Polyphosphate Nanoparticles: Balancing Energy Requirements in Tissue Regeneration Processes.

Nanoparticles of a particular, evolutionarily old inorganic polymer found across the biological kingdoms have attracted increasing interest in recent years not only because of their crucial role in metabolism but also their potential medical applicability: it is inorganic polyphosphate (polyP). This ubiquitous linear polymer is composed of 10-1000 phosphate residues linked by high-energy anhydride bonds. PolyP causes induction of gene activity, provides phosphate for bone mineralization, and serves as an energy supplier through enzymatic cleavage of its acid anhydride bonds and subsequent ATP formation. The biomedical breakthrough of polyP came with the development of a successful fabrication process, in depot form, as Ca- or Mg-polyP nanoparticles, or as the directly effective polymer, as soluble Na-polyP, for regenerative repair and healing processes, especially in tissue areas with insufficient blood supply. Physiologically, the platelets are the main vehicles for polyP nanoparticles in the circulating blood. To be biomedically active, these particles undergo coacervation. This review provides an overview of the properties of polyP and polyP nanoparticles for applications in the regeneration and repair of bone, cartilage, and skin. In addition to studies on animal models, the first successful proof-of-concept studies on humans for the healing of chronic wounds are outlined.

Biomimetic Polyphosphate Materials: Toward Application in Regenerative Medicine.

In recent years, inorganic polyphosphate (polyP) has attracted increasing attention as a biomedical polymer or biomaterial with a great potential for application in regenerative medicine, in particular in the fields of tissue engineering and repair. The interest in polyP is based on two properties of this physiological polymer that make polyP stand out from other polymers: polyP has morphogenetic activity by inducing cell differentiation through specific gene expression, and it functions as an energy store and donor of metabolic energy, especially in the extracellular matrix or in the extracellular space. No other biopolymer applicable in tissue regeneration/repair is known that is endowed with this combination of properties. In addition, polyP can be fabricated both in the form of a biologically active coacervate and as biomimetic amorphous polyP nano/microparticles, which are stable and are activated by transformation into the coacervate phase after contact with protein/body fluids. PolyP can be used in the form of various metal salts and in combination with various hydrogel-forming polymers, whereby (even printable) hybrid materials with defined porosities and mechanical and biological properties can be produced, which can even be loaded with cells for 3D cell printing or with drugs and support the growth and differentiation of (stem) cells as well as cell migration/microvascularization. Potential applications in therapy of bone, cartilage and eye disorders/injuries and wound healing are summarized and possible mechanisms are discussed.

Polyphosphate in Antiviral Protection: A Polyanionic Inorganic Polymer in the Fight Against Coronavirus SARS-CoV-2 Infection.

Polyanions as polymers carrying multiple negative charges have been extensively studied with regard to their potential antiviral activity. Most studies to date focused on organic polyanionic polymers, both natural and synthetic. The inorganic polymer, polyphosphate (polyP), despite the ubiquitous presence of this molecule from bacteria to man, has attracted much less attention. More recently, and accelerated by the search for potential antiviral agents in the fight against the pandemic caused by the coronavirus SARS-CoV-2, it turned out that polyP disrupts the first step of the viral replication cycle, the interaction of the proteins in the virus envelope and in the cell membrane that are involved in the docking process of the virus with the target host cell. Experiments on a molecular level using the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the cellular angiotensin converting enzyme 2 (ACE2) receptor revealed that polyP strongly inhibits the binding reaction through an electrostatic interaction between the negatively charged centers of the polyP molecule and a cationic groove, which is formed by positively charged amino acids on the RBD surface. In addition, it was found that polyP, due to its morphogenetic and energy delivering activities, enhances the antiviral host innate immunity defense of the respiratory epithelium. The underlying mechanisms and envisaged application of polyP in the therapy and prevention of COVID-19 are discussed.

Polyphosphate in Chronic Wound Healing: Restoration of Impaired Metabolic Energy State.

