum conditions.

[0082] Preferably, the manner of the second impurity removal is to maintain at 60-140 °C for 5-30 min under an inert atmosphere.

[0083] Preferably, the conditions of the polymerization reaction include: the reaction temperature is 100-200 °C, preferably 120-160 °C; the reaction time is 3-6 h, preferably 4-5 h. The polymerization reaction can be carried out under an inert atmosphere.

[0084] In the present invention, the inert atmosphere can be a nitrogen atmosphere or an argon atmosphere, etc.

[0085] The product obtained from the polymerization reaction can be purified to obtain a purified multi-arm polyethylene glycol-polyester block copolymer. The manner of purification can be, for example, first dissolving in chloroform to obtain a polymer solution, and then dropping the polymer solution into ether. The precipitated solid is dried to obtain the purified multi-arm polyethylene glycol-polyester block copolymer.

[0086] Preferably, the operation of step S1 makes the residual amount of the monomer used to prepare the polymer in the multi-arm polyethylene glycol-polyester block copolymer be 1 wt% or less, the catalyst residual amount be 150 ppm or less, and the organic solvent residual amount be 1000 ppm or less by weight.

[0087] In the present invention, in step S2, the multi-arm polyethylene glycol-polyester block copolymer is mixed with an inorganic mineral and subjected to a shaping treatment to obtain a porous scaffold; among them, the two can be mixed in an organic solvent. Preferably, the inorganic mineral is dispersed in an organic solvent and ultrasonically mixed, and then the multi-arm polyethylene glycol-polyester block copolymer is added and stirred to obtain a mixture. The inorganic mineral can be ultrasonically treated in an organic solvent for 20-40 min first, and then the multi-arm polyethylene glycol-polyester block copolymer is added and mixed evenly, and then the obtained material after removing the solvent is used for the shaping treatment. In the present invention, the conditions of the ultrasonic treatment are not particularly limited as long as the inorganic mineral can be evenly dispersed in the organic solvent. Similarly, the stirring conditions after adding the multi-arm polyethylene glycol-polyester block copolymer are not particularly limited. For example, it can be stirred at room temperature for 10-14 h. The organic solvent can be chloroform or a mixed solvent of chloroform / dimethylformamide, and in the mixed solvent of chloroform / dimethylformamide, the mass ratio of chloroform to dimethylformamide is preferably 1:1-6.

[0088] Preferably, in step S2, the forming method is electrospinning and / or 3D printing.

[0089] In the present invention, the forming method of electrospinning is exemplarily described. Among them, the method of electrospinning is as follows: The mixture obtained by mixing the multi-arm polyethylene glycol-polyester block copolymer and the inorganic mineral is added into a syringe (for example, the needle can be 22G). An aluminum disk is placed 10 - 20 cm below the needle, and a high voltage of 10 - 25 kV is applied between the aluminum disk and the needle. The mixture is extruded at a rate of 0.5 - 1.5 mL / h. To ensure the uniformity of the scaffold, the aluminum disk is rotated by a certain angle (45 - 90°) every 10 - 15 minutes during the process. The obtained multi-arm polyethylene glycol-polyester block copolymer scaffold is dried in a vacuum oven and reserved for use.

[0090] In the present invention, the inner diameter of the needle, the distance between the needle and the aluminum disk, the pressure between the aluminum disk and the needle, the extrusion rate of the mixture, the rotation angle and method of the aluminum disk are all conventional methods. The above data are only for exemplary illustration of the operation and do not limit it.

[0091] Further, exemplarily, the method of 3D printing is as follows: The mixture obtained by mixing the multi-arm polyethylene glycol-polyester block copolymer and the inorganic mineral is poured into a Teflon mold, placed in a fume hood for 12 h, and then completely dried in an oven and cut into small pieces. The small pieces of the mixture are made into filaments with a capillary rheometer, and the filaments are printed into a composite material of the required size by a 3D printer.

[0092] In the present invention, the placement time after the mixture is poured into the Teflon mold, the size of the small pieces, the size of the filaments, and the size of the composite material are only for exemplary illustration of the operation and do not limit it.

[0093] In the present invention, exemplarily, the conditions for 3D printing are set as follows: the line spacing is 0.6 - 1 mm, the ambient temperature is 3 - 5 °C, the printing speed is 80 - 100 mm / s, the nozzle temperature is 150 - 180 °C, and the platform temperature is 20 - 50 °C.

[0094] Preferably, by weight, the forming conditions are such that the content of the catalyst in the multi-arm polyethylene glycol-polyester block copolymer, calculated as a metal element, is not more than 150 ppm.

