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(54) Title: Nanoparticle of an Ion-Substituted Calcium Phosphate Compound for Use in Biomedical Applications (56) Published Publications: WO 2007137606 Al · WO 2005082780 Al · GB 2316940 A · HOFFMANN, I. et al: Precipitation of Carbonated Calcium Phosphate Powders from a High SBF Solution, In: Key Engineering Materials, 2007, Vols. 330-332, pp. 59-62, ISSN 1013-9826. · GOLDEN, DC et al: Nutrient-Substituted Hydroxyapatites: Synthesis and Characterization, In: Soil Science Society of America Journal, 1999, Vol. 63, No. 3, pp.657-664, ISSN 0361-5995. WEERAPAT, PO. et al: Substitution of Manganese and Iron into Hydroxyapatite: Core / Shell Nanoparticles In: Materials Research Bulletin, 2008, Vol. 43, pp. 2137-2144, ISSN 0025-5408. · OKAZAKI, M. et al: Differences in Solubility of Two Types of Heterogeneous Fluoridated Hydroxyapatites, In: Biomaterials, 1998, Vol. 19, pp. 611-616, ISSN 0142-9612. · TAS, AC et al: Preparation of Zn-doped B-tricalcium Phosphate (Ca3 (PO4) 2) Bioceramics In: Materials Science and Engineering, Part C, Vol. 27, pp. 394-401, ISSN 0925-4931. · BIGI, A. et al: Strontium-Substituted Hydroxyapatite Nanocrystals, In: Inorganica Chimica Acta, 2007, Vol. 360, pp. 1009-1016, ISSN 0020-1693. · JHA, LJ et al: Preparation and Characterization of Fluoride-Substituted Apatites, In: Journal of Materials Science: Materials in Medicine, 1997, Vol. 8, pp. 185-191, ISSN 0957-4530. · MANJUBALA, I. et al: Synthesis and Characterization of Hydroxy / Fluoroapatite Solid Solution, In: Journal of Materials Science, 2001, Vol. 36, pp. 5481-5486, ISSN 0022-2461. · SANDIN K. et al: Formation of carbonated apatite particles from a supersaturated inorganic blood serum model, Included in: Journal of Materials Science: Materials in Medicine, 2009, Vol. 20, no. 8, p. 1677-1687. · MEDVECKY L. et al: Influence of manganese on stability and particle growth of hydroxyapatite in simulated body fluid, Included in: Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2006, Vol. 281, no. 1-3, p. 221-229.

(47) Summary:

A process for producing spherical nanoparticles of hydroxyapatite and ion-substituted hydroxyapatite. The process is based on precipitation of particles from a buffered solution under static, stirring or hydrothermal conditions. The invention further relates to the use of the formed materials and the material itself.

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Summary

A process for producing spherical nanoparticles of hydroxyapatite and ion-substituted hydroxyapatite. The process is based on precipitation of particles from a buffered solution under static, stirring or hydrothermal conditions. The invention further relates to the use of the formed materials and the material itself.

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Nanoparticle of an Ion Substituted Calcium Phosphate Compound for Use in Biomedical Applications

Technical area

The present invention relates to a biomaterial system and to a process for the manufacture thereof. In particular, the invention relates to a process for producing nanoparticles of ion-substituted hydroxyapatite (HA) with controlled morphology. The invention also relates to such a material composition for use in biomedical applications.

