SE1551460A1 - Method for manufacturing of spherical calcium phosphate particles - Google Patents

Abstract

The present invention relätes to particles wherein the particle comprises 40-70 weight% of calcium, 20-40 vveight% of phosphate and 5-30 weight% magnesium, and wherein the Ca/P weight ratio is in the range of 1.10 to 1.90, and wherein more than 80 % of the particles have a particle size between 200 to lOOOnm, and method for preparing the same.

Description

Patent applicationMETHOD FOR MANUFACTURING OF SPHERICAL CALCIUM PHOSPHATE PARTICLESTECHNICAL FIELD OF THE INVENTION The present invention relates to a method of producing spherical calciumphosphate particles containing magnesium. The invention further relates to acomposition comprising said particles and its use in dental and medicalapplications.

BACKGROUND OF THE INVENTION Calcium phosphates (CaP) and in particular hydroxyapatite (Ca10(PO4)6(OH) 2,HA) are widely used in medical and dental applications due to its similarity tothe mineral components ofbone and teeth. lt is non-toxic, biocompatible andbioactive, which means that it is not harmful and not recognized as a foreignbody, resulting in positive effects on bone remodeling. Hence, HA has beenwidely used in bone repair and as drug or gene delivery vehicle, catalyst, ionadsorption/ exchange agent, photoelectric reagent, etc. Resorbable micro- ornanoparticles (i.e. particles that can be dissolved in vivo) are of special interestfor a number of applications, such as bone void fillers, drug delivery vehicles,desensitization agents of dentinal tubules, etc.

The morphology, structure, and size of CaP particles can influence theirproperties in above-mentioned applications. Particles with spherical shape andlarge pore volume are good candidates for drug delivery vehicles, protein andion adsorption, and bone and teeth fillers. Hence, they have recently attractedincreased attention. CaP, such as HA, spontaneously grow like flakes, fibers orrods by wet chemical methods. Spherical CaP has previously been preparedusing structure directing agents, such as ion substituents, surfactants andbiomolecules. A present inventor showed in WO201 1/016772 that strontiumions affected the morphology of CaP to form hollow spheres, and showed furtherin WO2014/148997 that CaP hollow spheres can also be formed in the absenceof strontium ions.

Not all morphologies are convenient to serve as drug delivery particles, catalystsupport, ion adsorption/ exchange agent, etc. To make a drug delivery processefficient, high surface areas and porous structures are advantageous to adsorb asmuch active substance as possible. There is also the requirement ofbiocompatibility and proper interaction between carrier and substance. Oneproblem for the preparation of CaP particles is to control their size distributionand shape. Often the size distribution is wide and caused by the hexagonalsymmetry and the lattice parameters of CaP. Most likely an orientation along thec-axis and therewith a pin-like shape occurs.

Furthermore, it is an advantage to form the hollow CaP particles without the useof techniques requiring many process steps (like sacrificing phases) or through the use of additives or less biocompatible substitution ions, which mightjeopardize authorial approval for biomedical use.

SUMMARY OF THE INVENTION The object of the present invention is to provide CaP particles that are essentiallyspherical, and porous, and a method of preparing said particles. The method ofthe present invention is simpler, faster, more cost effective, and with a higheryield than methods disclosed by prior art ln a first aspect the present invention relates to a spherical particle wherein theparticle comprises 40-70 weight% of calcium, 20-40 weight% of phosphate and5-30 weight% magnesium, and wherein the Ca/P molar ratio is in the range of1.10 to 1.90, and wherein the particle size is less than 1 um. ln a second aspect the present invention relates to a method of preparing theparticles according to the present invention wherein the method comprises: a)providing an aqueous buffer solution of purified water having a pH of 2 to 10comprising sodium, potassium and phosphate ions, wherein the concentration ofsaid ions are 100 to 400 mM for sodium, 0-10 mM for potassium and 8-25 mMfor phosphate; b) adding calcium ions in the range of 1.5-10 mM, and magnesiumions in the range of 0.5-15 mM to the buffer solution and forming a mixture; c)heating the mixture of step b) at 60 to 120° C for at least 2 minutes; d) isolatingthe formed particles; and e) optionally washing the isolated particles using asuitable solvent. ln a third aspect the present invention relates to a bleaching chewing gumcomprising particles according to the present invention and a paste formingcompound, and wherein the particles further comprises a bleaching agent suchas a peroxide. ln a fourth aspect the present invention relates to particles obtained from themethod of the present invention. ln a fifth aspect the present invention relates to a chewing gum comprising theparticles or the composition according to the present invention. ln a sixth aspect the present invention relates to a composition comprisingspherical particles and, a paste forming compound; wherein the particlescomprises 40-70 weight% of calcium, 20-40 weight% of phosphate and 5-30weight% magnesium, and wherein the Ca/P weight ratio is in the range of 1.10 to1.90, and wherein the particle size is less than 1 um.