[0095] In a preferred embodiment of the present invention, the preparation method of the composite material includes the following steps:

[0096] The multi-arm polyethylene glycol (PEG) is heated at 100 - 150 °C under vacuum for 1 - 3 h to remove water and impurities. Then, in the presence of the catalyst after impurity removal, PEG and polyester monomers are subjected to a polymerization reaction at 100 - 200 °C for 4 - 5 h to obtain a multi-arm polyethylene glycol-polyester block copolymer.

[0097] The multi-arm polyethylene glycol-polyester block copolymer is added to chloroform to dissolve and obtain a polymer solution. The polymer solution is added dropwise to ether, and a solid is precipitated. The solid is dried under vacuum to obtain a purified multi-arm polyethylene glycol-polyester block copolymer.

[0098] The inorganic mineral is dissolved in chloroform. After ultrasonic treatment for 20 - 40 min, the purified multi-arm polyethylene glycol-polyester block copolymer is added. After stirring evenly at room temperature, it is processed into a porous scaffold.

[0099] In step S3, the active ingredient can be loaded onto the porous scaffold in a conventional manner in the art. For example, the porous scaffold can be soaked in a material containing the active ingredient, or the material containing the active ingredient can be dropped onto the porous scaffold.

[0100] The third aspect of the present invention provides a composite material prepared by the method described above.

[0101] The fourth aspect of the present invention provides the application of the composite material described above in the preparation of bone tissue repair materials.

[0102] The present invention will be described in detail below through examples.

[0103] PEG is purchased from sigma-aldrich. Unless otherwise specified, the molecular weight of PEG involved in the examples and comparative examples is 20,000.

[0104] Chloroform is purchased from sigma-aldrich.

[0105] Lactide is L-lactide and is purchased from sigma-aldrich.

[0106] Diethylzinc is purchased from sigma-aldrich.

[0107] Stannous octoate is purchased from sigma-aldrich.

[0108] Hydroxyapatite is purchased from sigma-aldrich.

[0109] The method for measuring the degradation rate is to put the scaffold into a 1×PBS buffer solution with a pH of 7.2, then place it on a shaker at 37 °C and 120 rpm. After taking it out at different time points, it is dried and weighed. When the polymer completely disappears, it is regarded as complete degradation, and the time at this time is the degradation time.

[0110] Example 1

[0111] This example is used to illustrate the preparation method of the composite material described in the present invention.

[0112] (1) Preparation of the porous scaffold

[0113] Add 1 g of hydrophilic four-armed polyethylene glycol (PEG) into a reaction flask, heat it at 100 °C under vacuum for 2 h to remove water and impurities. In an N 2 atmosphere, add 0.05 mL of a toluene solution of stannous octoate (100 mg / mL) into the reaction flask (the effective mass of the catalyst is 5 mg), and heat it at 100 °C under vacuum for 15 min to remove toluene and impurities; in an N 2 atmosphere, add 4.375 g of L-lactide into the reaction flask, and carry out a polymerization reaction with stirring at 130 °C for 4 h to obtain a crude product of four-armed polyethylene glycol-polyester block copolymer.

[0114] Add the crude product of four-armed polyethylene glycol-polyester block copolymer into 25 mL of chloroform and dissolve it overnight to obtain a polymer solution. Dropwise add the polymer solution into 250 mL of ether to precipitate a solid. Vacuum-dry the solid at 60 °C to obtain a purified four-armed polyethylene glycol-polyester block copolymer; it is measured that, by weight, the residual amount of tin element of the catalyst in the copolymer is 2.1 ppm, the monomer residual amount < 1 wt%, the residual amounts of toluene and chloroform are lower than 100 ppm, and the residual amount of ether is lower than 100 ppb.

[0115] Dissolve 0.82 g of hydroxyapatite in 5 mL of chloroform and ultrasonicate for 30 min, add 2.5 g of the purified four-armed polyethylene glycol-polyester block copolymer, mix evenly and stir for 12 h. Pour the mixture into a Teflon mold, and place it in a fume hood for 12 h to volatilize the solvent. Completely dry the mixture in an oven at 60 °C and cut it into small pieces. Extrude the small pieces through a capillary rheometer from a 1.75-mm hole at 160 °C to obtain filaments. Control the environmental temperature at 4 °C, the printing speed at 90 mm / s, the nozzle temperature at 160 °C, and the platform temperature at 30 °C, and print the filaments into a 10 mm × 10 mm × 4 mm porous scaffold with a 3D printer (set the cross-section of the printed line to be 0.4 mm × 0.4 mm and the line spacing to be 0.8 mm).

[0116] Among them, the molar ratio of the hydrophilic PEG segment to the lipophilic polyester segment, the number of arms of PEG, the content of the block copolymer, the content of the inorganic mineral, the melt index and the average pore size are shown in Table 1; the number-average molecular weight of PEG, the number-average molecular weight of the PEG segment, the number-average molecular weight of the polyester segment, and the number-average molecular weight of the multi-arm polyethylene glycol-polyester block copolymer are shown in Table 1 (continued); the molar ratio of PEG to the polyester monomer, the dosage of the catalyst relative to PEG, the content of the catalyst in the crude amphiphilic block copolymer, and the addition amount of the inorganic mineral are shown in Table 2.

