GB2600768A

GB2600768A - Method for preventing infections by respiratory viruses including SARS-CoV-2 through strengthening airway mucus function

Info

Publication number: GB2600768A
Application number: GB2017727.5A
Authority: GB
Inventors: Ernst Ludwig Georg Müller Werner; Dr Werner E G Müller Prof
Original assignee: Prof Dr Werner E G Mueller
Current assignee: Prof Dr Werner E G Mueller
Priority date: 2020-11-10
Filing date: 2020-11-10
Publication date: 2022-05-11
Also published as: WO2022100877A1; GB202017727D0

Abstract

Stable nanoparticles of inorganic polyphosphate (Ca-polyP) as well as soluble Na-polyP increase the expression of specific, anti-virally protective mucins involved in the physical-mechanical barrier function of the mucus layer on the epithelium. A striking effect is shown by Ca-polyP nanoparticles with a narrow size of about 90 nm that matches the size of respiratory virus particles such as the corona virus SARS-CoV-2, with a chain length of approximately 40 phosphate units. A synergistic effect on mucin expression is observed if these nanoparticles are applied in combination with the soluble Na-polyP which, additionally, inhibits the binding of the receptor binding domain of the SARS-CoV-2 spike protein to its cellular receptor ACE2. Soluble Na-polyP alone is somewhat less active. The inventive narrow-sized polyP-nanoparticles that are synthesized in the presence of polyethylene glycol are stable and can be used as a component of a formulated spray, rinse or cream.

Description

METHOD FOR PREVENTING INFECTIONS BY RESPIRATORY VIRUSES

INCLUDING SARS-COV-2 THROUGH STRENGTHENING AIRWAY MUCUS

FUNCTION

This invention concerns the unexpected finding that stable nanoparticles of the calcium salt of inorganic polyphosphate (polyP) as well as soluble polyP (sodium salt) increase the expression of specific, anti-virally protective mucins involved in the physical-mechanical barrier function of the mucus layer on the epithelium. The most striking effect is shown by polyP nanoparticles with a narrow size of about 90 nm that matches the size of respiratory virus particles such as the corona virus SARS-CoV-2, and a chain length of approximately 40 phosphate units. Surprisingly, a synergistic effect on mucin expression is observed if these nanoparticles are applied in combination with the soluble polyP which, additionally, inhibits the binding of the receptor binding domain of the SARS-CoV-2 spike protein to its cellular receptor ACE2. Soluble Na-polyP alone is somewhat less active. The inventive narrow-sized polyP-nanoparticles that are synthesized in the presence of polyethylene glycol are stable and can be used as a component of a nasal spray or mouth rinse or spray, as well as a skin cream or spray.

Background of Invention

The mucus of the respiratory tract as a part of the innate immunity system forms a physical protective barrier against microbes, including virus particles. The major components of the mucus, the mucins, physically enwrap the viruses and allow their removal via the ciliated cells prior to infection and onset of disease.

It is well known that respiratory infections are a leading cause of death. Infections by respiratory viruses can even reach pandemic dimensions like in the case of influenza or the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Both influenza and COVID-19 are contagious respiratory illnesses and are spread via the airborne route in small droplets where they can remain for long periods of time before they are inhaled into the respiratory tract. These viruses have to reach the epithelial cells of the respiratory tract via a bulky mucus layer, which forms a physical barrier for incoming viruses.

The major components of the mucus, the mucins, are high-molecular-weight glycoproteins. They are secreted by the goblet cells. The different mucins are divided into the group of secreted mucins and the tethered, cell surface-associated mucins (Thornton DJ, Rousseau K, McGuckin MA (2008) Structure and function of the polymeric mucins in airways mucus Ann Rev Physiol 70:459-486). The major secreted mucins are the five oligomeric, gel-forming mucins (MUC2, MUC5AC, MUC5B, TVIUC6, and MUC19), as well as two non-polymeric glycoproteins (MUC7 and MUC8). The gel-forming mucins are layered outside of the epithelial cell layer, the periciliary layer, and form the epithelial lining fluid within the mucus layer. The directed motion of this layer is driven by the cilia protruding from the ciliated cells; the flow velocity of the mucus is 4-10 mm/min (Hansson GC (2019) Mucus and mucins in diseases of the intestinal and respiratory tracts. J Intern Med 285:479-490). The tethered membrane-spanning mucins (MUC I, NIUC3A, NIUC3B, MUC4, MUC12, MUC13, MUC15, etc.) are associated with the cell surface. The monomeric mucins multimerize with their Nand C-terminal regions to a close meshed, often gelatinous network. -2 -

The respiratory viruses are using different receptors. For example, the corona virus, SARSCoV-2 infects cells that are carrying the angiotensin-converting enzyme 2 (ACE2) receptor whereas the middle-east respiratory syndrome coronavirus select cells with the surface receptor dipeptidyl peptidase 4 and the human coronavirus 229E binds to the aminopeptidase N receptor (Subbarao K, Mahanty S (2020) Respiratory virus infections: Understanding COV1D-19. Immunity 52:905-909). Therefore, a general strategy for protection against all types of respiratory viral infections that is solely based on inhibition of virus-receptor binding will not be possible. The strategy underlying the inventive method is based on the strengthening of the physical barrier function of the mucus, which is not restricted to a specific virus, but can be expected to be effective against respiratory viruses in general, as all of them have to pass the mucus layer.

Focusing on compounds that are preventing SARS-CoV-2 infection, inorganic polyphosphate (polyP) has recently been found to bind to the receptor-binding domain (RBD) of the spike (S)-proteins that decorate the virus and associate with ACE2, the cell membrane receptor of the target cells (Neufurth M, Wang XH, Tolba E, Lieberwirth I, Wang S, Schrader HC, Muller WEG (2020) The inorganic polymer, polyphosphate, blocks binding of SARS-CoV-2 spike protein to ACE2 receptor at physiological concentrations. Biochem Pharmacol 182:114215). This polymer causes a complete inhibition of the binding of the RBD to the ACE2 receptor at a concentration of 01 pg/mL (Muller WEG, Neufurth M, Schepler H, Wang S, Tolba E, Schrader HC, Wang XH (2020) The biomaterial polyphosphate blocks stoichiometrically binding of the SARS-CoV-2 S-protein to the cellular ACE2 receptor. Biomaterials Sci, DOT: 10.1039/d0bm01244k), a level that is much lower than the one present in the circulating blood, polyP prevents binding to ACE2 by interaction with the basic amino acids of RBD of the Si subunit of S-protein.

