GB2535450A - Synergistically acting amorphous calcium-polyphosphate nanospheres containing encapsulated retinol for therapeutic applications - Google Patents

Abstract

A method for the preparation of degradable amorphous retinoid/calcium-polyphosphate nanospheres comprises: (i) dissolving a retinoid and a calcium salt in an organic solvent; (ii) adding the retinoid calcium salt solution as produced in step (i) to an aqueous solution containing dissolved polyphosphate (polyp) salt and poly(ethylene glycol) or another lubricating coating material, and (iii) collecting washing with water , and drying of the nanospheres as formed. Preferably the retinoid is retinol. Preferably the chain length of the polyphosphate is in the range of about 3 to about 1000 phosphate units. Preferably the amorphous retinoid/calcium-polyphosphate nanospheres are for use in the treatment or prophylaxis of dermatological conditions, such as inflammatory skin disorders, acne, disorders of increased cell turnover such as psoriasis, skin cancers and photoaging. The amorphous retinol/calcium-polyphosphate nanospheres displayed the following: (i) Homogenous size in a range (~45 nm) optimal for cellular uptake by clathrin-mediated endocytosis; (ii) Synergistic effect of both components, retinol and polyphosphate, at non-effective concentrations of the individual components, on cell proliferation and collagen expression (types I, II, and III); (iii) Pronounced synergistic effect on expression of collagen type III; (iv) Disintegration by extracellular enzymatic hydrolysis; (v) Dual biological effect of polyP and retinol, both via the extracellular and via the intracellular route; and (vi) No cytotoxicity.

Description

SYNERGISTICALLY ACTING AMORPHOUS CALCIUM-POLYPHOSPHATE NANOSPHERES CONTAINING ENCAPSULATED RETENOL FOR THERAPEUTIC APPLICATIONS

This invention relates to a method for producing amorphous retinol/calcium-polyphosphate nanospheres (retinol/aCa-polyP-NS) which show unexpected properties: (i) Homogenous size in a range (-45 nm) optimal for cellular uptake by clathrin-mediated endocytosis; (ii) Synergistic effect of both components, retinol and polyphosphate, even at non-effective concentrations of the individual components, on cell proliferation and collagen expression (types I, II, and III); (iii) In particular, pronounced synergistic effect on expression of collagen type III; (iv) Disintegration by extracellular enzymatic hydrolysis; (v) Therefore, dual biological effect of polyP and retinol, both via the extracellular and via the intracellular route; and (vi) No cytotoxicity. The inventive retinol/aCa-polyP-NS can be used in the treatment or prophylaxis of a variety of dermatological conditions, including photoaging.

Background of the invention

Skin aging and collagen The epidermis of human skin is composed of keratinocytes of different proliferation and differentiation state, while the dermis is composed of three major types of cells, the fibroblasts, macrophages, and adipocytes. The most abundant protein in the skin and connective tissue, collagen type I, and the other fibrillar collagens, types III and V, are secreted as procollagens and then enzymatically processed to assemble to the triple helix configuration. The quantity and quality, integrity and biomechanical properties, of collagen decrease during skin aging, often accelerated by external factors (Pandel R, Polj'Sak B, Godic A, Dahmane R (2013) Skin photoaging and the role of antioxidants in its prevention. ISRN Dermatol 12;2013:930164).

Skin aging can be grouped into: i) Chronological or ntrinsic aging, and ii) Solar aging (photoaging).

Focusing on the photoaging process, it is well established that the aging skin is characterized by reduced amounts of collagen, accumulation of abnormal elastic fibers and, in parallel, increased quantities of glycosaminoglycans in the upper and mid dermis. The major reason for this imbalance in the extracellular matrix fibrillar meshwork is the occurrence of oxygen-derived species including free radicals (Gilchrest BA, Bohr VA. Aging process, DNA damage, and repair. FASEB J 1997;11:322-330).

Over 20 different collagen types have been distinguished in human tissues (Shoulders MD, Raines RT. Collagen structure and stability. Annu Rev Biochem 2009;78:929-958), among which the type I is the most abundant collagen of the human body, and especially present in tendons, skin, and cornea. Type II is very abundant in hyaline cartilage, while type III collagen is found in skin, blood vessels and intestine. It is well established that collagen is not only formed in fibroblasts but also in nonfibroblastic cell lines (Langness U, Udenfriend S. Collagen biosynthesis in nonfibroblastic cell lines. Proc Natl Acad Sci USA 1974;71:50-51). Interestingly, the same fibroblast cell can form simultaneously both types I and III collagen (Gay S, Martin GR, Muller PK, Timpl R, Kuhn K. Simultaneous synthesis of types I and III collagen by fibroblasts in culture. Proc Natl Acad Sci USA 1976;73:4037-4040). More recent studies revealed that the ratio between type I and type III skin collagen is differentially regulated during aging and injury (Cheng W, Yan-hua R, Fang-gang N, Guo-an Z. The content and ratio of type I and III collagen in skin differ with age and injury. African J Biotechnol 201 1;10: 2524-2529).

