STABILIZED AMORPHOUS CALCIUM PHOSPHATE DOPED WITH FLUORIDE IONS AND A PROCESS FOR PRODUCING THE SAME

Abstract

The invention relates to a process for the preparation of a citrate-coated amorphous calcium phosphate nanoparticle which comprises the following steps: 1) providing a first solution of a salt of calcium and a citrate salt wherein the molar ratio of citrate ion to calcium ion is in the range from 1 to 2 thus obtaining a clear first solution; 2) providing a second solution of a salt capable to give phosphate anion and a carbonate salt; 3) mixing together the first and the second solution at a pH in the range from 8 to 11; 4) precipitating the nanoparticle; and 5) drying the nanoparticle obtained from step 4). Preferably and advantageously the invention provides for the addition of a fluoride compound in step 2) for obtaining a fluorine-doped citrate-coated calcium phosphate nanoparticle or a nanoparticle agglomerate. The nanoparticle/nanoparticle agglomerate of the invention has a peculiar superficial area and a diameter that allow to use it as a biomaterial for dentistry application.

Claims

1. A process for the preparation of a citrate-coated amorphous calcium phosphate nanoparticle which comprises the following steps: 1) providing a first solution of a salt of calcium and a citrate salt wherein the molar ratio of citrate ion to calcium ion is in the range from 1 to 2 thus obtaining a clear first solution; 2) providing a second solution of a salt capable to give phosphate anion and a carbonate salt; 3) mixing together the clear first solution and the second solution at a pH in the range from 8 to 11; 4) precipitating the nanoparticle; and 5) drying the nanoparticle obtained from step 4).

2. The process according to claim 1, wherein the salt of calcium is made of an anion selected from the group consisting of chloride, nitrate, hydroxide, acetate, oxalate, lactate, preferably the anion is chloride.

3. The process according to claim 1, wherein the salt of citrate is made of a cation selected from the group consisting of sodium and potassium, preferably the cation is sodium.

4. The process according to claim 1, wherein the molar ratio of citrate ion to calcium ion is about 2.

5. The process according to claim 1, wherein the first solution of step 1) comprises at least one further salt selected from the group of: strontium salt, and magnesium salt.

6. The process according to claim 1, wherein the salt capable to give phosphate anion is a salt of phosphate, hydrogen phosphate or hydrogen phosphate, preferably made of a cation selected from the group consisting of sodium, potassium and ammonium.

7. The process according to claim 1, wherein the pH of step 3) is in the range from 8.5-10.7.

8. The process according to claim 1, wherein in the mixing step 3) the second solution is added to the clear first solution.

9. The process according to claim 1, wherein in step 4) the precipitation is carried out by providing sedimentation cycles by centrifugation removal of supernatant, collecting and washing the precipitate.

10. The process according to claim 1, wherein the drying step 5) is selected from freeze-drying, spray-drying and ventilated oven drying.

11. The process according to claim 1, wherein in step 1) a fluoride compound is added.

12. The process according to claim 11, wherein the fluoride compound is a fluoride of a cation selected from the group consisting of sodium and potassium.

13. A citrate-coated amorphous calcium phosphate nanoparticle obtainable by the process according to claim 1, wherein the drying step 5) is a freeze-drying step, being said nanoparticle characterized by a superficial area from 250 m.sup.2 g.sup.−1 to 360 m.sup.2 g.sup.−1 as measured with Brunauer-Emmett-Teller (BET) gas adsorption method and having a round-shaped morphology with a diameter in the range from 30 to 80 nm as measured by transmission electron microscopy (TEM) images.

14. A citrate-coated amorphous calcium phosphate nanoparticle agglomerate obtainable by the process according to claim 1, wherein the drying step 5) is a spray-drying step, being said nanoparticle agglomerate characterized by a superficial area from 2 m.sup.2 g.sup.−1 to 10 m.sup.2 g.sup.−1 as measured with Brunauer-Emmett-Teller (BET) gas adsorption method and having a round shaped morphology with a diameter in the range from 2 to 25 μm as measured by scanning electron microscopy (SEM).

15. A fluorine-doped citrate-coated amorphous calcium phosphate nanoparticle obtainable by the process according to claim 1, wherein the drying step 5) is a freeze-drying step, being said nanoparticle characterized by a superficial area from 250 m.sup.2 g.sup.−1 to 370 m.sup.2 g.sup.−1 as measured with Brunauer-Emmett-Teller (BET) gas adsorption method and having a round shaped morphology with a diameter in the range from 30 to 80 nm as measured by transmission electron microscopy (TEM) images.

16. A fluorine-doped citrate-coated amorphous calcium phosphate nanoparticle agglomerate obtainable by the process according to claim 1, wherein the drying step 5) is a spray-drying step, being said nanoparticle agglomerate characterized by a superficial area from 3 m.sup.2 g.sup.−1 to 10 m.sup.2 g.sup.−1 as measured with Brunauer-Emmett-Teller (BET) gas adsorption method and having a round shape morphology with a diameter in the range from 2 to 25 μm as measured by scanning electron microscopy (SEM).

