CN113666641B

CN113666641B - Multifunctional bioactive glass ceramic nano material and preparation method and application thereof

Info

Publication number: CN113666641B
Application number: CN202110870997.6A
Authority: CN
Inventors: 雷波; 牛雯
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2021-07-30
Filing date: 2021-07-30
Publication date: 2022-08-23
Anticipated expiration: 2041-07-30
Also published as: CN113666641A

Abstract

The invention discloses a multifunctional bioactive glass ceramics nano material and a preparation method and application thereof, wherein the method comprises the following steps: synthesizing BGN by respectively taking TEOS, TEP and calcium nitrate tetrahydrate as a silicon source, a phosphorus source and a calcium source based on a sol-gel template method; mixing BGN and molybdenum acetylacetonate, and performing hydrothermal reaction to obtain the molybdenum-doped bioactive glass ceramics nano material. The sol-gel template method and the hydrothermal method used in the invention are environment-friendly, convenient to operate and low in raw material cost; the prepared Mo-BGN has excellent photo-thermal property, oxidation resistance, biocompatibility, anti-inflammatory property and blood vessel promoting capability, and simultaneously, the Mo-BGN also shows antibacterial and anti-tumor properties under the irradiation of near-infrared laser, so the nano material has good application prospect in tissue regeneration after infection/tumor operation.

Description

Multifunctional bioactive glass ceramic nano 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 multifunctional bioactive glass ceramic nano material, and a preparation method and application thereof.

Background

The skin, as the first line of defense against viral entry, inevitably faces serious cancer and trauma threats. Although there are established treatment regimens for both skin tumor therapy and wound repair, the synergistic treatment of tumor and wound remains a significant clinical challenge. Particularly for the postoperative treatment of local solid tumors, not only is there a risk of tumor recurrence, but the chronic inflammatory microenvironment at the site of tumor residual also has a negative impact on the healing of the numerous tissue defects resulting from surgical resection of the tumor. In recent years, with the continuous and deep research of biomaterials, the development of degradable biological platforms with the purposes of treatment and repair is the key to the success of skin-related disease treatment. Although the currently developed multifunctional biomaterials have application advantages in the fields of tumor treatment and tissue repair, the superposition of multiple functional components increases the difficulty of preparation and degradation, and the treatment and repair efficiency is still to be improved.

Currently, in the use of synthetic biodegradable materials, Bioactive Glass Nanoparticles (BGNs) have significant advantages in biomedical applications including drug/gene delivery, tumor treatment, and tissue repair due to their controllable structure, excellent biodegradability, simple synthesis technology, and low preparation cost. However, the research and application of BGN are still in the primary stage at present, and difficulties and challenges of easy agglomeration, lack of modification sites and the like exist. However, with the complication of clinical requirements, by controlling the components of the BGN, designing multifunctional BGN containing other functional ions is an effective method for solving the application problem.

Disclosure of Invention

The invention aims to provide a multifunctional bioactive microcrystalline glass nano material and a preparation method thereof, the method is simple in process, and the obtained nano material has good biocompatibility, oxidation resistance and photo-thermal property and shows great application advantages in infection/tumor treatment and postoperative tissue repair.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

a preparation method of a multifunctional bioactive glass ceramic nano material comprises the following steps:

synthesizing bioactive glass nanoparticles by taking tetraethoxysilane, triethyl phosphate and calcium nitrate tetrahydrate as a silicon source, a phosphorus source and a calcium source respectively based on a sol-gel template method;

mixing the bioactive glass nano particles with molybdenum acetylacetonate, and carrying out hydrothermal reaction to obtain the molybdenum-doped bioactive glass ceramic nano material.

As a further improvement of the invention, the specific method for synthesizing the bioactive glass nanoparticles comprises the following steps:

adding a template agent into a solvent, stirring until the template agent is fully dissolved to obtain a mixed solution, adding tetraethoxysilane, adding an aqueous solution of triethyl phosphate and tetrahydrate calcium nitrate after full reaction, and fully reacting until the reaction is finished; and centrifuging and washing the reaction product, and calcining after freeze drying to obtain the bioactive glass nano-particles.

As a further improvement of the invention, the template agent is dodecylamine or bromohexadecylpyridine.

As a further improvement of the invention, the template agent is dodecylamine, and the dodecylamine is added into the mixed solution of the absolute ethyl alcohol and the deionized water and stirred until the dodecylamine is fully dissolved to obtain a mixed solution.

As a further improvement of the invention, the template agent is cetylpyridinium bromide, the cetylpyridinium bromide and urea are completely dissolved in a mixed solution containing cyclohexane and deionized water, isopropanol is added after full stirring, and the mixed solution is obtained after full stirring until the isopropanol is fully dissolved.

As a further improvement of the invention, the specific method of the hydrothermal reaction is as follows:

completely dispersing molybdenum acetylacetonate and bioactive glass nano particles in an ethanol solution according to the mass ratio of (3-18) to 1, and performing ultrasonic treatment to uniformly mix the particles; in the range of 160 to 200 ^o And C, carrying out hydrothermal reaction to obtain a precipitate, and freeze-drying the precipitate to obtain the molybdenum-doped bioactive glass ceramic nano material.

As a further improvement of the invention, the chemical composition of BGN is 80SiO ₂ -16CaO-4P ₂ O ₅ And 60SiO ₂ -36CaO-4P ₂ O ₅ 。

A multifunctional bioactive glass ceramics nano material is prepared by the method.

The microcrystalline glass nano material has a crystalline state and an amorphous state structure, and the valence state of Mo exists in two forms of +4 and + 6.

