Method for preparing hollow mesoporous hydroxyapatite loaded with nanosilver and sodium carboxymethylcellulose hydroxyapatite powder and hollow mesoporous hydroxyapatite powder loaded with nanosilver prepared by the method

Abstract

The application discloses a preparation method of hollow mesoporous hydroxyapatite microspheres loaded with nano-silver, and steps are as follows: carboxymethyl cellulose sodium calcium carbonate is synthesized, the micro-morphology of the calcium carbonate before and after loading is changed from rhombus to spherical shape, and then the hollow mesoporous hydroxyapatite microspheres are synthesized through a hydrothermal synthesis method. Polyethylene imine is covalently modified to the hollow mesoporous hydroxyapatite microspheres, at this time, the microspheres are positively charged due to exposed amino groups, and then the hollow mesoporous hydroxyapatite microspheres are electrostatically adsorbed with negatively charged nano-silver coated with sodium polystyrene sulfonate, so as to finally prepare the hollow mesoporous hydroxyapatite microspheres loaded with nano-silver. The preparation method has the advantages of simple process, low cost and wide application range. The prepared hollow mesoporous hydroxyapatite microspheres loaded with nano-silver have good antibacterial effect, good durability and repeatability. The synthesized product has high biological safety and certain antiviral effect. The hollow mesoporous hydroxyapatite microspheres loaded with nano-silver are suitable for fields such as antifouling coatings, and can greatly promote the development of the material field and the antibacterial field.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibacterial materials, in particular to a preparation method of hollow mesoporous hydroxyapatite loaded with nano-silver, and a sodium carboxymethyl cellulose hydroxyapatite powder, a hollow mesoporous hydroxyapatite powder loaded with nano-silver and applications prepared by the method. BACKGROUND

[0002] Hydroxyapatite (HAP) is the main inorganic component of human bone and teeth. Due to its excellent biocompatibility, bone conduction and bioactivity, it is widely used in bone repair, drug carriers, tissue engineering and environmental governance. With the cross development of material science and biomedicine, the morphology control (such as microspheres, nanoparticles, etc.) and functionalization of HAP have become a research hotspot. Hollow mesoporous hydroxyapatite microspheres have attracted much attention due to their large specific surface area and hollow structure, which have large drug loading capacity and strong adsorption performance. For example, the invention patent application No. 201510351925.5 discloses a synthesis method of hollow hydroxyapatite. However, the surface of the product is a thorn-like structure, and its specific surface area is smaller than that of the flower-like structure in this paper, and its loading capacity is lower. The invention patent No. 202411603637X also discloses a malic acid-regulated mesoporous hydroxyapatite microsphere, which is characterized by: by controlling the concentration of morphology regulator malic acid, adjusting the reaction time and temperature, adjusting the sphericity, particle size, surface micro-nano structure and crystallinity of the hydroxyapatite microspheres. The regulation is more delicate and complex, and the workload is large, which is difficult to grasp.

[0003] Various surfaces in our daily life are vulnerable to attack from microorganisms (bacteria, viruses, etc.) in the air and solution, which may endanger human health. There are potential harmful bacteria everywhere around us, for example: speaking, coughing, sneezing, and even breathing can produce water vapor droplets containing bacteria. Similarly, bacteria present in liquids (for example, drinking water and hospital catheter liquids) can attach to the inner surface of blood vessels and pipes and grow and reproduce. When the number of cells increases on the surface, microbial cells usually begin to form biofilms, which are composed of polysaccharide matrix embedded cells. This biofilm allows microbial cells to survive in harsh conditions, and the embedded cells have a maximum 1000-fold reduction in sensitivity to most antibiotics and other biocides, which can become a source of persistent infections.

[0004] Traditional antibacterial materials include natural antibacterial materials, organic antibacterial materials and inorganic antibacterial materials. Natural antibacterial materials are widely available, low in price, and have good biocompatibility and safety, but their antibacterial activity is relatively low, and their water solubility is poor, which limits their use in some application scenarios that require rapid dissolution and uniform dispersion. Organic antibacterial materials have a wide antibacterial spectrum and good antibacterial effect, but long-term use can easily make bacteria resistant, and may also cause some irritation to the human body during use. Inorganic antibacterial materials represented by metal oxides have high chemical stability and antibacterial durability. However, the antibacterial performance of these materials is largely dependent on their crystal structure and particle size, which is difficult to control accurately during preparation, resulting in unstable antibacterial effect. In addition, some inorganic antibacterial materials may pose potential hazards to the human body and the environment during use. In addition, the silver ions in traditional silver-based antibacterial materials are also unstable and can easily be converted into silver oxide, making it difficult to maintain a high level of antibacterial effect for a long time. Therefore, the synthesis of nano-silver for antibacterial purposes has the advantages of strong antibacterial ability, wide antibacterial spectrum, long-lasting antibacterial effect, low risk of drug resistance, strong penetration, low toxicity, and environmental friendliness.

[0005] Hydroxyapatite is selected as the carrier, and antibacterial silver is loaded therein. For example, a patent published in 2020 provides a preparation method of a flaky hydroxyapatite carrier and a supported nano-silver catalyst, characterized in that: first, flaky hydroxyapatite is synthesized, then it is soaked in a silver salt solution, and finally it is reduced in a hydrogen atmosphere to obtain the finished product. The rich hydroxyl groups on the surface of hydroxyapatite allow it to be modified by other functional groups. However, the synthesis is relatively complex and requires many processes. Secondly, silver nitrate is used as a good silver salt solution, but the silver adsorbed by the immersion method is released quickly and the effect is not long-lasting. For example, the invention patent with application number CN201410619102.1 also discloses a preparation and application of an immune sensor of silver ion-substituted hydroxyapatite, characterized in that: a certain proportion of silver nitrate solution and hydroxyapatite are mixed and stirred constantly to allow ion exchange reaction, and finally the finished product is obtained by centrifugal washing. The exchanged silver ions are limited, and can be completely released in a short period of time. If used in the field of antibacterial materials, the antibacterial time is short and the effect is not long-lasting. SUMMARY

[0006] In view of the deficiencies in the background art, the technical problem to be solved by the present application is to provide a preparation method of hollow mesoporous hydroxyapatite loaded with nano-silver. The synthesis process of the method is simple, easy to operate and low in cost, and the finished product has better antibacterial performance. The nano-silver can slowly and continuously release silver ions, which can prolong the antibacterial effect of nano-silver and improve the durability of the antibacterial material.

