Shell-sheddable dendritic polyglycerol sulfates loaded with sunitinib for inhibition of tumor angiogenesis.

Induced angiogenesis, a specific hallmark of cancer, plays a vital role in tumor progression and can be targeted by inhibitors like sunitinib. Sunitinib is a small hydrophobic molecule suffering from low bioavailability and a short half-life in the bloodstream. To overcome these drawbacks, suitable drug delivery systems need to be developed. In this work dendritic polyglycerol (dPG), a well-known polymer, was functionalized with a sheddable shell. Therefore, aliphatic chains of different lengths (C5, C9, C11) were coupled to dPG through a cleavable ester bond. To restore water solubility and improve tumor targeting, the surface was decorated with sulfate groups. The resulting shell-sheddable dPG sulfates were characterized and evaluated regarding their loading capacity and biocompatibility in cell culture. The nine-carbon chain derivative (dPG-TNS) was selected as the best candidate for further experiments due to its high drug loading capacity (20 wt%), and a sustained release in vitro. The cellular biocompatibility of the blank carrier up to 1 mg/mL was confirmed after 24 h incubation on HeLa cells. Furthermore, the shell-cleavability of dPG-TNS under different physiological conditions was shown in a degradation study over four weeks. The activity of sunitinib-loaded dPG-TNS was demonstrated in a tube formation assay on Human umbilical vein endothelial cells (HUVECs). Our results suggest that the drug-loaded nanocarrier is a promising candidate to be further investigated in tumor treatments, as it shows similar efficacy to free sunitinib while overcoming its limitations.

Mucin-Inspired Single-Chain Polymer (MIP) Fibers as Potent SARS-CoV-2 Inhibitors.

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.

Sulfated Hyperbranched and Linear Polyglycerols Modulate HMGB1 and Morphological Plasticity in Neural Cells.

The objective of this study was to establish if polyglycerols with sulfate or sialic acid functional groups interact with high mobility group box 1 (HMGB1), and if so, which polyglycerol could prevent loss of morphological plasticity in excitatory neurons in the hippocampus. Considering that HMGB1 binds to heparan sulfate and that heparan sulfate has structural similarities with dendritic polyglycerol sulfates (dPGS), we performed the experiments to show if polyglycerols can mimic heparin functions by addressing the following questions: (1) do dendritic and linear polyglycerols interact with the alarmin molecule HMGB1? (2) Does dPGS interaction with HMGB1 influence the redox status of HMGB1? (3) Can dPGS prevent the loss of dendritic spines in organotypic cultures challenged with lipopolysaccharide (LPS)? LPS plays a critical role in infections with Gram-negative bacteria and is commonly used to test candidate therapeutic agents for inflammation and endotoxemia. Pathologically high LPS concentrations and other stressful stimuli cause HMGB1 release and post-translational modifications. We hypothesized that (i) electrostatic interactions of hyperbranched and linear polysulfated polyglycerols with HMGB1 will likely involve sites similar to those of heparan sulfate. (ii) dPGS can normalize HMGB1 compartmentalization in microglia exposed to LPS and prevent dendritic spine loss in the excitatory hippocampal neurons. We performed immunocytochemistry and biochemical analyses combined with confocal microscopy to determine cellular and extracellular locations of HMGB1 and morphological plasticity. Our results suggest that dPGS interacts with HMGB1 similarly to heparan sulfate. Hyperbranched dPGS and linear sulfated polymers prevent dendritic spine loss in hippocampal excitatory neurons. MS/MS analyses reveal that dPGS-HMGB1 interactions result in fully oxidized HMGB1 at critical cysteine residues (Cys23, Cys45, and Cys106). Triply oxidized HMGB1 leads to the loss of its pro-inflammatory action and could participate in dPGS-mediated spine loss prevention. LPG-Sia exposure to HMGB1 results in the oxidation of Cys23 and Cys106 but does not normalize spine density.

Zona Pellucida like Domain Protein 1 (ZPLD1) Polymerization Is Regulated by Two Distinguished Hydrophobic Motifs.

