SE535259C2 - Nanoparticle of an Ion-Substituted Calcium Phosphate Compound for Use in Biomedical Applications - Google Patents

Description

20 25 30 535 259 2 nanoparticles, which means a high risk of production failure.

In addition, the surfactants need to be removed from the particles so as not to alter the biological response of the nanoparticles, i.e. risk of contamination. In addition, the surfactants can only be easily used for a certain shape and porosity of the nanoparticles.

Resorbable nanoparticles (i.e. particles that can be dissolved in vivo) are of particular interest for a number of applications, e.g. 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]. Hydroxyapatite 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 part [13], and photoelectric reagent [14] etc. The shape and composition of the described HAs are limited to the rods and fl sheets and the described composition of the nanoparticles has been HA or carbonized HA. It is obvious that the application of fl ak- and rod-like hydroxyapatite in drug delivery, catalyst support and ion adsorption / exchange agents is limited by limited morphology of the particles produced. Hydroxyapatite in bone is a multi-substituted calcium phosphate, including traces of CO32, F, Cl, Mg2 *, Sr2 *, Si4 +, Zn2 *, Ba2 *, Fef important role in bone formation and normal functions such as the solubility, surface chemistry and morphology of the material.There has been significant research interest in the ion-substituted hydroxyapatites but ion-substituted nanopathic apatites with controlled morphology have not been described previously.Such apatite structures Methods for making pure HA powders, spherical granules and bulk materials have been described in the prior art, such as 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, 1 7, 5824-5830.

To date, no method for producing spherical nanoparticles using a surfactant-free process has been described in the prior art. 10 15 20 25 30 535 259 Fluoride is found in bones and teeth in the bodies of vertebrates. It has been reported that the substitution of fluoride for OI-I positions and the formation of fluoride-substituted hydroxyapatite increase the acid resistance and mechanical properties of hydroxyapatite biocerams [18], and induce better biological response [19].

Silicon has been shown to be essential for normal bone and cartilage growth and development.

Synthetic hydroxyapatite, which contains trace amounts of silicon (Si) in its structures, shows markedly 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 physiological processes of the bone and connective tissue systems.

Silicon substitution promotes biological activity by converting the material surface to 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 stimulatory effects on cells of the bone and cartilage tissue systems [20].

Because Strontium is chemically and physically closely related to calcium, it is easy to introduce as a natural substitute for calcium in hydroxyapatite.

Strontium has been shown to have the effects of increased bone formation and reduced bone resorption, leading to increased bone mass and improved mechanical properties in bones in animals and humans. Strontium-substituted hydroxyapatite ceramics show better mechanical properties than pure hydroxyapatite, and increase the proliferation and differentiation of osteoblast cells in an in vitro study [2 1].

Our contemporary Swedish patent application No. 0900560-4 describes a process for the formation of a crystalline coating of an ion-substituted calcium phosphate on a substrate.

The present application is an object to provide a process for producing porous hydroxyapatite nanoparticles, in particular nanospheres, with ion substitution via a surfactant-free biomineralization process, the material and its use in biomedical applications and as drug carriers. The invention relates to the described difficulties with nanoparticles grown using surfactants, i.e. difficult production process, risk of contamination and limitation in morphology. The invention further relates to a process for an ion delivery material.

Description of the Invention The present invention particularly describes a process for the production of nanoparticles of ion-substituted HA with controlled morphology. The invention further describes 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 solution regions. This surfactant-free self-organizing process takes place in one solution and the dynamic rate of formation can be controlled by temperature, pH, composition and ion concentration. The invention provides a new technique for producing nanospheres of hydrodapatite, ion-substituted apatite (Sr, Si, F). This new technology is a surfactant-free self-organized process that does not require 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 T, HPO 2, Na +, K *, I-ICOf, Cl 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 wherein Ca: P is 1:10, that the solution has an initial pH in the range 2.0 to 10.0, preferably a pH between 6.0 and 8.0, - that the solution further comprises one of the ions Sr2 +, F- or Si4 +, and -that growth and self-organization of the nanoparticles takes place in the solution according to a , bc or d where: a. is a static or stirring process in which the solution comprises Sr is a hydrotennal process where l the solution comprises Sr 2 * in a concentration of 0.3 mM to 0.67 mM and where the process is carried out between 60 ° C and 100 ° C and gives spherical particles with a dense shell structure and a porous interior, c. concentration of 0.04mM to 0.22mM and where the process is carried out between 80 and 100 ° C and gives spherical porous particles, and d. is a hydrothermal process where the solution comprises Si4 + at a concentration of 6mM to 10mM and where the process is carried out between 80 and 100 ° C and gives spherical porous particles.

