GB2559163A - Formulation based on polyphosphate microparticles for topical treatment of difficult-to-heal wounds - Google Patents

Abstract

A formulation having wound healing properties, such as an acceleration of wound healing, comprises amorphous microparticles or nanoparticles of a salt of polyphosphate and a divalent cation, preferably calcium or magnesium. The salt may be formed by adding a solution of a salt of a divalent cation to an aqueous sodium polyphosphate solution, which has been adjusted to pH 10.0 through the addition of an alkali, such as sodium hydroxide. The formed particles may be stirred overnight and collected by filtration. Preferably the formulation also comprises an alginate or collagen. A wound dressing may comprise the nanoparticles or microparticles embedded in an electrospun meshwork consisting of polylactide (or its copolymers), polycaprolactone (or its copolymers), or another biocompatible polymer. The formulation may alternatively be provided as a powder or gel. Preferably, the formulation is used in the treatment of wounds in patients showing delayed wound healing, such as in the elderly, or diabetic patients.

Description

(54) Title of the Invention: Formulation based on polyphosphate microparticles for topical treatment of difficultto-heal wounds

Abstract Title: Amorphous polyphosphate having wound healing properties (57) A formulation having wound healing properties, such as an acceleration of wound healing, comprises amorphous microparticles or nanoparticles of a salt of polyphosphate and a divalent cation, preferably calcium or magnesium. The salt may be formed by adding a solution of a salt of a divalent cation to an aqueous sodium polyphosphate solution, which has been adjusted to pH 10.0 through the addition of an alkali, such as sodium hydroxide. The formed particles may be stirred overnight and collected by filtration. Preferably the formulation also comprises an alginate or collagen. A wound dressing may comprise the nanoparticles or microparticles embedded in an electrospun meshwork consisting of polylactide (or its copolymers), polycaprolactone (or its copolymers), or another biocompatible polymer. The formulation may alternatively be provided as a powder or gel. Preferably, the formulation is used in the treatment of wounds in patients showing delayed wound healing, such as in the elderly, or diabetic patients.

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FORMULATION BASED ON POLYPHOSPHATE MICROPARTICLES FOR TOPICAL TREATMENT OF DIFFICULT-TO-HEAL WOUNDS

This invention concerns the unexpected property of energy-rich inorganic polyphosphate (polyP) microparticles and wound dressings, wound powders or other preparations containing such particles to enhance healing of wounds, in particular wounds showing delayed or impaired healing such as wounds of diabetic patients. The inventive amorphous microparticles consisting of salts of polyP with divalent cations, preferably calcium ions or magnesium ions, either alone or in the form of a hybrid material together with collagen or alginate, turned out to upregulate the expression of collagen genes, in particular collagen type III, as well as the expression of the genes encoding for α-smooth muscle actin (α-SMA) and plasminogen activator inhibitor-1 (PAI-1), markers for granulation tissue formation, both in vitro and in animal model. The Ca-polyP microparticles were most effective (stimulation of collagen type III expression already after a very early 3 d incubation period) and, even more impressing, if present as a hybrid material together with collagen or alginate, strongly enhance the expression the βΐ integrin gene. The microparticles according to this invention markedly increase the rate of re-epithelialization of wounds with delayed healing and, incorporated into wound dressings, are the first materials that may act as supply of metabolic energy, useful in topical treatment especially of difficult-to-heal chronic wounds.

Background of the Invention

There is a worldwide increasing demand for new therapies of acute and, in particular, chronic wounds. Over 10 million people are affected by acute or chronic wounds and -300,000 people are hospitalized every year alone in the United States (Demidova-Rice TN, Hamblin MR, Herman IM (2012) Acute and impaired wound healing: pathophysiology and current methods for drug delivery, part 1: normal and chronic wounds: biology, causes, and approaches to care. Adv Skin Wound Care 25:304-314). Non-healing wounds affect 3-6 million people in the United States; persons above 65 years and older contribute to 85% for these patients and demand more than $3 billion per year (Mathieu D, Linke J-C, Wattel F (2006) Non-healing wounds. In: Handbook on hyperbaric medicine, Mathieu DE, editor.

Netherlands: Springer, pp 401-427; Menke NB, Ward KR, Witten TM, Bonchev DG, Diegelmann RF (2007) Impaired wound healing. Clin Dermatol 25:19-25).

The process of wound healing can be dissected into the following stages: coagulation/inflammation, formation of granulation tissue, production of new structures and tissue, and finally, remodeling. These complex processes are regulated by cytokines and growth factors and are decisively modulated by systemic conditions, e.g. diabetes.

During the coagulation/inflammatory phase, blood platelets adhere to damaged blood vessels and initiate a release reaction resulting in the initiation of the blood-clotting cascade. Blood platelets release an array of growth factors and cytokines as well as survival or apoptosisinducing agents. Major factors involved are the platelet-derived growth factor and the transforming growth factors Al and 2. In turn inflammatory cells, including macrophages and leukocytes, are attracted that release antimicrobial reactive oxygen species and proteases, which remove non-self bacteria and cell debris. In the following proliferative/granulation phase, tissue repair starts a process that is controlled by growth factors produced by invading epidermal and dermal cells that via autocrine, paracrine, and juxtacrine pathways induce and maintain cellular proliferation and cellular migration. The formation of granulation tissue allows epithelialization within the wound. During the final matrix remodeling and scar formation phase normal blood supply to the regenerated tissue is completed providing a suitable microenvironment for epidermal and dermal cell proliferation and migration and contributing to wound re-epithelialization and restoration.

The supply with metabolic energy to the regeneration zone of the wound is a critical parameter that influences wound healing kinetics. For example, delayed wound healing during aging and diabetes may be associated with and are consequences of arterial and venous insufficiencies (Falanga V (2005) Wound healing and its impairment in the diabetic foot. Lancet 366:1736-1743). The biochemical consequences of impaired vascularization are a reduced cell metabolism and supply of oxygen required for energy production by means of ATP (Guo S, Dipietro LA (2010) Factors affecting wound healing. J Dent Res 89:219-229). Until recently it remained obscure which metabolic energy storages exist in the extracellular space of the tissue. Now it becomes more and more obvious that polyphosphate (polyP), comprising up to several hundreds of phosphate units linked with “high energy phosphoanhydride bonds, contributes as “metabolic fuel” (Muller WEG, Tolba E, Feng Q,

Schroder HC, Markl JS, Kokkinopoulou M, Wang XH (2015) Amorphous Ca²⁺ polyphosphate nanoparticles regulate ATP level in bone-like SaOS-2 cells. J Cell Sci 128:2202-2207; Muller WEG, Tolba E, Schroder HC, Wang XH (2015) Polyphosphate: a morphogenetically active implant material serving as metabolic fuel for bone regeneration. Macromolec Biosci 15:1182-1197) to the establishment and maintenance of the extracellular structural and functional organization. polyP exists as a polymer of around 100 phosphate units in many cells as well as in the blood (Morrissey JH, Choi SH, Smith SA (2012) Polyphosphate: an ancient molecule that links platelets, coagulation, and inflammation. Blood 119:5972-5979). The major polyP store in mammals are the blood platelets which play a crucial role during all phases of wound healing, including coagulation, immune cell recruitment/inflammation, angiogenesis as well as in remodeling (Golebiewska EM, Poole AW (2015) Platelet secretion: From haemostasis to wound healing and beyond. Blood Rev 29:153-162).

