The sweetest polymer nanoparticles: opportunities ahead for glycogen in nanomedicine.

Most cells take simple sugar (α-D-glucose) and assemble it into highly dense polysaccharide nanoparticles called glycogen. This is achieved through the action of multiple coupled-enzymatic reactions, yielding the cellular store of polymerised glucose to be degraded in times of metabolic need. These nanoparticles can be readily isolated from various animal tissues and plants, and are commercially available on a large scale. Importantly, glycogen is highly water soluble, non-toxic, low-fouling, and biodegradable, making it an attractive nanoparticle for use in nanomedicine, for both diagnosing and treating disease. This concept has been pursued actively recently, with exciting results on a variety of fronts, especially for targeting specific tissues and delivering nucleic acid and peptide cargo. In this perspective, the role of glycogen in nanomedicine going forward is discussed, with opportunities highlighted of where these sugary nanoparticles fit into the problem of treating disease.

Characterization and applications of biomacromolecule structurally similar to glycogen as a dispersion aid and skin protection agent.

Glycogen is a naturally occurring or metabolically synthesized biological macromolecule found in a wide range of living organisms, including animals, microorganisms, and even plants. However, naturally sourced glycogen poses challenges for industrial use. This study focused on a biological macromolecule referred to as glycogen-like particles (GLPs), detailing the production methods and biological properties of these particles. In vitro enzymatic production of GLPs was successfully achieved. GLPs synthesized through a simultaneous enzymatic reaction using sucrose had significant changes in their structure and functionality based on the branching enzyme (BE) to amylosucrase (ASase) ratio. As this ratio increased, the GLPs developed higher molecular weights and greater density, solubility, and branching degree while reducing size and turbidity. Structural changes in these enzymes were not observed beyond a critical BE/ASase ratio. Uniformly dispersed curcumin powder was generated in 50 % (w/v) aqueous GLP solution, and the GLPs were non-toxic to human skin keratinocytes at a concentration of 2.5 mg/mL. GLPs with lower branching inhibited tyrosinase activity and melanin synthesis, while those with more long chains displayed effective UV-blocking. By manipulating the BE/ASase ratio, GLPs were shown to display diverse chemical structures and physical characteristics, suggesting their potential application in the food and cosmetics industries.

Fabrication of phytoglycogen-derived core-shell nanoparticles: Structure and characterizations.

The objective of this work was to investigate the fabrication of core-shell nanoparticles using phosphorylase-catalyzed chain extension of phytoglycogen, and to analyze the changes of structure and characterizations in detail. During the glucosylation reaction, the inorganic phosphate increased substantially up to 2.3 mg/mL in the initial 12 h, and then increased incrementally to 2.5 mg/mL at 24 h. The similar to trends was observed for increasing Mw and Rz over time, due to glucosyl transfers on the surface chain to form a corona around the phytoglycogen core with a larger size. Phosphorylase modification increases the percentages of longer chain fractions and the average chain length increased from degree of polymerization (DP) 11.6 to DP 48.2. The modified phytoglycogen exhibited the characteristic of B-type crystalline structure, indicating that the specific core-shell nanoparticle with inner amorphous nature and outer crystalline layer. The above results revealed that the potentiality of enzymatic chain elongation of phytoglycogen to design novel core-shell nanoparticle with tailor-made structure and functionality.

Comparative transcriptome analysis of diurnal alterations of liver glycogen structure: A pilot study.

Molecular mechanisms behind structural alterations between fragile and stable glycogen α particles in liver are not clear yet. In this pilot study, we re-examined the diurnal alterations of glycogen structure from the perspective of liver tissue transcriptome. By comparing the structures of liver glycogen from mice at 12 am, 8 am, 12 pm, and 8 pm (light-on: 6 am; light-off: 6 pm), we re-confirmed that the liver glycogen was fragile at 12 am and 8 am and stable at 12 pm and 8 pm as previously reported. The structural differences of glycogen particles at 12 am and 12 pm were thoroughly compared via transcriptomics. Differentially expressed genes (DEGs) with statistical significance were identified, while expression level of the gene ppp1r3g (log2Fold_Change = -6.368, P-value = 2.89E-04) that encoded PPP1R3G with glycogen binding domain was most significantly changed, which provided preliminary clues to the structural alterations of glycogen α particles during the diurnal cycle.

