Marine sulfated glycans inhibit the interaction of heparin with S-protein of SARS-CoV-2 Omicron XBB variant.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide COVID-19 pandemic, leading to 6.8 million deaths. Numerous variants have emerged since its outbreak, resulting in its significantly enhanced ability to spread among humans. As with many other viruses, SARS­CoV­2 utilizes heparan sulfate (HS) glycosaminoglycan (GAG) on the surface of host cells to facilitate viral attachment and initiate cellular entry through the ACE2 receptor. Therefore, interfering with virion-HS interactions represents a promising target to develop broad-spectrum antiviral therapeutics. Sulfated glycans derived from marine organisms have been proven to be exceptional reservoirs of naturally existing HS mimetics, which exhibit remarkable therapeutic properties encompassing antiviral/microbial, antitumor, anticoagulant, and anti-inflammatory activities. In the current study, the interactions between the receptor-binding domain (RBD) of S-protein of SARS-CoV-2 (both WT and XBB.1.5 variants) and heparin were applied to assess the inhibitory activity of 10 marine-sourced glycans including three sulfated fucans, three fucosylated chondroitin sulfates and two fucoidans derived from sea cucumbers, sea urchin and seaweed Saccharina japonica, respectively. The inhibitory activity of these marine derived sulfated glycans on the interactions between RBD of S-protein and heparin was evaluated using Surface Plasmon Resonance (SPR). The RBDs of S-proteins from both Omicrion XBB.1.5 and wild-type (WT) were found to bind to heparin, which is a highly sulfated form of HS. All the tested marine-sourced sulfated glycans exhibited strong inhibition of WT and XBB.1.5 S-protein binding to heparin. We believe the study on the molecular interactions between S-proteins and host cell glycosaminoglycans provides valuable insight for the development of marine-sourced, glycan-based inhibitors as potential anti-SARS-CoV-2 agents.

Synthesis of bioengineered heparin chemically and biologically similar to porcine-derived products and convertible to low MW heparin.

Heparins have been invaluable therapeutic anticoagulant polysaccharides for over a century, whether used as unfractionated heparin or as low molecular weight heparin (LMWH) derivatives. However, heparin production by extraction from animal tissues presents multiple challenges, including the risk of adulteration, contamination, prion and viral impurities, limited supply, insecure supply chain, and significant batch-to-batch variability. The use of animal-derived heparin also raises ethical and religious concerns, as well as carries the risk of transmitting zoonotic diseases. Chemoenzymatic synthesis of animal-free heparin products would offer several advantages, including reliable and scalable production processes, improved purity and consistency, and the ability to produce heparin polysaccharides with molecular weight, structural, and functional properties equivalent to those of the United States Pharmacopeia (USP) heparin, currently only sourced from porcine intestinal mucosa. We report a scalable process for the production of bioengineered heparin that is biologically and compositionally similar to USP heparin. This process relies on enzymes from the heparin biosynthetic pathway, immobilized on an inert support and requires a tailored N-sulfoheparosan with N-sulfo levels similar to those of porcine heparins. We also report the conversion of our bioengineered heparin into a LMWH that is biologically and compositionally similar to USP enoxaparin. Ultimately, we demonstrate major advances to a process to provide a potential clinical and sustainable alternative to porcine-derived heparin products.

Sulfated Glycans Inhibit the Interaction of MERS-CoV Receptor Binding Domain with Heparin.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus with high contagion and mortality rates. Heparan sulfate proteoglycans (HSPGs) are ubiquitously expressed on the surface of mammalian cells. Owing to its high negatively charged property, heparan sulfate (HS) on the surface of host cells is used by many viruses as cofactor to facilitate viral attachment and initiate cellular entry. Therefore, inhibition of the interaction between viruses and HS could be a promising target to inhibit viral infection. In the current study, the interaction between the receptor-binding domain (RBD) of MERS-CoV and heparin was exploited to assess the inhibitory activity of various sulfated glycans such as glycosaminoglycans, marine-sourced glycans (sulfated fucans, fucosylated chondroitin sulfates, fucoidans, and rhamnan sulfate), pentosan polysulfate, and mucopolysaccharide using Surface Plasmon Resonance. We believe this study provides valuable insights for the development of sulfated glycan-based inhibitors as potential antiviral agents.