While the synthesis of collagen I remains almost unchanged in human skin during lifetime, the extent of collagen III formation drastically drops by 70%. The differential regulation of collagen gene expression type I versus type III is understood to some extent. Due to the exceptionally long half-life of collagen, the fibrils undergo nonenzymatic glycation under formation of advanced glycation end products (AGEs), through which several signaling pathways and collagen types I and III gene expression become modulated (Tang M, Zhong M, Shang Y, Lin H, Deng J, Jiang H, Lu H, Zhang Y, Zhang W. Differential regulation of collagen types I and III expression in cardiac fibroblasts by AGEs through TRB3/MAPK signaling pathway. Cell Mol Life Sci 2008;65:2924-2932).

Retinoids and polyphosphates (polyP) Retinoids are structurally characterized by a P-ionone ring with a polyunsaturated side chain consisting of four isoprenoid moieties with a terminal alcohol, aldehyde, carboxylic acid or ester group. One prominent member of the retinoids is the diterpenoid and alcohol retinol (vitamin A). The corresponding aldehyde retinal plays an important role in the visual system (binding to opsin), while the metabolite retinoic acid has an essential function in regulation of growths of epithelial cells and bone tissue.

It is well established that retinoids are beneficial for skin regeneration, reconstitution of the collagen network, and protective against skin aging (reviewed in: Mukherjee S, Date A, Patravale V, Korting HC, Roeder A, Weindl G. Retinoids in the treatment of skin aging: an overview of clinical efficacy and safety. Clin Intery Aging 2006;1:327-348). In addition, retinol has been proposed to function as a stabilizer for biologically active skin preservatives (Nystrand G, Debois J. Retinol stabilized cleansing compositions; EP 995433 Al).

Inorganic polyphosphates (polyP) are nontoxic linear polymers of tens to hundreds of phosphate units linked together via high energy phosphoanhydride bonds (Kulaev IS, Vagabov V, Kulakovskaya T (2004) The Biochemistry of Inorganic Polyphosphates. New York: John Wiley & Sons Inc). They are found in a variety of organisms including bacteria, fungi, algae, plants and animals (Schroder HC, Muller WEG, eds (1999) Inorganic Polyphosphates -Biochemistry, Biology, Biotechnology. Prog Mol Subcell Biol 23:45-81). PolyP can be produced either chemically at high temperature, or metabolically / enzymatically at ambient temperature, e.g. in bacteria via polyphosphate kinases. The degradation of polyP is mediated by several exo-and endopolyphosphatases. In human, polyP has been demonstrated to be hydrolyzed by alkaline phosphatase (ALP) (Lorenz B, Schroder HC. Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase. Biochim Biophys Acta 2001;1547:254-261).

Previous studies revealed that polyP is biologically active: -PolyP induces collagen expression in bone cells (Hacchou Y, Uematsu T, Ueda 0, Usui Y, Uematsu 5, Takahashi M, Uchihashi T, Kawazoe Y, Shiba T, Kurihara 5, Yamaoka M, Furusawa K. Inorganic polyphosphate: a possible stimulant of bone formation. J Dent Res 2007;86:893-897).

- PolyP displays antibacterial effect especially against Gram-positive bacteria (Moon JH, Park JH, Lee JY. Antibacterial action of polyphosphate on Porphyromonas gingivalis. Antimicrob Agents Chemother 2011;55:806-812).

- PolyP is an effective additive for food preservation (Ohtake H, Wu H, Imazu K, Anbe Y, Kato J, Kuroda A. Bacterial phosphonate degradation, phosphite oxidation and polyphosphate accumulation. Resour Consery Recycl 1996;18:125-134).

- PolyP stimulates differentiation and mineralization of bone cells (reviewed in: Wang XH, Schroder HC, Wiens M, Ushijima H, Muller WEG. Bio-silica and bio-polyphosphate: applications in biomedicine (bone formation). Current Opinion Biotechnol 2012;23:570-578; Wang XH, Schroder HC, Muller WEG. Enzymatically synthesized inorganic polymers as morphogenetically active bone scaffolds: application in regenerative medicine. Int Rev Cell Mol Biol 2014;313:27-77).