Many pathological conditions are characterized by a deficiency of metabolic energy. A prominent example is nonhealing or difficult-to-heal chronic wounds. Because of their unique ability to serve as a source of metabolic energy, inorganic polyphosphates (polyP) offer the opportunity to develop novel strategies to treat such wounds. The basis is the generation of ATP from the polymer through the joint action of two extracellular or plasma membrane-bound enzymes alkaline phosphatase and adenylate kinase, which enable the transfer of energy-rich phosphate from polyP to AMP with the formation of ADP and finally ATP. Building on these findings, it was possible to develop novel regeneratively active materials for wound therapy, which have already been successfully evaluated in first studies on patients.

An unexpected biomaterial against SARS-CoV-2: Bio-polyphosphate blocks binding of the viral spike to the cell receptor.

No other virus after the outbreak of the influenza pandemic of 1918 affected the world's population as hard as the coronavirus SARS-CoV-2. The identification of effective agents/materials to prevent or treat COVID-19 caused by SARS-CoV-2 is an urgent global need. This review aims to survey novel strategies based on inorganic polyphosphate (polyP), a biologically formed but also synthetically available polyanionic polymeric material, which has the potential of being a potent inhibitor of the SARS-CoV-2 virus-cell-docking machinery. This virus attaches to the host cell surface receptor ACE2 with its receptor binding domain (RBD), which is present at the tips of the viral envelope spike proteins. On the surface of the RBD an unusually conserved cationic groove is exposed, which is composed of basic amino acids (Arg, Lys, and His). This pattern of cationic amino acids, the cationic groove, matches spatially with the anionic polymeric material, with polyP, allowing an electrostatic interaction. In consequence, the interaction between the RBD and ACE2 is potently blocked. PolyP is a physiological inorganic polymer, synthesized by cells and especially enriched in the blood platelets, which releases metabolically useful energy through enzymatic degradation and coupled ADP/ATP formation. In addition, this material upregulates the steady-state-expression of the mucin genes in the epithelial cells. We propose that polyP, with its two antiviral properties (blocking the binding of the virus to the cells and reinforcing the defense barrier against infiltration of the virus) has the potential to be a novel protective/therapeutic anti-COVID-19 agent.

Inorganic Polyphosphates As Storage for and Generator of Metabolic Energy in the Extracellular Matrix.

Inorganic polyphosphates (polyP) consist of linear chains of orthophosphate residues, linked by high-energy phosphoanhydride bonds. They are evolutionarily old biopolymers that are present from bacteria to man. No other molecule concentrates as much (bio)chemically usable energy as polyP. However, the function and metabolism of this long-neglected polymer are scarcely known, especially in higher eukaryotes. In recent years, interest in polyP experienced a renaissance, beginning with the discovery of polyP as phosphate source in bone mineralization. Later, two discoveries placed polyP into the focus of regenerative medicine applications. First, polyP shows morphogenetic activity, i.e., induces cell differentiation via gene induction, and, second, acts as an energy storage and donor in the extracellular space. Studies on acidocalcisomes and mitochondria provided first insights into the enzymatic basis of eukaryotic polyP formation. In addition, a concerted action of alkaline phosphatase and adenylate kinase proved crucial for ADP/ATP generation from polyP. PolyP added extracellularly to mammalian cells resulted in a 3-fold increase of ATP. The importance and mechanism of this phosphotransfer reaction for energy-consuming processes in the extracellular matrix are discussed. This review aims to give a critical overview about the formation and function of this unique polymer that is capable of storing (bio)chemically useful energy.

Polyketides from the marine-derived fungus Aspergillus falconensis: In silico and in vitro cytotoxicity studies.

Fermentation of the marine-derived fungus Aspergillus falconensis, isolated from sediment collected from the Red Sea, Egypt on solid rice medium containing 3.5% NaCl yielded a new dibenzoxepin derivative (1) and a new natural isocoumarin (2) along with six known compounds (3-8). Changes in the metabolic profile of the fungus were induced by replacing NaCl with 3.5% (NH4)2SO4 that resulted in the accumulation of three further known compounds (9-11), which were not detected when the fungus was cultivated in the presence of NaCl. The structures of the new compounds were elucidated by HRESIMS and 1D/2D NMR as well as by comparison with the literature. Molecular docking was conducted for all isolated compounds on crucial enzymes involved in the formation, progression and metastasis of cancer which included human cyclin-dependent kinase 2 (CDK-2), human DNA topoisomerase II (TOP-2) and matrix metalloproteinase 13 (MMP-13). Diorcinol (7), sulochrin (9) and monochlorosulochrin (10) displayed notable stability within the active pocket of CDK-2 with free binding energy (ΔG) equals to -25.72, -25.03 and -25.37 Kcal/mol, respectively whereas sulochrin (9) exerted the highest fitting score within MMP-13 active center (ΔG = -33.83 Kcal/mol). In vitro cytotoxic assessment using MTT assay showed that sulochrin (9) exhibited cytotoxic activity versus L5178Y mouse lymphoma cells with an IC50 value of 5.1 µM and inhibition of migration of MDA-MB 231 breast cancer cells at a concentration of 70 µM.