An application of multifunctional bioactive glass ceramics nano material as a repairing material for tissue regeneration after infection/tumor operation.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a preparation method of a multifunctional bioactive glass ceramics nano material for tissue regeneration after infection/tumor operation, aiming at the defects of single function, limited surface modification sites and the like of the traditional bioactive glass material, the method firstly synthesizes BGN particles by a classical sol-gel template method or an improved template method, then obtains a multivalent molybdenum doped bioactive glass ceramics material (Mo-BGN) by taking human microelement molybdenum as doping ions and through hydrothermal reaction; the microcrystalline glass material acts on an infected wound or a postoperative wound of a tumor in an in-situ coating mode to play the multifunctional functions of antibiosis, anti-tumor and tissue healing. The ion doping technology used by the invention has the advantages of rich doped ions, simple doping process, controllable product morphology and the like, and the related template synthesis technology and hydrothermal synthesis method are safe and environment-friendly, convenient to operate and low in cost. The experimental results prove that: the molybdenum-doped bioactive material prepared by the method is calcium molybdate (CaMoO) ₄ ) Molybdenum dioxide (MoO) ₂ ) The microcrystalline glass composed of the amorphous glass has stronger photo-thermal heating capacity and excellent oxidation resistance, and the good biocompatibility, photo-thermal anti-tumor and photo-thermal antibacterial effects can effectively kill tumor cells and bacteria, promote the migration of endothelial cells and show a certain effect of promoting tissue repair. The BGN used in the invention has good biocompatibility and degradability, and is endowed with additional antibacterial and antitumor functions through doping of molybdenum, and the synthesized Mo-BGN not only can not cause immunogenic reaction, but also can play corresponding functions in the inflammation stage and the proliferation stage related to wound healing through antibacterial and vascularization promoting effects.

The invention also has the following advantages:

1) the BGN synthesis method used in the invention is a sol-gel template method, and the template-catalyst dodecylamine (DDA) or bromohexadecylpyridine (CPB) belongs to a surfactant, so that the BGN has good solubility, is cheap and is easy to obtain; particularly, BGN synthesized by using a template-catalyst CPB has a dendritic morphology, and a large specific surface area of the BGN provides abundant sites for ion doping.

2) According to the invention, the BGN is modified by utilizing the hydrothermal reaction of molybdenum acetylacetonate, the traditional bioactive material shows multifunctional bioactivity through a simple synthesis process, and compared with a nano composite material, the composite material has the advantages of simple process and stable performance.

3) The Mo-BGN has excellent photo-thermal property and oxidation resistance due to the doping of molybdenum ions; the reason for the photo-thermal property of the material is probably MoO in Mo-BGN ₂ Due to the oxygen vacancy structure of Mo-BGN, the oxidation resistance of the Localized Surface Plasmon Resonance (LSPR) effect can effectively capture free radicals.

4) The doping of Mo ions in the invention can not affect the inherent blood compatibility and cell compatibility of BGN, mainly because Mo element as a trace element in human body can not affect the normal metabolism of life.

5) The Mo-BGN prepared by the invention has biocompatibility and photothermal temperature (42) under laser irradiation ^o C) The temperature can selectively cause irreversible damage to bacteria and tumor cells, and does not affect the cell viability of normal tissue cells; the in vitro cell migration, anti-inflammation and angiogenesis promotion experiments show that the antioxidant activity and bioactive glass components (Si and Ca) of the Mo-BGN have continuous endothelial cell migration, anti-inflammation and angiogenesis promotion effects on the wound surface environment, and provide favorable conditions for the repair of infected wound surfaces and postoperative wound surfaces of tumors.

Drawings

FIG. 1 is a schematic structural diagram of a nanomaterial of the present invention;

FIG. 2 is a TEM (TEM) morphology photograph of 15Mo-BGN (molybdenum acetylacetonate: BGN =15: 1) of the multifunctional bioactive glass ceramics nano material synthesized by the present invention and used for tissue regeneration after infection/tumor operation;

FIG. 3 is the physicochemical characterization results of the 15Mo-BGN produced. Wherein, A is FTIR spectrum; b is an XRD spectrum; c is XPS spectrum; d is UV-Vis-NIR spectrum;

FIG. 4 shows the photo-thermal and oxidation resistance properties of 15Mo-BGN prepared in the invention. A is a change curve of the temperature of a sample irradiated by 808 nm laser along with irradiation time; b is a circulation stability curve; c is the UV-Vis spectrum of the DPPH solution after the DPPH solution is incubated for 30 minutes by the material; d is the oxidation resistance;

FIG. 5 shows that the bioactive glass-ceramic nanomaterial prepared by the invention can treat Staphylococcus aureus before and after laser actionS. aureusA), methicillin-resistant Staphylococcus aureus (S.) (MRSAB), human umbilical vein endothelial cells (HUVEC, C) and human malignant melanoma cells (a 375, D) toxicity, with "+" indicating laser irradiation;

FIG. 6 is a graph showing the anti-inflammatory and vasogenic effects of 15Mo-BGN prepared according to the invention. Wherein A is an expression result of inflammatory factor TNF-alpha; b is the expression result of inflammatory factor IL-1 beta; c is the expression result of the anti-inflammatory factor IL-10; d is relative vascular factor (CD 31) expression result;

fig. 7 shows the result of the tissue regeneration after infection/tumor operation of the bioactive glass-ceramic nanomaterial prepared by the invention. Wherein A isMRSAMacroscopic results of infected skin wound repair; b is the macroscopic result of the skin wound repair of the tumor partial resection type.

Detailed Description

The invention is described in further detail below with reference to the accompanying drawings:

the invention aims to prepare the bioactive glass ceramics nano material with good biocompatibility, antibiosis and anti-tumor properties, and realizes tissue regeneration after infection/tumor operation by utilizing the photo-thermal and oxidation resistance of the material.

Bioactive Glass Nanoparticles (BGNs) have been widely used in the biomedical field because of their controlled biodegradation, good biocompatibility, and low cost. However, BGN has no antibacterial and antitumor properties, so that the biological function of the BGN is limited; the ion doping technology is one of important means for modifying BGN, and the main strategy is to introduce functional ions into BGN to realize the regulation and control of the characteristics of the BGN, such as light, heat, electricity, magnetism and the like.