17. A dentistry application method comprising the use of a nanoparticle or a nanoparticle agglomerate according to claim 13 as a biomaterial.

18. The dentistry application method according to claim 17 wherein the biomaterial is used as remineralization agent or as dentin desensitizer.

19. A dentistry application method comprising the use of a nanoparticle or a nanoparticle agglomerate according to claim 14 as a biomaterial.

20. The dentistry application method according to claim 19 wherein the biomaterial is used as remineralization agent or as dentin desensitizer.

21. A dentistry application method comprising the use of a nanoparticle or a nanoparticle agglomerate according to claim 15 as a biomaterial.

22. The dentistry application method according to claim 21 wherein the biomaterial is used as remineralization agent or as dentin desensitizer.

23. A dentistry application method comprising the use of a nanoparticle or a nanoparticle agglomerate according to claim 16 as a biomaterial.

24. The dentistry application method according to claim 23 wherein the biomaterial is used as remineralization agent or as dentin desensitizer.

Description

DESCRIPTION OF THE FIGURES

[0035] FIG. 1A shows TEM micrograph of ACP.sup.4 prepared in example 1; (inset: corresponding SAED pattern)

[0036] FIG. 1B shows XRD patterns of ACP.sup.4 prepared in example 1;

[0037] FIG. 1C shows FT-IR spectra of ACP.sup.4 prepared in example 1;

[0038] FIG. 2A shows TEM micrograph of F-ACP.sup.4 prepared in example 1; (inset: corresponding SAED pattern)

[0039] FIG. 2B shows XRD pattern of F-ACP.sup.4 prepared in example 1;

[0040] FIG. 2C shows FT-IR spectra of F-ACP.sup.4 prepared in example 1;

[0041] FIG. 3A shows TEM micrograph of ACP.sup.1, prepared in example 2; (inset: corresponding SAED pattern)

[0042] FIG. 3B shows XRD patterns of ACP.sup.2, ACP.sup.1, F-ACP.sup.1, F-ACP.sup.2 prepared in example 2;

[0043] FIG. 3C shows FT-IR spectra of ACP.sup.2, ACP.sup.1, F-ACP.sup.1, F-ACP.sup.2 prepared in example 2;

[0044] FIG. 4A reports cumulative Ca.sup.2+ release from ACP samples prepared in example 1 and example 2; data are expressed as mean±standard deviation (n=5);

[0045] FIG. 4B reports cumulative Ca.sup.2+ release from F-ACP samples prepared in example 1 and example 2; data are expressed as mean±standard deviation (n=5);

[0046] FIG. 4C reports cumulative F.sup.− from F-ACP samples prepared in example 1 and example 2; data are expressed as mean±standard deviation (n=5);

[0047] FIG. 5 shows XRD pattern of spray dried F-ACP.sup.1 of example 8;

[0048] FIG. 6 shows SEM micrographs at different magnification of spray dried F-ACP.sup.1 of example 8;

[0049] FIG. 7 shows XRD patterns of ACP.sup.2, ACP.sup.1, F-ACP.sup.2, F-ACP.sup.1 prepared in example 2, one year after synthesis, stored at room temperature of example 9;

[0050] FIG. 8 shows XRD patterns of SrF-ACP.sup.2, MgF-ACP.sup.2, SrMgF-ACP.sup.2, SrF-ACP.sup.1, MgF-ACP.sup.1, SrMgF-ACP.sup.1 prepared in example 2bis;

[0051] FIG. 9 shows FT-IR spectra of SrF-ACP.sup.2, MgF-ACP.sup.2, SrMgF-ACP.sup.2, SrF-ACP.sup.1, MgF-ACP.sup.1, SrMgF-ACP.sup.1 prepared in example 2bis;

[0052] FIG. 10A reports cumulative Ca.sup.2+ release from SrF-ACP.sup.2, MgF-ACP.sup.2, SrMgF-ACP.sup.2, SrF-ACP.sup.1, MgF-ACP.sup.1, SrMgF-ACP.sup.1 samples prepared in example 2bis; data are expressed as mean±standard deviation (n=5); and

[0053] FIG. 10B reports cumulative F.sup.− release from SrF-ACP.sup.2, MgF-ACP.sup.2, SrMgF—SrF-ACP.sup.1, MgF-ACP.sup.1, SrMgF-ACP.sup.1 samples prepared in example 2bis; data are expressed as mean±standard deviation (n=5).

DETAILED DESCRIPTION OF THE INVENTION

[0054] Therefore the invention relates to a process for the preparation of a citrate-coated amorphous calcium phosphate nanoparticle, which comprises the following steps:

1) providing a first solution of a salt of calcium and a citrate salt wherein the molar ratio of citrate ion to calcium ion is in the range from 1 to 2 thus obtaining a clear first solution;
2) providing a second solution of a salt capable to give phosphate anion and a carbonate salt;
3) mixing together the clear first solution and the second solution at a pH in the range from 8 to 11;
4) precipitating the nanoparticle; and
5) drying the nanoparticle obtained from step 4).