[0117] (2) Preparation of PLA-PEG-PLA polymer vesicles loaded with pT7T3D-PacI plasmid

[0118] The pT7T3D-PacI plasmid encoding bone morphogenetic protein BMP-2 was amplified using an Escherichia coli transformant, and after isolation and purification, its absorbance at 260 nm was measured using a UV-visible spectrophotometer. The concentration of the DNA plasmid was calculated by the Lambert-Beer law c = A260 / εb. Then the DNA aqueous solution was lyophilized and stored at -20 °C for later use.

[0119] 1 mL of the aqueous solution of pT7T3D-PacI plasmid (concentration 10 mg / mL) was poured into 4 mL of the chloroform solution of PLA-PEG-PLA (concentration 25 mg / mL) to obtain a water / oil emulsion. Then the emulsion was added dropwise into 20 mL of deionized water containing 0.4 wt% PVA using a syringe and stirred at room temperature until the chloroform completely evaporated, obtaining PLA-PEG-PLA polymer vesicles loaded with pT7T3D-PacI plasmid, with a vesicle size of about 200 nm.

[0120] (3) Preparation of the composite material

[0121] The four-arm PEG-PLA porous scaffold was immersed in the above polymer vesicle solution for 2 h to allow the vesicles to diffuse into the porous material, obtaining a porous scaffold with gene activity, namely the composite material. This composite material can be used for filling bone tissue defects. After the encapsulated pT7T3D-PacI plasmid enters bone tissue cells, it can express BMP-2, induce the differentiation of bone marrow stromal cells, and promote bone tissue repair and regeneration.

[0122] Example 2

[0123] This example is used to illustrate the preparation method of the composite material of the present invention.

[0124] (1) Preparation of the porous scaffold

[0125] 2 g of hydrophilic four-arm polyethylene glycol (PEG) was added to a reaction flask and heated at 100 °C under vacuum for 2 h to remove water and impurities. Under N 2In an atmosphere, 0.1 mL of a toluene solution of diethylzinc (100 mg / mL) was added to a reaction flask (the effective mass of the catalyst was 10 mg), and it was heated at 100 °C under vacuum for 15 min to remove toluene and impurities; in an N 2 atmosphere, 8.75 g of lactide was added to the reaction flask, and polymerization reaction was carried out with stirring at 130 °C for 5 h to obtain a crude product of tetra-arm polyethylene glycol-polyester block copolymer;

[0126] The crude product of tetra-arm polyethylene glycol-polyester block copolymer was added to 50 mL of chloroform and dissolved overnight to obtain a polymer solution. The polymer solution was added dropwise to 500 mL of ether, and a solid was precipitated. The solid was dried under vacuum at 60 °C to obtain a purified tetra-arm polyethylene glycol-polyester block copolymer;

[0127] 0.41 g of hydroxyapatite was dissolved in 50 mL of a 1:4 (v / v) dimethylformamide / chloroform mixed solvent and sonicated for 30 min. 1.25 g of the purified tetra-arm polyethylene glycol-polyester block copolymer was added. After mixing evenly, it was stirred for 12 h and loaded into a 5 mL syringe (the needle was 22G). An aluminum plate was placed 15 cm below. A high voltage of 12 kV was applied between the aluminum plate and the needle. The mixed solution was extruded at a speed of 0.7 mL / h. To ensure uniformity, the aluminum plate was rotated 90° every 15 min during the process, and the average pore size and porosity were controlled to be the same as those in Example 1.

[0128] Among them, the molar ratio of the hydrophilic PEG segment to the lipophilic polyester segment, the number of arms of PEG, the content of the block copolymer, the content of the inorganic mineral, the melt index and the average pore size are shown in Table 1; the number-average molecular weight of PEG, the number-average molecular weight of the PEG segment, the number-average molecular weight of the polyester segment, and the number-average molecular weight of the multi-arm polyethylene glycol-polyester block copolymer are shown in Table 1 (continued); the molar ratio of PEG to the polyester monomer, the dosage of the catalyst relative to PEG, the content of the catalyst in the amphiphilic block copolymer crude product, and the addition amount of the inorganic mineral are shown in Table 2.

