Morphogenetically Active Amorphous Calcium Polyphosphate Nanoparticles Containing Retinol For Therapeutic Applications

Abstract

This invention concerns a calcium polyphosphate material consisting of amorphous nanoparticles with a diameter of approximately between about 45 nm to about 0.25 μm that displays a considerable hardness (elastic modulus) of about 1.3 GPa. The inventive noncrystalline and biodegradable material that is produced under mild conditions, at room temperature, is morphogenetically active and preferably induces bone formation and the expression of the marker gene for osteoblast activity, alkaline phosphatase. In a preferred aspect, the invention concerns a method for producing amorphous retinol/calcium-polyphosphate nanospheres (retinol/aCa-polyP-NS) that show several unexpected properties and can be used in the treatment or prophylaxis of a variety of dermatological conditions, including photoaging.

Claims

1. A method for the production of a solid, degradable amorphous polyphosphate material having calcium counterions, wherein said method comprises the steps of: i) dissolving of a polyphosphate salt in an aqueous solvent to create a solution and adjusting the pH value of the solution to an alkaline value, ii) adding a calcium salt solution to said polyphosphate salt solution, and adjusting the pH value to an alkaline value, iii) optionally, washing with a solvent, for example ethanol, and iv) collecting of the particles formed, wherein steps i) to iii) are performed at ambient temperature, and wherein said material has a hardness similar to that of bone tissue, and is morphogenetically active.

2. The method, according to claim 1, used to prepare degradable amorphous retinoid/calcium-polyphosphate nanospheres, wherein a) in step i) a lubricating coating material is co-dissolved in said aqueous solvent, b) in step ii) a retinoid salt is co-dissolved in said solvent, and c) nanospheres as formed are collected.

3. The method according to claim 2, wherein the said retinoid is retinol.

4. The method according to claim 1, wherein said calcium salt is calcium chloride.

5. The method according to claim 1, wherein said polyphosphate salt is sodium polyphosphate.

6. The method according to claim 1, wherein the polyphosphate has a chain length of about 3 to about 1000 phosphate units.

7. The method according to claim 1, wherein the pH is adjusted to 10.

8. The method according to claim 1, wherein the calcium polyphosphate material is formed from sodium polyphosphate in the presence of calcium chloride at a stoichiometric ratio of 0.1 to 10 (phosphate to calcium).

9. The method according to claim 1, wherein the calcium polyphosphate material is obtained by addition of a solution containing 14 g/L of calcium chloride or 28 g/L of calcium chloride to a solution containing 10 g/L of sodium polyphosphate.

10. The method according to claim 1, wherein an emulsifier is added to the polyphosphate solution in order to avoid phase separation.

11. The method according to claim 2, wherein the retinoid/Ca-polyP nanospheres are obtained by addition of one part of a solution containing 2 g/L of retinol and 56 g/L of calcium chloride in absolute ethanol to two parts of a solution containing 10 g/L of sodium polyphosphate and 20 g/L of poly(ethylene glycol) in water.

12. The method according to claim 2, wherein said retinoid/Ca-polyP nanospheres are obtained in an essentially homogenous size optimal suitable for cellular uptake by clathrin-mediated endocytosis.

13. The method according to claim 1, further comprising the step of producing hard amorphous and morphogenetically active polyphosphate nanoparticles, and/or a material containing such nanoparticles.

14. A method for bone regeneration and repair, dental use, or drug delivery, wherein the method comprises the use of the nanoparticles or the material containing such nanoparticles produced according to claim 13.

15. A degradable amorphous retinoid/calcium-polyphosphate nanosphere, produced according to claim 2.

16. A method for the treatment or prophylaxis of a dermatological condition wherein said method comprises the use of the amorphous retinoid/calcium-polyphosphate nanospheres according to claim 15.

17. A cosmetic or therapeutic composition comprising an amorphous retinoid/calcium-polyphosphate nanospheres according to claim 15, formulated as a creme or ointment.

18. A method for drug delivery wherein said method comprises the use of the amorphous retinoid/calcium-polyphosphate nanospheres according to claim 15.

19. The method, according to claim 1, wherein the aqueous solvent is water.

20. The method, according to claim 2, wherein in step i) the lubricating coating is polyethylene glycol) polymer.

Description

[0046] The invention will now be described further in the following preferred examples, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties.

[0047] FIG. 1 shows the FTIR spectra of Na-polyP, Ca-polyP1 and Ca-polyP2; (A) wavenumbers 4000 to 600 cm.sup.−1 and (B) wavenumbers 1400 and 600 cm.sup.−1.

[0048] FIG. 2 shows the SEM micrographs taken from the following polyP powders: (A and B) Na-polyP, (C and D) Ca-polyP1 and (E and F) Ca-polyP2.