PolyP is a physiological, inorganic polymer that is synthesized in any body cell, especially in blood platelets. This polymer has been found to act as extracellular generator of metabolic energy (Muller WEG, Schroder HC, Wang XH (2019) Inorganic polyphosphates as storage for and generator of metabolic energy in the extracellular matrix. Chem Rev 119:1233712374). PolyP, if present in the soluble form but not as nanoparticles, is degraded by the alkaline phosphatase (ALP). The polyP nanoparticles are more resistant to ALP hydrolysis.

The mucus/mucin layer which is composed of the secreted MUC5AC has the major role to bind to virus particles and to drive the mucus clearance system out of the lung. In contrast, the periciliary layer is surrounded by the tethered mucins with the major transmembrane MUC I. This heavily 0-linked glycosylated mucin species surrounds the cilia of the ciliated cells. Consequently, MUCI with its negatively charged sugar branches prevents the entry of most viruses into the periciliary layer because at neutral pH, the surfaces of most viruses are likewise negatively charged due to their isoelectric point below 7 (Michen B, Graule T (2010) Isoelectric points of viruses. J Appl Microbiol 109:388-397).

In the inventive method, calcium-polyP nanoparticles (Ca-polyP nanoparticles) were synthesized from polyP with a chain length of approximately 40 phosphate units. In the presence of polyethylene glycol and at a defined concentration ratio between polyP and Ca' ions and a defined time period of the dropwise addition of the Ca' solution to the polyP solution, stable narrow-sized nanoparticles of a size of about 90 nm are obtained. Surprisingly, the inventor found that these polyP-nanoparticles, if applied in combination with soluble polyP (sodium salt) are able to increase the expression specific mucin genes in a synergistic manner. polyP-nanoparticles with a size of around 310 nm or 482 nm are less effective and do not show this synergistic activity with soluble polyP. Consequently, these -3 -nanoparticles with size of about 90 nm will strengthen the physical-mechanical antiviral barrier function of the mucus based on the mucin network. In addition, the binding of the receptor binding domain of the spike protein of SARS-CoV-2 to its cellular receptor protein, ACE2, is inhibited by polyP and, even more, by a combination of polyP nanoparticles (90 nm) and polyP. The stable, narrow-sized polyP-nanoparticles allow the preparation of a nasal spray or mouth rinse or spray, as well as a skin cream or spray containing a suspension of the nanoparticles The state-of-the-art of polyP has been described, for example, in: -Mtiller WEG, Schroder HC, Wang XII (2019) Inorganic polyphosphates as storage for and generator of metabolic energy in the extracellular matrix. Chem. Rev. 119:12337-12374

Summary of the invention

This invention describes a novel method for the preparation of narrow-sized polyP nanoparticles that are strengthening the defense function of the mucus barrier against pathogenic respiratory virus by induction of mucin production and energy (ATP) delivery. These particles can be used for prevention but also therapy of respiratory virus infections, in particular infections caused by the corona virus SARS CoV-2.

Surprisingly, the inventor found that the polyP-nanoparticles are able to increase the expression specific mucin genes, whereby the polyP-nanoparticles with a narrow size of around 91 nm are superior to the nanoparticles with a size of around 310 nm or 482 nm. Interestingly, this size of around 91 nm is very similar the size of a virus particle like SARSCoV-2, which is -100 nm (Bar-On'VIM, Flamholz A, Phillips It, Milo R (2020) SARS-CoV-2 (COVID-19) by the numbers. Elife 9:e57309). This size matches with the dimensions of the particles entering the cells by clathrin-and caveolae endocytosis pathway. This correspondence is very striking, although at present the mechanisms of entry of SARS-CoV into endothelial cells is not yet clear, both a clathrin-and caveolae-independent and a -dependent uptake is discussed (Wang FI, Yang P, Liu K, Guo F, Zhang Y, Zhang G, Jiang C (2008) SARS coronavirus entry into host cells through a novel clathrin-and caveolaeindependent endocytic pathway. Cell Res 18:290-301; Glebov 00 (2020) Understanding SARS-CoV-2 endocytosis for COVID-19 drug repurposing. FEBS J. doi: 10.1111/febs.15369).

Most surprising was the finding that non-particulate soluble polyP (Na-polyP) acts synergistically in stimulating the mucin gene expression if applied in combination with the polyP-nanoparticles with a narrow size of around 90 nm but not the nanoparticles with a size of around 310 nm.

The inventive narrow-sized polyP-nanoparticles are synthesized in the presence of polyethylene glycol and at a defined concentration ratio between polyP and Ca' ions and a defined time period of the dropwise addition of the Ca' solution to the polyP solution. These nanoparticles are stable and can be used as a component of a nasal spray or mouth rinse or spray, as well as a skin cream or spray. -4 -

In the inventive nanoparticulate form the polyP is stabilized towards attack and immediate degradation by the mucus associated ALP activity, so these nanoparticles can penetrate the mucus layer and reach the cells of the respiratory epithelium.

In addition, the polyanionic polymer, polyP, as soon as released from the particles can block the binding of the vints (SARS-00V-2) via its spike-protein receptor binding domain to the host cell surface receptor ACE2 and deliver ATP necessary for airway epithelium and mucus clearance function. An increased inhibitory effect is shown by a combination of the soluble polyP and the 90 nm polyP nanoparticles.

Principle: A schematic presentation of the principle of the inventive method is shown in Figure 1. The airway epithelium is characterized by two cells types: Ciliated cells and secretory cells. On the surface of the airway epithelium, the periciliary fluid layer and the epithelial lining fluid are present. Both the ciliated cells and the secretory cells contain at their outer surfaces the two enzymes, which are involved in ATP production from polyP, the ALP and ADK. The ATP produced then elicits the expression of the genes encoding for NIUC1 and MUC5AC. The mucus entraps and removes the viral particles (clearance mechanism of the mucus).

