1

Glycogen as a Building Block for Advanced Biological Materials

Quinn A. Besford, Francesca Cavalieri,* and Frank Caruso*

Dr. Q. A. Besford†, Dr. F. Cavalieri, Prof. F. Caruso

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, and the

Department of Chemical Engineering, The University of Melbourne, Parkville, Victoria 3010,

Australia

E-mail: francesca.cavalieri@unimelb.edu.au, fcaruso@unimelb.edu.au

Dr. F. Cavalieri

Dipartimento di Scienze e Tecnologie Chimiche, Università degli Studi di Roma Tor Vergata,

via della Ricerca Scientifica 1, 00133, Rome, Italy

†Present address: Department of Nanostructured Materials, Leibniz Institute of Polymer

Research, Hohe Str. 6, D-01069 Dresden, Germany

Keywords: glycogen, nanoparticles, polysaccharides, therapeutic materials, biodegradable

materials

Biological nanoparticles found in living systems possess distinct molecular architectures and

diverse functions. Glycogen is a unique biological polysaccharide nanoparticle fabricated by

nature thorough a bottom-up approach. The biocatalytic synthesis of glycogen has evolved over

millions of years to form a nanometer-sized dendrimer-like structure (20–150 nm) with a highly

branched surface and a dense core. This makes glycogen markedly different from other natural

linear or branched polysaccharides and particularly attractive as a platform for biomedical

applications. Glycogen is inherently biodegradable, nontoxic, and can be functionalized with

diverse surface and internal motifs for enhanced biofunctional properties. Recently, there has

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been growing interest in glycogen as a natural alternative to synthetic polymers and

nanoparticles in a range of applications. Herein, the recent literature on glycogen in the

material-based sciences, including its use as a constituent in biodegradable hydrogels and fibers,

drug delivery vector, tumor targeting and penetrating nanoparticle, immunomodulator, vaccine

adjuvant, and contrast agent, is reviewed. The various methods of chemical functionalization

and physical assembly of glycogen nanoparticles into multicomponent nanodevices, that

advance glycogen toward a functional therapeutic nanoparticle from nature and back again, are

discussed in detail.

1. Introduction

Innovations in the preparation of biofunctional polymeric nanoparticles for biomedical use have

primarily focused on synthetic polymers.[1] Complex multifunctional nanoparticles endowed

with targeting molecules, drugs, prodrugs, tracking moieties, and imaging agents have been

developed.[2] The nanoparticle fabrication routes may require multiple starting chemical

components, organic solvents, and at times laborious difficult-to-control manufacturing

processes. This can lead to complex drug delivery systems that are challenging to scale up and

therefore have limited clinical and commercial translation. Although numerous nanoparticles

have been widely developed and used for various applications, including biomedical

applications[2] there is a growing interest in nontoxic and degradable alternatives to first-

generation synthetic nanomaterials. Ideally the synthesis of such nanoparticles needs to be cost

effective, simple, green, reproducible, scalable, and the constitutive building blocks of the

nanoparticles should be nontoxic, functional, biodegradable and available on a large scale.

Naturally occurring nanoparticles could be such a valid alternative. These nanoparticles include

intracellular structures, such as magnetosomes[3] and glycogen, and extracellular assemblies

such as lipoproteins and viruses.[3] Among these, glycogen is the most abundant and versatile

biological nanoparticle, which fulfils the requirements for the fabrication of nanostructured

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biomaterials. Glycogen is nature’s prime nanoparticle that exists in most organisms, from

bacteria and archaea to humans,[4, 5] as a vital component of the cellular energy machinery. It is

a highly branched polysaccharide comprising repeating units of glucose connected by linear α-

D-(1,4) glycosidic linkages with α-D-(1,6) branching, structured into roughly spherical

nanoparticles that have a high water solubility and molecular weight.

The use of polysaccharides as components for advanced materials has been an attractive

concept for decades.[6, 7] A key motive is that polysaccharides, as a natural biomaterial, are

endowed with a level of biodegradability and biocompatibility. In addition, they can be easily

modified chemically or biochemically to produce functional derivatives, which important s for

various therapeutic applications.[8] The structures of polysaccharides vary considerably but span

from linear to highly branched polymers with molecular weights ranging from the thousands to

the millions, composed of mono- or disaccharides, or short-chain oligomers bound together by

glycosidic linkages.[9] There is a rich history of research on the use of polysaccharides in a range

of therapeutic applications including: (i) soft tissue engineering,[10, 11] where the materials can

be designed to mimic the mechanical properties of the extracellular matrix in the tissue; (ii) as

drug, protein, or gene delivery systems,[7, 12, 13] where polysaccharides have a large number of

reactive groups[14] that allow for tunable properties[12] such as pH-responsiveness;[15] and (iii)

as nanoprobes for cellular and tissue imaging.[16, 17] Of the available polysaccharides, the most

studied biopolymers for use as therapeutic materials are arguably derivatives or composites of

cellulose,[18] chitosan,[19] hyaluronic acid,[20] and/or alginate.[21] Despite the wealth of literature

on polysaccharide materials, there are few studies on the use of the glycogen as a functional

material.[22] Research into the role of glycogen in physiological processes has a long and rich

history. More than 160 years ago, Claude Bernard reported to the Société de Biologie in Paris

the isolation of glycogen from liver tissue including a preliminary study of its chemical and

physical properties,[23] a remarkable feat of the times, to which our understanding of this

polysaccharide has only blossomed since. Various methods have been developed to isolate

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glycogen from living organisms, primarily for determining the structure and amount of

glycogen accumulated in biological samples,[24-26] rather than for using glycogen as a

biopolymer in diverse applications.