The application of polyP as a skin preservative is hampered by the fact that the salt Na-polyP is readily dissolved. The approaches to fabricate polyP-containing biomaterials that are more resistant and simultaneously bioactive included a calcination process. However, during those processes polyP is either disintegrated or transformed into a crystalline state and by that becomes inactive (Pilliar RM, Filiaggi MJ, Wells JD, Grynpas MD, Kandel RA. Porous calcium polyphosphate scaffolds for bone substitute applications -in vitro characterization. Biomaterials 2001;22:963-972; Ding YL, Chen YW, Qin YJ, Shi GQ, Yu XX, Wan CX. Effect of polymerization degree of calcium polyphosphate on its microstructure and in vitro degradation performance. J Mater Sci Mater Med 2008;19:1291-1295).

Previously, the inventors described a material consisting of a Ca: salt of polyP that is amorphous, nano-sized, biodegradable and retains the morphogenetic activity of the inorganic polymer (Muller WEG, Tolba E, SchrOder HC, Wang SF, Glal3er G, Munoz-Espi R, Link T, Wang XH. A new polyphosphate calcium material with morphogenetic activity. Materials Lett 2015, in press; Patent application GB 1420363.2; 17-November 2014. Polyphosphate calcium nanoparticles with morphogenetic activity. Inventor: Muller WEG). The polyP nanoparticles that form this nanoparticulate material are termed amorphous Ca-phosphate nanoparticles [aCa-polyP-N13].

The inventors now succeeded to include retinol into those nanoparticles and thereby to fabricate nanospheres, consisting of retinol inclusions encapsulated within Ca-polyP shells. These nanospheres are termed amorphous Ca-polyP/retinol nanospheres [retinol/aCa-polyPNS]. The inventors unexpectedly found that this new, inventive material, the retinol/aCapolyP-NS, causes collagen type III expression in an unexpected high extent, at concentrations at which the single components, retinol and aCa-polyP-NP, are biologically inactive.

The following patent applications on polyP are relevant: - GB1406840.7. Morphogenetically active hydrogel for bioprinting of bioartificial tissue. Inventor: Muller WEG.

- GB1403899.6. Synergistic composition comprising quercetin and polyphosphate for treatment of bone disorders. Inventor: Muller WEG.

Detailed description of the invention

This invention relates to a method for the fabrication of nanospheres that are composed of (i) Cat' together with (ii) polyP to form nanoparticles, aCa-polyP-NP, and together with (iii) retinol that is encapsulated by those nanoparticles to form nanospheres. -4 -

The inventive method is based on a previously developed method for fabrication of nanoparticles from Na-polyP and calcium salts that (i) retain after particle formation their amorphous state and (ii) display the morphogenetic activity known from Na-polyP (Muller WEG, Tolba E, Schroder HC, Wang SF, GlaBer G, Muiloz-Espi R, Link T, Wang XH. A new polyphosphate calcium material with morphogenetic activity. Materials Lett 2015, in press; Patent application GB1420363.2; 17-November 2014. Polyphosphate calcium nanoparticles with morphogenetic activity. Inventor: Muller WEG).

The polyP material formed by the latter particles, aCa-polyP-NP, is characterized by the following properties. This material: a) is amorphous (non-crystalline) b) has an unusual hardness (e.g. elastic modulus of 1.3 GPa [Ca-polyP2 particles with a phosphorus:calcium ration of 1:2]) c) consists of nanoparticles with a diameter of about 0.2 um (Ca-polyP2 particles) d) can be prepared under mild conditions (room temperature) e) is morphogenetically active (induction of bone alkaline phosphatase activity and bone hydroxyapatite formation) 0 is biodegradable (degradation by alkaline phosphatase) Unexpectedly, the inventors found that processing of these nanoparticles with retinol and poly(ethylene glycol) [PEG] results in the formation of nanospheres, retinol/aCa-polyP-NS, which show the following unexpected and advantageous properties: 1. The nanospheres according to this invention, retinol/aCa-polyP-NS, are highly homogenous in size (size -45 nm). This size is optimal for endocytotic cellular uptake (Zhang S, Li J, Lykotrafitis G, Bao G, Suresh S. Size-dependent endocytosis of nanoparticles. Adv Mater 2009;21:419-424).

In contrast, the nanoparticles, aCa-polyP-NP, are large (> 50 um sized) brick-like particles.

2. Both components of the nanospheres, retinol and polyP, act synergistically: if given together at "non-effective" concentrations, a strong increase in proliferation of cells occurs.

3. Both components of the nanospheres, retinol and polyP, cause a highly synergistic effect on the expression of collagen types I and II, and especially collagen type III.

Those effects are already observed at concentrations of retinol and aCa-polyP-NP, that do not display any biological effect if administered alone.

Even though the application of retinol at concentrations of < 1% (36 mM) is assessed as a safe cosmetic ingredient higher concentrations might display adverse effects, e.g. inhibition of responses to viral or chemical carcinogens (see: Final report on the safety assessment of retinyl palmitate and retinol. J Americ Coll Toxicol 1987;6:279-319). Therefore, the nanospheres according to the invention open a more safe application of this compound in cosmetics.