[0007] The application discloses a preparation method of hollow mesoporous hydroxyapatite microspheres loaded with nano-silver.

[0008] Preferably, the preparation of the hollow mesoporous hydroxyapatite microspheres comprises the following steps:

[0009] S1: anhydrous calcium chloride and sodium carboxymethyl cellulose are weighed and dispersed into distilled water, and are uniformly ultrasonically dispersed; the two solutions are mixed and magnetically stirred for 8-12 min;

[0010] S2: anhydrous sodium carbonate is weighed and dispersed into distilled water, and after being uniformly dispersed, is rapidly added into the above mixed solution, and stirring is continuously performed for 24-36 min;

[0011] S3: after the reaction is completed, the precipitated matter generated in the reaction is centrifugally washed with deionized water for 2-3 times, and is washed with anhydrous ethanol for 2-3 times;

[0012] S4: the precipitated matter is placed in an oven and is dried at a temperature of 50-70 DEG C, so that the prepared sodium carboxymethyl cellulose calcium carbonate microspheres are obtained, and are recorded as CMC-CaCO3;

[0013] S5: dodecahydrate disodium hydrogen phosphate is weighed and dissolved in distilled water, the prepared sodium carboxymethyl cellulose calcium carbonate microspheres are added into the solution, and after being uniformly ultrasonically dispersed, the liquid is transferred into a polytetrafluoroethylene reaction kettle, and is hydrothermally reacted at a temperature of 110-170 DEG C for 2.4-4 h;

[0014] S6: after the polytetrafluoroethylene reaction kettle is cooled, the precipitated matter generated in the reaction is centrifugally washed with deionized water for 2-3 times, and is washed with anhydrous ethanol for 2-3 times;

[0015] S7: the precipitated matter is placed in an oven and is dried at a temperature of 50-72 DEG C, so that the prepared hollow mesoporous hydroxyapatite microspheres are obtained, and are recorded as CMC-HAP.

[0016] Preferably, the addition amount of calcium chloride powder in 10 mL of the calcium chloride solution is 1.11 g, the addition amount of carboxymethyl cellulose powder in 10 mL of the carboxymethyl cellulose solution is 60 mg, the addition amount of sodium carbonate powder in 20 mL of the sodium carbonate solution is 1.06 g, when the sodium carbonate solution is added into the mixed solution, the solution is rapidly changed into a milky white colloidal liquid, and then 0.6 g of dried calcium carbonate powder is added into 15 mL of deionized water containing 5 g of dodecahydrate disodium hydrogen phosphate, and after being uniformly stirred, is transferred into a polytetrafluoroethylene reaction kettle.

[0017] Preferably, the method for preparing polyethyleneimine hydroxyapatite comprises the following steps:

[0018] S1: Weigh sodium carboxymethylcellulose hydroxyapatite microsphere powder and ultrasonically disperse it in a centrifuge tube filled with methanol solvent;

[0019] S2: Weigh EDC, disperse it in an appropriate amount of methanol, add a small amount of triethylamine, mix well, transfer it to the above solution, and react on a shaker for 24-36 minutes;

[0020] S3: Add methanol containing polyethyleneimine and continue the reaction on a shaker for a total of 12 hours;

[0021] S4: After the reaction is completed, the product is thoroughly washed with distilled water and anhydrous ethanol and centrifuged. The supernatant is discarded and then placed in a vacuum drying oven for drying. The resulting reaction product is polyethyleneimine hydroxyapatite, which is recorded as CMC-HAP-PEI.

[0022] Preferably, 10 mL of methanol is added to every 500 mg of sodium carboxymethyl cellulose hollow mesoporous hydroxyapatite powder, the ratio of EDC to methanol and triethylamine is 1g:5ml:2.3ml, and 5 ml of methanol is added to every 250 mg of polyethyleneimine. After the reaction is completed, the product is washed with distilled water and centrifuged 2 to 3 times, and the product is washed with anhydrous ethanol and centrifuged 2 to 3 times.

[0023] The preferred method for preparing the sodium polystyrene sulfonate-coated nanosilver comprises the following steps:

[0024] S1: Disperse silver nitrate in distilled water to prepare a silver nitrate solution, and transfer it to a light-proof conical flask;

[0025] S2: Place the silver nitrate solution on a magnetic stirrer and stir, add the sodium polystyrene sulfonate solution dropwise, and stir the prepared solution for 30 minutes;

[0026] S3: Weigh sodium borohydride and dissolve it in distilled water. Add it dropwise into the reaction system at a rate of 1 drop per 10 seconds and continue stirring for 2 hours.

[0027] S4: After the reaction is completed, stirring is turned off and the product can be stored away from light for several months and is recorded as Ag-PSS.

[0028] Preferably, the amount of silver nitrate solution added per 46 mL is 16.9 mg, the ratio of sodium polystyrene sulfonate to distilled water is 30 μl:2 mL, and the ratio of sodium borohydride to distilled water is 3.78 mg:2 mL.

[0029] Preferably, the method for preparing the hollow mesoporous hydroxyapatite loaded with nanosilver comprises the following steps:

[0030] S1: the polyethylene imine hydroxyapatite powder is weighed, and is ultrasonically dispersed in a centrifuge tube containing a nano-silver solution;

[0031] S2: the centrifuge tube is shaken on a shaking table for 6 hours in dark;

[0032] S3: after the reaction is completed, the centrifuge tube is washed several times by using distilled water, until the supernatant is colorless and transparent, the supernatant is discarded, and the obtained reaction product is dried in a vacuum drying oven, and the obtained reaction product is a hollow mesoporous hydroxyapatite loaded with nano-silver, which is denoted as CMC-HAP-PEI@Ag.