For application of polyP in human therapy, a formulation for polyP needed to be developed in which polyP is released in a slower rate and which prevents rapid enzymatic hydrolysis by ALP. Furthermore, the polyP preparations must be amorphous, in order to be biologically active. This task has been achieved by the fabrication of this polymer as an amorphous Ca²⁺ complex in 100 to 300 nm large microparticles (Muller WEG, Tolba E, Schroder HC, Wang S, GlaBer G, Munoz-Espi R, Link T, Wang XH (2015) A new polyphosphate calcium material with morphogenetic activity. Materials Lett 148:163-166). In this form polyP retains its propensity to undergo enzymatic hydrolysis and shows morphogenetic activity (Wang XH, Schroder HC, Muller WEG (2014) Enzymatically synthesized inorganic polymers as morphogenetically active bone scaffolds: application in regenerative medicine. Int Rev Cell Mol Biol 313:27-77).

Here the inventors surprisingly found that polyP if present in a suitable formulation(s) has a beneficial effect on the early phase of wound healing, accelerating the healing process in particular of wound with delayed healing. This unexpected effect was demonstrated both in vitro and in vivo experiments (mouse model). Four different forms of amorphous polyP microparticles have been fabricated: polyP in the form of a calcium salt and a magnesium salt (calcium polyphosphate microparticles, “Ca-polyP-MP”; and magnesium polyphosphate microparticles, “Mg-polyP-MP”) as well as a hybrid material together with collagen (collagen/calcium polyphosphate microparticles, “col/polyP-MP”) or alginate (alginate/calcium polyphosphate microparticles, “alginate/polyP-MP”). Collagen has been selected as additional component since this structural macromolecule has been proven to act as scaffold for wound dressings and facilitates cell attachment, growth and differentiation. Alginate has been selected because of its hydrogel forming properties. The animal experiments were performed both with normal (C57BL/6) and diabetic mice. As morphogenetic parameters for the effect of the polyP particles in vivo the potency to induce gene expression of collagen has been chosen. More specific, the expression of collagen type I (dominant in skin and vascular ligature) and the reticulate type III (reticular fibers) was determined; these types of collagen are critical for wound healing. In addition, integrin 1β has been selected because of the fundamental role of this protein in regulation of cell adhesions during re-epithelialization and granulation tissue formation in wound repair (Koivisto L, Heino J, Hakkinen L, Laijaval H (2014) Integrins in wound healing. Adv Wound Care 3:762783). Moreover, the steady-state-expression levels for a-smooth muscle actin (α-SMA) and for plasminogen activator inhibitor-1 (PAI-1), both genes are strongly upregulated during wound healing, were determined. Furthermore the increase of the proliferative marker Ki-67 was determined by immunohistochemistry; this marker protein is especially abundant during granulation phase.

The data underlying this invention convincingly show that in particular “Ca-polyP-MP” and its hybrid materials together with collagen and alginate, “col/polyP-MP” and “alginate/polyPMP”, positively accelerate the kinetics of wound healing and are beneficial for the topical treatment of wounds.

The state-of-the-art of polyP has been described, for example, in: Muller WEG, Tolba E, Schroder HC, Wang XH (2015) Polyphosphate: a morphogenetically active implant material serving as metabolic fuel for bone regeneration. Macromolec Biosci 15:1182-1197.

Summary of the invention

The inventor produced four different formulations for polyphosphate (polyP): (i) amorphous microparticles consisting of the magnesium salt of polyphosphate (“Mg-polyP-MP”; average size of 170±65 nm), amorphous microparticles consisting of the calcium salt of polyphosphate (“Ca-polyP-MP”; average size of 450±170 nm), and a polyphosphate hybrid material consisting of the calcium salt of polyphosphate and collagen (“col/polyP-MP”; average size of

0.45±0.19 mm) or the calcium salt of polyphosphate and alginate (alginate/calcium polyP microparticles, “alginate/polyP-MP”).

Unexpectedly, the inventor found that these amorphous polyP microparticles significantly improved wound healing, in particular of wounds showing delayed wound healing. The calcium polyP microparticles were most effective, in particular as a hybrid material together with collagen or alginate. PolyP alone (without divalent cations and not present as amorphous microparticles) was ineffective.

Animal experiments (excisional wound model) were performed using normal (C57BL/6) mice as well as diabetic (db/db) mice which show delayed wound healing. The effect of topical application of the inventive materials on healing of excisional wounds with a diameter of 8 mm was determined.

The results revealed that already after a healing period of 7 d “Ca-polyP-MP” significantly improved the score of re-epithelialization in C57BL/6 mice from 31% (control) to 72% (polyP microparticle-treated). The re-epithelialization rate with “Mg-polyP-MP” and “col/polyP-MP” was lower (42% and 44%, resp.), while “Na-polyP” was ineffective. In db/db mice, both “NapolyP”, “Ca-polyP-MP” and “col/polyP-MP” increased the rate of re-epithelialization to a score of 42%, 30% and 44%, resp. (control, 23%). Even more distinct were the effects on wound healing at day 13 post-infliction. In diabetic mice which showed only 48% reepithelialization (controls), treatment with “Ca-polyP-MP” or “col/polyP-MP” resulted in complete re-epithelialization of the wound (score of 100%), like in wildtype mice after the same interval.

In addition, it was surprisingly found, using the mouse model, that these polyP microparticles cause a significant increase of the steady-state-expression of the genes encoding for collagen type III (COL-ΠΙ) and plasminogen activator inhibitor-1 (PAI-1) already after a 7 d-healing period. After a longer incubation period (13 d), an upregulation of the expression both of collagen type I (COL-Py COL-ΠΙ, a-smooth muscle actin (a-SMA) and PAI-1 was determined. α-SMA and PAI-1 are marker proteins for granulation tissue formation in the regenerating wound. In addition, a strong increase in number of Ki67-positive proliferating cells was observed in both “Ca-polyP-MP”- and “col/polyP-MP”-treated wounds.

These results were supported by in vitro experiments. All four amorphous polyP microparticles significantly upregulate the steady-state-expression of collagen genes, in particular COL-ΠΙ, in mouse calvaria MC3T3-E1 cells, while - again - the sodium salt of polyP has no significant effect. Even more important, the calcium polyP microparticles even stimulated the expression of COL III already at a very early incubation period of 3 d and, if present as a hybrid material together with collagen or alginate, also the expression the /// integrin gene.

The data underlying this invention show that, in particular, “Ca-polyP-MP” and “col/polyPMP” or “alginate/polyP-MP” (acting as supply of metabolic energy), incorporated into wound dressings are beneficial in wound healing and especially useful for topical treatment of wounds showing in delayed wound healing.