Glycogen is currently obtained commercially though extraction processes from animal tissues

or sweet corn.[24] To impart functionality to glycogen nanoparticles, numerous methods

involving carbohydrate chemistry and biochemistry can be used, including synthetic and

enzymatic methods. Modifications can be performed either on the surface or the interior of the

particles. Chemical modifications can be preferentially performed in aqueous environments

under mild conditions and using nontoxic components. The polysaccharide chains can be

functionalized[27] and the nanoparticles remain partially biodegradable.[28] Together, these

properties potentially make glycogen nanoparticles well suited for use as a functional

nanomaterial in therapeutic applications. Given that glycogen already exists in organisms, the

use of glycogen as a material for the therapeutic treatment of disease is an interesting concept.

Herein, we review the literature on advancing glycogen nanoparticles, taken from biology,

toward therapeutic and biomedical applications.

This review first provides a detailed description of the role of glycogen in biology (Section 2),

its branching and physicochemical properties (Section 3), and methods to functionalize

glycogen both enzymatically (Section 4.1) and synthetically (Section 4.2). This is followed by

a review of the material applications of glycogen, including its interactions with inorganic

nanoparticles (Section 5.1), and its use in forming macroscopic materials (Section 5.2). We then

discuss its use as a solubilizing agent for therapeutic drugs (Section 5.3), as a nanocarrier for

therapeutic nucleic acids and we discuss the in vivo behavior of glycogen-based constructs. We

conclude this review by highlighting the future outlook of glycogen nanoparticles for use in

material-based applications (Section 6).

2. Glycogen in Biology

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In biology, glycogen is a key regulator of blood glucose homeostasis and cellular hydration and

is part of a constant cycle of synthesis and degradation depending on the metabolic state of the

tissue. In mammals, the liver and skeletal muscle are the two major deposits of glycogen,

constituting ~5–10 and ~2% of the hydrated weight of the organs,[29] respectively. Other organs,

including the heart[25] and brain,[30] also produce the polysaccharide but to a lesser extent.

Recently, the presence of glycogen has been reported in human milk, where its concentration

correlates to the level of inflammation in mastitis milk.[31]

Figure 1. Reaction schematic describing the biological synthesis of glycogen from the protein core. UDPG, uridine diphosphate glucose.

Glycogen is primarily synthesized by the cooperative action of three enzymes, namely glycogen

synthase, glycogenin, and glycogen-branching enzyme, using uridine diphosphate glucose as

the glucose donor (Figure 1).[32] Briefly, a glycogenin dimer initiates the polymer synthesis

through autoglucosylation of a tyrosine residue (Tyr195),[33] leading to an α-1,4-linked chain of

8–12 glucose units, which is subsequently converted into nanoparticles (20–50 nm in diameter)

through coordinated actions of glycogen synthase and glycogen branching enzyme.[32, 34] The

full description of glycogen synthesis and degradation, which is too broad to cover in the present

review, has been comprehensively reviewed elsewhere.[4, 35]

Cellular volume is correlated to the modulation of glycogen synthesis,[36] as storing glucose as

a part of glycogen reduces the osmotic stress on the cell.[30, 37] For example, approximately 400

mM of free glucose can be stored in 0.01 μM of liver glycogen.[38] The structure of glycogen has

been described as highly optimized toward its cellular function, with its branched structure

allowing for a dense compartmentalization of free glucose, providing the tissue with a readily

accessible form of energy.[37, 39] Further to its optimized structure, the subcellular localization

of glycogen is suggested to provide a substrate for specific cellular functions; skeletal muscle

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glycogen is highly ordered within intramuscular triglyceride deposits and mitochondria, placing

the polysaccharide in close proximity to its site of use.[39, 40]

3. Structure and Physicochemical Properties of Glycogen Nanoparticles

The structure of glycogen in solution can be described at three levels, with the first two levels

constituting randomly joined branches and branches linked together into roughly spherical

particles, called β-particles (Figure 2A).[41] From enzymatic degradation experiments, the

average chain length within the β-particles comprises 10–15 glucose units.[24, 42] For some

glycogen sources, typically the liver[43] and heart,[25] there is a third level of structure where

these β-particles are covalently joined[44] into supramolecular complex particles called α-

particles (Figure 2A). These α-particles hint at a tissue-specific structure–function relationship

as α- and β-particles are enzymatically degraded to release glucose at different rates.[45, 46]

Transmission electron microscopy (TEM) of glycogen particles from different sources

demonstrates significant morphological differences in the structure of glycogen (Figure 2B–E).