[0055] The step 1) of the process consists in providing a first solution of a salt of calcium and of a citrate salt, wherein the molar ratio of citrate ion to calcium ion is in the range from 1 to 2. The first solution so obtained is clear.

[0056] The salt of calcium is preferably made of an anion selected from the group consisting of chloride, nitrate, hydroxide, acetate, oxalate, lactate, more preferably the anion is chloride.

[0057] The salt of citrate is preferably made of a cation selected from the group consisting of sodium and potassium, more preferably the cation is sodium.

[0058] More preferably the molar ratio of citrate ion to calcium ion is about 1.

[0059] Still more preferably, the molar ratio of citrate ion to calcium ion is about 2.1n a preferred embodiment, the first solution of step 1) of the process according to the invention comprises at least one further salt selected from the group of: strontium salt, and magnesium salt.

[0060] The strontium salt is preferably made of an anion selected from the group consisting of chloride, nitrate, hydroxide, acetate, oxalate, lactate, more preferably the anion is chloride.

[0061] The magnesium salt is preferably made of an anion selected from the group consisting of chloride, nitrate, hydroxide, acetate, oxalate, lactate, more preferably the anion is chloride.

[0062] Step 2) consists in providing a second solution of a salt capable to give phosphate anion and a carbonate salt.

[0063] Preferably the ratio between the carbonate anion and phosphate is in the range from 1 to 1.66.

[0064] Preferably the salt capable to give phosphate anion is a salt of phosphate, hydrogen phosphate or hydrogen phosphate. The salt capable to give phosphate anion is preferably made of a cation select from the group consisting of sodium, potassium and ammonium, more preferably the cation is sodium.

[0065] Step 3) consists in mixing together the first and the second solution at a pH in the range from 8 to 11, preferably 8.5-10.7.

[0066] In an advantageous aspect the ratio of the first and the second solution is in the range 1:1 to 1:1.5.

[0067] According to the invention the mixing step 3) is carried out after the first solution is clear. Preferably the second solution is added to the clear first solution for the mixing step.

[0068] The step 4) consists in precipitating the nanoparticle.

[0069] The step of precipitation can advantageously be carried out by providing sedimentation cycles by centrifugation, after which the removal of supernatant can be carried out according to well-known methods. As soon as the precipitate is collected, it can be washed with preferably ultrapure water. The wet precipitate is then dried according to drying methods known in the art.

[0070] Step 5) consists in a drying step of the precipitated nanoparticle of the invention. The drying step can be carried out with any suitable means known in the art. Preferably the drying step can be selected from freeze-drying, spray-drying and ventilated oven drying. This latter is preferably carried out after washing with ethanol and at a temperature of about 40° C.

[0071] In a preferred aspect the drying step 5) is a freeze-drying step.

[0072] In another aspect the invention concerns a citrate-coated amorphous calcium phosphate nanoparticle obtainable by the process according to the invention, wherein the drying step 5) is a freeze-drying step, being said nanoparticle characterized by a superficial area from 250 m.sup.2 g.sup.−1 to 360 m.sup.2 g.sup.−1, preferably from 270 m.sup.2 g.sup.−1 to 360 m.sup.2 g.sup.−1, as measured with Brunauer-Emmett-Teller (BET) gas adsorption method and having a round-shaped morphology with a diameter in the range from 30 to 80 nm as measured by transmission electron microscopy (TEM) images.

[0073] In a first embodiment hence, the superficial area of the nanoparticle is in the range from 250 m.sup.2 g.sup.−1 to 360 m.sup.2 g.sup.−1, preferably from 270 m.sup.2 g.sup.−1 to 360 m.sup.2 g.sup.−1, as measured with Brunauer-Emmett-Teller (BET) gas adsorption method by using powdered samples and a Sorpty 1750 (Carlo Erba, Milan Italy) and said nanoparticle has preferably a spherical shape with a diameter in the range from 30 to 80 nm. All the instruments used for determining the diameter are instruments capable to have transmission electron microscopy (TEM) images.

[0074] In a further advantageous aspect the drying step 5) is a spray-drying step.

[0075] In another aspect the invention concerns a citrate-coated amorphous calcium phosphate nanoparticle agglomerate obtainable by the process according to the invention, wherein the drying step 5) is a spray-drying step, being said nanoparticle agglomerate characterized by a superficial area from 3 m.sup.2 g.sup.−1 to 10 m.sup.2 g.sup.−1 as measured with Brunauer-Emmett-Teller (BET) gas adsorption method and having a round-shaped morphology with a diameter in the range from 2 to 25 μm as measured by scanning electron microscopy (SEM).