The following patent applications on polyP are deemed relevant: GB1420363.2. Morphogenetically active calcium polyphosphate nanoparticles. Inventor: Muller WEG; GB 1406840.7. Morphogenetically active hydrogel for bioprinting of bioartificial tissue. Inventors: Muller WEG, Schroder HC, Wang XH; PCT/EP2015/054523. Synergistic composition comprising quercetin and polyphosphate for treatment of bone disorders. Inventors: Muller WEG, Schroder HC, Wang XH; PCT/EP2015/076222. Morphogenetically active calcium polyphosphate nanoparticles containing encapsulated retinol for therapeutic applications. Inventor: Muller WEG; PCT/EP2015/076468. Bioactive wound dressing and teeth coating based on morphogenetically active amorphous calcium polyphosphate. Inventor: Muller WEG; and PCT/EP2015/076172. Amorphous inorganic polyphosphate-calciumphosphate and carbonate particles as morphogenetically active coatings and scaffolds. Inventor: Muller WEG.

Detailed description of the invention

Previously the inventor described a method for the fabrication of amorphous calciumpolyphosphate nanoparticles or microparticles (“Ca-a-polyP-MP”) (Muller WEG, Tolba E, Schroder HC, Wang S, GlaBer G, Munoz-Espi R, Link T, Wang XH (2015) A new polyphosphate calcium material with morphogenetic activity. Materials Lett 148:163-166). These particles can be used as a bone regeneration material. They are (i) amorphous and (ii) biologically active (Muller WEG, Tolba E, Schroder HC, Wang XH (2015) Polyphosphate: a morphogenetically active implant material serving as metabolic fuel for bone regeneration. Macromolec Biosci 15:1182-1197). The properties of these nanoparticles or microparticles have been disclosed in patent application GB1420363.2. They are superior compared to conventional polyP preparations for application as a bone regeneration material (e.g., GB 1406840.7. Morphogenetically active hydrogel for bioprinting of bioartificial tissue [Inventors: Muller WEG, Schroder HC, Wang XH]; and GB 1403899.6. Synergistic composition comprising quercetin and polyphosphate for treatment of bone disorders [Inventors: Muller WEG, Schroder HC, Wang XH]).

Now the inventor unexpectedly found that these energy-rich polyP nanoparticles and microparticles show wound healing properties. These particles enhance the healing of wounds, in particular of wounds showing delayed or impaired healing such as wounds of diabetic patients or elderly patients.

The following formulation according to this invention show wound healing properties:

A. A formulation for amorphous microparticles or nanoparticles consisting of a salt formed from polyP and a divalent cation, prepared by a method involving the following steps:

a. Dissolution of sodium polyphosphate (Na-polyP) in distilled water and adjustment to pH 10.0 with sodium hydroxide solution.

b. Dissolution of a salt of the divalent cation in distilled water.

c. Dropwise addition of the solution of the divalent cation to the polyP solution and keeping the pH at 10 with sodium hydroxide solution.

d. Formation of the particles preferably by stirring of the suspension overnight at room temperature.

e. Collection of the particles preferably by filtration, washing with aqueous ethanol, and drying.

In the context of the present invention, any numeric value as given shall mean both the exact value as well as a variation thereof by +/- 10 percent including the values in between, unless explicitly stated otherwise. The term “about” shall also mean both the exact value as well as a variation thereof by +/- 10 percent, also including the values in between.

The divalent cation can be magnesium or calcium. The chain length of the polyP can be 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 40 phosphate units.

The polyP nanoparticles or microparticles are characterized by a stoichiometric ratio of 0.5 between divalent cation and polyP (based on one phosphate unit).

B. A formulation containing collagen as additional component and prepared by a method involving the following steps:

a. Addition of a suspension of collagen to an aqueous sodium polyphosphate (NapolyP) solution.

b. Maintaining the developing suspension at pH 9 (with NaOH) and stirring for 4 h at room temperature.

c. Injection of the resulting suspension into a calcium chloride bath in ethanol: acetone.

d. Stirring of the suspension overnight, collection of the particles by filtration, washing in acetone, and drying at room temperature.

In the most preferred example of this method, 20 mL of a suspension of collagen containing 0.2 g of fibrous material (referred to protein content) is added to 50 mL of an aqueous NapolyP solution (containing 0.2 g of solid salt) and the resulting suspension is filled into a syringe (aperture of 2 mm), clamped into an automatic pump, and injected with a speed of 1 mL/min into a calcium chloride bath composed of ethanol:acetone [1:2 v/v] containing 3 g of calcium chloride dihydrate per 100 mL.

C. A formulation containing alginate as additional component and prepared by a method involving the following steps:

a. Addition of a Na-alginate solution to an aqueous sodium polyphosphate (NapolyP) solution.

b. Maintaining the developing suspension at pH 9 (with NaOH) and stirring for 4 h at room temperature.

c. Injection of the resulting suspension into a calcium chloride bath in aqueous ethanol.

d. Stirring of the suspension overnight, collection of the particles by filtration, washing, and drying at room temperature.

In the most preferred example of this method, 20 mL of a 7.5% Na-alginate solution in 0.9% NaCl is added to 50 mL of an aqueous Na-polyP solution (containing 0.2 g of solid salt) and the resulting suspension is filled into a syringe (aperture of 2 mm), clamped into an automatic pump, and injected with a speed of 1 mL/min into a calcium chloride bath composed of 70% (v/v) ethanol containing 2.5 g of calcium chloride per 100 mL.

The average size of the amorphous microparticles formed from the magnesium salt of polyP is in the range of 600 to 30 nm and of the amorphous microparticles formed from the calcium salt of polyP in the range of 900 to 60 nm and of the polyP hybrid material with collagen in the range of 1000 to 100 pm, preferably in the range of 235 to 105 nm (Mg-polyP microparticles), 620 to 280 nm (Ca-polyP microparticles) and 640 to 260 pm (Ca-polyP collagen hybrid microparticles) with an average size of 170 nm (Mg-polyP microparticles), 450 nm (Ca-polyP microparticles) and 450 pm (Ca-polyP collagen hybrid microparticles).

The inventive formulation can be applied for the fabrication of a wound powder or wound gel containing the nanoparticles or microparticles as components.

The inventive nanoparticles or microparticles can be embedded into an electrospun meshwork consisting polylactide or its copolymers, polycaprolactone or its copolymers, or another biocompatible polymer.

The nanoparticles or microparticles can be used as components of a wound dressing.

The inventive formulation or a wound powder, wound gel, electrospun meshwork or wound dressing containing the nanoparticles or microparticles can be applied for the treatment of wounds, in particular wounds showing delayed wound healing or wounds of patients predisposed to such wounds like old or diabetic patients.

The inventive amorphous calcium polyphosphate or magnesium polyphosphate nanoparticles or microparticles, either alone or in the form of a hybrid material together with collagen, strongly upregulate the expression of the genes encoding collagen types I and III, as well as the expression of the genes encoding for α-smooth muscle actin (α-SMA) and plasminogen activator inhibitor-1 (PAI-1), markers for granulation tissue formation, both in vitro and in vivo (animal model).

