CN112168980B - Gene-activated antibacterial bioactive bone repair material and preparation method and application thereof - Google Patents

Gene-activated antibacterial bioactive bone repair material and preparation method and application thereof Download PDF

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CN112168980B
CN112168980B CN202010873905.5A CN202010873905A CN112168980B CN 112168980 B CN112168980 B CN 112168980B CN 202010873905 A CN202010873905 A CN 202010873905A CN 112168980 B CN112168980 B CN 112168980B
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glycerol
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CN112168980A (en
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雷波
郭旖
王敏
周丽
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Xian Jiaotong University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/48—Polymers modified by chemical after-treatment

Abstract

The invention provides a gene-activated antibacterial bioactive bone repair material and a preparation method and application thereof, wherein the preparation method comprises the following steps: dissolving glycerol in chloroform, adding triethylamine and acryloyl chloride, and reacting to obtain a GTA solution; adding the polypeptide into deionized water, stirring for dissolving, adding a GTA solution, and stirring for reacting to obtain a GEPL polymer; adding the GEPL polymer and the miRNA gene into a HEPES buffer solution according to the mass ratio of (0.8-50) to 1 to form a stable nano compound, thus obtaining the gene activated antibacterial bioactive material bone repair material (GEPL-miRNA). The GEPL-miRNA shows good biocompatibility, stability and higher gene transfection efficiency in vivo and in vitro, and simultaneously, the GEPL-miRNA also shows good broad-spectrum antibacterial property, so the polymer has good application prospect in gene therapy.

Description

Gene-activated antibacterial bioactive bone repair material and preparation method and application thereof
Technical Field
The invention belongs to the technical field of degradable biomedical materials, and particularly relates to a gene-activated antibacterial bioactive bone repair material and a preparation method and application thereof.
Background
Gene therapy based on microRNAs is a continuous research hotspot in the field of tissue regeneration in recent years, and the design and development of efficient, safe and multifunctional vectors are the key points of gene therapy. The most commonly used vectors at present are viral vectors and traditional cationic polymer non-viral vectors (liposomes and polyethyleneimine), but most of the vectors do not have antibacterial and biodegradable capabilities and have potential problems of tumorigenicity, immunogenicity, limited packaging capability, difficult preparation method and the like, so that the gene therapy is difficult to be widely applied clinically. However, the non-viral polypeptide-based vectors can solve the above problems well, and have the advantages of simple synthesis, good biocompatibility of raw materials, and low cost, so the design and development of the non-viral polypeptide-based vectors are receiving more and more attention in gene therapy.
The polypeptide plays an important role in the treatment of many diseases, wherein a small molecule short peptide with transport capacity is called a cell transmembrane peptide, which is composed of 5-30 amino acid residues and can not only penetrate a cell membrane by itself but also carry various exogenous substances such as proteins, nucleic acids and nanoparticles into a cell in a covalent or non-covalent binding mode. The most commonly used today are cationic transmembrane peptides, which are rich in fragments of arginine, lysine and histidine residues. Wherein, the natural transmembrane polypeptide epsilon-polylysine (EPL) synthesized by the microorganism has good biocompatibility, biodegradability and broad-spectrum antibacterial activity. EPL-based biodegradable scaffolds and hydrogels have been used for anti-infection, tumor therapy, promotion of wound healing, and bone regeneration. Because the binding ability of EPL itself to genes is limited, the use of EPL directly as a gene vector is greatly limited. If the surface charge of the EPL can be improved by further modifying the EPL, the application of the EPL in organisms can be greatly improved. In addition, glycerol is widely present in daily necessities and human bodies, has good water solubility, biodegradability, good biocompatibility and low price, and is commonly used for modification of biomaterials. To date, studies of non-viral vectors based on EPL and glycerol have not been reported.
Disclosure of Invention
The first purpose of the invention is to provide a preparation method of a gene activated antibacterial bioactive bone repair material, the method has simple process, and the prepared polymer has good biocompatibility, stability and gene transfection efficiency in vivo and in vitro.
The second aim of the invention is to provide a gene activated antibacterial bioactive bone repair material.