The following patents and patent applications on polyP are deemed relevant: -Patent EP 15794143.6. Morphogenetically active amorphous calcium polyphosphate nanoparticles for therapeutic applications. Inventor: Muller WEG. 11/2015.

Patent US 15/527,479. Morphogenetically active amorphous calcium polyphosphate nanoparticles for therapeutic applications. Inventor: Muller WEG. 11/2015.

CN 201580067850. Morphogenetically active amorphous calcium polyphosphate nanoparticles for therapeutic applications. Inventor: Muller WEG. 11/2015.

GB 2007810.1. Method for blocking binding of SARS-CoV-2 spike protein to its cellular receptor ACE2. Inventor: Muller WEG. 5/2020.

Detailed description of the invention

Previously the inventor disclosed a procedure for preparation of silica/polyphosphate nanoparticles that contain a silica core (GB 2007810.1. Method for blocking binding of SARS-CoV-2 spike protein to its cellular receptor ACE2. Inventor: Midler WEG. 5/2020). These particles were found to inhibit the binding of the receptor binding domain (RBD) of the SARS CoV-2 spike protein to its cellular receptor protein ACE2. An effect on mucin expression or the production of ATO required for mucus function (clearance mechanism of mucus) has not been reported In addition, the inventor disclosed a procedure for preparation of amorphous Ca-polyP nanoparticles (Patent EP 15794143.6. Morphogenetically active amorphous calcium polyphosphate nanoparticles for therapeutic applications. Inventor: Moller WEG. 11/2015. Patent US 15/527,479. Morphogenetically active amorphous calcium polyphosphate nanoparticles for therapeutic applications. Inventor: Muller WEG. 11/2015. CN 201580067850. Morphogenetically active amorphous calcium polyphosphate nanoparticles for therapeutic applications. Inventor: Muller WEG. 11/2015). However, the Ca-polyP -5 -nanoparticles according to EP 15794143.6, US 15/527,479 and CN 201580067850 show a brought size distribution and cannot be used for inhibiting of SARS-CoV-2 S-protein -ACE2 interaction according to GB 2007810.1 The present invention discloses a procedure for preparation of narrow-sized small polyP nanoparticles that induce the expression of mucins and can be used for the delivery of metabolic energy in the form of ATP Surprisingly, these nanoparticles act synergistically on mucin expression in combination with soluble polyP which additionally prevents binding of the SARS-CoV-2 spike-protein to the cellular receptor ACE2.

These inventive narrow-sized polyphosphate nanoparticles are characterized by the following properties: 1. The particles prepared according to the inventive procedure can be fabricated in an adjustable size range, not achieved by hitherto disclosed procedures 2. The adjustable small size of the nanoparticles according to the inventive procedure (about 90 nm) is in the same range as the size of virus (SARS-00V-2) particles and the size of particles that can be taken up by cells via the clathrin-and caveolae en docytosi s pathway.

3. The nanoparticles prepared according to the inventive procedure are less susceptible to hydrolysis by the enzyme ALP present in the mucus layer than polyP encapsulated into silica, following a co-precipitation procedure (disclosed in GB 2007810.1. Method for blocking binding of SARS-CoV-2 spike protein to its cellular receptor ACE2. Inventor: Muller WEG. 5/2020). The latter particles with a diameter of -150 to 200 nm have protruding polyP chains which are not included into the core particles, and therefore can be easily hydrolyzed by the ALP.

4. Therefore, the inventive narrow-sized polyP nanoparticles can permeate the mucus layer without significant degradation to reach the cells of the respiratory epithelium, where they can be taken up and induce the expression of mucins and become metabolized to ATP (via the membrane associated ALP and ADK), necessary for the function of the ciliated cells in the clearance mechanisms of the mucus layer.

The inventive method for preparation of the narrow-sized polyP nanoparticles that strengthen the physical virus-protective function of the respiratory mucus layer consists of the following steps: 1 Preparation of an alkaline aqueous solution containing a soluble polyP salt; 2 Preparation of an alkaline aqueous solution containing a divalent cation salt, 3 Supplementation of the polyP salt solution with polyethylene glycol; 4 Drop-wise addition of the solution of the divalent cation salt solution to the polyethylene glycol supplemented polyP salt solution, and adjusting the pH value to alkaline values; Collection of the polyP nanoparticles thereby formed by centrifugation; and 6 Washing the polyP nanoparticle pellet with ethanol-water and air-drying; All steps of this method are performed at ambient temperature. The soluble polyP salt consists of polyP and a monovalent cation -6 -The soluble polyP salt is preferably Na-polyP.

The divalent cation salt is magnesium chloride or calcium chloride. The pH of the alkaline aqueous solution is preferably pH 10.

The chain length of the polyP is in the range of about 2 to about 1000 phosphate units, preferably in the range of about 3 to about 100 phosphate units, and most preferred about 40 phosphate units.

The concentration of the polyP salt is in the range of about 1 to 25 g/L, preferably in the range of about 3 to about 10 mg/L, and most preferred 5 g/L.

The concentration of the divalent cation salt is in the range of about Ito 25 g/L, preferably in the range of about 3 to about 10 mg/L, and most preferred 5 g/L.

The polyethylene glycol is is preferably polyethylene glycol 200 (average molar mass 200 g/mol).

The polyethylene glycol concentration is is preferably 0.5% weight percent.

The volume ratio of the divalent cation salt solution to the polyethylene glycol supplemented polyP salt solution is in the range of about 1:5 to 5:1, preferably in the range of about 1:2 to about 2:1, and most preferred 1:1.

The solution of the divalent cation salt solution is added drop-wise to the polyethylene glycol supplemented polyP salt solution over a period of about 0.3 to about 10 h, preferably about 1 to about 3 h, and most preferred 2 h. The most preferred properties in terms of inducing expression of mucin genes are found for narrow-sized polyP nanoparticles with a size of about 90 nm.