The inventive amorphous calcium polyphosphate nanoparticles or microparticles, if present as a hybrid material together with collagen, even strongly upregulate the expression of the collagen type III gene in a very early phase (after a 3 d exposure period).

The inventive amorphous calcium polyphosphate nanoparticles or microparticles, in the form of a hybrid material either together with collagen or alginate, also strongly stimulate the expression of the genes encoding for integrin 1β, important for the regulation of cell adhesions during wound repair (re-epithelialization and granulation tissue formation).

The inventive amorphous calcium polyphosphate or magnesium polyphosphate nanoparticles or microparticles, either alone or in the form of a hybrid material together with collagen, markedly increase the rate of re-epithelialization of wounds with delayed healing.

By contrast, the sodium salt of polyphosphate has only a slight or no effect on reepithelialization and the expression of the genes mentioned above.

The beneficial properties of the amorphous polyphosphate microparticles according to this invention can be demonstrated in animal experiments. Animal experiments of the effect of topical application of polyP on healing of excisional wounds (diameter of 8 mm) in both normal (C57BL/6) mice and diabetic (db/db) mice (with delayed wound healing) revealed that already after a healing period of 7 d “Ca-polyP-MP” significantly improved the score of reepithelialization in C57BL/6 mice from 31% (control) to 72% (polyP microparticle-treated). The re-epithelialization rate with “Mg-polyP-MP” and “col/polyP-MP” was lower (42% and 44%, resp.), while “Na-polyP” was ineffective. In db/db mice, both “Na-polyP”, “Ca-polyPMP” and “col/polyP-MP” increased the rate of re-epithelialization to a score of 42%, 30% and 44%, resp. (control, 23%). Even more distinct were the effects on wound healing at day 13 post-infliction. In diabetic mice which showed only 48% re-epithelialization (controls), treatment with “Ca-polyP-MP” or “col/polyP-MP” resulted in complete re-epithelialization of the wound (score of 100%), like in wildtype mice after the same interval. Determination of the effect of the polyP particles on the expression of COL-I and COL-ΠΙ genes, as well as the transcript levels for a-SMA and PAI-1, markers for granulation tissue formation, in tissue from the regenerating wound revealed a significant increase of the steady-state-expression of COL-ΠΙ and PAI-1 after the 7 d-healing period, while all four marker genes become upregulated after a longer incubation period (13 d). In addition, a strong increase in number of Ki67-positive proliferating cells was determined in “Ca-polyP-MP”- and “col/polyP-MP”treated wounds.

The amorphous polyP microparticles according to this invention were also found to upregulate significantly the steady-state-expression of COL-I and COL-ΠΙ in vitro, in mouse calvaria MC3T3-E1 cells. The sodium salt of polyP has no significant effect.

The inventive amorphous calcium polyphosphate or magnesium polyphosphate nanoparticles or microparticles, either alone or in the form of a hybrid material together with collagen, incorporated into wound dressings, are the first materials that may act as supply of metabolic energy, useful in topical treatment especially of difficult-to-heal chronic wounds.

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. In the Figures listing,

Figure 1 shows the morphology of “Ca-polyP-MP” and “Mg-polyP-MP” microparticles; SEM analysis. (A to C) “Ca-polyP-MP” and (D to F) “Mg-polyP-MP”.

Figure 2 shows electron microscopic images of “col/polyP-MP” particles; A to D: REM and E_and F: SEM. (A and B) At a lower magnification (REM) the particles appear as globular to disc like particles. (C) The intact particles have on their surfaces ball-like protrusions which (D) proved to be gas bubbles in broken particles. (E and F) At SEM magnification it can be identified that the scaffold material is built of a collagen framework around which nanoparticles are arranged. (F) At the higher magnification it can be resolved that the nanoparticles have a homogeneous morphology with a diameter of ~30 nm.

Figure 3 shows the EDX analysis of “col/polyP-MP”. (A) SEM analysis and (B) EDX spectrum. The prominent element peaks (C, N, Ο, P and Ca) are marked. The Au peak originates from the gold surface after sputtering.

Figure 4 shows the kinetics of expression of the collagen type III (COL-ΠΙ) gene in MC3T3E1 cells exposed to 50 pg/mL of “Na-polyP”, “Mg-polyP-MP”, “Ca-polyP-MP” or “col/polyP-MP”; the controls received no additional component. After an incubation period of 3 days and 7 days the cells were harvested, their RNA was extracted and the steady-state levels of collagen expression were determined by qRT-PCR. The expression levels are correlated with the one of the housekeeping gene GAPDH. * p < 0.01 (n = 6 parallel experiments; Student’s /-test) with respect to the values of the controls.

Figure 5 shows the gene expression levels of βΐ integrin in MC3T3-E1 cells exposed to 50 pg/mL of “col/polyP-MP” (left panel) and 50 pg/mL of “alginate/polyP-MP” (right panel), compared to the effects of 50 pg/mL of “Na-polyP”, “Mg-polyP-MP” and “Ca-polyP-MP”; the controls received no additional component. After a 3 d incubation period the cells were harvested, their RNA was extracted and the steady-state levels of βΐ integrin expression were determined by qRT-PCR. The expression levels are correlated with the one of the housekeeping gene GAPDH. * p < 0.01 (n = 6 parallel experiments; Student’s /-test) with respect to the values of the controls.

Figure 6 shows the effects of different polyP formulations on wound healing kinetics (7 d after setting the wound), measured on the basis of re-epithelialization (in percent) which is calculated by dividing the degree of re-epithelialization (in mm) by the wound diameter (mm) x 100. For the study both wild-type (C57BL/6) and db/db mice were included. The values for both the controls (without polyP) and the different polyP formulations, “Na-polyP”, “MgpolyP-MP”, “Ca-polyP-MP” and “col/polyP-MP”, are given. The data are presented as mean ± SEM. The significance is *p < 0.05; n = 6 animals (Student’s /-test).

Figure 7 shows the effects of the tested materials on wound re-epithelialization in C57BL/6 and db/db mice on day 13. The data are presented as mean ± SEM.

Figure 8 shows the upregulation of the collagen genes, procollagen type la (COL-I) and collagen type III al (COL-ΠΙ), in tissue from wild-type animals treated with the microparticles “Ca-polyP-MP” and “col/polyP-MP”. The healing period was 7 d. The genes encoding procollagen type la (COL-I) and a-smooth muscle actin (α-SMA) remained unchanged. The significance (Box plot characteristics) are given by comparing the controls (no polyP added) with the values measured for the “Ca-polyP-MP” and “col/polyP-MP” treated wounds. The horizontal bars within the boxes indicate the median value; * p < 0.005 (n = 6 animals).

Figure 9 shows the changes of the steady-state expression of the genes COL-I and COL-III as well as of the genes a-SMA and PAI-1 in wild-type animals after treatment with polyP microparticles. The healing period was 13 d. In all four series of experiments the expression levels in tissue from regenerating wounds that were treated with polyP microparticles (“CapolyP-MP” and “col/polyP-MP”) significantly increased; * p < 0.005 (n = 6 animals).