The third purpose of the invention is to apply the gene activated antibacterial bioactive bone repair material as a nano compound in the preparation of the nano compound for promoting osteogenic differentiation and bone defect healing.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
a preparation method of a gene activated antibacterial bioactive bone repair material comprises the following steps:
dissolving glycerol in chloroform, adding triethylamine and acryloyl chloride for substitution reaction to prepare glycerol-acryloyl chloride; the molar ratio of the glycerol to the anhydrous chloroform to the triethylamine to the acryloyl chloride is 1 (3-5) to (3-5);
carrying out Michael addition reaction on epsilon-polylysine and the glycerol-acryloyl chloride, wherein the molar ratio of the epsilon-polylysine to the glycerol-acryloyl chloride is (3-5) to 1; reacting to obtain a glycerol-polylysine polymer;
and adding the glycerol-polylysine polymer and the miRNA gene into a HEPES buffer solution according to the mass ratio of (0.8-50) to 1 to form a stable nano compound, thus obtaining the gene-activated antibacterial bioactive material bone repair material.
As a further improvement of the method, the temperature of the substitution reaction is 45-55 ℃, and the reaction is carried out for 40-60 hours under the stirring condition.
As a further improvement of the invention, the post-treatment method for obtaining the glycerol-acryloyl chloride comprises the following steps:
after the reaction is finished, HCl and NaHCO are used in sequence3、H2O, washing the mixture; the washed glycerol-acryloyl chloride solution was spin-evaporated and stored in a vacuum desiccator.
As a further improvement of the invention, the temperature of the Michael addition reaction is 45-55 ℃, and the reaction lasts 40-60 hours.
As a further improvement of the invention, the glycerol-polylysine polymer obtained by the reaction is purified and freeze-dried by a dialysis tube in sequence.
As a further improvement of the invention, the pH value of the HEPES buffer solution is 7-7.5; the nano-composite is formed by incubation in water bath at a suitable temperature for the gene.
As a further improvement of the invention, the miRNA gene is miR-29a, miR-29b, miR-26a, antimiR-138, miR-21, miR-221, miR-103, miR-27a, miR-335, miR-22, miR-196, miR-17, miR-31, miR-133, miR-135, miR-378 or miR-34.
As a further improvement of the present invention, the structural formula of the glycerol-acryloyl chloride is:
Figure GDA0003213057770000031
the structural formula of the glycerol-polylysine polymer is as follows:
Figure GDA0003213057770000032
a gene-activated antibacterial bioactive bone repair material is prepared by the preparation method.
The gene activated antibacterial bioactive bone repair material is applied to preparation of a nano compound for promoting osteogenic differentiation and bone defect healing.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a preparation method of a gene-activated antibacterial bioactive bone repair material, aiming at the defects of poor biocompatibility, poor stability, low gene transfection efficiency and the like of the existing gene vector, wherein the method takes natural metabolites of human body, namely glycerol and acryloyl chloride, as monomers and obtains glycerol-acryloyl chloride (GTA) through substitution reaction; the polymer and epsilon-polylysine (EPL) are subjected to Michael addition reaction to obtain the glycerol-polylysine polymer (GEPL). The preparation method is simple, no organic solvent is left, and the used raw materials are green and environment-friendly, are convenient to operate and have low cost. The experimental results prove that: the gene-activated antibacterial bioactive material bone repair material (GEPL-miRNA) prepared by the method has good biocompatibility, stability and higher gene transfection efficiency in vitro and in vivo, can effectively carry genes (miRNA) to enter cells, and shows a certain biological effect. The glycerol, the acryloyl chloride and the EPL used in the invention have good biocompatibility, antibacterial property and degradability, the surface charge of the EPL is increased by introducing the glycerol modified micromolecules, and the glycerol modifies the cation transmembrane peptide (EPL) and can reduce the toxicity thereof, thereby improving the cell compatibility and transfection rate, increasing the stability of the cation transmembrane peptide in blood plasma, reducing the clearance of the compound by a reticuloendothelial system (RES) and prolonging the retention time of the compound in blood circulation.