Heparin Specifically Inhibits CRISPR/Cas12 Activation, Enabling Ultrasensitive Heparin Detection and Gene Editing Regulation.

Heparin is a highly sulfated linear glycosaminoglycan that is used as an anticoagulant to prevent and treat thrombotic diseases. Herein, we find that heparin specifically inhibits the activation of the Cas12 protein through the competitive binding of heparin and crRNA to Cas12. Studies illustrate that heparin's high molecular weight and strong negative charge are critical parameters for its inhibitory effect. This unexpected finding was engineered for the detection of heparin, affording a low detection limit of 0.36 ng/mL for fluorometric quantification. We further developed a rapid lateral flow-based system named HepaStrip (heparin strip), allowing heparin monitoring in clinical samples within 20 min. Finally, in vivo investigations revealed that heparin can regulate gene editing with the clusters of the regularly spaced short palindromic repeat (CRISPR)/Cas12 system in Escherichia coli. Heparin blocks the formation of Cas12-crRNA ribonucleoprotein, allowing the application of CRISPR for rapid and field-deployable detection of heparin and unleashing the potential use of heparin in future anti-CRISPR applications.

Improving impact of heparan sulfate on the endothelial glycocalyx abnormalities in atherosclerosis as revealed by glycan-protein interactome.

Endothelial dysfunction induced by oxidative stress is an early predictor of atherosclerosis, which can cause various cardiovascular diseases. The glycocalyx layer on the endothelial cell surface acts as a barrier to maintain endothelial biological function, and it can be impaired by oxidative stress. However, the mechanism of glycocalyx damage during the development of atherosclerosis remains largely unclear. Herein, we established a novel strategy to address these issues from the glycomic perspective that has long been neglected. Using countercharged fluorescence protein staining and quantitative mass spectrometry, we found that heparan sulfate, a major component of the glycocalyx, was structurally altered by oxidative stress. Comparative proteomics and protein microarray analysis revealed several new heparan sulfate-binding proteins, among which alpha-2-Heremans-Schmid glycoprotein (AHSG) was identified as a critical protein. The molecular mechanism of AHSG with heparin was characterized through several methods. A heparan analog could relieve atherosclerosis by protecting heparan sulfate from degradation during oxidative stress and by reducing the accumulation of AHSG at lesion sites. In the present study, the molecular mechanism of anti-atherosclerotic effect of heparin through interaction with AHSG was revealed. These findings provide new insights into understanding of glycocalyx damage in atherosclerosis and lead to the development of corresponding therapeutics.

ß-Glucosidase on clay minerals: Structure and function in the synthesis of octyl glucoside.

ß-Glucosidase is a biological macromolecule that catalyzes the hydrolysis of various glycosides and oligosaccharides. It may also be used to catalyze the synthesis of glycosides under suitable conditions. Carrier-bound ß-glucosidase can enhance the enzymatic activity in the synthesis of glycosides in organic solvent solutions, although the molecular mechanism regulating activity is yet unknown. This study investigated the impact of utilizing montmorillonite (Mmt), attapulgite (Attp), and kaolinite (Kao) as carriers on the activity of ß-glucosidase from Prunus dulcis (PdBg). When Attp was used as carriers, the molecular dynamic (MD) simulations found the distance between pNPG and the active site residues E183 and E387 was minimally impacted by the adsorptions, hence PdBg maintained about 81.3 ± 0.89 % of its native activity. Out of the three clay minerals, the relative activity of PdBg loaded on Mmt was the lowest because of the highest electrostatic energy. The substrate channel of PdBg on Kao is directed towards the surface, limiting the accessibility of substrates. Secondary structure and conformation studies revealed that the conformational stability of PdBg in solvent solutions was enhanced by coupling to Attp. Unlike dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) and 1,2-dimethoxyethane (DME), tert-butanol (t-BA) did not penetrate into the active site of PdBg interfering with its binding to the substrate. The maximum yield of n-octyl-ß-glucoside (OGP) synthesis catalyzed by Attp-immobilized PdBg reached 48.3 %.