The most striking effect on expression of mucin genes is observed if these narrow-sized polyP nanoparticles are applied in a synergistically acting combination with soluble polyP.

The inventive narrow-sized polyP nanoparticles can be applied, either alone or combination with soluble polyP, for the preparation of antiviral nasal spray, mouth rinse, mouth spray, chewing gum, eye drops, or skin cream and skin spray.

An example is the application of the inventive method for the preparation of a narrow-sized polyP nanoparticle-containing virus-protective nasal spray consisting of propylene glycol, 0.9% NaCI (pH stabilized with calcium carbonate).

A further example is the application of the inventive method for the preparation of a narrow-sized polyP nanoparticle-containing virus-protective skin cream consisting of urea (5 g), Unguentum emulsificans aquosum (89 g), lactic acid 90% (1 g), sodium lactate solution 50% (4 g) and preservatives (sorbic acid E200 0,045% final, and potassium sorbate E202 0,062% final). -7 -

A further example is the application of the inventive method for the preparation of a narrow-sized polyphosphate nanoparticle-containing virus-protective chewing gum consisting of soluble sodium polyphosphate (5% [w/w]), chicle gum from the sapodilla tree (25% [w/w]), xylitol (57.5% [w/w]), and vegetable glycerol (86%) (12.5% [w/w]).

A further example is the application of the inventive method for the preparation of a narrow-sized polyphosphate nanoparticle-containing virus-protective eye drops containing hyaluronic acid, heparin, ectoin, vitamin A and/or Euphrasia extract.

A further aspect of the inventive method concerns the application of the inventive narrow-sized polyP nanoparticles, either alone or in the inventive synergistically acting combination with soluble polyP, or of soluble polyphosphate alone, for prevention and therapy of respiratory virus infections.

A further aspect of the inventive method concerns the application of the inventive narrow-sized polyP nanoparticles, either alone or in the inventive synergistically acting combination with soluble polyP, or of soluble polyphosphate alone, for prevention and therapy of coronavirus SARS CoV-2 infections.

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 a schematic illustration of the morphogenetic activity of polyP (polyphosphate) on the airway epithelium. The two cells types, the ciliated cells and the secretory cells compose the airway epithelium. On its surface both the periciliary fluid layer and the epithelial lining fluid are organized (upper panel). With the directed flow of the mucus the polyP particles are transported both to the secretory and the ciliated cells (middle panel). Both cell types comprise on their outer surfaces the two enzymes, ALP and ADK, which hydrolyze polyP. The liberated free energy is (partially) harvested in the form of ADP (ALP) which undergoes subsequently up-phosphorylation to ATP (ADK). The polyP, if administrated in the form of narrow-sized nanoparticles of about 90 nm, similar to the size of the virus particles, is taken up by the cells and elicits gene induction and release of the mucins, MUC1 and MUC5AC.

Figure 2 shows the morphology of the different size fractions of Ca-polyP NP; SEM. The following fractions were used: (A) "Ca-polyP-NP-cross" (Ca-polyP with a broad size distribution and an average size of 482 nm), (B) -Ca-polyP-NP-91" (Ca-polyP with a narrow size distribution and an average size of 91 nm) and (C) "Ca-polyP-NP-310" (Ca-polyP with a narrow size distribution and an average size of 310 nm). The globular "Ca-polyP-NP-cross" particles have a more irregular morphology, while the "Ca-polyP-NP-91" and the "Ca-polyPNP-310" fractions are more homogenous.

Figure 3 shows the size determination of the particles using dynamic light-scattering spectra of the Ca-polyP NP fractions "Ca-polyP-NP-cross", "Ca-polyP-NP-91" and "Ca-polyP-NP310". At least 200 particles were measured for each sample to obtain the size distribution. The experiments were performed with a Zetasizer Nano ZS90 at a scattering angle 0 = 900.

Figure 4 shows the viability/growth of A549 cells was assessed with the IVITT assay. The cells were exposed to Na-polyP and also against the two size-restricted polyP NP fractions -8 - "Ca-polyP-NP-91" and "Ca-polyP-NP-310". The incubation period was 3 d. Then the assays were subjected to MTT, followed by a intensity determination of the formed fonnazan dye in a microplate reader at 450 nm. The data represent means ± SD (n=10). The significances within an individual group (*; p< 0.01) were calculated.

Figure 5 shows the microscopic aspects of the A549 cells after a 3 d incubation with NapolyP and using the indicated concentrations The cells were stained with the Draq5 dye for nuclear structures and Calcein AM for the viability.

Figure 6 shows the gene expression level for AILIC I and MUCSAC in A549 cells after exposure to 100 jig mL-I of soluble Na-polyP, "Ca-polyP-NP-91" or "Ca-polyP-NP-310" during an incubation period of 3 d and 6 d, respectively. After the termination of the incubation the RNA was extracted from the assays and subjected to qRT-PCR. Then, the expression levels were determined and correlated with the expression level of the house keeping gene GAPDH. Standard errors of the means (SEM) are indicated (n = 5 experiments per time point). Within on incubation time point (horizontal line) the significance has been calculated; *p <0.05.

Figure 7 shows the effect of the different NP preparations (administered with 100 ig mL-1), "Ca-polyP-NP-cross", "Ca-polyP-NP-91" and "Ca-polyP-NP-310" on (A) MUC1 and (B) MUC5AC gene expression in A549 cells. An assay without polyP (control) run in parallel. n = 5; *p< 0.05.

Figure 8 shows the increased level of /14(IC/ gene expression if Na-polyP is administered in combination with "Ca-polyP-NP-91" The polyP samples alone were tested at the concentration listed In the combination experiments 0 or 10 jig mL-1 of Na-polyP was added to assays with 10 gg mL-1 "Ca-polyP-NP-91" or with 10 Mg mL-1 "Ca-polyP-NP-310". n = 5; *p< 0.05.

Figure 9 shows the reduction of the binding affinity between viral RBD and the cellular ACE2 by Na-polyP (100 jig mli). The effect if "Ca-polyP-NP-91" (100 jig mli) on the binding is statistically not significant. However, after co-addition of 10 jig of "Ca-polyP-NP-91" to 50 jig mL-I of Na-polyP an even stronger inhibition is measured, compared to the one seen with 100 jig Na-polyP alone (n = 3, *p <0.05).