The gene-activated antibacterial bioactive material bone repair material (GEPL-miRNA) prepared by the method has the advantages of simple preparation method process, environment-friendly raw materials, low cost, effective combination of the gene (miRNA), effective prevention of degradation of the gene by nuclease in an organism, effective release of the gene from the material under the action of polyanion (heparin sodium), good biocompatibility, stability and high gene transfection efficiency of the GEPL-miRNA in vitro and in vivo, and good broad-spectrum antibacterial property of the GEPL-miRNA, so that the polymer has good application prospect in gene therapy.
Drawings
FIG. 1 is a structural formula of each monomer and polymer in the synthesized gene-activated antibacterial bioactive material bone repair material;
FIG. 2 is a 1H-NMR spectrum of the GTA modified small molecule and GEPL polymer prepared;
FIG. 3 shows the antibacterial property of the gene-activated bioactive bone repair material (GEPL-miRNA) prepared by the present invention;
FIG. 4 is a determination of cytotoxicity of the gene-activated antibacterial bioactive material bone repair material (GEPL-miRNA) prepared by the invention on adipose mesenchymal stem cells (ADSC, FIG. 4A) and bone marrow mesenchymal stem cells (BMSC, FIG. 4B);
FIG. 5 shows the in vitro gene transfection effect of the gene-activated bioactive bone repair material (GEPL-miRNA) prepared by the present invention;
FIG. 6 shows the biological effect of in vivo gene transfection of the gene-activated bioactive antibacterial bone repair material (GEPL-miRNA) prepared by the present invention.
Detailed Description
The invention is described in further detail below with reference to the accompanying drawings:
the invention relates to a preparation method of a gene activated antibacterial bioactive bone repair material, which comprises the following steps:
1) adding glycerol into chloroform, stirring and dissolving at room temperature, adding triethylamine, and then dropwise adding acryloyl chloride into the mixture, wherein the molar ratio of the glycerol to the triethylamine is 1 (3-5) to (3-5). Then, the reaction temperature is increased to 45-55 ℃ and stirred for 40-60 hours. After the reaction is finished, the mixture is washed by different reagents in sequence. Finally, the GTA solution was spin-evaporated and stored in a vacuum desiccator for further use;
2) adding polypeptide epsilon-polylysine into deionized water at 45-55 ℃, dissolving and stirring for 30 minutes, adding GTA modified micromolecule, and continuously stirring for 40-60 hours, wherein the molar ratio of the polypeptide epsilon-polylysine to GTA is (3-5): 1. Finally, the resulting GEPL polymer was purified with a dialysis tube (mwco1000) for 3 days. After freeze-drying, the final GEPL polymer is collected and stored for further use;
3) adding the GEPL polymer and the gene into 10 mu l of 50mM HEPES buffer solution with the pH value of 7-7.5 according to the mass ratio of (0.8-50) to 1, and incubating in a water bath at 37 ℃ for 20-40 minutes to form a stable nano compound, thereby obtaining the gene activated antibacterial bioactive material bone repair material.
Wherein the structural formula of the polymer is as follows:
Figure GDA0003213057770000061
the invention aims to provide a preparation method of a gene-activated antibacterial bioactive bone repair material which has good biocompatibility, stability and higher gene transfection efficiency in vivo and in vitro, and can carry genes to enter cells and generate certain biological effect.
Because of biodegradability, good biocompatibility, antibacterial properties, and low cost, epsilon-polylysine (EPL) has been widely used in various fields such as food, industry, and medicine. However, the EPL has weak self positive charge and is extremely unstable in a serum environment, so that the application of the EPL as a gene carrier is limited; glycerol, which is a non-toxic and non-irritant small molecule compound, has good hydrophilicity and stability, can improve the stability of the EPL if the EPL is modified by the glycerol, and the modification of the glycerol on the cation transmembrane peptide can reduce the toxicity and the immunogenicity of the glycerol, thereby improving the compatibility and the transfection rate of cells, increasing the stability of the cation transmembrane peptide in blood plasma, reducing the clearance of a complex by a reticuloendothelial system (RES), and prolonging the retention time of the complex in blood circulation; epsilon-polylysine (EPL) contains amino groups, so that the epsilon-polylysine has a certain positive charge, can be weakly combined with miRNA, and is unstable in serum environment, thereby causing low transfection efficiency.