Molybdenum (Mo) is an important trace element in human bodies and has good biological effect on different tissues. Molybdenum is a trace element essential to living body, is very important for normal metabolism of life, and is in high valence state (Mo) ⁶⁺ ) Molybdenum MoO in reduced valence state, associated with bone and tooth growth ₂ Can be used as a photo-thermal agent to show controllable temperature rising effect under Near Infrared (NIR) stimulation. On the other hand, Mo is a typical valence-variable element, Mo ⁶⁺ And Mo ⁴⁺ Respectively has the tissue growth promoting capacity and the photo-thermal performance. Can be prepared by mixing Mo ⁶⁺ And Mo ⁴⁺ Single-component multifunctional Mo-BGN is designed by introducing BGN, and material support is provided for tumor/infected photothermal therapy and tissue repair. The Mo-BGN is synthesized by BGN and molybdenum acetylacetonate through hydrothermal reaction, has the advantages of strong antioxidant activity, high photothermal conversion efficiency, low toxicity, biodegradability and the like, and if the morphology of the initial BGN can be controlled and the dosage of the molybdenum acetylacetonate can be adjusted in the synthesis process, the efficiency of the Mo-BGN material on infection/tumor treatment and postoperative tissue repair can be greatly improved.

If the multivalent Mo is doped into the BGN network, the BGN can be endowed with photothermal treatment and repair promotion functions. Therefore, the invention realizes the doping of the multivalent Mo by utilizing the hydrothermal reaction, designs and synthesizes the Mo-BGN with strong antioxidant activity, high photothermal conversion efficiency and low toxicity. And the physical and chemical properties, biocompatibility, in-vivo and in-vitro photo-thermal properties and the function and mechanism of the synthesized multivalent Mo-doped bioactive glass nano-particles in the wound healing process caused by bacterial infection and tumor excision are researched.

Therefore, the first purpose of the invention is to provide a preparation method of a multifunctional bioactive microcrystalline glass nano material, which comprises the following steps:

1) synthesizing BGN by a sol-gel template method: anhydrous ethanol and deionized water were added to a 250 mL round bottom flask in a 3:1 molar ratio and mixed well followed by the addition of 10 g template-catalyst dodecylamine (DDA) at 40 ^o Stirring in the environment of C until DDA is fully dissolved, and then stirring continuously until 4 mL of n-siliconEthyl phosphate (TEOS) was added dropwise to the above mixed solution, and after 30 minutes of reaction, triethyl phosphate (TEP) was added dropwise to the above reaction system, and stirring was continued for 15 minutes, and 1 mL of 5 mol L of TEP was added ^-1 Aqueous solution of calcium nitrate tetrahydrate at 40 ^o C, reacting for 3 hours in an environment; centrifugally collecting reaction products, washing the reaction products by absolute ethyl alcohol and deionized water, and freeze-drying the reaction products by 600-650 DEG C ^o Calcining the mixture for 3-10 hours in a muffle furnace of the step C to obtain a BGN product;

2) preparing Mo-BGN by a hydrothermal method: molybdenum acetylacetonate and BGN were completely dispersed in 10 mL of a 75% ethanol solution in a mass ratio of (3-18): 1. Secondly, the solution is subjected to ultrasonic treatment for 20-40 minutes, and the molybdenum acetylacetonate and BGN are uniformly mixed. Then directly transferring the solution into a 25 mL high-pressure reaction kettle at 160-200% ^o And C, preserving heat for 8-12 hours in an oven. Cooling the high-pressure reaction kettle to room temperature, taking out the suspension in the polytetrafluoroethylene lining, centrifuging at high speed to obtain a precipitate, and freeze-drying the precipitate to obtain a molybdenum-doped bioactive glass ceramic nano material (Mo-BGN);

the invention further improves the following steps:

the chemical composition of BGN in the step 1) can be 80SiO ₂ -16CaO-4P ₂ O ₅ And 60SiO ₂ -36CaO-4P ₂ O ₅ 。

Lauryl amine (DDA) or bromohexadecylpyridine (CPB) may be used as a templating agent in step 1). If CPB is used, the corresponding synthesis process is modified as follows: 1.25 g of CPB and 0.75 g of urea were completely dissolved in a mixed solution containing 37.5 mL of cyclohexane and 37.5 mL of deionized water, vigorously stirred for 15 minutes, then 1.15 g of isopropanol was added, and the mixture was stirred at 25 deg.C ^o Continuing stirring for 2 hours in the environment C; subsequently, 3.40 mL of TEOS was added dropwise to the mixed solution while maintaining stirring, and after 30 minutes of reaction, the temperature was raised to 70 ^o C. Stirring was continued for 7.5 hours, and after the reaction temperature stabilized, 0.77 mL of TEP and 1 mL of an aqueous solution of calcium nitrate tetrahydrate were added in that order at 30-minute intervals. After further stirring for 16 hours, a white bioactive glass sol was obtained. Then, the gel separated by centrifugation is washed with acetone, ethanol and deionized water respectivelyAnd preparing a BGN precursor. After the precursor is freeze-dried, the temperature is 600-650 DEG C ^o And C, calcining for 3-10 hours to remove residual organic matters and obtain the BGN with the radial morphology.

The molar ratio of molybdenum acetylacetonate to BGN in step 2) is 3:1, 9:1, 15:1 and 18: 1.

The second purpose of the invention is to provide the multivalent molybdenum-doped bioactive material prepared based on the method, which is a multifunctional bioactive glass ceramic nanomaterial, and the reaction mechanism and the product structure of the nanomaterial are shown in fig. 1.