Background of the Invention

Biomineralization is a natural self-assembly process for the preparation of nanomaterials of varied morphology with or without biomolecules as a structural conducting aid [1-3] For example, the nucleation and growth of hydroxyapaptite (Caio 2, HA), found in bones and teeth, is controlled by some specific biomolecules that induce the formation of hierarchical structure [4]. Mimicking biological behavior to produce materials with specific morphologies and structures has attracted increased attention due to its unique physical, chemical and biological properties and potential applications in advanced functional materials. Now on the preparation of nanomaterials with different morphologies such as spheres, fibers and rods, nucleation structure and mesoporous structure similar to the biomineralization process are focused on self-organization achieved through the use of surfactants and biomolecules [5], the surfactants guide and stabilize the morphologies of the nanomaterials. If there are no surfactants that contribute to the growth of the crystals, the crystals will grow along in a specific plane with the lowest surface tension and the morphology cannot be controlled during a biomineralization process. For example, hydroxyapatite grows spontaneously as flakes or fibers / rods oriented along the crystal c-axis from a supersaturated solution [6]. Self-organization manipulation of the hierarchical architecture of biominerals without surfactants at microscale and nanoscale levels is still not possible. It is known that surfactants can selectively inhibit the growth of crystals along a specific axis and then alter its evolution of morphologies [5, 7]. The use of surfactants leads to many steps in the production of

535 259 nanoparticles, which means a high risk of production failure. In addition, the surfactants need to be removed from the particles in order not to change the biological response of the nanoparticles, ie the risk of contamination. In addition, the surfactants can only be readily used for a certain shape and porosity of the nanoparticles.

Resorbable nanoparticles (ie, dissolvable particles in vivo) are of particular interest for a number of applications, such as fillers for bone cavities, drug carriers, desensitization of dentin tubules.

Natural bone mineral is a multi-substituted hydroxyapatite (HA) that is bioactive, biocompatible and osteoconductive [8 - 10]. Hydroxyapatitis has been widely used in bone healing. In addition, hydroxyapatite has also been used in drug / gene delivery [8, 11], catalyst [12], ion adsorption / exchange agent [13], and photoelectric reagent [14] etc. The shape and composition of the HA described are limited to the rods and flakes and the described composition of the nanoparticles has been HA or carbonized HA. It is obvious that the application of flake and rod-like hydroxyapatite in drug delivery, catalyst support and ion adsorption / exchange agents is limited by the limited morphology of the particles produced. Hydroxyapatite in bone is a multisubstituted calcium phosphate, containing traces of CO3²-, F-, Cl ·, Mg²⁺, Sr²⁺, Si⁴⁺, Zn²⁺, Ba²⁺, Fe³⁺, etc. [15-17]. These ionic substitutions play an important role in bone formation and normal functions such as the solubility, surface chemistry and morphology of the material. There has been a significant research interest in the ion-substituted hydroxy apatites, but non-ion-controlled ion-substituted apatites with controlled morphology have not been described previously. Such apatite structures can be used to promote bone growth in bone defects, in fillers for cavities in bone, in dental applications and in drug carriers. Methods for producing pure HA powders, spherical granules and bulk materials have been described in the prior art, e.g. US 5858318, US 7326464, US 5702677 and "Morphologically Controlled Synthesis of Hydroxyapatite with Partial Substitution of Fluorine", Hui Gang Zhang, Qingshan Zhu, Yong Wang, Chem. Mater. 2005, 17, 5824-5830.

To date, no method of producing spherical nanoparticles using a surfactant-free process has been described in the prior art.

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Fluoride is found in bones and teeth in the bodies of vertebrates. It has been reported that the substitution of fluoride for OH positions and formation of fluoride-substituted hydroxyapatite enhances the oxygen resistance and mechanical properties of hydroxyapatite biokerams [18], and induces better biological response [19].

Silicon has been found to be essential for normal bone and cartilage growth and development. Synthetic hydroxyapatite, which includes trace amounts of silicon (Si) in its structures, exhibits significantly increased biological performance compared to stoichiometric hydroxyapatite [20]. The improvement in biological performance can be attributed to the Si-induced changes in the material properties and also to the direct effects of Si in the physiological processes of the bone and connective tissue systems. Silicon substitution promotes biological activity by converting the material surface into a biologically equivalent hydroxyapatite by increasing the solubility of the material by generating a more electronegative surface and by creating a finer microstructure. Release of Si complexes to the extracellular medium and the presence of Si at the material surface can induce additional dose-dependent stimulation effects on cells of the bone and cartilage tissue systems [20].