In preferred embodiments of the invention, the concentration of calcium ions may be in the range of 0.01-25 10-3M, the concentration of magnesium ions may be in the range of 0.0 1- 10 10M, the concentration of sodium ions may be in the range of 0.01-1420 10 -3M, the concentration of potassium ions may be in the range of 0.01-1420 10-3M, the concentration of chloride ions may be in the range of 0.01-1030 10-3M, the concentration of phosphate ions may be in the range of 0.01-10-3M , the concentration of carbonates can be in the range 0.01-270 10 ~ 3M, the concentration of sulphate ions can be in the range 0.0 1-5 10 ~ 3M, the concentration of ions to be substituted is lower than 0.6 10-3M for Sr 2+, 10 10 -3 M for Si 4 * and 0.2 10 ~ 3 M for F: The diameter of the obtained hollow / porous nanospheres is 10-500 nm. The morphology of the nanospheres can be hollow, porous and porous nuclei with a dense shell. The smallest unit can be fl similar nanoparticles or needle-like nanoparticles that together form a larger particle (hair described as a nanosphere). Thus, in the nanospheres formed, they are not single crystals but formed by 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 range of nanoparticles that can be produced with the invention, your applications can be foreseen. (1) Drug delivery system with sustained and controlled release behavior. Non-limiting examples of drugs include antibiotics, anti-inflammatory, proteins, cancer-treating and drugs for the treatment of pain. The loading of the drug can be done using any method known to those 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 the methods known to those skilled in the art. Two non-limiting examples involve soaking the particles in blood plasma and densifying it into the defect or delivering the particles using a carrier liquid or gel via a syringe. (3) Filling particles in injectable biomaterials, where non-limiting examples include: PMMA bone cement, bioceramics (eg calcium phosphates, calcium sulphates), glass ionomer cement, (4) Heavy metal ion adsorption regeneration, (5) Gene delivery systems, (6) Densensitization of dentin tubulis. The N anospheres are suitable for filling the open dentin tubulis.

Sample Figure Figure 1 illustrates the morphology of SrHA after treatment at 60 ° C for one week using 0.06 mM Sr-doped PBS, Figure 2 illustrates the morphology of SrHA after treatment at 60 ° C. ° 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 by use of 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 agitation 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 part, some examples of the invention will be described more in the with reference to the drawings.

Example 1: Preparation of porous strontium-substituted HA nanospheres (static process) Porous hydroxyapatite with strontium-substituted nanospheres was prepared from an Sr-doped supernatant phosphate solution containing Ca , Mg ”. To resemble a body fluid sounding, the pH of the Sr-doped phosphate buffered saline was checked to 7.4 before the next treatment.

The initial ratio of Sr2 *: Ca2 *: 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 progress of the crystals' growth and self-organization, the treatment was carried out 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 changed with the altered concentration of strontium ion in 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 with a size of about 1 μm (fi g. 1). After the initial concentration of Sr in the PBS solution is increased to 0.15 mM, the sphere size decreases to 100-300 nm and the morphology appears spherical with a hollow core and a porous shell (fi g. 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 (fi g. 3). Even if 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 (fi g. S).

Example 2: Pelletization of porous strontium-substituted HA nanospheres (stirring process) 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 that is more uneven than that from the non-stirring process and a particle size of about 200-400 nm. 10 15 20 25 30 535 259 I fi g. 7, the morphology of SrHA after stirring at 60 ° C is illustrated using 0.6 mM Sr-doped PBS for one day.

Example 3: Preparation of porous strontium-substituted HA nanospheres (hydrothermal process) 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. Via a hydrothermal process, the production time is greatly shortened from one week to one hour.

I fi g. 8, the morphology of SrHA after hydrothermalization at 100 ° C is illustrated using 0.6 mM Sr-doped PBS for one hour.

