Triple-target stimuli-responsive anti-COVID-19 face mask with physiological virus-inactivating agents

Conventional face masks to prevent SARS-CoV-2 transmission are mostly based on a passive filtration principle. Ideally, anti-COVID-19 masks should protect the carrier not only by size exclusion of virus aerosol particles, but also be able to capture and destroy or inactivate the virus. Here we present the proof-of-concept of a filter mat for such a mask, which actively attracts aerosol droplets and kills the virus. The electrospun mats are made of polycaprolactone (PCL) a hydrophilic, functionalizable and biodegradable polyester, into which inorganic polyphosphate (polyP) a physiological biocompatible, biodegradable and antivirally active polymer (chain length, ∼40 P(i) units) has been integrated. A soluble Na-polyP as well as amorphous calcium polyP nanoparticles (Ca-polyP-NP) have been used. In this composition, the polyP component of the polyP-PCL mats is stable in aqueous protein-free environment, but capable of transforming into a gel-like coacervate upon contact with divalent cations and protein like mucin present in (virus containing) aerosol droplets. In addition, the Ca-polyP-NP are used as a carrier of tretinoin (all-trans retinoic acid) which blocks the function of the SARS-CoV-2 envelope (E) protein, an ion channel forming viroporin. The properties of this novel mask filter mats are as follows: First, to attract and to trap virus-like particles during the polyP coacervate formation induced in situ by aerosol droplets on the spun PCL fibers, as shown here by using SARS-CoV-2 mimicking fluorescent nanoparticles. Second, after disintegration the NP by the aerosol-mucus constituents, to release polyP that binds to and abolishes the function of the receptor binding domain of the viral spike protein. Third, to destroy the virus by releasing tretinoin, as shown by the disruption of virus-mimicking liposomes with the integrated recombinant viral viroporin. It is proposed that these properties, which are inducible (stimuli responsive), will allow the design of antiviral masks that are smart

Molekularbiologische Charakterisierung der beiden Untereinheiten des Mega-Hämocyanins der Schnecke Melanoides tuberculata

Bei dem 2010 von unserer Arbeitsgruppe entdeckten Mega-Hämocyanin handelt es sich um einen stark abgewandelten Typ des respiratorischen Proteins Hämocyanin, bestehend aus zwei flankierenden regulären Dekameren und einem zentralen Mega-Dekamer. Diese sind aus zwei immunologisch verschiedenen Untereinheiten mit ~400 bzw. ~550 kDa aufgebaut, die in unserer Arbeitsgruppe bereits proteinbiochemisch charakterisiert wurden. Im Zuge dieser Untersuchungen konnte zudem eine 3D-Rekonstruktion des Oligomers (13,5 MDa) mit einer Auflösung von 13Å erstellt werden. Das Ziel der vorliegenden Arbeit war die Aufklärung der Primärstruktur beider Polypeptide bei der Schnecke Melanoides tuberculata (MtH). Es gelang, die cDNAs der beiden Untereinheiten vollständig zu sequenzieren. Die zu typischen Dekameren assemblierende MtH400-Untereinheit umfasst 3445 Aminosäuren und besitzt eine theoretische Molekularmasse von 390 kDa. Nach dem Signalpeptid von 23 Aminosäuren Länge folgen die für Gastropoden-Hämocyanine typischen funktionellen Einheiten FU-a bis FU-h. Insgesamt verfügt die MtH400-Untereinheit über sechs potentielle N-Glykosylierungsstellen. Die MtH550-Untereinheit, welche mit 10 Kopien das Mega-Dekamer bildet, umfasst 4999 Aminosäuren und besitzt eine theoretische Molekularmasse von 567 kDa. Damit handelt es sich bei dieser Untereinheit um die zweitgrößte jemals bei einem Protein detektierte Polypeptidkette. Die MtH550-Untereinheit besteht aus einem Signalpeptid von 20 Aminosäuren Länge und den typischen Wand-FUs (FU-a bis FU-f). Daran anschließend folgen sechs weitere Varianten der FU-f (FU-f1 bis FU-f6). Die MtH550-Untereinheit verfügt über insgesamt zwölf potentielle N-Glykosylierungsstellen. Anhand der ermittelten Primärstrukturdaten wird klar, dass der auffällig vergrößerte Kragenbereich des Mega-Dekamers aus je 10 Kopien der FU-f1 bis FU-f6 besteht. Die ermittelten Sequenzdaten der beiden MtH-Untereinheiten weisen im Vergleich zu anderen Hämocyanin Sequenzen einige sehr charakteristische Indels sowie unübliche N-Glykosylierungsstellen auf. Es war zudem möglich, anhand einer molekularen Uhr den Entstehungszeitpunkt des Mega-Hämocyanins zu datieren (145 ± 35 MYA). Sowohl die Topologie als auch die berechneten Trennungszeitpunkte des an allen Verzweigungen gut unterstützten Stammbaums stimmen mit den bisher publizierten und auf Hämocyanindaten basierenden molekularen Uhren überein.Previously our group discovered, in cerithioid snails, an unusually complex type of the respiratory protein hemocyanin. This tridecameric mega-hemocyanin is built from two different types of decamer: two flanking decamers of the typical gastropod hemocyanin type, and a central mega-decamer. Each decamer type is built from a specific subunit: ~400 kDa in case of the regular decamer and ~550 kDa in case of the mega-decamer. The two subunit types have been analyzed biochemically, and furthermore, a cryoEM-based 3D-reconstruction of the oligomer (13.5 MDa) with 13Å resolution was provided.rnThe goal of this work was the elucidation of the primary structure from both subunits of the mega-hemocyanin of Melanoides tuberculata. The cDNAs coding for the two subunits could be completely sequenced. The MtH400-subunit comprises 3445 amino acids and has a molecular mass of 390 kDa as predicted from the sequence. It encompasses a signal peptide (23 amino acids) and the eight typical functional units FU-a to FU-h hitherto observed in molluscan hemocyanins. Moreover, it exhibits six potential attachment sites for N-glycans. In contrast, the MtH550-subunit comprises 4999 amino acids and has a predicted molecular mass of 567 kDa. This is the second largest polypeptide ever reported. It encompasses a signal peptide (20 amino acids) and the adjacent canonical functional units FU-a to FU-f which are assumed to form the cylinder wall of the decamer. Instead of the collar-forming functional units FU-g and FU-h as in the MtH400-subunit, the MtH550-subunit possesses C-terminally six variants of FU-f, termed FU-f1 to FU-f6. It exhibits a total of twelve potential N-glycosylation sites, partially at unusual positions. The different functional units of both subunit types show all structural features required for reversible oxygen binding.rnThe major branches of phylogenetic trees calculated from the now available sequence data are well bootstrap-supported and fit the branching pattern of earlier trees of molluscan hemocyanin. Based on a calculated molecular clock, the phylogenetic origin of mega-hemocyanin could be dated back 145 ± 35 million years.r

