(19) Europ3isches Patentamt

European Patent Office

Office européen des brevets

111111111111111111111111111111111111111111111111111111111111111111111111111

(11) EP 3 177 273 81

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) lnt Cl. : of the grant of the patent: A61K 911912006·01) A61K 6100 !20o6.01J

20.03.2019 Bulletin 2019/12 A61K 9151 12006·01) A61L 2410012006·01)

(86) lnternational application number: (21) Application number: 15748187.0 PCT/EP2015/066651

(22) Date of filing : 21.07.2015 (87) lnternational publication number:

WO 2016/012452 (28.01.2016 Gazette 2016/04)

(54) PROCESS FOR OBTAINING FLUORIDE-DOPED CITRATE-COATED AMORPHOUS CALCIUM PHOSPHATE NANOPARTICLES

VERFAHREN ZUR GEWINNUNG VON FLUORIDDOTIERTEN CITRATBESCHICHTETEN AMORPHEN CALCIUMPHOSPHATNANOPARTIKELN

PROCÉDÉ POUR OBTENIR DES NANOPARTICULES DE PHOSPHATE DE CALCIUM AMORPHE REVETUES DE CITRATE DOPÉES AU FLUORURE

(84) Designated Contracting S tates: AL AT BE BG CH CY CZ DE DK EE ES Fl FR GB GR HR HU lE IS IT Ll L T LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30) Priority: 21.07.2014 ES 201431091

(43) Date of publication of application : 14.06.2017 Bulletin 2017/24

(73) Proprietors: • Consejo Superior de Investigaciones Científicas

(CSIC) 28006 Madrid (ES)

• Consiglio Nazionale Delle Ricerche 00185 Roma (IT)

(72) lnventors: • DELGADO LÓPEZ, José Manuel

E-18100 Armilla (Granada) (ES) • GÓMEZ MORALES, Jaime

E-18100 Armilla (Granada) (ES) • FERNÁNDEZ PENAS, Raquel

E-18100 Armilla (Granada) (ES) • IAFISCO, Michele

1-48018 Faenza (IT) • TAMPIERI, Anna

"'C""" 1-48018 Faenza (IT) m M r.... N r....

• PANSERI, Silvia 1-48018 Faenza (IT)

(74) Representative: Pons Glorieta Ruben Dario 4 28010 Madrid (ES)

(56) References cited: CN-A- 101 428 779 US-A 1- 2006 11 O306

• JOSÉ MANUEL DELGADO-LÓPEZ ET AL: "Crystallization of bioinspired citrate-functionalized nanoapatite with tailored carbonate content", ACTA BIOMATERIALIA, vol. 8, no. 9, 1 September 2012 (2012-09-01), pages 3491-3499, XP55217943, ISSN: 1742-7061, DOI: 10.1 016/j.actbio.2012.04.046 cited in the application

• DOROZHKIN ET AL: "Amorphous calcium (ortho)phosphates", ACTA BIOMATERIALIA, ELSEVIER, AMSTERDAM, NL, vol. 6, no. 12, 1 December 2010 (2010-12-01), pages 4457-4475, XP027423368, ISSN: 1742-7061, DOI: 10.1016/J.ACTBI0.2010.06.031 [retrieved on 2010-07-04] cited in the application

• HUI YANG ET AL: "A systematic examination of the morphology of hydroxyapatite in the presence of citrate", RSC ADVANCES, vol. 3, no. 45, 1 January 2013 (2013-01-01), page 23184, XP055217923, DOI: 10.1039/c3ra44839h

r.... Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent "'C""" Bulletin, any person m ay give notice to the European Patent Office of opposition to that patent, in accordance with the M lmplementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been

D.. paid . (Art. 99(1) European Patent Convention) . w Printed by Jouve, 75001 PARIS (FR)

EP 3 177 273 B1

Description

SECTOR AND OBJECT OF THE INVENTION

5 [0001] Biomaterials of interest in biomedicine (i.e., drug delivery nanocarriers and bone regeneration) and dentistry.

