CN116370705B - Folic acid modified noble metal-based carbon nanotube composite hydroxyapatite bone repair stent material and preparation method thereof - Google Patents
Folic acid modified noble metal-based carbon nanotube composite hydroxyapatite bone repair stent material and preparation method thereof Download PDFInfo
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- CN116370705B CN116370705B CN202310398642.0A CN202310398642A CN116370705B CN 116370705 B CN116370705 B CN 116370705B CN 202310398642 A CN202310398642 A CN 202310398642A CN 116370705 B CN116370705 B CN 116370705B
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- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 title claims abstract description 174
- 239000002041 carbon nanotube Substances 0.000 title claims abstract description 94
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 92
- 239000011724 folic acid Substances 0.000 title claims abstract description 87
- 235000019152 folic acid Nutrition 0.000 title claims abstract description 87
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 title claims abstract description 86
- 229960000304 folic acid Drugs 0.000 title claims abstract description 86
- 229910021393 carbon nanotube Inorganic materials 0.000 title claims abstract description 76
- 239000000463 material Substances 0.000 title claims abstract description 55
- 229910052588 hydroxylapatite Inorganic materials 0.000 title claims abstract description 50
- 229910000510 noble metal Inorganic materials 0.000 title claims abstract description 46
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 title claims abstract description 43
- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 33
- 239000002131 composite material Substances 0.000 title claims abstract description 26
- 230000008439 repair process Effects 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 51
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- 238000003756 stirring Methods 0.000 claims description 35
- 238000005406 washing Methods 0.000 claims description 23
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
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- 238000001035 drying Methods 0.000 claims description 15
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 14
- 238000009210 therapy by ultrasound Methods 0.000 claims description 14
- 238000001291 vacuum drying Methods 0.000 claims description 14
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 9
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- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 9
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
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- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 6
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 3
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- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
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- KDLHZDBZIXYQEI-UHFFFAOYSA-N palladium Substances [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 1
- PIBWKRNGBLPSSY-UHFFFAOYSA-L palladium(II) chloride Chemical compound Cl[Pd]Cl PIBWKRNGBLPSSY-UHFFFAOYSA-L 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
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- 235000019731 tricalcium phosphate Nutrition 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/12—Phosphorus-containing materials, e.g. apatite
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/025—Other specific inorganic materials not covered by A61L27/04 - A61L27/12
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/08—Carbon ; Graphite
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Transplantation (AREA)
- Dermatology (AREA)
- Medicinal Chemistry (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Inorganic Chemistry (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention relates to the technical field of bone scaffold materials, and discloses a folic acid modified noble metal-based carbon nano tube composite hydroxyapatite bone repair scaffold material and a preparation method thereof. The FA/PM-CNTs/HAP scaffold material prepared by the invention mainly takes hydroxyapatite, noble metal doped carbon nano tube and folic acid as main synthesis raw materials, and the surface of the carbon nano tube is modified by acid treatment and noble metal doped, so that the surface of the carbon nano tube has larger specific surface area, and the subsequent modification by the composite hydroxyapatite and folic acid is beneficial to improving the biocompatibility of the scaffold and promoting the osteogenesis induction effect of bone environment, thereby being beneficial to the development of bone repair scaffolds.
Description
Technical Field
The invention relates to the technical field of bone scaffold materials, in particular to a folic acid modified noble metal-based carbon nano tube composite hydroxyapatite bone repair scaffold material and a preparation method thereof.
Background
Bone tissue defects are important diseases affecting the health and life of people, when the bone defect amount is too large, the bone tissue cannot heal by only relying on the repair capability of the bone tissue itself, and bone repair stent materials are required to be implanted to perform structural substitution on lesion sites so as to exert mechanical support and promote the regeneration of the bone tissue. In recent years, as the demands for bone quality and prognosis are continuously improved, researchers have made innovative demands for scaffold materials for bone repair treatment.
Carbon Nanotubes (CNTs) are tubular nano-sized graphite crystals having excellent mechanical properties, electrical conductivity, and structural stability and functional group modifier properties. In recent years, the carbon nanotubes are gradually and deeply researched in the field of bone tissue repair, and can form a composite scaffold with common bone tissue engineering materials such as hydroxyapatite, tricalcium phosphate, high polymer, collagen and the like, so that the mechanical property and biological property of the materials are further optimized, and bone regeneration is induced, and a new strategy is provided for tissue engineering bone repair. However, the data show that the carbon nanotubes may have potential toxicity to cells and often do not play an effective role, so the development of composite stent repair materials based on the carbon nanotubes still needs to be continuously optimized.
Disclosure of Invention
The invention aims at overcoming the defects in the prior art, and provides a folic acid modified noble metal-based carbon nano tube composite hydroxyapatite bone repair stent material, which consists of hydroxyapatite, noble metal doped carbon nano tubes and folic acid, wherein the mass ratio is 5: (0.1-1): (0.1-1), wherein the mass ratio of the carbon nano tube to the noble metal is 1: (0.001-0.015).