Therefore, the GEPL polymer is obtained by utilizing the Michael addition reaction of modified glycerol to epsilon-polylysine (EPL), and then the GEPL and miRNA form a gene-activated antibacterial bioactive material bone repair material (GEPL-miRNA) under a proper condition, so that the GEPL-miRNA has good biocompatibility, stability and higher gene transfection efficiency in vivo and in vitro, has excellent broad-spectrum antibacterial performance, can reduce the risk of infection when being applied in vivo, and is a gene-activated antibacterial bioactive material bone repair material for gene therapy.
For better understanding of the present invention, the present invention will be described in detail with reference to the following embodiments, but the present invention is not limited to the following examples.
Example 1
1) The preparation method of the GTA modified micromolecule comprises the following steps: 10mmol of glycerol is added into 40mL of anhydrous chloroform, stirred and dissolved at room temperature, and then 45mmol of triethylamine is added. Then is subjected to ice bathUnder the conditions, 45mmol of acryloyl chloride was added dropwise to the mixture. The reaction temperature was increased to 50 ℃ and stirred for 48 hours. After the reaction is finished, HCl and NaHCO are used in sequence3、H2And O, washing the mixture. Finally, the GTA solution was spin-evaporated and stored in a vacuum desiccator for further use;
2) the preparation method of the GEPL polymer comprises the following steps: 10mmol of epsilon-polylysine is added into deionized water at 50 ℃ for dissolving and stirring, and after 30 minutes, 3mmol of GTA modified micromolecule is added and continuously stirred for 48 hours. Finally, the resulting GEPL polymer was purified with a dialysis tube (mwco1000) for 3 days. After freeze-drying, the final GEPL polymer is collected and stored for further use;
3) the gene activated antibacterial bioactive material bone repair material comprises: the GEPL polymer and the gene (miRNA) are added into 10 mu l of HEPES buffer solution with the concentration of 50mM and the pH value of 7.4 according to the mass ratio of 30:1, and the mixture is incubated in a water bath at 37 ℃ for 30 minutes to form a stable nano-composite, so that the gene activated antibacterial bioactive material bone repair material is obtained.
The gene-activated antibacterial bioactive material bone repair material (GEPL-miRNA) prepared by the method can be effectively combined with the gene (miRNA), and the formed compound can effectively prevent the gene from being degraded by nuclease in an organism, and the gene can be effectively released from the material under the action of polyanion (heparin sodium).
Fig. 1 is structural formulas of various monomers and polymers in the gene-activated antibacterial bioactive material bone repair material synthesized in example 1 of the present invention, wherein a is a structural formula of glycerol, B is a structural formula of acryloyl chloride, C is a structural formula of epsilon-polylysine, D is a structural formula of glycerol-modified small molecule (GTA), and E is a structural formula of GEPL polymer.
Fig. 2 is a 1H-NMR spectrum of the resulting glycerol-modified small molecule (GTA) and GEPL polymers, and from fig. 2A, it can be seen that multiple peaks for methine (-CH-) and methylene (-CH2) are at 5.4 and 4.3-4.4ppm, respectively, assigned to glycerol, and multiple peaks for-CH 2 ═ CH-are at 5.9, 6.1, and 6.4ppm, respectively, assigned to acryloyl chloride, indicating the occurrence of these peaks, indicating successful synthesis of GTA-modified small molecules. Furthermore, in FIG. 2B, the presence of 1.2, 1.4 and 1.7ppm of methylene (-CH2) peaks for EPL, and 2.3 and 2.7ppm of proton (-HN-CH2-COO-) peaks indicate that EPL has been successfully grafted onto GTA modified small molecules and formed new GEPL polymers.
FIG. 3 shows the broad-spectrum antibacterial property of the gene-activated bioactive bone repair material (GEPL-miRNA). From FIG. 3, it can be seen that EPL and GEPL have 99.99% killing rate against gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Escherichia coli), indicating that GEPL has good broad-spectrum antibacterial properties. GEPL did not destroy the antimicrobial capacity of EPL during the synthesis.
Fig. 4 is a determination of cytotoxicity of the gene-activated antibacterial bioactive material bone repair material (GEPL-miRNA) prepared by the present invention on adipose mesenchymal stem cells (ADSC, fig. 4A) and bone marrow mesenchymal stem cells (BMSC, fig. 4B). It can be seen that the GEPL-miRNA has particularly low cytotoxicity and better biocompatibility than the PEI 25 kD-miRNA.