[0002] The object of the invention is a process for obtaining amorphous calcium phosphate nanoparticles coated with citrate (a molecule that forms part of the organic phase of the bone) and doped with fluoride. This material has a wide

range of applications in the field of biomedicine (i.e. drug delivery nanocarriers and bone regeneration) dueto its excellent biodegradability and bioactivity, in addition to promoting cell adhesion and osteogeneration. Likewise, it has multiple

1o applications in dentistry, where it may be used in toothpastes, mouthwashes, fluoride varnishes, chewing gum and gels

as a remineralising agent of dentine and enamel. [0003] The process is based on two solutions formed by calcium chloride and sodium citrate on the one hand, and sodium monohydrogenophosphate and sodium carbonate with a fluoride compound on the other, which are mixed at

room temperature. With respect to the prior art, it has the advantages that the process is eco-efficient, and eco-friendly 15 since it does not leave any acidic residue (strong acids are not used in the synthesis), it is synthesised in a single stage

(on using the sodium citrate as a reactive agent in the synthesis) and it is the first time that a citrate-coated and fluoride­

doped amorphous calcium phosphate is obtained, thereforewith a stronger remineralising action than amorphous calcium phosphate.

20 STA TE OF THE ART

[0004] The amorphous phase is a less frequent form of mineral calcium phosphate (CaP) in living organisms. Amor­phous calcium phosphate (ACP) has been found in the mitrochondria of eukaryotic and prokaryotic ce lis and is considered

the precursor stage in the formation process ofthe mineral phase of bone, nanocrystalline carbonate-apatite. The surface

25 of this bone apatite has recently been found to be coated with citrate. ACP also acts as an intermediate phase in the preparation of various CaPs using different methods. This material has a wide range of applications in the field of

biomedicine due to its interesting properties such as excellent bioactivity, facilitates cell adhesion and also favours osteoconductivity and osteogeneration . Likewise, it has multiple applications in dentistry, where it may be used in tooth­paste, mouthwashes, chewing gum, gels and fluoride varnishes as a remineralising agent of dentine and enamel.

30 [0005] Document CN1 01428779A relates to a nanometer materials and the preparation thereof, in particular to a

hydroxyapatite with a hollow nanometer structure and a preparation method thereof. The outer appearance of the hydroxyapatite with the hollow nanometer structure is elliptic, spindle-shaped or hexagonal prism-shaped; and the

nanometer structure is a hollow structure. [0006] Document US2006/11 0306A 1 discloses nano-particles of calcium and phosphorous compounds made in a

35 highly pure generally amorphous state by spray drying a weak a cid solution of said compound and evaporating the liquid from the atomized spray in a heated column followed by collection of the precipitated particles.

[0007] W098/40406 discloses a product composed of amorphous calcium phosphate stabilised with casein, a phos­phoprotein present in milk. This product is currently used as an abrasive material in toothpastes, chewing gum and in tooth whitening post-treatments. However, its efficiency in preventing caries and remineralising damaged varnish has

40 not yet been demonstrated. ACP is also used in compound polymeric materials as a filler material in the preparation of

dental pieces. ACP stimulates tooth repair particularly dueto the fact that Ca and phosphate ions are relea sed in response to acid pHs generated by bacteria! plaque, which are deposited on the tooth structure in the form of hydroxyapatite,

regenerating enamel (mainly composed of crystalline hydroxyapatite). Table 1 summarises the m a in applications of ACP

as a biomaterial. 45

Table 1. Amorphous calcium phosphates used as biomaterials.

Type of material Application Effect

Cements

50

Bone replacement

Dentistry

Hardening agent Absorbable with high surface reactivity Source of Ca2+ and P043- ions

Coatings

55 Mineral/organic

compounds

Metallic

protheses

Tooth

remineralisation

Bone

replacement

lncreases biodegradability and its biological activity

lmproves its mechanical properties

Release of Ca2+ and P043- ions, increasing their biological activity

2

5

10

15

20

25

30

35

40

45

50

55

EP 3 177 273 B1

(continued)

Type of material Application Effect

Aqueous suspension Release of genes Absorbable and biocompatible pH-dependent stability

[0008] Some of these applications are described in J. Zhao et al., Amorphous calcium phosphate and its application in dentistry; Chemistry Central Journal (2011 ), 5:40 (doi: 1 0.1186/1752-153X-5-40). [0009] As regards the preparation of ACP, it is known in several modalities obtained from soluble Ca2+ and P043­

precursors at adequate pHs for precipitation, commonly using soluble precursors whose cation does not give rise to

other species that could interfere a posteriori in the composition of the final product, such as Ca(OHh, H3P04 , phosphate or ammonium hydrogenophosphate. Ca(N03h is frequently used. [0010] The function of citric acid as a Ca2+ cation complexer is also known, which is also acceptable from the phar­

