Linear Polyglycerol Research Articles

Synthesis and Comparison of Linear Polymannosides for Direct Binding with Escherichia coli

AbstractHere, the synthesis of linear polyglycerols bearing multiple copies of mono and dimannosides (LPG40Man0.60 and LPG40(Manα1,2Man)0.60) is demonstrated. A method based on label‐free microscale thermophoresis is optimized to determine the direct binding affinity of multivalent mannosides for Escherichia coli (E. coli) strain ORN178 that produces the fimbriae protein FimH. It is observed that the LPG40(Manα1,2Man)0.60 exhibits only a modest onefold improvement in binding as compared to LPG40Man0.60. Nevertheless, both the multivalent mannosides display remarkably very low nm binding constant (Kd) in contrast to the high µm Kd of the single α‐d‐methylmannoside for intact E. coli ORN178 particles. Furthermore, in an adhesion‐inhibition assay, both multivalent mannosides show 50% inhibition of bacteria adhesion to the HT‐29 colon cells at low µm concentrations.

Development of Functional Filter Materials for Virus Protective Face Masks

AbstractWearing face masks during pandemics is an important protective measure against the spreading of virus‐related infectious diseases. Nevertheless, the risk of indirect transmission of virus by handling masks is one of the earliest concerns. This problem can be minimized by supplementing the masks’ textile structure with virus protective coatings. Therefore, in this concept, suitable techniques for manufacturing virus protective filter media should be evaluated. In this study, nonwoven polyamide 6 (PA6) filter material is functionalized with negatively charged linear polyglycerol sulfate (LPGS) as a virus binding functional group. Two coating conditions are investigated in which the direct covalent coating with LPGS has emerged as the optimum coating method, showing no damage to the PA6 nanofiber structure. The uncoated PA6 and LPGS‐coated PA6 filter materials exhibited virus particle filtration efficiencies of 95% and 94% for airborne feline coronavirus, 98% and 86% for airborne equine herpesvirus 1(EHV‐1), respectively. However, the SARS‐CoV‐2 absorption assay in solution indicates that the LPGS coating reduces viral titres up to 71% when incubating with the LPGS‐coated PA6 filter media for one‐hour. Thus, such an effect is not seen for uncoated PA6 materials. These findings confirm the suitability of LPGS coating as a suitable platform for suppression the spreading of viruses in different pandemics.

Mussel‐Inspired Polyglycerol Coatings for Surface Modification with Tunable Architecture

AbstractMussel‐inspired coatings, known for their outstanding substrate‐independent adhesive capabilities, have numerous potential applications in materials science and biomedical fields. To improve the understanding of how these polymers’ molecular structure and chemical composition affect their coating mechanisms and resulting coating properties, herein three mussel‐inspired polymers are developed: dendritic polyglycerol with 40% catechol groups and 60% amines (dPG40), linear polyglycerol with 80% catechols and 20% amines (lPG80), and finally lPG40 with 40% catechols and 60% amines. After a series of characterizations, it is found that chemical surface modification with a monolayer coating can be easily achieved with lPG40, and that robust and well‐defined nano‐ to micro‐structural surface coatings are possible with lPG80 and dPG40. Tunable properties are found to include not only coating speed, but coating thickness, roughness, and surficial topography. This diverse suite of controllable attributes enables mussel‐inspired polyglycerol (MiPG) coatings to satisfy a wide‐range of applications on multiple materials.

Dual-Action Heteromultivalent Glycopolymers Stringently Block and Arrest Influenza A Virus Infection In Vitro and Ex Vivo.

Here, we demonstrate the concerted inhibition of different influenza A virus (IAV) strains using a low-molecular-weight dual-action linear polymer. The 6'-sialyllactose and zanamivir conjugates of linear polyglycerol are optimized for simultaneous targeting of hemagglutinin and neuraminidase on the IAV surface. Independent of IAV subtypes, hemagglutination inhibition data suggest better adsorption of the heteromultivalent polymer than homomultivalent analogs onto the virus surface. Cryo-TEM images imply heteromultivalent compound-mediated virus aggregation. The optimized polymeric nanomaterial inhibits >99.9% propagation of various IAV strains 24 h postinfection in vitro at low nM concentrations and is up to 10000× more effective than the commercial zanamivir drug. In a human lung ex vivo multicyclic infection setup, the heteromultivalent polymer outperforms the commercial drug zanamivir and homomultivalent analogs or their physical mixtures. This study authenticates the translational potential of the dual-action targeting approach using small polymers for broad and high antiviral efficacy.