[0049] FIG. 3 shows the biological properties of the Ca-polyP material, developed here. (A) SaOS-2 cells remained without phosphate material (none), or were incubated with 10 μg/mL of Na-polyP, Ca-polyP1, Ca-polyP2, HA, or β-TCP for 7 d in the presence of the activation cocktail OC. Then the coverslips with the cells were stained with Alizarin Red S. (B) Expression level of ALP in SaOS-2 cells, in response to exposure to phosphate material. After an 1 or 7 d incubation period the cells were harvested and RNA was extracted and subjected to qRT-PCR analysis. The steady-state-levels of the ALP transcripts were measured and normalized to the expression of GAPDH. Data are expressed as mean values ±SD for four independent experiments; each experiment was carried out in duplicate. Differences between the groups were evaluated using unpaired t-test. *p<0.05. (C) Degradation of polyP in vitro, using the 16.5% polycrylamide/7 M urea gel electrophoresis technique. The cells were incubated with 50 μg/ml of solid Ca-polyP2 either in the presence of SaOS-2 cells and medium/serum or PBS [phosphate buffered saline]; after an incubation period of 1 or 7 d, samples were taken for chain length determination. Synthetic polyP markers with an average chain length of 80, 40 and 3 units were run in parallel.

[0050] FIG. 4 shows the preparation of retinol/aCa-polyP-NSs. (A) A retinol solution was added drop-wise to (B) a Na-polyP solution, containing PEG. (C) An emulsion was formed that contained (D) the retinol/aCa-polyP-NS, composed of Ca.sup.2+, polyP and retinol. (E) Nanospheres, lacking (left) and containing retinol (right) are shown. Further details are in “Methods”.

[0051] FIG. 5 shows the influence of aCa-polyP-NP versus Na-polyP on MC3T3 cell growth. The assays were composed of either Na-polyP (cross-hatched bars) or aCa-polyP-NP (filled bars) at concentrations between 1 and 30 μg/ml. After terminating the cultivation after 72 h the assays were subjected to the XTT assay and the absorbance at 650 nm was determined. Data represent means±SD of ten independent experiments (*P<0.01).

[0052] FIG. 6 shows the synergistic effect of retinol and aCa-polyP on the proliferation propensity. The concentration of aCa-polyP-NP had been kept constant (3 μg/ml) in all assays. At this concentration no effect on cell growth is seen. Addition of retinol in the range of 0.3 to 30 μM likewise did not affect the growth of the cells (left hatched bars). After co-addition of aCa-polyP-NP with retinol (right hatched bars) a strong growth-inducing effect is measured. The effect is synergistic at concentrations of retinol with higher than 1 μg/ml. *P<0.01.

[0053] FIG. 7 shows the florescence intensities of the nanoparticles and nanospheres at an excitation of 470 nm and an emission of 525 nm. While the nanoparticles, aCa-polyP-NP (A), show only background fluorescence (B), the retinol-containing nanospheres retinol/aCa-polyP-NS (C) are lighting up with a bright green fluorescence.

[0054] FIG. 8 shows the stability of polyP in the retinol/aCa-polyP-NS nanospheres after incubation (1 to 5 d) (A) in PBS or (B) in medium/serum supplemented with MC3T3-E1 cells in the standard incubation assay. Then aliquots were taken for chain length determination. Synthetic polyP markers with an average chain length of 80, 40 and 3 units were run in parallel.

[0055] FIG. 9 shows the expression levels of the different collagen types, collagen type I (COL-I), collagen type II (COL-II), collagen type III (COL-III) and collagen type V (COL-V) in MC3T3-E1 cells exposed to either retinol or the nanoparticles aCa-polyP-NP alone or in combination; one concentration of aCa-polyP-NP (3 μg/ml) and two concentrations of retinol (1 and 3 μM) were tested. The concentrations of retinol are given in μM; aCa-polyP-NP is added at a concentration of 3 μg/ml. After a 4 d incubation period the MC3T3-E1 cells were harvested, their RNA extracted and the steady-state-levels of collagen expression were determined by RT-qPCR using the GAPDH gene as house-keeping gene as reference. The expression values of the transcripts in the RNA from cells are given as ratios between the transcripts of treated (retinol and/or aCa-polyP-NP) to untreated cells (no additional component). The results are means from 5 parallel experiments; *P<0.01.

[0056] FIG. 10 shows the expression levels of the different types of collagen (COL-I, COL-II, COL-III and COL-V) in MC3T3-E1 cells. The steady-state expression values are normalized to the expression of the house-keeping gene GAPDG and are given as ratios between treated (retinol/aCa-polyP-NS) and untreated cells. *P<0.01.

[0057] FIG. 11 shows the expression level of collagen type III gene in cells exposed to retinol alone, Na-polyP, aCa-polyP-NP1 nanoparticles (1:1 ratio between phosphate and Ca.sup.2+) and aCa-polyP-NP (1:2 ratio between phosphate and Ca.sup.2+) in the absence or presence of retinol. In the last series the assays were composed with retinol, aCa-polyP-NP and 20 μM of the inhibitor of the clathrin-mediated endocytosis triflupromazine (TFP). The expression of collagen type III is correlated with the house-keeping gene GAPDH. *P<0.01.

EXAMPLES

[0058] In the following examples, the inventive method described only for polyP molecules with a chain length of 30 to 50 phosphate units. Similar results can be obtained by using polyP molecules with lower and higher chain lengths, such as between 100 to 20 units.