maceutical viewpoint, in addition to, for example, other polycarboxylic acids such as tartaric a cid. For this reason, these

acids are also used to stabilise amorphous compositions of ACP. This is set out in the claims of publication W003059304, where citric a cid is proposed, among other che late formers with the Ca2+ cation, in the proportion 0.1% to 5% by weight

in a preparation containing ACP combined with a phosphopeptide. [0011] JP2001169121 propases the use of citric acid as a stabiliser of ACP already formed by precipitation from

phosphoric acid and calcium hydroxide, subjecting it to subsequent milling in the presence of the aforementioned citric

a cid. [0012] Therefore, non e ofthese publications mentions a preparation such as the process objectofthe present invention, in which citrate is added as a reactive agent for ACP precipitation (in a single-stage process) and notas a stabiliser in

a phase subsequent to precipitation (two-stage process).

[0013] The revisions performed by Dorozhkin S. V. [Nanosized and nanocrystalline calcium orthophsphates, Acta Biomaterialia (201 0), No. 6 (3), 715-734]; Combes C. and Rey C. [Amorphous calcium phosphates: synthesis, properties

and uses in biomaterials, Acta Biomaterialia (201 0), No.6 (9), 3362-3378] and another revision by Dorozhkin S. V. [Amorphous calcium phosphates, Acta Biomaterialia (2010), No.6 (12), 4457-4475 disclose several wet processes, but in which the same conditions, process stages and reactive agents of the process object of the present invention are not

applied. In fact, citric acid is often envisaged as a dispersant agent in these preparations and occasionally the carbonate

anion, with similar functions. [0014] The publication of J.M. Delgado-Lopez et al. Crystallization of bioinspired citrate-functionalized nanoapatite with tailored carbonate content (Acta Biomaterialia (2012) No. 8, page 3491) establishes an apatite anda citrate-coated

nanocrystalline carbonate-apatite precipitation process. The substantial differences in the state of the art between the process object of the present invention and this documentare as follows:

(1) Precipitation temperature. (2) Precipitation of citrate-coated and fluoride-doped ACP nanoparticles as a stable phase. (3) There is no maturity process of the precipitate.

(4) Apatite or any other crystalline phase of the calcium phosphate is not formed in the precipitate.

BRIEF DESCRIPTION OF THE INVENTION

[0015] A first object ofthe present invention is a process for obtaining fluoride-doped citrate-coated amorphous calcium phosphate (FACP) comprising:

the preparation of a CaCI2 solution at a concentration comprised between 0.08 M and 0.12 M and Na3C6H50 7 (sodium citrate) ata concentration comprised between 0.35 M and 0.50 M;

the preparation of a second solution formed by Na2HP04 ata concentration comprised between 0.1 OM and 0.15 M with Na2C03 0.2 M and a fluoride compound; mixture under stirring of the two solutions prepared in the previous stages in the proportion 1:1 v/v ata pH comprised between 8.3 and 8. 7 (adjusted, forexample, with HCI) and at room temperature for a time period of less than 2 minutes;

three successive sedimentation cycles by centrifugation, removal of the supernatant and washing of the precipitate

with ultrapure water; and freeze-drying of the wet precipitate.

[0016] In a preferred embodiment of the invention, the concentrations of the reactive agents used in the first solution

are 0.1 M for CaCI2 and 0.4 M for Na3C6H50 7 and the concentrations used for the second solution are 0.12 M for

3

EP 3 177 273 B1

5

Na2HP04 and 0.2 M for Na2C03.

[0017] The fluoride compound is selected from among CaF2, NaF or KF and is added toa concentration comprised between 0.01 M and 0.1 M. In a preferred embodiment, the fluoride compound is CaF2 and is added toa concentration of 0.05 M. [0018] Another object of the present invention is constituted by fluoride-doped amorphous calcium phosphate nano­particles obtained by the previous process, having a spherical shape and size comprised between 30 nm and 80 nm, as well as the following Na, Ca, P, citrate, carbonate, fluoride and structural water content comprised:

1o

15

between 3.1% and 3.5% by weight of Na between 27.0% and 27.4% by weight of Ca between 37.0% and 37.8% by weight of P between 3.5% and 5.0% by weight of citrate between 5.4% and 7.0% by weight of carbonate

between 6% and 10% by weight of water between 2% and 5% by weight of fluoride

[0019] [0020]