[0033] The application further provides a carboxymethyl cellulose sodium hydroxyapatite powder, which is prepared by using the preparation method.

[0034] The application further provides a hollow mesoporous hydroxyapatite powder loaded with nano-silver, which is prepared by using the preparation method.

[0035] The application has the following technical effects: first, calcium carbonate microspheres are synthesized, then the hollow mesoporous hydroxyapatite microspheres are generated by using the calcium carbonate microspheres as a template through a hydrothermal reaction, and the product has a negative charge due to the participation of carboxymethyl cellulose sodium. Then, polyethylene imine (PEI) is allowed to modify the hollow mesoporous hydroxyapatite microspheres in an organic solution, so that the product has a positive charge due to the exposed amino groups. Then, the nano-silver coated with sodium polystyrene sulfonate and having a negative charge is synthesized, and according to the principle of layer-by-layer assembly, the nano-silver is loaded into the hollow mesoporous hydroxyapatite, so that the nano-silver is uniformly distributed, the product has antibacterial and antiviral effects, the carrier has a hollow structure and a mesoporous surface, has low density, large specific surface area, high loading capacity and slow release, and has good biocompatibility, and is a good carrier for loading nano-silver.

[0036] The nano-silver mainly relies on the released silver ions to produce the sterilization activity, but when the bacteria directly contact the surface of the microspheres, the loaded nano-silver can play a role by destroying the bacterial cell membrane through physical action or producing reactive oxygen species (ROS). The high specific surface area of the mesoporous structure also increases the contact probability of bacteria and the surface of the material. The material can be used to create a sterilization surface, and the particles with contact and release sterilization activity can be uniformly dispersed in polar / non-polar solvents, so as to be compatible with polar / non-polar film-forming matrices (such as TPU, PP, etc.), and the solid surface formed after blending can effectively kill bacteria by using the contact, release or both mechanisms, prevent the bacteria from being adsorbed on the surface, and prevent the formation of biofilm which can easily lead to infection events. It has been proved that the sterilization surface can kill most bacteria deposited on it by gas and attached from an aqueous solution, including gram-positive bacteria, gram-negative bacteria, fungi, etc. Specifically, the antibacterial performance is evaluated by spraying a bacterial cell aqueous suspension on the surface, then air-drying and counting the number of cells remaining alive (i.e. capable of growing colonies), and the obtained surface can kill up to 99% of gram-positive and gram-negative bacteria deposited in aerosol or aqueous suspension.

[0037] The application also provides application of the hollow mesoporous hydroxyapatite loaded with nano-silver in preparation of an antibacterial material or an antibacterial device or a composite antibacterial film of an organic polymer. The antibacterial material can serve as an antibacterial coating and be applied to paint, a mask and a gas mask; can be combined with an organic polymer material to prepare an antibacterial composite material; can be combined with resin and be applied to paint; can be combined with plastic and a polymer resin and be applied to a filter screen, a shell of an electrical appliance, food material and transmission, a reverse osmosis membrane, a microfiltration membrane, an ultrafiltration membrane and a nanofiltration membrane; can be combined with silicone rubber and latex and be applied to an interventional catheter (such as a cardiovascular interventional catheter and a urinary catheter); can be combined with rubber and fiber and be applied to clothing and a mask; and can be used to coat the surface of common objects (such as a door handle, a children's toy, a computer keyboard and a telephone) contacted by people in daily life and can be used in the fields of medical consumables (such as a urinary catheter, an esophageal tube and a tracheal tube), textiles, paint and packaging materials, so that they have antibacterial properties and cannot spread bacterial infection. The material has a very wide application field. BRIEF DESCRIPTION OF DRAWINGS

[0038] The application has the following drawings:

[0039] Figure 1 The application relates to a hollow mesoporous hydroxyapatite microsphere and a synthesis route of the hollow mesoporous hydroxyapatite microsphere loaded with nano-silver;

[0040] Figure 2 The drawings are scanning electron microscope images of CaCO3 (a), CMC-CaCO3 (b) and CMC-HAP (c);

[0041] Figure 3 Fourier transform infrared spectrograms of CaCO3 (a), CMC-CaCO3 (b), HAP (c), CMC-HAP (d), CMC (e);

[0042] Figure 4 X-ray diffraction patterns of CaCO3 (a), CMC-CaCO3 (b), CMC-HAP (c);

[0043] Figure 5 Transmission electron micrograph of CMC-HAP;

[0044] Figure 6 N2 adsorption-desorption curve, specific surface area and pore size distribution diagram of CMC-HAP;

[0045] Figure 7 Zeta diagram of CMC-HAP-PEI, Ag-PSS, CMC-HAP-PEI@Ag;

[0046] Figure 8 X-ray diffraction patterns of CMC-HAP (a), CMC-HAP-PEI@Ag (b);

[0047] Figure 9 Elemental mapping diagram of CMC-HAP-PEI@Ag;

[0048] Figure 10 Cell survival rate diagram of CMC-HAP-PEI@Ag at different concentrations;

[0049] Figure 11 Bacterial survival rate of S. aureus, E. coli, C. albicans, MRSA treated with PBS phosphate buffer, different concentrations of CMC-HAP-PEI and CMC-HAP-PEI@Ag;

[0050] Figure 12 PBS phosphate buffer (a), growth of colonies of CMC-HAP-PEI at 50 μg / ml (b); growth of various colonies of CMC-HAP-PEI@Ag at 1 μg / ml (c), 5 μg / ml (d), 10 μg / ml (e), 30 μg / ml (f), 50 μg / ml (g);

[0051] Figure 13The virus titers of PRV viruses in DMEM medium, CMC-HAP-PEI at 400 μg / ml and 500 μg / ml, and CMC-HAP-PEI@Ag at 400 μg / ml and 500 μg / ml. DETAILED DESCRIPTION

[0052] In order to further clarify the technical means and effects adopted by the present application to achieve the predetermined inventive purposes, the specific embodiments, structures, features and effects according to the present application are described in detail below in combination with the drawings and preferred embodiments.

