Mucins are the key component of the defensive mucus barrier. They are extended fibers of very high molecular weight with diverse biological functions depending strongly on their specific structural parameters. Here, we present a mucin‐inspired nanostructure, produced via a synthetic methodology to prepare methacrylate‐based dendronized polysulfates (MIP‐1) on a multi gram‐scale with high molecular weight (MW=450 kDa) and thiol end‐functionalized mucin‐inspired polymer (MIP) via RAFT polymerization. Cryo‐electron tomography (Cryo‐ET) analysis of MIP‐1 confirmed a mucin‐mimetic wormlike single‐chain fiber structure (length=144±59 nm) in aqueous solution. This biocompatible fiber showed promising activity against SARS‐CoV‐2 and its mutant strain, with a remarkable low half maximal (IC₅₀) inhibitory concentration (IC₅₀=10.0 nM). Additionally, we investigate the impact of fiber length on SARS‐CoV‐2 inhibition by testing other functional polymers (MIPs) of varying fiber lengths.

High Mobility Group Box 1 (HMGB1) is a central pro-inflammatory mediator released from damaged or stressed cells, where it activates receptors such as the Receptor for Advanced Glycation Endproducts (RAGE). Dendritic polyglycerol sulfate (dPGS), a hyperbranched polyanionic polymer, is known for its anti-inflammatory activity. In this study, we examined how dPGS modulates HMGB1-driven signaling in RAW 264.7 macrophages and human microglia. Recombinant human HMGB1 expressed in Escherichia coli (E. coli) was purified by nickel-nitrilotriacetic acid (Ni-NTA) and heparin chromatography. Proximity ligation assays (PLA) revealed that dPGS significantly disrupted HMGB1/RAGE interactions, particularly under lipopolysaccharide (LPS) stimulation, thereby reducing inflammatory signaling complex formation. This correlated with reduced activation of the nuclear factor kappa B (NF-κB) pathway, demonstrated by decreased nuclear translocation and transcriptional activity. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (RT-qPCR) showed that dPGS suppressed HMGB1- and LPS-induced transcription of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) and Griess assays confirmed reduced TNF-α secretion and nitric oxide production. Electron paramagnetic resonance (EPR) spectroscopy further showed that dPGS altered HMGB1/soluble RAGE (sRAGE) complex dynamics, providing mechanistic insight into its receptor-disruptive action.

HMGB1, a nuclear DNA-binding protein, can be secreted by activated immune cells or passively released from damaged cells. In such cases, HMGB1 functions as an alarmin that activates the immune system. Excessive inflammation may lead to pathogenesis, whereas this response can be dampened by polyanion binding, which impedes further receptor recognition. Moreover, HMGB1 is known to form liquid droplets in the cellular environment─a phase separation directly linked to its proper function. While the A-Box domain is believed to be primarily responsible for heparin binding due to its conserved binding site, the association and phase separation behavior of HMGB1 may be mediated by the B-box domain, owing to its extended hydrophobic regions. In this study, we first demonstrated that the B-box protein forms 30 nm large self-associates while maintaining its structure. Next, using molecularly sensitive EPR spectroscopy, we showed that the presence of these protein associates significantly enhances interactions with heparin. Notably, the local conformational changes induced by heparin are similar in both individual protein chains and their self-associated forms. To explain this effect, AlphaFold modeling was employed, revealing that the formation of protein multimers induces charge redistribution, resulting in an extended positively charged region that enhances electrostatic attraction to negatively charged polyanions such as heparin.

Mucine sind eine Schlüsselkomponente der defensiven Schleimbarriere. Es handelt sich um ausgedehnte Fasern mit sehr hohem Molekulargewicht, deren vielfältige biologische Funktionen stark von ihren spezifischen Strukturparametern abhängen. Hier stellen wir eine Mucin‐inspirierte Nanostruktur basierend auf dendronisierten Polysulfaten auf Methacrylatbasis (MIP‐1) mit hohem Molekulargewicht (MW=450 kDa) vor. Das Thiol‐End‐funktionalisierte Mucin‐inspiriertem Polymer (MIP) wurde durch RAFT‐Polymerisation im Multi‐Gramm‐Maßstab hergestellt. Die Kryo‐Elektronentomographie‐Analyse (Kryo‐ET) von MIP‐1 bestätigte eine Mucin‐artige, wurmartige einkettige Faserstruktur (Länge=144±59 nm) in wässriger Lösung. Diese biokompatible Faser zeigte eine vielversprechende Aktivität mit einer bemerkenswert niedrigen mittleren inhibitorischen Konzentration (IC₅₀, mit IC₅₀=10.0 nM) gegenüber SARS‐CoV‐2 und dessen Mutantenstamm. Darüber hinaus untersuchen wir die Auswirkungen der Faserlänge auf die Hemmung gegenüber SARS‐CoV‐2, indem wir andere funktionelle Polymere (MIPs) mit unterschiedlichen Faserlängen miteinander vergleichen.