Figure 10 shows the quantification of proliferating cells after staining with anti-Ki67 antibodies. Slices stained for Ki67-positive cells were counted in the transitional epidermis region and correlated with a defined area, n = 6 untreated controls and polyP (“Ca-polyP-MP” and “col/polyP-MP”)-treated wounds.

Examples

In the following examples, the inventive method described only for polyP molecules with a chain length of 40 phosphate units. Similar results can be obtained by using polyP molecules with lower and higher chain lengths, such as between 10 to 100 units.

Amorphous polyP microparticles

For both the in vivo and the in vitro tests Na-polyP powder and the microparticles “Mg-polyPMP”, “Ca-polyP-MP” and “col/polyP-MP” were prepared. The size of the globular “CapolyP-MP” particles varied between 100 nm and 800 nm with an average of 450±170 nm (n = 60) (Figure 1A to C). Only at higher magnification the surface of the particles showed pores of sizes around 10 nm.

A similar morphology is characteristic for the likewise globular “Mg-polyP-MP” microparticles (Figure ID to F). These particles are more homogeneous than the “Ca-polyP14

MP” with an average of 170±65 nm. Broken particles revealed that the particles are close to homogeneous.

The “col/polyP-MP” are less spherical than the other two particles. The particles size distribution is fairly homogeneous with an average diameter of 0.45±0.19 mm (Figure 2A,B). The morphology varies from almost globular to close to disc like. Already at a REM magnification it is seen that the intact particles show ball-like protrusions (Figure 2C) that proved to be gas bubbles (Figure 2D). At the higher SEM magnification it reveals that the solid material that surrounds the gas vesicles is built of a collagen scaffold around which the polyP particles are arranged (Figure 2E). At higher magnification the size of the polyP nanoparticles can be determined with -30-50 nm (Figure 2F).

For the analysis of the elements, present in the “col/polyP-MP” particles, EDX spectroscopy was applied. As documented in Figure 3, the spectrum shows the characteristic signals for C, N, Ο, P and Ca. The C, N O signal peaks can be attributed to the collagen framework and the P and Ca peaks to Ca-polyP. In previous studies it is reported that both the “Ca-polyP-MP” (Muller WEG, Tolba E, Schroder HC, Wang S, GlaBer G, Munoz-Espi R, Link T, Wang XH (2015) A new polyphosphate calcium material with morphogenetic activity. Materials Letters 148:163-166; Muller WEG, Tolba E, Schroder HC, Diehl-Seifert B, Wang XH (2015) Retinol encapsulated into amorphous Ca²⁺ polyphosphate nanospheres acts synergistically in MC3T3E1 cells. Eur J Pharm Biopharm 93:214-223) and the “Mg-polyP-MP” particles are amorphous (Muller WEG, Ackermann M, Tolba E, Neufurth M, Wang S, Schroder HC, Wang XH (2016) A bio-imitating approach to fabricate an artificial matrix for cartilage tissue engineering using magnesium-polyphosphate and hyaluronic acid. RSC Adv. 6:88559-88570). It has also been verified that the “col/polyP-MP” have this state.

Potency of polyP to change the steady-state expression of collagen gene and integrin βΐ gene in MC3T3-E1 cells in vitro

Mouse calvaria MC3T3-E1 cells were cultivated in medium/serum as described under “Methods”; the seeding cell concentration was 5 x 10³ cells/well [24-well plates]. The cultures received either no additional component (controls) or were exposed to 50 pg/mL of “Na-polyP”, “Mg-polyP-MP”, “Ca-polyP-MP” or “col/polyP-MP” and incubated for 3 d or 7

d. Then the cells were harvested, RNA was extracted which was subjected to PCR analysis to assess the expression levels for collagen type III. The results revealed that even after a short incubation period of 3 d the steady-state-expression of collagen type III becomes significantly upregulated (by about 4-fold) compared to the control (Figure 4). There was a small but not significant effect on gene expression caused by “Mg-polyP-MP” and “Ca-polyP-MP”. After a prolonged incubation period (7 d), a significant increase in the steady-state-expression of collagen type III (to about 2-fold) is measured with all three microparticle formulations, “MgpolyP-MP”, “Ca-polyP-MP” and “col/polyP-MP” (Figure 4). It is striking that, under the conditions used, “Na-polyP” did not change the expression levels for the collagen gene compared to the controls.

Even more interesting, exposure of the cells to “col/polyP-MP” also caused a marked and significant increase in the expression levels for the integrin βΐ gene even after a short incubation period of 3 d (Figure 5 left panel). The transcript levels of integrin βΐ were found to be enhanced to approximately the same degree (about 3- to 4-fold) after incubation of the cells for 3 d in the presence of “alginate/polyP-MP” (Figure 5 right panel). The effects of “Mg-polyP-MP” and “Ca-polyP-MP” were less significant (Figure 5 left and right panel).

Effect of polyP application on re-epithelialization in C57BL/6 and db/db mice

Groups of 6 specimens each of C57BF/6 and db/db mice were used for the study. Each mouse received one defined wound with a diameter of 8 mm. The wounds were supplied daily with 50 mg of the respective samples immediately after setting the wound. After a healing period of 7 d (Figure 6) or 13 d (Figure 7) the experimental animals were analyzed for the degree of re-epithelialization.

As described before (Zhao G, Hochwalt PC, Usui MF, Underwood RA, Singh PK, James GA, Stewart PS, Fleckman P, Olerud JE (2010) Delayed wound healing in diabetic (db/db) mice with Pseudomonas aeruginosa biofilm challenge: a model for the study of chronic wounds. Wound Repair Regen 18:467-477), also in the present study a delayed wound healing in diabetic (db/db) mice is observed if compared to normal mice. The results revealed that after a 7 d healing period (Figure 6) the C57BF/6 mice recovered with a 31% score of reepithelialization (controls), while the db/db mice showed only 23% re-epithelialization. Already at day 7 some polyP formulations showed a distinct beneficial effect on the wound healing kinetics. Statistical significant is the effect of “Ca-polyP-MP” in C57BF/6 mice with a score of re-epithelialization of 72%; somewhat lower is the positive wound healing effect of “Mg-polyP-MP” (40%) and “col/polyP-MP” (44%). In contrast, “Na-polyP” proved to be ineffective. Very distinctive is also the beneficial effect of polyP in db/db mice. In this model “Na-polyP” increased the rate of wound healing to 42%, if checked by the rate of reepithelialization. Likewise significant is the effect of “Ca-polyP-MP” (30% reepithelialization) and “col/polyP-MP” (44%) in diabetic mice.

Very pronounced is also the wound healing velocity after the 13 days healing period. While in the experiments with normal mice both the controls and the polyP-treated wounds reepithelialized already to 100%, the diabetic mice (controls) showed only 48% reepithelialization (Figure 7). In this series of experiments with both “Ca-polyP-MP” (score of 100%) and “col/polyP-MP” (100%) the epithelium totally covered the wound.