The β-particle units are typically 20–50 nm in diameter,[24] whereas the larger supramolecular

α-particles are polydisperse with a mean diameter of ~150 nm accompanied with the smaller

disperse β-particles.[45] Drochmans[47], in 1962, and Orpin et al.,[48] in 1976, reported electron

microscopy images and opalescence curves of rat liver and Moniezia expansa glycogen,

respectively, in a range of solutions of increasing pH (from less than 2 up to 7.4). The studies

confirmed the dissociation of the α-particles into β-particles at acidic pH, which is consistent

with recent dynamic light scattering data.[45]

The most common schematic structure of glycogen is drawn as a regular tree- or dendrimer-

like structure that contains tiers of glucose chains, or branches, ordered into a self-similar, or

fractal structure.[37, 49] However, recent small-angle X-ray scattering data of glycogen β-

particles[45] have allowed the application of robust polymer branching models that describe the

evolution of branching as a function of particle radius. This has shown that glycogen β-particles

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are best described as randomly hyperbranched polymer particles,[45, 50, 51] rather than fractal

structures, with a radius of gyration that scales with the logarithm of the molecular weight (i.e.,

Rg ~ log M).[51, 52] This has recently been complemented by Monte Carlo simulations that

suggest that glycogen particles have a higher density toward the core of the particles than at the

periphery (Figure 2F).[53] This type of random hyperbranching represents a natural principle for

energy storage in compact macromolecules.[54]

The degree of branching is an important parameter of hyperbranched polymers because it

directly correlates to the density of the polymer structure and the number and location of the

end groups,[55] which for glycogen can be directly probed by 1H NMR spectroscopy.[56] For

example, comparison of the H1-6 to the H1-4 proton signal and the H4 to the H1-4 signal

Figure 2. (A) Schematic describing the three basic levels of glycogen structure: randomly joined branches, which on a larger scale form β-particles, that are joined on a larger scale to form α-particles. Adapted with permission.[41] Copyright 2010, American Chemical Society. Transmission electron microscopy (TEM) images of purified glycogen from (B) rat liver, (C) rabbit liver, (D) slipper limpet, and (E) human skeletal muscle. Reproduced with permission.[24] Copyright 2009, Elsevier. (F) Monte Carlo simulation of the 3D conformation of a glycogen β-particle when the equilibrium state of growth is reached. Reproduced with permission.[53] Copyright 2019, Elsevier. (G) Atomic force microscopy images of phytoglycogen nanoparticles on mica. The inset shows an isolated nanoparticle in water, with a line profile through its center, indicating its flattened nature (horizontal axis is 90 nm and range of the vertical axis is 12.5 nm). Adapted with permission.[57] Copyright 2016, American Chemical Society.

of bovine liver glycogen reveals the particles to have 9% branching and 12% chain ends defined

as a percentage per monomer unit (Table 1).[28] It is worth noting that this approximation

assumes that all protons in the glycogen nanoparticles can be probed by NMR spectroscopy.

NMR studies of large molecules and polymers, such as glycogen, are typically complicated by

an increased line width, which for glycogen is associated with a slower tumbling of chains in

the inner core of the particle. This broadens the spectrum of the inner chains to the extent that

it is unobservable, even with a high-resolution NMR spectrometer. Hence, the evaluation of

chain ends and branching by NMR spectroscopy is likely only representative of the outer shell

of the glycogen nanoparticle.

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Various methods have been developed to isolate pure glycogen particles from tissue extracts.

A type of purification procedure typically involves boiling the glycogen in ethanol for several

hours,[58] which likely results in the dismantling of larger α-particulate glycogen into the smaller

subunits. In more recent years, gentle differential centrifugation methods have been developed

that allow for isolation of intact α-particles along with their associated proteins.[24, 59] In contrast,

commercial preparation methods likely use harsher conditions that may result in the more

disperse β-particulate glycogen. The ζ-potential of most commercial glycogen particles is

negative, depending on the source, spanning from neutral up to about −11 mV (Table 1).[28, 45]

This charge is indicative of a small amount of residual phosphate from the biological

synthesis/degradation routes. Typically, the stoichiometry of glycogen phosphorylation is low,

ranging from 1 phosphate per 500 glucose units to 1 phosphate per several thousand glucose

units, depending on the glycogen source and the nature of the study.[60]

Glycogen can be readily obtained commercially from oyster, slipper limpet, bovine and rabbit

liver,[24, 56] and sweet corn (termed phytoglycogen).[57, 61, 62] This availability makes glycogen

well positioned for use in a material-based application. Figure 2B–E shows TEM images of

purified glycogen from different animal sources and Figure 2G shows atomic force microscopy

images of commercial phytoglycogen. Glycogen from these different sources have been also

characterized in our laboratory in terms of physical properties such as particle size, degree of

branching and chain ends, molecular weight, protein content and degradability (Table 1).

Typically, the different biodegradability by α-amylase (Table 1), in terms of both extent[28] and

kinetics,[45] correlates to the enzymatic penetrability of the particles. In vivo, the degradability

is dictated by numerous other α-glucosidase enzymes. For brevity, herein, we restrict our

attention to commercially available or synthesizable glycogens and broadly refer to any roughly

spherical hyperbranched α-glucan as glycogen but note that some unique properties of

glycogen-based materials are likely correlated to a specific glycogen source.