FTIR Analyses

[0059] The two phosphate materials, Ca-polyP1 and Ca-polyP2, were characterized by FTIR and compared with the spectrum obtained with Na-polyP (FIG. 1). The complete spectra between the wavenumbers 4000 and 600 cm.sup.−1 are shown in FIG. 1A, while segments between 1400 and 600 cm.sup.−1 are given in FIG. 1B. The band near 1250 cm.sup.−1 is assigned to the asymmetric stretching mode of the two non-bridging oxygen atoms bonded to phosphorus atoms in the PO.sub.2 metaphosphate units, ν.sub.as(PO.sub.2).sup.−. The weak band at 1190 cm.sup.−1 is the PO.sub.2 symmetric stretching mode ν.sub.s(PO.sub.2).sup.−. The absorption bands close to 1083 and 999 cm.sup.−1 are assigned to the asymmetric and symmetric stretching modes of chain-terminating PO.sub.3 groups [ν.sub.as(PO.sub.3).sup.2− and ν.sub.s(PO.sub.3).sup.2−]. The absorption band near 864 cm.sup.−1 is attributed to the asymmetric stretching modes of the P—O—P linkages, ν.sub.as (P—O—P) and the partially split band centered around 763 cm.sup.−1 is due to the symmetric stretching modes of these linkages, ν.sub.s (P—O—P).

[0060] The comparison of the spectra of Na-polyP and Ca-polyP shows that the polyP features are seen in the 1300-1000 cm.sup.−1 region. There, most of the chemical characteristics of polyP chains are found. In all three samples a similar pattern is seen, reflecting that the polyP chain backbones are not broken down during the reaction with the Ca.sup.2+ ions. However, the peaks are shifted in the Ca-polyP1 and Ca-polyP2 samples if compared the Na-polyP spectrum. This shifting is also seen for other bands in the Ca-polyP spectra; they even increase by increasing the Ca.sup.2+ content in the polyP sample (from Ca-polyP1 to Ca-polyP2). Moreover, it has been reported that the adsorptions bands near 1100-1000 cm.sup.−1 are attributed to the ionic stretching mode of the P—O— group, the shifting as well as broadening of this peak of the Ca-polyP samples is attributed to the formation of P—O••M.sup.+(+) (where M is Ca.sup.2+). In conclusion, the IR spectra confirm the interaction between Ca.sup.2+ and polyP and the formation of Ca-PolyP.

[0061] XRD analyses were performed with both Ca-polyP1 and Ca-polyP2; the patterns showed no sign of crystallinity, like Na-polyP (FIG. 1C).

Morphology of polyP Samples

[0062] The three samples, Na-polyP, Ca-polyP1 and Ca-polyP2, were analyzed by SEM (FIG. 2). The Na-polyP particles, of a non-regular shapes, often show a tapered morphology (FIGS. 2A and B). The sizes of the particles vary between 1 and 300 μm with an average size of ≈100 μm. Likewise non-regular shapes show the Ca-polyP1 particles (FIGS. 2C and D). They are smaller than the Na-polyP particles with an average diameter of ≈4 μm. Even smaller are the Ca-polyP2 particles with an average diameter of ≈0.2 μm (FIGS. 2 E and F).

PolyP-Induced Mineralization of SaOS-2 Cells

[0063] The cells were incubated with 10 μg/mL of Na-polyP, Ca-polyP1, Ca-polyP2, HA, or β-TCP in the presence of the activation cocktail OC for 7 d. Then the coverslips onto which the cells had been cultivated were removed and stained with Alizarin Red S, as described under Methods. Eye-inspection revealed that the intensity of the color reaction, which reflects the extent of minerals being present in the samples, is highest for Ca-polyP1 and for Ca-polyP2. The degree of color reaction is lower for Na-polyP, β-TCP and HA; the intensities of those samples are only slightly higher, compared to the control (FIG. 3A).

Expression Level of ALP

[0064] Since the Alizarin Red S color reaction might not be sensitive enough due to cross-reactivity with exogenously added grains, the steady-state-expression level of ALP in SaOS-2 cells was quantified by qRT-PCR. The inventors determined previously that the expression level of this enzyme is a reliable marker for the polyP-induced activation of bone cells (Müller W E G, Wang X H, Diehl-Seifert B, Kropf K, Schloβmacher U, Lieberwirth I, Glasser G, Wiens M and Schröder H C. Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca.sup.2+ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomaterialia 2011j; 7:2661-2671). Therefore, the inventors subjected the cells, after incubation with the different phosphate samples and in the presence of the OC activation cocktail to qRT-PCR analysis. The data revealed that at day 1 the expression level of ALP is statistically not different between the different polyphosphate samples used. However, after an incubation period of 7 d the steady-state-expression levels of ALP in the cells exposed to Ca-polyP1 and Ca-polyP2 are significantly higher (≈0.10 expression units compared to the one of the reference GAPDH) than those measured for Na-polyP, β-TCP or HA (≈0.055 expression units). The expression levels of the latter three assays are not significantly higher than the one seen in control assays (no phosphate sample added); FIG. 3B.

Hardness of the Ca-polyP2 Material

[0065] Determination of the mechanical properties (elastic modulus) of the Ca-polyP2 biopolymer was performed with a ferrule-top cantilever and found to be of ≈1.3 GPa, close to values measured for trabecular tissue that is surrounding human bone with 6.9 GPa (Zysset P K, Guo X E, Hoffler C E, Moore K E, Goldstein S A. Elastic modulus and hardness of cortical and trabecular bone lamellae measured by nanoindentation in the human femur. J Biomech 1999; 32:1005-1012).