A role for decorin in improving motor deficits after traumatic brain injury.

Traumatic brain injury (TBI) is the leading cause of death and disability due to injury worldwide. Extracellular matrix (ECM) remodeling is known to significantly contribute to TBI pathophysiology. Glycosaminoglycans, which are long-chain, variably sulfated polysaccharides abundant within the ECM, have previously been shown to be substantially altered after TBI. In this study, we sought to delineate the dynamics of glycosaminoglycan alterations after TBI and discover the precise biologic processes responsible for observed glycosaminoglycan changes after injury. We performed state-of-the art mass spectrometry on brain tissues isolated from mice after TBI or craniotomy-alone. We observed dynamic changes in glycosaminoglycans at Day 1 and 7 post-TBI, with heparan sulfate, chondroitin sulfate, and hyaluronan remaining significantly increased after a week vis-à-vis craniotomy-alone tissues. We did not observe appreciable changes in circulating glycosaminoglycans in mice after experimental TBI compared to craniotomy-alone nor in patients with TBI and severe polytrauma compared to control patients with mild injuries, suggesting increases in injury site glycosaminoglycans are driven by local synthesis. We subsequently performed an unbiased whole genome transcriptomics analysis on mouse brain tissues 7 days post-TBI and discovered a significant induction of hyaluronan synthase 2, glypican-3, and decorin. The functional role of decorin after injury was further examined through multimodal behavioral testing comparing wild-type and Dcn-/- mice. We discovered that genetic ablation of Dcn led to an overall negative effect of TBI on function, exacerbating motor impairments after TBI. Collectively, our results provide a spatiotemporal characterization of post-TBI glycosaminoglycan alterations in the brain ECM and support an important adaptive role for decorin upregulation after TBI.

New insights into the binding of PF4 to long heparin oligosaccharides in ultralarge complexes using mass spectrometry.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a serious complication caused by heparin drugs. The ultralarge complexes formed by platelet factor 4 (PF4) with heparin or low molecular weight heparins (LMWHs) are important participants in inducing the immune response and HIT. OBJECTIVES: We aim at characterizing the interaction between PF4 and long-chain heparin oligosaccharides and providing robust analytical methods for the analysis of PF4-heparin complexes. METHODS: In this work, the characteristics of PF4-enoxaparin complexes after incubation in different molar ratios and concentrations were analyzed by multiple analytical methods, especially liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry with multiple reaction monitoring were developed to qualitatively and quantitatively monitor heparin oligosaccharides and PF4 in HIT-inducing complexes. RESULTS: The results showed that the largest proportion of ultralarge complexes formed by PF4 and enoxaparin was at a specific molar ratio, ie, a PF4/enoxaparin ratio of 2:1, while the ultralarge complexes contained PF4 tetramer and enoxaparin at a molar ratio of approximately 2:1. CONCLUSION: A binding model of PF4 and enoxaparin in ultralarge complexes is proposed with one heparin oligosaccharide chain (∼ dp18) bound to 2 PF4 tetramers in different morphologies to form ultralarge complexes, while PF4 tetramer is surrounded by multiple heparin chains in smaller complexes. Our study provides new insights into the structural mechanism of PF4-LMWH interaction, which help to further understand the mechanism of LMWH immunogenicity and develop safer heparin products.

Synthesis of multivalent sialyllactose-conjugated PAMAM dendrimers: Binding to SARS-CoV-2 spike protein and influenza hemagglutinin.