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Table 1. Comparison the physicochemical properties of four common commercial glycogen

sources.

Property Source

BG RG OG PG

Particle size [nm] 19 ± 4a) 80 ± 35a) 55 ± 20a) 82 ± 1b)

M [Da] 3.6 × 105 a) 8.6 × 106 a) 5.2 × 106 a) 1.8 × 107 b)

ζ-potential [mV] -11 ± 7a) -2 ± 3a) 0 ± 4a) -1 ± 1b)

Degree of branching [%] 9a) 5a) 5a) 3b)

Degree of chain ends [%] 12a) 22a) 7a) 15b)

Protein content [%] 0.5 ± 0.2b) 0.3 ± 0.1b) 0.8 ± 0.1b) 0.16 ± 0.04b)

Degradability by α-amylasec)

[%]

40 ± 10b) 3 ± 1b) na 5 ± 3b)

BG, bovine liver; RG, rabbit liver; OG, oyster; PG, phytoglycogen; na, not available. a) Data

taken from Wojnilowicz et al.[28] b) Unpublished data. c) For the phenol-sulfuric acid assay, 0.7

mg mL-1 solution of glycogen was treated with α-amylase in PBS pH 7.4 (0.5 U mg-1) for 3 h.

BG, RG, and OG were purchased from Sigma-Aldrich (St Louis, MO, USA) and PG was

purchased from Mirexus Inc (Canada).

Water can interact with the polar groups of polysaccharides, where the strength of interaction

depends on the branching and chain architecture of the polysaccharide. This interaction can

lead to interesting solvation phenomena that have use, for example, in moisturizers, rheological

modifiers, and emulsifiers.[63, 64] Like other polysaccharides, glycogen has a very well hydrated

structure.[57, 65] From a biological perspective, glycogen constitutes a high proportion of wet

mass of various organs. Grossutti and Dutcher[64] have reported attenuated total reflection

infrared spectroscopy measurements of phytoglycogen, hyaluronic acid, and chitosan films as

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a function of relative humidity. In contrast to the comparable hydration water structure

displayed by the two linear polysaccharides, that of phytoglycogen was significantly different,

suggesting that the high degree of branching in phytoglycogen allows for much more well-

ordered water clusters. Nickels et al.[57] used neutron scattering to interpret the hydration of

small dense phytoglycogen nanoparticles, achieved through determination of the neutron

scattering length density (NSLD) match point of PG nanoparticles (i.e., the ratio of H2O to D2O

where the scattering from the molecule equals that of the solvent), which allowed for the

calculation of molecular volume per glucose monomer. This in turn was used in calculations

for the approximate number of water molecules associated with each glucose monomer

(obtained through a best fit of the NSLD at 100 % D2O), which suggested that the particles

contained approximately 23 water molecules per glucose monomer. This number is

significantly higher than that observed for other oligo- and polysaccharides, indicating that the

phytoglycogen nanoparticles, when fully hydrated, contain a mass of water that is equal to about

250% of its dry mass. Shamana et al.[66] quantified the softness of phytoglycogen nanoparticles

by performing equilibrium osmotic pressure measurements on concentrated dispersions; the

findings revealed that the particles had a compressional modulus of about 15 kPa, which is

comparable to that of other soft colloidal particles such as microgels and star polymers.[67]

Phytoglycogen nanoparticle dispersions at a concentration of 32% w/w have been achieved,[66]

which corresponds to an effective volume fraction of 1.13. This volume fraction is significantly

larger than the maximum volume fraction of a hard sphere glass, further indicating the soft

nature of the phytoglycogen particles. It is likely that at such concentrations, the domains of the

macromolecules overlap owing to chain interpenetration, leading to transient physical gels.[68]

These data have been used in simple models of polymer structure, suggesting that the

phytoglycogen particles contain chains that collapse onto a compact structure.[69] This

highlights the strong influence of chain architecture on the hydration of polysaccharides,

particularly with respect to hydration forces.[70]

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4. Functionalizing Glycogen Nanoparticles

4.1. Enzymatic Modification

Post-enzymatic modifications of glycogen nanoparticles have been performed in vitro for

preparing differently branched structures, leading to different material properties. Putaux et

al.[71] reported the enzymatic chain extension of oyster glycogen by a recombinant

amylosucrase from Neisseria polysaccharea, which from sucrose catalyzes the synthesis of

linear α-(1,4) glycosidic chains and releases fructose. The resulting particles displayed

significantly increased diameters, 4–5 times greater than the initial particle, observed from TEM

images, depending on the feedstock sucrose-to-glycogen ratio. Once the chains reached a

certain length, controlled by the incubation time with the enzyme, the glucan chains formed

double helical segments. Small glucose heptamers, maltoheptaose, have been shown to form

left-handed helices in solution.[72] The double helical segments on the extended glycogen

particles subsequently form insoluble crystallites (Figure 3A and B). Figure 3C and D shows

TEM images of the product after short and long incubation times, respectively, indicating the

formation of fibrous-like structures from the glycogen particles.