The multifunctional bioactive microcrystalline glass nano material prepared by the method has Mo ⁶⁺ With Mo ⁴⁺ The tissue growth promoting and photo-thermal heating performance. When the Mo-BGN is applied to the body in an in-situ coating mode, the Mo-BGN not only can promote various wounds caused by bacterial infection and surgical tumor excision to heal, but also can effectively inhibit tumor recurrence. Especially in the treatment after tumor resection, the temperature rise of the material under the mediation of laser can lead the wound environment to reach 42 ^o C, this temperature is sufficient to kill tumor cells and bacteria, but promotes endothelial cell migration. Meanwhile, the oxidation resistance caused by a large number of oxygen vacancies in the Mo-BGN has an obvious promotion effect on tissue repair.

The third purpose of the invention is to provide the application of the multifunctional bioactive glass ceramics nano material as a repairing material for tissue regeneration after infection/tumor operation.

The result shows that the multifunctional microcrystalline glass with bioactivity is synthesized by an in-situ hydrothermal method. The Mo-BGN has a nanoparticle shape with good dispersibility; the physical and chemical structure characterization result shows that the Mo-BGN has CaMoO ₄ -MoO ₂ Microcrystalline glass structure of-BGN, Mo ions in Mo ⁴⁺ /Mo ⁶⁺ Is in the form of Mo-BGN; due to MoO in the Mo-BGN structure ₂ The crystal form and rich oxygen vacancy, Mo-BGN has selective photo-thermal anticancer, antibacterial, free radical scavenging, antioxidant, anti-inflammatory and good angiogenesis activity. Mo-BGN not only can effectively improve the tissue reconstruction of infected wound and tumor excision defect, but also can inhibit tumor recurrence, and has good effectThe organization security of. The work provides a good strategy for designing multifunctional bioactive nano materials with simple components to treat the tissue regeneration of complex disease injury.

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) Synthesizing BGN by a classical sol-gel template method: anhydrous ethanol and deionized water were added to a 250 mL round bottom flask in a 3:1 molar ratio and mixed well, followed by the addition of 10 g of template-catalyst DDA at 40 ^o Stirring in the environment C until DDA is fully dissolved, then dripping 4 mL of Tetraethoxysilane (TEOS) into the mixed solution under the condition of continuous stirring, dripping TEP into the reaction system after reacting for 30 minutes, continuing stirring for 15 minutes, adding 1 mL of 5 mol L ^-1 Aqueous solution of calcium nitrate tetrahydrate at 40 ^o C, reacting for 3 hours in an environment; collecting reaction product by centrifugation, washing with anhydrous ethanol and deionized water, freeze drying, and treating with 650 deg.C ^o Calcining the mixture in a muffle furnace of the C furnace for 3 hours to obtain a BGN product;

2) preparing Mo-3BGN by a hydrothermal method: molybdenum acetylacetonate and BGN were completely dispersed in 10 mL of 75% ethanol solution at a mass ratio of 3: 1. Secondly, the solution is treated by ultrasonic for 30 minutes to ensure that the molybdenum acetylacetonate and BGN are uniformly mixed. The solution was then transferred directly to a 25 mL autoclave at 180 deg.C ^o And C, keeping the temperature in the oven for 10 hours. And (3) taking out the suspension in the polytetrafluoroethylene lining along with the cooling of the high-pressure reaction kettle to room temperature, performing high-speed centrifugation to obtain a precipitate, and performing freeze drying on the precipitate to obtain the molybdenum-doped bioactive glass ceramics nano material (Mo-3 BGN).

Example 2

1) Synthesizing BGN by a classical sol-gel template method: anhydrous ethanol and deionized water were added to a 250 mL round bottom flask in a 3:1 molar ratio and mixed well, followed by the addition of 10 g of template-catalyst DDA at 40 ^o Stirring in the environment C until DDA is fully dissolved, and then dripping 4 mL of Tetraethoxysilane (TEOS) into the environment under the condition of continuous stirringAfter the above mixed solution reacted for 30 minutes, TEP was added dropwise to the above reaction system, and stirring was continued for 15 minutes, followed by addition of 1 mL of 5 mol L ^-1 Aqueous solution of calcium nitrate tetrahydrate at 40 ^o C, reacting for 3 hours in an environment; collecting reaction product by centrifugation, washing with anhydrous ethanol and deionized water, freeze drying, and processing with 630 deg.C ^o Calcining the mixture in a muffle furnace of the C for 5 hours to obtain a BGN product;

2) preparing Mo-3BGN by a hydrothermal method: molybdenum acetylacetonate and BGN were completely dispersed in 10 mL of 75% ethanol solution at a mass ratio of 9: 1. Secondly, the solution is treated by ultrasonic for 30 minutes to ensure that the molybdenum acetylacetonate and BGN are uniformly mixed. The solution was then transferred directly to a 25 mL autoclave at 180 deg.F ^o And C, keeping the temperature in the oven for 10 hours. And (3) cooling the high-pressure reaction kettle to room temperature, taking out the suspension in the polytetrafluoroethylene lining, centrifuging at a high speed to obtain a precipitate, and freeze-drying the precipitate to obtain the molybdenum-doped bioactive glass ceramic nano material (Mo-9 BGN).

Example 3

1) Synthesizing BGN by a classical sol-gel template method: anhydrous ethanol and deionized water were added to a 250 mL round bottom flask in a 3:1 molar ratio and mixed well, followed by the addition of 10 g of template-catalyst DDA at 40 ^o Stirring in the environment C until DDA is fully dissolved, then dripping 4 mL of Tetraethoxysilane (TEOS) into the mixed solution under the condition of continuous stirring, dripping TEP into the reaction system after reacting for 30 minutes, continuing stirring for 15 minutes, adding 1 mL of 5 mol L ^-1 Aqueous solution of calcium nitrate tetrahydrate at 40 ^o C, reacting for 3 hours in an environment; collecting reaction product by centrifugation, washing with anhydrous ethanol and deionized water, freeze drying, and purifying with 620 ^o Calcining the mixture in a muffle furnace of the C furnace for 6 hours to obtain a BGN product;

2) preparing Mo-15BGN by a hydrothermal method: molybdenum acetylacetonate and BGN were completely dispersed in 10 mL of 75% ethanol solution at a mass ratio of 15: 1. Secondly, the solution is treated by ultrasonic for 30 minutes to ensure that the molybdenum acetylacetonate and BGN are uniformly mixed. The solution was then transferred directly to a 25 mL autoclave at 180 deg.F ^o And C, keeping the temperature in the oven for 10 hours. Along with the cooling of the high-pressure reaction kettle to the roomAnd (3) taking out the suspension in the polytetrafluoroethylene lining, centrifuging at a high speed to obtain a precipitate, and freeze-drying the precipitate to obtain the molybdenum-doped bioactive glass ceramic nano material (Mo-15 BGN).