[0129] (2) Preparation of liposomes loaded with MMP-2 siRNA

[0130] Design the matrix metalloproteinase MMP-2 siRNA sequence (the sense strand sequence is 5'-CUGCAAACAGGACAUUGUA-3', and the antisense strand sequence is 5'-UACAAUGUCCUGUUUGCAG-3'). Synthesize the designed sequence using a synthesizer, add ammonia water, and heat at 60 °C for 3 h for ammonolysis. Then place the sample in a vacuum centrifugal concentrator and heat at 60 °C for 40 min until the ammonia water is completely removed. The deaminated RNA is purified using high-performance liquid chromatography (HPLC), and its absorbance at 260 nm is measured using an ultraviolet-visible spectrophotometer. The concentration of RNA is calculated by the Lambert-Beer law c = A260 / εb. Finally, lyophilize the RNA aqueous solution and store it at -20 °C for later use.

[0131] Add 1 mL of DOTAP (10 mM) and 1 mL of cholesterol (10 mM) to a 25 mL round-bottom flask, and evaporate the solvent to dryness under vacuum using a rotary evaporator. Add 2 mL of deionized water to the flask and sonicate to obtain a liposome solution. Mix the sense strand and antisense strand of siRNA in a molar ratio of 1:1. Take 12.5 μg of RNA and mix it with 12.5 μg of sodium hyaluronate, dissolve them in 100 μL of deionized water, add 13.5 μg of protamine to the water, and incubate at room temperature for 10 min. Then add 60 μL of the liposome solution and incubate at room temperature for 10 min to form liposomes loaded with MMP-2 siRNA.

[0132] (3) Preparation of the composite material

[0133] Drop the above liposome solution onto a four-arm PEG-PLA porous scaffold and let it stand for 2 h to allow the liposomes to diffuse into the porous material, obtaining a porous scaffold with gene activity, that is, the composite material. This composite material can be used for ulcer repair in diabetic patients. The encapsulated MMP-2 siRNA can effectively reduce the excessive MMP level in the patient's body and promote wound healing.

[0134] Example 3

[0135] This example is used to illustrate the preparation method of the composite material described in the present invention.

[0136] (1) Preparation of the porous scaffold

[0137] Add 1 g of hydrophilic four-arm polyethylene glycol (PEG) to a reaction flask, heat at 100 °C under vacuum for 2 h to remove water and impurities, and under N 2 atmosphere, add 0.05 mL of a toluene solution of stannous octoate (100 mg / mL) to the reaction flask (the effective mass of the catalyst is 5 mg), and heat at 100 °C under vacuum for 15 min to remove toluene and impurities; under N 2In an atmosphere, 8.75 g of L-lactide was added to a reaction flask, and polymerization reaction was carried out with stirring at 130 °C for 4 h to obtain a crude product of four-armed polyethylene glycol-polyester block copolymer;

[0138] The crude product of four-armed polyethylene glycol-polyester block copolymer was added to 50 mL of chloroform and dissolved overnight to obtain a polymer solution. The polymer solution was added dropwise to 500 mL of ether, and a solid was precipitated. The solid was vacuum dried at 60 °C to obtain a purified four-armed polyethylene glycol-polyester block copolymer;

[0139] 3.3 g of calcium phosphate was dissolved in 20 mL of chloroform and ultrasonicated for 30 min. 10 g of the purified four-armed polyethylene glycol-polyester block copolymer was added. After mixing evenly, it was stirred for 12 h. The mixture was poured into a Teflon mold and placed in a fume hood for 12 h to allow the solvent to volatilize. The mixture was completely dried in an oven at 60 °C and then cut into small pieces, and extruded through a 1.75-mm hole at 160 °C by a capillary rheometer to obtain filaments. The filaments were printed into a porous scaffold of 10 mm × 10 mm × 4 mm by a 3D printer. (The printing conditions are the same as in Example 1)

[0140] Among them, the molar ratio of the hydrophilic PEG segment to the lipophilic polyester segment, the number of arms of PEG, the content of the block copolymer, the content of the inorganic mineral, the melt index, and the average pore size are shown in Table 1; the number-average molecular weight of PEG, the number-average molecular weight of the PEG segment, the number-average molecular weight of the polyester segment, and the number-average molecular weight of the multi-armed polyethylene glycol-polyester block copolymer are shown in Table 1 (continued); the molar ratio of PEG to the polyester monomer, the dosage of the catalyst relative to PEG, the content of the catalyst in the amphiphilic block copolymer crude product, and the addition amount of the inorganic mineral are shown in Table 2.

[0141] (2) Preparation of PCL-PEG-PCL polymer vesicles loaded with KGF plasmid

[0142] Keratinocyte growth factor KGF was expressed in the commercial blank vector gWiz TM to obtain a plasmid encoding KGF. The absorbance at 260 nm was measured using a UV-visible spectrophotometer, and the concentration of the DNA plasmid was calculated by the Lambert-Beer law c = A260 / εb. Then the DNA aqueous solution was freeze-dried and stored at -20 °C for later use.