In order to achieve the above purpose, the invention provides a preparation method of a folic acid modified noble metal-based carbon nano tube composite hydroxyapatite bone repair stent material, which specifically comprises the following steps:
(1) Noble metal doped modified carbon nanotubes (PM-CNTs): dispersing the carbon nano tube into 10M hydrochloric acid solution, carrying out ultrasonic treatment for 25min each time, standing for 5min, and carrying out ultrasonic treatment-standing for 6 times until a suspension of 0.01g/mL is formed; then washing the carbon nanotubes with deionized water to ph=7 and ultrasonically dispersing the carbon nanotubes with deionized water to form a suspension of 0.01 g/mL; then adding 10mM noble metal solution dropwise into the carbon nano tube suspension at 650-800rpm/min, stirring for 10min, then performing ultrasonic treatment for 10min, and performing stirring-ultrasonic circulation for 9 times; then adding 1M NaOH solution under magnetic stirring to adjust the pH to 8, adding sodium borohydride, stirring for 5-10h, washing with deionized water to pH=7, and drying overnight in a vacuum drying oven at 50 ℃;
(2) Composite hydroxyapatite (PM-CNTs-HAP): dispersing hydroxyapatite in deionized water, stirring for 20min, adding PM-CNTs prepared in the step (1), stirring for 20min, transferring to a reaction kettle, and performing hydrothermal reaction at 180-200 ℃ for 24h; washing with deionized water to ph=7 after the reaction is finished, and then drying overnight in a vacuum drying oven at 50 ℃;
(3) Folic acid modified noble metal-based carbon nanotubes-hydroxyapatite (fa@pm-CNTs-HAP): dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and Folic Acid (FA) in dimethyl sulfoxide to prepare a folic acid solution of 0.02g/mL, and then adding all PM-CNTs-HAP prepared in the step (2) into the folic acid solution under the conditions of light shielding and water bath heating and stirring at 80-95 ℃ for reaction for 5-12h; then washed 3 times with deionized water and absolute ethanol, respectively, to ph=7 and dried overnight in a vacuum oven at 50 ℃.
Preferably, the ultrasonic temperature in the step (1) is 4 ℃, and the ultrasonic power is 150-200W.
Preferably, the noble metal solution in the step (1) is one of chloroplatinic acid, chloroauric acid, chloropalladac acid or silver nitrate solution.
Preferably, the mass ratio of the carbon nanotubes, the noble metal solution and the sodium borohydride in the step (1) is 1g: (0.24-8.83) mL:2g.
Preferably, the mass to volume ratio of the HAP, the PM-CNTs and the deionized water in the step (2) is 5g: (0.1-1) g:100mL.
Preferably, the mass ratio of EDC, NHS, FA in the step (3) is 1:1:8.
the invention has the advantages that:
the FA@PM-CNTs-HAP scaffold material prepared by the invention mainly uses hydroxyapatite, noble metal doped carbon nanotubes and folic acid as main synthesis raw materials, and the three materials provide good effects for constructing a bone repair scaffold. Wherein the hydroxyapatite component is similar to the inorganic component in the bone matrix, and has good biocompatibility; folic acid exists in bone marrow tissue in vivo, plays an important role in protein synthesis, cell division and growth process, and has good biocompatibility; according to the invention, the surface of the carbon nano tube is modified by acid treatment and noble metal doping, so that the carbon nano tube has a larger specific surface area, and is favorable for subsequent composite hydroxyapatite and folic acid modification. The composition of the materials is beneficial to improving the biocompatibility of the scaffold, and also can promote the osteogenesis induction of the bone environment, which is beneficial to the development of the bone repair scaffold.
Drawings
FIG. 1 is an X-ray diffraction pattern of example 1 and comparative example 1 of the present invention, wherein FIG. 1 (a) is an X-ray diffraction pattern of example 1, and FIG. 1 (b) is an X-ray diffraction pattern of comparative example 1;
fig. 2 is a transmission electron microscope image of example 1 and comparative example 1 of the present invention, wherein fig. 2 (a) is a transmission electron microscope image of example 1, and fig. 2 (b) is a transmission electron microscope image of comparative example 1;
FIG. 3 is an infrared spectrum test chart of example 1, comparative example 1 and comparative example 3 of the present invention;
FIG. 4 is a graph showing cytotoxicity test conducted in example 1 and comparative examples 2 to 3 of the present invention;
FIG. 5 is a graph showing the results of the detection of the bone formation-related gene expression in example 1 and comparative examples 3 to 4 of the present invention, FIG. 5 (a-c) is a graph showing the results of the quantitative PCR detection of OPG, OCN, OSX gene in this order, and FIG. 5 (d) is a graph showing the data of alkaline phosphatase activity.