FIG. 5 shows the in vitro gene transfection results of the gene-activated bioactive bone repair material (GEPL-miRNA) prepared by the present invention. FIG. 5A blue light indicates nuclei, FIG. 5B green light is a green fluorescent protein expressed in cells after miRNA transfection, FIG. 5C is a combined graph of FIGS. 5A and B, and FIG. 5D is an enlarged view of a single cell. As can be seen, the GEPL polymer can carry genes to enter cells smoothly and enable the genes to be expressed, and the results provide the possibility that the GEPL polymer is expected to be used as a gene carrier.
FIG. 6 shows the biological results of miR-29b gene transfection in vivo of the gene-activated antibacterial bioactive material bone repair material (GEPL-miRNA) prepared by the invention. This is a micro-CT picture of the rat skull at each 3mm defect at 12 weeks after injection of the GEPL-miR-29b complex. From the pictures we can clearly see that the defects of the group of GEPL-miR-29b complex have been almost completely repaired, however, the blank group, EPL and liposome group have obvious defects. These results provide the possibility that the GEPL polymer is expected to be a gene vector for clinical use.
Example 2
1) The preparation method of the GTA modified micromolecule comprises the following steps: 10mmol of glycerol is added into 35mL of anhydrous chloroform, stirred and dissolved at room temperature, and then 50mmol of triethylamine is added. Then, 45mmol of acryloyl chloride was added dropwise to the mixture under ice-bath conditions. The reaction temperature was increased to 45 ℃ and stirred for 60 hours. After the reaction is finished, HCl and NaHCO are used in sequence3、H2And O, washing the mixture. Finally, the GTA solution was spin-evaporated and stored in a vacuum desiccator for further use;
2) the preparation method of the GEPL polymer comprises the following steps: 10mmol of epsilon-polylysine is added into deionized water at 50 ℃ for dissolving and stirring, and after 30 minutes, 2.8mmol of GTA modified micromolecule is added and continuously stirred for 48 hours. Finally, the resulting GEPL polymer was purified with a dialysis tube (mwco1000) for 3 days. After freeze-drying, the final GEPL polymer is collected and stored for further use;
3) the gene activated antibacterial bioactive material bone repair material comprises: the GEPL polymer and the gene (miRNA) are added into 10 mu l of HEPES buffer solution with the concentration of 50mM and the pH value of 7.0 according to the mass ratio of 20:1, and the mixture is incubated in a water bath at 37 ℃ for 30 minutes to form a stable nano-composite, so that the gene activated antibacterial bioactive material bone repair material is obtained.
Example 3
1) The preparation method of the GTA modified micromolecule comprises the following steps: 10mmol of glycerol is added into 45mL of anhydrous chloroform, stirred and dissolved at room temperature, and then 40mmol of triethylamine is added. Then 40mmol of acryloyl chloride was added dropwise to the mixture under ice-bath conditions. The reaction temperature was increased to 55 ℃ and stirred for 40 hours. After the reaction is finished, HCl and NaHCO are used in sequence3、H2And O, washing the mixture. Finally, the GTA solution was spin-evaporated and stored in a vacuum desiccator for further use;
2) the preparation method of the GEPL polymer comprises the following steps: 10mmol of epsilon-polylysine is added into deionized water at the temperature of 55 ℃ for dissolving and stirring, and after 30 minutes, 2.5mmol of GTA modified micromolecule is added and continuously stirred for 40 hours. Finally, the resulting GEPL polymer was purified with a dialysis tube (mwco1000) for 3 days. After freeze-drying, the final GEPL polymer is collected and stored for further use;
3) the gene activated antibacterial bioactive material bone repair material comprises: the GEPL polymer and the gene (miRNA) are added into 10 mul of HEPES buffer solution with the mass ratio of 10:1 and the pH value of 50mM and 7.5, and the mixture is incubated in a water bath at 37 ℃ for 30 minutes to form a stable nano-composite, so that the gene activated antibacterial bioactive material bone repair material is obtained.
