PL229015B1 - Method for obtaining hydroxyapatite nanoparticles - Google Patents

Description

REPUBLIC

POLAND

(12) PATENT DESCRIPTION ₍ i ₉₎ PL ₍ n) 229015 (13) B1 (51) Int.CI.

(21) Filing number: 404820 _{CQ1B 25/32 (200601)}

A61L 27/32 (2006.01)

Patent Office of the Republic of Poland (22) Application date: 23.07.2013 (54)

Method for obtaining hydroxyapatite nanoparticles (73) The right holder of the patent:

WARSZAWSKA POLITECHNIKA, Warszawa, PL (43) Application was announced:

02/02/2015 BUP 03/15 (45) The following was announced about the grant of the patent:

30.05.2018 WUP 05/18 (72) Inventor (s):

TOMASZ CIACH, Warsaw, PL

EWA DUSZYŃSKA, Ostrów Mazowiecka, PL

MICHAŁ WOJASIŃSKI, Wołomin, PL (74) Representative:

thing, pat. Grażyna Padee m

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PL 229 015 B1

Description of the invention

The subject of the invention is a method of obtaining hydroxyapatite nanoparticles in the form of nanoplates, nanowires, nanospheres, in the process of wet chemical precipitation, with the use of a dispersing agent.

Human bone can be considered as an example of an inorganic-organic composite, containing about 70% by weight of inorganic crystals, mainly hydroxyapatite with the chemical formula Ca5 (PO4) 3OH, and 30% by weight of an organic matrix, mainly type I collagen [Osteoblast mineralization with composite nanofibrous substrate for bone tissue regeneration, Venugopal JR, Giri Dev VR, Senthilram T, Sathiskumar D, Gupta D, Ramakrishna S., Cell Biology International, 35, 2011]. Currently, hydroxyapatite for biomedical applications, such as drug carriers, filling bone defects, preparation of implants, is most often produced using the following chemical reaction:

5Ca ² + + 3PO4 ^3- + OH ^- = Ca5 (PO4) 3OH. (Formula 1)

Water-soluble calcium salts such as nitrate or chloride are most commonly used as the source of calcium ions, and water-soluble ammonium, sodium or calcium phosphates are used as the source of phosphate ions. The reaction is usually carried out in a neutral environment, at room or elevated temperature.

The reaction according to the above equation yields hydroxyapatite with the appropriate Ca / P molar ratio equal to 1.67, which is a value characterizing hydroxyapatite of natural origin [Calcium phosphates as substitution of bone tissues, VelletRegi M, Gonzalez-Calbet JM, Progress in Solid State Chemistry, 32, 2004]. Thick hydroxyapatite has a number of applications in bone replacement and is used to repair bone damage in dental and orthopedic cavities, immediate tooth replacement, augmentation of alveolar edges, as a pulp capping material, and in maxillofacial reconstruction [Synthesis of stoichiometric nano crystalline hydroxyapatite by ethanol- based solgel technique at low temperature, Kuriakose TA, Kalkura SN, Palanichamy M, Arviuoli D, Dierks K, Bocelli G et al., Journal of Crystal Growth, 263, 2004].

Unfortunately, the crystals of hydroxyapatite precipitated in purely aqueous salt solutions are relatively large, with sizes in the order of several dozen or even up to several hundred micrometers. Moreover, the crystals obtained in this way are completely insoluble and are not reconstructed in the human body. Natural bone, on the other hand, is a dynamic structure; it is constantly dissolved and built up by bone cells, so the use of hydroxyapatite that is insoluble by bone cells interferes with this process. In order to solve this problem, a number of techniques have been developed to obtain hydroxyapatite in the form of nanocrystals, or hydroxyapatite containing various additives have been used to make it susceptible to cellular remodeling. Techniques for producing nanoparticles of hydroxyapatite by modifying the course of the reaction represented by formula 1, using UV radiation, microwaves, often in combination with high temperature and significant pressure (hydrothermal process) or using laser ablation on finished crystals have been described. Classically used techniques for producing nanohydroxyapatite are based on processes such as: sol-gel synthesis, solid state reactions, coprecipitation, hydrothermal reactions, synthesis in microemulsions, mechanochemical synthesis and various wet chemistry techniques, including wet chemical precipitation. All the previously presented methods for producing nanohydroxyapatite require appropriate process temperature settings, ranging from 25 ° C to even 500 ° C, appropriate pH settings of the reaction mixture, ranging from 4.5 to 11. In some cases, increased process pressure is required, even up to 40 atmospheres, or toxic surfactants are used.