Detailed Description
The following detailed description of the invention provides specific embodiments with reference to the accompanying drawings.
Example 1: the present example prepared FA@Au-CNTs-HAP with an Au loading of 1% in the Au-CNTs.
(1) Noble metal doped modified carbon nanotubes (Au-CNTs): dispersing 2g of carbon nano tube into 10M hydrochloric acid solution, performing ultrasonic treatment at 4 ℃ and 150W for 25min, standing for 5min, and performing ultrasonic treatment-standing circulation for 6 times until a suspension of 0.01g/mL is formed; then washing the carbon nanotubes with deionized water to ph=7 and ultrasonically dispersing the carbon nanotubes with deionized water to form a suspension of 0.01 g/mL; then, 4.86mL of 10mM chloroauric acid solution is added dropwise into the carbon nano tube suspension at the rotation speed of 700rpm/min, the stirring is carried out for 10min, the ultrasonic treatment is carried out for 10min, and the stirring-ultrasonic circulation is carried out for 9 times; then adding 1M NaOH solution under magnetic stirring to adjust the pH to 8, adding 4g of sodium borohydride, stirring for 10 hours, washing with deionized water to pH=7, and drying overnight in a vacuum drying oven at 50 ℃;
(2) Composite hydroxyapatite (Au-CNTs-HAP): dispersing 5g of hydroxyapatite in 100mL of deionized water, stirring for 20min, adding 1gAu-CNTs prepared in the step (1), stirring for 20min, transferring to a reaction kettle, and performing hydrothermal reaction at 200 ℃ for 24h; washing with deionized water to ph=7 after the reaction is finished, and then drying overnight in a vacuum drying oven at 50 ℃;
(3) Folic acid modified noble metal-based carbon nanotubes-hydroxyapatite (fa@au-CNTs-HAP): dissolving 0.1g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), 0.1g of N-hydroxysuccinimide (NHS) and 0.8g of Folic Acid (FA) in 50mL of dimethyl sulfoxide to prepare 0.02g/mL of folic acid solution, and then adding all Au-CNTs-HAP prepared in the step (2) into the folic acid solution under the conditions of light shielding and water bath heating and stirring at 90 ℃ for reaction for 12 hours; then washed 3 times with deionized water and absolute ethanol, respectively, to ph=7 and dried overnight in a vacuum oven at 50 ℃.
Comparative example 1
The difference between comparative example 1 and example 1 is that the carbon nanotubes are doped with noble metals, but are not modified by folic acid, and are not compounded with hydroxyapatite, and the rest steps are consistent with the preparation process of the material of example 1, and the finally prepared material is named as Au-CNTs.
Comparative example 2
The difference between comparative example 2 and example 1 is that the carbon nanotubes are undoped with noble metals, the rest steps are consistent with the preparation process of the material of example 1, and the finally prepared material is named FA@CNTs-HAP.
Comparative example 3
The difference between comparative example 3 and example 1 of the present invention is that folic acid is not modified, the rest steps are the same as the preparation process of the material of example 1, and the finally prepared material is named as Au-CNTs-HAP.
Comparative example 4
The difference between comparative example 4 and example 1 is that the carbon nanotubes doped with noble metal are not compounded, the rest steps are consistent with the preparation process of the material of example 1, and the finally prepared material is named FA@HAP.
Example 2: the present example prepared FA@Au-CNT-/HAP with an Au loading of 0.1% in Au-CNTs.
(1) Noble metal doped modified carbon nanotubes (Au-CNTs): dispersing 2g of carbon nano tube into 10M hydrochloric acid solution, performing ultrasonic treatment at 4 ℃ and 170W for 25min, standing for 5min, and performing ultrasonic-standing circulation for 6 times until a suspension of 0.01g/mL is formed; then washing the carbon nanotubes with deionized water to ph=7 and ultrasonically dispersing the carbon nanotubes with deionized water to form a suspension of 0.01 g/mL; then 0.48mL of 10mM chloroauric acid solution is dropwise added into the carbon nano tube suspension at the rotation speed of 800rpm/min, the stirring is carried out for 10min, the ultrasonic treatment is carried out for 10min, and the stirring-ultrasonic circulation is carried out for 9 times; then adding 1M NaOH solution under magnetic stirring to adjust the pH to 8, adding 4g of sodium borohydride, stirring for 10 hours, washing with deionized water to pH=7, and drying overnight in a vacuum drying oven at 50 ℃;
(2) Composite hydroxyapatite (Au-CNTs-HAP): dispersing 5g of hydroxyapatite in 100mL of deionized water, stirring for 20min, adding 0.1-gAu-CNTs prepared in the step (1), stirring for 20min, transferring to a reaction kettle, and performing hydrothermal reaction at 180 ℃ for 24h; washing with deionized water to ph=7 after the reaction is finished, and then drying overnight in a vacuum drying oven at 50 ℃;
(3) Folic acid modified noble metal-based carbon nanotubes-hydroxyapatite (fa@au-CNTs-HAP): dissolving 0.05g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), 0.05g of N-hydroxysuccinimide (NHS) and 0.4g of Folic Acid (FA) in 25mL of dimethyl sulfoxide to prepare 0.02g/mL of folic acid solution, and then adding all Au-CNTs-HAP prepared in the step (2) into the folic acid solution under the conditions of light shielding and water bath heating and stirring at 85 ℃ for reaction for 10 hours; then washed 3 times with deionized water and absolute ethanol, respectively, to ph=7 and dried overnight in a vacuum oven at 50 ℃.