4. The nanospheres according to this invention can be disintegrated by enzymatic hydrolysis in the extracellular space. The main component within the nanospheres, -5 -polyP, undergoes degradation by the exo-phosphatase, most likely by the alkaline phosphatase which is involved in the extracellular degradation of polyP (Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth 1, Glasser G, Wiens M, Schroder NC. Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Cap-level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomaterialia 2011j;7:2661-2671). In consequence, not only polyP is released extracellularly but also retinol, as sketched in Fig. 11.

The nanospheres according to this invention have the appropriate size to be taken up by a clathrin-mediated endocytosis process. Their biological activity can be blocked with the endocytosis inhibitor triflupromazine. This property of the nanospheres is advantageous under conditions during which the transmembrane retinol transporter is down-regulated (resulting in epidermal thickening).

6 In consequence, the nanospheres show a dual biological effect of the two active components, polyP and retinol -both via the extracellular route (activation of cell membrane-bound receptors) and via the transmembrane / intracellular route (through endocytosis).

7. The nanospheres according to this invention are not cytotoxic.

This inventive material, consisting of amorphous retinoid/Ca-polyP nanospheres, which are superior compared to the individual (retinoid and polyP) components, can be prepared according to this invention as follows: a) Dissolution of a retinoid and a calcium salt in an organic solvent, b) Slow addition of the retinoid calcium salt solution to an aqueous polyP solution, and c) Collection and drying of the nanospheres formed after washing with water.

This procedure is performed at room temperature, under avoidance of light.

As an example, the preparation of the amorphous retinol/Ca-polyP nanospheres (retinol/aCapolyP-NS) can be carried out using Na-polyP as a polyP salt (chain length: 30 phosphate units) and calcium chloride as a calcium salt as follows.

a) Preparation of a solution containing 100 mg of retinol and 2.8 g of CaC12 in 50 ml absolute ethanol (= Retinol / calcium solution).

b) Preparation of a polyP solution containing 1 g of polyP in 100 ml water; in order to avoid a phase separation and to stabilize the emulsion, 2 g of poly(ethylene glycol) [PEG] are added to the Na-polyP solution (= PolyP solution).

c) Drop-wise addition of the Retinol calcium solution to the PolyP solution.

d) Stirring of the emulsion formed for 6 h. e) Collection of the nanospheres formed by filtration and washing with water to remove excess of calcium ions and unreacted components. 0 Drying the particles at room temperature overnight.

The chain lengths of the polyP molecules can be in the range 3 to up to 1000 phosphate units. Optimal results were achieved with polyP molecules with an average chain length of approximately 30 phosphate units. -6 -

A further aspect of the invention concerns a material such as a creme or ointment containing such retinol/calcium-polyphosphate nanospheres (retinol/aCa-polyP-NS) obtained by one of the methods described above.

The technology according to this invention can be applied for the fabrication of nanospheres or a material containing these nanospheres, such as a crème or ointment, to be used in the treatment or prophylaxis of dermatological conditions such as inflammatory skin disorders, acne, disorders of increased cell turnover like psoriasis, skin cancers, and photoaging.

A further aspect of the invention concerns the application of the inventive nanospheres in drug delivery.

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.

Figure 1 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 Cat, polyP and retinol. (E) Nanospheres, lacking (left) and containing retinol (right) are shown. Further details are in "Methods".

Figure 2 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 pg/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).

Figure 3 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 pM likewise did not affect the growth of the cells (left hatched bars). After co-addition of aCapolyP-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 pg/ml. * P < 0.01.

Figure 4 shows the light microscopic inspection of the density of the MC3T3 cells after an incubation period of 72 h in (A) the absence of the test compounds, (B) the presence of 3 jig/m1 of aCa-polyP-NP, (C) the presence of 10 jiM retinol, or (D) in assays with the two components together (3 pg/ml of aCa-polyP-NP and 10 pM retinol). The striking synergistic action of the two compounds together is obvious.

Figure 5 shows the different morphologies and compositions of the polyP salts. The NapolyP salts are large bricks (A and B), while the nanoparticles of aCa-polyP-NP (D and E) and the nanospheres, composed of aCa-polyP-NP and retinol, the retinol/aCa-polyP-NS [retaCa-polyP-NS], are spheres (G and H). The EDX spectra for Na-polyP show signals for Na, P and 0 (C), while the aCa-polyP-NP nanoparticles have additionally a strong signal for Ca, while the one for Na is almost negligible (F). (I) Retinol/aCa-polyP-NS nanospheres have a distinct peak for C, which is likewise large like the 0 signal. A,B, D,E, G,H SEM analysis; C, 0, 1 EDX spectra. -7 -

Figure 6 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/aCapolyP-NS (C) are lighting up with a bright green fluorescence.