Degradation of polyP In Vitro

[0066] In one series of experiments, the SaOS-2 cells were incubated with 50 μg/ml of solid Ca-polyP2 and incubated in the standard assay for 1 d or 7 d either in the presence of SaOS-2 cells and medium/serum or in PBS. Then samples were taken and assayed for the chain length of polyP (Lorenz B, Schröder H C. Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase. Biochim Biophys Acta 2001; 1547:254-261). The gels were stained with o-toluidine blue. The results (FIG. 3C) show that Ca-polyP2, added to the assays, does not undergo hydrolytic degradation during the 7 d long incubation period if dissolved in PBS. In contrast the average chain length of Ca-polyP2 drops from an average chain length of 40 to around 3 P.sub.i units if the polymer is incubated in the assays which contained SaOS-2 cells and medium/serum. This result indicates that Ca-polyP2 is prone to hydrolysis by ALP (exopolyphosphatase), but also to other polyP hydrolyzing enzymes, endophosphatases, that exist in serum.

Methods

Polyphosphate

[0067] The sodium polyphosphate (Na-polyP of an average chain of 40 phosphate units) used in the Examples has been obtained from Chemische Fabrik Budenheim (Budenheim; Germany). Na-polyphosphate (Na-polyP) with an average chain length of 30 phosphate units (NaPO.sub.3).sub.n can also be obtained, for example, from Merck Millipore ((#106529; Darmstadt; Germany), all-trans retinol, for example, from Sigma (#95144; ≧97.5%, M.sub.r 286.45; Sigma, Taufkirchen; Germany)

Preparation of Calcium Polyphosphate Nanospheres

[0068] Ten g of Na-polyP are dissolved in 500 ml of distilled water and the pH is adjusted to 10 with 1 M NaOH. A solution of 14 g of calcium chloride dihydrate or 28 g of CaCl.sub.2 in 250 ml is added slowly, dropwise (1 ml/min) to the Na-polyP solution, adjusting steadily the pH to 10 at room temperature. The suspension formed is stirred for 4 hr. Then the particles formed are collected by washing twice with ethanol while filtering through a 0.45 μm filter (e.g., Nalgene Rapid-Flow). Then the particles are dried at 50° C. or 60° C. The Ca-polyP material obtained by addition of 14 g of CaCl.sub.2 is named “Ca-polyP1”, and the one with 28 g of CaCl.sub.2 is termed “Ca-polyP2”. aCa-polyP-NP contains a ratio: phosphate:Ca.sup.2=2.

[0069] The amorphous retinol/Ca-polyP nanospheres, retinol/aCa-polyP-NS, are prepared by a process under avoidance of light. A retinol solution (100 mg/50 ml absolute ethanol), containing 2.8 g of CaCl.sub.2 (FIG. 4A), is prepared and added drop-wise to a Na-polyP solution (1 g in 100 ml water; FIG. 4B). In order to avoid a phase separation and to stabilize the emulsion 2 g of poly(ethylene glycol) [PEG] (for example: #P5413; Sigma-Aldrich; average mol wt 8,000) are added to the Na-polyP solution. The emulsion formed (FIG. 4C) is stirred for 6 h. The particles formed (FIG. 4D) are collected by filtration and washed three times with water to remove excess of calcium ions and unreacted components. Then the particles are dried at room temperature overnight; in contrast to the nanospheres, formed without retinol, those which contain this ingredient have a light yellow color (FIG. 4E).

[0070] The preparation of the aCa-polyP-NP has been described (Müller W E G, Tolba E, Schröder H C, Wang S F, Glaβer G, Muñoz-Espí R, Link T, Wang X H. A new polyphosphate calcium material with morphogenetic activity. Materials Lett 2015, in press). These nanoparticles are prepared by precipitation of Na-polyP with Ca.sup.2+ in a stoichiometric ratio of 1:2. The polyP nanospheres, likewise amorphous, are produced from an ethanolic solution of retinol with CaCl.sub.2 (FIG. 4A) that is added to a solution of Na-polyP with PEG (FIG. 4B), during which a suspension of retinol/aCa-polyP-NS is formed (FIG. 4D). The nanospheres are collected and have a slightly yellow color, in contrast to the nanoparticles that lack retinol (FIG. 4F).

Chemical Characterization by FTIR

[0071] Fourier transformed infrared (FTIR) spectroscopy in the attenuated total reflectance (ATR) mode is applied, using, for example, the Varian 660-IR spectrometer with Golden Gate ATR auxiliary (Agilent). Spectra between the wavenumbers 4000 and 600 cm.sup.−1 are recorded.

Scanning Electron Microscopy and Energy Dispersive X-Ray Spectroscopy

[0072] For the scanning electron microscopic (SEM) analyses, for example, a HITACHI SU 8000 can be employed at low voltage (<1 kV; analysis of near-surface organic surfaces). EDX spectroscopy can be performed, for example, with an EDAX Genesis EDX System attached to a scanning electron microscope (Nova 600 Nanolab) operating at 10 kV with a collection time of 30-45 s. Areas of approximately 10 μm.sup.2 were analyzed by EDX.