Example 4

1) Synthesizing BGN by a classical sol-gel template method: anhydrous ethanol and deionized water were added to a 250 mL round bottom flask in a 3:1 molar ratio and mixed well, followed by the addition of 10 g of template-catalyst DDA at 40 ^o Stirring in the environment C until DDA is fully dissolved, then dripping 4 mL of Tetraethoxysilane (TEOS) into the mixed solution under the condition of continuous stirring, dripping TEP into the reaction system after reacting for 30 minutes, continuing stirring for 15 minutes, adding 1 mL of 5 mol L ^-1 Aqueous solution of calcium nitrate tetrahydrate at 40 ^o C, reacting for 3 hours in an environment; collecting reaction product by centrifugation, washing with anhydrous ethanol and deionized water, freeze drying, and purifying with 650 deg.C ^o Calcining the mixture in a muffle furnace of the step C for 3 hours to obtain a BGN product;

2) preparing Mo-18BGN by a hydrothermal method: molybdenum acetylacetonate and BGN were completely dispersed in 10 mL of 75% ethanol solution at a mass ratio of 18: 1. Secondly, the solution is treated by ultrasonic for 30 minutes to ensure that the molybdenum acetylacetonate and BGN are uniformly mixed. The solution was then transferred directly to a 25 mL autoclave at 180 deg.F ^o And C, keeping the temperature in the oven for 10 hours. And (3) cooling the high-pressure reaction kettle to room temperature, taking out the suspension in the polytetrafluoroethylene lining, centrifuging at a high speed to obtain a precipitate, and freeze-drying the precipitate to obtain the molybdenum-doped bioactive glass ceramic nano material (Mo-18 BGN).

Example 5

1) Synthesizing BGN by an improved sol-gel template method: 1.25 g of CPB and 0.75 g of urea were completely dissolved in a mixed solution containing 37.5 mL of cyclohexane and 37.5 mL of deionized water, vigorously stirred for 15 minutes, then 1.15 g of isopropanol was added, and the mixture was stirred at 25 deg.C ^o Stirring for 2 hours in the environment C; subsequently, 3.40 mL of TEOS was added dropwise to the mixed solution while maintaining stirring, and after 30 minutes of reaction, the temperature was raised to 70 ^o C. Stirring is continued for 7.5 hours, and 0.77 mL of TEP and 1 mL of TEP are added in sequence every 30 minutes after the reaction temperature is stableAn aqueous solution of calcium nitrate hydrate. After further stirring for 16 hours, a white bioactive glass sol was obtained. And then washing the gel separated by centrifugation with acetone, ethanol and deionized water for three times respectively to prepare the BGN precursor. After freeze-drying the precursor, at 600 ^o Calcining for 10 hours under C to remove residual organic matters and obtain BGN with radial morphology;

2) preparing Mo-3BGN by a hydrothermal method: molybdenum acetylacetonate and BGN were completely dispersed in 10 mL of 75% ethanol solution at a mass ratio of 3: 1. Secondly, the solution is treated by ultrasonic for 30 minutes to ensure that the molybdenum acetylacetonate and BGN are uniformly mixed. The solution was then transferred directly to a 25 mL autoclave at 180 deg.C ^o And C, keeping the temperature in the oven for 10 hours. And (3) cooling the high-pressure reaction kettle to room temperature, taking out the suspension in the polytetrafluoroethylene lining, centrifuging at a high speed to obtain a precipitate, and freeze-drying the precipitate to obtain the molybdenum-doped bioactive glass ceramic nano material (Mo-3 BGN).

Example 6

1) Synthesizing BGN by an improved sol-gel template method: 1.25 g of CPB and 0.75 g of urea were completely dissolved in a mixed solution containing 37.5 mL of cyclohexane and 37.5 mL of deionized water, vigorously stirred for 15 minutes, then 1.15 g of isopropanol was added, and the mixture was stirred at 25 deg.C ^o Stirring for 2 hours in the environment C; subsequently, 3.40 mL of TEOS was added dropwise to the mixed solution while maintaining stirring, and after 30 minutes of reaction, the temperature was raised to 70 ^o C. Stirring was continued for 7.5 hours, and after the reaction temperature stabilized, 0.77 mL of TEP and 1 mL of an aqueous solution of calcium nitrate tetrahydrate were added in that order at 30-minute intervals. After further stirring for 16 hours, a white bioactive glass sol was obtained. And then washing the gel separated by centrifugation with acetone, ethanol and deionized water for three times respectively to prepare the BGN precursor. After freeze-drying the precursor, at 600 ^o Calcining for 10 hours at the temperature of C to remove residual organic matters and obtain BGN with radial morphology;

2) preparing Mo-9BGN by a hydrothermal method: molybdenum acetylacetonate and BGN were completely dispersed in 10 mL of 75% ethanol solution at a mass ratio of 9: 1. Secondly, the solution is treated by ultrasonic for 30 minutes to ensure that the molybdenum acetylacetonate and BGN are uniformly mixed. Followed byThe solution was transferred directly to a 25 mL autoclave at 180 deg.C ^o And C, keeping the temperature in the oven for 10 hours. And (3) cooling the high-pressure reaction kettle to room temperature, taking out the suspension in the polytetrafluoroethylene lining, centrifuging at a high speed to obtain a precipitate, and freeze-drying the precipitate to obtain the molybdenum-doped bioactive glass ceramic nano material (Mo-9 BGN).