Entrapment of Proteins Within Columns for High-Performance Affinity Chromatography.

Entrapment is a noncovalent immobilization method that enables a large biological binding agent, such as a protein, to be put within a support without modifying the structure of the binding agent. This chapter describes an on-column entrapment method that can be used with proteins and HPLC-grade silica to prepare columns for high-performance liquid chromatography. In this method, a protein is trapped within a dihydrazide-activated silica support by using oxidized glycogen as a capping agent. This method allows the protein to be placed within the support in a soluble form and with little or no loss of activity. The approach and reagents needed for this method are described in this chapter, along with some applications reported for columns that have been made using on-column protein entrapment.

Triggering the nanophase separation of albumin through multivalent binding to glycogen for drug delivery in 2D and 3D multicellular constructs.

Engineered nanoparticles for the encapsulation of bioactive agents hold promise to improve disease diagnosis, prevention and therapy. To advance this field and enable clinical translation, the rational design of nanoparticles with controlled functionalities and a robust understanding of nanoparticle-cell interactions in the complex biological milieu are of paramount importance. Herein, a simple platform obtained through the nanocomplexation of glycogen nanoparticles and albumin is introduced for the delivery of chemotherapeutics in complex multicellular 2D and 3D systems. We found that the dendrimer-like structure of aminated glycogen nanoparticles is key to controlling the multivalent coordination and phase separation of albumin molecules to form stable glycogen-albumin nanocomplexes. The pH-responsive glycogen scaffold conferred the nanocomplexes the ability to undergo partial endosomal escape in tumour, stromal and immune cells while albumin enabled nanocomplexes to cross endothelial cells and carry therapeutic agents. Limited interactions of nanocomplexes with T cells, B cells and natural killer cells derived from human blood were observed. The nanocomplexes can accommodate chemotherapeutic drugs and release them in multicellular 2D and 3D constructs. The drugs loaded on the nanocomplexes retained their cytotoxic activity, which is comparable with the activity of the free drugs. Cancer cells were found to be more sensitive to the drugs in the presence of stromal and immune cells. Penetration and cytotoxicity of the drug-loaded nanocomplexes in tumour mimicking tissues were validated using a 3D multicellular-collagen construct in a perfusion bioreactor. The results highlight a simple and potentially scalable strategy for engineering nanocomplexes made entirely of biological macromolecules with potential use for drug delivery.

Structural elements determining the transglycosylating activity of glycoside hydrolase family 57 glycogen branching enzymes.

Glycoside hydrolase family 57 glycogen branching enzymes (GH57GBE) catalyze the formation of an α-1,6 glycosidic bond between α-1,4 linked glucooliogosaccharides. As an atypical family, a limited number of GH57GBEs have been biochemically characterized so far. This study aimed at acquiring a better understanding of the GH57GBE family by a systematic sequence-based bioinformatics analysis of almost 2500 gene sequences and determining the branching activity of several native and mutant GH57GBEs. A correlation was found in a very low or even no branching activity with the absence of a flexible loop, a tyrosine at the loop tip, and two ß-strands.

Differentiated structure of synthetic glycogen-like particle by the combined action of glycogen branching enzymes and amylosucrase.