Figure 7 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.

Figure 8 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-k) 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 pg/m1) and two concentrations of retinol (1 and 3 04) were tested. The concentrations of retinol are given in 0/1; aCa-polyP-NP is added at a concentration of 3 ps/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 GAI'DH 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.

Figure 9 shows the expression levels of the different types of collagen (COL-I, COL-II, COL-III and COL-J) 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. Sc P < 0.01.

Figure 10 shows the expression level of collagen type III gene in cells exposed to retinol alone, Na-polyP, aCa-polyP-NPI nanoparticles (1:1 ratio between phosphate and Ca2+) and aCa-polyP-NP (1:2 ratio between phosphate and Cat) in the absence or presence of retinol. In the last series the assays were composed with retinol, aCa-polyP-NP and 20 0/1 of the inhibitor of the clathrin-mediated endocytosis triflupromazine (TLP). The expression of collagen type III is correlated with the house-keeping gene GAPDH. * P < 0.01.

Figure 11 shows a scheme explaining the synergistic pathway elicited by polyP and retinol, the main components of the nanospheres retinol/aCa-polyP-NS, during collagen type III gene expression. The nanospheres undergo metabolism both extracellularly ("Extracellular disintegration") and intracellularly via clathrin-mediated endocytosis (Nanospheres endocytosis"). In the first route the Ca-polyP nanospheres undergo enzymatic hydrolysis via the alkaline phosphatase (ALP). The released Ca2+ and the inorganic phosphate are taken up by the cells and are postulated to activate the mTOR pathway and in turn the metabolic state of the cells. Retinol acts after binding to transthyretin (TTR) and the retinol binding protein (RBP) and transport via the STRA6 receptor. Intracellularly the complex binds to cellular retinol binding protein and undergoes oxidation to retinal by alcohol dehydrogenase (ADH4). The retinaldehyde dehydrogenases (RALDH) oxidizes retinal to retinoic acid that binds to the cellular retinoic acid binding receptor (CRABP) which in turn activates the retinoic acid receptor (RAR) and/or the retinoid X receptor (RXR). In turn, the gene collagen type III undergoes strong expression. In the second route, the nanospheres are taken up by a clathrinmediated pathway and undergo intracellular disassembly followed by the described biological activity for polyP and retinol. -8 -

Examples

In the following examples, only an inventive method for polyP molecules with an average chain length of 30 phosphate units is described. Similar results can be obtained by using polyP molecules with shorter and longer chain lengths.

Effect of aCa-polyP-NP and retinol on cell growth In the first series of experiments Na-polyP (complexed with Ca2+) was tested towards aCapolyP-NP (Fig. 2). 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 ng/ml, which even increases at 30 µg/ml.

Addition of retinol at concentrations between 0.3 and 30 p.M and in the absence of the aCapolyP-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 mg/ml, a strong and significant increase in the proliferation propensity is measured (Fig. 3). 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. 3). At a concentrations of > 1 RM retinol a significant increase in the proliferation rate is seen which reaches a value of 290% at 10 nNI, 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.

This synergistically-acting effect of the two test components can also be followed microscopically (Fig. 4). In the absence of any of the components the density of the MC3T3 cells after the 72 h incubation is only low (Fig. 4A). Addition of aCa-polyP-NP (3 µg/ml) alone did not change markedly the density of the cells, attached to the substrate (Fig. 4B). Likewise low is the number of cells onto the plastic surface if 10 pM retinol is added (Fig. 4C). However, if the two components [3 mg/m1 of aCa-polyP-NP and 10 p,M retinol] are added together the cells form an (almost) confluent cell monolayer (Fig. 4D), supporting the synergistic action of aCa-polyP-NP and retinol.

Preparation of Ca-polyP nanoparticles and Ca-polyP/retinol nanospheres The preparation of the aCa-polyP-NP has been described (Muller WEG, Tolba E, Schroder HC, Wang SF, GlaBer G, Mutioz-Espi R, Link T, Wang XH. A new polyphosphate calcium material with morphogenetic activity. Materials Lett 2015, in press; Patent application GB 1420363.2; 17-November 2014. Polyphosphate calcium nanoparticles with morphogenetic activity. Inventor: Muller WEG). These nanoparticles are prepared by precipitation of NapolyP with Ca2+ in a stoichiometric ratio of 1:2. The polyP nanospheres, likewise amorphous, are fabricated from an ethanolic solution of retinol with CaC12 (Fig. 1A) that is added to a solution of Na-polyP with PEG (Fig. 1B), during which a suspension of retinol/aCa-polyP-NS is formed (Fig. 1D). The nanospheres are collected and have a slightly yellow color, in contrast to those nanoparticles, lacking retinol (Fig. 1 F).