Caged Dexamethasone/Quercetin Nanoparticles, Formed of the Morphogenetic Active Inorganic Polyphosphate, are Strong Inducers of MUC5AC.

Inorganic polyphosphate (polyP) is a widely distributed polymer found from bacteria to animals, including marine species. This polymer exhibits morphogenetic as well as antiviral activity and releases metabolic energy after enzymatic hydrolysis also in human cells. In the pathogenesis of the coronavirus disease 2019 (COVID-19), the platelets are at the frontline of this syndrome. Platelets release a set of molecules, among them polyP. In addition, the production of airway mucus, the first line of body defense, is impaired in those patients. Therefore, in this study, amorphous nanoparticles of the magnesium salt of polyP (Mg-polyP-NP), matching the size of the coronavirus SARS-CoV-2, were prepared and loaded with the secondary plant metabolite quercetin or with dexamethasone to study their effects on the respiratory epithelium using human alveolar basal epithelial A549 cells as a model. The results revealed that both compounds embedded into the polyP nanoparticles significantly increased the steady-state-expression of the MUC5AC gene. This mucin species is the major mucus glycoprotein present in the secreted gel-forming mucus. The level of gene expression caused by quercetin or with dexamethasone, if caged into polyP NP, is significantly higher compared to the individual drugs alone. Both quercetin and dexamethasone did not impair the growth-supporting effect of polyP on A549 cells even at concentrations of quercetin which are cytotoxic for the cells. A possible mechanism of the effects of the two drugs together with polyP on mucin expression is proposed based on the scavenging of free oxygen species and the generation of ADP/ATP from the polyP, which is needed for the organization of the protective mucin-based mucus layer.

Aged Mice Devoid of the M3 Muscarinic Acetylcholine Receptor Develop Mild Dry Eye Disease.

The parasympathetic nervous system is critically involved in the regulation of tear secretion by activating muscarinic acetylcholine receptors. Hence, various animal models targeting parasympathetic signaling have been developed to induce dry eye disease (DED). However, the muscarinic receptor subtype (M1-M5) mediating tear secretion remains to be determined. This study was conducted to test the hypothesis that the M3 receptor subtype regulates tear secretion and to evaluate the ocular surface phenotype of mice with targeted disruption of the M3 receptor (M3R-/-). The experimental techniques included quantification of tear production, fluorescein staining of the ocular surface, environmental scanning electron microscopy, assessment of proliferating cells in the corneal epithelium and of goblet cells in the conjunctiva, quantification of mRNA for inflammatory cytokines and prooxidant redox enzymes and quantification of reactive oxygen species. Tear volume was reduced in M3R-/- mice compared to age-matched controls at the age of 3 months and 15 months, respectively. This was associated with mild corneal epitheliopathy in the 15-month-old but not in the 3-month-old M3R-/- mice. M3R-/- mice at the age of 15 months also displayed changes in corneal epithelial cell texture, reduced conjunctival goblet cell density, oxidative stress and elevated mRNA expression levels for inflammatory cytokines and prooxidant redox enzymes. The findings suggest that the M3 receptor plays a pivotal role in tear production and its absence leads to ocular surface changes typical for DED at advanced age.

A synthetic biology approach for the fabrication of functional (fluorescent magnetic) bioorganic-inorganic hybrid materials in sponge primmorphs.

