Hydroxyapatite composite nano-material, preparation thereof and application of hydroxyapatite composite nano-material in antibacterial and antibacterial bone repair drugs

Abstract

The invention provides a hydroxyapatite composite nanomaterial, preparation thereof and application of the hydroxyapatite composite nanomaterial in antibacterial and antibacterial bone repair drugs. The preparation method of the composite nano material comprises the following steps: fully dissolving calcium nitrate tetrahydrate and polyacrylic acid in deionized water, and adjusting the pH value to 9-10 to obtain a viscous solution; adding a diammonium hydrogen phosphate aqueous solution, and reacting to obtain a suspension; dialyzing the suspension liquid to obtain hydroxyapatite dispersion liquid; and adding a mixed solution of a carboxyl activator, a carboxyl condensing agent, alkali and phthalocyanine, reacting, dialyzing and drying. According to the invention, polyacrylic acid is selected as a polymer medium to help hydroxyapatite crystals to grow and assemble into spherical nanoparticles and prevent aggregation of the spherical nanoparticles, and silicon phthalocyanine is further used for modification to obtain multifunctional nanoparticles, so that the stability and dispersity of the system are improved, and the system has photodynamic therapy and photothermal therapy properties, and has good application prospects. The phototherapy antibacterial bone repair medicine is a novel bone repair medicine with a phototherapy antibacterial effect, can effectively promote osteogenesis and has an excellent antibacterial capability.

Description

Technical Field

[0001] The invention relates to a hydroxyapatite composite nano material and its preparation and application in antibacterial and antibacterial bone repair medicines, belonging to the field of functional materials. Background Art

[0002] Loss of bone tissue due to trauma, infection, tumor resection, and congenital disease can severely damage the skeletal system and pose a long-standing threat to human health. Furthermore, postoperative infection is the greatest risk factor for nonunion of bone defects in clinical practice. Therefore, selecting the appropriate bone repair material for bone grafting is crucial. With the continuous advancement of technology, in vivo studies have demonstrated that bioresorbable bone substitutes can reduce the risk of common complications such as inflammation and osteonecrosis caused by long-term implantation of bioinert materials. However, the implantation of various composite materials during bone defect treatment is highly susceptible to bacterial infection. These bacteria invade the wound through injured soft tissue and rapidly proliferate, leading to symptoms such as osteitis or osteonecrosis. Furthermore, bacterial infection can reduce the osteoinductive capacity of the site, thereby slowing new bone formation. However, current clinical treatment options are limited to continuous, high-dose antibiotics and surgical debridement of damaged tissue. Effective treatments for biofilms and infection have yet to be developed, leaving patients susceptible to secondary bone infection and bone defects. Therefore, if a bone repair material with its own anti-infection ability can be constructed, it can not only effectively destroy bacterial biofilm and eliminate the source of infection, but also restore bone metabolic balance, which will have great significance and application value for society and clinical treatment.

[0003] In recent years, photodynamic therapy and photothermal therapy have gradually begun to be used in fields such as antibacterial therapy. The mechanism of action of photodynamic therapy is to irradiate drugs with light of a specific wavelength. The reactive oxygen species generated bind to the bacterial cell membrane, DNA and other components to inactivate them, thereby achieving an antibacterial effect; the mechanism of action of photothermal therapy is to irradiate the affected area with near-infrared laser. The photothermal agent in the tissue absorbs the light energy and quickly converts it into heat, causing a local thermal effect without damaging normal tissue. The superposition of photodynamic therapy and photothermal therapy will greatly increase the lethality of bacteria and enhance the therapeutic effect. In addition, both photodynamic therapy and photothermal therapy have the advantages of being minimally invasive, reproducible, having few toxic side effects, and having little effect on normal cells and tissues. Therefore, synergistic photodynamic therapy / photothermal therapy drugs have great application potential in antibacterial treatment.

[0004] Hydroxyapatite is a bioactive ceramic material. As the primary inorganic component of human and vertebrate bones and teeth, it chemically bonds with natural bone tissue in vivo. Upon degradation, it releases harmless ions, promoting bone regeneration and rebuilding damaged tissue. Hydroxyapatite typically has a hexagonal crystal structure, offering a large surface area and high chemical inertness, making it suitable for drug adsorption or loading. Hydroxyapatite exhibits excellent biocompatibility and osteoinductivity, making it a common delivery system for drugs, genes, and tissue engineering scaffolds. Consequently, hydroxyapatite is widely used in the preparation of bone tissue scaffolds. However, hydroxyapatite has a strong tendency to aggregate, resulting in poor dispersion in media and even the potential for vascular occlusion, posing potential risks to the human body. Furthermore, most inorganic particles lack active groups for targeted modification or fluorescent labeling, significantly hindering cellular internalization and in vivo tracking, leading to poor therapeutic efficacy.