The term "water" relates in this aspect of the invention both to adsorbed water and structural water. In a third aspect, another object of the invention is the use of nanoparticles in applications such as:

2o

25

transport of biomolecules and/or drugs biomaterials in orthopaedic applications in dentistry, preferably as a material for preparing cements for filling and/or sealing root Canals and dental repairs, oras a component of toothpastes, chewing gums, mouthwashes, fluoride varnishes and gels to favoring the rem­ineralisation of enamel through the gradual ralease of calcium, phosphate and fluoride ions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]

30

35

Figure 1 shows transmission electron microscopy (TEM) images of the citrate-coated ACP (a) and FACP (b) nan­

oparticles. The selected area electron diffraction patterns (SAED) obtained for each of the nanoparticles are also shown as insets. The left inset in A shows a TEM image of a single nanoparticle. Panels e and d represent ACP and FACP X-ray energy dispersive (EDS) spectra, respectively. Figure 2 shows nanoparticles X-ray diffractograms (a) and Raman spectra (b). Figure 3 shows MTI cell proliferation assays conducted on human osteoblasts incubated for 1, 3 and 7 days with ACP nanoparticles (1 00 fLg/mL, 500 fLg/mL, 1,000 fLg/mL). *p ~ 0:05; ***p ~ 0:001; n =3.

EMBODIMENT OF THE INVENTION

40 [0022] ACP nanoparticles were obtained by a precipitation process by mixing two solutions containing:

(i) 0.1 M CaCI2 + 0.4 M Na3C6H50 7 and (ii) 0.12 M Na2HP04 + 0.2M Na2C03

45

50

in the proportion 1:1 v/v, a total of 200 mi and adjusting the pH to 8.5 with HCI at ambient temperature. [0023] When the mixture takes on a milky appearance (approximately 30 s after mixing), the particles are subjected to three successive sedimentation cycles by centrifugation, removal of the supernatant and washing of the precipitate with ultrapure water (MilliQ©, Millipore). Subsequently, the wet precipitate is freeze-dried and the particles are subse­

quently characterised. [0024] In arder to obtain these fluoride-doped particles, CaF2 0.05 M is added to the solution (ii).

Characterisation techniques

55

[0025] The nanoparticles were analysed using a Scanning Transmission Electron Microscope (STEM Philips CM 20) operating at 80 kV. This equipment also allowed the acquisition of selected area electron diffraction (SAED) patterns and X-ray energy dispersive (EDS) spectra. For these analyses, the freeze-dried samples were dispersad in ultrapure water and then a few drops of this suspension were depositad on conventional copper gratings. [0026] The amount of Ca and P was quantified using optical emission spectroscopy (ICP-OES) using a Liberty 200

4

EP 3 177 273 B1

(Varian, Australia) spectrometer. To this end, the freeze-dried samples were dissolved in concentrated ultrapure nitric

a cid ( 1% v/v). [0027] The thermogravimetric analysis (TGA) was performed using a SDT Q 600 system (TA lnstruments, New Castle, DE, USA) under a constant flow of nitrogen (1 00 ml.min-1) and increasing the temperature up to 1 ,200°C at intervals

5 of 10°C.min-1.

[0028] The X-ray diffraction patterns were acquired using an X-Pert PRO (PANalytical) diffractometer equipped with a PIXcel detector operating at 45 kV and 40 mA, with incident Cu Ka radiation (A.= 1,5418 Á). Variable spectral bandwidths

(anti-scatter) having a radiation length of 1Omm were used. The 28 range was varied from 5° to 70° with increments in

28 of 0.039. 1o [0029] The Raman spectra were obtained using a LabRAMHR spectrometer (Jobin-Yvon, Horiba, Japan). This unit

is equipped with a diode laser asan excitation so urce (A.=532 nm) anda Peltier-cooled CCD detector (1 026 x 256 pixels). The spectra were obtained with a spectral resolution of 3 cm-1 . [0030] The quantity of fluoride in the samples was quantified by X-ray fluorescence spectroscopy (XRF) using a

PHILIPS Magix Pro (PW-2440) spectrometer. Additionally, the fluoride content was also determined by spectrophotom­15 etrycomplexed with zirconyl chlorideand eriochrome cyanine Rand measuring theabsorbance ofthe complexat570 mm .