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) and influenza viruses have spread around the world at an unprecedented rate. Despite multiple vaccines, new variants of SARS-CoV-2 and influenza have caused a remarkable level of pathogenesis. The development of effective antiviral drugs to treat SARS-CoV-2 and influenza remains a high priority. Inhibiting viral cell surface attachment represents an early and efficient means to block virus infection. Sialyl glycoconjugates, on the surface of human cell membranes, play an important role as host cell receptors for influenza A virus and 9-O-acetyl-sialylated glycoconjugates are receptors for MERS, HKU1 and bovine coronaviruses. We designed and synthesized multivalent 6'-sialyllactose-counjugated polyamidoamine dendrimers through click chemistry at room temperature concisely. These dendrimer derivatives have good solubility and stability in aqueous solutions. SPR, a real-time analysis quantitative method for of biomolecular interactions, was used to study the binding affinities of our dendrimer derivatives by utilizing only 200 micrograms of each dendrimer. Three SARS-CoV-2 S-protein receptor binding domain (wild type and two Omicron mutants) bound to multivalent 9-O-acetyl-6'-sialyllactose-counjugated and 6'-sialyllactose-counjugated dendrimers bound to a single H3N2 influenza A virus's HA protein (A/Hong Kong/1/1968), the SPR study results suggest their potential anti-viral activities.

Regional and disease-specific glycosaminoglycan composition and function in decellularized human lung extracellular matrix.

Decellularized lung scaffolds and hydrogels are increasingly being utilized in ex vivo lung bioengineering. However, the lung is a regionally heterogenous organ with proximal and distal airway and vascular compartments of different structures and functions that may be altered as part of disease pathogenesis. We previously described decellularized normal whole human lung extracellular matrix (ECM) glycosaminoglycan (GAG) composition and functional ability to bind matrix-associated growth factors. We now determine differential GAG composition and function in airway, vascular, and alveolar-enriched regions of decellularized lungs obtained from normal, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) patients. Significant differences were observed in heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA) content and CS/HS compositions between both different lung regions and between normal and diseased lungs. Surface plasmon resonance demonstrated that HS and CS from decellularized normal and COPD lungs similarly bound fibroblast growth factor 2, but that binding was decreased in decellularized IPF lungs. Binding of transforming growth factor ß to CS was similar in all three groups but binding to HS was decreased in IPF compared to normal and COPD lungs. In addition, cytokines dissociate faster from the IPF GAGs than their counterparts. The differences in cytokine binding features of IPF GAGs may result from different disaccharide compositions. The purified HS from IPF lung is less sulfated than that from other lungs, and the CS from IPF contains more 6-O-sulfated disaccharide. These observations provide further information for understanding functional roles of ECM GAGs in lung function and disease. STATEMENT OF SIGNIFICANCE: Lung transplantation remains limited due to donor organ availability and need for life-long immunosuppressive medication. One solution, the ex vivo bioengineering of lungs via de- and recellularization has not yet led to a fully functional organ. Notably, the role of glycosaminoglycans (GAGs) remaining in decellularized lung scaffolds is poorly understood despite their important effects on cell behaviors. We have previously investigated residual GAG content of native and decellularized lungs and their respective functionality, and role during scaffold recellularization. We now present a detailed characterization of GAG and GAG chain content and function in different anatomical regions of normal diseased human lungs. These are novel and important observations that further expand knowledge about functional GAG roles in lung biology and disease.

Ganoderenic acid D-loaded functionalized graphene oxide-based carrier for active targeting therapy of cervical carcinoma.

Ganoderenic acid D (GAD), extracted from the Chinese herb Ganoderma lucidum, was loaded onto a graphene oxide-polyethylene glycol-anti-epidermal growth factor receptor (GO-PEG-EGFR) carrier to develop a targeting antitumor nanocomposite (GO-PEG@GAD). The carrier was fabricated from PEG and anti-EGFR aptamer modified GO. Targeting was mediated by the grafted anti-EGFR aptamer, which targets the membrane of HeLa cells. Physicochemical properties were characterized by transmission electron microscopy, dynamic light scattering, X-ray powder diffraction, and Fourier transform infrared spectroscopy. High loading content (77.3 % ± 1.08 %) and encapsulation efficiency (89.1 % ± 2.11 %) were achieved. Drug release continued for approximately 100 h. The targeting effect both in vitro and in vivo was confirmed by confocal laser scanning microscopy (CLSM) and imaging analysis system. The mass of the subcutaneous implanted tumor was significantly decreased by 27.27 ± 1.23 % after treatment with GO-PEG@GAD compared with the negative control group. Moreover, the in vivo anti-cervical carcinoma activity of this medicine was due to activation of the intrinsic mitochondrial pathway.