[0005] Therefore, it is of great significance to develop a bone repair material based on hydroxyapatite with good dispersibility, stability and antibacterial effect. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention provides a hydroxyapatite composite nanomaterial, its preparation, and its application in antibacterial and antimicrobial bone repair drugs. The present invention uses polyacrylic acid as a polymer medium to help hydroxyapatite crystals grow and assemble into spherical nanoparticles while preventing their aggregation. The multifunctional nanoparticles are further modified with silicon phthalocyanine, improving the stability and dispersibility of the system. These nanoparticles also possess both photodynamic therapy and photothermal therapy properties. This new class of bone repair drugs with phototherapeutic and antimicrobial properties effectively promotes osteogenesis and exhibits excellent antibacterial properties.

[0007] The technical solutions of the present invention are as follows: A hydroxyapatite composite nanomaterial, wherein the hydroxyapatite composite nanomaterial is a hydroxyapatite spherical nanoparticle whose surface is sequentially coated with polyacrylic acid and phthalocyanine from the inside out; the phthalocyanine has the following structure: .

[0008] According to the present invention, preferably, the weight average molecular weight of the polyacrylic acid is 1000-3000, preferably 2000.

[0009] According to the present invention, the microstructure of the hydroxyapatite composite nanomaterial is preferably nanoparticle-shaped, with an average particle size of 20 to 300 nm, preferably 100 to 200 nm.

[0010] The preparation method of the hydroxyapatite composite nanomaterial comprises the following steps: (1) Calcium nitrate tetrahydrate and polyacrylic acid are fully dissolved in deionized water, and the pH is adjusted to 9-10 to obtain a viscous solution; an aqueous solution of diammonium hydrogen phosphate is added to react to obtain a suspension; the suspension is dialyzed to obtain a hydroxyapatite dispersion; (2) A mixture of a carboxyl activator, a carboxyl condensing agent, a base and phthalocyanine is added to a hydroxyapatite dispersion, and the mixture is reacted, dialyzed and dried to obtain a hydroxyapatite composite nanomaterial.

[0011] According to the preferred embodiment of the present invention, in step (1), the mass ratio of polyacrylic acid to deionized water is 1:500-1000, preferably 1:960; the mass ratio of calcium nitrate tetrahydrate to deionized water is 1:350-450, preferably 1:400-410, and further preferably 1:407.

[0012] Preferably, according to the present invention, in step (1), calcium nitrate tetrahydrate and polyacrylic acid are added to deionized water and stirred at room temperature for 1 to 3 hours to fully dissolve.

[0013] Preferably, according to the present invention, in step (1), ammonia water with a mass concentration of 25-28% is used to adjust the pH.

[0014] According to the preferred embodiment of the present invention, in step (1), the aqueous solution of diammonium hydrogen phosphate is added dropwise to the system under stirring conditions.

[0015] According to the preferred embodiment of the present invention, in step (1), in the diammonium hydrogen phosphate aqueous solution, the mass ratio of diammonium hydrogen phosphate to deionized water is 1:250-350, preferably 1:303; the molar ratio of calcium nitrate tetrahydrate to diammonium hydrogen phosphate is 1:0.5-0.8, preferably 1:0.6.

[0016] According to the present invention, preferably, in step (1), the reaction temperature is room temperature, the reaction time is 12 to 24 hours, and the reaction is carried out under stirring conditions.

[0017] According to a preferred embodiment of the present invention, in step (1), the dialysis method is as follows: the suspension is placed in a dialysis bag and dialyzed against deionized water at room temperature for 1 to 5 days, and then dialyzed against an organic solvent at room temperature for 1 to 7 days; the molecular weight cut-off of the dialysis bag used for dialysis is 500-2000 Da. Preferably, the organic solvent is N,N-dimethylformamide, methanol, or ethanol, more preferably N,N-dimethylformamide.

[0018] According to the preferred embodiment of the present invention, in step (2), the carboxyl activator is N-hydroxysuccinimide (NHS); the mass ratio of the carboxyl activator to the polyacrylic acid in step (1) is 1 to 5:1, preferably 1 to 2:1, and more preferably 1.23:1.

[0019] According to the preferred embodiment of the present invention, in step (2), the carboxyl condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl); the mass ratio of the carboxyl condensing agent to the polyacrylic acid in step (1) is 3 to 35:1, preferably 4 to 5:1, and more preferably 4.1:1.

[0020] According to the preferred embodiment of the present invention, in step (2), the base is N,N-diisopropylethylamine (DIPEA); the molar ratio of the base to phthalocyanine is 10-120:1, preferably 90-110:1, and more preferably 100:1.

[0021] According to the present invention, preferably, in step (2), the mass ratio of phthalocyanine to the polyacrylic acid in step (1) is 1 to 4:1, preferably 1 to 2:1, and more preferably 1.2:1.

[0022] According to the preferred embodiment of the present invention, in step (2), the solvent used for the mixed solution of carboxyl activator, carboxyl condensing agent, base and phthalocyanine is N,N-dimethylformamide, methanol or ethanol; and the concentration of phthalocyanine in the mixed solution of carboxyl activator, carboxyl condensing agent, base and phthalocyanine is 4-5 g / L.