In the wet chemical precipitation method, it is necessary to use a reagent dispersant in the reaction medium to prevent the formation of large crystals. Known art protocols for the preparation of nanohydroxyapatite by wet chemical precipitation use dispersants including: surfactants such as cetyltrimethylammonium bromide (CTAB) [Hydrothermal synthesis of hydroxyapatite nanopowders using cationic surfactant as a template, Wang Y, Zhangha S, Wei K, Z N, Chen J, Wang X. Materials Letters 60, 2006], emulsifiers such as ethanolamine [Effects of synthesis conditions on the morphology of hydroxyapatite nanoparticles produced by wet chemical process, Wang P, Li C, Gong H, Jiang X, Wang H, Li K. Powder Technology

PL 229 015 B1

203, 2010], polymer matrices from polymers such as polyvinyl alcohol (PVA), polyethylene glycol (PEG) [In situ synthesis and characterization of nano-size hydroxyapatite in poly (vinyl alcohol) matrix, Mollazadeh S, Javadpour J, Khavandi A Ceramics International 33, 2007] and other chemicals such as e.g. citric acid, amino acids, (ethylenedinitrile) tetraacetic acid (EDTA), and the like [Nanoscale hydroxyapatite particles for bone tissue engineering, Zhou H, Lee J. Acta Biomaterialia 7 , 2011].

The so far known techniques for obtaining nanohydroxyapatite require the use of high temperatures in combination with high pressure, toxic emulsifiers or dispersants, non-biodegradable polymers. This entails the need for long-term product purification and a large amount of waste, with the risk of always having a toxic effect from residual chemical additives.

The aim of the invention was to develop a new method of obtaining hydroxyapatite nanoparticles by wet chemical precipitation under mild conditions, using a fully biocompatible emulsifier. The aim of the invention was also to obtain the possibility to control the morphology of the produced hydroxyapatite. This object is achieved by carrying out the reaction represented by the formula I in a reaction system in which lecithin is used as the dispersing agent.

The method of obtaining nanoparticles of hydroxyapatite by wet chemical precipitation method consists in reacting a water-soluble calcium salt and a water-soluble phosphate in an aqueous solution, with a molar ratio of calcium atoms to phosphorus atoms of approximately Ca / P 1.67 and in the presence of % of the dispersant, and according to the invention it is characterized in that lecithin is used as the dispersant in an amount of 0.05 wt. % to 20.00 wt.%, the reaction is carried out at a temperature ranging from 20 ° C to 95 ° C, preferably 60 ° C, with a pH of 7 to 14, preferably 10.

Preferably, calcium salts readily soluble in water, such as nitrate, acetate, chloride, lactate (e.g. Ca (NO3) 2 4H2O) are used as the source of calcium Ca ^{2+ in the reaction system.}

Preferably, easily water-soluble ammonium, sodium, potassium phosphates (e.g. (NH4) 2HPO4) are used as the source of PO4 ^{3- phosphate in the reaction system.}

Preference is given to using lecithin of natural origin, such as soybean or chicken egg lecithin.

Preferably, lecithin is added at a concentration of 0.05% to about 1.5% to obtain nanoplates; at a concentration of 1.5% to about 3% to obtain nanowires; in a concentration of 3% to 20% to obtain nanospheres.

In particular, the method of the invention is carried out by adding an aqueous solution of lecithin in demineralized water to the aqueous solution of the calcium ion source compound in demineralised water. The reaction mixture is heated to a temperature in the range from 20 ° C to 95 ° C, preferably 60 ° C, and the pH is adjusted in the range from 6 to 14, preferably about 10. After stable conditions are obtained, an aqueous solution is added to the reaction mixture. of phosphate, such that the stoichiometric amount is added over a period of up to 24 hours, preferably 1-2 hours. The solution is then cooled to ambient temperature and centrifuged to isolate the product. After decanting, the obtained product is washed with an organic solvent (hexane, gasoline, ethanol, acetone, isopropanol) or water to remove residual lecithin from the product. A small residue of lecithin (0.01% -2%) does not adversely affect the properties of the product, and even improves them. The obtained product is dried at ambient or elevated temperature.

The obtained product of the synthesis reaction was characterized by X-ray diffraction (XRD) and by means of Fourier transform infrared spectrophotometry (FTIR).