XRD Analyses

[0073] X-ray diffraction (XRD) is performed using established procedures (Fischer V, Lieberwirth I, Jakob G, Landfester K, Muñoz-Espí R. Metal oxide/polymer hybrid nanoparticles with versatile functionality prepared by controlled surface crystallization. Adv Funct Mat 2013; 23:451-466).

Cells and Cell Culture Conditions

[0074] Human osteogenic sarcoma cells, SaOS-2 cells, are used for the experiments and cultivated in McCoy's medium with fetal calf serum [FCS] (Wiens M, Wang X H, Schloβmacher U, Lieberwirth I, Glasser G, Ushijima H, Schröder H C, Müller W E G. Osteogenic potential of bio-silica on human osteoblast-like (SaOS-2) cells. Calcif Tissue Intern 2010; 87:513-524). Cultivation of the cells is performed in 24-well plates; 3×10.sup.4 cells are seeded per well. After an initial incubation period of 3 d, the cultures are supplemented either with 10 μg/mL of solid Na-polyP, supplemented with CaCl.sub.2 in a 2:1 stoichiometric ratio, in order to compensate for the chelating activity of polyP as described (Müller W E G, Wang X H, Diehl-Seifert B, Kropf K, Schloβmacher U, Lieberwirth I, Glasser G, Wiens M, Schröder H C. Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca.sup.2+ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomaterialia 2011; 7; 2661-26719), or with the two polyP samples, prepared here, with Ca-polyP1 or with Ca-polyP2. In comparison also nano-hydroxyapatite (HA; for example: 677418 Sigma-Aldrich) or β-TCP (for example: 13204 Sigma-Aldrich) had been included in the test series. As controls none of those polymers is added. Then the cells are continued to be incubated in the presence of the osteogenic cocktail [OC], containing 10 nM dexamethasone, 5 mM β-glycerophosphate and 50 mM ascorbic acid. After a 7 d incubation period the cells are either stained with Alizarin Red S to assess the extent of mineralization (Schröder H C, Borejko A, Krasko A, Reiber A, Schwertner H, Müller W E G. Mineralization of SaOS-2 cells on enzymatically (silicatein) modified bio active osteoblast-stimulating surfaces. J Biomed Mater Res B Appl Biomater 2005; 75:387-392) or subjected to qRT-PCR [quantitative real-time RT-PCR] analysis (Wiens M, Wang X H, Schloβmacher U, Lieberwirth I, Glasser G, Ushijima H, Schröder H C, Müller W E G. Osteogenic potential of bio-silica on human osteoblast-like (SaOS-2) cells. Calcif Tissue Intern 2010; 87:513-524).

[0075] Mouse calvaria cells MC3T3-E1 cells (ATCC-CRL-2593) are used for the experiments and cultivated in a-MEM (Gibco—Invitrogen) containing 20% fetal calf serum [FCS] (Gibco). The medium contains 2 mM L-glutamine, 1 mM Na-pyruvate and 50 μg/ml of gentamycin. The cells are incubated in 25 cm.sup.2 flasks or in 24-well plates (Greiner Bio-One) in an incubator 37° C. and 5% CO.sub.2. Reaching 80% confluency, the cells are detached using trypsin/EDTA and continuously subcultured at a density of 5.Math.10.sup.3 cells/ml. The cells are seeded at a density of 5.Math.10.sup.3 cells/well. Medium/serum change was every 3 d.

[0076] The aCa-polyP-NP are added at the indicated concentration to the cultures, usually 3 μg/ml. Retinol is added in parallel; it is dissolved at 1 mg/ml ethanol and then diluted in DMSO [dimethyl sulfoxide] at the indicated concentrations. In one series of experiments Na-polyP, stoichiometrically complexed with Ca.sup.2+ (molar ratio of 2:1/phosphate monomer:Ca.sup.2+; Müller W E G, Wang X H, Diehl-Seifert B, Kropf K, Schloβmacher U, Lieberwirth I, Glasser G, Wiens M, Schröder H C. Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca.sup.2+ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomaterialia 2011; 7:2661-2671) is studied in parallel.

Transcripts Expression

[0077] For qRT-PCR reactions the primer pair Fwd: 5′-TGCAGTACGAGCTGAACAGGAACA-3′ (SEQ ID NO: 1) [nt.sub.1141 to nt.sub.1164] and Rev: 5′-TCCACCAAATGTGAAGACGTGGGA-3′ (SEQ ID NO: 2) [nt.sub.1418 to nt.sub.1395; PCR product length 278 bp] can be used for the quantification of the alkaline phosphatase [ALP] transcripts (accession number NM_000478.4). The expression level of the ALP can be normalized to the one of the reference gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase; NM_002046.3) using the primer pair Fwd: 5′-CCGTCTAGAAAAACCTGCC-3′ (SEQ ID NO: 3) [nt.sub.845 to nt.sub.863] and Rev: 5′-GCCAAATTCGTTGTCATACC-3′ (SEQ ID NO: 4) [nt.sub.1o59 to nt.sub.1o78; 215 bp].