Analysis of in vitro cell culture

[0031] The biological response of the nanoparticles was evaluated using human osteoblast celllines (MG-63, Lanza,

2o ltaly). The cells were cultured in DMEM/F12 medium (PAA, Austria), containing 10% offetal bovine serum (FBS) and

streptomycin-penicillin (100 U/mL-100f.Lg/mL) at 3rc andina C02 atmosphere (5%). Subsequently, the cells were separated from their medium by trypsinisation and then centrifuged and resuspended . The Trypan blue exclusion test was used to count the live ce lis (cell viability test). The ce lis were deposited on 96-well plates with a density of 3.0x1 03

cells per well. Twenty-four hours later, three different concentrations of citrate-coated ACP nanoparticles were added

25 to the cell culture (100 f.LQ/ml, 500 f.LQ/ml, 1,000 f.LQ/ml), previously sterilized by 25 kGy Y radiation . The cells were incubated under standard conditions (3rC, 5% C02) for 1, 3 and 7 days. The culture medium was renewed every three

days. All these assays were conducted in a laminar flow cabinet.

MTT cytotoxicity and cell viability 30

[0032] The MTT method [3-(4.5-dimethylthiazol-2-yl)-2 .5-diphenyltetrazolium bromide] was used to determine the possible toxic effect of the nanoparticles. This assay is based on the metabolic reduction of 3-(4.5-dimethylthiazol-2-yl)­2.5-diphenyltetrazolium bromide (MTT) by the mitochondrial enzyme succinate dehydrogenase in a blue-coloured com­

pound (formazan), whose concentration may be colorimetrically determined, making it possible to determine the mito­35 chondrial functionality of the treated cells.

[0033] The ce lis, after being in contact with the nanoparticles for 1, 3 and 7 days, were incubated in MTT disolved in

PBS (5 mg mL-1) in the pro portian 1:1 O for 2 hours at 3rC. The ce lis were then incubated with 200 f.LI of dimethyl sulfoxide (Sigma)for 15min todissolvetheformazan crystals.A Multiskan FC Microplate (Thermo Scientific) spectrometer was used to measure absorbance, which is directly proportional to the number of metabolically active cells, at 570 nm.

40 Three samples were analysed for each of the time intervals studied (1, 3 and 7 days).

Results

[0034] TEM images (Fig . 1) indicate that both the non-doped samples, ACP (A), and doped samples, FCAP (B), are 45 spherical nanoparticles with sizes comprised between 30 nm and 80 nm. Also, the absence of diffraction points in the

SAED patterns evidences their amorphous nature. In turn, the EDS spectra confirm that they are composed only of Ca and P. The F peak in the doped particle spectrum that should appear around 0.68 KeV is not observed, possibly beca use it is overlapped by the oxygen peak (0.2 KeV), which is by far more intense. The absence of peaks in the X-ray diffraction

patterns confirms the amorphous nature of these materials (Fig. 2A). Raman spectra are also typical of amorphous 50 calcium phosphates, as the m a in peak appears at 952 cm-1, slightly shifted with respect to the m a in crystalline hydroxya­

patite peak (961 cm­ 1). The chemical composition of the ACP and FACP materials obtained by TGA, ICP and X-ray fluorescence has already been described earlier.

[0035] The biological response of the nanoparticles was studied using osteoblast ce lis (MG-63). Three different nan­

oparticle concentrations (1 00, 500 and 1,000 f.LQ/ml) were added to the culture medium and, after a certain incubation 55 period (1, 3 or 7 days), the number of metabolically active cells was quantified by MTT assays (Fig. 3). An increase in

cell proliferation was observed in all cases (even for the highest concentration) after 1 to 7 days of incubation. Also, for the lowest concentration studied, cell growth is comparable to that observed by the cells in the absence of nanoparticles

(control). However, increasing the concentration, cell growth is much less significant than in the control, possibly dueto

5

EP 3 177 273 B1

the fact that they are excessively high nanoparticle concentrations. Despite this, the cell viability and morphology assays

(not shown) obtained very similar results for all the concentrations studied. These results clearly indicate that the nan­oparticles are completely biocompatible in contact with this human osteoblast cellline.