Example 7

1) Synthesizing BGN by an improved sol-gel template method: 1.25 g of CPB and 0.75 g of urea were completely dissolved in a mixed solution containing 37.5 mL of cyclohexane and 37.5 mL of deionized water, vigorously stirred for 15 minutes, then 1.15 g of isopropanol was added, and the mixture was stirred at 25 deg.C ^o Stirring for 2 hours in the environment C; subsequently, 3.40 mL of TEOS was added dropwise to the mixed solution while maintaining stirring, and after 30 minutes of reaction, the temperature was raised to 70 ^o C. Stirring was continued for 7.5 hours, and after the reaction temperature stabilized, 0.77 mL of TEP and 1 mL of an aqueous solution of calcium nitrate tetrahydrate were added in that order at 30-minute intervals. After further stirring for 16 hours, a white bioactive glass sol was obtained. And then washing the gel separated by centrifugation with acetone, ethanol and deionized water for three times respectively to prepare the BGN precursor. After freeze-drying the precursor, at 600 ^o Calcining for 10 hours under C to remove residual organic matters and obtain BGN with radial morphology;

2) preparing Mo-15BGN by a hydrothermal method: molybdenum acetylacetonate and BGN were completely dispersed in 10 mL of 75% ethanol solution at a mass ratio of 15: 1. Secondly, the solution is treated by ultrasonic for 30 minutes to ensure that the molybdenum acetylacetonate and BGN are uniformly mixed. The solution was then transferred directly to a 25 mL autoclave at 180 deg.C ^o And C, keeping the temperature in the oven for 10 hours. And (3) taking out the suspension in the polytetrafluoroethylene lining along with the cooling of the high-pressure reaction kettle to room temperature, performing high-speed centrifugation to obtain a precipitate, and performing freeze drying on the precipitate to obtain the molybdenum-doped bioactive glass ceramics nano material (Mo-15 BGN).

Example 8

1) Synthesizing BGN by an improved sol-gel template method: 1.25 g of CPB and 0.75 g of urea were completely dissolved in a mixed solution containing 37.5 mL of cyclohexane and 37.5 mL of deionized water, vigorously stirred for 15 minutes and then added with 1.15 g of isopropyl alcoholAt 25 ^o Stirring for 2 hours in the environment C; subsequently, 3.40 mL of TEOS was added dropwise to the mixed solution while maintaining stirring, and after 30 minutes of reaction, the temperature was raised to 70 ^o C. Stirring was continued for 7.5 hours, and after the reaction temperature stabilized, 0.77 mL of TEP and 1 mL of an aqueous solution of calcium nitrate tetrahydrate were added in that order at 30-minute intervals. After further stirring for 16 hours, a white bioactive glass sol was obtained. And then washing the gel separated by centrifugation with acetone, ethanol and deionized water for three times respectively to prepare the BGN precursor. After freeze-drying the precursor, at 600 ^o Calcining for 10 hours at the temperature of C to remove residual organic matters and obtain BGN with radial morphology;

2) preparing Mo-18BGN by a hydrothermal method: molybdenum acetylacetonate and BGN were completely dispersed in 10 mL of 75% ethanol solution at a mass ratio of 18: 1. Secondly, the solution is treated by ultrasonic for 30 minutes to ensure that the molybdenum acetylacetonate and BGN are uniformly mixed. The solution was then transferred directly to a 25 mL autoclave at 180 deg.C ^o And C, keeping the temperature in the oven for 10 hours. And (3) cooling the high-pressure reaction kettle to room temperature, taking out the suspension in the polytetrafluoroethylene lining, centrifuging at a high speed to obtain a precipitate, and freeze-drying the precipitate to obtain the molybdenum-doped bioactive glass ceramic nano material (Mo-18 BGN).

The multifunctional bioactive glass ceramics nano material (Mo-BGN) for tissue regeneration after infection/tumor operation, which is prepared by the invention, has inherent photothermal antioxidant antibacterial anti-inflammatory activity due to the special microcrystalline structure, oxygen vacancy and bioactive components. When Mo-BGN acts on infected wound or postoperative wound of tumor, the material has biocompatible photothermal temperature (42) under the mediation of 808 nm laser ^o C) The temperature can selectively kill tumor cells and various bacteria, but shows excellent cell compatibility to normal cells around the wound surface. In addition, the photothermal, antioxidant and bioactive glass components (bioactive Si and Ca) of Mo-BGN have a sustained anti-inflammatory and angiogenic effect on the wound environment, which makes it a significant advantage in applications for tissue regeneration after infection/tumor surgery, as analyzed in detail below in combination with experimental data.

FIG. 2 is a TEM morphology photograph of the multifunctional bioactive glass ceramics nano material 15Mo-BGN (molybdenum acetylacetonate: BGN =15: 1) synthesized by the invention and used for tissue regeneration after infection/tumor operation. As can be seen from TEM photographs with different magnifications, the hydrothermal reaction does not affect the dispersibility of the nanoparticles, and the original spherical morphology and particle size distribution range (200-300 nm) of BGN can be maintained.