Zona Pellucida Like Domain 1 Protein (ZPLD1) is a main component of the cupula, a gelatinous structure located in the labyrinth organ of the inner ear and involved in vestibular function. The N-glycosylated protein is likely able to organize high-molecular-weight polymers via its zona pellucida (ZP) module, which is common for many extracellular proteins that self-assemble into matrices. In this work, we confirmed that ZPLD1 can form multimers while setting up a cellular model leveraging Madin-Darby canine kidney (MDCK) cells to study protein polymerization. We identified two motifs within ZPLD1 which regulate its polymerization and follow previously published conserved regions, identified across ZP proteins. Mutational depletion of either one of these modules led to diminished or abnormal polymer formation outside of the cells, likely due to altered processing at the plasma membrane. Further, intracellular polymer formation was observed. Proteolytic cleavage during secretion, separating the regulatory motif located distinct of the ZP module from the mature monomer, seems to be necessary to enable polymerization. While the molecular interactions of the identified motifs remain to be proven, our findings suggest that ZPLD1 is a polymer forming ZP protein following an orchestrated mechanism of protein polymerization to finally build up a gelatinous hydrogel.

Establishment and Characterization of hTERT Immortalized Hutchinson-Gilford Progeria Fibroblast Cell Lines.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging syndrome caused by a dominant mutation in the LMNA gene. Previous research has shown that the ectopic expression of the catalytic subunit of telomerase (hTERT) can elongate the telomeres of the patients' fibroblasts. Here, we established five immortalized HGP fibroblast cell lines using retroviral infection with the catalytic subunit of hTERT. Immortalization enhanced the proliferative life span by at least 50 population doublings (PDs). The number of cells with typical senescence signs was reduced by 63 + 17%. Furthermore, the growth increase and phenotype improvement occurred with a lag phase of 50-100 days and was not dependent on the degree of telomere elongation. The initial telomeric stabilization after hTERT infection and relatively low amounts of hTERT mRNA were sufficient for the phenotype improvement but the retroviral infection procedure was associated with transient cell stress. Our data have implications for therapeutic strategies in HGP and other premature aging syndromes.

Imaging Keratan Sulfate in Ocular Tissue Sections by Immunofluorescence Microscopy and LA-ICP-MS.

Carbohydrate-specific antibodies can serve as valuable tools to monitor alterations in the extracellular matrix resulting from pathologies. Here, the keratan sulfate-specific monoclonal antibody MZ15 was characterized in more detail by immunofluorescence microscopy as well as laser ablation ICP-MS using tissue cryosections and paraffin-embedded samples. Pretreatment with keratanase II prevented staining of samples and therefore demonstrated efficient enzymatic keratan sulfate degradation. Random fluorescent labeling and site-directed introduction of a metal cage into MZ15 were successful and allowed for a highly sensitive detection of the keratan sulfate landscape in the corneal stroma from rats and human tissue.

Synthesis and functionalization of dendritic polyglycerol-based nanogels: application in T cell activation.

The concept of multivalency finds various applications in the fields of chemistry and biology, relying on the principle that multiple weak interactions can lead to strong adhesive forces. Polymeric carriers are promising tools to translate these properties into the field of biomedicine, especially upon functionalization by active biomolecules, such as antibodies. In this study we report on the synthesis of dendritic polyglycerol (dPG) and dPG-based nanogels (NGs) as platforms for the multivalent display of molecules and their potential application as carrier units. Macromolecules based on dPG were synthesized and NGs were generated by strain-promoted azide-alkyne cycloaddition (SPAAC) by inverse nanoprecipitation under mild conditions. Scale-up screening rendered a reproducible method for a batch size of up to 50 mg for the formation of NGs in a size range of 150 nm with narrow dispersity. Dye-labelled bovine serum albumin (FITC-BSA) was chosen as a model protein and showed successful conjugation to the carriers, while the protein's secondary structure was not affected. Consequently, cyanine-5-amine (Cy5-NH2) and avidin (Av) were conjugated in order to exploit the strong avidin-biotin interaction, facilitating the directed attachment of a myriad of biotinylated (bio)molecules. As a proof-of-concept, the biotinylated monoclonal antibodies (mAbs) α-CD3 and α-CD28 were attached to the platforms and their capability to activate T cells was assessed. Experiments were performed with a Jurkat reporter cell line which expresses green fluorescent protein (GFP) upon activation, providing a rapid and reliable readout by flow cytometry. Carriers clearly outperformed conventional compounds for activation (i.e. antibodies crosslinked with anti-IgG antibody) at significantly lower dosages. These findings could be confirmed by confocal laser scanning microscopy (CLSM), showing accumulation of the functional nanoplatforms at the cell surface and cytoplasmic GFP expression (>95% activation of cells for the multivalent conjugates at 10 µg mL-1 compared to 37% activation with conventionally crosslinked mAbs at 25 µg mL-1), whereas carriers without mAbs could not activate cells. As the attachment of biotinylated molecules to the functional nanoplatforms is straightforward, the results obtained show the great potential of our platforms for a broad range of applications.