[0076] In a first embodiment hence, the superficial area of the nanoparticle agglomerate is in the range from 3 m.sup.2 g.sup.−1 to 10 m.sup.2 g.sup.−1 as measured with Brunauer-Emmett-Teller (BET) gas adsorption method by using powdered samples and a Sorpty 1750 (Carlo Erba, Milan Italy) and said nanoparticle agglomerate has preferably a spherical shape and a diameter in the range from 2 to 25 μm. All the instruments used for determining the diameter are instruments for scanning electron microscopy (SEM).

[0077] In a further and preferred aspect of the invention allows to obtain a fluorine-doped citrate-coated amorphous calcium phosphate nanoparticle by providing of adding a fluoride compound in the second solution of step 2).

[0078] Preferably the fluoride compound is a fluoride of a cation selected from the group consisting of sodium and potassium.

[0079] Therefore in another and preferred aspect the invention concerns a fluorine-doped citrate-coated amorphous calcium phosphate nanoparticle obtainable by the process according to the invention and comprising the addition of a fluoride compound in step 2), wherein the drying step 5) is a freeze-drying step, being said nanoparticle characterized by a superficial area from 250 m.sup.2 g.sup.−1 to 370 m.sup.2 g.sup.−1, preferably from 270 m.sup.2 g.sup.−1 to 370 m.sup.2 g.sup.−1, as measured with Brunauer-Emmett-Teller (BET) gas adsorption method and having a round shape with a diameter in the range from 30 to 80 nm as measured by transmission electron microscopy (TEM) images.

[0080] In a first embodiment hence, the superficial area of fluorine-doped citrate-coated amorphous calcium phosphate nanoparticle is in the range from 250 m.sup.2 g.sup.−1 to 370 m.sup.2 g.sup.−1, preferably from 270 m.sup.2 g.sup.−1 to 370 m.sup.2 g.sup.−1, as measured with Brunauer-Emmett-Teller (BET) gas adsorption method by using powdered samples and a Sorpty 1750 (Carlo Erba, Milan Italy) and said nanoparticle has preferably a spherical shape with a diameter in the range from 30 to 80 nm. All the instruments used for determining the diameter are instruments capable to have transmission electron microscopy (TEM) images.

[0081] In another and preferred aspect the invention concerns a fluorine-doped citrate-coated amorphous calcium phosphate nanoparticle agglomerate obtainable by the process according to the invention and comprising the addition of a fluoride compound in step 2), wherein the drying step 5) is a spray-drying step, being said nanoparticle agglomerate characterized by a superficial area from 3 m.sup.2 g.sup.−1 to 10 m.sup.2 g.sup.−1 as measured with Brunauer-Emmett-Teller (BET) gas adsorption method and having a round-shape with a diameter in the range from 2 to 25 μm as measured by scanning electron microscopy (SEM).

[0082] The superficial area is measured with Brunauer-Emmett-Teller (BET) gas adsorption method by using powdered samples and a Sorpty 1750 (Carlo Erba, Milan Italy).

[0083] In a further aspect the invention concerns the use of a particle of the invention as a biomaterial for use in dentistry applications. Preferably the biomaterial is used for the dental hard tissues remineralization or as dentin desensitizer, where in this latter case its action is preferably to fill and occlude the dentinal tubules.

[0084] In a still further aspect the invention concerns the use of a particle of the invention as a biomaterial in orthopaedic applications.

EXPERIMENTAL PART

Materials

[0085] Calcium chloride dihydrate (CaCl.sub.2.2H.sub.2O, ≥99.0% pure), sodium citrate tribasic dihydrate (Na3(C6H5O7).2H.sub.2O, ≥99.0% pure (hereafter named Na3(Cit)), sodium phosphate dibasic dihydrate (Na.sub.2HPO.sub.4.2H.sub.2O, ≥99.0% pure), strontium chloride hexahydrate (SrCl2.6H2O, ≥99.0% pure), magnesium chloride hexahydrate (MgCl2.6H2O, ≥99.0% pure), sodium carbonate monohydrate (Na.sub.2CO.sub.3.2H.sub.2O, ≥99.0% pure), sodium fluoride (NaF, ≥99.0% pure), potassium chloride (KCl≥99.5% pure), potassium thiocyanate (KSCN≥98.0% pure), sodium carbonate monobasic (NaHCO.sub.3, ≥99.7% pure) and lactic acid (C.sub.3H.sub.6O.sub.3≥90.0% pure) were purchased from Sigma Aldrich (St. Luis, Mo., USA). All the solutions were prepared with ultrapure water (0.22 μS, 25° C., MilliQ©, Millipore).

Instruments and Methods of Evaluation

[0086] X-ray diffraction (XRD) patterns of the samples reported in Table 1 were recorded on a D8 Advance diffractometer (Bruker, Karlsruhe, Germany) equipped with a Lynx-eye position sensitive detector using Cu Kα radiation (λ=1.54178 Å) generated at 40 kV and 40 mA. Spectra were recorded in the 2θ range from 10 to 60° with a step size (28) of 0.021 and a counting time of 0.5 s.