Morphology and chemical elemental of Ca-polyP nanoparticles and Ca-polyP/retinol nanospheres The Na-polyP particles show a brick stone morphology (Fig. 5A and B). The size of the building bricks is > 50x50x50 gm. The EDX spectra show signals, (almost) exclusively for 0 (oxygen), Na (sodium) and P (phosphorous); only a small peak corresponding to C (carbon) is seen; Fig. 5C.

In contrast to the morphology of the Na-polyP particles, the aCa-polyP-NP are spheres (Fig. 5D and E). 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 (Ka peak at 3.7 keV and the Kh peak at 4.0 keV). Only a weak signal for Na is observed (Fig. 5F); in contrast the Na peak in the Na-polyP salt is almost as high as the one for P (Fig. 5C).

The retinol/aCa-polyP-NS, fabricated from aCa-polyP-NP and retinol, are like the Ca-polyP nanoparticles globular (Fig. 56 and H). 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 (Fig. 5I).

Presence of retinol in retinol/aCa-polyP-NS Retinol exhibits fluorescence properties with maximum absorbance and emission at 326 nm and 520 nm (cyclohexane) (Tanumihardjo SA, Howe TA. Twice the amount of a-carotene isolated from carrots is as effective as 0-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. 6C), show a bright green fluorescence (Fig. 6D), while the nanoparticles, aCa-polyP-NP (Fig. 6A), lacking retinol, show only a slight background fluorescence (Fig. 6B).

Retinol was determined quantitatively using the SbC13-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 To determine if polyP within the retinol/aCa-polyP-NS is prone to hydrolysis by phosphatases the nanospheres were incubated in PBS (Fig. 7A, lanes a-c) or in medium/serum and cells (Fig. 7B, 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. 7A, lanes a-c), while the size of polyP within the retinol/aCa-polyP-NS progressively decrease from 30 units after 1 d (Fig. 7B, lane a) to = 20 units after 3 d (lane b) and even to less that 1-3 units after 5 d (lane c).

-10 -Co-incubation of retinol with aCa-polyP-NP on collagen expression MC3T3-E1 cells were incubated with 3 pg/m1 of aCa-polyP-NP in the absence or presence of retinol (Fig. 8). After a 5 d incubation period the cells were collected and subjected to RTOCR 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.

The results are shown in Fig. 8. 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 pM or 3 pM) and nanoparticles (3 [(gimp, are added separately. These increases are statistically not significant. However, if retinol is added together with 3 pg/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 pg/ml of aCa-polyP-NP and 1 RNI retinol is for collagen type I 4.8-fold and with 3 p.M retinol 9.4-fold; for type II 31.7-fold with 1 pM retinol or 55.8-fold with 3 pM 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 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. 9 as ratios between the collagen steady-state-values in treated cells (0.3 pg/m1 to 10 µg/ml) and the values determined in untreated cells. The results show (Fig. 9), that the expression levels for collagen Ope I, collagen type II and of collagen type III are at concentration higher than > 3 pg/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 The effect of 3 gM 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-ciPCR (Fig. 10). 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 pNI retinol the level increased significantly to 0.25+0.03. Na-polyP, stoichiometrically complexed to Ca-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-in an 1:1 molar ratio, aCa-polyP-NP I, 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 pM 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 Ca2+ have a brick-like morphology with edge lengths of 4 gm, while the aCa-polyP-NP nanoparticles are globular/spherical with a size <100 nm.

Methods Material s Na-polyphosphate (Na-polyP) with an average chain length of 30 phosphate units (NaP03)" can be obtained, for example, from Merck Millipore ((#106529; Darmstadt; Germany), all-trans retinol, for example, from Sigma (#95144; >97.5%, M, 286.45; Sigma, Taufkirchen; Germany).

Preparation of Ca-polyP nanoparticles and Ca-polyP/retinol nanospheres The amorphous Ca-phosphate nanoparticles, aCa-polyP-NP, are prepared, following a described procedure (Muller WEG, Tolba E, Schroder HC, Wang SF, GlaBer G, Mufioz-Espi R, Link T, Wang XH. A new polyphosphate calcium material with morphogenetic activity. Materials Lett 2015, in press; Patent application GB 1420363.2; 17-November 2014. Polyphosphate calcium nanoparticles with morphogenetic activity. Inventor: Muller WEG). In brief, 2.8 g of CaC12 in 30 ml distilled water is added drop-wise to 1 g of Na-polyP, dissolved in 50 ml distilled water at room temperature. During the procedure the pH is adjusted to 10.0 with a NaOH aqueous solution. After stirring for 12 h the nanoparticles are collected by filtration through Nalgene Filter Units (pore size 0.45 gm; Cole-Parmer). The resulting aCapolyP-NP (ratio:phosphate:Ca2 = 2) are dried at 50°C.