Example 3: the present example prepared FA@Ag-CNTs-HAP with an Ag loading of 1.5%.
(1) Noble metal doped modified carbon nanotubes (Ag-CNTs): dispersing 2g of carbon nano tube into 10M hydrochloric acid solution, performing ultrasonic treatment at 200W at 4 ℃ for 25min, standing for 5min, and performing ultrasonic-standing circulation for 6 times until a suspension of 0.01g/mL is formed; then washing the carbon nanotubes with deionized water to ph=7 and ultrasonically dispersing the carbon nanotubes with deionized water to form a suspension of 0.01 g/mL; then 17.66mL of 10mM silver nitrate solution is added dropwise into the carbon nano tube suspension at the rotation speed of 800rpm/min, the stirring is carried out for 10min, the ultrasonic treatment is carried out for 10min, and the stirring-ultrasonic circulation is carried out for 9 times; then adding 1M NaOH solution under magnetic stirring to adjust the pH to 8, adding 4g of sodium borohydride, stirring for 10 hours, washing with deionized water to pH=7, and drying overnight in a vacuum drying oven at 50 ℃;
(2) Composite hydroxyapatite (Ag-CNTs-HAP): dispersing 5g of hydroxyapatite in 100mL of deionized water, stirring for 20min, adding 0.5g of Ag-CNTs prepared in the step (1), stirring for 20min, transferring to a reaction kettle, and performing hydrothermal reaction at 200 ℃ for 24h; washing with deionized water to ph=7 after the reaction is finished, and then drying overnight in a vacuum drying oven at 50 ℃;
(3) Folic acid modified noble metal-based carbon nanotubes-hydroxyapatite (fa@ag-CNTs-HAP): dissolving 0.125g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), 0.125g of N-hydroxysuccinimide (NHS) and 1g of Folic Acid (FA) in 62.5mL of dimethyl sulfoxide to prepare 0.02g/mL of folic acid solution, and then adding all Ag-CNTs-HAP prepared in the step (2) into the folic acid solution under the condition of light shielding and heating and stirring at 80 ℃ for reaction for 6 hours; then washed 3 times with deionized water and absolute ethanol, respectively, to ph=7 and dried overnight in a vacuum oven at 50 ℃.
Example 4: the present example prepared FA@Pt-CNTs-HAP with a Pt loading of 0.2%.
(1) Noble metal doped modified carbon nanotubes (Pt-CNTs): dispersing 2g of carbon nano tube into 10M hydrochloric acid solution, performing ultrasonic treatment at 4 ℃ and 170W for 25min, standing for 5min, and performing ultrasonic-standing circulation for 6 times until a suspension of 0.01g/mL is formed; then washing the carbon nanotubes with deionized water to ph=7 and ultrasonically dispersing the carbon nanotubes with deionized water to form a suspension of 0.01 g/mL; then 0.96mL of 10mM chloroplatinic acid solution is dropwise added into the carbon nano tube suspension at the rotating speed of 650rpm/min, stirring is carried out for 10min, then ultrasonic is carried out for 10min, and stirring-ultrasonic circulation is carried out for 9 times; then adding 1M NaOH solution under magnetic stirring to adjust the pH to 8, adding 4g of sodium borohydride, stirring for 10 hours, washing with deionized water to pH=7, and drying overnight in a vacuum drying oven at 50 ℃;
(2) Composite hydroxyapatite (Pt-CNTs-HAP): dispersing 5g of hydroxyapatite in 100mL of deionized water, stirring for 20min, adding 1gPt-CNTs prepared in the step (1), stirring for 20min, transferring to a reaction kettle, and performing hydrothermal reaction at 180 ℃ for 24h; washing with deionized water to ph=7 after the reaction is finished, and then drying overnight in a vacuum drying oven at 50 ℃;
(3) Folic acid modified noble metal-based carbon nanotubes-hydroxyapatite (fa@pt-CNTs-HAP): dissolving 0.125g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), 0.125g of N-hydroxysuccinimide (NHS) and 1g of Folic Acid (FA) in 62.5mL of dimethyl sulfoxide, and then adding all Pt-CNTs-HAP prepared in the step (2) into folic acid solution under the condition of light-proof environment and water bath heating and stirring at 95 ℃ for reaction for 6 hours; then washed 3 times with deionized water and absolute ethanol, respectively, to ph=7 and dried overnight in a vacuum oven at 50 ℃.