[0087] Fourier transform infrared (FT-IR) spectroscopy analyses were carried out on a Nicolet 5700 spectrometer (Thermo Fisher Scientific Inc., Waltham, Mass., USA) with a resolution of 2 cm.sup.−1 by accumulation of 64 scans covering the 4000 to 400 cm.sup.−1 range, using the KBr pellet method.

[0088] Transmission electron microscopy (TEM) and selected area electron diffraction (SAED) evaluation was performed with Tecnai F20 microscope (Fei Corp., Hillsboro, Oreg., USA) operating at 120 kV. The powder samples were ultrasonically dispersed in ultrapure water and then a few droplets of the slurry were deposited on 200 mesh copper TEM grids covered with thin amorphous carbon films and incubated for several minutes.

[0089] Quantification of Ca and P, Mg, and Sr was carried out by inductively coupled plasma atomic emission (ICP-OES) spectrometer (Agilent Technologies 5100 ICP-OES, Santa Clara, Calif., USA) while F was quantified with a fluoride ion electrode (Intellical™ ISEF121, Hach Lange, Loveland, Colo., USA). Samples were prepared dissolving an aliquot of powder in a 1 wt. % HNO.sub.3 solution.

[0090] Thermogravimetry analyses (TGA) were performed using a STA 449 Jupiter (Netzsch GmbH, Selb, Germany) apparatus. About 10 mg of sample was weighted in a platinum crucible and heated from room temperature to 1200° C. under air flow with a heating rate of 10° C./min.

[0091] Brunauer-Emmett-Teller (BET) gas adsorption method was employed to measure the specific surface area (SSA) of powdered samples using a Sorpty 1750 (Carlo Erba, Milan Italy).

[0092] Scanning electron microscopy (SEM) evaluation was performed employing a Sigma NTS Gmbh (Carl Zeiss, Oberkochen, Germany). The powder samples were mounted on aluminum stubs using carbon tape, and prior the analyses sputter coated with gold in a Sputter Coater E5100 (Polaron Equipment, Watford, Hertfordshire, UK) under argon at 10.sup.−3 mbar for 4 minutes with a sputtering current of 30 mA.

Example 1

Preparation of the Nanoparticle of the Prior Art WO2016/016012452

[0093] Dry powder ACP (amorphous calcium phosphate) was synthesized by mixing two solutions (1:1 v/v, 200 ml total) at room temperature of (i) 100 mM CaCl.sub.2)+400 mM Na.sub.3(Cit) and (ii) 120 mM Na.sub.2HPO.sub.4+200 mM Na.sub.2CO.sub.3. The pH was adjusted to 8.5 with HCl solution. When the mixture became milky, the particles were washed three times with ultrapure water by centrifugation at 5000 rpm for 15 min thermostated at 4° C. and then freeze-dried overnight at −50° C. under a vacuum of 3 mbar.

[0094] F-ACP samples were prepared similarly to ACP while 50 mM NaF.sub.2 was added to the solution (ii).

Example 2

Preparation of the Nanoparticle of the Invention

[0095] Following the same preparation of example 1, samples of ACP and F-ACP (doped using 50 mM NaF.sub.2) were also prepared decreasing the initial molar Cit/Ca ratio to 2 and 1 (hereafter coded as ACP.sup.2, F-ACP.sup.2 and ACP.sup.1, F-ACP.sup.1, respectively).

Example 2Bis

[0096] Preparation of the Nanoparticle of the Invention with Mg and Sr

[0097] Following the preparation of example 2, samples of SrF-ACP, MgF-ACP and SrMgF-ACP were also prepared similarly to F-ACP while 5 mM SrCl.sub.2 or 40 mM MgCl.sub.2 or both were added to the solution (i). The initial molar Cit/Ca ratio of 2 and 1 (hereafter coded as SrF-ACP.sup.2, MgF-ACP.sup.2, SrMgF-ACP.sup.2 and SrF-ACP.sup.1, MgF-ACP.sup.1, SrMgF-ACP.sup.1, respectively) was used.

[0098] Codes of the samples and concentration of the chemical reactants used for the preparation of example 1 and example 2 are reported in the following Table 1.

TABLE-US-00001 TABLE 1 NaF CaCl.sub.2 Na.sub.3(Cit) Na.sub.2HPO.sub.4 Na.sub.2CO.sub.3 MgCl.sub.2 SrCl.sub.2 Sample Cit/Ca (mM) (mM) (mM) (mM) (mM) (mM) (mM) ACP.sup.4 4 — 100 400 120 200 — — F-ACP.sup.4 4 50 100 400 120 200 — — ACP.sup.2 2 — 100 200 120 200 — — F-ACP.sup.2 2 50 100 200 120 200 — — ACP.sup.1 1 — 100 100 120 200 — — F-ACP.sup.1 1 50 100 100 120 200 — — SrF-ACP.sup.2 2 50 100 200 120 200 — 5 MgF-ACP.sup.2 2 50 100 200 120 200 40 — SrMgF-ACP.sup.2 2 50 100 200 120 200 40 5 SrF-ACP.sup.1 1 50 100 100 120 200 — 5 MgF-ACP.sup.1 1 50 100 100 120 200 40 — SrMgF-ACP.sup.1 1 50 100 100 120 200 40 5

[0099] The samples of the prior art have a molar ratio between citrate and calcium of 4, while the samples of the present invention were prepared with a molar ratio in the range from 1 to 2.