Morphogenetically active scaffold for osteochondral repair (polyphosphate/alginate/N,O-carboxymethyl chitosan)

Here we describe a novel bioinspired hydrogel material that can be hardened with calcium ions to yield a scaffold material with viscoelastic properties matching those of cartilage. This material consists of a negatively charged biopolymer triplet, composed of morphogenetically active natural inorganic polyphosphate (polyP), along with the likewise biocompatible natural polymers N,O-carboxymethyl chitosan (N,O-CMC) and alginate. The porosity of the hardened scaffold material obtained after calcium exposure can be adjusted by varying the pre-processing conditions. Various compression tests were applied to determine the local (nanoindentation) and bulk mechanical properties (tensile/compression test system for force measurements) of the N,O-CMC-polyP-alginate material. Determinations of the Young’s modulus revealed that the stiffness of this comparably water rich (and mouldable) material increases during successive compression cycles to values measured for native cartilage. The material not only comprises viscoelastic properties suitable for a cartilage substitute material, but also displays morphogenetic activity. It upregulates the expression of genes encoding for collagen type II and aggrecan, the major proteoglycan within the articular cartilage, in human chondrocytes, and the expression of alkaline phosphatase in human bone-like SaOS-2 cells, as revealed in RT qPCR experiments. Further, we demonstrate that the new polyP-based material can be applied for manufacturing 3D solid models of cartilage bone such as of the tibial epiphyseal plate and the superior articular cartilage surface. Since the material is resorbable and enhances the activity of cells involved in regeneration of cartilage tissue, this material has the potential to be used for artificial articular cartilage implants

Inorganic Polyphosphate: Coacervate Formation and Functional Significance in Nanomedical Applications

Heinz C Schröder,1 Meik Neufurth,1 Huan Zhou,2 Shunfeng Wang,1 Xiaohong Wang,1 Werner E G Müller1 1ERC Advanced Investigator Group, Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany; 2School of Health Sciences and Biomedical Engineering, Heibei University of Technology, Tianjin, People’s Republic of ChinaCorrespondence: Heinz C Schröder; Werner E G Müller, ERC Advanced Investigator Group, Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Duesbergweg 6, Mainz, 55128, Germany, Tel +49 6131 392 5791 ; +49 6131 392 5910, Email [email protected]; [email protected]: Inorganic polyphosphates (polyP) are long-chain polymers of orthophosphate residues, which, depending on the external conditions, can be present both physiologically and synthetically in either soluble, nanoparticulate or coacervate form. In recent years, these polymers have received increasing attention due to their unprecedented ability to exhibit both morphogenetic and metabolic energy delivering properties. There are no other physiological molecules that contain as many metabolically utilizable, high-energy bonds as polyP, making these polymers of particular medical interest as components of advanced hydrogel scaffold materials for potential applications in ATP-dependent tissue regeneration and repair. However, these polymers show physiological activity only in soluble form and in the coacervate phase, but not as stable metal-polyP nanoparticles. Therefore, understanding the mechanisms of formation of polyP coacervates and nanoparticles as well as their transformations is important for the design of novel materials for tissue implants, wound healing, and drug delivery and is discussed here.Keywords: polyphosphate nanoparticles, phase separation, biomaterial, metabolic energy, morphogenetic activity, tissue regeneratio

Morphogenetically-Active Barrier Membrane for Guided Bone Regeneration, Based on Amorphous Polyphosphate

We describe a novel regeneratively-active barrier membrane which consists of a durable electrospun poly(ε-caprolactone) (PCL) net covered with a morphogenetically-active biohybrid material composed of collagen and inorganic polyphosphate (polyP). The patch-like fibrous collagen structures are decorated with small amorphous polyP nanoparticles (50 nm) formed by precipitation of this energy-rich and enzyme-degradable (alkaline phosphatase) polymer in the presence of calcium ions. The fabricated PCL-polyP/collagen hybrid mats are characterized by advantageous biomechanical properties, such as enhanced flexibility and stretchability with almost unaltered tensile strength of the PCL net. The polyP/collagen material promotes the attachment and increases the viability/metabolic activity of human mesenchymal stem cells compared to cells grown on non-coated mats. The gene expression studies revealed that cells, growing onto polyP/collagen coated mats show a significantly (two-fold) higher upregulation of the steady-state-expression of the angiopoietin-2 gene used as an early marker for wound healing than cells cultivated onto non-coated mats. Based on our results we propose that amorphous polyP, stabilized onto a collagen matrix, might be a promising component of functionally-active barrier membranes for guided tissue regeneration in medicine and dentistry