Example 5: the present example prepared FA@Pd-CNTs-HAP with a Pd loading of 1% in Pd-CNTs.
(1) Noble metal doped modified carbon nanotubes (Pd-CNTs): dispersing 2g of carbon nano tube into 10M hydrochloric acid solution, performing ultrasonic treatment at 4 ℃ and 150W for 25min, standing for 5min, and performing ultrasonic-standing circulation for 6 times until a suspension of 0.01g/mL is formed; then washing the carbon nanotubes with deionized water to ph=7 and ultrasonically dispersing the carbon nanotubes with deionized water to form a suspension of 0.01 g/mL; then 5.03mL of 10mM palladium chloride acid solution is added dropwise into the carbon nano tube suspension at the rotation speed of 700rpm/min, the stirring is carried out for 10min, the ultrasonic treatment is carried out for 10min, and the stirring-ultrasonic circulation is carried out for 9 times; then adding 1M NaOH solution under magnetic stirring to adjust the pH to 8, adding 4g of sodium borohydride, stirring for 10 hours, washing with deionized water to pH=7, and drying overnight in a vacuum drying oven at 50 ℃;
(2) Composite hydroxyapatite (Pd-CNTs-HAP): dispersing 5g of hydroxyapatite in 100mL of deionized water, stirring for 20min, adding the Pd-CNTs prepared in the step (1), stirring for 20min, transferring to a reaction kettle, and performing hydrothermal reaction at 180 ℃ for 24h; washing with deionized water to ph=7 after the reaction is finished, and then drying overnight in a vacuum drying oven at 50 ℃;
(3) Folic acid modified noble metal-based carbon nanotubes-hydroxyapatite (fa@pd-CNTs-HAP): dissolving 0.0125g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), 0.0125g of N-hydroxysuccinimide (NHS) and 0.1g of Folic Acid (FA) in 6.25mL of dimethyl sulfoxide, and then adding all Pd-CNTs-HAP prepared in the step (2) into the folic acid solution under the condition of light shielding and water bath heating and stirring at 80 ℃ for reaction for 5 hours; then washed 3 times with deionized water and absolute ethanol, respectively, to ph=7 and dried overnight in a vacuum oven at 50 ℃.
Example 6: characterization of materials-X-ray diffraction (XRD) testing
To further characterize the inventive materials, XRD tests were performed on the samples prepared in inventive example 1 and comparative example 1. In the test of the experiment, the scanning angle is 10-80 degrees, and the scanning speed is 5 degrees/min. After the test is finished, the spectrogram is analyzed by using Jade software.
As shown in FIG. 1 (a), a large number of diffraction peaks of HAP and diffraction peaks of CNTs were found, which indicates that both were successfully compounded, but no diffraction peak of gold was found, which is attributable to the lower gold loading in the carbon nanotubes, and the lower ratio of Au-CNTs in FA@Au-CNTs-HAP. To further illustrate the loading of Au onto carbon nanotubes, comparative example 1 was also XRD characterized, specifically as shown in fig. 1 (b), the (111) and (311) crystal plane diffraction peaks of gold were observed at 38.2 ° and 77.5 °, respectively, which also illustrates that Au nanoparticles were loaded in fa@au-CNTs-HAP material.
Example 7: material characterization-scanning electron microscope (TEM) testing
To further characterize the inventive materials, TEM testing was performed on the samples prepared in inventive example 1 and comparative example 1.
As shown in fig. 2 (a), the sample of example 1 (fa@au-CNTs-HAP) has a tubular structure as a whole, and uses carbon nanotubes as a core, and hydroxyapatite is uniformly coated on the outer side of the carbon nanotubes, and is uniformly distributed as a whole (the circled portions in the figure are hydroxyapatite nanoplatelets or nanorods). To further characterize the structure of carbon nanotubes, comparative example 1 (Au-CNTs) was examined, as shown in fig. 2 (b), with carbon nanotubes having a tube diameter of about 60nm and gold nanoparticles (3-5 nm) clearly visible on the tube, the gold nanoparticles were uniformly supported on the carbon nanotubes, which also demonstrated that example 1 (fa@au-CNTs-HAP) successfully achieved the recombination of gold, hydroxyapatite and carbon nanotubes from the side.