Figure 10 shows the ATP release kinetics in A549 cells (luciferin-luciferase-based assay), growing onto the collagen matrix, COL-HG, or after addition of mucin (100 pg/mL mucin; COL/MUC-HG) or mucin and polyP (100 iig/mL mucin and 10 iig/mL polyP; COL/MUC/polyP-HG) After an incubation period of 0, 30 and 60 min aliquots were taken and assayed for the extracellular ATP concentration. The ATP concentrations were determined in the culture medium (n = 10; *p <0.001).

Examples

In the following examples, the inventive method described only for polyP molecules with chain lengths 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.

Preparation of Ca-polyP nanoparticles -9 -In order to obtain amorphous Ca-polyP NP a 2:1 molar ratio between CaC12 and Na-polyP (based on phosphate) was applied; during the fabrication the pH was adjusted to 10. For a cross and faster preparation, "Ca-polyP-NP-cross", higher concentrations of the polyP and CaC12 solutions were selected. Also the reaction time was shorter with 30 min. For the fabrication of the larger size particles, "Ca-polyP-NP-310", the concentrations of the starting solutions remained identical but the reactions time was increased to 60 min. To obtain a more homogeneous and small-size particle distribution the solutions for polyP and Can') were less concentrated and the reaction was extended to 120 h. Morphology and size of the particles The NP of Ca-polyP have a globular size with a different degree of symmetry. The "CapolyP-NP-cross" particles comprise different size dimension, measuring form 150 to 700 nm with an average of -470 nm (n=6) (Figure 2A). In contrast, the smaller "Ca-polyP-NP-91" particles are very homogeneously globular with an -95 nm size (Figure 2B). Likewise homogenous but less globular are the larger particles ranging around 350 nm (Figure 2C).

The more accurate sizes of the particles were determined by dynamic light scattering measurements after dispersion in ultrapure water (Figure 3). The "Ca-polyP-NP-cross" sample shows a slightly negatively-skewed with a 6 of 0.1 (Box GEP, Cox DR (1964) An analysis of transformations. J R Statist Soc B (Methodological) 26:211-252). The median is calculated with 482 nm and a standard deviation of 310 nm (Figure 3A). The small particles, "Ca-polyP-NP-91", show an almost normal, symmetrical distribution with a median of 91 nm and a standard deviation of 158 nm (Figure 3B). The large particles, -Ca-polyP-NP-310", show again a skewed distribution curve with a slightly positively-skewness with a 6 of 0.05. The median is 310 nm and the standard deviations are 180 nm.

In the experimental series with soluble Na-polyP additional Ca2-was added to the assay in order to compensate for the complexation property of the polymer. Applying the MTT assay a significant increase of the viability was seen between the concentration range of 10 pg mL-1 to 100 pg mL-1; a maximum is measured at 10 pg mL-1 (Figure 4). In the incubation period was 3 d. Using the small size particles, "Ca-polyP-NP-91", a likewise significant higher viability is seen at 10 and 30 pg mL4. In contrast, no significant change of the growth rate of the cells were measured at the concentration range chosen (Figure 4).

Representative microscopic aspects of the cells, incubated for 3 d, and using the three concentrations of Na-polyP, of 3 pg mL-1 (Figure 5A), 30 pg mL-1 (Figure 5B) and of 100 pg mL-1 (Figure 5C). The cells were stained with Draq5 (fluorescence labeling of DNA) and with Calcein AM (cell viability). After the 3 d incubation only a slightly scattered distribution is seen at 3 pg m1_,-1, while at the higher concentrations more dense accumulation of cells appear.

Expression levels of mucins in assays with individual polyP fractions The A549 cells were exposed against Na-polyP, or "Ca-polyP-NP-91" and "Ca-polyP-NP310", respectively, at concentrations of 100 pg ml]'. In a separate series the cells remained without polyP (controls). The cultures were incubated for 3 d or 6 d. RNA from seeding cultures or from those exposed to the compounds were extracted and subjected for the qRTPCR (Figure 6). For AdUC 1 in all four samples an increase of the expression with -50% is measured after a 3 d incubation period. Within this 3 d groups, the levels of four parallel samples are closely together. Only the sample exposed to Na-polyP and "Ca-polyP-NP-91" are significantly higher. However, after a 6 d incubation the expression of cells exposed to -10 -Mg mL-1 Na-polyP (by 90%) or to "Ca-polyP-NP-9I" (by 46%) are significantly higher compared to the controls or to "Ca-polyP-NP-310".

For AlUC5AC (Figure 6B) a similar expression level is measured. Within the 3 d group the levels are not significantly different. However, after 6 d again the expressions of the Na-polyP and the "Ca-polyP-NP-91" samples are significant, with 130% and with 75%, higher compared to the controls.

A direct comparison of the gene-inducing activities of the three different, fabricated polyP NP factions, "Ca-polyP-NP-cross", "Ca-polyP-NP-91" and 310, is given in Figure 7. A concentration of 100 jig mil' is applied. /11/1/(17 gene expression is not altered during the 3 d incubation time point (Figure 7A). Only after 6 d the cells exposed to "Ca-polyP-NP-cross" and to "Ca-polyP-NP-91" express MUC/ significantly higher, by 32% and 65%, compared to the controls or to the "Ca-polyP-NP-310" preparation. Also for the AIIIIC5AC expression only the "Ca-polyP-NP-91" sample (both at day 3 and day 6) and the "Ca-polyP-NP-cross" fraction (at day 6) are significantly inducing (Figure 7B).

Expression levels of mucins in the presence polyP combinations Lower concentrations of polyP have been selected to determine a possible synergistic effect between the different polyP samples (Figure 8). Soluble Na-polyP cause a significantly induce MUG/ expression at a concentration of 30 pg mL-1 and higher, while at the lower concentration of 10 jig mL-1 the increase is not significant. Interestingly, 10 jig mL-1 of "CapolyP-NP-91" cause a slightly intensified expression already at 10 jig mL-1 "Ca-polyP-NP310" elicits only a change within the range of variation.