[0143] 1 mL of the aqueous solution of KGF plasmid (concentration: 10 mg / mL) was poured into 4 mL of the chloroform solution of PCL-PEG-PCL (concentration: 25 mg / mL) to obtain a water / oil emulsion. Then the emulsion was added dropwise to 20 mL of deionized water containing 0.4 wt% PVA with stirring at room temperature until the organic solvent completely volatilized to obtain PCL-PEG-PCL polymer vesicles loaded with KGF plasmid.

[0144] (3) Preparation of the composite material

[0145] Immerse the four-arm PEG-PLA porous scaffold in the above polymer vesicle solution for 2 h to allow the vesicles to diffuse into the porous material, obtaining a porous scaffold with gene activity, i.e., the composite material. This composite material can be used for the repair of skin wounds. After the encapsulated KGF plasmid enters the skin tissue cells, it can express KGF, improve the epidermal cell regeneration rate and keratinocyte proliferation rate, and promote wound healing.

[0146] Example 4

[0147] This example is used to illustrate the preparation method of the composite material of the present invention.

[0148] According to the method of Example 1, the difference is that the hydrophilic four-arm polyethylene glycol is replaced with an equimolar amount of hydrophilic three-arm polyethylene glycol. The results are shown in Table 1.

[0149] Among them, the molar ratio of the hydrophilic PEG segment to the lipophilic polyester segment, the number of arms of PEG, the content of the block copolymer, the content of the inorganic mineral, the melt index and the average pore size are shown in Table 1; the number-average molecular weight of PEG, the number-average molecular weight of the PEG segment, the number-average molecular weight of the polyester segment and the number-average molecular weight of the multi-arm polyethylene glycol-polyester block copolymer are shown in Table 1 (continued); the molar ratio of PEG to the polyester monomer, the amount of the catalyst relative to PEG, the content of the catalyst in the crude amphiphilic block copolymer and the addition amount of the inorganic mineral are shown in Table 2.

[0150] Example 5

[0151] This example is used to illustrate the preparation method of the composite material of the present invention.

[0152] According to the method of Example 1, the difference is that the number-average molecular weight of the hydrophilic four-arm polyethylene glycol is 100000 g / mol.

[0153] Among them, the molar ratio of the hydrophilic PEG segment to the lipophilic polyester segment, the number of arms of PEG, the content of the block copolymer, the content of the inorganic mineral, the melt index and the average pore size are shown in Table 1; the number-average molecular weight of PEG, the number-average molecular weight of the PEG segment, the number-average molecular weight of the polyester segment and the number-average molecular weight of the multi-arm polyethylene glycol-polyester block copolymer are shown in Table 1 (continued); the molar ratio of PEG to the polyester monomer, the amount of the catalyst relative to PEG, the content of the catalyst in the crude amphiphilic block copolymer and the addition amount of the inorganic mineral are shown in Table 2.

[0154] Example 6

[0155] This example is used to illustrate the preparation method of the composite material of the present invention.

[0156] According to the method of Example 1, except that the amount of hydroxyapatite is 0.25 g, such that the mass fraction of the inorganic mineral is 9 wt%.

[0157] Among them, the molar ratio of the hydrophilic PEG segment to the lipophilic polyester segment, the number of arms of PEG, the content of the block copolymer, the content of the inorganic mineral, the melt index and the average pore size are shown in Table 1; the number-average molecular weight of PEG, the number-average molecular weight of the PEG segment, the number-average molecular weight of the polyester segment, and the number-average molecular weight of the multi-arm polyethylene glycol-polyester block copolymer are shown in Table 1 (continued); the molar ratio of PEG to the polyester monomer, the amount of the catalyst relative to PEG, the content of the catalyst in the crude amphiphilic block copolymer, and the addition amount of the inorganic mineral are shown in Table 2.

[0158] Comparative Example 1

[0159] This comparative example is used to illustrate the preparation method of the reference composite material.

[0160] According to the method of Example 1, except that the amount of hydrophilic tetra-armed polyethylene glycol is 13.5 g, such that the molar ratio of hydrophilic tetra-armed polyethylene glycol to L-lactide monomer is 1:45.

[0161] Among them, the molar ratio of the hydrophilic PEG segment to the lipophilic polyester segment, the number of arms of PEG, the content of the block copolymer, the content of the inorganic mineral, the melt index and the average pore size are shown in Table 1; the number-average molecular weight of PEG, the number-average molecular weight of the PEG segment, the number-average molecular weight of the polyester segment, and the number-average molecular weight of the multi-arm polyethylene glycol-polyester block copolymer are shown in Table 1 (continued); the molar ratio of PEG to the polyester monomer, the amount of the catalyst relative to PEG, the content of the catalyst in the crude amphiphilic block copolymer, and the addition amount of the inorganic mineral are shown in Table 2.

[0162] Comparative Example 2

[0163] This comparative example is used to illustrate the preparation method of the reference composite material.