[0053] The preparation method of the hollow mesoporous hydroxyapatite microspheres loaded with nano-silver provided by the present application comprises the following steps: synthesizing the hollow mesoporous hydroxyapatite microspheres by a hydrothermal synthesis method, covalently modifying polyethyleneimine (PEI) to the hollow mesoporous hydroxyapatite microspheres to form polyethyleneimine hydroxyapatite, and then electrostatically adsorbing nano-silver with negative charge.

[0054] The preparation of the hollow mesoporous hydroxyapatite microspheres in the above method comprises the following steps:

[0055] S1: weigh anhydrous calcium chloride and sodium carboxymethyl cellulose, respectively disperse them in distilled water, and uniformly ultrasonically disperse them; mix the two solutions, and magnetically stir for 8-12 min, and the optimal magnetic stirring time is 10 min;

[0056] S2: weigh anhydrous sodium carbonate, disperse it in distilled water, quickly add it to the above mixed solution after uniform dispersion, and continue to stir for 24-36 min, and the optimal stirring time is 30 min;

[0057] S3: after the reaction is completed, centrifugally wash the generated precipitate with deionized water for 2-3 times, and then wash it with anhydrous ethanol for 2-3 times;

[0058] S4: place the precipitate in an oven, dry it at a temperature of 50-70°C (and the optimal temperature is 60°C), and thus the prepared sodium carboxymethyl cellulose calcium carbonate microspheres are obtained, which are denoted as CMC-CaCO3;

[0059] S5: weigh dodecahydrate sodium phosphate dibasic, dissolve it in distilled water, add the above generated sodium carboxymethyl cellulose calcium carbonate microspheres (CMC-CaCO3) to the solution, uniformly ultrasonically disperse them, and then transfer the liquid to a polytetrafluoroethylene reaction kettle, and hydrothermally react at a temperature of 110-170°C (and the optimal temperature is 140°C) for 2.4-4 h, and the optimal reaction time is 3 h;

[0060] S6: after the polytetrafluoroethylene reaction kettle is cooled, centrifugally wash the generated precipitate with deionized water for 2-3 times, and then wash it with anhydrous ethanol for 2-3 times;

[0061] S7: The precipitated material is placed in an oven and dried at a temperature of 50-72°C (optimum temperature 60°C), thus obtaining the prepared hollow mesoporous hydroxyapatite microspheres (i.e. sodium carboxymethyl cellulose hydroxyapatite), denoted as CMC-HAP.

[0062] In the above steps, the amount of calcium chloride powder added is 1.11 g per 10 mL of calcium chloride solution, and the amount of carboxymethyl cellulose powder added is 60 mg per 10 mL of carboxymethyl cellulose solution; the amount of sodium carbonate powder added is 1.06 g per 20 mL of sodium carbonate solution; when the sodium carbonate solution is added to the mixed solution, the solution quickly turns into a milky white gelatinous liquid; in the next step, 0.6 g of dry calcium carbonate powder is added to 15 mL of deionized water containing 5 g of dodecahydrate sodium hydrogen phosphate, and after stirring until uniform, it is transferred to a polytetrafluoroethylene reaction kettle.

[0063] The above method for preparing polyethyleneimine hydroxyapatite includes the following steps:

[0064] S1: Weigh the sodium carboxymethyl cellulose hydroxyapatite microspheres powder (CMC-HAP) and ultrasonically disperse it in a centrifuge tube containing methanol solvent;

[0065] S2: Weigh the EDC and disperse it in an appropriate amount of methanol, add a small amount of triethylamine, mix well, and transfer it to the above solution, and react on a shaking table for 24-36 min (optimum reaction time 30 min);

[0066] S3: Add methanol containing polyethyleneimine (PEI) and continue to react on the shaking table for a total of 12 h;

[0067] S4: After the reaction is complete, the product is washed and centrifuged with distilled water and anhydrous ethanol, the supernatant is discarded, and it is placed in a vacuum drying oven for drying, the drying temperature can be 60°C, and the resulting reaction product is polyethyleneimine hydroxyapatite, denoted as CMC-HAP-PEI.

[0068] In the above steps, 10 mL of methanol is added to 500 mg of sodium carboxymethyl cellulose hollow mesoporous hydroxyapatite powder (CMC-HAP), the ratio of EDC to methanol to triethylamine is 1 g: 5 mL: 2.3 mL, 5 mL of methanol is added to 250 mg of polyethyleneimine (PEI), and after the reaction is complete, the product is washed and centrifuged with distilled water 2-3 times and with anhydrous ethanol 2-3 times.

[0069] The above method for preparing polyethyleneimine hydroxyapatite includes the following steps:

[0070] S1: Disperse silver nitrate in distilled water to form a silver nitrate solution and transfer it to a light-resistant conical flask;

[0071] S2: The silver nitrate solution is placed on a magnetic stirrer and stirred at a stirring speed of 300 rpm / min -1） , and the prepared solution is stirred for 30 min.

[0072] S3: Sodium borohydride is weighed and dissolved in distilled water, and is added dropwise into the above reaction system at a speed of 10 s per drop, and stirring is continued for 2 h.

[0073] S4: After the reaction is completed, stirring is stopped, and the product can be stored for several months in the dark, and is recorded as Ag-PSS.

[0074] In the above steps, the amount of the silver nitrate solution added is 16.9 mg per 46 mL, the ratio of the polystyrene sodium sulfonate to the distilled water is 30 μl:2 ml, and the ratio of the sodium borohydride to the distilled water is 3.78 mg:2 ml.