[0023] According to the present invention, preferably, in step (2), the reaction temperature is room temperature, the reaction time is 12 to 72 hours, preferably 60 to 72 hours, and the reaction is carried out under stirring and inert gas protection. Preferably, the inert gas is nitrogen or argon.

[0024] According to a preferred embodiment of the present invention, in step (2), the dialysis method is as follows: the reaction solution is placed in a dialysis bag, dialyzed in N,N-dimethylformamide at room temperature for 1 to 7 days, and then dialyzed in deionized water at room temperature for 1 to 7 days; preferably, the reaction solution is dialyzed in N,N-dimethylformamide at room temperature for 2 to 5 days, and then dialyzed in deionized water at room temperature for 1 to 3 days; the molecular weight cut-off of the dialysis bag used for dialysis is 500-2000Da.

[0025] According to the preferred embodiment of the present invention, in step (2), the drying is freeze-drying, the freeze-drying temperature is -60 to -50°C, and the freeze-drying time is 1 to 7 days, preferably 2 to 3 days.

[0026] According to the present invention, the preparation method of the phthalocyanine is an existing technology and can be prepared with reference to patent document CN115241379A; the polyacrylic acid can be purchased commercially or prepared according to the existing technology.

[0027] Application of the above hydroxyapatite composite nanomaterial in the preparation of antibacterial drugs and / or antibacterial bone repair drugs.

[0028] Preferably, according to the present invention, the hydroxyapatite composite nanomaterial is used in the preparation of antibacterial drugs or / and antibacterial bone repair drugs for synergistic photodynamic therapy and photothermal therapy.

[0029] The reaction scheme of the present invention is as follows: The structure of Formula I is shown below: The technical features and beneficial effects of the present invention are as follows: The present invention uses polyacrylic acid as a polymer medium to help hydroxyapatite crystals grow and assemble into spherical nanoparticles while preventing aggregation. The nanoparticles are then modified with silicon phthalocyanine to obtain multifunctional nanoparticles. The preparation method is simple, and the raw materials are readily available and inexpensive, making it suitable for industrial production.

[0030] 2. The polyacrylic acid of the present invention contains a large number of carboxyl groups, which can be used as 2+ The polyacrylic acid-modified hydroxyapatite has excess carboxyl groups on its surface, which can form covalent bonds with the axial amino groups of phthalocyanine, thereby improving the uniformity, stability, and dispersibility of the system.

[0031] 3. The present invention provides a novel composite nanomaterial. The phthalocyanine-hydroxyapatite composite nanomaterial, as a novel antibacterial and / or antimicrobial bone repair material for combined photodynamic therapy and photothermal therapy, exhibits excellent stability, dispersibility, biocompatibility, and bioactivity. This overcomes the shortcomings of the inorganic nanomaterial hydroxyapatite, such as its tendency to aggregate, poor dispersibility, and difficulty in long-term metabolism in the body. It reduces damage to healthy human tissue and facilitates renal clearance. Furthermore, because hydroxyapatite closely resembles natural bone composition and exhibits excellent biocompatibility, it can effectively inhibit the inflammatory response surrounding the implant after bone grafting surgery, promoting wound healing and osteogenesis.

[0032] 4. The phthalocyanine-hydroxyapatite composite nanomaterial of the present invention has a good singlet oxygen yield, enabling photodynamic sterilization. It also has an excellent photothermal conversion efficiency (η = 30.85%) in the near-infrared region, enabling photothermal sterilization. The composite material of the present invention can simultaneously perform photodynamic therapy and photothermal therapy, improving the therapeutic effect. It falls into the category of antibacterial bone repair materials that combine near-infrared synergistic photodynamic therapy and photothermal therapy, effectively promoting osteogenesis and possessing excellent antibacterial properties.

[0033] 5. The present invention controls the particle size of hydroxyapatite by regulating the amount of polymer added, the concentration of the precursors (calcium nitrate and diammonium hydrogen phosphate) and the reaction time, thereby achieving controllable preparation of nanoparticles, and preparing nanoparticles with small particle size, regular morphology, uniform particle size and uniform dispersion, which is conducive to further compounding with phthalocyanine to prepare composite nanomaterials with excellent effects; if the above conditions are not suitable, the obtained nanoparticles will have a large particle size and poor dispersibility, and their application effect will be poor. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is an atomic force microscope (AFM) photograph of the phthalocyanine-hydroxyapatite composite nanomaterial prepared in Example 1.

[0035] Figure 2 This is a scanning electron microscope (SEM) photograph of the phthalocyanine-hydroxyapatite composite nanomaterial prepared in Example 1.

[0036] Figure 3 is a scanning electron microscope (SEM) photograph of the hydroxyapatite nanoparticles in Example 1.

[0037] Figure 4 This is a UV-visible absorption spectrum of the phthalocyanine-hydroxyapatite composite nanomaterial prepared in Example 1 in N,N-dimethylformamide solution, where the abscissa is the wavelength and the ordinate is the absorption intensity.