One to one comparison of cell-free synthesized erythropoietin conjugates modified with linear polyglycerol and polyethylene glycol

With more than 20 Food and Drug Administration (FDA)-approved poly (ethylene glycol) (PEG) modified drugs on the market, PEG is the gold standard polymer in bioconjugation. The coupling improves stability, efficiency and can prolong blood circulation time of therapeutic proteins. Even though PEGylation is described as non-toxic and non-immunogenic, reports accumulate with data showing allergic reactions to PEG. Since PEG is not only applied in therapeutics, but can also be found in foods and cosmetics, anti-PEG-antibodies can occur even without a medical treatment. Hypersensitivity to PEG thereby can lead to a reduced drug efficiency, fast blood clearance and in rare cases anaphylactic reactions. Therefore, finding alternatives for PEG is crucial. In this study, we present linear polyglycerol (LPG) for bioconjugation as an alternative polymer to PEG. We report the conjugation of LPG and PEG by click-chemistry to the glycoprotein erythropoietin (EPO), synthesized in a eukaryotic cell-free protein synthesis system. Furthermore, the influence of the polymers on EPOs stability and activity on a growth hormone dependent cell-line was evaluated. The similar characteristics of both bioconjugates show that LPGylation can be a promising alternative to PEGylation.

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.

Functionalized Fullerene for Inhibition of SARS-CoV-2 Variants.

As virus outbreaks continue to pose a challenge, a nonspecific viral inhibitor can provide significant benefits, especially against respiratory viruses. Polyglycerol sulfates recently emerge as promising agents that mediate interactions between cells and viruses through electrostatics, leading to virus inhibition. Similarly, hydrophobic C60 fullerene can prevent virus infection via interactions with hydrophobic cavities of surface proteins. Here, two strategies are combined to inhibit infection of SARS-CoV-2 variants in vitro. Effective inhibitory concentrations in the millimolar range highlight the significance of bare fullerene's hydrophobic moiety and electrostatic interactions of polysulfates with surface proteins of SARS-CoV-2. Furthermore, microscale thermophoresis measurements support that fullerene linear polyglycerol sulfates interact with the SARS-CoV-2 virus via its spike protein, and highlight importance of electrostatic interactions within it. All-atom molecular dynamics simulations reveal that the fullerene binding site is situated close to the receptor binding domain, within 4nm of polyglycerol sulfate binding sites, feasibly allowing both portions of the material to interact simultaneously.

Virus removal from aqueous environments with polyelectrolyte coatings on a polypropylene fleece

AbstractThe adsorption of viruses from aqueous solution is frequently performed to detect viruses. Charged filtration materials capture viruses via electrostatic interactions, but lack the specificity of biological virus‐binding substances like heparin. Herein, we present three methods to immobilize heparin‐mimicking, virus‐binding polymers to a filter material. Two mussel‐inspired approaches are used, based on dopamine or mussel‐inspired dendritic polyglycerol, and post‐functionalized with a block‐copolymer consisting of linear polyglycerol sulfate and amino groups as anchor (lPGS‐b‐NH2). As third method, a polymer coating based on lPGS with benzophenone anchor groups is tested (lPGS‐b‐BPh). All three methods yield dense and stable coatings. A positively charged dye serves as a tool to quantitatively analyze the sulfate content on coated fleece. Especially lPGS‐b‐BPh is shown to be a dense polymer brush coating with about 0.1 polymer chains per nm2. Proteins adsorb to the lPGS coated materials depending on their charge, as shown for lysozyme and human serum albumin. Finally, herpes simplex virus type 1 (HSV‐1) and severe acute respiratory syndrome coronavirus type 2 (SARS‐CoV‐2) can be removed from solution upon incubation with coated fleece materials by about 90% and 45%, respectively. In summary, the presented techniques may be a useful tool to collect viruses from aqueous environments.

Polymer selection impacts the pharmaceutical profile of site-specifically conjugated Interferon-α2a.

Conjugation of poly(ethylene glycol) (PEG) to biologics is a successful strategy to favorably impact the pharmacokinetics and efficacy of the resulting bioconjugate. We compare bioconjugates synthesized by strain-promoted azide-alkyne cycloaddition (SPAAC) using PEG and linear polyglycerol (LPG) of about 20kDa or 40kDa, respectively, with an azido functionalized human Interferon-α2a (IFN-α2a) mutant. Site-specific PEGylation and LPGylation resulted in IFN-α2a bioconjugates with improved in vitro potency compared to commercial Pegasys. LPGylated bioconjugates had faster disposition kinetics despite comparable hydrodynamic radii to their PEGylated analogues. Overall exposure of the PEGylated IFN-α2a with a 40kDa polymer exceeded Pegasys, which, in return, was similar to the 40kDa LPGylated conjugates. The study points to an expanded polymer design space through which the selected polymer class may result in a different distribution of the studied bioconjugates.