In one series of experiments, described in Examples, amorphous Ca-phosphate nanoparticles have been prepared that are characterized by a phosphate:Ca2 ratio of 1 (Muller WEG, Tolba E, Schroder HC, Wang S, GlaBer G, NIuftoz-Espi R, Link T, Wang XH. A new polyphosphate calcium material with morphogenetic activity. Materials Lett 2015; in press); these particles are designated "aCa-polyP-NP1". They are also amorphous.

The amorphous retinol/Ca-polyP nanospheres, retinol/aCa-polyP-NS, are prepared by a new fabrication process, under avoidance of light. A retinol solution (100 mg/50 ml absolute ethanol), containing 2.8 g of CaC12 (Fig. 1A), is prepared and added drop-wise to a Na-polyP solution (1 g in 100 ml water; Fig. 1B). 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. 1C) is stirred for 6 h. The particles formed (Fig. 1D) 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. 1E).

Chemical characterization by FTIR Fourier transformed infrared (FTIR) spectroscopy can be applied to prove the polymer character of the polyP preparations. FTIR spectroscopy in the attenuated total reflectance (ATR) mode can be performed, for example, using the Varian 660-IR spectrometer with Golden Gate ATR auxiliary (Agilent). Spectra between the wavenumbers 4000 and 600 cm-1 are recorded.

XRD analyses X-ray diffraction (XRD) analysis can be used to verify the amorphous state of the polyP nanoparticles. XRD is performed using established procedures (Fischer V, Lieberwirth I, Jakob G, Landfester K, NItthoz-Espi R. Metal oxide/polymer hybrid nanoparticles with -12 -versatile functionality prepared by controlled surface crystallization. Adv Funct Mat 2013;23:451-466).

Scanning electron microscopy and energy dispersive X-ray spectroscopy For the scanning electron microscopic (SEM) analyses, for example, a HITACHI SU 8000 electron microscope can be employed. 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 pm" were analyzed by EDX.

Cells and cell culture conditions 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 p.g/ml of gentamycin. The cells are incubated in 25 cm2 flasks or in 24-well plates (Greiner Bio-One) in an incubator 37°C and 5% CO). Reaching 80% confluency, the cells are detached using trypsin/EDTA and continuously subcultured at a density of 5.10' cells/ml. The cells are seeded at a density of 5.103 cells/well. Medium/serum change was every 3 d.

The aCa-polyP-NP are added at the indicated concentration to the cultures, usually 3µg/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 Can (molar ratio of 2:1 / phosphate monomer:Can; Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, Schlamacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC. Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Can level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomaterialia 2011; 7:2661-2671) is studied in parallel.

Cell proliferation -cell viability assays 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 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 GB, Parrish DB. 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 xg. 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% SbC13 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-VisNIR spectrophotometer.

Enzymatic degradation of polyP in the retinol/aCa-polyP-NS nanospheres 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 NIC3T3-E1 cells and incubated -13 -for 1 d, 3 d or 5 d at 37°C. Finally aliquots of 50 gl are taken, kept at pH 3, and assayed for the chain length of polyP (for example, see: Lorenz B, Schroder HC. 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 The technique of reverse transcription-quantitative real-time polymerase chain reaction (RTqPCR) 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. 1) [nt4003 to nt4022] and Rev: 5'-GGTGGACATTAGGCGCAGGAAG-3' (SEQ ID NO. 2) [nt4i46 to nta 125; product size 144 bp]; type II alpha 2 (Mus Coll a2; NM_007743) Fwd: 5'-AACACCCCAGCGAAGAACTCATAC-3' (SEQ ID NO. 3) [nt3789 to nt3812] and Rev: 5'-TTCCTTGGAGGACACCCCTTCTAC-3' (SEQ ID NO. 4) [nt3908 to nt3885; size 120 bp]; type III, alpha 1 (Mus Col3a1; NM 009930) Fwd: 5'-GCTGTTTCAACCACCCAATACAGG-3' (SEQ ID NO. 5) [nt4764 to nt4787] and Rev: 5'-CTGGTGAATGAGTATGACCGTTGC-3' (SEQ ID NO. 6) [nt4,41 to nt4,18; size 178 bp]; type IV, alpha 1 (Mus Col4al; NM 009931) Fwd: 5'-AACGTCTGCAACTTCGCCTCC-3' (SEQ ID NO. 7) [nt4752 to nt4772] and Rev: 5'-TGCTTCACAAACCGCACACC-3' (SEQ ID NO. 8) [nt4886 to nt4867; size 135 bp]; and type V, alpha 1 (Mus Col5a1; NM_015734)Fwd: 5'-AGTCCCTTCCTGAAGCCTGTCC-3' (SEQ ID NO. 9) [nt7iio to nt7131] and Rev: 5'-GCACACACACAGAGATTAGCACC-3' (SEQ ID NO. 10) [nt7265 to nt7243; 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. 11) [num° to nt220] and Rev: 5'-AGACTCCACGACATACTCAGCAC-3' (SEQ ID NO. 12) [nt338 to nt316; 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, values the expression of the respective transcripts is calculated.