Polysulfates Block SARS-CoV-2 Uptake through Electrostatic Interactions*.

Here we report that negatively charged polysulfates can bind to the spike protein of SARS-CoV-2 via electrostatic interactions. Using a plaque reduction assay, we compare inhibition of SARS-CoV-2 by heparin, pentosan sulfate, linear polyglycerol sulfate (LPGS) and hyperbranched polyglycerol sulfate (HPGS). Highly sulfated LPGS is the optimal inhibitor, with an IC50 of 67 µg mL-1 (approx. 1.6 µm). This synthetic polysulfate exhibits more than 60-fold higher virus inhibitory activity than heparin (IC50 : 4084 µg mL-1 ), along with much lower anticoagulant activity. Furthermore, in molecular dynamics simulations, we verified that LPGS can bind more strongly to the spike protein than heparin, and that LPGS can interact even more with the spike protein of the new N501Y and E484K variants. Our study demonstrates that the entry of SARS-CoV-2 into host cells can be blocked via electrostatic interactions, therefore LPGS can serve as a blueprint for the design of novel viral inhibitors of SARS-CoV-2.

Understanding the Interaction of Polyelectrolyte Architectures with Proteins and Biosystems.

The counterions neutralizing the charges on polyelectrolytes such as DNA or heparin may dissociate in water and greatly influence the interaction of such polyelectrolytes with biomolecules, particularly proteins. In this Review we give an overview of studies on the interaction of proteins with polyelectrolytes and how this knowledge can be used for medical applications. Counterion release was identified as the main driving force for the binding of proteins to polyelectrolytes: Patches of positive charge become multivalent counterions of the polyelectrolyte and lead to the release of counterions from the polyelectrolyte and a concomitant increase in entropy. This is shown from investigations on the interaction of proteins with natural and synthetic polyelectrolytes. Special emphasis is paid to sulfated dendritic polyglycerols (dPGS). The Review demonstrates that we are moving to a better understanding of charge-charge interactions in systems of biological relevance. Research along these lines will aid and promote the design of synthetic polyelectrolytes for medical applications.

Rational Design of a DNA-Scaffolded High-Affinity Binder for Langerin.

Binders of langerin could target vaccines to Langerhans cells for improved therapeutic effect. Since langerin has low affinity for monovalent glycan ligands, highly multivalent presentation has previously been key for targeting. Aiming to reduce the amount of ligand required, we rationally designed molecularly defined high-affinity binders based on the precise display of glycomimetic ligands (Glc2NTs) on DNA-PNA scaffolds. Rather than mimicking langerin's homotrimeric structure with a C3-symmetric scaffold, we developed readily accessible, easy-to-design bivalent binders. The method considers the requirements for bridging sugar binding sites and statistical rebinding as a means to both strengthen the interactions at single binding sites and amplify the avidity enhancement provided by chelation. This gave a 1150-fold net improvement over the affinity of the free ligand and provided a nanomolar binder (IC50 =300 nM) for specific internalization by langerin-expressing cells.