In the combination experiments, using as a basis 10 jig mL-1 of "Ca-polyP-NP-91-alone and then in combination 10 jig mL-1 of "Ca-polyP-NP-91" together with 10 jig mL-1 of Na-polyP a significant increase of MUG/ expression is measured and the value comes to 174% (Figure 8). Addition of "Ca-polyP-NP-310" to Na-polyP does not cause a similar strong effect.

Inhibition of the binding between the RBD and the ACE2 by polyP An ELISA-like assay was used for the determination of the binding strength between the viral RBD and the ACE2 (Figure 9). The strength of the binding in the controls, not containing polyP, was set to 100%. After addition of 100 jig mL-1 of Na-polyP to the RBD the binding affinity between the RBD and the ACE2 was reduced to 54.8+4.9, while the "Ca-polyP-NP91" sample (100 pg mL-1) caused only a non-significant effect on this interaction. However, if the reduced concentration of 10 jig mL-1 of "Ca-polyP-NP-91" was added to 50 jig mL-1 of Na-polyP even a slightly higher inhibition with 47+4.1% was measured.

Increased release of ATP from A549 cells in response to polyP PolyP also increases the production of ATP as revealed in the following experiment. As shown in Figure 10, plating alveolar basal epithelial A549 cells on a collagen hydrogel (COL-HG) does not affect the ATP export from A549 cells during the 60 min incubation period in vitro. Addition of mucin to this matrix, COL/MUC-HG, caused a slight, but significant increase of the ATP release (by 25%) from the cells. However, after addition of 10 p.g/mL of polyP, as in COL/MUC/poly-HG, a sharp induction of ATP release, by 2.3-fold, from the cells is measured. Addition of the ALP inhibitor levamisole (LEV; I mM) causes a significant reduction of the ATP release in A549 cells only if these cells are cultured on the polyPcontaining matrix. A similar reduction is seen if the cells were preincubated with the ADK inhibitor (P1,P5-di(adenosine-5') pentaphosphate (Ap5A; 40 gM). These data strongly suggest that the cell-associated enzymes ADK and ALP, in concert with polyP, are involved in the generation of extracellular ATP.

Methods Materials Na-polyphosphate (Na-polyP) with an average chain length of 40 P, units (polyP40) can be obtained, for example, from Chemische Fabrik Budenheim (Budenheim; Germany), polyethylene glycol 200 (average molar mass 200 g/mol), mucin from bovine submaxillary glands (type I-S), MTT (thiazolyl blue tetrazolium bromide) and calcein AM from Sigma (Taufkirchen, Germany), Ham's F-12K (Kaighn's) medium and Draq5 dye from Gibco/Thermo Fisher Scientific (Dre'eich; Germany).

Preparation of Ca-polyP nanoparticl es Amorphous Ca-polyP nanoparticles (Ca-polyP-NP) are prepared using a modified procedure (addition of polyethylene glycol 200 and dropwise addition of the CaC12 solution over a defined time period) of the method described (Muller WEG, Tolba E, Schroder HC, Wang 5, Glal3er G, Mulloz-Espi R, Link T, Wang XH (2015) A new polyphosphate calcium material with morphogenetic activity. Mater Left 148:163-166). A 2:1 molar ratio between CaC12 and Na-polyP (based on phosphate) is applied and the pH is adjusted to 10.

Ca-polyP-NP-cross: For the cross reaction 10 g of Na-polyP is dissolved in 1 L distilled water. This solution was supplemented with 0.5 % (wt/vol) polyethylene glycol 200 (average molar mass 200 g/mol). Calcium chloride dihydrate (38.6 g; for example, from Sigma-Aldrich) is dissolved in 1 L distilled water. The CaC12 solution is slowly and dropwise added to the polyP solution during a 30 min period of time. After stirring for 12 h the nanoparticles (NP) are collected by filtration, washed twice with ethanol and then three-time with water and dried at 50 °C. The particles are collected; "Ca-polyP-NP-cross".

Ca-polyP-NP-310: For the preparation of the large (310 nm size) NP fraction the same concentration ratio between CaC12 and polyP is used and the dropwise mixing process is increased to 60 min. The particles were collected and dried; "Ca-polyP-NP-310".

Ca-polvP-NP-91: For the small, 91 nm large NP preparation the 38.6 g of CaC12 are dissolved in 2 L and the 10 g Na-polyP dissolved in likewise 2 L. The addition process is extended to 2 h. The resulting particles are collected and dried; "Ca-polyP-NP-91".

Microscopic analyses The scanning electron microscopic (SEM) images can be performed, for example, with an HITACHI 5U8000 microscope (Hitachi, Krefeld; Germany) is used. The light microscopical images can be taken, for example, with a VT-IX-600 Digital Microscope from Keyence (NeuIsenburg; Germany).

Particle size determination The size of the particles can be determined, for example, with the Zetasizer Nano ZS90 (Malvern Instruments, Malvern; United Kingdom). The particles are dispersed in ultrapure water.

Cell culture In the experiments shown in Examples, A549 cells, a human lung (carcinoma) line, are used. These cells can be obtained, for example, from Sigma The cells are cultivated in Ham's F- -12 - 12K (Kaighn's) medium, supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 4 mM glutamine. The cells are incubated in 96-well plates/24-well plates in a humidified atmosphere of 5% CO2 in air (37°C). Every 3 to 4 d the medium/serum is replaced. If the cells are incubated with additional compounds for a longer period of time, half of the medium/serum is replaced after 3 d with new medium/serum containing the original components with the concentration used for the inoculum For the MTT-viability study the incubation with polyP, both soluble Na-polyP or NP-bound Ca-polyP are applied, the indicated concentrations are used. In the series with Na-polyP additional Ca' (molar ratio of 2 with respect to phosphate monomer [in the polyP] to 1 Ca') are added to the assays in order to compensate for the complexation potential of the polyanionic polymer.