FIG. 3 is the physicochemical characterization results of the 15Mo-BGN produced. As can be seen by the FTIR spectra shown in A in FIG. 3, 1632, 1100 and 801 cm ^-1 The infrared absorption peak is attributed to the characteristic absorption peak of BGN, 945 cm ^-1 The absorption band at (b) corresponds to the shear vibration of Mo-O-Mo. As can be seen from B in FIG. 3, 23.0 ^o The diffraction package at (b) corresponds to amorphous silica, 2 θ =25.3, 37.0, 38.6 and 48.1 ^o The diffraction peak at (A) corresponds to CaMoO ₄ Diffraction peaks of (101), (112), (204), and (220) crystal planes of (JCPDS 29-0351); 2 θ =27.5, 36.1 and 54.4 ^o The diffraction peak at (A) corresponds to MoO ₂ (JCPDS No. 29-0351) and (311). The product prepared by Mo doping was proved to be crystalline (CaMoO) ₄ With MoO ₂ ) And amorphous state. This product, which has both crystalline and amorphous structures, is called a glass-ceramic. In FIG. 3C, 4 characteristic peaks can be seen, with binding energies of 233.9 eV and 231.8 eV for Mo 3d _3/2 Can be respectively assigned to Mo ⁶⁺ And Mo ⁴⁺ While Mo 2d at 229.6 eV and 228.1eV _5/2 Peaks correspond to Mo ⁶⁺ And Mo ⁴⁺ It is shown that Mo in 15Mo-BGN exists in two forms, namely +4 and + 6. The UV-Vis-NIR absorption spectrum shown in D in FIG. 3 shows that the material has an absorbance in the near infrared wavelength range of 400-1000 nm. Therefore, the photo-thermal temperature rising performance of the 15Mo-BGN material can be researched by using near-infrared laser with wavelength of 808 nm.

FIG. 4 shows the photo-thermal and oxidation resistance properties of 15Mo-BGN prepared in the invention. As can be seen from A in FIG. 4, the sample pairs 808 nm (0.8W cm) ^-2 10 minutes) the absorption of the laser depends on the concentration of the sample. After laser irradiation, 100- ^-1 The temperature of 15Mo-BGN can reach 42 ^o C. The photothermal cycling stability results shown as B in fig. 4 also illustrate that the sample also maintains a stable and sustained ability to heat up after undergoing five cycles of "laser on-laser off". The controllable photothermal capacity of 15Mo-BGN determines the application value of the compound in the fields of tumor treatment and efficient sterilization. As shown in fig. 4, C, 15Mo-BGN showed similar antioxidant performance to ascorbic acid (VC, positive control). Comparing the oxidation resistance of the materials in each group (D in figure 4), the materials are found to be 5 microgram mL ^-1 The DPPH & ltSUB & gt can be eliminated by 50.9% under the concentration of the (D & ltSUB & gt), which indicates that 15Mo-BGN has strong free radical capture capacity. The presence of oxygen vacancies is presumed to be the main cause of its high antioxidant activity.

FIG. 5 shows the pair of bioactive glass-ceramic nanomaterials prepared by the invention before and after laser actionS. aureusBacteria, bacteria,MRSABacteria, HUVEC cells and A375 cytotoxicity assay results. From the antibacterial results (A and B in FIG. 5), it can be seen that after 12 hours of exposure to the 15Mo-BGN sample, neither strain was significantly inhibited compared to the control without laser (Blank group). The cytocompatibility results shown in FIGS. 5C and D show that the material was between 0 and 250. mu.g mL without laser irradiation ^-1 There was no significant cytotoxicity on both a375 and HUVEC cells in the concentration range, but laser irradiation was sufficient to kill more than 80% of a375 tumor cells, whereas near-infrared laser had no effect on HUVEC cell viability. The results show that the 15Mo-BGN can effectively kill bacteria and skin tumor cells under laser irradiation without affecting normal tissue cells.

FIG. 6 is a graph showing the anti-inflammatory and vasogenic effects of 15Mo-BGN prepared according to the invention. FIG. 6A shows the expression result of inflammatory factor TNF-alpha; in FIG. 6, B is the result of expression of IL-1 β, an inflammatory factor; in FIG. 6, C is the result of expression of the anti-inflammatory factor IL-10; in FIG. 6, D is the relative vascular factor (CD 31) expression result. As shown in A-C in FIG. 6, 15Mo-BGN significantly inhibited the expression of TNF- α and IL-1 β, but increased the expression of IL-10, as compared to the blank control. This result demonstrates that 15Mo-BGN can effectively inhibit inflammation. This significant anti-inflammatory effect is attributed to the multi-valence Mo doping, the oxygen vacancy structure of 15Mo-BGN facilitates the capture of free radicals. While free radicals are effectors of inflammatory reactions, oxygen radicals can induce inflammatory reactions via PRRS and non-PRRS pathways. Therefore, the inflammation is effectively relieved by consuming free radicals through 15Mo-BGN, and the skin wound surface repair caused by infection is promoted. In addition, as shown by D in FIG. 6, the expression of CD31 was higher in the 15Mo-BGN group than in the control group, indicating that it had good angiogenic ability.

Fig. 7 shows the result of the tissue regeneration after infection/tumor operation of the bioactive glass-ceramic nanomaterial prepared by the invention. In FIG. 7A isMRSAThe macroscopic result of the infected skin wound repair is shown in the figure, and compared with a control group, the 15Mo-BGN + group has the fastest wound recovery rate within 14 days; in FIG. 7, B is the macroscopic result of the tumor partially resected skin wound repair, and it can be seen from the figure that the 15Mo-BGN + group wound irradiated by near infrared is gradually closed within 14 days, and no obvious tumor recurrence occurs. The results show that the 15Mo-BGN has the effects of resisting bacteria, inhibiting tumor recurrence and promoting wound repair, and the results prove that the Mo-BGN is expected to be used as a repair material for tissue regeneration after infection/tumor operation.