Platelet-derived growth factor receptor ß activation and regulation in murine myelofibrosis.

There is prevailing evidence to suggest a decisive role for platelet-derived growth factors (PDGF) and their receptors in primary myelofibrosis. While PDGF receptor ß (PDGFRß) expression is increased in bone marrow stromal cells of patients correlating with the grade of myelofibrosis, knowledge on the precise role of PDGFRß signaling in myelofibrosis is sparse. Using the Gata-1low mouse model for myelofibrosis, we applied RNA sequencing, protein expression analyses, multispectral imaging and, as a novel approach in bone marrow tissue, an in situ proximity ligation assay to provide a detailed characterization of PDGFRß signaling and regulation during development of myelofibrosis. We observed an increase in PDGFRß and PDGF-B protein expression in overt fibrotic bone marrow, along with an increase in PDGFRß-PDGF-B interaction, analyzed by proximity ligation assay. However, PDGFRß tyrosine phosphorylation levels were not increased. We therefore focused on regulation of PDGFRß by protein tyrosine phosphatases as endogenous PDGFRß antagonists. Gene expression analyses showed distinct expression dynamics among PDGFRß-targeting phosphatases. In particular, we observed enhanced T-cell protein tyrosine phosphatase protein expression and PDGFRß-T-cell protein tyrosine phosphatase interaction in early and overt fibrotic bone marrow of Gata-1low mice. In vitro, T-cell protein tyrosine phosphatase (Ptpn2) knockdown increased PDGFRß phosphorylation at Y751 and Y1021, leading to enhanced downstream signaling in fibroblasts. Furthermore, Ptpn2 knockdown cells showed increased growth rates when exposed to low-serum growth medium. Taken together, PDGF signaling is differentially regulated during myelofibrosis. Protein tyrosine phosphatases, which have so far not been examined during disease progression, are novel and hitherto unrecognized components in myelofibrosis.

dPGS Regulates the Phenotype of Macrophages via Metabolic Switching.

The synthetic compound dendritic polyglycerol sulfate (dPGS) is a pleiotropic acting molecule but shows a high binding affinity to immunological active molecules as L-/P-selectin or complement proteins leading to well described anti-inflammatory properties in various mouse models. In order to make a comprehensive evaluation of the direct effect on the innate immune system, macrophage polarization is analyzed in the presence of dPGS on a phenotypic but also metabolic level. dPGS administered macrophages show a significant increase of MCP1 production paralleled by a reduction of IL-10 secretion. Metabolic analysis reveals that dPGS could potently enhance the glycolysis and mitochondrial respiration in M0 macrophages as well as decrease the mitochondrial respiration of M2 macrophages. In summary the data indicate that dPGS polarizes macrophages into a pro-inflammatory phenotype in a metabolic pathway-dependent manner.

Sulfated Dendritic Polyglycerol Is a Potent Complement Inhibitor.

The complement system is a powerful mechanism of the innate immune defense system. Dysregulation may contribute to several diseases. Heparin is a known regulator of the complement system, but its application is limited due to its anticoagulative activity. A promising alternative is the synthetic analogue dendritic polyglycerol sulfate (dPGS). Although dPGS-mediated inhibition of the classical and alternative pathway has been roughly described previously, here we analyzed the effects of dPGS regarding the three pathways at different levels of the proteolytic cascades for the first time. Regarding the final outcome (membrane attack complex formation), IC50 values for dPGS varied between the alternative (900 nM), the classical (300 nM), and the lectin pathway (60 nM). In a backward approach, processing of proteins C5 and C3 via the respective convertase was analyzed by ELISA to narrow down dPGS targets. A dose-dependent reduction of C5a and C3a levels was detected. Further, the analysis via surface plasmon resonance revealed novel dPGS binding proteins; the pro-inflammatory anaphylatoxins C3a and C5a and the classical pathway activator C1q showed down to nanomolar binding affinities. The fully synthetic multivalent polymer dPGS seems to be a promising candidate for the further development to counteract excessive complement activation in disease.