Polyethers Based on Short-Chain Alkyl Glycidyl Ethers: Thermoresponsive and Highly Biocompatible Materials.

The polymerization of short-chain alkyl glycidyl ethers (SCAGEs) enables the synthesis of biocompatible polyethers with finely tunable hydrophilicity. Aliphatic polyethers, most prominently poly(ethylene glycol) (PEG), are utilized in manifold biomedical applications due to their excellent biocompatibility and aqueous solubility. By incorporation of short hydrophobic side-chains at linear polyglycerol, control of aqueous solubility and the respective lower critical solution temperature (LCST) in aqueous solution is feasible. Concurrently, the chemically inert character in analogy to PEG is maintained, as no further functional groups are introduced at the polyether structure. Adjustment of the hydrophilicity and the thermoresponsive behavior of the resulting poly(glycidyl ether)s in a broad temperature range is achieved either by the combination of the different SCAGEs or with PEG as a hydrophilic block. Homopolymers of methyl and ethyl glycidyl ether (PGME, PEGE) are soluble in aqueous solution at room temperature. In contrast, n-propyl glycidyl ether and iso-propyl glycidyl ether lead to hydrophobic polyethers. The use of a variety of ring-opening polymerization techniques allows for controlled polymerization, while simultaneously determining the resulting microstructures. Atactic as well as isotactic polymers are accessible by utilization of the respective racemic or enantiomerically pure monomers. Polymer architectures varying from statistical copolymers, di- and triblock structures to star-shaped architectures, in combination with PEG, have been applied in various thermoresponsive hydrogel formulations or polymeric surface coatings for cell sheet engineering. Materials responding to stimuli are of increasing importance for "smart" biomedical systems, making thermoresponsive polyethers with short-alkyl ether side chains promising candidates for future biomaterials.

A Simple and Robust Method to Prepare Polyelectrolyte Brushes on Polymer Surfaces

AbstractA simple and robust method is presented to immobilize a heparin‐analog polyelectrolyte on inert hydrophobic surfaces. It is demonstrated that an amphiphilic block copolymer consisting of linear polyglycerol sulfate (lPGS) and a benzophenone modified anchor block can be bound to polystyrene surfaces in a facile dip‐coating procedure. The chaotropic salt guanidinium chloride is used to overcome the aggregation of the polymer as well as the repulsion between highly hydrated sulfate groups and the polystyrene surface. Irradiation with UV light tethers the polymer chains covalently to the surface. The resulting coating exhibits an aggregate morphology that resembles the aggregation behavior in solution, with a coating thickness of 8 nm. The behavior of the surfaces is dominated by the polyelectrolyte brush coating. They swell and collapse in response to different ionic strengths of the surrounding medium, and bind proteins via electrostatic interactions. The coating is stable toward physiological conditions over the course of several weeks. Coated surfaces bind to proteins of the complement cascade when in contact with dilute blood serum, which results in a decrease of complement activity to 78 ± 4%. The coating procedure can also be applied to other nonactivated polymer surfaces, as demonstrated on a polypropylene fleece.

Selective ring-opening polymerization of glycidyl esters: a versatile synthetic platform for glycerol-based (co)polyethers

Well-defined (isotactic) linear polyglycerols and glycerol-based copolyethers are obtained by controlled and chemoselective ring-opening (co)polymerizations of glycidyl butyrates.

Linear Polyglycerol for N-terminal-selective Modification of Interleukin-4

Polymer conjugation to biologics is of key interest to the pharmaceutical industry for the development of potent and long acting biotherapeutics, with poly(ethylene glycol) (PEG) being the gold standard. Within the last years, unwanted PEG-related side effects (immunological reactions, antibody formation) arose, therefore creating several attempts to establish alternative polymers with similar potential to PEG. In this article, we synthesized N-terminal bioconjugates of the potential therapeutic human interleukin-4 (hIL-4 WT) with linear polyglycerol (LPG) of 10 and 40 kDa and compared it with its PEG analogs of same nominal weights. Polyglycerol is a highly hydrophilic polymer with good biocompatibility and therefore represents an alternative polymer to PEG.Both polymer types resulted in similar conjugation yields, comparable hydrodynamic sizes and an unaltered secondary structure of the protein after modification. LPG- and PEG-bioconjugates remained stable in human plasma, whereas binding to human serum albumin (HSA) decreased after polymer modification. Furthermore, only minor differences in bioactivity were observed between LPG- and PEG-bioconjugates of same nominal weights. The presented findings are promising for future pharmacokinetic evaluation of hIL-4-polymer bioconjugates.