[0164] According to the method of Example 1, except that the hydrophilic tetra-armed polyethylene glycol is replaced with an equimolar amount of hydrophilic linear polyethylene glycol (number of arms is 2).

[0165] Among them, the molar ratio of the hydrophilic PEG segment to the lipophilic polyester segment, the number of arms of PEG, the content of the block copolymer, the content of the inorganic mineral, the melt index and the average pore size are shown in Table 1; the number-average molecular weight of PEG, the number-average molecular weight of the PEG segment, the number-average molecular weight of the polyester segment, and the number-average molecular weight of the multi-arm polyethylene glycol-polyester block copolymer are shown in Table 1 (continued); the molar ratio of PEG to the polyester monomer, the amount of the catalyst relative to PEG, the content of the catalyst in the crude amphiphilic block copolymer, and the addition amount of the inorganic mineral are shown in Table 2.

[0166] Comparative Example 3

[0167] This comparative example is used to illustrate the preparation method of the reference composite material.

[0168] According to the method of Example 1, except that the amount of hydrophilic tetra-arm polyethylene glycol is 4.05 g, so that the molar ratio of hydrophilic tetra-arm polyethylene glycol to L-lactide monomer is 1:150.

[0169] Among them, the molar ratio of hydrophilic PEG segment to lipophilic polyester segment, the number of arms of PEG, the content of block copolymer, the content of inorganic mineral, the melt index and the average pore size are shown in Table 1; the number-average molecular weight of PEG, the number-average molecular weight of PEG segment, the number-average molecular weight of polyester segment and the number-average molecular weight of multi-arm polyethylene glycol-polyester block copolymer are shown in Table 1 (continued); the molar ratio of PEG to polyester monomer, the amount of catalyst relative to PEG, the content of catalyst in the crude amphiphilic block copolymer and the addition amount of inorganic mineral are shown in Table 2.

[0170] Table 1

[0171]

[0172] PEG: polyester a : refers to the molar ratio of hydrophilic PEG segment to lipophilic polyester segment

[0173] Table 1 (continued)

[0174]

[0175]

[0176] Table 2

[0177] Number <![CDATA[PEG: Polyester b > Catalyst dosage / wt% Catalyst content / ppm Inorganic mineral additive amount / wt% Example 1 1:608 0.5 93 24.8 Example 2 1:608 0.5 93 24.8 Example 3 1:1216 0.5 51 24.8 Example 4 1:608 0.5 95 24.8 Example 5 1:3040 0.5 95 24.8 Example 6 1:608 0.5 93 9 Comparative Example 1 1:45 0.04 56 24.8 Comparative Example 2 1:608 0.5 98 24.8 Comparative Example 3 1:150 0.12 72 24.8

[0178] PEG: polyester b : refers to the molar ratio of hydrophilic PEG to lipophilic polyester monomer

[0179] Test Example 1

[0180] The porous scaffolds (without loading active ingredients) prepared in the examples and comparative examples were tested for modulus, melt strength and performance, and the results are shown in Table 3.

[0181] Table 3

[0182] Number Compression modulus (kPa) Melt strength (mN) Degradation time (weeks) Example 1 280 20 16 Example 2 300 20 18 Example 3 410 29 22 Example 4 200 15 16 Example 5 260 18 15 Example 6 250 18 16 Comparative Example 1 30 2 6 Comparative Example 2 120 11 13 Comparative Example 3 90 5 12

[0183] From the above results, it can be seen that Examples 1-6 of the four-armed lactic acid block copolymer of the present invention have higher number-average molecular weight, compression modulus and melt strength. Among them, the compression modulus is not less than 200 kPa, the melt strength is not less than 15 mN, and the catalyst content is less than 100 ppm.

[0184] In Comparative Example 1 and Comparative Example 3, due to the excessive or too small molar ratio of hydrophilic PEG and lipophilic polyester monomers, the strength is reduced, and it cannot be well applied to bone tissue repair. In Comparative Example 2, due to the low mechanical strength of linear polyethylene glycol (arm number is 2), it also cannot be well applied to bone tissue repair.

[0185] Test Example 2

[0186] This test example is used to illustrate the evaluation results of the porous scaffolds and composite materials described in Example 1 and Comparative Example 1.

[0187] (1) Biocompatibility of four-armed PEG-PLA scaffold

[0188] After culturing endothelial cells HUVECs in the prepared four-armed PEG-PLA scaffold for 3 days, the cells were stained with live and dead stains.

[0189] According to statistics, after culturing the porous scaffold described in Example 1, 99% of the cells were still viable, higher than 96% of Comparative Example 1, proving that the porous scaffold has better biocompatibility. The biocompatibility of the porous scaffolds prepared in other examples is basically the same as that of Example 1.

