RU2614772C1 - Method for producing nanohydroxyapatite - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method for producing nanohydroxyapatite comprises mixing in the reactor the four-watered calcium nitrate solution of Ca(NO₃)₂⋅4H₂O with the concentration of 0.01-0.15 mol/l with the aqueous solution of orthophosphoric acid H₃PO₄ with the concentration of 1.4-1.5 mol/l at a molar ratio of Ca/P=5/3, thermostating the resulting solutions mixture under constant stirring with a stirrer rotating at the speed of 170-200 rev/min, until reaching the temperature equilibrium with the external thermostatic jacket heated to the temperature of 33-37°C, adding sodium hydroxide solution NaOH with a concentration of 1 mol/l, heated to 37-42°C, until reaching the acidity index of pH=7, maintaining the molar ratio of Ca/Na=1.25-1.30. Moreover, 2/3 of the sodium hydroxide solution is added directly to the reactor center at a flow rate of 7-10 m/s, the remaining amount - with portions of 50 ml, and then the resulting sol is maintained at a temperature of 37°C and the stirrer switched off for one hour, removed from the reactor, centrifuged and washed three times with bidistilled water.

EFFECT: simplifying the production and reducing the production time.

2 cl, 4 dwg, 1 tbl, 2 ex

Description

The invention relates to inorganic chemistry, relates to a method for producing nanohydroxyapatite, which can be used in medicine for the production of medical materials that stimulate the restoration of bone defects, including dentistry.

Stoichiometric hydroxyapatite is a bio-inorganic compound with the formula Ca ₅ (PO ₄ ) ₃ OH, with increased biological compatibility due to the presence in the chemical composition of the compound of isomorphically incorporated sodium ions, which is the mineral basis of hard mammalian tissues (teeth, bones). Due to its high isomorphic capacity (the ability to incorporate atoms of other chemical elements without changing the structure), the chemical composition of this compound can be modified to achieve improved biological and physical properties of the final materials. The most commonly used chemical modifiers are Na, K, CO ₃ , S, etc.

A known method of producing hydroxyapatite (RU 9301269 A, class C01B 25/32, publ. March 20, 1996), comprising mixing a suspension of calcium hydroxide with an aqueous solution of phosphoric acid with successive passage of the reaction mixture through two zones, while maintaining in the first zone a pH of 10.0-11.0 and a flow velocity of 0.8-1.5 m / s, and in the second zone, the suspension is diluted 400-500 times and returned to the first zone, providing 4-5-fold circulation the reaction mixture in a closed cycle for 10-20 minutes The product is filtered off and dried. The method provides an increase in the yield of hydroxyapatite while improving its quality and increasing the purity of the phase composition.

The disadvantage of this method is its multi-stage, the complexity of the hardware design, high energy consumption of obtaining a suspension (paste) of hydroxylapatite, as well as the inability to prepare a product with particle sizes close to nanoscale.

A known method of producing a finely divided single-phase hydroxyapatite (RU 2165389 C1, class C01B 25/32, publ. 04/20/2001), including the synthesis of calcium phosphate, its treatment with alcohol at a volume ratio of calcium phosphate solution: alcohol equal to 1: 0.001-1 mixing it with an alcohol solution of alkali having a volume ratio of an aqueous solution of alkali: alcohol 1: 0.001-1, to a pH of 10-12, separating, washing and drying the precipitate of the product at a temperature of not more than 60 ° C. According to this method, when water-alcohol solutions of calcium phosphate and alkali are used as a reaction mixture, it is possible to obtain finely divided hydroxylapatite powders with a particle size of up to 0.3-1.0 μm. The introduction of alcohol in the reaction medium in this method is intended solely to prevent particle agglomeration, which allows to obtain finely divided hydroxylapatite with a particle size of 0.3-1 μm.

The disadvantage of this method is that the particles of the resulting product are not nanoscale.

A known method of producing finely divided hydroxylapatite of high purity (RU 2149827 C1, class C01B 25/32, publ. 05.27.2000), which includes obtaining finely divided hydroxylapatite in the form of a suspension by pouring a 6-9% solution of phosphoric acid at a rate of 70- 120 ml / min to a 20-55% suspension of calcium hydroxide, pre-treated with ultrasound at a frequency of 9-15 kHz for 1-2 hours. The formation of particles of hydroxylapatite with a size of 1-5 microns occurs in the sump for 20-24 hours. The concentration of hydroxylapatite in the finished suspension is 320-350 g / l.