Since strontium is chemically and physically closely related to calcium, it is easy to introduce as a natural substitution for calcium in hydroxyapatite. Strontium has been proven to have the effects of increased bone formation and reduced bone resorption leading to increased bone mass and improved mechanical properties of bones in animals and humans. Strontium-substituted hydroxyapatite ceramics exhibit better mechanical properties than pure hydroxyapatite, enhancing the proliferation and differentiation of osteoblast cells in an in vitro study [21].

In our contemporary Swedish patent application no. 0900560-4, a process for the formation of a crystalline coating of an ion-substituted calcium phosphate on a substrate is described.

In the present application, an object is to provide a process for producing porous hydroxyapatite nanoparticles, especially nanospheres, with ion substitution via a

535 259 surfactant-free biomineralization process, the material and its use in biomedical applications and as drug carriers. The invention relates to the described nanoparticle growth difficulties when using surfactants, ie difficult production process, risk of contamination and restriction in morphology. The invention further relates to a process for an ion-supply material.

Description of the invention

In particular, the present invention discloses a process for producing nanoparticles of ion-substituted HA with controlled morphology. The invention further discloses a material composition which can be used in biomedical applications as described below.

The manufacturing process is based on a self-organizing process without the use of surfactants to produce hollow or porous nanoparticles or combinations thereof. The structure of the nanospheres can be controlled by adjusting the concentration of substitution ions in the growth solutions. This surfactant-free self-organizing process takes place in a solution and the dynamic formation rate can be controlled by temperature, pH, composition and ion concentration. The invention provides a new technique for producing nanospheres of hydroxyapatite, ion-substituted apatite (Sr, Si, F). This new technology is a surfactant-free soul-organized process that does not need template management.

The bio-like process uses supersaturated phosphate buffered solution containing calcium ion and other substitution ions (Sr, Si and F). This buffered solution contains ions such as Ca.²⁺, HPO4², Na⁺, K⁺, HCO3-, Cl ·, Mg²⁺, SO4²-, Sr²⁺, SiO₃²', Zn²⁺, Ba²⁺, Fe³⁺ and F-.

Accordingly, the present invention relates to a process for preparing nanoparticles of an ion-substituted calcium phosphate compound comprising the steps of:

- providing an aqueous solution comprising calcium ions, magnesium ions, sodium ions, potassium ions, chloride ions and phosphate ions and where Ca: P is 1:10,

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the solution has an initial pH in the range of 2.0 to 10.0, preferably a pH between 6.0 and 8.0,

the solution further comprising one of the ions Sr²⁺, F or Si⁴⁺, and that the growth and self-organization of the nanoparticles takes place in the solution according to a, b, c or d where:

a. is a static or stirring process where the locking comprises Sr²⁺ at a concentration of 0.3mM to 0.67mM and that the solution has a temperature in the range of 37-60 ° C which gives spherical hollow particles,

b. is a hydrothermal process wherein the solution comprises Sr²⁺ at a concentration of 0.3mM to 0.67mM and where the process is carried out between 60 ° C and 100 ° C to give spherical particles with a dense shell structure and a porous interior,

c. is a hydrothermal process wherein the solution comprises F- at a concentration of

0.04mM to 0.22mM and where the process is carried out between 80 and 100 ° C to produce spherical porous particles, and

d. is a hydrothermal process wherein the solution comprises Si⁴⁺ at a concentration of 6 mM to 10 mM and where the process is carried out between 80 and 100 ° C to give spherical porous particles.

In preferred embodiments of the invention, the concentration of calcium ions may be in the range of 0.01-25. ³M, the concentration of magnesium ions can be in the range 0.01-15 ³M, the concentration of sodium ions can be in the range of 0.01-1420³M, the concentration of potassium ions can be in the range of 0.01-1420³M, the concentration of chloride ions can be in the range 0.01-1030 ³M, the concentration of phosphate ions can be in the range of 0.01-10 ³M, the concentration of carbonate ions can be in the range 0.01-270-10³M, the concentration of sulfate ions can be in the range of 0.01-5 ³M, the concentration of ions to be substituted is less than 0.6 ³M for Sr²⁺, 10

10-³M for Si⁴⁺ and 0.2 ³M for F-.