Glycogen-like particles (GLPs) were built up from sucrose by applying de novo one-pot enzymatic process of amylosucrase (ASase; 6 U·mL-1) and glycogen branching enzymes (GBEs; 0.001 and 0.005 U·mL-1). Due to different chain-length transferring patterns of GBEs, structurally differentiated GLPs were synthesized. Yields of GLPs synthesized at pH 7.0 and 30 °C were improved by increasing the GBE/ASase ratio. Branching degrees of GLPs obviously was increased along with the ratio of GBEs, of which result was directly supported by shortened branch-chain length with greater GBE activity. Long branch chains seemed to play as efficient acceptor molecules to bind newly transferred branch chains especially at lower ratio of GBE/ASase, resulting in greater molecular weight and size of GLP with higher proportion of them. Molecular weight, size, and density of GLPs were ranged from 7.37 × 105 to 1.94 × 108 g·mol-1, from 23.70 to 52.65 nm, and from 7.99 to 374.32 g·mol-1·nm-3, respectively. By increasing GBE/ASase ratio, more compact GLP architecture was fabricated due to increased weight and reduced size with exception of a unique GBE. GLPs were efficiently synthesized by two different glycosyltransferases, and their chemical structures were controllable by source and ratio of GBEs due to their different branch-chain transferring specificity.

Crystal structures of glycogen-debranching enzyme mutants in complex with oligosaccharides.

Debranching is a critical step in the mobilization of the important energy store glycogen. In eukaryotes, including fungi and animals, the highly conserved glycogen-debranching enzyme (GDE) debranches glycogen by a glucanotransferase (GT) reaction followed by a glucosidase (GC) reaction. Previous work indicated that these reactions are catalyzed by two active sites located more than 50 Šapart and provided insights into their catalytic mechanisms and substrate recognition. Here, five crystal structures of GDE in complex with oligosaccharides with 4-9 glucose residues are presented. The data suggest that the glycogen main chain plays a critical role in binding to the GT and GC active sites of GDE and that a minimum of five main-chain residues are required for optimal binding.

Ultrasound-Assisted Microencapsulation of Soybean Oil and Vitamin D Using Bare Glycogen Nanoparticles.

Ultrasonically synthesized core-shell microcapsules can be made of synthetic polymers or natural biopolymers, such as proteins and polysaccharides, and have found applications in food, drug delivery and cosmetics. This study reports on the ultrasonic synthesis of microcapsules using unmodified (natural) and biodegradable glycogen nanoparticles derived from various sources, such as rabbit and bovine liver, oyster and sweet corn, for the encapsulation of soybean oil and vitamin D. Depending on their source, glycogen nanoparticles exhibited differences in size and 'bound' proteins. We optimized various synthetic parameters, such as ultrasonic power, time and concentration of glycogens and the oil phase to obtain stable core-shell microcapsules. Particularly, under ultrasound-induced emulsification conditions (sonication time 45 s and sonication power 160 W), native glycogens formed microcapsules with diameter between 0.3 µm and 8 µm. It was found that the size of glycogen as well as the protein component play an important role in stabilizing the Pickering emulsion and the microcapsules shell. This study highlights that native glycogen nanoparticles without any further tedious chemical modification steps can be successfully used for the encapsulation of nutrients.

Porous particles and novel carrier particles with enhanced penetration for efficient pulmonary delivery of antitubercular drugs.

This study aimed to design dry powder inhaler formulations using a hydrophilic polymeric polysaccharide, phytoglycogen (PyG), as a multi-functional additive that increases the phagocytic activity of macrophage-like cells and enhances pulmonary delivery of drugs. The safety and usefulness of PyG were determined using in vitro cell-based studies. Dry powder inhaler formulations of an antitubercular drug, rifampicin, were fabricated by spray drying with PyG. The cytotoxicity, effects on phagocytosis, particle size, and morphology were evaluated. The aerosolization properties of the powder formulations were evaluated using an Andersen cascade impactor (ACI). Scanning electron microscope images of the particles on each ACI stage were captured to observe the deposition behavior. PyG showed no toxicity in A549, Calu-3, or RAW264.7 cell lines. At concentrations of 0.5 and 1 g/L, PyG facilitated the cellular uptake of latex beads and the expression of pro-inflammatory cytokine genes in RAW264.7 cells. Formulations with outstanding inhalation potential were produced. The fine particle fraction (aerodynamic size 2-7 µm) of the porous particle batch reached nearly 60%, whereas in the formulation containing wrinkled carrier particles, the extra-fine particle fraction (aerodynamic particle size < 2 µm) was 25.0% ± 1.7%. The deposition of porous and wrinkled particles on individual ACI stages was distinct. The inclusion of PyG dramatically improved the inhalation performance of porous and wrinkled powder formulations. These easily inhaled immunostimulatory carrier particles may advance the state of research by enhancing the therapeutic effect and alveolar delivery of antitubercular drugs.