Determination of the Hardness

[0078] The hardness of the polyphosphate material Ca-polyP2 can be determined, for example, by a ferruled optical fiber-based nanoindenter as described (Chavan D, Andres D, Iannuzzi D. Ferrule-top atomic force microscope. II. Imaging in tapping mode and at low temperature. Rev Sci Instrum 2011; 82:046107. doi: 10.1063/1.3579496).

PolyP Degradation In Vitro

[0079] In one series of experiments the SaOS-2 cells are incubated with 50 μg/ml of solid Ca-polyP or Ca-polyP2 and incubated in the standard assay for 4 d. Then samples (50 μl) are taken and assayed for the chain length of polyP (for example, see: Lorenz B, Schröder H C. Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase. Biochim Biophys Acta 2001; 1547:254-261). The gels are stained with o-toluidine blue.

Effect of aCa-polyP-NP and Retinol on Cell Growth

[0080] In a first series of experiments Na-polyP (complexed with Ca.sup.2+) was tested towards aCa-polyP-NP (FIG. 5). It is seen that during the 72 h incubation period the concentration of viable cells in the assays with Na-polyP did not significantly change within a concentration range 1 to 30 μg/ml if compared with the control assays (not containing polyP). In contrast, if the aCa-polyP-NP are added instead a significant increase of the concentration of viable cells is seen at 10 μg/ml, which even increases at 30 μg/ml.

[0081] Addition of retinol at concentrations between 0.3 and 30 μM and in the absence of the aCa-polyP-NP did not change significantly the growth rate of the MC3T3 cells. However, if the nanoparticles are added to the retinol-treated cells at a concentration of 3 μg/ml, a strong and significant increase in the proliferation propensity is measured (FIG. 6). The concentration of aCa-polyP-NP was kept constant with 3 μg/ml in the assays, while retinol was co-added with 0.3 to 30 μg/ml to the cells (FIG. 6). At a concentrations of ≧1 μM retinol a significant increase in the proliferation rate is seen which reaches a value of 290% at 10 μM, compared to the controls (100%; without retinol and plus/minus 3 μg/ml of aCa-polyP-NP). These findings imply that retinol and aCa-polyP-NP act synergistically on the proliferation potency of the cells.

[0082] This synergistically-acting effect of the two test components can also be followed microscopically. In the absence of any of the components the density of the MC3T3 cells after the 72 h incubation is only low. Addition of aCa-polyP-NP (3 μg/ml) alone did not change markedly the density of the cells, attached to the substrate. Likewise low is the number of cells onto the plastic surface if 10 μM retinol is added. However, if the two components [3 μg/ml of aCa-polyP-NP and 10 μM retinol] are added together the cells form an (almost) confluent cell monolayer, supporting the synergistic action of aCa-polyP-NP and retinol.

Morphology and Chemical Elemental of Ca-polyP Nanoparticles and Ca-polyP/Retinol Nanospheres

[0083] The Na-polyP particles show a brick stone morphology. The size of the building bricks is >50×50×50 μm. The EDX spectra show signals, (almost) exclusively for O (oxygen), Na (sodium) and P (phosphorous); only a small peak corresponding to C (carbon) is found. In contrast to the morphology of the Na-polyP particles, the aCa-polyP-NP are spheres. After counting of 150 particles the average size (diameter) of the nanoparticles is 96±28 nm. The EDX spectra show, in addition to the elements for 0 and P also Ca (K.sub.α peak at 3.7 keV and the K.sub.β peak at 4.0 keV). Only a weak signal for Na is observed; in contrast the Na peak in the Na-polyP salt is almost as high as the one for P.

[0084] The retinol/aCa-polyP-NS, produced from aCa-polyP-NP and retinol, are like the Ca-polyP nanoparticles globular. However, their size is significantly smaller with a diameter of 45±29 nm. In the EDX spectrum it is obvious that the peak, corresponding to C and originating from retinol, is significantly higher than in the spectrum of the Ca-polyP nanoparticles; the height in the EDX signal for the element C exceeds even the one for 0.

Presence of Retinol in Retinol/aCa-polyP-NS

[0085] Retinol exhibits fluorescence properties with maximum absorbance and emission at 326 nm and 520 nm (cyclohexane) (Tanumihardjo S A, Howe J A. Twice the amount of α-carotene isolated from carrots is as effective as β-carotene in maintaining the vitamin A status of Mongolian gerbils. J Nutr 2005; 135:2622-2626). In turn, retinol can be identified by fluorescence microscopy at an excitation of 470 nm and an emission of 525 nm. The images reveal that the nanospheres, retinol/aCa-polyP-NS (FIG. 7C), show a bright green fluorescence (FIG. 7D), while the nanoparticles, aCa-polyP-NP (FIG. 7A), lacking retinol, show only a slight background fluorescence (FIG. 7B).

[0086] Retinol was determined quantitatively using the SbCl.sub.3-based spectroscopic technique. A suspension with 100 mg of retinol/aCa-polyP-NS nanospheres was extracted with chloroform/methanol and the released retinol determined spectrophotometrically. Applying this approach, the retinol content of the retinol/aCa-polyP-NS was determined to be 23±7% (6 parallel determinations). This figure implies that retinol undergoes an accumulation within the nanospheres that had been formed in 10% retinol-polyP starting ratio (100 mg of retinol per 1 g of polyP).