Molecular Insights into Site-Specific Interferon-α2a Bioconjugates Originated from PEG, LPG, and PEtOx.

Conjugation of biologics with polymers modulates their pharmacokinetics, with polyethylene glycol (PEG) as the gold standard. We compared alternative polymers and two types of cyclooctyne linkers (BCN/DBCO) for bioconjugation of interferon-α2a (IFN-α2a) using 10 kDa polymers including linear mPEG, poly(2-ethyl-2-oxazoline) (PEtOx), and linear polyglycerol (LPG). IFN-α2a was azide functionalized via amber codon expansion and bioorthogonally conjugated to all cyclooctyne linked polymers. Polymer conjugation did not impact IFN-α2a's secondary structure and only marginally reduced IFN-α2a's bioactivity. In comparison to PEtOx, the LPG polymer attached via the less rigid cyclooctyne linker BCN was found to stabilize IFN-α2a against thermal stress. These findings were further detailed by molecular modeling studies which showed a modulation of protein flexibility upon PEtOx conjugation and a reduced amount of protein native contacts as compared to PEG and LPG originated bioconjugates. Polymer interactions with IFN-α2a were further assessed via a limited proteolysis (LIP) assay, which resulted in comparable proteolytic cleavage patterns suggesting weak interactions with the protein's surface. In conclusion, both PEtOx and LPG bioconjugates resulted in a similar biological outcome and may become promising PEG alternatives for bioconjugation.

Polyglycerol-Based Mucus-Inspired Hydrogels.

The mucus layer is a hydrogel network that covers mucosal surfaces of the human body. Mucus has important protective properties that are related to its unique rheological properties, which are based on mucins being the main glycoprotein constituents. Mucin macromolecules entangle with one another and form a physical network that is instrumental for many important defense functions. Mucus derived from various human or animal sources is poorly defined and thus not suitable for many application purposes. Herein, a synthetic route is fabricated to afford a library of compositionally defined mucus-inspired hydrogels (MIHs). MIHs are synthesized by thiol oxidation to render disulfide bonds between the crosslinker ethoxylated trimethylolpropane tri(3-mercaptopropionate) (THIOCURE ETTMP 1300) and the linear precursors, dithiolated linear polyglycerol (LPG(SH)2 ) or polyethylene glycol (PEG(SH)2 ) of different molecular weights. The mixing ratio of linear polymers versus crosslinker and the length of the linear polymer are varied, thus delivering a library of compositionally defined mucin-inspired constructs. Their viscoelastic properties are determined by frequency sweeps at 25 and 37 °C and compared to the corresponding behavior of native human mucus. Here, MIHs composed of a 10:1 ratio of LPG(SH)2 and ETTMP 1300 are proved to be the best comparable to human airway mucus rheology.

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.

Prolonged activity of exenatide: Detailed comparison of Site-specific linear polyglycerol- and poly(ethylene glycol)-conjugates

Exenatide is a small therapeutic peptide being currently used in clinic for the treatment of diabetes mellitus type II, however, displaying a short blood circulation time which makes two daily injections necessary. Covalent polymer modification of a protein is a well-known approach to overcome this limitation, resulting in steric shielding, an increased size and therefore a longer circulation half-life. In this study, we employed site-selective C-terminal polymer ligation of exenatide via copper-catalyzed azide-alkyne-cycloaddition (CuAAC) to yield 1:1-conjugates of either poly(ethylene glycol) (PEG) or linear polyglycerol (LPG) of different molecular weights. Our goal was to compare the impact of the two polymers on size, structure and activity of exenatide on the in vitro and in vivo level. Both polymers did not alter the secondary structure of exenatide and expectedly increased its hydrodynamic size, where the LPG-versions of exenatide showed slightly smaller values than their PEG-analogs of same molecular weight. Upon conjugation, GLP-1 receptor activation was diminished, however, still enabled maximum receptor response at slightly higher concentrations. Exenatide modified with a 40 kDa LPG (Ex-40-LPG) showed significant reduction of the blood glucose level in diabetic mice for up to 72 h, which was comparable to its PEG-analog, but 9-fold longer than native exenatide (8 h).