During evolution, sponges (Porifera) have honed the genetic toolbox and biosynthetic mechanisms for the fabrication of siliceous skeletal components (spicules). Spicules carry a protein scaffold embedded within biogenic silica (biosilica) and feature an amazing range of optical, structural, and mechanical properties. Thus, it is tempting to explore the low-energy synthetic pathways of spiculogenesis for the fabrication of innovative hybrid materials. In this synthetic biology approach, the uptake of multifunctional nonbiogenic nanoparticles (fluorescent, superparamagnetic) by spicule-forming cells of bioreactor-cultivated sponge primmorphs provides access to spiculogenesis. The ingested nanoparticles were detected within intracellular vesicles resembling silicasomes (silica-rich cellular compartments) and as cytosolic clusters where they lent primmorphs fluorescent/magnetic properties. During spiculogenesis, the nanoparticles initially formed an incomplete layer around juvenile, intracellular spicules. In the mature, extracellular spicules the nanoparticles were densely arranged as a surface layer that rendered the resulting composite fluorescent and magnetic. By branching off the conventional route of solid-state materials synthesis under harsh conditions, a new pathway has been opened to a versatile platform that allows adding functionalities to growing spicules as templates in living cells, using nonbiogenic nanoscale building blocks with multiple functionalities. The magnet-assisted alignment renders this composite with its fluorescent/magnetic properties potentially suitable for application in biooptoelectronics and microelectronics (e.g., microscale on-chip waveguides for applications of optical detection and sensing).

Morphogenetic (Mucin Expression) as Well as Potential Anti-Corona Viral Activity of the Marine Secondary Metabolite Polyphosphate on A549 Cells.

The mucus layer of the nasopharynx and bronchial epithelium has a barrier function against inhaled pathogens such as the coronavirus SARS-CoV-2. We recently found that inorganic polyphosphate (polyP), a physiological, metabolic energy (ATP)-providing polymer released from blood platelets, blocks the binding of the receptor binding domain (RBD) to the cellular ACE2 receptor in vitro. PolyP is a marine natural product and is abundantly present in marine bacteria. Now, we have approached the in vivo situation by studying the effect of polyP on the human alveolar basal epithelial A549 cells in a mucus-like mucin environment. These cells express mucins as well as the ectoenzymes alkaline phosphatase (ALP) and adenylate kinase (ADK), which are involved in the extracellular production of ATP from polyP. Mucin, integrated into a collagen-based hydrogel, stimulated cell growth and attachment. The addition of polyP to the hydrogel significantly increased cell attachment and also the expression of the membrane-tethered mucin MUC1 and the secreted mucin MUC5AC. The increased synthesis of MUC1 was also confirmed by immunostaining. This morphogenetic effect of polyP was associated with a rise in extracellular ATP level. We conclude that the nontoxic and non-immunogenic polymer polyP could possibly also exert a protective effect against SARS-CoV-2-cell attachment; first, by stimulating the innate antiviral response by strengthening the mucin barrier with its antimicrobial proteins, and second, by inhibiting virus attachment to the cells, as deduced from the reduction in the strength of binding between the viral RBD and the cellular ACE2 receptor.

Polyketide Derivatives from Mangrove Derived Endophytic Fungus Pseudopestalotiopsis theae.

Chemical investigation of secondary metabolites from the endophytic fungus Pseudopestalotiopsis theae led to the isolation of eighteen new polyketide derivatives, pestalotheols I-Q (1-9) and cytosporins O-W (15-23), together with eight known analogs (10-14 and 24-26). The structures of the new compounds were elucidated by HRMS and 1D and 2D NMR data, as well as by comparison with literature data. Modified Mosher's method was applied to determine the absolute configuration of some compounds. Compound 23 showed significant cytotoxicity against the mouse lymphoma cell line L5178Y with an IC50 value of 3.0 µM. Furthermore, compounds 22 and 23 showed moderate antibacterial activity against drug-resistant Acinetobacter baumannii (ATCC BAA-1605) in combination with sublethal colistin concentrations.

Biomimetic Alginate/Gelatin Cross-Linked Hydrogels Supplemented with Polyphosphate for Wound Healing Applications.