5

Claims

1. A process for obtaining citrate-coated and fluoride-doped amorphous calcium phosphate nanoparticles comprising:

10 - the preparation of a CaCI2 solution ata concentration com prised between 0.08 M and 0.12 M and Na3C6H50 7 ata concentration comprised between 0.35 M and 0.50 M; - the preparation of a second solution formed by Na2HP04 ata concentration comprised between 0.1 O M and

0.15 M with Na2C03 0.2 Manda fluoride compound;

- mixture under agitation of the two solutions prepared in the previous stages in the proportion 1:1 v/v ata pH 15 comprised between 8.3 and 8.7 at ambient temperature for a time period of less than 2 minutes;

- three successive sedimentation cycles by centrifugation, removal of the supernatant and washing of the

precipitate using ultrapure water; and - freeze-drying of the wet precipitate.

2o 2. A process, according to claim 1, characterised in that the concentrations of the reactive agents used for the first

solution are 0.1 M for CaCI2 and 0.4 M for Na3C6H50 7.

3. A process, according to any one of claims 1 and 2, characterised in that the concentrations used for the second

solution are 0.12 M for Na2HP04 and 0.2 M for Na2C03.

25

4. A process, according to any one of claims 1 to 3, characterised in that the fluoride compound is selected from

among CaF2, NaF and KF and is added toa concentration comprised between 0.01 M and 0.1 M.

5. A process, according to claim 4, characterised in that the fluoride compound is CaF2 which is added toa concen­30 tration of 0.05 M.

6. Citrate-coated and fluoride-doped amorphous calcium phosphate nanoparticles obtained by means of a process as

defined in claims 1 to 5, characterised in that they have a spherical shape and a size comprised between 30 nm and 80 nm, meas u red by transmission electron microscopy (TEM) images and Na, Ca, P, citrate, carbonate, fluoride

35 and water content comprised:

- between 3.1% and 3.5% by weight of Na - between 27.0% and 27.4% by weight of Ca - between 37.0% and 37.8% by weight of P

40 - between 3.5% and 5.0% by weight of citrate

- between 5.4% and 7.0% by weight of carbonate - between 6% and 1 0% by weight of water

- between 2% and 5% by weight of fluoride.

45 7. Citrate-coated and fluoride-doped amorphous calcium phosphate nanoparticles, as defined in claim 6, for use as

vehicles for biomolecules, drugs or both.

8. Citrate-coated and fluoride-doped amorphous calcium phosphate nanoparticles, as defined in claim 6, for use as

biomaterials in orthopaedic applications. 50

9. Citrate-coated and fluoride-doped amorphous calcium phosphate nanoparticles, as defined in claim 6, for use for dentistry applications.

1O. Nanoparticles, as defined in claim 9, for use as a material for preparing cements for filling, sealing or both operations

55 in root canals and dental repairs.

11. Nanoparticles, as defined in claim 9, for use as a component of toothpastes, chewing gums, mouthwashes, fluoride

varnishes and gels for favouring the remineralisation of enamel through the gradual release of calcium, phosphate

6

EP 3 177 273 B1

and fluaride ians.

Patentansprüche 5

1. Verfahren zur Gewinnung van Citrat-beschichteten und Fluarid-datierten amarphen Calciumphasphat-Nanaparti­keln, falgendes umfassend:

- Herstellen einer CaCirlosung bei einer Kanzentratian zwischen 0,08 MundO, 12M und Na3C6H50 7 bei einer 10 Kanzentratian zwischen 0,35 M und 0,50 M;

- Herstellen einer zweiten Losung, die durch Na2HP04 bei einer Kanzentratian zwischen O, 1 O M und O, 15 M mit Na2C03 0,2 M und einer Fluaridverbindung gebildet wird; - Mischen, unter Rühren, der beiden in den varangegangenen Stufen hergestellten Losungen im Verhaltnis 1:1

vlv bei einem pH-Wert zwischen 8,3 und 8,7 bei Umgebungstemperatur für einen Zeitraum van weniger als 2 15 Minuten;

- drei aufeinanderfalgende Sedimentatianszyklen durch Zentrifugatian, Entfernung des Überstandes und Wa­schen des Niederschlags mit Reinstwasser; und

- Gefriertracknung des nassen Niederschlags.

2o 2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass die Kanzentratianen der für die erste Losung ver­wendeten reaktiven Mittel O, 1 M für CaCI2 und 0,4 M für Na3C6H50 7 betragen.

3. Verfahren nach einem der Ansprüche 1 und 2, dadurch gekennzeichnet, dass die für die zweite Losung verwen­

deten Kanzentratianen 0,12 M für Na2HP04 und 0,2 M für Na2C03 betragen. 25

4. Verfahren nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, dass die Fluaridverbindung ausgewahlt

ist aus CaF2, NaF und KF und zu einer Kanzentratian zwischen 0,01 M und O, 1 M hinzugefügt wird.