[0038] Figure 5 This is the UV-visible absorption spectrum of the deionized water solution of phthalocyanine-hydroxyapatite composite nanomaterial prepared in Example 1, where the abscissa is the wavelength and the ordinate is the absorption intensity.

[0039] Figure 6 This is a temperature change curve of phthalocyanine-hydroxyapatite composite nanomaterial aqueous solutions of different concentrations prepared in Example 1 under 808 nm laser irradiation, with the abscissa representing time and the ordinate representing temperature.

[0040] Figure 7 This is a temperature change curve of the aqueous solution of phthalocyanine-hydroxyapatite composite nanomaterial prepared in Example 1 under 808nm laser irradiation at different power densities, with the abscissa representing time and the ordinate representing temperature.

[0041] Figure 8 This is the photothermal response curve of the aqueous solution of phthalocyanine-hydroxyapatite composite nanomaterial prepared in Example 1 under 808nm laser irradiation, with the abscissa representing time and the ordinate representing temperature.

[0042] Figure 9This is the singlet oxygen test UV-visible spectrum of the phthalocyanine-hydroxyapatite composite nanomaterial prepared in Example 1 dispersed in N,N-dimethylformamide with 1,3-diphenylisobenzofuran (DPBF) as a quencher; the abscissa is wavelength and the ordinate is absorbance.

[0043] Figure 10 This is the antibacterial effect of the phthalocyanine-hydroxyapatite composite nanomaterial prepared in Example 1 on Staphylococcus aureus.

[0044] Figure 11 This is the antibacterial effect of the phthalocyanine-hydroxyapatite composite nanomaterial prepared in Example 1 on Escherichia coli.

[0045] Figure 12 This is the cell proliferation effect of the phthalocyanine-hydroxyapatite composite nanomaterial prepared in Example 1 on mouse embryonic osteoblasts.

[0046] Figure 13 This is the alkaline phosphatase staining effect of the phthalocyanine-hydroxyapatite composite nanomaterial prepared in Example 1 on mouse embryonic osteoblasts.

[0047] Figure 14 This is the Alizarin Red S staining effect of the phthalocyanine-hydroxyapatite composite nanomaterial prepared in Example 1 on mouse embryonic osteoblasts.

[0048] Figure 15 is a scanning electron microscope (SEM) photograph of the hydroxyapatite prepared in Example 2.

[0049] Figure 16 This is a scanning electron microscope (SEM) photograph of the hydroxyapatite prepared in Example 3.

[0050] Figure 17 This is a scanning electron microscope (SEM) photograph of the hydroxyapatite composite nanomaterial prepared in Example 4. DETAILED DESCRIPTION

[0051] The present invention will be further described below with reference to specific embodiments and drawings, but is not limited thereto.

[0052] The raw materials involved in the present invention are all commercially available analytical reagents.

[0053] The phthalocyanine used in the examples can be prepared with reference to patent document CN115241379A.

[0054] Example 1 Preparation of hydroxyapatite composite nanomaterials Polyacrylic acid (37.5 mg, Mw=2000) and calcium nitrate tetrahydrate (88.5 mg, 0.375 mmol) were added to 36 mL of deionized water and stirred at room temperature for 1 hour. The pH was adjusted to 10 with 25-28% aqueous ammonia to obtain a viscous solution. To this viscous solution, diammonium hydrogen phosphate (29.7 mg, 0.225 mmol) dissolved in 9 mL of deionized water was added dropwise with stirring. The solution was stirred at room temperature for 12 hours to obtain a suspension. The suspension was placed in a dialysis bag (molecular weight cut-off: 1000 Da) and dialyzed against deionized water at room temperature for 24 hours. The hydroxyapatite dispersion was then dialyzed against N,N-dimethylformamide at room temperature for 24 hours to obtain a hydroxyapatite dispersion. To 52 mL of the resulting hydroxyapatite dispersion was added a solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (153.4 mg, 0.8 mmol), N-hydroxysuccinimide (46 mg, 8 mmol), N,N-diisopropylethylamine (1024 μL, 6 mmol), and phthalocyanine (45.0 mg, 0.06 mmol) in 10 mL of N,N-dimethylformamide. The mixture was stirred at room temperature under a nitrogen atmosphere for 3 days. The resulting mixed solution was placed in a dialysis bag (molecular weight cutoff: 1000 Da) and dialyzed against N,N-dimethylformamide at room temperature for 72 h, followed by 48 h of dialysis against deionized water at room temperature. The liquid in the dialysis bag was dried in a vacuum freeze dryer (-60°C for 2 days) to yield 77.5 mg of hydroxyapatite composite nanomaterial.

[0055] The AFM photos of the obtained composite nanomaterials are shown in Figure 2. Figure 1 As shown in the SEM photos Figure 2 As shown in the figure, the particle size of the obtained product is less than 200nm.