Inhibition of Herpes Simplex Virus Type 1 Attachment and Infection by Sulfated Polyglycerols with Different Architectures.

Inhibition of herpes simplex virus type 1 (HSV-1) binding to the host cell surface by highly sulfated architectures is among the promising strategies to prevent virus entry and infection. However, the structural flexibility of multivalent inhibitors plays a major role in effective blockage and inhibition of virus receptors. In this study, we demonstrate the inhibitory effect of a polymer scaffold on the HSV-1 infection by using highly sulfated polyglycerols with different architectures (linear, dendronized, and hyperbranched). IC50 values for all synthesized sulfated polyglycerols and the natural sulfated polymer heparin were determined using plaque reduction infection assays. Interestingly, an increase in the IC50 value from 0.03 to 374 nM from highly flexible linear polyglycerol sulfate (LPGS) to less flexible scaffolds, namely, dendronized polyglycerol sulfate and hyperbranched polyglycerol sulfate was observed. The most potent LPGS inhibits HSV-1 infection 295 times more efficiently than heparin, and we show that LPGS has a much reduced anticoagulant capacity when compared to heparin as evidenced by measuring the activated partial thromboplastin time. Furthermore, prevention of infection by LPGS and the commercially available drug acyclovir were compared. All tested sulfated polymers do not show any cytotoxicity at concentrations of up to 1 mg/mL in different cell lines. We conclude from our results that more flexible polyglycerol sulfates are superior to less flexible sulfated polymers with respect to inhibition of HSV-1 infection and may constitute an alternative to the current antiviral treatments of this ubiquitous pathogen.

The Application of Dual‐Layer, Mussel‐Inspired, Antifouling Polyglycerol‐Based Coatings in Ventricular Assist Devices

AbstractContinuous‐flow ventricular assist devices (VADs) have established themselves as a lifesaving therapy option in patients with severe cardiovascular disease. Unfortunately, complications with VADs resulting from the shear‐induced formation of surface blood clots are common. In the current work, an antifouling coating based on the combination of mussel‐inspired dendritic polyglycerol (MI‐dPG) and linear polyglycerol (lPG) is tested for its cell‐repelling properties, biocompatibility, and complement activating properties. Furthermore, the adhesion and activation of blood platelets are tested under static and flow conditions. The adhesion and proliferation of two cell types are studied by means of LIVE/DEAD cell staining, and it is clearly observed that the lPG‐functionalized MI‐dPG coating prevents cell adhesion. Additionally, no cell mortality is observed on all substrates, indicating the biocompatibility of the tested coatings. All coatings show lower (or equal) complement‐activating properties than bare titanium, which is considered a highly biocompatible material. Most importantly, the lPG‐functionalized system prevents the adhesion and activation of blood platelets under static and flow conditions. Finally, a prototype VAD is successfully coated with MI‐dPG under flow conditions. In the current study, the efficient lPG‐functionalization of the MI‐dPG coating is proved to obtain cell‐ and platelet‐repelling surfaces.

Mussel-Inspired Multivalent Linear Polyglycerol Coatings Outperform Monovalent Polyethylene Glycol Coatings in Antifouling Surface Properties.

Biofouling constitutes a major challenge in the application of biosensors and biomedical implants, as well as for (food) packaging and marine equipment. In this work, an antifouling surface coating based on the combination of mussel-inspired dendritic polyglycerol (MI-dPG) and an amine-functionalized block copolymer of linear polyglycerol (lPG-b-OA11, OA = oligo-amine) was developed. The coating was compared to a MI-dPG surface which was postfunctionalized with commercially available amine-terminated polyethylene glycol (HO-PEG-NH2) of similar molecular weight. In the current work, these coatings were compared in their chemical stability, protein fouling characteristics, and cell fouling characteristics. The lPG-b-OA11-functionalized coating showed high chemical stability in both phosphate buffered saline (PBS) and sodium dodecyl sulfate (SDS) solutions and reduced the adhesion of fibrinogen from human plasma with 99% and the adhesion of human serum albumin with 96%, in comparison to the bare titanium dioxide substrate. Furthermore, the proliferation of human umbilical vein endothelial cells (HUVECs) was reduced with 85% when the lPG-b-OA11 system was compared to bare titanium dioxide. Additionally, a reduction of 94% was observed when the lPG-b-OA11 system was compared to tissue culture polystyrene.