5. Verfahren nach Anspruch 4, dadurch gekennzeichnet, dass die Fluaridverbindung CaF2 ist, das zu einer Kan­30 zentratian van 0,05 M zugesetzt wird.

6. Citrat-beschichtete und Fluarid-datierte amarphe Calciumphasphat-Nanapartikel, die nach einem Verfahren gemar..

den Ansprüchen 1 bis 5 gewannen werden, dadurch gekennzeichnet, dass sie eine kugelformige Form und eine Gror..e zwischen 30 nm und 80 nm aufweisen, gemessen durch Transmissianselektranenmikraskapie (TEM)-Bilder

35 und Na, Ca, P, Citrat, Carbanat, Fluarid und Wassergehalt:

- zwischen 3,1 Gew.-% und 3,5 Gew.-% Na - zwischen 27,0 Gew.-% und 27,4 Gew.-% Ca

- zwischen 37,0 Gew.-% und 37,8 Gew.-% P 40 - zwischen 3,5 Gew.-% und 5,0 Gew.-% Citrat

- zwischen 5,4 Gew.-% und 7,0 Gew.-% Carbanat

- zwischen 6 Gew.-% und 1 O Gew.-% Wasser - zwischen 2 Gew.-% und 5 Gew.-% Fluarid.

45 7. Citrat-beschichtete und Fluarid-datierte amarphe Calciumphasphat-Nanapartikel naeh Anspruch 6 zur Verwendung

als Trager für Biamaleküle, Arzneimittel ader beides.

8. Citrat-beschichtete und Fluarid-datierte amarphe Calciumphasphat-Nanapartikel naeh Anspruch 6 zur Verwendung

als Biamaterialien in arthapadischen Anwendungen. 50

9. Citrat-beschichtete und Fluarid-datierte amarphe Calciumphasphat-Nanapartikel naeh Anspruch 6 zur Verwendung in zahnarztlichen Anwendungen.

10. Nanapartikel nach Anspruch 9 zur Verwendung als Material zur Herstellung van Zementen zum Füllen, Versiegeln

55 ader für beide Operatianen in Wurzelkanalen und Zahnreparaturen.

11. Nanapartikel nach Anspruch 9 zur Verwendung als Bestandteil van Zahnpasten, Kaugummis, Mundwasser, Flua­

ridlacken und Gelen zur Forderung der Remineralisierung des Schmelzes durch die allmahliche Freisetzung van

7

EP 3 177 273 B1

Calcium-, Phosphat- und Fluoridionen.

Revendications 5

1. Procédé pour l'obtention de nanoparticules de phosphate de calcium amorphe enrobées de citrate et dopées a la

fluorure comprenant:

- la préparation d'une solution CaCI2 a une concentration comprise entre 0,08 M et O, 12 M et Na3C6H50 7 a 10 une concentration comprise entre 0,35 M et 0,50 M ;

- la préparation d'une deuxiéme solution constituée de Na2HP04 a une concentration comprise entre O, 1O M et O, 15 M avec Na2C03 0,2 M et un compasé de fluorure; - mélange sous agitation des deux solutions préparées dans les étapes précédentes dans la proportion 1:1 v/v

aun pH compris entre 8,3 et 8, 7 aune température ambiante pour une période de temps inférieure a2 minutes ; 15 - trois cycles de sédimentation successifs par centrifugation, élimination du surnageant et lavage du précipité

en utilisant de l'eau ultrapure ; et

- lyophilisation du précipité humide.

2. Procédé, selon la revendication 1, caractérisé en ce que les concentrations des agents réactifs utilisés pour la

2o premiére solution sont O, 1 M pour CaCI2 et 0,4 M pour Na3C6H50 7.

3. Procédé, selon l'une quelconque des revendications 1 et 2, caractérisé en ce que les concentrations utilisées pour la deuxiéme solution sont O, 12 M pour Na2HPO4 et 0,2 M pour Na2C03.

25 4. Procédé, selon l'une quelconque des revendications 1 a3, caractérisé en ce que le compasé de fluorure est choisi parmi CaF2, NaF et KF et est ajouté a une concentration comprise entre 0,01 M et O, 1 M.