Designing Electrical Stimulation Platforms for Neural Cell Cultivation Using Poly(aniline): Camphorsulfonic Acid.

Electrical stimulation is a powerful strategy to improve the differentiation of neural stem cells into neurons. Such an approach can be implemented, in association with biomaterials and nanotechnology, for the development of new therapies for neurological diseases, including direct cell transplantation and the development of platforms for drug screening and disease progression evaluation. Poly(aniline):camphorsulfonic acid (PANI:CSA) is one of the most well-studied electroconductive polymers, capable of directing an externally applied electrical field to neural cells in culture. There are several examples in the literature on the development of PANI:CSA-based scaffolds and platforms for electrical stimulation, but no review has examined the fundamentals and physico-chemical determinants of PANI:CSA for the design of platforms for electrical stimulation. This review evaluates the current literature regarding the application of electrical stimulation to neural cells, specifically reviewing: (1) the fundamentals of bioelectricity and electrical stimulation; (2) the use of PANI:CSA-based systems for electrical stimulation of cell cultures; and (3) the development of scaffolds and setups to support the electrical stimulation of cells. Throughout this work, we critically evaluate the revised literature and provide a steppingstone for the clinical application of the electrical stimulation of cells using electroconductive PANI:CSA platforms/scaffolds.

The Sea Cucumber Thyonella gemmata Contains a Low Anticoagulant Sulfated Fucan with High Anti-SARS-CoV-2 Actions against Wild-Type and Delta Variants.

In this work, we isolated two new sulfated glycans from the body wall of the sea cucumber Thyonella gemmata: one fucosylated chondroitin sulfate (TgFucCS) (17.5 ± 3.5% kDa) and one sulfated fucan (TgSF) (383.3 ± 2.1% kDa). NMR results showed the TgFucCS backbone composed of [→3)-ß-N-acetylgalactosamine-(1→4)-ß-glucuronic acid-(1→] with 70% 4-sulfated and 30% 4,6-disulfated GalNAc units and one-third of the GlcA units decorated at the C3 position with branching α-fucose (Fuc) units either 4-sulfated (65%) or 2,4-disulfated (35%) and the TgSF structure composed of a tetrasaccharide repeating unit of [→3)-α-Fuc2,4S-(1→2)-α-Fuc4S-(1→3)-α-Fuc2S-(1→3)-α-Fuc2S-(1→]n. Inhibitory properties of TgFucCS and TgSF were investigated using SARS-CoV-2 pseudovirus coated with S-proteins of the wild-type (Wuhan-Hu-1) or the delta (B.1.617.2) strains and in four different anticoagulant assays, comparatively with unfractionated heparin. Molecular binding to coagulation (co)-factors and S-proteins was investigated by competitive surface plasmon resonance spectroscopy. Among the two sulfated glycans tested, TgSF showed significant anti-SARS-CoV-2 activity against both strains together with low anticoagulant properties, indicating a good candidate for future studies in drug development.

Interactions of heparin with key glycoproteins of human respiratory syncytial virus.

Introduction: The unexpected surge of respiratory syncytial virus (RSV) cases following pandemic phase of COVID-19 has drawn much public attention. Drawing on the latest antiviral research, revisiting this heightened annual outbreak of respiratory disease could lead to new treatments. The ability of sulfated polysaccharides to compete for a variety of viruses binding to cell surface heparan sulfate, suggests several drugs that might have therapeutic potential for targeting RSV-glycosaminoglycan interactions. Methods: In the current study, the binding affinity and kinetics of two RSV glycoproteins (RSV-G protein and RSV-F protein) to heparin were investigated by surface plasmon resonance. Furthermore, solution competition studies using heparin oligosaccharides of different lengths indicated that the binding of RSV-G protein to heparin is size-dependent, whereas RSV-F protein did not show any chain length preference. Results and discussion: The two RSV glycoproteins have slightly different preferences for heparin sulfation patterns, but the N-sulfo group in heparin was most critical for the binding of heparin to both RSV-G protein and RSV-F protein. Finally, pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for their inhibition of the RSV-G protein and RSV-F protein-heparin interaction, and both highly negative compounds showed strong inhibition.