Example 8: characterization of materials-Infrared Spectroscopy (FTIR) testing
To further characterize the inventive materials, FTIR testing was performed on the samples prepared in inventive example 1 and comparative example 2. The experiment is carried out before the testAnd tabletting the sample, and detecting the characteristic functional groups of the sample by a Fourier transform infrared instrument. The test wavelength range is 4500-4000cm -1 Resolution of 5cm -1 。
As shown in FIG. 3, the infrared spectra of comparative example 1 (Au-CNTs) showed stretching vibration peaks (2922 cm) representing C-H, respectively -1 ) C-H bending vibration peak (1433 cm) -1 ) C=O stretching vibration peak (1630 cm) -1 ) In addition, it was found to be 3439cm -1 Characteristic peaks of-OH appear at the positions, which indicate that the surface of the carbon nano tube is rich in hydroxyl groups after being subjected to acid treatment. Representative PO appears on the infrared spectrum of comparative example 3 (Au-CNTs-HAP), respectively 4 3- The antisymmetric stretching vibration peak of the P-O bond in the group (1033 cm -1 )、PO 4 3- Bending vibration peak of group O-P-O bond (604 cm) -1 And 558cm -1 ) C=o stretching vibration peak (1640 cm) -1 ) C-H bending vibration peak (1378 cm) -1 ) Stretching vibration peak of C-H (2806 cm) -1 ) Characteristic peak of-OH (3151 cm) -1 ). The infrared spectrum of example 1 (FA/Au-CNTs/HAP) shows characteristic peaks representing both carbon nanotubes and hydroxyapatite, and characteristic peaks of FA, specifically including-NH 2 Stretching vibration absorption peak (3421 cm) -1 ) -NH stretching vibration absorption peak (3324 cm) -1 ) Amide (c=o absorption peak (1694 cm -1 ) Telescopic vibration absorption of benzene ring skeleton (1606 cm) -1 ，1485cm -1 ). The above results demonstrate that the material prepared in example 1 successfully composited folic acid, carbon nanotubes and hydroxyapatite. In addition, the infrared spectrum of example 1 (FA/Au-CNTs/HAP) was found to be significantly blue-shifted compared to that of comparative examples 1 (Au-CNTs) and 3 (Au-CNTs-HAP), which also demonstrates that the amount of hydroxyl groups in the composite material is greatly increased, which is beneficial to the increase of the surface activity thereof.
Example 9: characterization of materials-Nitrogen adsorption desorption (BET) test
To further characterize the inventive materials, the samples prepared in inventive example 1, comparative examples 1-3 were subjected to a nitrogen adsorption desorption (BET) test. The specific surface area and the pore diameter of the material are tested by adopting a nitrogen adsorption-desorption instrument, the material is firstly degassed for 12 hours at 200 ℃, and the material is tested in a liquid nitrogen environment at-196 ℃ after water and pollutants adsorbed on the surface are removed, wherein specific data are shown in the following table:
table 1: material specific surface area data sheet
As shown in Table 1, examples 1 to 5 all have relatively high specific surface areas compared with comparative example 1 (Au-CNTs) and comparative example 4 (FA@HAP), and the specific surface areas are overall distributed in the range of 180.6 to 258.2m 2 In the range of/g, this suggests that PM-CNTs and HAP are compounded to improve the dispersibility of the material, thereby increasing the specific surface area. Example 2 (fa@au-CNT-HAP) and example 4 (fa@pt-CNT-HAP) have relatively small specific surface areas and are similar to those of comparative example 2 (fa@cnts-HAP), which can be attributed to the lower noble metal doping amount. In addition, comparative example 3 (Au-CNTs-HAP) was found to have a specific surface area similar to that of example 1 (FA@Au-CNTs-HAP), which suggests that folic acid modification has little effect on the specific surface area of the material.
Example 10: cytotoxicity test
To further investigate the feasibility of the materials of the present invention for use in bone repair scaffolds, cytotoxicity assays were performed on the materials prepared in inventive example 1 and comparative examples 2-3. The osteoblasts used in this experiment were human SV40 transfected osteoblasts (hFOB 1.19) purchased from North NaBian BNCC255176, and were used as test subjects for cytotoxicity. The cell culture broth used in this experiment was CM9-1 broth comprising 90% DMEM-H/F12, 10% FBS and 0.3mg/mL G418; wherein the DMEM-H/F12 is prepared by mixing a DMEM high-sugar culture solution containing glutamine and sodium pyruvate with an F12 culture solution according to a ratio of 1:1. The specific steps of toxicity detection experiments are as follows:
(1) Inoculating hFOB1.19 cells into a 96-well culture plate, wherein the volume of each well is 100 mu l, 5000 cells are arranged in each well, and then adding CM9-1 culture solution to suspend the cells; the cells were then subjected to 5% CO at 37 ℃ 2 Culturing in a cell culture box with 95% air for 24 hours;
(2) 200mg of the materials prepared in example 1 and comparative examples 2 to 3 were immersed in a CM9-1 culture solution, and after 24 hours, the culture solution was extracted, followed by a sterilization treatment with an ultraviolet lamp for 1 hour for use; sucking out the stock culture solution of the cultured cells, washing with PBS for 1 time, adding 90 μl of fresh culture solution and 10 μl of leaching solution into the well plate, and placing the 96 well plate into 5% CO at 37deg.C 2 Culturing in a cell incubator with 95% air for 24 hours, 48 hours and 72 hours respectively;
(3) After the leaching solution acts on hFOB1.19 cells for 24 hours, 48 hours and 72 hours, the original culture solution is sucked out again, 90 mu l of fresh culture solution and 10 mu l of CCK-8 reagent are injected after PBS cleaning, and then the cells are incubated for 3 hours in a cell incubator; after the incubation, the absorbance at 450nm was measured with a microplate reader.