The diameter of hollow / porous nanospheres obtained is 10-500 nm. The morphology of the nanospheres can be hollow, porous and porous core with a dense shell. The smallest unit may be flake-like nanoparticles or similar nanoparticles that together form a larger particle (herein described as a nanosphere). Thus,

535 The formed nanospheres are not single crystals but are formed by several smaller units of ion-substituted HA. The self-organizing process can be in a static process, a stirring process and a hydrothermal process. The formation time is typically within 1 hour to 4 weeks but longer or shorter formation times can also form particles.

Based on the wide selection of nanoparticles that can be produced by the invention, several applications can be predicted.

(1) Drug delivery system with sustained and controlled release behavior. Non-limiting examples of drugs include antibiotics, anti-inflammatory, proteins, cancer treatment and pain management drugs. The loading of the drug can be done by using any method known to the person skilled in the art. Non-limiting examples include soaking and charging during the manufacture of the particles or a combination of the two.

(2) The nanospheres can be used in bone and tooth repair and regeneration. The particles can be delivered to the bone defect using methods known to those skilled in the art. Two non-limiting examples include soaking the particles in blood plasma and densifying it into the defect or delivering the particles using a carrier fluid or gel via a syringe.

(3) Fill particles in injectable biomaterials, where non-limiting examples include: PMMA bone cement, biocerams (e.g. calcium phosphates, calcium sulphates), glass ionomer cement, (4) Heavy metal ion adsorption regeneration, (6) Densensitization systems for dentin tubulitis. The nanospheres are suitable for filling the open dentin tubulis.

Description of the figures

Figure 1 illustrates the morphology of SrHA after treatment at 60 ° C for one week using 0.06 mM Sr-doped PBS.

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Figure 2 illustrates the morphology of SrHA after treatment at 60 ° C for one week using 0.15 mM Sr-doped PBS.

Figure 3 illustrates the morphology of SrHA after treatment at 60 ° C for one week using 0.3 mM Sr-doped PBS. The arrows in the figure illustrate hollow particles,

Figure 4 illustrates the morphology of SrHA after treatment at 60 ° C for one week using 0.6 mM Sr-doped PBS.

Figure 5 illustrates the morphology of SrHA after treatment at 60 ° C for two weeks using 0.6 mM Sr-doped PBS.

Figure 6 illustrates TEM images of SrHA particles obtained using 0.6 mM Sr-doped PBS at 60 ° C for one week.

Figure 7 illustrates the morphology of SrHA after stirring at 60 ° C using 0.6 mM Sr-doped PBS for one day,

Figure 8 illustrates the morphology of SrHA after hydrothermalized at 100 ° C using 0.6 mM Sr-doped PBS for one hour.

Figure 9 illustrates the morphology of FHA after hydrothermalized at 100 ° C using (a) 0.04 mM and (b) 0.2 mM F-doped PBS for 24 hours;

Figure 10 illustrates the morphology of SiHA after hydrothermalized at 100 ° C using 6 mM Si-doped PBS for 24 hours, and

Example

In the following, some examples of the invention will be described in more detail with reference to the drawings.

Example 1: Production of porous strontium-substituted HA nanospheres (static process)

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Porous hydroxyapatite with strontium-substituted nanospheres was prepared from a Sr-doped supersaturated phosphate solution containing Ca²⁺, HPO4²-, Na⁺, K⁺, Cl ·, Mg²⁺. To resemble body fluid sounding, the pH of the Sr-doped phosphate buffered saline solution was checked to 7.4 before the next treatment.²⁺: C²⁺: HPO4²'was x: 1: 10 (x: from 0 to 0.67).

Hydroxyapatite with strontium substitution crystallized, grew and self-organized in the supersaturated solution. To increase the course of crystalline growth and self-organization, the treatment was performed at 37 ° C or 60 ° C in a static oven.