[0075] The preparation method of the hollow mesoporous hydroxyapatite loaded with nano-silver includes the following steps:

[0076] S1: Polyethylene imine hydroxyapatite (CMC-HAP-PEI) powder is weighed and is ultrasonically dispersed in a centrifuge tube containing a nano-silver solution (Ag-PSS).

[0077] S2: The centrifuge tube is shaken on a shaking table in the dark for 6 h.

[0078] S3: After the reaction is completed, the material is washed and centrifuged several times with distilled water until the supernatant is colorless and transparent, and then is placed in a vacuum drying oven for drying, with a drying temperature of 60 ℃. The obtained reaction product is the hollow mesoporous hydroxyapatite loaded with nano-silver, and is recorded as CMC-HAP-PEI@Ag.

[0079] In the above steps, 30 mL of the nano-silver solution (PSS-Ag) is added to 300 mg of the polyethylene imine hydroxyapatite powder, and after the reaction is completed, the material is washed and centrifuged with distilled water until the supernatant is colorless.

[0080] The present application discloses a carboxymethyl cellulose sodium hydroxyapatite powder in the form of white powder, and the preparation steps are as follows:

[0081] S1: Anhydrous calcium chloride and carboxymethyl cellulose sodium are weighed and are dispersed in distilled water, and are ultrasonically dispersed uniformly; the two solutions are mixed and are magnetically stirred for 8-12 min, and the optimal magnetic stirring time is 10 min.

[0082] S2: Anhydrous sodium carbonate is weighed and is dispersed in distilled water, and after being uniformly dispersed, is quickly added into the above mixed solution, and stirring is continued for 24-36 min, and the optimal stirring time is 30 min.

[0083] S3: After the reaction is completed, the precipitate generated by the reaction is washed 2 to 3 times with deionized water by centrifugation, and then washed 2 to 3 times with anhydrous ethanol;

[0084] S4: placing the precipitated material in an oven and drying it at a temperature of 50°C-70°C (optimum temperature 60°C) to obtain prepared sodium carboxymethyl cellulose calcium carbonate microspheres, which are recorded as CMC-CaCO3;

[0085] S5: Weigh disodium hydrogen phosphate dodecahydrate and dissolve it in distilled water. Add the sodium carboxymethyl cellulose calcium carbonate microspheres (CMC-CaCO3) generated above to the solution. After ultrasonic homogenization, transfer the liquid to a polytetrafluoroethylene reactor and perform a hydrothermal reaction at 110°C-170°C (optimum temperature 140°C) for 2.4-4 hours, with an optimal reaction time of 3 hours.

[0086] S6: After the polytetrafluoroethylene reactor is cooled, the precipitated material generated by the reaction is washed 2 to 3 times with deionized water by centrifugation, and then washed 2 to 3 times with anhydrous ethanol;

[0087] S7: The precipitated material is placed in an oven and dried at a temperature of 50°C-72°C (optimal temperature 60°C) to obtain the prepared hollow mesoporous hydroxyapatite microspheres (i.e., sodium carboxymethyl cellulose hydroxyapatite), which are recorded as CMC-HAP.

[0088] In the above steps, the amount of calcium chloride powder added to 10 mL of calcium chloride solution is 1.11 g, and the amount of carboxymethyl cellulose powder added to 10 mL of carboxymethyl cellulose solution is 60 mg; the amount of sodium carbonate powder added to 20 mL of sodium carbonate solution is 1.06 g; when the sodium carbonate solution is added to the mixed solution, the solution quickly turns into a milky white colloidal liquid; next, 0.6 g of dry calcium carbonate powder is added to 15 mL of deionized water containing 5 g of disodium hydrogen phosphate dodecahydrate, stirred evenly, and then transferred to a polytetrafluoroethylene reactor.

[0089] The sodium carboxymethyl cellulose hydroxyapatite powder material disclosed in the present invention is a micron material with a hollow mesoporous microsphere morphology, which can be applied in the following fields: a. Drug delivery field: sustained and controlled release of drugs to protect drug activity; b. Medical aesthetics field: as a carrier to load growth factors, promote skin repair and regeneration, and can also be used as a filling material; c. Orthopedics field: can be used as a scaffold material for bone tissue engineering; d. Dentistry field: can be loaded with periodontal growth factors or antibacterial drugs for the repair and regeneration of periodontal tissue, and can also be used for surface modification of dental implants.

[0090] The present invention provides a hollow mesoporous hydroxyapatite powder loaded with nanosilver, which is in the form of a light yellow powder. The preparation steps are as follows:

[0091] S1: weigh the polyethylenimine hydroxyapatite (CMC-HAP-PEI) powder, and ultrasonically disperse in a centrifuge tube containing a nano-silver solution (Ag-PSS);

[0092] S2: avoid light and shake on a shaker for 6h;

[0093] S3: after the reaction, wash several times with distilled water by centrifugation until the supernatant is colorless and transparent, discard the supernatant, and then place in a vacuum drying oven (60℃) to dry, and the obtained reaction product is a hollow mesoporous hydroxyapatite loaded with nano-silver, denoted as CMC-HAP-PEI@Ag;

[0094] In the above steps, 30 mL of nano-silver solution (PSS-Ag) is added to every 300 mg of polyethylenimine hydroxyapatite powder, and after the reaction is completed, the material is washed and centrifuged with distilled water until the supernatant is colorless.

[0095] In the above preparation method, heparin, hyaluronic acid, sodium alginate and other anionic polymers can be used to replace carboxymethyl cellulose sodium, and the addition amount is 50-80 mg, and the rest of the reaction is the same as that of carboxymethyl cellulose sodium.

[0096] The hollow mesoporous hydroxyapatite powder loaded with nano-silver can be used to prepare antibacterial materials or antibacterial appliances or composite antibacterial films of organic polymers.