5. Procédé, selon la revendication 4, caractérisé en ce que le compasé de fluorure est CaF2 qui est ajouté a une

concentration de 0,05 M. 30

6. Nanoparticules de phosphate de calcium amorphe enrobées de citrate et dopées a la fluorure obtenues au moyen d'un procédé tel que défini dans les revendications 1 a5, caractérisées en ce qu'elles ont une forme sphérique

et une taille comprise entre 30 nm et 80 nm, mesurée par des images de microscopie électronique en transmission (MET) et une teneur en Na, Ca, P, citrate, carbonate, fluorure et eau comprise:

35

- entre 3,1 % et 3,5 % en poids de Na

-entre 27,0 % et 27,4% en poids de Ca -entre 37,0 % et 37,8% en poids de P

- entre 3,5 % et 5,0 % en poids de citrate 40 -entre 5,4% et 7,0 %en poids de carbonate

- entre 6 % et 1 O% en poids d'eau

-entre 2 % et 5 % en poids de fluorure.

7. Nanoparticules de phosphate de calcium amorphe enrobées de citrate et dopées a la fluorure, telles que définies 45 dans la revendication 6, pour leur utilisation en tant que véhicules pour les biomolécules, les médicaments o u les deux.

8. Nanoparticules de phosphate de calcium amorphe enrobées de citrate et dopées a la fluorure, telles que définies dans la revendication 6, pour leur utilisation en tant que biomatériaux dans les applications orthopédiques.

so 9. Nanoparticules de phosphate de calcium amorphe enrobées de citrate et dopées a la fluorure, telles que définies

dans la revendication 6, pour leur utilisation pour les applications de dentisterie.

1O. Nanoparticules, telles que définies dans la revendication 9, pour leur utilisation en tant que matériau pour la prépa­ration de ciments pour l'obturation, le scellement ou les deux opérations dans les canaux radiculaires et les répa­

ss r~~nsden~iffis.

11. Nanoparticules, telles que définies dans la revendication 9, pour leur utilisation en tant que composant de dentifrices, chewing-gums, bains de bouche, vernis et gels fluorés pour favoriser la reminéralisation de l'émail par la libération

8

5

10

15

20

25

30

35

40

45

50

55

EP 3 177 273 B1

graduelle d'ions de calcium, de phosphate et de fluorure.

9

EP 3 177 273 B1

Ca CaC) D)

Ni"

o p o p

cu•

o 2 4 6 8 10 o 2 4 8 8 10

Energy (KeV) Energy (KeV)

FIG. 1

10

EP 3 177 273 B1

A) 2-f/J...

e:5 20 o->.... 'ii.i 15 e Q)... e:

10 20 30 40 50 60 29 (degrees)

800 1000 1200 1400 2800 3000 3200400 600

Raman displacement (cm-1)

FIG. 2

11

70

EP 3 177 273 81

... D Ce lis (control) ,-----, -E e: tlr'!tk..--. ~ 100 J..IQ/mL... o 1'""""'1 ,..... • 500 J.Jg/mLlO

• 1000 J.Jg/mL -CD o ,-----,e: ca .e ' ­o 0,5en .e <(

0,0 ...... n, -\ ~ ~ ~ ~ ~ ~

FIG. 3

12

EP 3 177 273 B1

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader's convenience only. lt does not form part of the European patent document. E ven though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims a/1/iabi/ity in this regard.

Patent documents cited in the description

CN 101428779 A [0005] US 200611 0306 A 1 [0006]

WO 9840406 A [0007]

Non-patent literature cited in the description

J. ZHAO et al. Amorphous calcium phosphate and its application in dentistry. Chemistry Central Journal, 2011, vol. 5, 40 [0008] DOROZHKIN S. V. Nanosized and nanocrystalline calcium orthophsphates. Acta Biomaterialia, 201 O, vol. 3 (6), 715-734 [0013]

COMBES C. ; REY C. Amorphous calcium phos­phates: synthesis, properties and uses in biomateri­als. Acta Biomaterialia, 201 O, vol. 9 (6), 3362-3378 [0013]

WO 03059304 A [001 O] JP 2001169121 B [0011]

DOROZHKIN S. V. Amorphous calcium phosphates. Acta Biomaterialia, 2010, vol. 12 (6), 4457-4475 [0013] J.M. DELGADO-LOPEZ et al. Crystallization of bio­inspired citrate-functionalized nanoapatite with tai­lored carbonate content. Acta Biomateria/ia, 2012, 3491 [0014]

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