Susceptibility of polyP in the Retinol/aCa-polyP-NS Nanospheres

[0087] To determine if polyP within the retinol/aCa-polyP-NS is prone to hydrolysis by phosphatases the nanospheres were incubated in PBS (FIG. 8A, lanes a-c) or in medium/serum and cells (FIG. 8B, lanes a-c) in the standard assay for 1 to 5 d. Then aliquots were taken and analyzed for the intactness of the polyP polymer. The data revealed that the average chain length of 30 P, units in the samples incubated in PBS did not change within the incubation period of 5 d (FIG. 8A, lanes a-c), while the size of polyP within the retinol/aCa-polyP-NS progressively decrease from 30 units after 1 d (FIG. 8B, lane a) to 20 units after 3 d (lane b) and even to less that 1-3 units after 5 d (lane c).

Co-Incubation of Retinol with aCa-polyP-NP on Collagen Expression

[0088] MC3T3-E1 cells were incubated with 3 μg/ml of aCa-polyP-NP in the absence or presence of retinol (FIG. 9). After a 5 d incubation period the cells were collected and subjected to RT-qPCR analyses. The aim of the study was to elucidate if the aCa-polyP-NP modulate the expression of the different fibrillar collagen gene for type I, type II, type III and type V. the expression of the type IV basement collagen was not included since the expression level in the MC3T3-E1 cells was, even after incubating the cells with retinol, too low.

[0089] The results are shown in FIG. 9. The expression levels of the different collagen genes are given as ratio between the levels in cells exposed to either retinol or nanoparticles alone or in combination and the level measured in cells not incubated with those components. It is seen that the expression levels of collagen type I, type II, type III or type V only slightly change between 0.95 and 1.8-fold if the two components, retinol (1 μM or 3 μM) and nanoparticles (3 μg/ml), are added separately. These increases are statistically not significant. However, if retinol is added together with 3 μg/ml of aCa-polyP-NP significant increases of the expression of the collagen type I to type III levels are seen; only the changes of collagen type V are not significant. The induction level of the different collagen genes in MC3T3-E1 cells incubated with 3 μg/ml of aCa-polyP-NP and 1 μM retinol is for collagen type I 4.8-fold and with 3 μM retinol 9.4-fold; for type II 31.7-fold with 1 μM retinol or 55.8-fold with 3 μM retinol; for type III 88.7-fold (127.4-fold) and for type V 1.3-fold (2.1-fold).

Effect of the Retinol/aCa-polyP-NS Nanospheres on Collagen Expression

[0090] In a final series of experiments the MC3T3-E1 cells were exposed to different concentrations of the retinol-containing nanospheres, retinol/aCa-polyP-NS. The expression levels are given in FIG. 10 as ratios between the collagen steady-state-values in treated cells (0.3 μg/ml to 10 μg/ml) and the values determined in untreated cells. The results show (FIG. 10), that the expression levels for collagen type I, collagen type II and of collagen type III are at concentration higher than >3 μg/ml retinol/aCa-polyP-NS significantly higher than the one measured for 0.3 or 1 μg/ml.

Comparative Gene Activating Effect of Retinol with Nanoparticles and Nanospheres

[0091] The effect of 3 μM retinol on Na-polyP and on different nanoparticles as well as on the retinol/nanospheres was tested in a comparative way. The expression level of collagen type III was determined by RT-qPCR (FIG. 11). The expression values are correlated to the expression of the house-keeping gene GAPDH. In the absence of any additional component the collagen type III expression level was 0.17±0.02, while in the presence of 3 μM retinol the level increased significantly to 0.25±0.03. Na-polyP, stoichiometrically complexed to Ca.sup.2+ in a molar ratio of 2:1 with the phosphate monomer forms, caused a steady-state-expression of 0.28±0.04; addition of retinol did not significantly alter the level 0.20±0.03. The nanoparticles formed from Na-polyP and Ca.sup.2+ in an 1:1 molar ratio, aCa-polyP-NP1, caused in the absence of retinol a transcript level of 0.21±0.04 and in the presence of retinol 0.31±0.05. However, if retinol is added to the aCa-polyP-NP an increase of the expression from 0.44±0.07 to 8.7±0.93 is determined. If the inhibitor of the clathrin-mediated endocytosis triflupromazine is co-added at a 20 μM concentration the retinol-induced collagen type III expression is reduced to 3.1±0.5. To mention here, is that recently we could establish that nanoparticles fabricated in a 1:1 stoichiometric ratio between phosphate and Ca.sup.2+ have a brick-like morphology with edge lengths of ≈4 μm, while the aCa-polyP-NP nanoparticles are globular/spherical with a size <100 nm.

Cell Proliferation—Cell Viability Assays

[0092] Cell proliferation can be determined, for example, by a colorimetric method based on the tetrazolium salt XTT (Cell Proliferation Kit II; Roche). The absorbance is determined at 650 nm and subtracted form the background values (500 nm). In the experiments described in Examples, the viable cells have been determined after 72 h.