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

SEQUENCE LISTING

<110> MULLER, WERNER E.G.

<120> SYNERGISTICALLY ACTING AMORPHOUS CALCIUM-POLYPHOSPHATE NANOSPHERES CONTAINING ENCAPSULATED RETINOL FOR THERAPEUTIC APPLICATIONS <130> M32788GB <160> 12 <170> PATENTIN VERSION 3.5 <210> 1 <211> 21 <212> DNA <213> MUS MUSCULUS <400> 1

TACATCAGCC CGAACCCCAA G

<210> 2 <211> 22 <212> DNA <213> MUS MUSCULUS <400> 2

GGTGGACATT AGGCGCAGGA AG

<210> 3 <211> 24 <212> DNA <213> MUS MUSCULUS <400> 3 AACACCCCAG CGAAGAACTC ATAC 24 <210> 4 <211> 24 <212> DNA <213> MUS MUSCULUS <400> 4 TTCCTTGGAG GACACCCCTT CTAC 24 <210> 5 <211> 24 <212> DNA <213> MUS MUSCULUS <400> 5 GCTGTTTCAA CCACCCAATA CAGG 24 <210> 6 <211> 24 <212> DNA <213> MUS MUSCULUS <400> 6 CTGGTGAATG AGTATGACCG TTGC 24 <210> 7 <211> 21 <212> DNA <213> MUS MUSCULUS <400> 7

AACGTCTGCA ACTTCGCCTC C

<210> 8 <211> 20 <212> DNA <213> MUS MUSCULUS <400> 8 T GC T T CACAA ACC GCAC AC C 20 <210> 9 <211> 22 <212> DNA <213> MUS MUSCULUS <400> 9

AGTCCCTTCC TGAAGCCTGT CC

<210> 10 <211> 23 <212> DNA <213> MUS MUSCULUS <400> 10 GCACACACAC AGAGATTACC ACC 23 <210> 11 <211> 21 <212> DNA <213> MUS MUSCULUS <400> 11 TCACGGCAAA TTCAACGGCA C 21 <210> 12 <211> 23 <212> DNA <213> MUS MUSCULUS <400> 12 AGACTCCACG ACATACTCAG CAC 23 -1 -

Claims (13)

1. CLAIMS1. A method for the preparation of degradable amorphous retinoid/calcium-polyphosphate nanospheres, comprising i) dissolving a retinoid and a calcium salt in an organic solvent, ii) adding of said retinoid calcium salt solution as produced in step a) to an aqueous solution containing a dissolved polyphosphate (polyP) salt and poly(ethylene glycol) polymer or another lubricating coating material, and iii) collecting, washing with water, and drying of the nanospheres as formed.
2. 2. The method according to claim 1, wherein the said retinoid is retinol
3. 3. The method according to any of claims 1 and 2, wherein the calcium salt is calcium chloride.
4. 4. The method according to any of claims 1 to 3, wherein the said polyphosphate salt is sodium polyphosphate.
5. 5. The method according to any of claims 1 to 4, wherein the chain length of the polyphosphate is in the range of about 3 to about 1000 phosphate units, preferably in the range of about 10 to about 100 phosphate units, and most preferred about 30 phosphate units.
6. 6. The method according to any of claims 1 to 5, wherein an emulsifier is added to the polyphosphate solution in order to avoid phase separation.
7. 7. The method according to claim 6, wherein the emulsifier is poly(ethylene glycol).
8. 8. The method according to any of claims 1 to 7, 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.
9. 9. The method according to any of claims 1 to 8, wherein said retinoid/Ca-polyP nanospheres are obtained in an essentially homogenous size optimal for cellular uptake by clathrinmediated endocytosis, such as, for example at about 45 nm +/-5 nm in diameter.
10. 10. Degradable amorphous retinoid/calcium-polyphosphate nanospheres produced according to any one of claims 1 to 9.
11. Ii. The amorphous retinoid/calcium-polyphosphate nanospheres according to claim 10 for use in the treatment or prophylaxis of dermatological conditions, such as inflammatory skin disorders, acne, disorders of increased cell turnover like psoriasis, skin cancers, and photoaging.
12. 12. Use of the amorphous retinoid/calcium-polyphosphate nanospheres according to claim 10 in cosmetic or therapeutic compositions, such as, for example as a component in a creme or ointment.
13. 13. Use of the amorphous retinoid/calcium-polyphosphate nanospheres according to claim 10 for drug delivery.