[0097] The technical steps of the present application will be described one by one with reference to the drawings as follows:

[0098] Referring to Figure 1 The present application provides a simple preparation method of hollow mesoporous hydroxyapatite, and the steps are as follows:

[0099] I. Preparation of carboxymethyl cellulose sodium calcium carbonate (CMC-CaCO3):

[0100] Take 1.11 g of anhydrous calcium chloride, disperse in 10 ml of distilled water, take 60 mg of sodium carboxymethyl cellulose, disperse in 10 ml of distilled water, mix ultrasonically, and stir magnetically at room temperature for 10 min (speed 800 rpm). After stirring evenly, quickly add 1.06 g of anhydrous sodium carbonate dispersed in 20 ml of distilled water, continue to stir at room temperature for 30 min (speed 800 rpm). After the reaction is completed, the product is washed with deionized water and anhydrous ethanol for 3 times respectively to remove the unreacted part, and then dried in a 60℃ oven to obtain sodium carboxymethyl cellulose calcium carbonate microspheres, which is recorded as CMC-CaCO3.

[0101] II. Preparation of sodium carboxymethyl cellulose hydroxyapatite (CMC-HAP):

[0102] Take 5 g of dodecahydrate sodium phosphate dibasic, disperse in 15 ml of distilled water, ultrasonically disperse evenly, take 0.6 g of CMC-CaCO3 from the above reaction product, add to the solution, continue to ultrasonically disperse, then transfer the liquid to a polytetrafluoroethylene reaction kettle, and react at 140℃ for 3 h. After the polytetrafluoroethylene reaction kettle is cooled, wash with deionized water and anhydrous ethanol for 3 times respectively to remove the unreacted part, and then dry in a 60℃ oven to obtain sodium carboxymethyl cellulose hydroxyapatite powder, which is white powder, recorded as CMC-HAP.

[0103] The obtained product is characterized by scanning electron microscope (SEM), Figure 2 The scanning electron microscope images of CaCO3 (a), CMC-CaCO3 (b), and CMC-HAP (c) are shown in Figure 1, Figure 2 From which it can be clearly observed that under the action of sodium carboxymethyl cellulose, the morphology of calcium carbonate is changed from rhombus to microspherical shape, and the surface of hydroxyapatite generated by hydrothermal reaction is mesoporous structure, and the results show that the preparation of mesoporous hydroxyapatite is successful.

[0104] The product is characterized by Fourier transform infrared spectrometer (FTIR), Figure 3 The FTIR spectra of CaCO3 (a), CMC-CaCO3 (b), HAP (c), and CMC-HAP (d) are shown in Figure 2, and the a curve is the infrared spectrum of CaCO3. From the structure of inorganic matter, when CMC is not contained, most of the particles are calcite aggregates; in the b curve, a peak of 745 cm -1 appears, which is the bending vibration characteristic peak of vaterite, 712 cm -1 is the in-plane bending vibration peak of aragonite, and 874 cm -1is the carboxyl face out-of-bending vibration peak of calcite, so b is the calcite and aragonite ball vaterite doping state. The cd curve is the FTIR spectrum of HAP, which is consistent with the corresponding peak position, which proves the successful synthesis of hydroxyapatite. In addition, the peak of 1640 cm -1 in d curve appears, which is consistent with the stretching vibration peak of C=O double bond of carboxyl group of e curve CMC, which proves that CMC exists in HAP.

[0105] The obtained product is characterized by X-ray diffraction pattern (XRD), Figure 4 X-ray diffraction patterns of CaCO3 (a), CMC-CaCO3 (b), CMC-HAP (c), in the absence of CMC, a figure corresponds to the diffraction peak (012), (104), (110), (113), (202), (018) and (116) is the diffraction peak of calcite CaCO3. In the presence of CMC, in addition to the diffraction peak of calcite, the other peaks (004) (110) (112) (114) and (300) in b figure indicate the existence of spherical crystal, which means that the crystal is composed of calcite and aragonite ball vaterite; At the same time, the diffraction peak in b figure obviously deviates to high θ angle, which indicates that there is organic doping, which proves the existence of CMC. There is only the characteristic peak of hydroxyapatite in c figure, which indicates that there is no CaCO3 residue, and the wide peak is caused by the low crystallinity of the formed hydroxyapatite. The detected structure is very similar to biological hydroxyapatite, which indicates the successful synthesis of hydroxyapatite.

[0106] The obtained product is observed by transmission electron microscope (TEM) for microstructure, which can be seen that the product is a microsphere with a diameter of about 3-5 μm, and has a hollow structure, thus confirming the successful synthesis of hollow mesoporous hydroxyapatite microspheres.

[0107] The mesoporous structure and the corresponding BJH pore size distribution curve of the obtained product are further demonstrated by nitrogen adsorption-desorption isotherm. CMC-HAP can be confirmed as type IV isotherm, with a H3 type hysteresis loop. The H3 type hysteresis loop does not show limited adsorption at high P / P0 because the aggregation of particles leads to the formation of crack-shaped pores, which can also be seen from the TEM picture. Through the analysis of adsorption-desorption isotherm, the BET surface area and pore volume of CMC-HAP are calculated to be 24.5908 m 2 / g and 0.114585 cm 3 / g, respectively. The BJH average pore size is 18.6387 nm with a single peak. These nanopores greatly increase the specific surface area and biological activity, making them better applied in the field of drug release and other fields.

[0108] III. Preparation of polyethyleneimine hydroxyapatite (CMC-HAP-PEI):

[0109] Take 500 mg of the above-synthesized sodium carboxymethyl cellulose hydroxyapatite (CMC-HAP), take 1 g of EDC and 250 mg of polyethyleneimine (PEI) dispersed in 20 ml of methanol, add 2.3 ml of triethylamine, and shake on a shaker (180 rpm) for 12 h. After the reaction is completed, the product is washed with deionized water and anhydrous ethanol for 3 times respectively to remove the unreacted part, and then dried in a 60°C oven to obtain the covalently grafted PEI hollow mesoporous hydroxyapatite, which is denoted as CMC-HAP-PEI.