Example 3: Evaluation of the Physical Characteristics and of the Compositions of the Particles of ACP.SUP.4 .Prepared in Example 1

[0100] Dry powder ACP.sup.4 prepared in example 1 was evaluated for physical properties with the instruments above reported.

[0101] In FIG. 1A TEM micrograph of ACP.sup.4 is reported, revealing round shaped nanoparticles rather than faceted and angular shape typical of crystalline CaP, with sizes ranging between 20 and 50 nm. The SAED pattern collected for such nanoparticles (top right inset in FIG. 1A) demonstrates their amorphous nature due to the presence of diffuse wings rather than spots.

[0102] XRD pattern of ACP.sup.4 (FIG. 1B) reveals a broad band at about 30° (2θ) typical of a phase without a long range periodic regularity confirming the non-crystalline structure of ACP.sup.4 and excluding the presence of HA and others CaP crystalline phases. FT-IR spectrum (FIG. 1C) displays broad, unresolved bands characteristic of CaP having amorphous structure. In particular, the adsorption bands at about 560 and 1050 cm.sup.−1 were associated to the bending and stretching modes of phosphate groups, respectively; those at about 870 cm.sup.−1 and in the range 1400-1500 cm.sup.−1 were attribute to the carbonate ions, while the band at about 1605 cm.sup.−1 was assigned to the adsorbed water as well as the stretching of COO.sup.− of citrate. Therefore, FIGS. 1A, 1B, 1C confirmed the spherical shape of nanoparticles made of amorphous calcium phosphate.

Example 4: Evaluation of the Physical Characteristics and of the Compositions of the Particles of F-ACP Prepared in Example 1

[0103] With reference to example 1, in a first step the reagents concentration as well as the Cit/Ca ratio were kept constant respect to the protocol used for the preparation of ACP.sup.4, while NaF was added to dope ACP.sup.4 (hereafter called F-ACP.sup.4)). TEM image of F-ACP.sup.4 (FIG. 2A) displays round shaped particles having size and morphology similar to that of ACP.sup.4. The SAED pattern collected for such nanoparticles (top right inset in FIG. 2A) demonstrates their amorphous nature due to the presence of diffuse wings rather than spots. XRD patterns of F-ACP.sup.4 (FIG. 2B) show the same broad diffraction peak at about 30° (2θ) of that recorded for ACP.sup.4 indicating that the presence of F.sup.− ions did not cause the precipitation of fluoride salt or other crystalline CaP phases. FT-IR spectra of F-ACP.sup.4 also display broad, unresolved bands similar to those reported in the FT-IR spectrum of ACP.sup.4 (FIG. 2C).

Example 5: Evaluation of the Physical Characteristics and of the Compositions of the Particles of ACP and F-ACP Prepared in Example 2

[0104] As above reported, two ACP samples were prepared changing the nominal Cit/Ca ratio of the reagents according to the invention, that was set to 4 according to the prior art, to 2 or 1 (hereafter called ACP.sup.2 and ACP.sup.1, respectively) according to the invention in order to evaluate the effect of the molar ratio Cit/Ca on the chemical-physical features of these amorphous materials. Moreover, the same amount of NaF used for the synthesis of F-ACP.sup.4 was employed to dope ACP.sup.2 and ACP.sup.1, and these samples were called F-ACP.sup.2 and F-ACP.sup.1 respectively.

[0105] TEM images of ACP.sup.2 (not shown) and ACP.sup.1 (FIG. 3A) display round shaped particles having size and shape comparable to ACP.sup.4. The SAED pattern collected for ACP.sup.1 (top right inset in FIG. 3A) demonstrates their amorphous nature due to the presence of diffuse wings rather than spots. Also in this case the addition of F.sup.− did not induce changes in the size and morphology in comparison to the un-doped counterparts. The XRD patterns of ACP.sup.2, ACP.sup.1, F-ACP.sup.2 and F-ACP.sup.1 (FIG. 3B) showed the same broad diffraction band characteristic of a pure amorphous phase. FT-IR spectra of ACP.sup.2, ACP.sup.1, F-ACP.sup.2 and F-ACP.sup.1 (FIG. 3C) also displayed broad, unresolved bands similar to those reported in the other FT-IR spectra.

[0106] The particles of ACP or F-ACP according to the invention hence were round shaped and had dimensions similar to those of the prior art.