[0056] The suspension before adding phthalocyanine was dialyzed in deionized water at room temperature for 48 hours (molecular weight cut-off: 1000Da), and then dried and freeze-dried in a vacuum freeze dryer (-60℃, 2 days) to obtain solid hydroxyapatite particles. The SEM image of the obtained hydroxyapatite nanoparticles is shown in Figure 2. Figure 3 As shown in the figure, the obtained hydroxyapatite nanoparticles are nanosphere particles with uniform particle size; it can be seen that the addition of phthalocyanine not only destroys the agglomeration of hydroxyapatite nanoparticles, but also greatly reduces the size of the particles.

[0057] Test example The hydroxyapatite composite nanomaterial prepared in Example 1 was tested as follows: 1. UV-visible spectrum absorption test The obtained hydroxyapatite composite nanomaterial was dissolved in N,N-dimethylformamide to prepare solutions of different concentrations, and their UV-visible spectral absorption was tested in turn. The results are as follows: Figure 4 As shown, from Figure 4 It can be seen that the hydroxyapatite composite nanomaterial has good solubility in N,N-dimethylformamide and has the characteristic absorption peak of phthalocyanine.

[0058] The obtained hydroxyapatite composite nanomaterials were dissolved in deionized water to prepare solutions of different concentrations. Figure 5 As shown. Figure 5 It can be seen that the hydroxyapatite composite nanomaterial has good solubility in water, has the characteristic absorption peak of phthalocyanine, has absorption in the entire ultraviolet-near infrared band, and has enhanced absorption in the near-infrared region.

[0059] 2. Photothermal response effect test The obtained hydroxyapatite composite nanomaterials were prepared into solutions of different concentrations in neutral deionized water and the 2 The 808nm laser was irradiated for 10 minutes, starting from 0s, and the temperature was recorded every 30s using an infrared thermal imager. The temperature change curve is shown in the figure below. Figure 6 shown.

[0060] The obtained hydroxyapatite composite nanomaterial was prepared into a solution with a concentration of 20 μg / mL in neutral deionized water, and irradiated with an 808 nm laser at different power densities for 10 min. The time was started from 0 s, and the temperature was recorded every 30 s with an infrared thermal imager. The temperature change curve is shown in the figure below. Figure 7 shown.

[0061] from Figure 6 、 Figure 7 It can be seen that the maximum heating temperature of hydroxyapatite composite nanomaterials is positively correlated with the concentration and the power density of the laser, with obvious temperature changes and good photothermal effect.

[0062] 3. Photothermal conversion efficiency test The obtained hydroxyapatite composite nanomaterial was prepared into a solution with a concentration of 20 μg / mL in neutral deionized water and heated with 1.25 W / cm 2 After irradiation with 808nm laser for 16 minutes, the temperature was naturally cooled for 12 minutes. The time was counted from 0 seconds and the temperature was recorded every 30 seconds using an infrared thermal imager. The results are as follows: Figure 8 shown.

[0063] After calculation, the hydroxyapatite composite nanomaterial of Example 1 has excellent photothermal conversion efficiency, and the photothermal conversion efficiency reaches 30.85% under the irradiation of 808 nm laser.

[0064] 4. Singlet oxygen test The obtained hydroxyapatite composite nanomaterial was prepared into a solution with a concentration of 10 μg / mL in N,N-dimethylformamide and the solution was prepared by a 610 nm (0.2 mW / cm 2 ) halogen lamp with cut-off filter for 60s, Figure 9 It can be seen that the hydroxyapatite composite nanomaterial has stable singlet oxygen generation ability and has good photodynamic therapy effect.

[0065] 5. Antibacterial test The obtained hydroxyapatite composite nanomaterials were prepared into solutions of different concentrations in deionized water, mixed with Staphylococcus aureus, and then heated at 3W / cm 2 808nm laser irradiation for 3min as photothermal treatment, or 0.2mW / cm 2 660nm LED light irradiation for 3min as photodynamic treatment, or 3W / cm 2 After irradiation with 808nm laser for 3min, 0.2mW / cm 2 The cells were irradiated with 660nm LED light for 3 minutes as photothermal / photodynamic synergistic treatment, and the bacteria were counted after one day of culture. Figure 10 It can be seen that the hydroxyapatite composite nanomaterial has a good photodynamic and photothermal synergistic antibacterial effect on Staphylococcus aureus.

[0066] The obtained hydroxyapatite composite nanomaterials were prepared into solutions of different concentrations in deionized water, mixed with Escherichia coli, and heated at 3W / cm 2 808nm laser irradiation for 3min as photothermal treatment, or 0.2mW / cm 2 660nm LED light irradiation for 3min as photodynamic treatment, or 3W / cm 2 After irradiation with 808nm laser for 3min, 0.2mW / cm 2 The 660nm LED light was irradiated for 3 minutes as a photothermal / photodynamic synergistic treatment. Bacteria were counted after one day of culture. The results are as follows: Figure 11 As shown. Figure 11 It can be seen that the hydroxyapatite composite nanomaterial has good photodynamic and photothermal synergistic antibacterial effects on Escherichia coli.