Expression of selected genes in tissue from the regenerating wound by qRT-PCR

RNA was extracted from FFPE-fixed tissue samples and then subjected to qRT-PCR. As a marker for cell migration and tissue re-organization the expression of the genes encoding for procollagen type la (COL-I) and collagen type III al (COL-ΠΙ) have been chosen. As a measure for the granulation efficiency the genes a-smooth muscle actin (α-SMA) and plasminogen activator inhibitor-1 (PAI-1) have been selected. The studies were performed with wild type mice. As summarized in Figure 8 it became evident that after the 7 dincubation period the transcript levels for the collagen marker gene COL-ΠΙ and for PAI-1 significantly upregulate in tissue taken from the wound area that has been treated with powder composed of polyP microparticles (“Ca-polyP-MP” and “col/polyP-MP”). With respect to the a-SMA gene or COL-I gene no significant change is seen.

After the longer healing period of 13 d the expression of all four marker genes for wound healing and tissue regeneration becomes upregulated by up to 1.6-fold (compared to the controls) for COL-I, to 4.6-fold for COL-ΠΙ, 1.5-fold for a-SMA and 5.1-fold for PAI-1 (Figure 9).

Proliferation activity in the transitional epidermis region

Microscope slices were reacted with anti-Ki67 antibodies and the identified Ki67-positive cells were determined in a grid square of 3 mm². As documented in Figure 10 the number of proliferating cells (Ki67-positive) is low in the untreated controls (327±58); significantly higher is the number in “Ca-polyP-MP”- (884±129) and “col/polyP-MP”-treated wounds (740±122).

Methods

Materials

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).

Rat tail collagen type I, used in the Examples, has been obtained from Shenzhen Lando Biomaterials (Shenzhen; China).

Mg-polyP microparticles

Amorphous Mg-polyP particles is prepared from Na-polyP in the presence of a surplus of divalent ions (Mg²⁺). The final stoichiometric ratio between these ions and the polyP polymer (with reference to one charged phosphate unit) is close to 0.5. The procedure is outlined in brief.

The Mg-polyP particles (“Mg-polyP-MP”) are basically prepared as described (Muller WEG, Ackermann M, Tolba E, Neufurth M, Wang S, Schroder HC, Wang XH (2016) A bioimitating approach to fabricate an artificial matrix for cartilage tissue engineering using magnesium-polyphosphate and hyaluronic acid. RSC Adv 6:88559-88570). In short, 3.86 g of MgCl₂ · 6 H₂O are dissolved in 25 mL of distilled water and added dropwise to 1 g of NapolyP, likewise in 25 mL of distilled water. During particle formation the suspension is kept at pH 10 and then stirred for 12 h. The microparticles formed can be collected by filtration, washed with ethanol and dried at 50°C.

Ca-polyP microparticles

Amorphous Ca-polyP particles are prepared from Na-polyP in the presence of a surplus of divalent ions (Ca²⁺)· The final stoichiometric ratio between these ions and the polyP polymer (with reference to one charged phosphate unit) is close to 0.5. The procedure is outlined in brief.

The Ca-polyP particles (“Ca-polyP-MP”) are fabricated from 2.8 g of CaCl₂ · 2 H₂O [25 mL of distilled water] and 1 g of Na-polyP [25 mL water] at room temperature (Muller WEG, Tolba E, Schroder HC, Wang S, GlaBer G, Munoz-Espi R, Link T, Wang XH (2015) A new polyphosphate calcium material with morphogenetic activity. Materials Letters 148:163-166;

Muller WEG, Tolba E, Schroder HC, Diehl-Seifert B, Wang XH (2015) Retinol encapsulated into amorphous Ca²⁺ polyphosphate nanospheres acts synergistically in MC3T3-E1 cells. Eur J Pharm Biopharm 93: 214-223). The microparticles are formed in the pH 10 suspension during the 12 h stirring period. Then the microparticles can be collected by filtration, washed with ethanol and dried at 50°C.

Collagen-polyP hybrid particles

Collagen-polyP hybrid particles (“col/polyP-MP”) are prepared as follows. A suspension of 20 mL of collagen (containing 0.2 g of fibrous material, referred to the protein content which can be determined, for example, by the Bradford procedure [Lopez JM, Imperial S, Valderrama R, Navarro S (1993) An improved Bradford protein assay for collagen proteins. Clin Chim Acta 220:91-100]) is added to 50 mL of aqueous Na-polyP solution (containing 0.2 g of solid salt). The developing suspension is kept at pH 9 (with NaOH) and stirred for 4 h at room temperature. The resulting suspension is filled into a syringe (aperture of 2 mm), clamped into an automatic pump, and injected with a speed of 1 mL/min into a CaCl₂ bath (3 g of CaCl₂ · 2 H₂O per 100 mL) composed of ethanol:acetone [1:2 v/vj. Ethanol and acetone prevent shrinkage of the particles, reduce the surface tension and extracted water. The suspension is stirred overnight. The particles can be collected by filtration and washed twice in acetone. Then the particles can be dried at room temperature.

Alginate-polyP hybrid particles

Alginate-polyP hybrid particles (“alginate/polyP-MP”) are prepared following a similar procedure as described collagen-polyP hybrid particles with the following modifications. A solution (20 mL) containing 7.5% (w/v) Na-alginate, dissolved in 0.9% NaCl, is added to 50 mL of aqueous Na-polyP solution (containing 0.2 g of solid salt). The developing suspension is kept at pH 9 (with NaOH) and stirred for 4 h at room temperature. The resulting suspension is filled into a syringe (aperture of 2 mm), clamped into an automatic pump, and injected with a speed of 1 mL/min into a CaCl₂ bath (2.5 g of CaCl₂ per 100 mL) composed of 70% (v/v) ethanol. The suspension is stirred overnight. The particles can be collected by filtration and washed twice in acetone. Then the particles can be dried at room temperature. Prior to use all particles are sieved through a 500 pm mesh net.

Microstructure analyses

Scanning electron microscopic (SEM) imaging can be performed, for example, using a Hitachi SU-8000 electron microscope. Reflection electron microscope (REM) can be performed, for example, in a Philips XL30 microscope at 15 KeV and 21 μ A. The samples are coated with 20-25 A gold in argon atmosphere.

For energy-dispersive X-ray (EDX) spectroscopy an ED AX Genesis EDX System attached to the scanning electron microscope (Nova 600 Nanolab) can be used, for example, using the operation mode 10 kV and a collection time of 30-45 s.

Cultivation ofMC3T3-El cells

Mouse calvaria cells MC3T3-E1 (ATCC-CRL-2593) are cultivated in α-MEM medium (Gibco/Invitrogen) enriched with 20% fetal calf serum (FCS; Gibco). In addition, the medium contains 2 mM L-glutamine, 1 mM Na-pyruvate and 50 pg/mL of gentamicin. In the experiments described under Examples, for the gene expression studies the cells were cultivated in 24-well plates (Greiner Bio-One). They were seeded at a density of 5 x 10³ cells/well. After an incubation period of 4 d the cells were harvested and subjected to PCR analysis.