As shown in FIG. 4, the survival rate of the FA/Au-CNTs/HAP stent material prepared in example 1 on hFOB1.19 cells under the actions of 24h,48h and 72h is higher than that of comparative examples 2-3, and particularly, the survival rate of the FA/Au-CNTs/HAP stent material leaching solution on hFOB1.19 cells under the action of 72h is still not lower than 93% and is 19% higher than that of comparative examples 2-3; whereas the Au-CNTs/HAP stent material prepared in comparative example 3 has the worst cell viability with respect to it. This is attributable to the improved biocompatibility of the FA/Au-CNTs/HAP scaffold by the modification of folic acid. Meanwhile, it was also found that comparative example 2 (FA/CNTs/HAP) with folate modification, which had cell viability in 24 hours similar to that of example 1, but significantly decreased with time, decreased to the same level as comparative example 3 (Au-CNTs/HAP) at 72 hours. This is because the carbon nanotubes in comparative example 2 (FA/CNTs/HAP) were not modified with gold to have a smaller specific surface area, and compared with example 1, folic acid could not be supported in a large amount, or folic acid was only floating on the surface of the material and unstable, thus reducing the modification effect of folic acid. Therefore, the FA/Au-CNTs/HAP stent material has good biocompatibility and is the result of the combined action of noble metal-gold doped modified carbon nano tube, hydroxyapatite composite and folic acid modification.
Example 11: osteogenic related gene expression detection
To further investigate the feasibility of the materials of the present invention for use in bone repair scaffolds, osteogenic related gene expression assays were performed on the materials prepared in inventive example 1 and comparative examples 3-4.
(1) Selecting SD rats of 2 weeks old, and extracting and culturing mesenchymal stem cells (BMSCs) by using a whole bone marrow adherent culture method;
(2) Osteogenic induced differentiation: adding the bracket slice into the osteogenesis inducing liquid, carrying out ultrasonic treatment for 10min, and culturing for 24h to prepare the osteogenesis inducing leaching liquid. Then adding BMSCs cells into a pore plate for culturing, sucking a culture medium after the cell fusion degree reaches 70%, changing the culture medium into an osteogenesis induction leaching solution, changing the solution once every 2 days, and detecting and analyzing osteogenesis related genes OPG, OCN, OSX by using an RT-PCR method when the induction is carried out to 3 days, wherein the primer design is shown in the following table.
| Gene | Forward primers | Reverse primers |
| GAPDH | ATGACTCTACCCACGGCAAG | TACTCAGCACCAGCATCACC |
| OPG | CTGTGCACTCCTGGTGTTCTT | GTAGCGCCCTTCCTCACATT |
| OCN | GAGGACCCTCTCTCTGCTCA | GGTAGCGCCGGAGTCTATTC |
| OSX | AGGATTGGATCTGAGTGAGCC | CATAGTGAGCTTCTTCCTGGG |
As shown in FIGS. 5 (a-c), BMSCs cells were cultured in a medium containing an osteogenic induction liquid for 3 days, and then, the expression of an osteogenic related gene was detected by real-time fluorescent quantitative PCR. Compared with the blank group, the FA/Au-CNTs/HAP group of example 1 had 3.0-fold increased OPG expression, 4.2-fold increased OCN expression and 5.5-fold increased OSX expression. This demonstrates that FA/Au-CNTs/HAP can effectively increase the expression of osteogenesis-related genes.
(3) Alkaline phosphatase activity staining is carried out when induction is carried out for 7 days, osteogenic induction liquid is removed, PBS is gently washed for 1 time, PBS is sucked off, cell lysate is added for full lysis, and supernatant is centrifugally taken and used for alkaline phosphatase detection; adding working solution and chromogenic substrate solution according to the instruction for detection; then using a multifunctional enzyme-labeled instrument to detect the absorbance of the solution at 405nm wavelength; the alkaline phosphatase activity in the sample was calculated from the total protein amount in the supernatant measured by the BCA method.