The disadvantages of this technical solution are the long process time, as well as the fact that the particle size of hydroxylapatite in this suspension is 1-5 microns, i.e. they cannot be classified as nanoscale.

A known method of producing hydroxyapatite (RU 2505479 C1, class C01B 25/32, C01F 11/00, AK 6/033, publ. 01/27/2014), which involves obtaining hydroxyapatite by mixing a 1-1.5% aqueous suspension of calcium carbonate saturated with carbon dioxide at a concentration of 1-1.5 g / l, and 1-1.5% aqueous solution of sodium hydrogen phosphate at a temperature of 20-37 ° C, with constant stirring and at a molar ratio of CaCO ₃ / Na ₂ HPO ₄ = 5-7.

The disadvantage of this method is the use of calcium carbonate due to the fact that carbonate ions are embedded in the structure of hydroxyapatite, significantly affecting the mechanical properties of the future material.

A known method of producing nanohydroxyapatite (RU 2402483 C2, class C01B 25/32, A61L 27/00, B82B 3/00, publ. 10.27.2010), based on the precipitation of the product from solutions of calcium salts and alkali metal and / or ammonium phosphates in the presence of a biopolymer, for example gelatin or starch, a concentration of 0.1-1 wt. %

The disadvantages of the method are the long process time (more than 24 hours), the need for high-temperature exposure (600-700C), as well as obtaining a product with highly variable particle sizes (5-50 nm).

A method of producing nanohydroxyapatite (RU 2457174 C1, class C01B 25/32, B82B 1/00, B82Y 40/00, publ. July 27, 2012), considers the preparation of a product by reacting an organic derivative of calcium with an organic derivative of phosphorus in an organic polar solvent at atomic the ratio of calcium to phosphorus in the range of 1.6-1.8.

A significant disadvantage of this method is the high cost and danger to a living organism of reagents and solvents.

The closest in technical essence and the result to the proposed invention is a method for producing nanosized hydroxylapatite, protected by patent RU 2342938 C1, cl. A61K 33/42, A61K 33/06, A61K 9/10, A61K 6/033, A61L 27/12, B82B 3/00, C01B 25/32, publ. 01/10/2009, adopted for the closest analogue (prototype).

The prototype method includes pouring a solution of orthophosphoric acid into a solution of calcium hydroxide, exposure to ultrasound for 1-2 hours, adding to the colloid nanosized hydroxylapatite with a concentration of 5-40 ± 2% ethyl or isopropyl alcohol to a concentration of hydroxylapatite in an alcohol solution from 0.2 to 20 %, and exposure to ultrasound at a frequency of 10-50 kHz after adding alcohol. In this case, the synthesis of hydroxylapatite is carried out in a saturated solution of calcium hydroxide, decanted after daily settling from the precipitated aggregates of Ca (OH) ₂ , by adding at a rate of 1.5-2.2 ml / min per liter of alkaline solution of a 10-20% phosphoric acid solution acid with constant stirring until the pH of the reaction mixture is not lower than 10.5 ± 0.5; mix for 20-30 minutes, stand for 1-2 hours and decanted clarified upper layer; the concentration of the resulting colloidal hydroxylapatite solution is increased from 1.5-2 to 5-30% by evaporation at a temperature not exceeding 60 ° C or from 1.5-2 to 40 ± 2% by cryoprocessing, including complete freezing followed by thawing at a temperature no more than 60 ° C and decantation of a liquid phase. The synthesis of hydroxylapatite in this way allows to obtain hydroxylapatite in the form of elongated crystallites with a size of 10-20 nm, oriented in a certain way and arranged in chains, including parallel and agglomerated, whose sizes do not exceed 200 nm in length and 40 nm in width, this shape of the crystals contributes the formation of stable coagulation structures. The obtained alcohol colloid (gel) of nanosized high-purity hydroxylapatite with a predetermined concentration can be used for the production of medical materials that stimulate the restoration of bone tissue defects, including in dentistry.

The disadvantages of the prototype method is the need to significantly complicate the installation by supplementing it with an ultrasonic wave generator and cryostation, as well as multi-stage and a fairly long process time.

The objective of the invention is to create a new method for producing nanohydroxyapatite in one stage.

The technical result from the use of the invention is to simplify manufacturing, reduce production time.