The cells are double stained with the Draq5 dye for nuclear staining and Calcein AM for the viability representation The growth/viability of the A549 cells can be assessed, for example, by the colorimetric MTT assay. The starting cell concentration is 7x103 cells mL-1 medium/serum and the incubation period is 3 d. After termination, the cells are sequentially incubated with MTT (1 jag mL71; 2 h) and with 20% SDS in 50% dimethyl-formamide for 24 h. The formazan grains are dissolved followed by the determination of the optical density 450 nm. Ten parallel experiments are performed. In the Examples, the results from 10 parallel assays are given.

Quantitative real-time polvmerase chain reaction The expression of mucin genes is quantitated using the qRT-PCR procedure. The A549 cells are exposed to either the soluble Na-polyP, or the two size-restricted polyP fractions, "CapolyP-NP-91" and "Ca-polyP-NP-310", respectively. In a separate experiment the cells remain without a polyP preparation (controls). The polyP samples are added as individual substance at concentrations given with the experiments, or in in combination. The cells are seeded at a density of 7x103 cells mL-1 medium/serum and incubated for 3 d or 6 d. Then the cells are harvested and RNA is extracted from them and used for the qRT-PCR reaction. The following primer pairs are used: for the human Alt/Cl, expressed in the periciliary layer (Xu M, Wang X (2017) Critical roles of mucin-1 in sensitivity of lung cancer cells to tumor necrosis factor-alpha and dexamethasone. Cell Biol Toxicol 33:361-371), (Accession number; P15941) Fwd: 5'-AATTGACTCTGGCCTTCCGA-3' and Rev: 5'-TGCCACCATTACCTGCAGAA-3', and for human MUC5AC (Damiano 5, Sasso A, De Felice B, Di Gregorio I, La Rosa G, Lupoli GA, Belfiore A, Mondola P, Santillo M (2018) Quercetin increases /141/(72 and Al(IC5AC gene expression and secretion in intestinal goblet cell-like LS174T via PLC/PKCa/ERK1-2 pathway. Front Physiol 9:357) (U06711) Fwd: 5PTCCGGCCTCATCTTCTCC-3' and Rev: 5'-ACTTGGGCACTGGTGCTG-3'. After termination of the reaction the expression levels are correlated with the one of the reference housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase; NM 002046.3) with the primer pair Fwd: 5'-ACTTTGTGAAGCTCATTTCC-3' and Rev: 5PTTGCTGGGGCTGGTGGTCCA-3'. The amplifications can be performed, for example, in an iCycler (Bio-Rad, Hercules, CA; USA), and the mean Ct values and the efficiencies are calculated applying the iCycler software (Bio-Rad). In the Examples, the estimated PCR efficiencies are 95103%.

Determination of extracellular ATP concentration The release of ATP can be quantified, for example by applying the luciferin-luciferase-based Enlighten assay (Promega, Madison; WI) Briefly, the A549 cells are grown in 24-well plates -13 -to 90-100% contluency. Then the cells are transferred into Ham's medium without serum and incubation is continued for additional 30 min or 60 mM at the cell density of 106 cells/mL The following matrices are used for cell cultivation; COL-HG, COL/MUC-HG (100 ug/mL of mucin) or COL/MUC/PolyP-HG (100 pg/mL of mucin and 10 pg/mL of polyP). Subsequently, a sample of 0 5 mL is collected and transferred into chilled polypropylene tubes and centrifuged (12,000 x g; 5 min). Aliquots (100!IL) are taken from the supernatant and measured in the luciferin-luciferase assay. From the standard curve the ATP level is read. The ATP concentrations are given as pmo1/106 cells. Where mentioned under Examples, the cells are pre-incubated with 40 1iA4 Ap5A (inhibitor of ADK) or 1 mIVI levamisol (inhibitor of the ALP) for 10 min prior to cell seeding.

The hydrogel matrices are prepared as follows.

Collagen-hydrogel (COL-HG): Bovine collagen is dissolved in 0.1 M acetic acid (pH 3.6) at a concentration of 10 mg/mL. After adjusting the pH to 7.4 (with NaOH) the solution is brought to 5 mg/mL and supplemented with 10 mM MgC12; COL-HG.

Collagen/mucin-hydrogel (COL/MUC-HG): An aliquot of 1 mL of collagen solution (pH 3.6) is supplemented with 1 mL of mucin (concentration between 0 and 1 mg/mL [final]; average concentration in the human saliva -200 pg/mL) and the mixture is brought to 10 mM MgC12; COL/MUC-HG. The pH is adjusted to 7.4 (with NaOH).

Collagen/mucin/polyP-hydrogel (COL/MUC/polyP-HG): The collagen -mucin (200 pg/mL) mixture is supplemented with Na-polyP (concentration range of 0 to 100 pg/mL [final]). MgC12 was stoichiometrically added (molar ratio of 2 [with respect to phosphate monomer] to 1 [Me]) to compensate for any effect caused by the chelating activity of polyP; COL/MUC/polyP-HG.

Binding inhibition assay of SARS-CoV-2 spike (S)-Protein [RBD] to ACE2 For example, the Screening Assay Kit (BPS Bioscience/Tebu-bio, Offenbach; Germany) can be used for the quantitation of the binding strength between the RED of the viral S-protein to the ACE2 cell surface receptor. The recombinant ACE2 (50 ng/well) is attached to the bottom of the 96 well plate. The recombinant RBD/Sl-protein (100 ng/well), labeled with biotin, is incubated with the polyP test formulation for a period of 60 min (23°C) in 10 mM HEPES buffer, pH 7.0. Then the RBD is added to the well, coated with ACE2, and the complex formation between the RED and the ACE2 is detected with streptavidin-HRP (horseradish peroxidase) using the ITRP substrate. Finally the complex is quantitated on the basis of the chemiluminescence using, for example, the Perkin Elmer-Wallac victor 3 V multi-label microplate reader (Perkin-Elmer, Waltham, MA; USA). The values for the blank (consisting only of the immuno buffer) are subtracted from the readings. The values obtained for the samples are correlated with the positive controls (containing only the buffers and the labeled S-protein), which are set to 100%.

