Process for obtaining fluoride-doped citrate-coated amorphous calcium phosphate nanoparticles

Abstract

Process for obtaining fluoride-doped citrate-coated amorphous calcium phosphate nanoparticles. This material has applications in biomedicine due to its biodegradability and bioactivity; it also promotes cell adhesion and osteogeneration. In dentistry, it may be used in toothpastes, mouthwashes, chewing gums, gels and fluoride varnishes as a remineralising agent of dentine and enamel. It is based on two solutions formed by calcium chloride and sodium citrate on the one hand, and by sodium monohydrogenophosphate and sodium carbonate with a fluoride compound on the other, which are mixed at room temperature. The process is eco-efficient and eco-friendly, as it does not leave any acid residue; it consists of a single stage and it is the first time that an amorphous calcium phosphate coated with citrate and doped with fluoride, which enhances its remineralising action, is obtained.

Claims

1. A process for obtaining fluoride-doped citrate-coated amorphous calcium phosphate nanoparticles comprising: preparing a first solution comprising CaCl.sub.2 at a concentration comprised between 0.08 M and 0.12 M and Na.sub.3C.sub.6H.sub.5O.sub.7 at a concentration comprised between 0.35 M and 0.50 M; preparing a second solution comprising Na.sub.2HPO.sub.4 at a concentration comprised between 0.10 M and 0.15 M, Na.sub.2CO.sub.3 at a concentration of 0.2 M and a fluoride compound; mixing under stirring the two solutions prepared in the previous stages in the proportion 1:1 v/v at a pH comprised between 8.3 and 8.7 at ambient temperature for a time period of less than 2 minutes; performing three successive sedimentation cycles of the mixture of the two solutions formed in the previous step by centrifugation, removal of the obtained supernatant and washing of the obtained precipitate using ultrapure water; and freeze-drying the wet precipitate obtained in the previous step.

2. The process according to claim 1, characterised in that the concentrations used for the first solution are 0.1 M for CaCl.sub.2 and 0.4 M for Na.sub.3C.sub.6H.sub.5O.sub.7.

3. The process according to claim 1, characterised in that the concentrations used for the second solution are 0.12 M for Na.sub.2HPO.sub.4 and 0.2 M for Na.sub.2CO.sub.3.

4. The process according to claim 1, characterised in that the fluoride compound is selected from among CaF.sub.2, NaF and KF and is added to a concentration comprised between 0.01 M and 0.1 M.

5. The process according to claim 4, characterised in that the fluoride compound is CaF.sub.2 which is added to a concentration of 0.05 M.

6. Citrate-coated and fluoride-doped amorphous calcium phosphate nanoparticles obtained by a process comprising: preparing a first solution comprising CaCl.sub.2 at a concentration comprised between 0.08 M and 0.12 M and Na.sub.3C.sub.6H.sub.5O.sub.7 at a concentration comprised between 0.35 M and 0.50 M; preparing a second solution comprising Na.sub.2HPO.sub.4 at a concentration comprised between 0.10 M and 0.15 M, Na.sub.2CO.sub.3 at a concentration of 0.2 M, and a fluoride compound; mixing under stirring the two solutions prepared in the previous stages in the proportion 1:1 v/v at a pH comprised between 8.3 and 8.7 at ambient temperature for a time period of less than 2 minutes; performing three successive sedimentation cycles of the mixture of the two solutions formed in the previous step by centrifugation, removal of the obtained supernatant and washing of the obtained precipitate using ultrapure water; and freeze-drying the wet precipitate obtained in the previous step, characterised in that they have a spherical shape and a size comprised between 30 nm and 80 nm and sodium, calcium, and phosphate, citrate, carbonate, fluoride and water content comprised: between 3.1% and 3.5% by weight of sodium between 27.0% and 27.4% by weight of calcium between 37.0% and 37.8% by weight of phosphate (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, and between 2% and 5% by weight of fluoride.

7. A vehicle for biomolecules, drugs, or both comprising the citrate-coated and fluoride-doped amorphous calcium phosphate nanoparticles as defined in claim 6.

8. A biomaterial comprising the fluoride-doped citrate-coated amorphous calcium phosphate nanoparticles as defined in claim 6.

9. The biomaterial according to claim 8, wherein the biomaterial is orthopaedic or dental.

10. A composition comprising the citrate-coated and fluoride-doped amorphous calcium phosphate nanoparticles as defined in claim 6.

11. The composition according to claim 10, wherein the composition is toothpaste, chewing gum, mouthwash, fluoride varnish, or gel.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows transmission electron microscopy (TEM) images of the citrate-coated ACP (a) and FACP (b) nanoparticles. 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 c and d represent ACP and FACP X-ray energy dispersive (EDS) spectra, respectively.