[0110] Four, preparation of sodium polystyrene sulfonate coated silver nanoparticles (AgNPs-PSS):

[0111] Take 16.9 mg of silver nitrate and place it in a 100 mL light-proof conical flask. Add 46 ml of distilled water and ultrasonically dissolve it. Take 30 μl of sodium polystyrene sulfonate (PSS) and add it to 2 mL of distilled water, ultrasonically disperse it uniformly, and then add it dropwise to the silver nitrate solution. Stir at a speed of 300 r min -1 for 30 min. Take 3.8 mg of sodium borohydride and completely dissolve it in 2 mL of distilled water, then slowly add it dropwise to the above reaction system. As the sodium borohydride solution is added dropwise, the mixed solution quickly changes from colorless to brownish yellow. Continue stirring at a speed of 300 r min -1 for 2 h. The PSS-coated silver nanoparticles solution is obtained and labeled as Ag-PSS.

[0112] Five, preparation of silver-loaded hollow mesoporous hydroxyapatite (CMC-HAP-PEI@Ag):

[0113] Take 100 mg of dried CMC-HAP-PEI material in a centrifuge tube, add 10 mL of the above AgNPs-PSS solution, and shake at room temperature in the dark for 6 h. After the reaction is completed, centrifuge (8000 r min-1, 3 min), discard the yellow liquid on top, and continue washing until the wash liquid becomes colorless. Remove the AgNPs-PSS that is not loaded onto the material, and dry at 60°C overnight to obtain the silver-loaded hollow mesoporous hydroxyapatite material in the form of a light yellow powder, which is denoted as CMC-HAP-PEI@Ag.

[0114] The product is characterized by zeta potential, Figure 7The zeta potential graph of CMC-HAP, CMC-HAP-PEI, Ag-PSS, CMC-HAP-PEI@Ag, because a large number of imine groups are on the polyethyleneimine, the potential of the particles changes from -20 mv to 24.9 mv before and after the modification of polyethyleneimine. The synthesized nano-silver is negatively charged due to the coating of polystyrene sulfonic acid sodium, and the potential is -36.3, and the potential of the final product is 3.83. The results show that the preparation of hollow mesoporous hydroxyapatite loaded with nano-silver is successful.

[0115] The obtained product is characterized by X-ray diffraction pattern (XRD), Figure 8 The XRD pattern of CMC-HAP-PEI, CMC-HAP-PEI@Ag, the (111), (200), (220) in the figure are the peaks of silver elements, and the detected structure is very similar to the silver element, indicating the successful loading of nano-silver.

[0116] The obtained product is characterized by mapping in transmission electron microscopy, Figure 9 The element mapping of hollow mesoporous hydroxyapatite loaded with nano-silver, it can be seen from the figure that the silver element is uniformly distributed on the surface of the material, and the results show that the preparation of hollow mesoporous hydroxyapatite loaded with nano-silver is successful.

[0117] The obtained product is detected by cck-8 kit for cell toxicity, Figure 10 The relationship between material concentration and cell survival rate, it can be seen from the figure that the cell survival rate is greater than 90% at a concentration of 500 μg / ml and below, which proves that it is a biological safety concentration range below the concentration.

[0118] Technical effect verification:

[0119] The antibacterial test of CMC-HAP-PEI and CMC-HAP-PEI@Ag prepared by the above preparation method on Staphylococcus aureus, Escherichia coli, Candida albicans and methicillin-resistant Staphylococcus aureus:

[0120] The sample was incubated with 10 6 CFU mL -1 of bacterial suspension in PBS phosphate buffer for 24 h, and 100 µL of the suspension was coated on solid culture medium and cultured for 24 h; after incubation at 37°C for 24 h, the colonies on the agar medium were counted, and the bacterial survival rate was defined as the colony forming units on the agar plate relative to the control group, 10 6 CFU mL -1 of bacterial suspension in PBS phosphate buffer as a blank control.

[0121] The experimental results are shown in Figures 11-13CMC-HAP-PEI does not have obvious inhibitory effect on bacteria at microgram dose, and the bacterial killing rate of CMC-HAP-PEI@Ag group is 99%, the lethal dose of Staphylococcus aureus is 50 mu g / ml, the lethal dose of Escherichia coli is 5 mu g / ml, the lethal dose of Candida albicans is 50 mu g / ml, and the lethal dose of methicillin-resistant Staphylococcus aureus is 10 mu g / ml.

[0122] The application shows a method for constructing hollow mesoporous hydroxyapatite microspheres loaded with nano-silver, which kills bacteria through the loaded nano-silver; here, the antibacterial effect and antibacterial spectrum are verified through Staphylococcus aureus, Escherichia coli, Candida albicans, methicillin-resistant Staphylococcus aureus and the like, and the results prove that the hollow mesoporous hydroxyapatite loaded with nano-silver has good antibacterial effect. On the one hand, the high specific surface area and porous structure of the hollow mesoporous material enable it to adsorb more bacteria, enrich the bacteria around the material, thereby increasing the contact opportunity between the nano-silver and the bacteria and improving the antibacterial efficiency. On the other hand, the antibacterial mechanism of the nano-silver loaded on the hollow mesoporous material can be further optimized. The nano-silver can not only release silver ions to interfere with the physiological functions of bacteria, but also can destroy the structure and integrity of the cell membrane through direct contact with the bacterial cell membrane, thereby inhibiting the growth and reproduction of bacteria. The synthesis method has simple process, low cost and wide application range; the antibacterial durability, repeatability are good; the synthesis product has high biological safety and can be applied to the fields of orthopedics, dentistry, medical aesthetics and the like, which will greatly promote the development of the antibacterial field.

[0123] The above is only a preferred embodiment of the present application, and is not intended to limit the present application in any form. Although the present application has been disclosed as above with a preferred embodiment, it is not intended to limit the present application, and any person skilled in the art can make some changes or modifications to the above disclosed technical content to obtain equivalent embodiments with equivalent changes, without departing from the technical solution of the present application. Any simplification, modification, equivalent change and modification of the above embodiments made according to the technical essence of the present application, without departing from the technical solution of the present application, are still within the scope of the technical solution of the present application.