BRIEF DESCRIPTION OF FIGURES Figure 1. SEM image of calcium phosphate hollow spheres.

Figure 2. SEM image of calcium phosphate hollow spheres.

Figure 3. SEM image of calcium phosphate hollow spheres.Figure 4. SEM image of calcium phosphate hollow spheres.Figure 5. SEM image of calcium phosphate hollow spheres.Figure 6. SEM image of calcium phosphate hollow spheres.

Figure 7 SEM image of calcium phosphate hollow spheres.

DETAILED DESCRIPTION OF INVENTION The chemical formula for stoichiometric hydroxyapatite (HA) is Ca10(P04)6(0H)2,but for the purpose ofthis application many variations can be used. The presentinvention is mainly described in terms calcium phosphates (CaP), which includesbut is not limited to: ß-tricalcium phosphate (ß-TCP) dicalcium phosphatedihydrate (DCPD), octacalcium phosphate (OCP), tricalcium phosphate (TCP),and amorphous calcium phosphate (ACP) or any derivative thereof.

The present application discloses a method of preparing spherical particles ofCaP without the use oftemplates or sintering steps. lnstead the presentinvention is a method that is driven by ion concentration and temperature.Compared to previous ion doped CaP spheres, the present invention discloses amethod to produce spheres that are smaller in size and at a higher yield point. Bydeveloping a method that produces more CaP spheres per unit volume ofmixture, the present inventors facilitates a production method of CaP spheresthat is more cost and energy effective than what has been described in the priorart.

The particles The particles of the present invention comprise 40-70 weight% of calcium (Ca),20-40 weight% of phosphate (P04) and 5-30 weight% magnesium (Mg). Theparticles may contain other ions as well. ln one embodiment the concentration of magnesium is 3-15 weight%. ln anotherembodiment the concentration of magnesium is 1-10 weight%. ln oneembodiment the particles comprise 55-65 weight% of calcium, 25-35 weight% ofphosphate and 3-15 weight% of magnesium. The Ca/P molar ratio may bebetween 1.10 and 1.90, for example 1.10 and 1.70. ln one embodiment the ratiois 1.30 to 1.70. ln one embodiment the ratio is 1.40 to 1.50. The particles maycontain trace amounts of potassium and/or sodium since they are used in thebuffer solution as counter ions.

The mean particle size, the diameter, should be small, not more than 1 um. Thisto provides a higher surface area per unit mass, which also makes it easier to fillvoids for example dentinal tubules. The size should not be too small since itmakes it harder to make a formulation suitable as e.g. a chewing gum. Without being bound by theory, the size is also believed to influence the particles abilityto stick to the teeth. ln one embodiment the particles are 1 um or less, or 700 nmor less, or 400 nm or less (mean particle size). ln one embodiment the meanparticle size is 200 to 1000 nm. ln one embodiment the particles are 10 nm ormore, or 50 nm or more, or 100 nm or more, or 300 nm or more, or 450 nm ormore. ln one embodiment the mean particle size is between 200-500 nm. ln one embodiment the particles have a hollow core and a shell and wherein theparticle comprises 40-70 weight% of calcium, 20-40 weight% of phosphate and5-30 weight% magnesium, and wherein the Ca/P weight ratio is in the range of1.10 to 1.90, preferably 1.10 to 1.70, and wherein more than 80 % of theparticles have a particle size between 200 to 1000nm The method The synthesis is performed in an aqueous buffer solution having a pH of 2-10,preferably a pH of 6-10, or pH 6-9, preferably 6.5 to 8 or more preferably a pH of7.0-7.5, comprising calcium, phosphate, magnesium, potassium and sodium ions.The pH value ofthe solutions before and after precipitation is stable.