Glycogen can also be enzymatically degraded in vitro to yield smaller molecular weight species

by sequential trimming of glucan molecules from the “outside-in”, which also allows for real-

time analysis of degradation products from the glycogen particles and fine structural details,

including molecular density with radius and chain length patterns.[61, 73]

Figure 3. (A) Scheme describing the possible growth mechanism and structural transformation of glycogen particles with enzymatically extended glucan chains as a function of time. (B) X-ray diffraction patterns of glycogen after incubation with amylosucrase under high sucrose/glycogen ratio conditions at varying incubation times of 2 h 45 min, 7 h 20 min, and 23 h. TEM images of the product obtained after incubation for (C) 2 h 45 min and (D) 7 h 20 min (D) under high sucrose/glycogen ratio conditions. Scale bars are 100 nm. Adapted with permission.[71] Copyright 2006, American Chemical Society. Micro-computed tomography images of a calvarial bone defect in mice after 14 days in the presence of (E) bone morphogenetic proteins (BMP-2) and (F) a combination of BMP-2 with enzymatically synthesized glycogen (ESG). Reproduced with permission.[74] Copyright 2019, Wiley.

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Immunofluorescence staining of GLUT-1 (FITC) and ESG (Texas Red) for MC3T3-E1 cells (G), in the presence of ESG (H), and in the presence of ESG and Phloretin (I). Within 30 min the ESG is incorporated into the cytoplasm (H) (arrows), however in the presence of Phloretin (a GLUT-1 inhibitor), the ESG particles are mainly aggregated on the surface of the cell membrane (I) (arrows). Reproduced with permission.[74]

Glycogen-like hyperbranched α-glucans have been reported to form in vitro upon action of a

branching enzyme on short-chain amylose,[75] as well as the tandem action of a branching

enzyme and an amylosucrase enzyme on sucrose units,[76] for example. These in vitro

synthesized glycogens have different properties from native glycogen particles in terms of

molecular weight, size distribution, and degradability. Unlike the one-pot approach in vitro,[76,

77] the in vivo synthesis of glycogen is tightly regulated by cascade signaling and allosteric

enzyme regulation.[4, 34] Both enzymatically synthesized glycogen (ESG) and natural glycogens

tend to have structures that are consistent with a randomly hyperbranched polymer, as

determined from small-angle X-ray scattering.[45, 78] ESGs have found use in a variety of

applications. For example, immersion of extracted teeth into an ESG solution prior to

replantation in mice can improve the pulpal healing process by stimulating the differentiation

of hard-tissue-forming cells, possibly by activation of the stem/progenitor cells.[79] Other work

has shown that ESG pellets that are grafted with bone morphogenetic proteins into mouse

calvarial defects afford significant new bone formation (Figure 3E and F). Mitani et al.[80] have

shown that oral administration of ESG suppresses dextran sulfate sodium- and 2,4,6-

trinitrobenzenesulfonic acid-induced colitis and oxidative stress in mice, suggesting that ESG

could prevent inflammatory bowel disease. Interestingly, as natural glycogen is typically only

present in the cytoplasm, there is limited data on the uptake pathway of exogenous glycogen

nanoparticles into cells.[17, 28] A recent study by Ida-Yonemochi et al.[74] showed that uptake of

ESG into MC3T3-E1 cells was accompanied with an increase in expression of the GLUT-1

transporter protein, and when this protein was inhibited, the ESG particles mainly aggregated

on the cell surface, with some particles internalized by endocytosis (Figure 3G–I). Yasuda et

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al.[81] used models of the intestinal barrier (Caco-2 human intestinal cells) to investigate the

metabolism of ESG in the intestinal tract. ESG partially degraded to so-called “resistant

glycogen”, which can subsequently activate the immune system through stimulation of the

intestinal epithelium.

4.2. Synthetic Modification of Glycogen Nanoparticles

The rational design of glycogen nanoparticles for diverse applications, from nanomedicine to

biodegradable films, requires the precise engineering of the chemical, physical, and structural

properties of glycogen to allow tuning of the surface and bulk material properties. These include

hydrophobicity, colloidal stability, toxicity, and degradability. For example, glycogen

nanoparticles engineered to be active in biological contexts should be designed to optimize

interactions with proteins, hydrolytic enzymes, and cellular membranes. Glycogen

nanoparticles engineered to be functional components of composite materials must interact and

be compatible for blending with other synthetic or natural polymers. Leveraging the intrinsic

properties of glycogen, the use of simple chemistry to conjugate functional moieties will be key

to controlling these interactions.

Besides the enzymatic modification approaches discussed above, synthetic modification of

glycogen is becoming a promising strategy to engineer new functional glycogen. Like other

polysaccharides,[14] glycogen has three active hydroxyl groups, with each hydroxyl group in the

glucose molecule possessing a different reactivity. This difference in reactivity is essential to

consider in designing the chemical synthesis of a specific α-glucan.[82] In most instances, the

hydroxyl groups at the C2 and C3 positions behave as secondary alcohols, whereas the hydroxyl

group at the C6 position acts as a primary alcohol.[14] However, unlike amylose, glycogen has

a core-initiated, highly branched structure that likely contributes to a different solubility and

strongly affects its reactivity.