(2) FIG. 2 shows nanoparticles X-ray diffractograms (a) and Raman spectra (b).

(3) FIG. 3 shows MTT cell proliferation assays conducted on human osteoblasts incubated for 1, 3 and 7 days with ACP nanoparticles (100 ?g/mL, 500 ?g/mL, 1,000 ?g/mL). *p 0:05; ***p 0:001; n=3.

EMBODIMENT OF THE INVENTION

(4) ACP nanoparticles were obtained by a precipitation process by mixing two solutions containing: (i) 0.1 M CaCl.sub.2+0.4 M Na.sub.3C.sub.6H.sub.5O.sub.7 and (ii) 0.12 M Na.sub.2HPO.sub.4+0.2M Na.sub.2CO.sub.3
in the proportion 1:1 v/v, a total of 200 ml and adjusting the pH to 8.5 with HCl at ambient temperature.

(5) 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 subsequently characterised.

(6) In order to obtain these fluoride-doped particles, CaF.sub.2 0.05 M is added to the solution (ii).

(7) Characterisation Techniques

(8) 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 dispersed in ultrapure water and then a few drops of this suspension were deposited on conventional copper gratings.

(9) The amount of Ca and P was quantified using optical emission spectroscopy (ICP-OES) using a Liberty 200 (Varian, Australia) spectrometer. To this end, the freeze-dried samples were dissolved in concentrated ultrapure nitric acid (1% v/v).

(10) The thermogravimetric analysis (TGA) was performed using a SDT Q 600 system (TA Instruments, New Castle, Del., USA) under a constant flow of nitrogen (100 mL.Math.min.sup.?1) and increasing the temperature up to 1,200? C. at intervals of 10? C..Math.min.sup.?1.

(11) 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 K? radiation (A=1,5418 ?). Variable spectral bandwidths (anti-scatter) having a radiation length of 10 mm were used. The 2? range was varied from 5? to 70? with increments in 2? of 0.039.

(12) The Raman spectra were obtained using a LabRAMHR spectrometer (Jobin-Yvon, Horiba, Japan). This unit is equipped with a diode laser as an excitation source (?=532 nm) and a Peltier-cooled CCD detector (1026?256 pixels). The spectra were obtained with a spectral resolution of 3 cm.sup.?1.

(13) 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 spectrophotometry complexed with zirconyl chloride and eriochrome cyanine R and measuring the absorbance of the complex at 570 mm.

(14) Analysis of In Vitro Cell Culture

(15) The biological response of the nanoparticles was evaluated using human osteoblast cell lines (MG-63, Lonza, Italy). The cells were cultured in DMEM/F12 medium (PAA, Austria), containing 10% of fetal bovine serum (FBS) and streptomycin-penicillin (100 U/mL-100 ?g/mL) at 37? C. and in a CO.sub.2 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 cells (cell viability test). The cells were deposited on 96-well plates with a density of 3.0?10.sup.3 cells per well. Twenty-four hours later, three different concentrations of citrate-coated ACP nanoparticles were added to the cell culture (100 ?g/mL, 500 ?g/mL, 1,000 ?g/mL), previously sterilized by 25 kGy ? radiation. The cells were incubated under standard conditions (37? C., 5% CO.sub.2) for 1, 3 and 7 days. The culture medium was renewed every three days. All these assays were conducted in a laminar flow cabinet.

(16) MTT Cytotoxicity and Cell Viability

(17) 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 compound (formazan), whose concentration may be colorimetrically determined, making it possible to determine the mitochondrial functionality of the treated cells.

(18) The cells, after being in contact with the nanoparticles for 1, 3 and 7 days, were incubated in MTT dissolved in PBS (5 mg mL.sup.?1) in the proportion 1:10 for 2 hours at 37? C. The cells were then incubated with 200 ?l of dimethyl sulfoxide (Sigma) for 15 min to dissolve the formazan 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. Three samples were analysed for each of the time intervals studied (1, 3 and 7 days).

(19) Results

(20) TEM images (FIG. 1) indicate that both the non-doped samples, ACP (A), and doped samples, FCAP (B), are 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 because 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 calcium phosphates, as the main peak appears at 952 cm.sup.?1, slightly shifted with respect to the main crystalline hydroxyapatite peak (961 cm.sup.?1). The chemical composition of the ACP and FACP materials obtained by TGA, ICP and X-ray fluorescence has already been described earlier.

(21) The biological response of the nanoparticles was studied using osteoblast cells (MG-63). Three different nanoparticle concentrations (100, 500 and 1,000 ?g/ml) were added to the culture medium and, after a certain incubation 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 due to 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 cell line.