The concentration of calcium ions may be in the range of 1.5-10 mM, theconcentration of magnesium ions may be in the range of 1-15 mM, and theconcentration of phosphate ions can be in the range 8-25 mM. ln oneembodiment the calcium ion concentration is 1.5 to less than 5 mM, or 1.5 to 3mM, and in another embodiment the magnesium ion concentration is 1 to lessthan 5 mM, or 1.5 to 3 mM ln one embodiment the Ca:Mg molar ratio is from 1:3to 4:1, for example 1:2 to 3:1 , or 1:1 to 2:1.

The sodium (Na) and potassium (K) ions are believed to stabilize the buffer andacts as counter ions. These ions are not expected to be found in the formedparticles or at least not in any larger amounts. ln one embodiment the concentration of sodium ions in the solution is in therange of 0.01-1420 mM and the concentration of potassium ions is in the rangeof 0.01-1420 mM. The concentration ofsodium may be 100 to 400mM and theconcentration of potassium ions may be 0.08 to 10, or 0.5 to 5 mM. The sodiumions may be added as NaCl or Na2HP04 or as a combination, and potassium ionsmay be added as KCl or KH2P04 or as a combination. ln one embodiment theNa:K molar ratio is more than 23:1, preferably more than 30:1, more preferablymore than 35:1. Without being bound by any theory it is believed that thepresence of magnesium ions and the elevated temperature of the solutionpromotes the formation of a hollow structure. ln one embodiment the molar ratio of sodium:potassium:phosphate is 100-400:0-10:8-25.

The molar ratio between Ca and P should be close to 1:10, for example 1:9.0 to1:11, or 1:9.5 to 1:10.7, or 1:10 to 1:10.5.

The water used to prepare the aqueous buffer solution should be purified water.The water may be deionized, distilled, double distilled or ultra-pure water. Forexample the water may be Milli-Q®.

As mentioned above, the method of the present invention is driven by ionconcentration and temperature. This means that if the ion concentrations arewrong or the temperature is too low, spheres and especially hollow spheres willnot be formed or at least not formed within a reasonable period oftime. Thetemperature in the present invention is at least 60°C, or at least 70°C, or at least80°C. The temperature may be 90°C or more, 100°C or more, or 120°C or less.. Apreferred temperature range is from about 60°C to 120°C, or 70°C to 110°C, orfrom about 80°C to 100°C. The method could be a static process, stirring orshaking process or a hydrothermal process.

The particles may be isolated using any suitable technique for example filtering,evaporation, centrifugation or combinations thereof. The method of the presentinvention facilitates a very short synthesis time but it is also believed that timewill influence the particle size distribution. The synthesis time may be 1 minute,but it may be a couple of hours. ln one embodiment the synthesis time is at least2 minutes, or at least 5 minutes, or at least 10 minutes, or at least 30 minutes, orat least 1 hour, or at least 2 hours, or at least 6 hours. The obtained particles willhave essentially a TCP structure, such as ß-TCP. ln one embodiment the methodcomprises heating at 60 to 120° C for at least 5 minutes; Applications The particles of the present invention may be used as a bleaching agent or pasteor chewing gum for teeth. A problem with prior art has been that the bleachingalso degrades the enamel, resulting in increased tooth sensitivity and gingivalirritation. The present invention aims at delivering a bleaching agent locallyusing the particles of the present invention, which will act to remineralize thetooth during and/or after the bleaching step. The preparation of a bleachingagent with the particles of the present invention comprises: -providing particles of the present invention and a solution of a bleachingadditive for example a peroxide solution; -mixing the particles and the solution, for example a peroxide solution, and;-isolating the bleaching additive containing particles.

The peroxide used may be hydrogen peroxide or carbamide peroxide or amixture thereof. Other non-limiting examples of tooth bleaching additivesinclude; sodium percarbonate, sodium chlorite, sodium perborate,peroxymonosulphate, peroxide plus metal catalysts and oxireductase enzymes.The concentrations of peroxides in the solution may be 0.1-60wt% such as 1wt% or more, or 5 wt% or more, or 10 wt% or more or 15 wt% or more, or 20wt% or more, or 25 wt% or more, but 55 wt% or less, or 50 wt% or less, or 45wt% or less, or 40 wt% or less. For example the concentration may be 10-60wt%for carbamide peroxide, and 0.1-35 wt% for example for hydrogen peroxide suchas 10-35wt% for hydrogen peroxide.