[0190] (2) Study on the release of DNA from the composite material

[0191] The operations were carried out respectively according to the methods described in Example 1 and Comparative Example 1, except that the porous scaffold was directly immersed in 20 mL of an aqueous solution of pT7T3D-PacI plasmid (concentration 0.5 mg / mL) for 2 h to obtain a porous scaffold loaded with naked plasmid.

[0192] The composite materials prepared in Example 1 and Preparation Example 1 and the corresponding porous scaffolds loaded with naked plasmid were respectively immersed in 10 mL of 1×PBS buffer solution, and 10 μL of the buffer solution was taken at a specific time to detect the DNA content therein, so as to explore the release behavior of pT7T3D-PacI plasmid in the scaffold. Figure 1 and Figure 2 are respectively the release curves of pT7T3D-PacI plasmid (including naked and vesicle-encapsulated plasmids) in the composite materials of Example 1 and Comparative Example 1.

[0193] It can be seen that the naked DNA in Example 1 was completely released on the 6th day, while the DNA encapsulated in vesicles was completely released on the 25th day. In Comparative Example 1, the naked DNA was completely released on the 4th day, and the DNA encapsulated in vesicles was completely released on the 18th day. This indicates that the composite material of the present invention can significantly delay the release rate of the active ingredient, achieving a sustained-release effect, and thus can play a role more continuously.

[0194] (3) In vitro expression of pT7T3D-PacI plasmid

[0195] The composite materials were respectively tested for transfection of human mesenchymal stem cells hMSC. The results are shown in Table 4. The protein expression efficiency of the pT7T3D-PacI plasmid of the composite material corresponding to Example 1 of the present invention is higher, indicating that the composite material of the present invention is more conducive to entering cells, thereby improving the gene expression effect.

[0196] In addition, the protein expression efficiency of the pT7T3D-PacI plasmid encapsulated in PEG-PLA vesicles was significantly improved, even reaching 4.8 times, far exceeding the expected effect. This also proves that DNA encapsulated in a nanocarrier is more conducive to entering cells, thereby improving the gene expression effect.

[0197] Table 4

[0198] BMP-2 in vitro protein expression (pg / mg protein) Naked DNA DNA encapsulated in vesicles Example 1 120 580 Comparative Example 1 85 430

[0199] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A composite material, characterized in that: The composite material comprises a porous support and an active component loaded on the porous support; Wherein, the porous scaffold comprises a multi-arm polyethylene glycol-polyester block copolymer and an inorganic mineral; Wherein, the active ingredient comprises a nucleic acid drug and a carrier for delivering the nucleic acid drug; Wherein, in the multi-arm polyethylene glycol-polyester block copolymer, the molar ratio of the monomer structure of the multi-arm polyethylene glycol unit and the polyester segment is 1:50-10000.

2. The composite material according to claim 1, wherein Based on the weight of the porous support, the content of the multi-arm polyethylene glycol-polyester block copolymer is 30-95wt%, preferably 50-90wt%, and the content of the inorganic mineral is 5-70wt%, preferably 10-50wt%; and / or Wherein, in the multi-arm polyethylene glycol-polyester block copolymer, the molar ratio of the monomer structure of the multi-arm polyethylene glycol unit and the polyester segment is 1:200-2000; and / or The active ingredient loading is less than 5 wt % based on the weight of the porous support; and / or In the active ingredient, the weight ratio of the nucleic acid drug to the carrier is 1:1-20, preferably 1:3-12; and / or In the multi-arm polyethylene glycol-polyester block copolymer, the residual amount of the monomer used to prepare the polymer is less than 1wt%, the residual amount of the catalyst is less than 150ppm, and the residual amount of the organic solvent is less than 1000ppm.

3. The composite material according to claim 1 or 2, wherein: The number average molecular weight of the multi-arm polyethylene glycol-polyester block copolymer is 12000-1200000 g / mol, preferably 100000-300000 g / mol; and / or Wherein, the porosity of the porous support is above 40%, preferably 50-80%; and / or The porous support has a melt index of 1-50, preferably 5-30 at 190°C and 2.16 kg load; and / or Wherein, the compression modulus of the porous scaffold is 50-500 kPa, preferably 200-350 kPa; and / or Wherein, the melt strength of the porous support is 3-30 mN, preferably 15-25 mN; and / or Wherein, the average pore size of the porous scaffold is 200-800 μm.

4. The composite material according to any one of claims 1 to 3, wherein: The multi-arm polyethylene glycol unit is derived from multi-arm polyethylene glycol, and the number of arms of the multi-arm polyethylene glycol is 3-12, preferably 4-8; and / or The monomer unit of the polyester chain segment comes from a polyester monomer, and the polyester monomer is lactide; The inorganic mineral is selected from at least one of calcium phosphate, hydroxyapatite, bioglass and calcium carbonate; Preferably, the number average molecular weight of the multi-arm polyethylene glycol is 2000-200000 g / mol, more preferably 10000-50000 g / mol; Preferably, the number average molecular weight of the polyester segment is 10,000-1,000,000 g / mol, preferably 90,000-250,000 g / mol.