Identification of Retinol

[0093] The green fluorescence of retinol is recorded with a fluorescence light microscope at an excitation of 470 nm and an emission of 525 nm. A quantitative analysis of retinol in the nanospheres can be performed using a colorimetric assay (Subramanyam G B, Parrish D B. Colorimetric reagents for determining vitamin A in feeds and foods. J Assoc Off Anal Chem 1976; 59:1125-1130). A suspension of 100 mg of retinol/aCa-polyP-NS is mixed with 0.45 ml of a chloroform/methanol solvent mixture (2:1; v/v) and centrifuged for 3 min at 4,200×g. Then a 0.15 ml aliquot of the organic solvent layer, containing the extracted retinol, is transferred into a reaction tube; 1 ml of 20% SbCl.sub.3 solution is added drop-wise. Finally, the absorbance of the solution is measured at 620 nm immediately by using, for example, a UV-VIS spectrophotometer Varian Cary 5G UV-Vis-NIR spectrophotometer.

Enzymatic Degradation of polyP in the Retinol/aCa-polyP-NS Nanospheres

[0094] A suspension of 50 μg/ml of retinol/aCa-polyP-NS is dissolved/suspended either in PBS [phosphate buffered saline] or in medium/serum, containing MC3T3-E1 cells and incubated for 1 d, 3 d or 5 d at 37° C. Finally aliquots of 50 μl are taken, kept at ≈pH 3, and assayed for the chain length of polyP (for example, see: Lorenz B, Schröder H C. Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase. Biochim Biophys Acta 2001; 1547:254-261). The gels can be stained, for example, with o-toluidine blue.

Reverse Transcription-Quantitative Real-Time PCR Analyses

[0095] The technique of reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) can be applied to determine the gene expression level of the different types of collagens in the MC3T3-E1 cells. The cells are incubated in medium/serum for 5 d in the absence or presence of retinol, aCa-polyP-NP or retinol/aCa-polyP-NS, as indicated with the respective experiment described under Examples. Then the cells are collected and the isolated RNA is subjected to RT-qPCR. The following primer pairs, matching with the respective mouse collagen types, can be used. Collagen type I alpha 1 (Mus Collal; NM_007742) Fwd: 5′-TACATCAGCCCGAACCCCAAG-3′ (SEQ ID NO. 5) [nt.sub.4003 to nt.sub.4023] and Rev: 5′-GGTGGACATTAGGCGCAGGAAG-3′ (SEQ ID NO. 6) [nt.sub.4146 to nt.sub.4125; product size 144 bp]; type II alpha 2 (Mus Col1a2; NM_007743) Fwd: 5′-AACACCCCAGCGAAGAACTCATAC-3′ (SEQ ID NO. 7) [nt.sub.3789 to nt.sub.3812] and Rev: 5′-TTCCTTGGAGGACACCCCTTCTAC-3′ (SEQ ID NO. 8) [nt.sub.39o8 to nt.sub.3885; size 120 bp]; type III, alpha 1 (Mus Col3a1; NM_009930) Fwd: 5′-GCTGTTTCAACCACCCAATACAGG-3′ (SEQ ID NO. 9) [nt.sub.4764 to nt.sub.4787] and Rev: 5′-CTGGTGAATGAGTATGACCGTTGC-3′ (SEQ ID NO. 10) [nt.sub.4941 to nt.sub.4918; size 178 bp]; type IV, alpha 1 (Mus Col4a1; NM_009931) Fwd: 5′-AACGTCTGCAACTTCGCCTCC-3′ (SEQ ID NO. 11) [nt.sub.4752 to nt.sub.4772] and Rev: 5′-TGCTTCACAAACCGCACACC-3′ (SEQ ID NO. 12) [nt.sub.4886 to nt.sub.4867; size 135 bp]; and type V, alpha 1 (Mus Col5a1; NM_015734) Fwd: 5′-AGTCCCTTCCTGAAGCCTGTCC-3′ (SEQ ID NO. 13) [nt.sub.7110 to nt.sub.7131] and Rev: 5′-GCACACACACAGAGATTAGCACC-3′ (SEQ ID NO. 14) [nt.sub.7265 to nt.sub.7243; size 156 bp]. As the reference gene the GAPDH can be used [glyceraldehyde 3-phosphate dehydrogenase (Mus GAPDH; NM_008084) Fwd: 5′-TCACGGCAAATTCAACGGCAC-3′ (SEQ ID NO. 15) [nt.sub.200 to nt.sub.220] and Rev: 5′-AGACTCCACGACATACTCAGCAC-3′ (SEQ ID NO. 16) [nt.sub.338 to nt.sub.316; size 139 bp]. The amplification can be performed, for example, in an iCycler (Bio-Rad) with the respective iCycler software. After determination of the C.sub.t values the expression of the respective transcripts is calculated.

[0096] In the Examples, the expression levels of the respective collagen genes have been determined and the values measured for the genes in cells, not exposed to either retinol or the nanoparticles/nanospheres, have been set to 1. Then the ratios between the levels in the cells, exposed to retinol or the nanoparticles/nanospheres alone or together, have been calculated and plotted.

Statistical Analysis

[0097] The results can be statistically evaluated using the paired Student's t-test.