EXAMPLESExample 1: CaP particles were prepared as follows: NaCl, KCl, CaClg, MgClg, KH2P04, andNa2HP04 were dissolved in water at a molar ratio of 185:0.29:2:1.25:2.231:12.05to form a phosphate buffered saline solution. The solution was heated understirring to a temperature of 85°C, where it was held for 40 min. During this timethe CaP particles will precipitate from the solution. The particles were thenfiltered out of solution and dried before examination in SEM. At this ionconcentration and under the described process parameters, spherical particleswith a mean diameter of roughly 300 nm were produced. See Figure 1.

Example 2: CaP particles were prepared as follows: NaCl, KCl, CaClg, MgClg, KH2P04, andNa2HP04 were dissolved in water at a molar ratio of 170:0.08:1.5:1.5:1.5:8.1 toform a phosphate buffered saline solution. The solution was heated understirring to a temperature of 85°C, where it was held for 40 min. During this timethe CaP particles will precipitate from the solution. The particles were thenfiltered out of solution and dried before examination in SEM. At this ionconcentration and under the described process parameters, spherical particleswith a mean diameter of roughly 560 nm were produced. See Figure 2.

Example 3: CaP particles were prepared as follows: NaCl, KCl, CaClg, MgClg, KH2P04, andNa2HP04 were dissolved in water at a molar ratio of 200:0.5:2.5:1.5:2.963:16 toform a phosphate buffered saline solution. The solution was heated understirring to a temperature of 85°C, where it was held for 40 min. During this timethe CaP particles will precipitate from the solution. The particles were thenfiltered out of solution and dried before examination in SEM. At this ionconcentration and under the described process parameters, spherical particleswith a mean diameter of roughly 230 nm were produced. See Figure 3.

Example 4: A full factorial screening series of experiments were made according to a designof experiments (DOE) software (MODDE, Umetrics), where the variables werethe molar concentrations of NaCl, KCl, CaClg, MgClg, and Na2HP04. The molarconcentration of KH2P04 was fixed to a ratio of 1:5.4 to Na2HP04. ln the screening experiments, CaP particles were prepared as follows: NaCl, KCl,CaClg, MgClg, KH2P04, and Na2HP04 were dissolved in water at designated molarconcentrations to form a phosphate buffered saline solution. The molarconcentrations varied as follows: NaCl: 100-170 mM; KCl: 0.1-5 mM; CaClg: 0.9-1.8 mM; MgClg: 0.1-1 mM; Na2HP04: 8.1-16 mM;KH2P04:1.5-2.963 mM.. Thesolution was heated under stirring to a temperature of 85°C, where it was heldfor 40 min. During this time the CaP particles precipitated from the solution. Theparticles were then filtered out of solution and dried before examination in SEM.

The particles from each experiment were rated according to morphology andsize, and the data was evaluated in the DOE software to identify statisticallyverified trends. The results ofthe screening revealed that certain ionconcentrations, or molar ratios between different ions within the definedlimitations, allowed formation of hollow and spherical CaP particles at higherconcentrations or ratios than previously described in prior art. The screeningalso revealed regions ofion concentrations or molar ratios where spheres didnot form. Figures 4 and 5 display successful and unsuccessful attempts at sphereformation, respectively, within the specified region of ion concentrations. Thescreening led to an extended experimental series, wherein a new window withyet higher ion concentrations was evaluated, which is further described inExample 5.

Example 5: Following the full factorial screening experiments described in Example 4, anoptimization series of experiments was made, also governed by DOE software. Aspreviously, the variables were the molar concentrations of NaCl, KCl, CaClg,MgClg, and Na2HP04. The molar concentration of KH2P04 was fixed to a ratio of125.4 'CO Na2HPO4. ln the optimization experiments, CaP particles were prepared as follows: NaCl,KCl, CaClg, MgClg, KH2P04, and Na2HP04 were dissolved in water at designatedmolar concentrations to form a phosphate buffered saline solution. The molarconcentrations varied as follows: NaCl: 170-200 mM; KCl: 0.08-0.5 mM; CaClg:1.5-2.5 mM;MgCl2:1-1.5 mM; Na2HP04: 8.1-16 mM;KH2P04:1.5-2.963 mM.. Thesolution was heated under stirring to a temperature of 85°C, where it was heldfor 40 min. During this time the CaP particles precipitated from the solution. Theparticles were then filtered out of solution and dried before examination in SEM.The particles from each experiment were rated according to size andmorphology, i.e. if spheres were formed or not. See Table 1. The data wasevaluated in the DOE software to identify statistically verified trends. The resultsof the evaluation revealed that, within the ion concentration design space,spheres were formed in nearly all areas, but having different sizes. lt alsorevealed an increase in yield of spheres, primarily according to the amount ofcalcium added.