In the present study, the fabrication of a biomimetic wound dressing that mimics the extracellular matrix, consisting of a hydrogel matrix composed of non-oxidized and periodate-oxidized marine alginate, was prepared to which gelatin was bound via Schiff base formation. Into this alginate/oxidized-alginate-gelatin hydrogel, polyP was stably but reversibly integrated by ionic cross-linking with Zn2+ ions. Thereby, a soft hybrid material is obtained, consisting of a more rigid alginate scaffold and porous structures formed by the oxidized-alginate-gelatin hydrogel with ionically cross-linked polyP. Two forms of the Zn-polyP-containing matrices were obtained based on the property of polyP to form, at neutral pH, a coacervate-the physiologically active form of the polymer. At alkaline conditions (pH 10), it will form nanoparticles, acting as a depot that is converted at pH 7 into the coacervate phase. Both polyP-containing hydrogels were biologically active and significantly enhanced cell growth/viability and attachment/spreading of human epidermal keratinocytes compared to control hydrogels without any adverse effect on reconstructed human epidermis samples in an in vitro skin irritation test system. From these data, we conclude that polyP-containing alginate/oxidized-alginate-gelatin hydrogels may provide a suitable regeneratively active matrix for wound healing for potential in vivo applications.

Self-Healing Properties of Bioinspired Amorphous CaCO3/Polyphosphate-Supplemented Cement.

There is a strong interest in cement additives that are able to prevent or mitigate the adverse effects of cracks in concrete that cause corrosion of the reinforcement. Inorganic polyphosphate (polyP), a natural polymer that is synthesized by bacteria, even those on cement/concrete, can increase the resistance of concrete to progressive damage from micro-cracking. Here we use a novel bioinspired strategy based on polyP-stabilized amorphous calcium carbonate (ACC) to give this material self-healing properties. Portland cement was supplemented with ACC nanoparticles which were stabilized with 10% (w/w) Na-polyP. Embedding these particles in the hydrated cement resulted in the formation of calcite crystals after a hardening time of 10 days, which were not seen in controls, indicating that the particles dissolve and then transform into calcite. While there was no significant repair in the controls without ACC, almost complete closure of the cracks was observed after a 10 days healing period in the ACC-supplemented samples. Nanoindentation measurements on the self-healed crack surfaces showed a similar or slightly higher elasticity at a lower hardness compared to non-cracked surfaces. Our results demonstrate that bioinspired approaches, like the use of polyP-stabilized ACC shown here, can significantly improve the repair capacity of Portland cement.

Polyphosphate as a donor of high-energy phosphate for the synthesis of ADP and ATP.

Here, we studied the potential role of inorganic polyphosphate (polyP) as an energy source for ADP and ATP formation in the extracellular space. In SaOS-2 cells, we show that matrix vesicles are released into the extracellular space after incubation with polyP. These vesicles contain both alkaline phosphatase (ALP) and adenylate kinase (AK) activities (mediated by ALPL and AK1 enzymes). Both enzymes translocate to the cell membrane in response to polyP. To distinguish the process(es) of AMP and ADP formation during ALP hydrolysis from the ATP generated via the AK reaction, inhibition studies with the AK inhibitor A(5')P5(5')A were performed. We found that ADP formation in the extracellular space occurs after enzymatic ATP synthesis. After exposure to polyP, a significant increase of the ADP level was observed, which is likely to be been catalyzed by ALP. This increase is not due to an intensified ATP release via exocytosis. The ATP level in the extracellular space of SaOS-2 cells is strongly increased in response to polyP, very likely mediated by the AK. We propose that the ALP and AK enzymes are involved in the extracellular ADP and ATP synthesis.

Cyclic heptapeptides from the soil-derived fungus Clonostachys rosea.

Three new cyclic heptapeptides (1-3) together with three known compounds (4-6) were isolated from a solid rice culture of the soil-derived fungus Clonostachys rosea. Fermentation of the fungus on white beans instead of rice afforded a new γ-lactam (7) and a known γ-lactone (8) that were not detected in the former extracts. The structures of the new compounds were elucidated on the basis of 1D and 2D NMR spectra as well as by HRESIMS data. Compounds 1 and 4 exhibited significant cytotoxicity against the L5178Y mouse lymphoma cell line with IC50 values of 4.1 and 0.1 µM, respectively. Compound 4 also displayed cytotoxicity against the A2780 human ovarian cancer cell line with an IC50 value of 3.5 µM. The preliminary structure-activity relationships are discussed.

Sesquiterpenoids from the Endophytic Fungus Rhinocladiella similis.