Optimization and Validation of an In Vitro Standardized Glycogen Phosphorylase Activity Assay.

Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway and a potential therapeutic target in the management of type 2 diabetes. It catalyzes a reversible reaction: the release of the terminal glucosyl residue from glycogen as glucose 1-phosphate; or the transfer of glucose from glucose 1-phosphate to glycogen. A colorimetric method to follow in vitro the activity of GP with usefulness in structure-activity relationship studies and high-throughput screening capability is herein described. The obtained results allowed the choice of the optimal concentration of enzyme of 0.38 U/mL, 0.25 mM glucose 1-phosphate, 0.25 mg/mL glycogen, and temperature of 37 °C. Three known GP inhibitors, CP-91149, a synthetic inhibitor, caffeine, an alkaloid, and ellagic acid, a polyphenol, were used to validate the method, CP-91149 being the most active inhibitor. The effect of glucose on the IC50 value of CP-91149 was also investigated, which decreased when the concentration of glucose increased. The assay parameters for a high-throughput screening method for discovery of new potential GP inhibitors were optimized and standardized, which is desirable for the reproducibility and comparison of results in the literature. The optimized method can be applied to the study of a panel of synthetic and/or natural compounds, such as polyphenols.

Nanoparticles composed of the tea polysaccharide-complexed cationic vitamin B12-conjugated glycogen derivative.

Tea polysaccharides exhibit multiple important bioactivities, but very few of them can be absorbed through the small intestine. To enhance the absorption efficacy of tea polysaccharides, a cationic vitamin B12-conjugated glycogen derivative bearing the diethylenetriamine residues (VB12-DETA-Gly) was synthesized and characterized using FTIR, 1H NMR, and UV-vis spectroscopy. An acidic tea polysaccharide (TPSA) was isolated from green tea. The TPSA/VB12-DETA-Gly complexed nanoparticles were prepared, which showed positive zeta potentials and were irregular spherical nanoparticles in the sizes of 50-100 nm. To enable the fluorescence and UV-vis absorption properties of TPSA, a Congo red residue-conjugated TPSA derivative (CR-TPSA) was synthesized. The interactions and complexation mechanism between the CR-TPSA and the VB12-DETA-Gly derivatives were investigated using fluorescence spectroscopy, resonance light scattering spectroscopy, and UV-vis spectroscopy. The results indicated that the electrostatic interaction could play a major role during the CR-TPSA and VB12-DETA-Gly-II complexation processes. The TPSA/VB12-DETA-Gly nanoparticles were nontoxic and exhibited targeted endocytosis for the Caco-2 cells, and showed high permeation through intestinal enterocytes using the Caco-2 cell model. Therefore, they exhibit potential for enhancing the absorption efficacy of tea polysaccharides through the small intestinal mucosa.

Glycogen nanoparticles as a potential corrosion inhibitor.

Biological macromolecules are proven to be potential green corrosion inhibitors because of their outstanding structural features and eco-friendliness. This study is aimed at enhancing their corrosion mitigation capabilities by converting them into nanoparticles. This is the first work where nanoparticles of biological macromolecules are exploited for corrosion mitigation studies. Glycogen nanoparticles (GLY-Np) were synthesized by microwave-mediated nanoprecipitation method and characterized by ATR-FTIR, XRD, UV-Visible Spectroscopy, FESEM analysis, EDX, TEM, and Zeta potential measurements. They are used as an eco-friendly inhibitor for corrosion control of zinc in sulfamic acid (NH2SO3H). The electrochemical study was a primary experimental tool employed for corrosion rate measurement. Conditions were optimized to obtain maximum inhibition efficiency by varying concentrations of inhibitor and temperature. Activation and thermodynamic parameters were evaluated and discussed in detail. A suitable adsorption isotherm was proposed to fit the experimental results. Adsorption of the inhibitor was confirmed by SEM, EDX, and AFM techniques. The inhibition efficiency of 92% was obtained for 0.02 gL-1 GLY-Np. Thus, GLY-Np turned out to be an effective green inhibition with economic benefits.