Example 4: Preparation of porous fluoride-substituted HA nanospheres (hydrothermal process) Porous hydroxyapatite with fluoride-substituted nanospheres was prepared from an F-doped supersaturated phosphate solution containing Ca 2+, HPO a body fluid (void sounding), the pI-I value of the F-doped phosphate-buffered saline was checked to 7.4 before the next treatment.

The initial ratio of F-zCa2 fl HPO42- was x: 1: 10 (x: from 0 to 0.22). Hydroxyapatite with chloride substitution crystallized, grew and self-organized in the supersaturated solution. The hydrothermalization was performed 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.

I fi g. 9, the morphology of FHA after hydrothermalization at 100 ° C is illustrated using 0.04 mM (fi g. 9a) and 0.2 mM (fi g. 9b) F-doped PBS for 24 hours. Example 5: Porous hydroxyapatite with silicate-substituted nanospheres Porous hydroxyapatite with silicate-substituted nanospheres was prepared from a Sy-doped supersaturated phosphate solution containing Ca 2+, HPO 4 23 Na *, K *, Cl-, Mg To resemble a 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-: Ca2 *: 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 crystal 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.

I fi g. The morphology of SiI-IA after hydrothermalized at 100 ° C using 6 mM Si-doped PBS for 24 hours is illustrated. 10 15 20 25 30 535 259 ll References l. Ozin GA. Morphogenesis of Biomineral and Morphosynthesis of Biomimetic Forms. Acc Chem Res 1997; 30: 17-27. 2. Dujardin E, Mann S. Bio-inspired Materials Chemistry. Adv Mater 2002; 141775-788. 3. Mann S. Molecular tectonics in biomineralization and bjiomimetic chemistry.

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J Mater Chem 2004; 14: 2l24-2 147. 8. Vallet-Regi M, Gonzalez-Calbet JM. Calcium phosphates as a substitution of bone tissues. Progress in Solid State Chemistry 2004; 32: 1-31. 9. Dorozhkin SV, Epple M. Bioligical and medical significance of calcium phosphates.

Angew Chem Int Ed 2002; 4l: 3130-3146. 10. Oliveira AL, Reis RL, Li P. Strontium-substituted apatite coating grown on Ti6A14V substrate through biomimetic Synthesis. J Biomed Mater Res Part B 2007; 83: 258-265. 1 1. Kumta PN, Sfeir C, Lee DH, Olton D, Choi D. Nanostructured calcium phosphates for biomedical applications: novel synthesis and characterization. Acta Biomaterials 2005; 1: 65-83. 12. Ho CM, Yu WY, Che CM. Ruthenium Nanoparticles Supported on Hydroxyapatite as an Efficient and Reeyclable Catalyst for Cis-Dihydroxylation and Oxidative Cleavage of Alkenes. Angew Chem Int Ed 2004; 43: 3303-3307. 13. Hamrnañ LEL, Laghzizil A, Barboux P, Lahlil K, Saoiabi A. Retention of or uoride ions from aqueous solution using Porous hydroxyapatite: Structure and conduction properties. J Hazard Mater 2004; 1 14: 41-44. 14. Yuki I, Yokomizo Y. Inorganic phosphate materials. Sensa Gyntsn 1981; 1: 23- 27. 10 15 535 259 12 15. M. Vallet-Regi JMGC. Calcium phosphate as a substitution of bone tissues. Progress in Solid State Chemistry 2004; 32: 1-31. 16. S.V. Dorozhkin ME. Bioligical and medical significance of calcium phosphates.

Angew Chem Int Ed 2002; 41: 3130-3146. 17. A.L. Oliveira RLR, P Li. Strontium-substituted apatite coating grown on Ti6A14V substrate through biomimetic Synthesis. J Biomed Mater Res Part B 2007; 83: 258-265. 18. KA. Gross LMR-L. Sintered hydroxy fl uorapatites. Part I: Sintering ability of precipitated solid solution powders. Biomatcrials 2004; 25: 1375-1384. 19. C. Robinson RCS, S.J. Brookes, S. Strafford, S.R. Wood, J. Kirkham. The Chemistry of Enamel Caries. Crit Rev Oral Biol Med 2000; 1 1:48 1-495. 20. A.M. Pietak JWR, M.J. Stott, M. Sayer. Silicon substitution in the calcium phosphate bioceramics. Biomatcrials 2007; 28: 4023-4032. 21. E. Landi AT, G. Celotti, S. Sprio, M. Sandri, G. Logroscino. Sr-substituted hydroxyapatite for osteoporotic bone replacement. Acta Biomatcrials 2007; 3: 961- 969.