Example 4
1) The preparation method of the GTA modified micromolecule comprises the following steps: 10mmol of glycerol is added into 40mL of anhydrous chloroform, stirred and dissolved at room temperature, and then 35mmol of triethylamine is added. Then 35mmol of acryloyl chloride was added dropwise to the mixture under ice-bath conditions. The reaction temperature was increased to 50 ℃ and stirred for 48 hours. After the reaction is finished, HCl and NaHCO are used in sequence3、H2And O, washing the mixture. Finally, the GTA solution was spin-evaporated and stored in a vacuum desiccator for further use;
2) the preparation method of the GEPL polymer comprises the following steps: 10mmol of epsilon-polylysine is added into deionized water at 50 ℃ for dissolving and stirring, and after 30 minutes, 2.2mmol of GTA modified micromolecule is added and continuously stirred for 48 hours. Finally, the resulting GEPL polymer was purified with a dialysis tube (mwco1000) for 3 days. After freeze-drying, the final GEPL polymer is collected and stored for further use;
3) the gene activated antibacterial bioactive material bone repair material comprises: the GEPL polymer and the gene (miRNA) are added into 10 mu l of HEPES buffer solution with 50mM and pH of 7.2 according to the mass ratio of 0.8:1, and the mixture is incubated in a water bath at 37 ℃ for 25 minutes to form a stable nano-composite, so that the gene activated antibacterial bioactive material bone repair material is obtained.
Example 5
1) The preparation method of the GTA modified micromolecule comprises the following steps: 10mmol of glycerol is added into 50mL of anhydrous chloroform, stirred and dissolved at room temperature, and then 45mmol of triethylamine is added. Then 50mmol of acryloyl chloride was added dropwise to the mixture under ice-bath conditions.The reaction temperature was increased to 50 ℃ and stirred for 48 hours. After the reaction is finished, HCl and NaHCO are used in sequence3、H2And O, washing the mixture. Finally, the GTA solution was spin-evaporated and stored in a vacuum desiccator for further use;
2) the preparation method of the GEPL polymer comprises the following steps: 10mmol of epsilon-polylysine is added into deionized water at 50 ℃ for dissolving and stirring, and after 30 minutes, 2mmol of GTA modified micromolecule is added and continuously stirred for 48 hours. Finally, the resulting GEPL polymer was purified with a dialysis tube (mwco1000) for 3 days. After freeze-drying, the final GEPL polymer is collected and stored for further use;
3) the gene activated antibacterial bioactive material bone repair material comprises: the GEPL polymer and the gene (miRNA) are added into 10 mu l of HEPES buffer solution with the mass ratio of 50:1 and the pH value of 50mM and 7.3, and the mixture is incubated in a water bath at 37 ℃ for 20 minutes to form a stable nano-composite, so that the gene activated antibacterial bioactive material bone repair material is obtained.
The gene-activated antibacterial bioactive material bone repair material (GEPL-miRNA) prepared by the method has the advantages of simple preparation method process, environment-friendly raw materials, low cost, effective combination of the gene (miRNA), effective prevention of degradation of the gene by nuclease in an organism, effective release of the gene from the material under the action of polyanion (heparin sodium), good biocompatibility, stability and high gene transfection efficiency of the GEPL-miRNA in vitro and in vivo, and good broad-spectrum antibacterial property of the GEPL-miRNA, so that the polymer has good application prospect in gene therapy.
The invention also has the following advantages:
(1) the glycerol used in the invention is an additive widely applied to food, industry and medical treatment, has good biocompatibility, is environment-friendly, and is cheap and easy to obtain.
(2) After the epsilon-polylysine (EPL) is grafted with the glycerol, the invention can effectively improve the combination of the epsilon-polylysine and a gene (miRNA) through electrostatic interaction, and stably enter cells, thereby finally achieving higher gene transfection effect.
(4) The gene-activated antibacterial bioactive material bone repair material (GEPL-miRNA) prepared by the preparation method disclosed by the invention has broad-spectrum gram-positive bacterium and gram-negative bacterium resistance capability.