The polyP samples were suspended in medium/serum at a stock solution of 3 mg mL' . Spray formulation For the application of polyP as a nasal spray, the polymer is dissolved in a concentration of 200 mg m1;1 in buffered saline with an antimicrobial agent. During the binding assay the concentration is diluted down to 100 jig Statistical analysis The skewness of the non-Gaussian distribution curves of the NP reported under Examples has been evaluated as published (Box GEP, Cox DR (1964) An analysis of transformations. J R Statist Soc B (Methodological) 26:211-252; Gunmathan S, Han SW, Dayem AA, Eppakayala -14 -V, Kim JH (2012) Oxidative stress-mediated antibacterial activity of graphene oxide and reduced graphene oxide in Pseudomonas aeruginosa. mt J Nanomedicine 7:5901-5914; Hu H, Zhao P, Liu J, Ke Q, Zhang C, Guo V, Ding H (2018) Lanthanum phosphate/chitosan scaffolds enhance cytocompatibility and osteogenic efficiency via the Wnt/P-catenin pathway. J Nanobiotechnology 16:98). The values reported under Examples represent the respective average ± standard deviations (a). Student t test can be applied to perform comparisons between two groups. The average values and a reported under Examples originate from at least three to six independent experiments. Values of p < 0.05 are considered statistically significant (*). The calculations can be performed, for example, with the GraphPad Prism 7.0 software (GraphPad Software, La Jolla; CA).

Claims

CLAIMS1. Method for preparation of narrow-sized polyphosphate nanoparticles in the size range of viral (such as SARS C0V-2) particles that strengthen the physical virus-protective function of the respiratory mucus layer, involving the following steps: a) Preparing an alkaline aqueous solution containing a soluble polyphosphate salt; b) Preparing an alkaline aqueous solution containing a divalent cation salt; c) Supplementing the polyphosphate salt solution with polyethylene glycol; d) Adding drop-wise the solution of the divalent cation salt solution to the polyethylene glycol supplemented polyphosphate salt solution, and adjusting the pH value to alkaline values; e) Collecting the polyphosphate nanoparticles thereby formed by centrifugation; and 0 Washing the polyphosphate nanoparticle pellet with ethanol-water and air-drying; wherein said method in steps a) to 0 is performed at ambient temperature.
2. The method according to claim 1 wherein the soluble polyphosphate salt is sodium polyphosphate.
3. The method according to claims 1 and 2 wherein the divalent cation salt is magnesium chloride or calcium chloride
4. The method according to claims 1 to 3 wherein the pH of the alkaline aqueous solution is pH 10.
5. The method according to any of claims I to 4, wherein the chain length of the polyphosphate is in the range of about 2 to about 1000 phosphate units, preferably in the range of about 3 to about 100 phosphate units, and most preferred about 40 phosphate units.
6. The method according to claims 1 to 5 wherein the concentration of the polyphosphate salt is in the range of about 1 to 25 g/L, preferably in the range of about 3 to about 10 mg/L, and most preferred 5 g/L.
7. The method according to claims 1 to 6 wherein the concentration of the divalent cation salt is in the range of about Ito 25 g/L, preferably in the range of about 3 to about 10 mg/L, and most preferred 5 g/L.
8. The method according to claims 1 to 7 wherein the polyethylene glycol is polyethylene glycol 200 (average molar mass 200 g/mol)
9. The method according to claims Ito 8 wherein the concentration of polyethylene glycol is 0.5% weight percent.
10. The method according to claims Ito 9 wherein the volume ratio of the divalent cation salt solution to the polyethylene glycol supplemented polyphosphate salt solution is in the range of about 1:5 to 5:1, preferably in the range of about 1:2 to about 2:1, and most preferred 1:1.
11. The method according to claims 1 to 10 wherein the solution of the divalent cation salt solution is added drop-wise to the polyethylene glycol supplemented polyphosphate salt solution over a period of about 0.3 to about 10 h, preferably about 1 to about 3 h, and most preferred 2 h.
12. Narrow-sized polyphosphate nanoparticles prepared according to any of claims 1 to 11 with a size of about 90 nm.
13. A synergistically acting preparation consisting of the narrow-sized polyphosphate nanoparticles according to claim 12 in combination with soluble polyphosphate.
14. The application of the narrow-sized polyphosphate nanoparticles according to claims 1 to 12, either alone or combination with soluble polyphosphate, for the preparation of antiviral nasal spray, mouth rinse, mouth spray, chewing gum, eye drops, or skin cream and skin spray.
15. The application of the method according claims 1 to 11 for the preparation of a narrow-sized polyphosphate nanoparticle-containing virus-protective nasal spray consisting of propylene glycol, 0.9% NaC1 (pH stabilized with calcium carbonate).
16. The application of the method according claims 1 to 11 for the preparation of a narrow-sized polyphosphate nanoparticle-containing virus-protective skin cream consisting of urea (5 g), Unguentum emulsificans aquosum (89 g), lactic acid 90% (1 g), sodium lactate solution 50% (4 g) and preservatives (sorbic acid E200 0,045% final, and potassium sorbate E202 0,062% final).
17. The application of the method according claims 1 to 11 for the preparation of a narrow-sized polyphosphate nanoparticle-containing virus-protective chewing gum consisting of soluble sodium polyphosphate (5% [w/w]), chicle gum from the sapodilla tree (25% [w/w]), xylitol (57.5% [w/w]), and vegetable glycerol (86%) (12.5% [vv-/w]).
18. The application of the method according claims 1 to 11 for the preparation of a narrow-sized polyphosphate nanoparticle-containing virus-protective eye drops containing hyaluronic acid, heparin, ectoin, vitamin A and/or Euphrasia extract.
19. The application of narrow-sized polyphosphate nanoparticles according to claim 12, or the combination of these nanoparticles with soluble polyphosphate according to claim 13, or of soluble polyphosphate alone for prevention and therapy of respiratory virus infections.
20. The application of narrow-sized polyphosphate nanoparticles according to claim 12, or the combination of these nanoparticles with soluble polyphosphate according to claim 13, or of soluble polyphosphate alone, for prevention and therapy of coronavirus SARS CoV-2 infections,