Ten new sesquiterpenoid derivatives, rhinomilisins A-J (1-10), along with six known analogues (11-16), were isolated from the mangrove-derived endophytic fungus Rhinocladiella similis. The structures of the new compounds were elucidated by their NMR and MS data, while the absolute configuration of 3 and 6 was determined by X-ray crystallographic analysis and Mosher's method, respectively. All isolated compounds (1-16) were evaluated for their cytotoxicity against the mouse lymphoma cell line L5178Y, and compounds 1, 7, and 15 showed moderate activity with IC50 values of 5.0, 8.7, and 24.4 µM, respectively.

Indole Diterpenoids from an Endophytic Penicillium sp.

A chemical investigation of the endophyte Penicillium sp. (strain ZO-R1-1), isolated from roots of the medicinal plant Zingiber officinale, yielded nine new indole diterpenoids (1-9), together with 13 known congeners (10-22). The structures of the new compounds were elucidated by 1D and 2D NMR analysis in combination with HRESIMS data. The absolute configuration of the new natural products 1, 3, and 7 was determined using the TDDFT-ECD approach and confirmed for 1 by single-crystal X-ray determination through anomalous dispersion. The isolated compounds were tested for cytotoxicity against L5178Y, A2780, J82, and HEK-293 cell lines. Compound 1 was the most active metabolite toward L5178Y cells, with an IC50 value of 3.6 µM, and an IC50 against A2780 cells of 8.7 µM. Interestingly, 1 features a new type of indole diterpenoid scaffold with a rare 6/5/6/6/6/6/5 heterocyclic system bearing an aromatic ring C, which is suggested to be important for the cytotoxic activity of this natural product against L5278Y and A2780 cells.

Brominated Azaphilones from the Sponge-Associated Fungus Penicillium canescens Strain 4.14.6a.

The fungus Penicillium canescens was isolated from the inner tissue of the Mediterranian sponge Agelas oroides. Fermentation of the fungus on solid rice medium yielded one new chlorinated diphenyl ether (1) and 13 known compounds (2-14). Addition of 5% NaBr to the rice medium increased the amounts of 4-6, while lowering the amounts of 8, 12, and 14. Furthermore, it induced the accumulation of 17 and two new brominated azaphilones, bromophilones A and B (15 and 16). Compounds 15 and 16 are the first example of azaphilones with the connection of a benzene moiety and the pyranoquinone core through a methylene group. The structures of the new compounds were elucidated based on the 1D and 2D NMR spectra as well as on HRESIMS data. The absolute configuration of the condensed bicyclic moiety of 15 and 16 was determined by sTDA ECD calculations. Compound 16 exhibited moderate cytotoxicity against the mouse lymphoma cell line L5178Y (IC50 8.9 µM), as well as against the human ovarian cancer cell line A2780 (IC50 2.7 µM), whereas the stereoisomer 15 was considerably less active.

Inorganic polyphosphate induces accelerated tube formation of HUVEC endothelial cells.

In this study, the effect of inorganic polyphosphate (polyP) on the initial phase of angiogenesis and vascularization was investigated, applying the HUVEC cell tube formation assay. PolyP is a physiological and high energy phosphate polymer which has been proposed to act as a metabolic fuel in the extracellular space with only a comparably low ATP content. The experiments revealed that polyP accelerates tube formation of human umbilical vein endothelial cells (HUVEC), seeded onto a solidified basement membrane extract matrix which contains polyP-metabolizing alkaline phosphatase (ALP) activity. This effect is abolished by co-addition of apyrase, which degrades ATP to AMP and inorganic phosphate. The assumption that ATP, derived from polyP, activates HUVEC cells leading to tube formation was corroborated by experiments showing that addition of polyP to the cells causes a strong rise of ATP level in the culture medium. Finally, we show that at a later stage of cultivation of HUVEC cells, after 3 d, polyP causes a strong enhancement of the expression of the genes encoding for the two major matrix metalloproteinases (MMPs) released by endothelial cells during tube formation, MMP-9 and MMP-2. This stimulatory effect is again abrogated by addition of apyrase together with polyP. From these results, we propose that polyP is involved either directly or indirectly in energy supply, via ALP-mediated transfer of energy-rich phosphate under ATP formation. This ATP is utilized for the activation and oriented migration of endothelial cells and for the matrix organization during the initial phases of tube formation.