The problem is achieved in that the method of producing nanohydroxyapatite involves mixing in a batch reactor, equipped with an external thermostatic jacket and an axial stirrer with a mechanical drive, a solution of four-water calcium nitrate Ca (NO ₃ ) ₂ ⋅ 4H ₂ O with a concentration of 0.01-0.15 mol / l with an aqueous solution of orthophosphoric acid H ₃ PO ₄ with a concentration of 1.4-1.5 mol / l, with a molar ratio Ca / P = 5/3, the temperature of the resulting mixture of solutions is thermostated with constant stirring with a stirrer rotating at a speed of 170- 200 rpm, to set temperature equilibrium with an external thermostatic jacket, heated to a temperature of 33-37 ° C, adding a solution of sodium hydroxide NaOH with a concentration of 1 mol / l heated to 37-42 ° C until a pH of pH 7 is reached, maintaining the molar ratio Ca / Na = 1.25-1.30, moreover, 2/3 of the sodium hydroxide solution is added directly to the center of the reactor at a flow rate of 7-10 m / s, the remaining amount is added in 50 ml portions, then the resulting sol is kept at 37 ° C when the mixer is off for an hour, remove tion from the reactor, centrifugation and washing three times with bidistilled water; the batch reactor is additionally equipped with a heating element and a pump.

In FIG. 1 is a schematic illustration of a batch reactor for implementing a process for producing nanohydroxyapatite.

In FIG. Figure 2 shows the IR spectrum of nanohydroxyapatite.

In FIG. Figure 3 shows an AFM photograph of the surface of nanohydroxyapatite (average particle size is 25 ± 3 nm).

In FIG. Figure 4 presents powder X-ray diffraction patterns of (a) nanohydroxyapatite and (b) microhydroxyapatite.

Structurally, a batch reactor for implementing the method for producing nanohydroxyapatite in FIG. 1 contains:

1 - inner vessel;

2 - external thermostatic jacket;

3 - axial mixer with a mechanical drive;

4 - heating element;

5 - pump.

The agitator with a mechanical drive 3 rotates at a speed of 170-200 rpm. The selected rotation speed of the mixer 3 provides optimal turbulence when mixing the reagents in the inner vessel 1 of the reactor. By means of the heating element 4, the water in the external thermostatic jacket 2 is heated to a temperature of 33-37 ° C. Using pump 5, water is circulated in the external thermostatic jacket 2.

The method of obtaining nanohydroxypatite on the proposed installation is as follows.

In the inner vessel 1 of the batch reactor, an aqueous solution of 4-aqueous calcium nitrate Ca (NO ₃ ) ₂ ⋅ 4H ₂ O with a concentration of 0.01-0.15 mol / l and an aqueous solution of orthophosphoric acid H ₃ PO ₄ with a concentration of 1 4-1.5 mol / l. The number of solutions is selected so as to provide a molar ratio Ca / P = 5/3 (stoichiometric ratio of the corresponding ions in hydroxyapatite). A decrease in the concentration of reagents below these limits leads, as a rule, to a mixture of calcium phosphates of various compositions. At the same time, an increase in concentrations above the specified limits leads to a significant enlargement of product particles and the prevalence of calcium orthophosphate in its composition.

The resulting mixture of solutions is thermostated with the mixer turned on until the temperature equilibrium is established with the water jacket 2 of the reactor.

Next, a sodium molar (NaOH) solution with a concentration of 1 mol / L, heated to 37-42 ° C, is added to the reactor until the pH value of the medium reaches pH = 7. In this case, the molar ratio Ca / Na = 1.25-1.30 is maintained. Moreover, 2/3 of the required amount of sodium hydroxide solution is added directly to the center of the reactor 1 at a flow rate of 7-10 m / s, the remaining amount - in portions of 50 ml. A decrease in the temperature of the process significantly affects the increase in reaction time, while its increase above the specified limit does not affect the quality of the product, but complicates the technical support of the process. The feed rate of the sodium hydroxide solution also has a significant effect on the phase composition of the product - at lower values, the main synthesis product is calcium orthophosphate.

With this approach to the synthesis in solution, the following chemical transformations are realized:

The obtained sol (a solution of nanoparticles in water) is kept at a temperature of 37 ° C and with the stirrer turned off for an hour, then it is removed from the reactor, centrifuged and washed three times with bidistilled water. The temperature of the product after the completion of the process is selected in such a way as to obtain a gel from hydroxyapatite nanocrystals, and not from its amorphous particles, on the one hand, and to avoid agglomeration of particles with long exposure times.

The total time for the synthesis process of the proposed method is 2 hours.

The resulting gel of hydroxyapatite nanoparticles contains 10-15% of the product.

The resulting gel can be used directly as a component of various products or can be dried and used as a nanosized powder for the production of ceramics.