[0067] 6. Osteoblast culture experiment The obtained hydroxyapatite composite nanomaterial was sterilized by ultraviolet light in a clean workbench for 30 minutes, and prepared into solutions with concentrations of 0, 5, 10, and 20 μg / mL in physiological saline, and co-cultured with adherent mouse embryonic osteoblasts in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. After removal, the culture medium was aspirated with a pipette, washed three times with PBS solution, and then DMEM osteogenic induction medium (DMEM culture medium with 1% double antibody, 10% FBS and osteogenic induction components) was added and placed in a constant temperature incubator at 37°C and 5% CO2 for culture. Each group with different concentrations was set to a culture time of 1, 3, and 5 days respectively. After removal, the culture medium was aspirated with a pipette, CCK-8 culture medium was added, and incubated in a constant temperature incubator at 37°C and 5% CO2 for 2 hours in the dark. The absorbance at 450 nm was measured with a spectrophotometer. Figure 11 The absorbance changes over time and concentration are shown in the image. In the presence of electron coupling reagent, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt in the CCK-8 kit is reduced by mitochondrial dehydrogenase to form a highly water-soluble orange-yellow formazan product. The color depth is linearly related to the cell number, so the OD value at a wavelength of 450nm can be used to indirectly reflect the number of living cells.

[0068] from Figure 12 It can be seen from the figure that after 1, 3 and 5 days of co-culture of hydroxyapatite composite nanomaterials with cells, the material has no inhibitory effect on cell proliferation. Therefore, it can be considered that hydroxyapatite composite nanomaterials have good biosafety.

[0069] 7. In vitro osteogenesis test (1) MC3T3 cells were seeded into 6-well cell culture plates (5 × 10 4 Cells were grown in a 6-well plate (100 cells / well) until the cell confluency reached 70-80%. The old culture medium was discarded using a pipette and osteogenic induction solution containing HAP-PAA-SiPc was added. The cells were then cultured for an additional 72 hours in a 37°C, 5% CO2 incubator. Four 6-well plates were used for each series of experiments, each containing a concentration range of 0, 5, 10, and 20 μg / mL of the hydroxyapatite composite nanomaterial osteogenic induction solution. Five days later, to evaluate the osteogenic potential of the cells, the culture medium was aspirated, the cells were washed three times with phosphate buffered saline, fixed with fixative for 20 minutes, and stained with the Beyotime BCIP / NBT alkaline phosphatase colorimetric kit for 30 minutes. Finally, the cells were washed several times with phosphate buffered saline to remove excess dye. The cells were photographed and observed.

[0070] Alkaline phosphatase is an exoenzyme of osteoblasts and one of the key biological indicators of early osteoblast differentiation. Under the catalysis of alkaline phosphatase, the alkaline phosphatase staining kit can form an insoluble blue-purple precipitate. Figure 13 It can be seen that the staining of the group containing hydroxyapatite composite nanomaterials is more obvious than that of the blank control group. With the increase of the concentration of hydroxyapatite composite nanomaterials, the intensity of blue-purple color increases. The hydroxyapatite composite nanomaterials with a concentration of 20 μg / mL have the darkest staining, indicating that the activity of alkaline phosphatase in the cells is higher, indicating that hydroxyapatite composite nanomaterials have a promoting effect on the differentiation of osteoblasts.

[0071] (2) MC3T3 cells were seeded into 6-well cell culture plates (5×10 4 Cells were plated in a 6-well plate (100 μg / mL) and the cells were plated. When the cell confluency reached 70-80%, the old culture medium was discarded using a pipette and osteogenic induction solution containing HAP-PAA-SiPc was added. The cells were then cultured in a 37°C, 5% CO2 incubator for an additional 72 hours. Four 6-well plates were used for each series of experiments, each containing a series of concentrations of 0, 5, 10, and 20 μg / mL of the hydroxyapatite composite nanomaterial osteogenic induction solution. Five days later, to evaluate the osteogenic potential of the cells, the solution was discarded from the 6-well plates using a pipette. The plates were gently washed three times with phosphate buffered saline (PBS). The cells were fixed with fixative for 20 minutes and stained with the Beyotime Alizarin Red colorimetric kit for 30 minutes. Finally, the plates were washed several times with phosphate buffered saline to remove excess dye. The staining was then observed and recorded.

[0072] Alizarin red is an anthraquinone derivative that can chelate with calcium salts to form an orange-red complex. Figure 14 As can be seen, as the concentration of the hydroxyapatite composite nanomaterial in the osteogenic induction solution increases, the results of Alizarin Red staining become redder. In the group with a 20 μg / mL concentration of the hydroxyapatite composite nanomaterial in the osteogenic induction solution, more obvious mineralized nodules were observed, with deep red staining and localized aggregation. These results indicate that the hydroxyapatite composite nanomaterial's promoting effect on calcium nodule formation in MC3T3 cells increases with increasing concentration.