Example 5 Bis

[0107] The XRD patterns of SrF-ACP.sup.2, MgF-ACP.sup.2, SrMgF-ACP.sup.2, SrF-ACP.sup.1, MgF-ACP.sup.1, SrMgF-ACP.sup.1 (FIG. 8) showed the same broad diffraction band characteristic of a pure amorphous phase. FT-IR spectra of SrF-ACP.sup.2, MgF-ACP.sup.2, SrMgF-ACP.sup.2, SrF-ACP.sup.1, MgF-ACP.sup.1, SrMgF-ACP.sup.1 (FIG. 9) also displayed broad, unresolved bands similar to those reported in the other FT-IR spectra.

Example 6: Chemical Composition of Samples Prepared in Example 1 and Example 2 and Example 2Bis

[0108] The chemical composition of the samples prepared in example 2 are summarized in Table 2.

TABLE-US-00002 TABLE 2 Calcium + Strontium + Magnesium/ Phosphate.sup.a Citrate.sup.c Carbonate.sup.c SSA.sub.BET.sup.d Sample Ca.sup.a (wt %) P.sup.a (wt %) (mol) F.sup.b (wt %) Mg.sup.a (wt %) Sr.sup.a (wt %) (wt %) (wt %) (m.sup.2g.sup.−1) ACP.sup.2 29.1 ± 1.0 13.2 ± 0.3 1.70 ± 0.02 — 2.2 ± 0.2 3.8 ± 0.4 287 ± 29 F-ACP.sup.2 32.1 ± 0.5 13.1 ± 0.2 1.89 ± 0.01 2.2 ± 0.1 2.0 ± 0.2 3.4 ± 0.3 328 ± 33 ACP.sup.1 28.0 ± 0.6 12.7 ± 0.2 1.70 ± 0.04 — 1.8 ± 0.2 3.2 ± 0.3 309 ± 31 F-ACP.sup.1 31.9 ± 0.8 13.1 ± 0.3 1.88 ± 0.01 1.8 ± 0.1 2.4 ± 0.2 3.1 ± 0.3 293 ± 29 SrF-ACP.sup.2 29.4 ± 0.1 12.9 ± 0.1 1.86 ± 0.02 2.5 ± 0.1 — 3.8 ± 0.1 2.3 ± 0.2 3.4 ± 0.3 287 ± 29 MgF-ACP.sup.2 27.3 ± 0.4 14.2 ± 0.2 1.82 ± 0.01 3.4 ± 0.1 3.7 ± 0.1 — 2.1 ± 0.2 3.2 ± 0.3 289 ± 29 SrMgF-ACP.sup.2 25.3 ± 0.3 13.2 ± 0.2 1.86 ± 0.03 4.1 ± 0.1 3.1 ± 0.1 3.6 ± 0.1 2.4 ± 0.2 3.5 ± 0.3 273 ± 27 SrF-ACP.sup.1 30.0 ± 0.9 13.4 ± 0.4 1.83 ± 0.01 2.2 ± 0.1 — 3.6 ± 0.1 2.2 ± 0.2 3.3 ± 0.3 352 ± 35 MgF-ACP.sup.1 26.7 ± 0.1 14.2 ± 0.1 1.80 ± 0.01 3.7 ± 0.1 3.8 ± 0.1 — 2.3 ± 0.2 3.6 ± 0.3 304 ± 30 SrMgF-ACP.sup.1 24.8 ± 0.2 13.7 ± 0.1 1.81 ± 0.01 3.7 ± 0.1 3.6 ± 0.1 3.1 ± 0.2 1.9 ± 0.2 3.4 ± 0.3 318 ± 32 .sup.aQuantified by ICP-OES; .sup.bQuantified by fluoride ion electrode; .sup.cQuantified by TGA; .sup.dCalculated from BET adsorption.

[0109] The SSA.sub.BET was determined also for the sample of prior art as prepared in example 1.

[0110] The following values were obtained:

ACP.sup.4 200±20 m.sup.2 g.sup.−1
F-ACP.sup.4 213±21 m.sup.2 g.sup.−1

[0111] The value of SSA.sub.BET resulted to be a feature differentiating the ACP and F-ACP obtained with the process of the prior art and the process of the invention.

[0112] TGA curve of the samples according to the invention mainly exhibits four weight losses, that can be attributed to adsorbed water (from room temperature to 150° C.), structural water (from 150 to 350° C.), citrate (from 350 to 700° C.) and carbonate (from 700 to 1000° C.). According to these losses the content of citrate and carbonate were estimated and reported in Table 2.

[0113] The calcium/phosphate ratio of ACP.sup.2 was similar to ACP′ while F-ACP.sup.2 and F-ACP.sup.1 show higher calcium contents and higher Ca/P ratios than their undoped counterparts. The calcium/phosphate ratio of F-ACP.sup.2 was similar to F-ACP.sup.1.

[0114] The calcium+strontium+magnesium/phosphate ratio of SrF-ACP.sup.2, MgF-ACP.sup.2, SrMgF-ACP.sup.2, SrF-ACP.sup.1, MgF-ACP.sup.1, SrMgF-ACP.sup.1 was similar to the value calculated for the samples F-ACP.sup.2 and F-ACP.sup.1. The content of citrate and carbonate didn't change among the samples doped with Sr and Mg and it was similar to value calculated for the samples F-ACP.sup.2 and F-ACP.sup.1. Interestingly, it was found that when Mg, alone or in combination with Sr, is included in the preparation, the amount of fluoride increased.