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A relatively simple, scalable, and extensively reviewed method for activating polysaccharides

for further modification is sodium periodate oxidation (Figure 4). Periodate oxidation is

generally performed in aqueous solvent in the dark.[83] Activation of glycogen via periodate

oxidation occurs rapidly with good yield (90–100 %) in aqueous systems.[27] The periodate

nominally cleaves the C2–C3 linkage within the glycosidic groups, subsequently yielding two

aldehydes.[84] Periodate oxidation has previously been used to determine the number of end-

groups in glycogen.[85]

Figure 4. Reaction schematic of various synthetic functionalization methods of (1,4)-α-D-glucopyranoside units of a glycogen chain segment. TEMPO, 2,2,6,6-Tetramethylpiperidinyloxyl; DMAP, 4-(Dimethylamino)pyridine; DMAc, Dimethylacetamide; TEA, Triethylamine; DMF, N,N-Dimethylformamide. Oxime linkages, TEMPO-mediated oxidation, and succinic anhydride addition have all been performed in our laboratory (unpublished).

Upon oxidation of glycogen by sodium periodate, the particles are extremely reactive toward

primary amines to form Schiff bases (R–C=NH–R′) (Figure 4). The use of Schiff bases can

endow the particles with pH-responsive properties, whereby the stability of Schiff bases

decreases with decreasing pH.[86] The Schiff bases can be reduced to secondary amines through

reduction with sodium cyanoborohydride,[87] thus enabling the formation of secondary amines

with optimized pKa values.[28] Perrone et al.[88] have developed glycogen-cysteamine conjugates

through reductive amination of cysteamine with oxidized glycogen. The conjugates displayed

prolonged mucosal residence times and a gel-like structure owing to covalent bond formation

between mucus and polymer, thus offering potential for use as a mucosal drug delivery system.

In addition, owing to the presence of positive charges on glycogen, the particle can be used as

a biological flocculant for extracting iron- and manganese ore-containing suspensions[89] or as

gene delivery vectors.[90]

Alternatively, the oxidized glycogen can react with amino-oxy-containing molecules, resulting

in the formation of oxime linkages (R–C=N–O–R′). Such linkages enable glycogen

15

modification without introducing new charges. Other synthetic methods for modifying

glycogen include etherification—reaction between the saccharide alcohol and an alkylating

agent in the presence of a base.[91] This method has been exploited for incorporating alkene and

alkyne moieties on glycogen.[92, 93] It is also possible to directly install carboxylate groups on

the C6 position through 2,2,6,6-tetramethylpiperidinyloxyl (TEMPO)-mediated oxidation of

glycogen for subsequent amide formation through (3-dimethylaminopropyl)-N′-

ethylcarbodiimide (EDC) chemistry with primary amine-containing groups. Carboxylate motifs

can also be introduced from the C6 position through reaction with succinic anhydride in the

presence of a base. Other synthetic methods include radical formation and subsequent cross-

linking with alkene-bearing monomers,[94] direct instalment of azido groups on the C6 position

that can be used for copper-catalyzed “click” chemistry, and functionalization with tosylate

groups for subsequent nucleophilic substitution reactions (Figure 4). These methods enable

tuning of the material properties of glycogen for a given application in terms of charge,

reactivity, solubility, material strength, and functionalization with cellular targeting motifs.

Once the periodate oxidation is used to “ring-open” the glycosidic units via periodate oxidation,

Smith degradation of the polysaccharide can occur via reduction by NaBH4 and hydrolysis with

diluted acid (Figure 5A). Bertoldo et al.[27] showed that glycogen nanoparticles that were

oxidized to different extents (i.e., varied amounts of NaIO4) underwent Smith degradation to

yield glycogens of smaller molecular weights, as determined by size exclusion chromatography

(SEC) (Figure 5D). The most-oxidized glycogen (~20% of glucose monomers) degraded to the

lowest molecular weight distribution, and the glycogens oxidized to a lesser extent displayed

convoluted SEC profiles with multiple distributions of species (Figure 5D). It is worth pointing

out that SEC analysis of the degraded glycogen nanoparticles is based on elution volume, which

has an inverse relation to the hydrodynamic volume of macromolecules, and no direct relation

to size as seen by light scattering or electron microscopy, for example. Interestingly, Bertoldo

et al. showed that when the reaction was performed in organic solvents, as opposed to aqueous

16

solvents, Smith degradation did not occur to such a large extent. Instead, the products formed

from periodate oxidation in methanol and N,N-dimethyl formamide (DMF) only showed a

minor decrease in the hydrodynamic volume when compared to the starting material (Figure

5E). This indicates that glycogen macromolecular regions that are not swollen by the solvent

(i.e., a poor solvent) are not accessible, leading to surface-constrained functionalization. In our

experience, commercial glycogen nanoparticles (see Table 1) typically can be suspended in

organic solvents, such as DMF and diethylacetamide, after prolonged incubation times at

elevated temperature. For example, incubating BG in diethylacetamide at 80 °C for 2 h results

in complete dissolution.