Na-polyP is added to the culture/serum, after stoichiometric complexation with Ca²⁺ (molar ratio of 2:l/phosphate monomer:Ca²⁺; Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca²⁺ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671). Prior to addition of the microparticles to the culture they are washed twice in medium for 3 min.

Animals

In the Examples, genetically diabetic male mice, BKS.Cg-m +Lepr^db/+Lepr^db (db/db), 6 and 7 weeks at arrival (Charles River) and the common inbred strain of laboratory mouse, male C57BL/6, 7 weeks at arrival (Charles River), have been used. The diabetic mice were markedly hyperglycemic (mean blood glucose: 527±9 mg/dL), compared to non-diabetic animals (205±9 mg/dL) with all details, given before (Tkalcevic V, Cuzic S, Pamham MJ, Pasalic I, Brajsa K (2009) Differential evaluation of excisional non-occluded wound healing in db/db mice. Toxicol Pathol. 37:183-192). For acclimatization a minimum of 5 consecutive days prior to the experiments in the laboratory animal house was chosen. All animals received a detailed physical examination from the resident veterinarian to confirm that the animals are in a good, adequate state of health. Daily observations were performed at the time of delivery of the animals, during the total period of acclimatization and also throughout the duration of the study.

Housing of the animals: Mice were kept in solid bottom cages (polysulfone Type III H; Tecniplast) having dimensions of 425 mm x 266 mm x 185 mm. The animals were kept on 34 cm thick ALPHA-dri dust free bedding (pure cellulose fiber, uniform particle size 5 mm sq, highly absorbent; LBS Serving (RH6 OUW). Each cage was provided with nestlet and Des Res Standard (for mice) 16 cm long x 12 cm wide x 8 cm high; LBS Serving. The animals were maintained under standard laboratory conditions [temperature 22±2°C, relative humidity 55±10%, 15-20 air changes per h, 12 h artificial lighting / 12 h darkness per day (7 a.m. lights on - 7 p.m. lights off)]. The animals had free access to food VRF1 (P) (Akronom KfT) and water distributed in bottles (Tecniplast) and filled with drinking water from the municipal water supply.

Permissions

All animal related research was conducted in accordance with 2010/63/EU and National legislation regulating the use of laboratory animals in scientific research and for other purposes (Official Gazette 55/13). An Institutional Committee on Animal Research Ethics (CARE-Zg) had overseen that animal related procedures were not compromising the animal welfare. All experiments conducted in studies described herein were performed under the Institutional ethics committee approval number: CAREZG_13-06-14_49 EP/2016. Approval number from Ministry of Agriculture, Republic of Croatia is KLASA: UP/I-322-01/1501/108, URBROJ: 525-10/0255-16-8.

Experimental procedures in wound healing studies

In the animal studies described under Examples, mice were divided into groups (six animals per group). The interscapular region was shaved, depilatory cream Veet (Slough; UK) was applied onto the shaved region and removed 2 min after application on day -2. The interscapular region was disinfected and, utilizing strictly aseptic procedures, a single fullthickness excisional wound of 8 mm in diameter was inserted midline with a sterile, disposable biopsy punch, thus exposing the underlying fascia muscularis as described (Wang

X, Ge J, Tredget EE, Wu Y (2013) The mouse excisional wound splinting model, including applications for stem cell transplantation. NatProtoc 8:302-309).

The test samples were applied in the powder form (100%) and directly spread into the wound beds, immediately post wounding. Then the polyP particles were subjected again daily to the wounds.

Postoperative pain control included daily s.c. injections of 4 mg/kg carprofen (5% Norocarp [Pfizer] - 50 mg/mL; a 1:20 dilution was made in sterile, deionised water; 30 pL were injected into an animal) for two days post wounding at day 0 until day 2. Skin samples were collected at time points specified in the study. Prior to skin sampling all animals were humanely killed with an overdose of ketamine (Taj Pharmaceuticals) / xylasine (KHBoddin) administered via the intra peritoneal route.

Sample analysis: wound excision and morphometry

In the animal studies described under Examples, after terminating the experiments the wound area and the surrounding healthy tissue were excised post mortem (1 x 2.5 cm, rectangular shape), clamped onto a strip of paper and stored in 10% formalin for histological assessment.

Morphometry. Slides cut from paraffin blocks were stained with hematoxylin-eosin. Morphometric evaluation of the degree of re-epithelialization was performed using a Zeiss Axioskop 2 Plus microscope and an Axiovision program (Zeiss). The magnification used was lOOx. Re-epithelialization is expressed as length of newly formed epithelium (given in mm) and percentage of the wound diameter covered with a new epithelial layer.

Immunohi stochemi stry

Immunohistochemical analysis was performed with 5 pm thick paraffin sections which had been de-paraffmized and transferred in 10 mM Na-citrate (pH 6.0) for 15 min at 120°C to preserve epitope structures. After washing with Tris-buffered saline (50 mM Tris-Cl, pH 7.6; 150 mM NaCl) the sections were blocked with Protein Block Serum-Free (Dako) and then incubated with rabbit polyclonal anti-Ki67 IgG (Leica Biosystems; dilution 1:500); the immunocomplexes were subsequently visualized with ChemMate Dako EnVision Detection Kit (peroxidase/DAB, rabbit/mouse). For the quantification of the immunohistochemical staining, images were taken at x20 magnification from representative areas. The numbers of positively stained (brown) cells per area were counted.

Gene expression studies

Animals: The technique of quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is applied to determine semi-quantitatively the effect of the polyP particles on wound healing. In the experiments described under Examples, Formalin-fixed paraffin-embedded (FFPE) tissue samples were used and RNA was extracted from those. The reactions were performed in 1.5 mF Eppendorf tubes and all incubation steps were performed in a thermal cycler (Thermomixer comfort; Eppendorf). Random primers were used in concentrations of 250 ng/reaction. RNA was mixed with primers together with 1 pL of 10 mM dNTPS and incubated for 5 min at 65°C and then cooled on ice for 1 min. To each sample the following components were added to the reaction: 4 pL 5xbuffer (Thermo Fisher Scientific), 1 pL 0.1 M DTT (dithiothreitol), 1 pL RNaseOUT (RNAse inhibitor; Thermo Fisher Scientific) and 1 pF Superscript III reverse transcriptase (Thermo Fisher Scientific). The mixture were incubated as follows: 5 min at 25°C, 60 min at 50°C and 15 min at 70°C. When cooled down the mixture was diluted 5x with RNase free water and used for qPCR. The following primer pairs for mouse genes were used: for procollagen type la (COF-I; AK075707) Fwd: 5’-AGGCTGACACGAACTGAGGT-3’ (SEQ ID NO: 1) and Rev: 5’ATGCACATCAATGTGGAGGA-3’ (SEQ ID NO: 2); collagen type III ol (COF-III; P08121) Fwd: 5’-GCTGTTTCAACCACCCAATACAGG-3’ (SEQ ID NO: 3) and Rev: 5’CTGGTGAATGAGTATGACCGTTGC-3’ (SEQ ID NO: 4); a-smooth muscle actin (a-SMA; NC 000074.6) Fwd: 5'-CAGGGAGTAATGGTTGGAAT-3' (SEQ ID NO: 5) and Rev: 5'TCTCAAACATAATCTGGGTCA-3' (SEQ ID NO: 6); plasminogen activator inhibitor-1 (PAI-1; NC 000071.6) Fwd: 5'-CTGCAGATGACCACAGCGGG-3' (SEQ ID NO: 7) and Rev: 5'-AGCTGGCGGAGGGCATGA-3' (SEQ ID NO: 8). The hypoxanthine phosphoribosyltransferase 1 gene was used as house-keeping gene (HPRT1; NM 013556.2) Fwd: 5’-TGGATACAGGCCAGACTTTG-3’ (SEQ ID NO: 9) and Rev: 5’GTACTCATTATAGTCAAGGGCATAT-3’ (SEQ ID NO: 10). All reactions were carried out at least in duplicates and the results were analyzed by a 2'^AACT method.