[0115] Without being bound to any theory the inventors deem that the higher surface areas were due to the process of the invention which provides for the molar ratio of citrate ion to calcium ion in the range from 1 to 2.

[0116] The above data demonstrate that the citrate-coated particles and fluorine-doped citrate-coated particles obtained by the process of the invention are different and so novel from the fluorine-doped citrate-coated particles of WO2016/012452.

Example 7: Ion Release in Artificial Saliva of Samples Prepared in Example 1 and Example 2

[0117] The application of ACP in tooth treatment products is based on the principle of releasing calcium and phosphate ions in order to generate a local supersaturation for triggering the enamel rem ineralization. Therefore, this effect has been tested in vitro. The in vitro ion release in acidic artificial saliva (a solution that mimics the human saliva after eating, without its macromolecular components) has been tested.

[0118] 200 mg of ACP or F-ACP powders prepared in the examples 1 and 2 were dispersed into 10 mL of artificial saliva prepared as modified Tani-Zucchi solution containing KCl 20 mM, KSCN 5.3 mM, Na.sub.2HPO.sub.4 1.4 mM, NaHCO.sub.315 mM, and lactic acid 10 mM. The suspension was maintained at 37° C. under shaking. At scheduled times, 8 ml of the supernatant (that was well separated from the solid phase by centrifugation at 5000 rpm for 15 min) was removed for Ca.sup.2+ and F.sup.− quantification by ICP-OES and fluoride ion electrode, respectively. After that, samples were rinsed with 8 ml of fresh artificial saliva and the suspension was maintained at 37° C. under shaking and treated as previously described at the next time point.

[0119] All the samples of examples 1 and 2 show a sustained release of Ca.sup.2+ and F.sup.− ions in the first two hours (FIG. 4). The samples with a Cit/Ca ratio according to the invention showed surprisingly higher ion release rates, thus revealing themselves as an improved and advantageous product with respect to the prior art.

[0120] Without being bound to any theory the inventors deem that this surprising effect was probably due by the peculiar features of the particles of the invention that had higher surface areas, due to the specific molar ratio used in the process for producing the nanoparticles.

Example 7Bis

[0121] The in vitro ion release in acidic artificial saliva of SrF-ACP.sup.2, MgF-ACP.sup.2, SrMgF-ACP.sup.2, SrF-ACP.sup.1, MgF-ACP.sup.1, SrMgF-ACP.sup.1 prepared according to example 2bis has been tested in the same conditions of Example 7.

[0122] All the samples of the example 2bis, similar to those of the examples 1 and 2, show a sustained release of Ca.sup.2+ and F.sup.− ions in the first two hours (FIGS. 10A and 10B). The samples of the example 2bis according to the invention showed ion release rates comparable to the samples of the example 2, thus revealing themselves as an improved and advantageous product with respect to the prior art.

Example 8: Preparation of the Nanoparticle of the Invention Agglomerated in Microparticles (Nanoparticle Agglomerate of the Invention)

[0123] To evaluate the feasibility to dry the samples of example 2 by a spray dryer without affecting their amorphous feature, ACP.sup.2, ACP.sup.1, F-ACP.sup.2 and F-ACP.sup.1 after washings and as obtained in Example 2 have been re-suspended in water at 3.5% w/v and dried by spray drying (Mini Spray Dryer B-290, Büchi Labortechnik AG, Switzerland) under the following conditions: nozzle diameter 0.7 mm, feed rate 3 ml/min, argon flow rate 450 I h.sup.−1, inlet temperature 120° C. and aspirator rate 70%. XRD pattern of the spray dried F-ACP.sup.1 powder (FIG. 5) showed only a broad band at about 30° (2θ), corroborating the fact that the amorphous phase is preserved. SEM micrographs of the spray dried F-ACP.sup.1 powder (FIG. 6) revealed that the sample consists of spherical particles of about 2-25 μm of diameter that in turn are composed of the agglomerated nanoparticles independently of the Cit/Ca ratio and presence of fluoride. The value of SSA.sub.BET of the dried powder was in the range 3-10 m.sup.2 g.sup.−1 independently of the Cit/Ca ratio and presence of fluoride.

Example 9: Dry Powder Stability of Samples Prepared in Example 2

[0124] ACP is unstable than the crystalline polymorphs of CaP, so it converts in the crystalline phase even in dry state reacting with atmospheric water. Therefore its use and handling is difficult unless a stable material is developed. The stability of ACP.sup.2, ACP.sup.1, F-ACP.sup.2 and F-ACP.sup.1 powders stored at room temperature has been evaluated analyzing its structure by collecting XRD up to one year (FIG. 7). Interestingly, the XRD pattern remained unchanged, establishing that the amorphous nature of all the samples is preserved during this period of time.