Automated Assembly of Starch and Glycogen Polysaccharides.

Polysaccharides are Nature's most abundant biomaterials essential for plant cell wall construction and energy storage. Seemingly minor structural differences result in entirely different functions: cellulose, a ß (1-4) linked glucose polymer, forms fibrils that can support large trees, while amylose, an α (1-4) linked glucose polymer forms soft hollow fibers used for energy storage. A detailed understanding of polysaccharide structures requires pure materials that cannot be isolated from natural sources. Automated Glycan Assembly provides quick access to trans-linked glycans analogues of cellulose, but the stereoselective installation of multiple cis-glycosidic linkages present in amylose has not been possible to date. Here, we identify thioglycoside building blocks with different protecting group patterns that, in concert with temperature and solvent control, achieve excellent stereoselectivity during the synthesis of linear and branched α-glucan polymers with up to 20 cis-glycosidic linkages. The molecules prepared with the new method will serve as probes to understand the biosynthesis and the structure of α-glucans.

Optimization of liver glycogen extraction when considering the fine molecular structure.

Liver glycogen is a branched glucose polymer that functions as a blood-sugar buffer in animals. Previous studies have shown that glycogen's molecular structure affects its properties. This makes it important to develop a technique that extracts and purifies a representative sample of glycogen. Here we aim to optimize the sucrose density gradient centrifugation method for preserving glycogen's molecular structure by varying the density of the sucrose solution. The preservation of glycogen's structure involves: 1) minimizing molecular damage and 2) obtaining a structurally representative sample of glycogen. The addition of a 10-minute boiling step was also tested as a means for denaturing any glycogen degrading enzymes. Lower sucrose concentrations and the introduction of the boiling step were shown to be beneficial in obtaining a more structurally representative sample, with the preservation of smaller glycogen particles and decreased glycogen chain degradation.

The dynamic changes of glycogen molecular structure in Escherichia coli BL21(DE3).

Diurnal alteration of glycogen molecular structure has been identified in healthy mice. Recently, both fragile (disintegration in dimethyl sulfoxide) and stable (not disintegrating in DMSO) glycogen particles were found in Escherichia coli. However, how glycogen structure changes dynamically in E. coli is not clear. The question examined here is whether fragile, stable glycogen α particles occur in bacteria, following a similar pattern as in mice. In this study, we examine the dynamic changes of glycogen molecular structure over 24-h in E. coli BL21(DE3), using transmission electron microscopy, size exclusion chromatography and fluorophore-assisted carbohydrate electrophoresis at representative time points. It was found that glycogen structure was mainly fragile at the synthesis stage and largely stable during the degradation stage. qRT-PCR results indicated that balance of anabolic and catabolic gene expression levels in glycogen metabolism could be a key factor affecting the fragility of glycogen α particles in bacteria.

Inhibition of glutaminolysis in combination with other therapies to improve cancer treatment.

Targeting glutamine catabolism has been attracting more research attention on the development of successful cancer therapy. Catalytic enzymes such as glutaminase (GLS) in glutaminolysis, a series of biochemical reactions by which glutamine is converted to glutamate and then alpha-ketoglutarate, an intermediate of the tricarboxylic acid (TCA) cycle, can be targeted by small molecule inhibitors, some of which are undergoing early phase clinical trials and exhibiting promising safety profiles. However, resistance to glutaminolysis targeting treatments has been observed, necessitating the development of treatments to combat this resistance. One option is to use synergy drug combinations, which improve tumor chemotherapy's effectiveness and diminish drug resistance and side effects. This review will focus on studies involving the glutaminolysis pathway and diverse combination therapies with therapeutic implications.