5. The composite material according to any one of claims 1 to 3, wherein: The nucleic acid drug is DNA and / or RNA; Preferably, the DNA is a plasmid encoding an active factor, and its length is within 1 million bp, more preferably 2000-100,000 bp; Preferably, the RNA is a small interfering RNA with a length of less than 100 bp, more preferably 15-35 bp; and / or The carrier is a lipid nanoparticle carrier or an amphiphilic block copolymer carrier.

6. A method for preparing a composite material, characterized in that: The method comprises the following steps: S1. In the presence of a catalyst after impurities are removed, the multi-arm polyethylene glycol after impurities are contacted with a polyester monomer and subjected to a polymerization reaction to obtain a multi-arm polyethylene glycol-polyester block copolymer; S2, mixing the multi-arm polyethylene glycol-polyester block copolymer with an inorganic mineral and subjecting the mixture to a molding process to obtain a porous scaffold; S3, loading the active ingredient onto the porous support to obtain the composite material; Wherein, the active ingredient comprises a nucleic acid drug and a carrier for delivering the nucleic acid drug; Wherein, the molar ratio of the multi-arm polyethylene glycol to the polyester monomer is 1:50-10000.

7. The method according to claim 6, wherein: The molar ratio of multi-arm polyethylene glycol to polyester monomer is 1:200-2000; and / or The amount of the catalyst is 0.1-1 wt %, preferably 0.2-0.6 wt % of the multi-arm polyethylene glycol; and / or The amounts of the components are such that, based on the weight of the porous support, the content of the multi-arm polyethylene glycol-polyester block copolymer is 30-95wt%, preferably 50-90wt%, and the content of the inorganic mineral is 5-70wt%, preferably 10-50wt%; and / or The amount of each component is such that the loading amount of the active ingredient is less than 5 wt % based on the weight of the porous support; and / or In the active ingredient, the weight ratio of the nucleic acid drug to the carrier is 1:1-20, preferably 1:3-12.

8. The method according to claim 6 or 7, wherein: The catalyst is selected from at least one of stannous isooctanoate, stannous octoate, tributylaluminum and diethylzinc; The multi-arm polyethylene glycol is derived from multi-arm polyethylene glycol, and the number of arms of the multi-arm polyethylene glycol is 3-12, preferably 4-8; and / or Preferably, the number average molecular weight of the multi-arm polyethylene glycol is 2000-200000 g / mol, more preferably 10000-50000 g / mol; The polyester monomer is lactide; Preferably, the number average molecular weight of the polyester segment is 10000-1000000 g / mol, preferably 90000-250000 g / mol; and / or The inorganic mineral is selected from at least one of calcium phosphate, hydroxyapatite, bioglass and calcium carbonate; and / or The nucleic acid drug is DNA and / or RNA; and / or Preferably, the DNA is a plasmid encoding an active factor, and its length is within 1 million bp, more preferably 2000-100,000 bp; Preferably, the RNA is a small interfering RNA with a length of less than 100 bp, more preferably 15-35 bp; and / or The carrier is a lipid nanoparticle carrier or an amphiphilic block copolymer carrier.

9. The method according to any one of claims 6 to 8, wherein: In step S1, the contacting and polymerization reaction comprises: performing a first impurity removal on the multi-arm polyethylene glycol, then adding a catalyst for a second impurity removal, and then adding a polyester monomer and performing a polymerization reaction to obtain a multi-arm polyethylene glycol-polyester block copolymer; Preferably, the first impurity removal method is to maintain the temperature at 60-140° C. for 1-4 hours under vacuum conditions; and / or Preferably, the second impurity removal method is to maintain the temperature at 60-140° C. for 5-30 min under an inert atmosphere; and / or Preferably, the conditions of the polymerization reaction include: reaction temperature of 100-200°C, more preferably 120-160°C; reaction time of 3-6h, more preferably 4-5h; Preferably, the operation of step S1 is such that, by weight, in the multi-arm polyethylene glycol-polyester block copolymer, the residual amount of the monomer used to prepare the polymer is less than 1wt%, the residual amount of the catalyst is less than 150ppm, and the residual amount of the organic solvent is less than 1000ppm.

10. The method according to any one of claims 6 to 9, wherein: In step S2, the molding method is electrospinning and / or 3D printing; preferably, the molding method is 3D printing; Preferably, the molding conditions are such that the catalyst content in the multi-arm polyethylene glycol-polyester block copolymer, calculated as metal elements, is no more than 150 ppm by weight.

11. A composite material prepared according to the method according to any one of claims 6 to 10.

12. Use of the composite material according to any one of claims 1 to 5 and 11 in preparing bone tissue repair materials.

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