Design and Synthesis of PEG-Oligoglycerol Sulfates as Multivalent Inhibitors for the Scavenger Receptor LOX-1.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a cell surface scavenger receptor. The protein is involved in binding and internalization of oxidized low-density lipoprotein (oxLDL), which leads under pathophysiological circumstances to plaque formation in arteries and initiation of atherosclerosis. A structural feature of LOX-1 relevant to oxLDL binding is the "basic spine" motif consisting of linearly aligned arginine residues stretched over the dimer surface. Inhibition of LOX-1 can be done by blocking these positively charged motifs. Here we report on the design, synthesis, and evaluation of a series of novel LOX-1 inhibitors having different numbers of sulfates and polyethylene glycerol (PEG) spacer. Two molecules, compounds 6b and 6d, showed binding affinity in the low nM range, i.e. 45.8 and 47.4 nM, respectively. The in vitro biological studies reveal that these molecules were also able to block the interaction of LOX-1 with its cognate ligands oxLDL, aged RBC, and bacteria.

Biodegradable Polyglycerol Sulfates Exhibit Promising Features for Anti-inflammatory Applications.

Inflammatory processes are beneficial responses to overcome injury or illness. Knowledge of the underlying mechanisms allows for a specific treatment. Thus, synthetic systems can be generated for a targeted interaction. In this context, dendritic polyglycerol sulfates (dPGS) have been investigated as anti-inflammatory compounds. Biodegradable systems are required to prevent compound accumulation in the body. Here we present biodegradable analogs of dPGS based on hyperbranched poly(glycidol- co-caprolactone) bearing a hydrophilic sulfate outer shell (hPG- co-PCLS). The copolymers were investigated regarding their physical and chemical properties. The cytocompatibility was confirmed using A549, Caco-2, and HaCaT cells. Internalization of hPG- co-PCLS by A549 and Caco-2 cells was observed as well. Moreover, we demonstrated that hPG- co-PCLS acted as a competitive inhibitor of the leukocytic cell adhesion receptor L-selectin. Further, a reduction of complement activity was observed. These new biodegradable dPGS analogs are therefore attractive for therapeutic applications regarding inflammatory diseases.

Droplet-Based Microfluidic Templating of Polyglycerol-Based Microgels for the Encapsulation of Cells: A Comparative Study.

Cell microencapsulation holds great promise as a therapeutic strategy for the controlled and sustained delivery of biologically relevant agents. The authors developed cell-laden microgel scaffolds with excellent long-term viabilities by combining bioorthogonal strain promoted azide-alkyne cycloaddition (SPAAC) and droplet-based microfluidic templating. Star-shaped polyglycerol hexaazide, α,ω-bis azido-linear polyglycerol or polyethylene glycol as well as dendritic polyglycerol-(polycyclooctyne) served as bioinert hydrogel precursors. The authors demonstrate for the first time the generation of entirely polyglycerol-based microcapsules with excellent stability and full retention of viability of the packed cells for longer than 3 weeks. As a result, our microgel particles could be used for long-term immunoisolation of cells enabling their study during encapsulation.

Interactions of Fullerene-Polyglycerol Sulfates at Viral and Cellular Interfaces.

Understanding the mechanism of interactions of nanomaterials at biointerfaces is a crucial issue to develop new antimicrobial vectors. In this work, a series of water-soluble fullerene-polyglycerol sulfates (FPS) with different fullerene/polymer weight ratios and varying numbers of polyglycerol sulfate branches are synthesized, characterized, and their interactions with two distinct surfaces displaying proteins involved in target cell recognition are investigated. The combination of polyanionic branches with a solvent exposed variable hydrophobic core in FPS proves to be superior to analogs possessing only one of these features in preventing interaction of vesicular stomatitis virus coat glycoprotein (VSV-G) with baby hamster kidney cells serving as a model of host cell. Interference with L-selectin-ligand binding is dominated by the negative charge, which is studied by two assays: a competitive surface plasmon resonance (SPR)-based inhibition assay and the leukocyte cell (NALM-6) rolling on ligands under flow conditions. Due to possible intrinsic hydrophobic and electrostatic effects of synthesized compounds, pico- to nanomolar half maximal inhibitory concentrations (IC50 ) are achieved. With their highly antiviral and anti-inflammatory properties, together with good biocompatibility, FPS are promising candidates for the future development towards biomedical applications.