The figure shows:

Fig. 1 - FTIR spectrum of the resulting product with respect to lecithin and commercial hydroxyapatite. The first spectrum is for lecithin, the second is for commercial crystalline hydroxyapatite, and the third is for nano-hydroxyapatite obtained according to the invention.

Fig. 2 - crystallographic characteristics of the obtained product, consistent with the data in the JCPDF file No. 09-0432, in the form of an XRD pattern

Fig. 3 - SEM photo of the product with nanoplate morphology obtained according to example 1.

Fig. 4 - SEM photo of the product with the morphology of nanowires and nanospheres, obtained according to example 2.

Fig. 5 - SEM photo of the product with nanosphere morphology with diameters from about 30 nm to about 70 nm, obtained according to example 3.

Fig. 6 - SEM photo of the product with nanosphere morphology with a diameter of about 30 nm to 100 nm, obtained according to example 4.

PL 229 015 B1

In the method according to the invention, the natural surfactant - lecithin, consisting mainly of phosphatidylcholines, phospholipid compounds with phosphate-bound choline, a component of the cell membrane, mitochondria, endoplasmic reticulum and cell nucleus wall - surrounds the emerging nuclei of hydroxyapatite crystals and blocks the possibility of further crystal growth. Such a reaction system allows the crystallization of hydroxyapatite to form nanoparticles with a morphology dependent on the concentration of the dispersing agent - lecithin - in the reaction system. In the process of formation of hydroxyapatite nanoparticles, lecithin is partially bound to the product or it is physically bound in the pores of the particles being formed. In the present invention, such a situation is regarded as advantageous, increasing the biocompatibility and bioactivity of the material.

The concentration of lecithin influences the morphology of the obtained product, which allows to control the shape of the obtained nanoparticles.

The use of lecithin as an emulsifier allows the process to be carried out in an aqueous solution and under relatively mild conditions. Lecithin is completely non-toxic, and its small residue in the obtained product does not cause negative effects, but improves the wettability of the obtained product, which increases its usability and facilitates the formation of bone implants from such powder. Lecithin is a component of the cell membrane, which increases the biocompatibility of the nanohydroxyapatite obtained in this way.

The method according to the invention is presented in more detail in the application examples.

P r z k ł a d 1

0.0075 moles (1.775 g) of Ca (NO3) 2 4H2O were dissolved in 25 ml of demineralized water. A 0.3% solution of lecithin was prepared in 37.5 ml of demineralized water. The lecithin solution was added dropwise to the calcium nitrate tetrahydrate solution. The pH of the resulting reaction mixture was adjusted to 10 and the mixture was set in a water bath to 60 ° C and stirred. A solution of 0.0046 mol (0.594 g) (NH4) 2HPO4 in demineralized water adjusted to pH 10 was added dropwise to the reaction mixture at a rate of 2 ml / h. After the dropwise addition, the reaction mixture was stirred for 1.5 hours at the reaction temperature of 60 ° C. C. Then the mixture was naturally cooled to ambient temperature, subjected to centrifugation, decantation. The resulting white product was washed five times with hexane to remove residual lecithin. The morphology of the obtained product was characterized by means of scanning electron microscopy (SEM) and is presented in Fig. 3. The obtained product with the morphology of nanoplatelets was several nanometers thick, and several to several dozen micrometers wide and long.

P r z k ł a d 2

0.0075 moles (1.775 g) of Ca (NO3) 2 4H2O were dissolved in 25 ml of demineralized water. A 1.5% solution of lecithin was prepared in 37.5 ml of demineralized water. The lecithin solution was added dropwise to the calcium nitrate tetrahydrate solution. The pH of the resulting reaction mixture was adjusted to 10 and the mixture was set in a water bath to 60 ° C and stirred. 15 ml of a solution of 0.0046 mol (0.594 g) (NH4) 2HPO4 in demineralized water adjusted to pH 10 at a rate of 2 ml / h was added dropwise to the reaction mixture. After dropwise addition, the reaction mixture was stirred for 1.5 hours at the reaction temperature of -60 ° C. Then the mixture was naturally cooled to ambient temperature, subjected to centrifugation, decantation. The resulting white product was washed five times with hexane to remove residual lecithin. The morphology of the obtained product was characterized by scanning electron microscopy (SEM) and is presented in Fig. 4. A product with the morphology of nanowires and nanospheres was obtained. Nanowires with a length of about 30 nm to about 180 nm and nanospheres with diameters from about 30 nm to 70 nm were obtained.