[0073] Example 2 Preparation of hydroxyapatite nanoparticles Polyacrylic acid (37.5 mg, Mw=2000) and calcium nitrate tetrahydrate (88.5 mg, 0.375 mmol) were added to 36 mL of deionized water and stirred at room temperature for 1 hour. The pH was adjusted to 10 with 25-28% aqueous ammonia to obtain a viscous solution. To this viscous solution, diammonium hydrogen phosphate (29.7 mg, 0.225 mmol) dissolved in 9 mL of deionized water was added dropwise with stirring. The mixture was stirred at 110°C for 12 hours to obtain a suspension. The suspension was placed in a dialysis bag (molecular weight cut-off: 1000 Da) and dialyzed against deionized water at room temperature for 48 hours. The liquid in the bag was then dried in a vacuum freeze dryer (-60°C for 2 days) to obtain hydroxyapatite particles.

[0074] The SEM images of the obtained hydroxyapatite nanoparticles are shown in Figure 2. Figure 15 As shown in the figure, hydroxyapatite is in the shape of rods, which are too large and have poor dispersion, making them unsuitable for application.

[0075] Example 3 Preparation of hydroxyapatite nanoparticles Polyacrylic acid (25 mg, Mw=2000) and calcium nitrate tetrahydrate (88.5 mg, 0.375 mmol) were added to 36 mL of deionized water and stirred at room temperature for 1 hour. The pH was adjusted to 10 with 25-28% aqueous ammonia to obtain a viscous solution. To this viscous solution, diammonium hydrogen phosphate (29.7 mg, 0.225 mmol) dissolved in 9 mL of deionized water was added dropwise with stirring. The solution was stirred at room temperature for 12 hours to obtain a suspension. The suspension was placed in a dialysis bag (molecular weight cut-off: 1000 Da) and dialyzed against deionized water at room temperature for 48 hours. The liquid in the dialysis bag was dried in a vacuum freeze dryer (-60°C for 2 days) to obtain hydroxyapatite particles.

[0076] The SEM images of the obtained hydroxyapatite nanoparticles are shown in Figure 2. Figure 16 As shown in the figure, due to the small amount of polyacrylic acid, the hydroxyapatite is partially aggregated and becomes spherical in different sizes. The size is too large to be used.

[0077] Example 4 Preparation of hydroxyapatite composite nanomaterials Polyacrylic acid (25 mg, Mw=2000) and calcium nitrate tetrahydrate (88.5 mg, 0.375 mmol) were added to 36 mL of deionized water and stirred at room temperature for 1 hour. The pH was adjusted to 10 with 25-28% ammonia water to obtain a viscous solution. To this viscous solution, diammonium hydrogen phosphate (29.7 mg, 0.225 mmol) dissolved in 9 mL of deionized water was added dropwise with stirring. The solution was stirred at room temperature for 12 hours to obtain a suspension. The suspension was placed in a dialysis bag (molecular weight cut-off: 1000 Da) and dialyzed against deionized water at room temperature for 24 hours. The hydroxyapatite dispersion was then dialyzed against N,N-dimethylformamide at room temperature for 24 hours to obtain a dispersion. To 52 mL of the resulting hydroxyapatite dispersion was added a solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (153.4 mg, 0.8 mmol), N-hydroxysuccinimide (46 mg, 8 mmol), N,N-diisopropylethylamine (1024 μL, 6 mmol), and phthalocyanine (45.0 mg, 0.06 mmol) in 10 mL of N,N-dimethylformamide. The mixture was stirred at room temperature under a nitrogen atmosphere for 3 days. The resulting mixed solution was placed in a dialysis bag (molecular weight cutoff: 1000 Da) and dialyzed against N,N-dimethylformamide at room temperature for 72 h, followed by 48 h of dialysis against deionized water at room temperature. The liquid in the dialysis bag was dried in a vacuum freeze dryer (-60°C for 2 days) to yield 69.4 mg of hydroxyapatite composite nanomaterial.

[0078] The SEM photos of the obtained composite nanomaterials are shown in Figure 17 As shown in the figure, due to the small amount of PAA, hydroxyapatite and phthalocyanine are connected through electrostatic adsorption, resulting in flake phthalocyanine, which is too large and has poor dispersion and cannot be used.

Claims

1. A hydroxyapatite composite nanomaterial, characterized in that: The hydroxyapatite composite nanomaterial is a hydroxyapatite spherical nanoparticle whose surface is sequentially coated with polyacrylic acid and phthalocyanine from the inside out; the phthalocyanine has the following structure: 。 2. The hydroxyapatite composite nanomaterial according to claim 1, characterized in that: The weight average molecular weight of the polyacrylic acid is 1000-3000, preferably 2000; the microscopic morphology of the hydroxyapatite composite nanomaterial is nanoparticle-shaped, and the average particle size is 20-300 nm, preferably 100-200 nm.