As shown in FIG. 5 (d), BMSCs cells were cultured in a medium containing an osteogenic induction solution for 7 days, and then, alkaline phosphatase activity was measured. The ALP protein expression level of example 1 (FA/Au-CNTs/HAP group) was significantly increased (< p < 0.001) and the ALP protein expression level of comparative example 3 (Au-CNTs/HAP group) was also significantly increased (< p < 0.01) compared to the blank group and comparative example 4. This demonstrates that carbon nanotubes can exert good osteoinductive effects and protein adsorption capacity in the FA/Au-CNTs/HAP scaffold. The HAP component is similar to the inorganic component in the bone matrix, and calcium and phosphorus ions released by the bracket cover the surface of the HAP component in the in-vivo dissolution process; when Au-CNTs are compounded with HAP, the Au-CNTs provide a large specific surface area and osteoinductive property for the scaffold; after FA is compounded with Au-CNTs/HAP, the FA provides good biocompatibility for the scaffold, and is further beneficial to bone cell growth.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.
Claims (6)
1. The folic acid modified noble metal-based carbon nanotube composite hydroxyapatite bone repair stent material is characterized by comprising hydroxyapatite, noble metal doped carbon nanotubes and folic acid, wherein the mass ratio of the hydroxyapatite to the noble metal doped carbon nanotubes to the folic acid is 5: (0.1-1): (0.1-1), wherein the mass ratio of the carbon nano tube to the noble metal is 1: (0.001-0.015), the preparation method of the folic acid modified noble metal-based carbon nano tube composite hydroxyapatite bone repair stent material comprises the following steps:
(1) Noble metal doped modified carbon nanotubes (PM-CNTs): dispersing the carbon nano tube into 10M hydrochloric acid solution, carrying out ultrasonic treatment for 25min each time, standing for 5min, and carrying out ultrasonic treatment-standing for 6 times until a suspension of 0.01g/mL is formed; then washing the carbon nanotubes with deionized water to ph=7 and ultrasonically dispersing the carbon nanotubes with deionized water to form a suspension of 0.01 g/mL; then adding 10mM noble metal solution dropwise into the carbon nano tube suspension at 650-800rpm/min, stirring for 10min, then performing ultrasonic treatment for 10min, and performing stirring-ultrasonic circulation for 9 times; then adding 1M NaOH solution under magnetic stirring to adjust the pH to 8, adding sodium borohydride, stirring for 5-10h, washing with deionized water to pH=7, and drying overnight in a vacuum drying oven at 50 ℃;
(2) Composite hydroxyapatite (PM-CNTs-HAP): dispersing hydroxyapatite in deionized water, stirring for 20min, adding PM-CNTs prepared in the step (1), stirring for 20min, transferring to a reaction kettle, and performing hydrothermal reaction at 180-200 ℃ for 24h; washing with deionized water to ph=7 after the reaction is finished, and then drying overnight in a vacuum drying oven at 50 ℃;
(3) Folic acid modified noble metal-based carbon nanotubes-hydroxyapatite (fa@pm-CNTs-HAP): dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and Folic Acid (FA) in dimethyl sulfoxide to prepare 0.02g/mL folic acid solution, heating and stirring in a water bath at 80-95 ℃ under a dark environment, dropwise adding PM-CNTs-HAP prepared in the step (2), and reacting for 5-12h; and then washing with deionized water and absolute ethyl alcohol for 3 times respectively, centrifuging and drying for standby.
2. The folic acid modified noble metal-based carbon nanotube composite hydroxyapatite bone repair stent material according to claim 1, wherein the ultrasonic temperature in the step (1) is 4 ℃, and the ultrasonic power is 150-200W.
3. The folic acid modified noble metal-based carbon nanotube composite hydroxyapatite bone repair scaffold material according to claim 1, wherein the noble metal solution in step (1) is one of chloroplatinic acid, chloroauric acid, chloropalladate or silver nitrate solution.
4. The folic acid modified noble metal-based carbon nanotube composite hydroxyapatite bone repair stent material according to claim 1, wherein the mass ratio of the carbon nanotubes to the noble metal solution to the sodium borohydride in the step (1) is 1g: (0.24-8.83) mL:2g.
5. The folic acid modified noble metal-based carbon nanotube composite hydroxyapatite bone repair scaffold material according to claim 1, wherein the mass to volume ratio of HAP, PM-CNTs and deionized water in step (2) is 5g: (0.1-1) g:100mL.
6. The folic acid modified noble metal-based carbon nanotube composite hydroxyapatite bone repair scaffold material according to claim 1, wherein the mass ratio of EDC, NHS, FA in the step (3) is 1:1:8.
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| CN104740638A (en) * | 2015-02-16 | 2015-07-01 | 南京工业大学 | Hydroxyapatite and folic acid co-modified carboxylated graphene oxide as well as preparation method and application thereof |
| CN104998301A (en) * | 2015-08-12 | 2015-10-28 | 河北工业大学 | Preparation method of carbon nano tube reinforced mesoporous hydroxyapatite composite material |
| CN106344927A (en) * | 2016-09-27 | 2017-01-25 | 大连理工大学 | Folate-modified medicine-carrying hydroxylapatite and preparation method thereof |
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