P r z k ł a d 3

0.0075 moles (1.775 g) of Ca (NO3) 2 4H2O were dissolved in 25 ml of demineralized water. A 3% solution of lecithin was prepared in 37.5 ml of demineralized water. The lecithin solution was added dropwise to the calcium nitrate tetrahydrate solution. The pH of the resulting reaction mixture was adjusted to 10 and the mixture was set in a water bath to 60 ° C and stirred. 15 ml of a solution of 0.0046 mol (0.594 g) (NH4) 2HPO4 in demineralized water adjusted to pH 10 at a rate of 2 ml / h was added dropwise to the reaction mixture. After dropwise addition, the reaction mixture was stirred for 1.5 hours at the reaction temperature of -60 ° C. Then the mixture was naturally cooled to ambient temperature, subjected to centrifugation, decantation. The resulting white product was washed five times with hexane to remove residual lecithin. The morphology of the obtained product was characterized by electron means

It was obtained from scanning microscopy (SEM) and shown in Fig. 5. The obtained product had nanosphere morphology with diameters from about 30 nm to about 70 nm.

P r z k ł a d 4

0.0075 moles (1.775 g) of Ca (NO3) 2 4H2O were dissolved in 25 ml of demineralized water. A 9% solution of lecithin in 37.5 ml of demineralized water was prepared. The lecithin solution was added dropwise to the calcium nitrate tetrahydrate solution. The pH of the resulting reaction mixture was adjusted to 10 and the mixture was set in a water bath to 60 ° C and stirred. 15 ml of a solution of 0.0046 mol (0.594 g) (NH4) 2HPO4 in demineralized water adjusted to pH 10 at a rate of 2 ml / h was added dropwise to the reaction mixture. After dropwise addition, the reaction mixture was stirred for 1.5 hours at the reaction temperature of -60 ° C. Then the mixture was naturally cooled to ambient temperature, subjected to centrifugation, decantation. The resulting white product was washed five times with hexane to remove residual lecithin. The morphology of the obtained product was characterized by scanning electron microscopy (SEM) and is presented in Fig. 6. The obtained product was nanospheres morphology with a diameter of about 30 nm to 100 nm.

P r z k ł a d 5

A 0.0075 mole portion of the hydrated calcium chloride was dissolved in 30 ml of demineralized water. A 10% solution of lecithin in 40 ml of deionized water was prepared.

The lecithin solution was added dropwise to the calcium chloride solution. The pH of the resulting reaction mixture was adjusted to 9, and the mixture was set in a water bath to 50 ° C and stirred. 15 ml of a solution of 0.0046 mol sodium phosphate in demineralized water having a pH adjusted to 9 was added dropwise to the reaction mixture. After the dropwise addition, the reaction mixture was stirred for one hour at a reaction temperature of 50 ° C. The mixture was then naturally cooled to ambient temperature and vortexed. The resulting white powder was washed twice with 95% ethanol to remove excess lecithin. A product with the morphology of nanospheres with diameters from 30 nm to 110 nm was obtained.

Claims (7)

Patent claims 1. The method of obtaining nanoparticles of hydroxyapatite by the wet chemical precipitation method, which consists in reacting a water-soluble calcium salt and a water-soluble phosphate in an aqueous solution, with the molar ratio of calcium atoms to phosphorus atoms being approximately Ca / P 1.67 and in the presence of a dispersant, characterized in that lecithin is used as the dispersant in an amount of 0.05 wt.%. % to 20.00 wt.%, the reaction is carried out at a temperature ranging from 20 ° C to 95 ° C, at a pH from 7 to 14. 2. The method according to p. 1, characterized in that the source of calcium ions Ca ² + in the reaction system used calcium nitrate, calcium acetate, calcium chloride, calcium lactate. 3. The method according to p. The process according to claim 1, characterized in that ammonium, sodium and potassium phosphates are used ^{as the source of PO4 3- phosphate in the reaction system.} 4. The method according to p. A method according to claim 1, characterized in that the lecithin of natural origin is used, preferably soy or chicken egg lecithin. 5. The method according to p. The method of claim 1, wherein the lecithin is added at a concentration of 0.05 wt.%. % up to 1.5 wt.% in order to obtain nanoplates. 6. The method according to p. The method of claim 1, wherein the lecithin is added at a concentration of 1.5 wt.%. % to about 3 wt.%. in order to obtain nanowires. 7. The method according to p. The method of claim 1, wherein the lecithin is added at a concentration of 3 wt.%. up to 20 wt.% to obtain nanospheres.