Claims (9)

10 15 20 25 30 535 259 | 3 PATENT REQUIREMENTS A process for the preparation of 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 wherein Ca: P is 1:10, - that the solution has an initial pH in the range 2.0 to 10.0, preferably a pH between 6.0 and 8.0, -that the solution further comprises one of the ions Sr2 *, F- or Si4 *, and -that 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 solution comprises Sr 2+ in a concentration of 0.3 mM to 0.67 mM and that the solution has a temperature in the range 37-60 ° C which gives spherical hollow particles, b. is a hydrothermal process where the solution comprises Sr2ti 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 having a dense shell structure and a porous interior, c. is one hydrothermal process where the solution comprises Fi a concentration of 0.04 mM to 0.22 mM and where the process is carried out between 80 and 100 ° C and gives spherical porous particles, and d. is a hydrothermal process where the solution comprises Si * in a concentration of 6 mM to 10 mM and where the process is carried out between 80 and 100 ° C and gives spherical porous particles. A method according to claim 1, wherein the formed nanoparticles have a diameter of 10-500 nm. A process according to any one of claims 1 or 2, wherein the concentration of calcium ions is in the range 0.01-25 x 10 ~ 3M. A process according to any one of claims 1 or 2, wherein the concentration of magnesium ions is in the range of 0.01-15 x 10-BM. 10 15 20 25 10. 11. 12. 13. 14. 1 535 259 IH A process according to any one of claims 1 or 2, wherein the concentration of sodium ions is in the range 0.01-1420 x 10-3M. A process according to any one of claims 1 or 2, wherein the concentration of potassium ions is in the range 0.01-1420 x 10 -3 M. A process according to claim 1 or 2, wherein the concentration of chloride ions is in the range 0.01-1030 X 10 -3 M. A process according to claim 1 or 2, wherein the concentration of phosphate ions is in the range 0.01-10 x 10 -3 M. A process according to claim 1 or 2, wherein the solution further comprises carbonate ions in the range 0.01-270 x 10 ~ 3M. A process according to claim 1 or 2, wherein the solution further comprises sulphate ions in the range 0.01-5 x 10 -3M. A method according to claim 1, wherein the concentration of Sri 'in the solution is 0.3 mM or more and less than 0.6 mM, preferably about 0.3 mM, whereby regular spherical Sr-substituted nanoparticles with a diameter of 200-500 nm , where each nanoparticle has a hollow core and a porous shell, is formed. A method according to claim 1, wherein the concentration of Sr nanoparticle has a dense shell and a porous core, formed. A method according to claim 1, wherein the concentration of F- in the solution is about 0.04-0.2 mM, whereby spherical porous F-substituted nanoparticles with a diameter of 300-500 nm are formed. A process according to claim 1, wherein the concentration of Si * is about 6 mM, whereby porous Si-substituted nanoparticles with a size of 200-500 nm are formed. A nanoparticle obtained by the process according to any one of claims 1 to 14. 1 6. 1 7. 1 8. 1 A nanoparticle according to claim 15, wherein the diameter of the nanoparticle is 10-500 IIIII. A nanoparticle according to claim 15, wherein the nanoparticles are spherical porous Sr-substituted nanoparticles with a diameter of 100-300 nm and with a hollow core and a porous shell. A nanoparticle according to claim 15, wherein the nanoparticles are regular spherical Sr-substituted nanoparticles with a diameter of 200-500 nm and with a hollow core and a porous shell. A nanoparticle according to claim 15, wherein the nanoparticles are Sr-substituted spherical nanoparticles with a diameter of 100-500 nm and with a dense shell and a porous core. A nanoparticle according to claim 15, wherein the nanoparticles are spherical porous F-substituted nanoparticles with a diameter of 300-500 nm. A nanoparticle according to claim 15, wherein the nanoparticles are porous Si-substituted nanoparticles with a size of 200-500 nm. A nanoparticle according to any one of claims 15-21 for use in a drug delivery system. Nanoparticle according to one of Claims 15 to 21 for use in bone and tooth repair and generation, respectively. A nanoparticle according to any one of claims 15-2 l for use in desensitizing the dentin tubulis.