The phase composition of the product was confirmed using a complex of methods of x-ray diffractometry (X-ray diffractometer Shimadzu XRD-6000) and IR spectroscopy (IR-Fourier spectrometer Shimadzu FTIR-8400s) (Fig. 2).

Nanoscale of the resulting product is achieved through the use of dilute solutions of reagents, high speed mixing of the reaction mixture; confirmed using an atomic force microscope AURIGA CrossBeam Workstation, Carl Zeiss (Fig. 3), as well as indirectly by line broadening on X-ray diffractograms obtained on a Shimadzu XRD-6000 X-ray diffractometer (Fig. 4).

Biological activity was confirmed in the framework of studies under State contract No. 209-ЗК-11 dated 08/31/2011. The experiments were carried out on human cells - fibroblasts. It was shown that samples of nanohydroxyapatite do not destroy cells in the culture, do not interfere with cell adhesion to the surface, do not affect cell proliferation in culture, do not interfere with the synthesis of fibronectin, and do not interfere with the dynamics of IL-6 formation (interleukin-6) (table 1).

The invention is illustrated by the following examples.

Example 1. A 0.15 M solution of tetrahydrous calcium nitrate (500 g per 14 liters of distilled water) and 865 ml of 1.47 M phosphoric acid, the amount of which is calculated by reaction stoichiometry, are placed in an internal vessel of a batch reactor. The resulting mixture of solutions is thermostated with the mixer turned on until the temperature equilibrium is established with the water jacket of the reactor. Then, 2/3 of the required amount of sodium hydroxide solution (1.1 L) is added to the reaction mixture, the remaining amount (0.55 L) is added in 50 ml portions. The obtained sol of nanohydroxyapatite is kept in the reactor with the stirrer switched off and thermostating conditions maintained for an hour. Thus obtained nanohydroxyapatite has a particle size of about 25 nm (Fig. 3), the absence of components of the starting reagents in accordance with the data of IR spectroscopy (Fig. 2), in particular NO ₃ groups. Radiographic data (Fig. 4) also demonstrate the absence of crystalline impurities of the starting reagents or by-products.

Example 2. Obtaining nanohydroxyapatite is carried out analogously to example 1, only instead of a semi-industrial reactor, a 200 ml beaker is used, the temperature of the reaction mixture is maintained using a laboratory thermostat such as Lauda Alpha A 24. A 0.15 M solution of four-water calcium nitrate is placed in a beaker (5 g 140 ml of distilled water) and 8.7 ml of 1.47 M phosphoric acid, the amount of which is calculated by stoichiometry of the reaction. The resulting mixture of solutions is thermostated with the mixer turned on until the temperature equilibrium is established with the water jacket of the reactor. Then, 2/3 of the required amount of sodium hydroxide solution (11 ml) is added to the reaction mixture, the remaining amount (5.5 ml) is added in 5 ml portions. The obtained sol of nanohydroxyapatite is kept in a glass in the absence of mixing and maintaining thermostating conditions for an hour.

The resulting nanohydroxyapatite is characterized by the same indices as in Example 1, which indicates a good scalability of the method (an increase in the volume of the reaction mixture by 2 orders of magnitude does not affect the features of the process and product characteristics).

Claims (2)

1. A method of producing nanohydroxyapatite, including mixing in a batch reactor equipped with an external thermostatic jacket and an axial stirrer with a mechanical drive, a solution of four-water calcium nitrate Ca (NO ₃ ) ₂ ⋅ 4H ₂ O with a concentration of 0.01-0.15 mol / l with an aqueous solution of orthophosphoric acid H ₃ PO ₄ with a concentration of 1.4-1.5 mol / l at a molar ratio Ca / P = 5/3; the temperature of the resulting mixture of solutions is thermostated with constant stirring with a stirrer rotating at a speed of 170-200 rpm to establish temperature equilibrium with in external thermostatic jacket, heated to a temperature of 33-37 ° C, the addition of a solution of sodium hydroxide NaOH heated to 37-42 ° C with a concentration of 1 mol / l until the pH value of the medium reaches pH = 7, maintaining the molar ratio Ca / Na = 1.25 -1.30, moreover, 2/3 of the sodium hydroxide solution is added directly to the center of the reactor at a flow rate of 7-10 m / s, the remaining amount is added in 50 ml portions, then the resulting sol is kept at 37 ° C with the mixer turned off within an hour, extraction from the reactor, centrifugation, etc. repeated washing with double-distilled water. 2. The method according to claim 1, characterized in that the batch reactor is additionally equipped with a heating element and a pump.

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