A toolbox approach for multivalent presentation of ligand-receptor recognition on a supramolecular scaffold.

A supramolecular toolbox approach for multivalent ligand-receptor recognition was established based on ß-cyclodextrin vesicles (CDVs). A series of bifunctional ligands for CDVs was synthesised. These ligands comprise on one side adamantane, enabling the functionalisation of CDVs with these ligands, and either mannose or sulphate group moieties on the other side for biological receptor recognition. The physicochemical properties of the host-guest complexes formed by ß-cyclodextrin (ß-CD) and adamantane were determined by isothermal titration calorimetry (ITC). Ligand-lectin interactions were investigated by surface plasmon resonance experiments (SPR) for the mannose ligands and the lectin Concanavalin A (ConA). Microscale thermophoresis (MST) measurements were applied for sulphate-dependent binding to L-selectin. In both cases, a multivalent affinity enhancement became apparent when the ligands were presented on the CDV scaffold. Furthermore, not only the clustering between our supramolecular mannosylated complex and Escherichia coli (E. coli), expressing the lectin FimH, was visualised by cryo-TEM, but also the competitive character to detach bound E. coli from a cell line, representing the uroepithelial cell surface, was demonstrated. In summary, a facile and effective supramolecular toolbox was established for various ligand-receptor recognition applications.

Anti-inflammatory properties of GLPss58, a sulfated polysaccharide from Ganoderma lucidum.

Sulfated polysaccharides exhibit various biological properties, including anti-coagulant, anti-oxidant, anti-viral, anti-cancer, anti-inflammatory and immune regulatory activities. In the present study, the anti-inflammatory properties of GLPss58, a sulfated polysaccharide from Ganoderma lucidum formed by chemical sulfation, were investigated. We found that GLPss58 inhibited L-selectin/sTyr-sLeX binding significantly, blocked the binding of anti-l-selectin antibodies to L-selectin on the surface of human peripheral blood lymphocytes, and inhibited the secondary lymphoid tissue chemokine-induced chemotactic invasion of HPBLs. In vivo studies in mice showed that lymphocyte homing from peripheral blood to spleen and lymph nodes was significantly inhibited by GLPss58. Furthermore, GLPss58 also inhibited the activation of complement systems and blocked the binding of TNF-α and IFN-γ to their antibodies. These results indicate that GLPss58 is able to inhibit not only the L-selectin-mediated inflammation, but also the complement system- and cytokines mediated-inflammation. Our results suggest that GLPss58 is a favorable potential anti-inflammatory agent.

Heterobifunctional Dyes: Highly Fluorescent Linkers Based on Cyanine Dyes.

Herein, we present a new synthetic route to cyanine-based heterobifunctional dyes and their application as fluorescent linkers between polymers and biomolecules. The synthesized compounds, designed in the visible spectral range, are equipped with two different reactive groups for highly selective conjugation under physiological conditions. By applying indolenine precursors with functionalized benzenes, we achieved water-soluble asymmetric cyanine dyes bearing maleimido and N-hydroxysuccinimidyl functionalities in a three-step synthesis. Spectroscopic characterization revealed good molar absorption coefficients and moderate fluorescence quantum yields. Further reaction with polyethylene glycol yielded dye-polymer conjugates that were subsequently coupled to the antibody cetuximab, often applied in cancer therapy. Successful coupling was confirmed by mass shifts detected by gel electrophoresis. Receptor-binding studies and live-cell imaging revealed that labeling did not alter the biological function. In sum, we provided a successful synthetic pathway to rigid heterobifunctional cyanine dyes that are applicable as fluorescent linkers, for example, for connecting antibodies with macromolecules. Our approach contributes to the field of bioconjugation chemistry, such as antibody-drug conjugates by combining diagnostic and therapeutic approaches.