(5) The solvent used in the invention is deionized water, and the gene-activated antibacterial bioactive material bone repair material (GEPL-miRNA) prepared by the method does not contain any organic solvent.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims (10)

1. A preparation method of a gene activated antibacterial bioactive bone repair material is characterized by comprising the following steps:
dissolving glycerol in chloroform, adding triethylamine and acryloyl chloride for substitution reaction to prepare glycerol-acryloyl chloride; the molar ratio of the glycerol to the anhydrous chloroform to the triethylamine to the acryloyl chloride is 1 (3-5) to (3-5);
carrying out Michael addition reaction on epsilon-polylysine and the glycerol-acryloyl chloride, wherein the molar ratio of the epsilon-polylysine to the glycerol-acryloyl chloride is (3-5) to 1; reacting to obtain a glycerol-polylysine polymer;
and adding the glycerol-polylysine polymer and the miRNA gene into a HEPES buffer solution according to the mass ratio of (0.8-50) to 1 to form a stable nano compound, thus obtaining the gene-activated antibacterial bioactive material bone repair material.
2. The method for preparing a gene-activated antibacterial bioactive bone repair material according to claim 1, wherein the temperature of the substitution reaction is 45-55 ℃, and the reaction is carried out for 40-60 hours under stirring.
3. The method for preparing a gene-activated antibacterial bioactive bone repair material according to claim 1, wherein the post-treatment method for obtaining glycerol-acryloyl chloride comprises:
after the reaction is finished, HCl and NaHCO are used in sequence3、H2O, washing the mixture; the washed glycerol-acryloyl chloride solution was spin-evaporated and stored in a vacuum desiccator.
4. The method for preparing a gene-activated antibacterial bioactive bone repair material according to claim 1, wherein the temperature of the Michael addition reaction is 45-55 ℃ and the reaction time is 40-60 hours.
5. The method for preparing a gene-activated antibacterial bioactive bone repair material according to claim 1 or 4, wherein the glycerol-polylysine polymer obtained by the reaction is sequentially subjected to dialysis tube purification and freeze-drying.
6. The method for preparing the gene-activated antibacterial bioactive bone repair material according to claim 1, wherein the pH of the HEPES buffer solution is 7-7.5; the nano-composite is formed by incubation in a water bath at a temperature suitable for the gene.
7. The preparation method of the gene-activated antibacterial bioactive bone repair material, according to claim 1, wherein the miRNA gene is miR-29a, miR-29b, miR-26a, antimiR-138, miR-21, miR-221, miR-103, miR-27a, miR-335, miR-22, miR-196, miR-17, miR-31, miR-133, miR-135, miR-378 or miR-34.
8. The method for preparing a gene-activated antibacterial bioactive bone repair material according to claim 1, wherein the structural formula of the glycerol-acryloyl chloride is as follows:
Figure FDA0003213057760000021
the structural formula of the glycerol-polylysine polymer is as follows:
Figure FDA0003213057760000022
9. a gene-activated antibacterial bioactive bone repair material, characterized by being prepared by the preparation method of any one of claims 1 to 8.
10. Use of the gene-activated antibacterial bioactive bone repair material of claim 9 for the preparation of a nanocomposite for promoting osteogenic differentiation and bone defect healing.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106823009A (en) * 2017-01-12 2017-06-13 广东泰宝医疗器械技术研究院有限公司 A kind of antibacterial bone renovating bracket material with induction repair and preparation method thereof
CN110755683A (en) * 2019-10-21 2020-02-07 西安交通大学 Silicon-based elastic nanofiber bone repair material and preparation method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106823009A (en) * 2017-01-12 2017-06-13 广东泰宝医疗器械技术研究院有限公司 A kind of antibacterial bone renovating bracket material with induction repair and preparation method thereof
CN110755683A (en) * 2019-10-21 2020-02-07 西安交通大学 Silicon-based elastic nanofiber bone repair material and preparation method and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Biodegradable antibacterial branched glycerol-polypeptide with efficient in vitro/in vitro miRNA-29b delivery for promoting osteogenic differentiation of stem cells and bone regeneration;Yi Guo等;《Chemical Engineering Journal》;20200924;127085 *
Characterization of PLL-g-PEG-DNA nanoparticles for the delivery of therapeutic DNA;RIMANN M等;《Bioconjugate Chemistry》;20081123;548-557 *
聚赖氨酸在基因治疗技术中的研究进展;李从欣等;《化学与生物工程》;20191231;1-4 *

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