MC3T3-E1 cells: The cells were harvested and RNA was extracted. Then the RNA was subjected to qRT-PCR analysis for collagen type I, alpha 1 (COL-I; NM 007742) using the primer pair Fwd: 5’-TACATCAGCCCGAACCCCAAG-3’ (SEQ ID NO: 11) and Rev: 5’23

GGTGGACATTAGGCGCAGGAAG-3’ (SEQ ID NO: 12); for collagen type III, alpha 1 (COL-ΠΙ; NM 009930) the pair Fwd: 5’-GCTGTTTCAACCACCCAATACAGG-3’ (SEQ ID NO: 13) and Rev: 5’-CTGGTGAATGAGTATGACCGTTGC-3’ (SEQ ID NO: 14); and for βΐ integrin (NM 017022.2) Fwd: 5'-TTGGTCAGCAGCGCATATCT-3' (SEQ ID NO: 15) and Rev: 5'- ATTCCTCCAGCCAATCAGCG-3' (SEQ ID NO: 16)). In this series of experiments the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH; NM_008084) was chosen as a reference gene; the primer pair Fwd: 5’TCACGGCAAATTCAACGGCAC-3’ (SEQ ID NO: 17) and Rev: 5’AGACTCCACGACATACTCAGCAC-3’ (SEQ ID NO: 18) was chosen. The determinations were performed in an iCycler (Bio-Rad, Hercules, CA; USA) with the respective iCycler software.

Statistical analyses

The gene expression studies for the incubation experiments in vitro as well as for the reepithelialization determinations in vivo are expressed as mean [± standard error of mean; independent two-sample Student’s /-test] (Petrie A, Watson P (2013) Statistics for veterinary and animal science. Wiley-Blackwell, Oxford, pp 85-99). The PCR studies forming the in vivo experiments are given as Box plot analyses.

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Claims (12)

1. A formulation having wound healing properties, such as an acceleration of wound healing, comprising amorphous microparticles or nanoparticles consisting of a salt formed from polyphosphate and a divalent cation and prepared by a method comprising the following steps:

a) Preparation and adjusting of an aqueous sodium polyphosphate solution to pH 10.0 with alkali, for example sodium hydroxide solution,

b) Preparation of an aqueous solution of a salt of the divalent cation;

c) Adding the solution of said divalent cation from b) slowly, e.g. dropwise to the polyphosphate solution of a), while maintaining the pH at 10 with alkali, for example sodium hydroxide solution,

d) Forming of the particles, preferably by stirring of the suspension overnight at room temperature; and

e) Collecting of the particles, preferably by filtration, washing with aqueous ethanol, and drying.

2. The formulation according to claim 1, wherein said divalent cation is magnesium or calcium.

3. The formulation according to claim 1 or 2, 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 40 phosphate units.

4. The formulation according to any one of claims 1 to 3, wherein the polyphosphate nanoparticles or microparticles have a stoichiometric ratio of 0.5 between divalent cation and polyphosphate (based on one phosphate unit).

5. A formulation having wound healing properties, such as an acceleration of wound healing, comprising amorphous microparticles or nanoparticles consisting of a salt formed from polyphosphate and a divalent cation and further comprising collagen or alginate, wherein said formulation is prepared by a method comprising the following steps:

i) Adding of a suitable suspension of collagen to an aqueous sodium polyphosphate solution;

ii) Maintaining the developing suspension at pH 9 with alkali, e.g. sodium hydroxide solution, and stirring for several hours, preferably 4 h, at room temperature;

iii) Injecting of the resulting suspension into a calcium chloride bath in ethanol: acetone;

iv) Stirring of the suspension overnight, collecting of the particles by filtration, washing in acetone, and drying at room temperature.

6. The formulation according to claim 5, wherein 20 mL of a suspension of collagen containing 0.2 g of fibrous material (referred to protein content) is added to 50 mL of an aqueous sodium polyphosphate solution (containing 0.2 g of solid salt) and the resulting suspension is filled into a syringe (aperture of 2 mm), clamped into an automatic pump, and injected with a speed of 1 mL/min into a calcium chloride bath composed of ethanol:acetone [1:2 v/v] containing 3 g of calcium chloride dihydrate per 100 mL.

7. The formulation according to claim 6, wherein 20 mL of a solution containing 7.5% sodium alginate in 0.9% sodium chloride is added to 50 mL of an aqueous sodium polyphosphate solution (containing 0.2 g of solid salt) and the resulting suspension is filled into a syringe (aperture of 2 mm), clamped into an automatic pump, and injected with a speed of 1 mL/min into a calcium chloride bath composed of 70% (v/v) ethanol containing 2.5 g of calcium chloride per 100 mL.

8. The formulation according to any one of claims 1 to 7, wherein the average size of the amorphous microparticles formed from the magnesium salt of polyphosphate is in the range of 600 to 30 nm and of the amorphous microparticles formed from the calcium salt of polyphosphate in the range of 900 to 60 nm and of the polyphosphate hybrid material with collagen in the range of 1000 to 100 pm, preferably in the range of 235 to 105 nm (magnesium polyphosphate microparticles), 620 to 280 nm (calcium polyphosphate microparticles) and 640 to 260 pm (calcium polyphosphate collagen hybrid microparticles) with an average size of 170 nm (magnesium polyphosphate microparticles), 450 nm (calcium polyphosphate microparticles) and 450 pm (calcium polyphosphate collagen hybrid microparticles).

9. The formulation according to any one of claims 1 to 8, wherein the nanoparticles or microparticles are embedded into an electrospun meshwork consisting polylactide or its copolymers, polycaprolactone or its copolymers, or another biocompatible polymer.

10. The formulation according to any one of claims 1 to 8, in the form of a wound powder or wound gel.

11. The formulation according to any one of claims 1 to 9, in the form of a wound dressing.

12. The formulation according to any one of claims 1 to 8 or the wound powder, wound gel, electrospun meshwork or wound dressing according to claim 9 to 11 for use the treatment of wounds and wound healing, in particular for wounds showing delayed wound healing or wounds of patients predisposed to such wounds, like elderly or diabetic patients.

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Application No: GB1701403.6