Figure 5. (A) Possible reaction mechanism of Smith degradation of glycogen nanoparticles suspended in water (B) and organic solvents (C). (D) Size exclusion chromatography (SEC) analysis of glycogen samples oxidized with periodate to different extents (labels refer to the amount of sodium periodate per glucose units in % mol: G1, 0.5; G2, 1.0; G3, 3.4; G4, 5.6; G5, 11.3; G6, 22.5). (E) SEC analysis of glycogen samples oxidized in water, methanol, and N,N-dimethyl formamide. Poly(ethylene glycol)–poly(ethylene oxide) (PEG–PEO) standards were used for the calibration. Reproduced and adapted with permission.[27] Copyright 2013, The Royal Society of Chemistry. 5. Glycogen as a Building Block of Nanostructured Materials

5.1. Interactions of Glycogen with Inorganic Particles

Hybrid systems that combine organic motifs with the electromagnetic properties of inorganic

nanoparticles hold potential for developing advanced multifaceted devices for sensing[95] and

optical devices.[96] Xiang et al.[97] reported that immersion of gold nanoparticles, AuNPs, with

a diameter of 20 nm in solutions of oyster glycogen (up to 0.8 μg mL−1) led to an increase of

the plasmon absorption of the AuNPs from 520 to 532 nm. This enhancement was possibly due

to an increased dielectric constant in the environment surrounding the AuNPs, where the AuNPs

aggregate on the surface of glycogen. In addition to this red shift, an increase of the plasmon

resonance light-scattering signal at 550 nm was observed, which was correlated to the glycogen

concentration in the range of 10–800 ng mL−1 with a detection limit of 2 ng mL−1. Addition of

other glycans or proteins did not lead to significant changes in the magnitude of the signal at

17

550 nm, demonstrating glycogen’s high affinity for the AuNPs.[98] These findings demonstrate

the potential of glycogen-inorganic constructs for probing glycogen–biomacromolecular

interactions at the nanomolar level.

Božanić et al.[99] synthesized 15 nm AuNPs in the presence of glycogen, whereupon the AuNPs

were aggregated on the surface of the glycogen particles (Figure 6A and B). Higher

concentrations of gold ions on the surface resulted in an increased number of AuNPs per

glycogen. Consequently, the distance between AuNPs decreased, resulting in a controlled shift

in the plasmonic band (Figure 6C). Similar silver nanoparticle-coated glycogen particles have

been developed with antimicrobial properties.[100] In a study by Kandimalla et al.[101] glycogen-

gold nanohybrids were fabricated to enhance the therapeutic potency of the hepatoprotective

agent silymarin by improving its solubility and gut permeation in Wistar rats. The nanohybrids

provided significant protection against CCl4-induced hepatic injury. In addition, the

nanohybrids were nontoxic and biocompatible to human erythrocytes (Figure 6D–G).

Figure 6. (A) Schematic and digital photograph describing the assembly of AuNPs on the surface of glycogen nanoparticles. (B) TEM image of uranyl-stained glycogen sample at an initial concentration of gold ions of 0.09 mM. (C) UV-visible absorption spectra of glycogen-stabilized AuNP colloids at different initial concentration of gold ions. Reproduced with permission.[99] Copyright 2013, The Royal Society of Chemistry. Field-emission scanning electron microscopy images of red blood cells treated with (D) distilled water, (E) saline, (F) silymarin-tethered AuNPs, and (G) silymarin-tethered AuNPs aggregated within glycogen. Reproduced under the terms of the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/).[10] Copyright 2017, Kandimalla et al; published by Dove Medical Press Limited.

5.2. Glycogen-Based Macroscopic Materials

The formation of materials from various polysaccharides has been utilized in tissue

engineering,[102] the process of regeneration of damaged tissue or reconstruction of organs by

adapting appropriate three-dimensional microenvironments for cell adhesion, proliferation,

followed by the formation of new tissues.[11] By using polysaccharides as key components, or

as additives, the material scaffolds can mimic parts of the natural extracellular matrices, which

18

are mainly composed of proteoglycans that are long-chain polysaccharides and fibrous proteins,

thus allowing the material to host new cells and improve their survival.[103] Rousseau and

Gagnieu[104] used periodate-oxidized glycogen as a collagen cross-linker to prepare

macroscopic films with defined cross-linking degrees for cellular adhesion and proliferation.

Such materials can be cytotoxic if aldehyde groups remain in the oxidized glycogen. However,

the degree of cytotoxicity can be alleviated with a single-step reduction treatment with sodium

borohydride.

Figure 7. (A) Reaction scheme for the base-catalyzed alkylation of glycogen hydroxyl groups with allyl and propargyl bromide. Scanning electron microscopy images of freeze-dried and cross-linked solutions of (B) 0.5 and (C) 5.0 wt.% alkylated glycogen. Morphology of human osteoblast-like cells on (D–F) day 1 and (G–I) day 7 after seeding on a control polystyrene dish (D,G) or at the base of a cultivation dish with arginine-glycine-aspartic acid-modified glycogen material prepared from a freeze-dried solution of 0.5 wt.% (E,H) or 5.0 wt.% (F,I). The cells were stained with Texas Red C2-maleimide and Hoechst. Reproduced with permission.[92] Copyright 2016, Elsevier.