SPR Sensor-Based Analysis of the Inhibition of Marine Sulfated Glycans on Interactions between Monkeypox Virus Proteins and Glycosaminoglycans.

Sulfated glycans from marine organisms are excellent sources of naturally occurring glycosaminoglycan (GAG) mimetics that demonstrate therapeutic activities, such as antiviral/microbial infection, anticoagulant, anticancer, and anti-inflammation activities. Many viruses use the heparan sulfate (HS) GAG on the surface of host cells as co-receptors for attachment and initiating cell entry. Therefore, virion-HS interactions have been targeted to develop broad-spectrum antiviral therapeutics. Here we report the potential anti-monkeypox virus (MPXV) activities of eight defined marine sulfated glycans, three fucosylated chondroitin sulfates, and three sulfated fucans extracted from the sea cucumber species Isostichopus badionotus, Holothuria floridana, and Pentacta pygmaea, and the sea urchin Lytechinus variegatus, as well as two chemically desulfated derivatives. The inhibitions of these marine sulfated glycans on MPXV A29 and A35 protein-heparin interactions were evaluated using surface plasmon resonance (SPR). These results demonstrated that the viral surface proteins of MPXV A29 and A35 bound to heparin, which is a highly sulfated HS, and sulfated glycans from sea cucumbers showed strong inhibition of MPXV A29 and A35 interactions. The study of molecular interactions between viral proteins and host cell GAGs is important in developing therapeutics for the prevention and treatment of MPXV.

Fucosylated heparan sulfate from the midgut gland of Patinopecten yessoensis.

The structural and functional relationships of glycosaminoglycans (GAGs) derived from marine organisms have been investigated, suggesting that marine invertebrates, particularly Bivalvia, are abundant sources of highly sulfated or branched GAGs. In this study, we identified a novel fucosylated heparan sulfate (Fuc-HS) from the midgut gland of the Japanese scallop, Patinopecten yessoensis. Scallop HS showed resistance to GAG-degrading enzymes, including chondroitinases and heparinases, and susceptibility to heparinases increased when scallop HS was treated with mild acid hydrolysis, which removes the fucosyl group. Moreover, 1H NMR detected significant signals near 1.2-1.3 ppm corresponding to the H-6 methyl proton of fucose residues and small H-3 (3.59 ppm) or H-2 (3.39 ppm) signals of glucuronate (GlcA) were detected, suggesting that the fucose moiety is attached to the C-3 position of GlcA in scallop HS. GC-MS detected peaks corresponding to 1, 3, 5-tri-O-acetyl-2, 4-di-O-methyl-L-fucitol and 1, 4, 5-tri-O-acetyl-2, 3-di-O-methyl-L-fucitol, suggesting that the fucose moiety is 3-O- or 4-O-sulfated. Furthermore, scallop HS showed anti-coagulant and neurite outgrowth-promoting (NOP) activities. These results suggest that the midgut gland of scallops is a valuable source of Fuc-HS with biological activities.

Dynamic Changes in Heparan Sulfate Nanostructure in Human Pluripotent Stem Cell Differentiation.

Heparan sulfate (HS) is a heterogeneous, cell-surface polysaccharide critical for transducing signals essential for mammalian development. Imaging of signaling proteins has revealed how their localization influences their information transfer. In contrast, the contribution of the spatial distribution and nanostructure of information-rich, signaling polysaccharides like HS is not known. Using expansion microscopy (ExM), we found striking changes in HS nanostructure occur as human pluripotent stem (hPS) cells differentiate, and these changes correlate with growth factor signaling. Our imaging studies show that undifferentiated hPS cells are densely coated with HS displayed as hair-like protrusions. This ultrastructure can recruit fibroblast growth factor for signaling. When the hPS cells differentiate into the ectoderm lineage, HS is localized into dispersed puncta. This striking change in HS distribution coincides with a decrease in fibroblast growth factor binding to neural cells. While developmental variations in HS sequence were thought to be the primary driver of alterations in HS-mediated growth factor signaling, our high-resolution images indicate a role for the HS nanostructure. Our study highlights the utility of high-resolution glycan imaging using ExM. In the case of HS, we found that changes in how the polysaccharide is displayed link to profound differences in growth factor binding.