3. The method for preparing the hydroxyapatite composite nanomaterial according to claim 1 or 2, comprising the steps of: (1) Calcium nitrate tetrahydrate and polyacrylic acid are fully dissolved in deionized water, and the pH is adjusted to 9-10 to obtain a viscous solution; an aqueous solution of diammonium hydrogen phosphate is added to react to obtain a suspension; the suspension is dialyzed to obtain a hydroxyapatite dispersion; (2) A mixture of a carboxyl activator, a carboxyl condensing agent, a base and phthalocyanine is added to a hydroxyapatite dispersion, and the mixture is reacted, dialyzed and dried to obtain a hydroxyapatite composite nanomaterial.

4. The method for preparing the hydroxyapatite composite nanomaterial according to claim 3, characterized in that: In step (1), one or more of the following conditions are included: i. The mass ratio of polyacrylic acid to deionized water is 1:500-1000, preferably 1:960; the mass ratio of calcium nitrate tetrahydrate to deionized water is 1:350-450, preferably 1:400-410, and more preferably 1:407; ii. Add calcium nitrate tetrahydrate and polyacrylic acid to deionized water and stir at room temperature for 1-3 hours to fully dissolve; iii. Use 25-28% ammonia water to adjust the pH; iv. The aqueous solution of diammonium hydrogen phosphate is added dropwise into the system under stirring; v. In the diammonium hydrogen phosphate aqueous solution, the mass ratio of diammonium hydrogen phosphate to deionized water is 1:250-350, preferably 1:303; the molar ratio of calcium nitrate tetrahydrate to diammonium hydrogen phosphate is 1:0.5-0.8, preferably 1:0.

6.

5. The method for preparing the hydroxyapatite composite nanomaterial according to claim 3, characterized in that: In step (1), one or more of the following conditions are included: i. The reaction temperature is room temperature, the reaction time is 12 to 24 hours, and the reaction is carried out under stirring conditions; ii. The dialysis method is as follows: the suspension is placed in a dialysis bag and dialyzed in deionized water at room temperature for 1 to 5 days, and then in an organic solvent at room temperature for 1 to 7 days; the dialysis bag used for dialysis has a molecular weight cutoff of 500-2000 Da; preferably, the organic solvent is N,N-dimethylformamide, methanol or ethanol, more preferably N,N-dimethylformamide.

6. The method for preparing the hydroxyapatite composite nanomaterial according to claim 3, characterized in that: In step (2), one or more of the following conditions are included: i. The carboxyl activator is N-hydroxysuccinimide (NHS); the mass ratio of the carboxyl activator to the polyacrylic acid in step (1) is 1 to 5:1, preferably 1 to 2:1, and more preferably 1.23:1; ii. The carboxyl condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl); the mass ratio of the carboxyl condensing agent to the polyacrylic acid in step (1) is 3 to 35:1, preferably 4 to 5:1, and more preferably 4.1:1; iii. The base is N,N-diisopropylethylamine (DIPEA); the molar ratio of the base to phthalocyanine is 10 to 120:1, preferably 90 to 110:1, and more preferably 100:1; iv. The mass ratio of phthalocyanine to the polyacrylic acid in step (1) is 1 to 4:1, preferably 1 to 2:1, and more preferably 1.2:1; v. The solvent used for the mixed solution of carboxyl activator, carboxyl condensing agent, base and phthalocyanine is N,N-dimethylformamide, methanol or ethanol; the concentration of phthalocyanine in the mixed solution of carboxyl activator, carboxyl condensing agent, base and phthalocyanine is 4-5g / L.

7. The method for preparing the hydroxyapatite composite nanomaterial according to claim 3, characterized in that: In step (2), the reaction temperature is room temperature, the reaction time is 12 to 72 hours, preferably 60 to 72 hours, and the reaction is carried out under stirring conditions and under the protection of an inert gas; preferably, the inert gas is nitrogen or argon.

8. The method for preparing the hydroxyapatite composite nanomaterial according to claim 3, characterized in that: In step (2), one or more of the following conditions are included: i. The dialysis method is as follows: the reaction solution is placed in a dialysis bag and dialyzed in N,N-dimethylformamide at room temperature for 1-7 days, and then dialyzed in deionized water at room temperature for 1-7 days; preferably, the reaction solution is dialyzed in N,N-dimethylformamide at room temperature for 2-5 days, and then dialyzed in deionized water at room temperature for 1-3 days; the molecular weight cut-off of the dialysis bag used for dialysis is 500-2000 Da; ii. Drying is freeze drying, the freeze drying temperature is -60 to -50°C, and the freeze drying time is 1 to 7 days, preferably 2 to 3 days.

9. Use of the hydroxyapatite composite nanomaterial according to claim 1 or 2 in the preparation of antibacterial drugs and / or antibacterial bone repair drugs.

10. The use according to claim 9, characterized in that The hydroxyapatite composite nanomaterial is used in the preparation of antibacterial drugs or / and antibacterial bone repair drugs for synergistic photodynamic therapy and photothermal therapy.

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