Claims

1. A method for preparing hollow mesoporous hydroxyapatite microspheres loaded with nanosilver, characterized by the following steps: The method comprises the following steps: The preparation of the hollow mesoporous hydroxyapatite microspheres comprises the following steps: S1: the anhydrous calcium chloride and sodium carboxymethyl cellulose are respectively dispersed in distilled water and uniformly ultrasonically dispersed; the two solutions are mixed and magnetically stirred for 8-12 min; S2: the anhydrous sodium carbonate is dispersed in distilled water, and after being uniformly dispersed, the mixed solution is quickly added into the above solution, and stirring is continuously carried out for 24-36 min; S3: after the reaction is completed, the generated precipitate is washed by centrifugation with deionized water for 2-3 times, and then washed with anhydrous ethanol for 2-3 times; S4: the precipitate is placed in an oven and dried at a temperature of 50-70 DEG C, so that the prepared sodium carboxymethyl cellulose calcium carbonate microspheres are obtained, which are denoted as CMC-CaCO3; S5: the dodecahydrate disodium hydrogen phosphate is dissolved in distilled water, and the prepared sodium carboxymethyl cellulose calcium carbonate microspheres (CMC-CaCO3) are added into the solution, and after being uniformly ultrasonically dispersed, the liquid is transferred into a polytetrafluoroethylene reaction kettle, and hydrothermal reaction is carried out at a temperature of 110-170 DEG C for 2.4-4 h; S6: after the polytetrafluoroethylene reaction kettle is cooled, the generated precipitate is washed by centrifugation with deionized water for 2-3 times, and then washed with anhydrous ethanol for 2-3 times; S7: the precipitate is placed in an oven and dried at a temperature of 50-72 DEG C, so that the prepared hollow mesoporous hydroxyapatite microspheres are obtained, which are denoted as CMC-HAP; The method for preparing the polyethyleneimine hydroxyapatite comprises the following steps: S1: the sodium carboxymethyl cellulose hydroxyapatite microsphere powder is ultrasonically dispersed in a centrifuge tube containing a methanol solvent; S2: the EDC is dispersed in a proper amount of methanol, a small amount of triethylamine is added, the mixture is uniformly mixed, and then is transferred into the above solution, and reaction is carried out on a shaking table for 24-36 min; S3: methanol containing polyethyleneimine is added, and the reaction is continuously carried out on the shaking table, and the total time is 12 h; S4: after the reaction is completed, the product is washed and centrifuged with distilled water and anhydrous ethanol, the supernatant is discarded, and then the product is dried in a vacuum drying box for standby use; the obtained reaction product is polyethyleneimine hydroxyapatite, which is denoted as CMC-HAP-PEI.

2. The method of claim 1, wherein the method is characterized by: The addition amount of the calcium chloride powder in 10 mL of the calcium chloride solution is 1.11 g, the addition amount of the carboxymethyl cellulose powder in 10 mL of the carboxymethyl cellulose solution is 60 mg, and the addition amount of the sodium carbonate powder in 20 mL of the sodium carbonate solution is 1.06 g; when the sodium carbonate solution is added into the mixed solution, the solution quickly becomes a milky white colloidal liquid; then, 0.6 g of the dried calcium carbonate powder is added into 15 mL of deionized water containing 5 g of the dodecahydrate disodium hydrogen phosphate, and the mixture is uniformly stirred and then transferred into a polytetrafluoroethylene reaction kettle.

3. The method of claim 1, wherein the method is characterized by: Add 10 mL of methanol to each 500 mg of carboxymethylcellulose sodium hollow mesoporous hydroxyapatite powder, the ratio of EDC to methanol and triethylamine is 1 g:5 mL:2.3 mL, add 5 mL of methanol to each 250 mg of polyethyleneimine, after the reaction is completed, wash the product with distilled water and centrifuge 2-3 times, wash the product with anhydrous ethanol and centrifuge 2-3 times.

4. The method of claim 1, wherein the method is characterized by: The preparation method of the sodium polystyrene sulfonate coated nanosilver comprises the following steps: S1: disperse silver nitrate into distilled water to form a silver nitrate solution, and transfer the solution to a light-proof conical flask; S2: stir the silver nitrate solution on a magnetic stirrer, and drop sodium polystyrene sulfonate solution, stir the prepared solution for 30 min; S3: weigh sodium borohydride and dissolve it in distilled water, drop it into the above reaction system at a speed of 10 s per drop, and continue to stir for 2 h; S4: after the reaction is completed, stop stirring, and store the solution in the dark for several months, and mark it as Ag-PSS.

5. The method of claim 4, wherein the method is characterized by: The amount of silver nitrate solution added is 16.9 mg per 46 mL, the ratio of sodium polystyrene sulfonate to distilled water is 30 μL:2 mL, and the ratio of sodium borohydride to distilled water is 3.78 mg:2 mL.

6. The method of claim 1, wherein the method is characterized by: The preparation method of the hollow mesoporous hydroxyapatite loaded with nanosilver comprises the following steps: S1: weigh polyethyleneimine hydroxyapatite powder, and ultrasonically disperse it in a centrifuge tube containing a sodium polystyrene sulfonate coated nanosilver solution; S2: shake the solution in the dark on a shaking table for 6 h; S3: after the reaction is completed, wash the product with distilled water and centrifuge several times until the supernatant is colorless and transparent, discard the supernatant, and then place the product in a vacuum drying oven for drying, and the obtained reaction product is hollow mesoporous hydroxyapatite loaded with nanosilver, and is marked as CMC-HAP-PEI@Ag.

7. A hollow mesoporous hydroxyapatite powder loaded with nanosilver, characterized by: The preparation method is prepared by using the preparation method of claim 6.

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