Table 1 Exp. Name [KCI] [Na2HPO4] N4 200 335 Y N6 170 16 1 231 Y N8 0.5 200 16 1 1.5 277 Y N10 0.5 170 8.1 1.5 2.5 275 Y 12.05 12.05 1.5 2 681 Y Example 6: Recycling of process water and counter ions in the solution is essential for a costand energy efficient production process of the CaP particles. A formulationretrieved from the DOE described in Example 5 was tested in a cyclic fashion,wherein the process water was re-used after the formed CaP particles had beenfiltered out. To this process water, after cooling down to room temperature,CaClg, MgClg, Na2HP04, and KH2P04 were added in succession to replenish thesolution similar to its original composition. This solution was then heated understirring to a temperature of 85°C and held for 40 min. The particles were thenfiltered out of solution and dried before examination in SEM. See Figure 6 forappearance of spheres obtained from the original solution. See Figure 7 forappearance of spheres obtained after recycling the process water and counterions. The results reveal that the process water and counter ions can be recycledto produce CaP spheres in a more cost and energy efficient way.

Example 7: Based on sample N7 found in Table 1, the effect of an increased amount ofmagnesium content was evaluated. CaP particles were prepared as follows: NaCl,KCl, CaClg, MgClg, KH2P04, and Na2HP04 were dissolved in water at a molar ratioof 200:0.08:2.5:2.5:2.963:16 to form a phosphate buffered saline solution. Thesolution was heated under stirring to a temperature of 85°C, where it was heldfor 40 min. The particles were then filtered out of solution and dried beforeexamination in SEM, see Figure 8. The experiment revealed that spheres couldsuccessfully be formed also with this elevated concentration of magnesium ions.

Example 8: The effect of a yet further increase in magnesium content was evaluated. CaPparticles were prepared as follows: NaCl, KCl, CaClg, MgClg, KH2PO4, and Na2HPO4were dissolved in water at a molar ratio of 200:0.08:2.5:10:2.231:16 to form aphosphate buffered saline solution. The solution was heated under stirring to atemperature of 85°C, where it was held for 40 min. The particles were thenfiltered out of solution and dried before examination in SEM, see Figure 9. Theexperiment revealed that spheres were formed also with this further elevatedconcentration of magnesium ions.

Claims (1)

CLAïh/ES

1. C41 Spherittai partitties tvherein the partittie eeniprises 40-79 iveigiitß/f; ofeaicitim, 29-49 vveightfi/íi of phespiiate and 5-36 iveigiitiiíi tnagiiesiuni, andwherein the Cat/P »fi/eight ratio is in the range of 1.10 to 1.96, and tvhereiniriore than 89 % of the partieies have a particie size hetta/eefi 266 to'iüiiiliiirin preferahiy ZÜG to 560 nin. 'Fhe particies ziccertiing to ciziini 1 i/xfherein the particies coinprises 8-12tfveighitfyfi of rnagriesinrn The tiarticies according to ciaiins 'i er 2 ifi/iiereiri tiie particies haveparticie size ef lšÛÛ-Miflnin. . A inetheti tií” preparirig the particies atteerding te Ciaini 1 tvhereiii the inetheti eeniprises: a. providing an ziqueous buffet seiutien of ptirified tvater having a pH of 6te 1G eeniprising stioiiuni, tiotassinrn and phospiiate ions, wherein theeencentratien ef said ions are 100 te liíšü fet sedinm, (i-Éifi iniví forpetassiuin anti 8-25 iniVi for phespiizite; h. adding caiciuni ions in the range ef frem 1.5 to iess than 10inM, andmagnesium ions in the range ef 6.545 iniVi te the buffer seiutien andferining a. inixture; c. heating the niixtnre ef step h) at 6G to 12Û° C for at ieast 5 minutes;ti. iseiating the fornied particies; ande. eptionaiiy tfxfashing the iseiateci particies iising a siiitahie seivent. The inetheti according te ciairn 4» tfvhereiri the rnagiiesiuni eeneentratioriis líi-12inii/i. 'The method accertiing to eiaini 4 er 5 wherein the temperature is 7G-90%. The niethod accerdiiig to ariy ene ef'ciaiins 4-5 wherein the iieating isdone for not more than 6G minutes.