Example 9

1) Synthesizing BGN by an improved sol-gel template method: 1.25 g of CPB and 0.75 g of urea were completely dissolved in a mixed solution containing 37.5 mL of cyclohexane and 37.5 mL of deionized water, vigorously stirred for 15 minutes, then 1.15 g of isopropanol was added, and the mixture was stirred at 25 deg.C ^o Stirring for 2 hours in the environment C; subsequently, 3.40 mL of TEOS was added dropwise to the mixed solution while maintaining stirring, and after 30 minutes of reaction, the temperature was raised to 70 ^o C. Stirring was continued for 7.5 hours, and after the reaction temperature stabilized, 0.77 mL of TEP and 1 mL of an aqueous solution of calcium nitrate tetrahydrate were added in that order at 30-minute intervals. After further stirring for 16 hours, a white bioactive glass sol was obtained. Then, the gel separated by centrifugation is washed with acetone, ethanol and deionized water three times respectively to prepare the BGN precursor. After freeze drying of the precursor, at 620 ^o Calcining for 6 hours under C to remove residual organic matters and obtain BGN with radial morphology;

2) preparing Mo-18BGN by a hydrothermal method: molybdenum acetylacetonate and BGN were completely dispersed in 10 mL of 75% ethanol solution at a mass ratio of 6: 1. Secondly, the solution is mixedAnd carrying out ultrasonic treatment for 30 minutes to ensure that the molybdenum acetylacetonate and the BGN are uniformly mixed. The solution was then transferred directly to a 25 mL autoclave at 160 deg.C ^o And C, keeping the temperature in the oven for 12 hours. And (3) cooling the high-pressure reaction kettle to room temperature, taking out the suspension in the polytetrafluoroethylene lining, centrifuging at a high speed to obtain a precipitate, and freeze-drying the precipitate to obtain the molybdenum-doped bioactive glass ceramic nano material (Mo-6 BGN).

Example 10

1) Synthesizing BGN by an improved sol-gel template method: anhydrous ethanol and deionized water were added to a 250 mL round bottom flask in a 3:1 molar ratio and mixed well, followed by the addition of 10 g of template-catalyst DDA at 40 ^o Stirring in C environment until DDA is fully dissolved, then dripping 4 mL of Tetraethoxysilane (TEOS) into the mixed solution under the condition of continuous stirring, dripping TEP into the reaction system after reacting for 30 minutes, continuing stirring for 15 minutes, adding 1 mL of 5 mol L ^-1 Aqueous solution of calcium nitrate tetrahydrate at 40 ^o C, reacting for 3 hours in an environment; collecting reaction product by centrifugation, washing with anhydrous ethanol and deionized water, freeze drying, and freeze drying at 640 deg.C ^o Calcining the mixture in a muffle furnace of the C furnace for 5 hours to obtain a BGN product;

2) preparing Mo-18BGN by a hydrothermal method: molybdenum acetylacetonate and BGN were completely dispersed in 10 mL of 75% ethanol solution at a mass ratio of 10: 1. Secondly, the solution is treated by ultrasonic for 30 minutes to ensure that the molybdenum acetylacetonate and BGN are uniformly mixed. The solution was then transferred directly to a 25 mL autoclave at 200 ^o And C, keeping the temperature in an oven for 8 hours. And (3) cooling the high-pressure reaction kettle to room temperature, taking out the suspension in the polytetrafluoroethylene lining, centrifuging at a high speed to obtain a precipitate, and freeze-drying the precipitate to obtain the molybdenum-doped bioactive glass ceramic nano material (Mo-10 BGN).

The multifunctional bioactive glass ceramic nano material (Mo-BGN) for tissue regeneration after infection/tumor operation, which is prepared by the invention, has the characteristics of simple preparation process, excellent photo-thermal performance, strong oxidation resistance and good biocompatibility, can effectively kill tumor cells and various bacteria under the action of laser, and can inhibit inflammation and promote vascularization, so the nano material has good application prospect in tissue regeneration after infection/tumor operation.

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

1. The preparation method of the multifunctional bioactive glass ceramic nano material is characterized by comprising the following steps:

mixing bioactive glass nano particles with molybdenum acetylacetonate, and carrying out hydrothermal reaction to obtain a molybdenum-doped bioactive glass ceramic nano material;

the specific method for synthesizing the bioactive glass nano-particles comprises the following steps:

adding a template agent into a solvent, stirring until the template agent is fully dissolved to obtain a mixed solution, adding ethyl orthosilicate, fully reacting, adding an aqueous solution of triethyl phosphate and calcium nitrate tetrahydrate, and fully reacting until the reaction is finished; centrifuging and washing the reaction product, and calcining after freeze drying to obtain bioactive glass nano particles;

the template agent is dodecylamine or bromohexadecylpyridine;

the specific method of the hydrothermal reaction comprises the following steps:

completely dispersing molybdenum acetylacetonate and bioactive glass nano particles in an ethanol solution according to the mass ratio of (3-18) to 1, and performing ultrasonic treatment to uniformly mix the particles; in the range of 160 to 200 ^o C, carrying out hydrothermal reaction to obtain a precipitate, and freeze-drying the precipitate to obtain a molybdenum-doped bioactive glass ceramic nano material;

2. The method for preparing the multifunctional bioactive glass ceramics nano material according to claim 1, wherein the template agent is dodecylamine, and the dodecylamine is added into the mixed solution of the absolute ethyl alcohol and the deionized water, and stirred until the mixed solution is fully dissolved to obtain the mixed solution.

3. The preparation method of the multifunctional bioactive glass-ceramic nanomaterial according to claim 1, wherein the template agent is cetylpyridinium bromide, the cetylpyridinium bromide and urea are completely dissolved in a mixed solution containing cyclohexane and deionized water, isopropyl alcohol is added after the mixture is fully stirred, and the mixed solution is obtained after the mixture is fully dissolved.

4. The method for preparing the multifunctional bioactive glass-ceramic nano material according to claim 1, wherein the chemical composition of the bioactive glass nano particles is 80SiO ₂ -16CaO-4P ₂ O ₅ And 60SiO ₂ -36CaO-4P ₂ O ₅ 。

5. A multifunctional bioactive glass ceramic nano material is characterized by being prepared by the method of any one of claims 1 to 4.

6. The application of the multifunctional bioactive glass ceramics nano material prepared by the method of any one of claims 1 to 4 as a repairing material for tissue regeneration after infection/tumor operation.