The process resulted in Sr-substituted HA nanospheres with a different shell structure and a porous interior and a particle size of about 100-1000 nm.

The morphologies and structures of Sr-substituted HA nanospheres were changed with the changed concentration of strontium ion into a phosphate buffered solution. When the initial concentration of Sr in the PBS solution is 0.06 mM, the SrHA sphere is an irregular spherical porous particle of the size of about 1 µm (Fig. 1). After increasing the initial concentration of Sr in the PBS solution to 0.15 mM, the sphere size decreases to 100-300 nm and the morphology looks spherical with a hollow core and a porous shell (Fig. 2). When the Sr ion concentration is increased to 0.3 mM, the particles look like regular spheres with a hollow core and a porous shell and the size is about 200-500 nm (Fig. 3). Although the treatment time is increased to two weeks, the morphology does not change, and the particles do not grow and the size is the same as the result after one week (Fig. 5).

Example 2: Manufacture of porous strontium-substituted HA nanospheres (stirring process)

The same experimental procedure as in Example 1 but the solution was magnetically stirred in a water bath at 37 ° C or 60 ° C.

The process resulted in Sr-substituted HA nanospheres with a hollow core and a shell more uneven than that of the non-stirring process and a particle size of about 200-400 nm.

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In Figure 7, the morphology of SrHA after stirring at 60 ° C is illustrated using 0.6 mM Sr-doped PBS for one day.

Example 3: Production of porous strontium-substituted HA nanospheres (hydrothermal process)

The same experimental procedure as in Example 1 but the solution was placed in an autoclave at 60 ° C, 80 ° C or 100 ° C.

The process resulted in Sr-substituted HA nanospheres with a dense shell structure and a porous interior and a particle size of about 200-500 nm. Through a hydrothermal process, the production time is greatly reduced from one week to one hour.

In Figure 8, the morphology of SrHA after hydrothermalized at 100 ° C is illustrated using 0.6 mM Sr-doped PBS for one hour.

Example 4: Production of Porous Fluoride-Substituted HA Nanospheres (Hydrothermal Process)

Porous hydroxyapatite with fluoride-substituted nanospheres was prepared from a doped supersaturated phosphate solution containing Ca²⁺, HPCU²', Na⁺, K⁺, Cl ·, Mg²⁺. To resemble body fluid sounding, the pH of the F-doped phosphate buffered saline solution was checked to 7.4 before the next treatment.

Starting ratio of F-: Approx²⁺: HPO4²'was x: 1: 10 (x: from 0 to 0.22). Hydroxyapatite with fluoride substitution crystallized, grew and self-organized in the supersaturated solution. The hydrothermalization was carried out at 60 ° C, 80 ° C or 100 ° C in an oven.

The process resulted in porous F-substituted HA nanospheres and a particle size of about 300-500 nm.

In Fig. 9, the morphology of FHA after hydrothermalized at 100 ° C is illustrated using 0.04 mM (Fig. 9a) and 0.2 mM (Fig. 9b) F-doped PBS for 24 hours.

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Example 5: Porous hydroxyapatite with silicate-substituted nanospheres

Porous hydroxyapatite with silicate-substituted nanospheres was prepared from a Sidopad supersaturated phosphate solution containing Ca²⁺, HPO4²·, Na⁺, K⁺, Cl-, Mg²⁺. To resemble body fluid sounding, the pH of the silicate-doped phosphate buffered saline was checked to 7.4 before the next treatment. The initial ratio of SiO3²: C²⁺: HPO4²'was x: 1: 10 (x: from 0 to 10). Hydroxyapatite with silicate substitution crystallized, grew and self-organized in the supersaturated solution. To increase the course of crystalline growth and self-organization, we placed the solution in an autoclave at 80 ° C or 100 ° C.

The process resulted in porous Si-substituted HA nanoparticles and a particle size of about 200-500 nm.

In Fig. 10, the morphology of SiHA is illustrated after hydrothermalized at 100 ° C using 6 mM Si-doped PBS for 24 hours.

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