Absolute pharmacokinetics of heparin in primates.

Heparin is a commonly used anticoagulant drug, derived from the tissues of animals including pigs, cows, and sheep. Measuring heparin concentration in plasma is challenging due to its complex molecular structure. Existing methods rely on measuring heparin's anticoagulant activity, which provides pharmacodynamic (PD) data but not pharmacokinetic (PK) data, measuring concentration over time. To overcome this limitation, we used liquid chromatography-mass spectrometry (LC-MS) and the multiple reaction monitoring (MRM) method to directly measure heparin's concentration in non-human primates after administering porcine, bovine, and ovine heparin. A protocol was developed to enable an MRM method for application to small plasma volumes without purification. The PK data obtained from LC-MS are then compared with the data obtained using the Heparin Red assay and the PD data determined using biochemical clinical assays. Results showed that LC-MS and Heparin Red assay measurements closely correlated with unfractionated heparin's biological activities, supporting the use of mass spectra and dye-binding assays to determine heparin levels in plasma. This study builds a way for the measurement of heparin concentration in plasma, which could lead to an improved understanding of heparin's metabolism and dosing safety.

Suramin binds and inhibits infection of SARS-CoV-2 through both spike protein-heparan sulfate and ACE2 receptor interactions.

SARS-CoV-2 receptor binding domains (RBDs) interact with both the ACE2 receptor and heparan sulfate on the surface of host cells to enhance SARS-CoV-2 infection. We show that suramin, a polysulfated synthetic drug, binds to the ACE2 receptor and heparan sulfate binding sites on the RBDs of wild-type, Delta, and Omicron variants. Specifically, heparan sulfate and suramin had enhanced preferential binding for Omicron RBD, and suramin is most potent against the live SARS-CoV-2 Omicron variant (B.1.1.529) when compared to wild type and Delta (B.1.617.2) variants in vitro. These results suggest that inhibition of live virus infection occurs through dual SARS-CoV-2 targets of S-protein binding and previously reported RNA-dependent RNA polymerase inhibition and offers the possibility for this and other polysulfated molecules to be used as potential therapeutic and prophylactic options against COVID-19.

One-Pot Self-Assembly of Core-Shell Nanoparticles within Fibers by Coaxial Electrospinning for Intestine-Targeted Delivery of Curcumin.

Nanotechniques for curcumin (Cur) encapsulation provided a potential capability to avoid limitations and improve biological activities in food and pharmaceutics. Different from multi-step encapsulation systems, in this study, zein-curcumin (Z-Cur) core-shell nanoparticles could be self-assembled within Eudragit S100 (ES100) fibers through one-pot coaxial electrospinning with Cur at an encapsulation efficiency (EE) of 96% for ES100-zein-Cur (ES100-Z-Cur) and EE of 67% for self-assembled Z-Cur. The resulting structure realized the double protection of Cur by ES100 and zein, which provided both pH responsiveness and sustained release performances. The self-assembled Z-Cur nanoparticles released from fibermats were spherical (diameter 328 nm) and had a relatively uniform distribution (polydispersity index 0.62). The spherical structures of Z-Cur nanoparticles and Z-Cur nanoparticles loaded in ES100 fibermats could be observed by transmission electron microscopy (TEM). Fourier transform infrared spectra (FTIR) and X-ray diffractometer (XRD) revealed that hydrophobic interactions occurred between the encapsulated Cur and zein, while Cur was amorphous (rather than in crystalline form). Loading in the fibermat could significantly enhance the photothermal stability of Cur. This novel one-pot system much more easily and efficiently combined nanoparticles and fibers together, offering inherent advantages such as step economy, operational simplicity, and synthetic efficiency. These core-shell biopolymer fibermats which incorporate Cur can be applied in pharmaceutical products toward the goals of sustainable and controllable intestine-targeted drug delivery.