Vetrik et al.[105] reported the formation of porous sponge-like fibrous structures by simply

freeze-drying glycogen solutions. The architecture of the resulting nanofibers was dependent

on the starting glycogen solution concentration, ranging between 0.1 and 5 wt.%. Recently,

Rabyk et al.[92] reported the modification of glycogen with hydrophobic allyl and propargyl

moieties under aqueous conditions (Figure 7A), which likely allows functionalization both

within and on the surface of the particles, followed by freeze drying and irradiation to cross-

link the allyl groups through radical polymerization. The morphology of the resulting water-

insoluble films was dependent on the solution concentration used prior to freeze drying: a

solution concentration of 0.5 wt.% resulted in a fibrous structure (Figure 7B), whereas a

solution concentration of 5 wt.% resulted in a sponge-like structure (Figure 7C). Before

irradiation (i.e., cross-linking), the films became water insoluble when more than 36% of the

hydroxyl groups in glycogen were modified. The degree of functionalization also influenced

the rate of enzymatic hydrolysis of glycogen and its subsequent biodegradability. The films

allowed cell growth (osteoblast-like MG 63 cells) under physiological-like conditions and

19

without noticeable signs of cell damage after 1 and 7 days (Figure 7D–I), highlighting the

potential of such materials for application in wound dressings.

Hydrogels composed of functionalized glycogen and other synthetic and natural polymers can

also be prepared. For example, mixtures of glycogen with poly(acrylic acid) and FeIII ions,

produce strong, elastic networks composed of hydrogen bonding and metal–ligand

interactions.[106] Such hydrogels have shown excellent self-healing abilities owing to the

presence of sacrificial bonds. A stimuli-responsive hydrogel has been obtained via free-radical

polymerization of glycogen, N-isopropylacrylamide and ethylene glycol dimethacrylate as a

cross-linker (cl-Gly/pNIPAm);[94] the hydrogel was applied for colon-targeted drug delivery.

The use of NIPAm monomers endows the hydrogel with a temperature-responsiveness,

displaying a lower critical solution temperature of 32.5–34 °C. Figure 8A and B shows field-

emission scanning electron microscopy images of glycogen and the cross-linked hydrogel

respectively. Glycogen displayed a smooth surface morphology, whereas the cross-linked

hydrogel had a complex 3D architecture. The hydrogel exhibited good compatibility toward

human mesenchymal stem cells, which enabled enhanced cell proliferation compared with cells

grown on standard tissue culture plates (Figure 8C and D). Furthermore, incubation of the

hydrogel with hen white lysozyme degraded the material, highlighting the biodegradable nature

of the hydrogel, thus allowing for a prolonged in vitro release of colon-specific drugs.

Figure 8. Field-emission scanning electron microscopy images of (A) glycogen (Gly) and (B) dried cl-Gly/pNIPAm hydrogel. (C) Cell viability results of control (tissue culture plate) and cl-Gly/pNIPAm 4 hydrogel determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay at day 1, 3, and 5. (D) Morphology of human mesenchymal stem cells assessed by rhodamine phalloidin and 4′,6-diamidino-2-phenylindole assay after incubation for 3 and 5 days. Reproduced with permission.[94] Copyright 2016, The Royal Society of Chemistry. (E) Schematic reaction for phosphorylase-catalyzed chain elongation of glycogen to form hydrogels, and digital photographs of the reaction solution, hydrogel, xerogel, and dry film. Glc-1-P, glucose 1-phosphate. Reproduced with permission.[107] Copyright 2009, Wiley-VCH.

20

Izawa et al.[107] found that the enzymatic phosphorylase-catalyzed chain elongation of glycogen,

using glucose 1-phosphate as feedstock, afforded the formation of junctions between opposing

amylose chains on different particles (Figure 8E), producing a stable hydrogel after

equilibration at room temperature. These hydrogels could be lyophilized to form xerogels, re-

hydrated to hydrogels, and cast as glycogen-based films (Figure 8E). By increasing the ratio of

glycogen to glucose 1-phosphate in the feedstock, harder xerogels were afforded, which is

likely a consequence of tighter junctions forming between particles within the cross-linked

networks. Coatings composed of cross-linked sodium alginate and glycogen by bridged

glutaraldehyde have been used to develop swelling-resistant membranes for highly aqueous

ethanol pervaporation, offering potential for the dehydration of ethanol–water solutions with

high water contents.[108] Three-component hydrogels consisting of glycogen, poly(acrylic acid),

and poly(acrylamide) have been shown to adsorb toxic metal ions (Cd2+, Pb2+, and Hg2+) and

organic dyes (methylene blue and methyl orange) from contaminated water with high efficiency

(90–98%).[109] Though, the commercial viability of using glycogen, in terms of volume, for

such systems is, to our knowledge, not established.

5.3. Glycogen-Based Platforms for In Vitro and In Vivo Delivery of Bioactive Agents

A variety of glycogen-based nanocarriers have been developed for protecting and prolonging

the efficacy of antimicrobial peptides,[110] poorly soluble drugs,[111] nucleic acids,[28] MRI

contrast agents,[112] and other active pharmaceutical ingredients (APIs).[113] A key advantage

that glycogen has over other polysaccharides and synthetic polymers, is that it does not require

multiple starting chemical components, cross-linkers, organic solvents, or laborious and

difficult-to-control lengthy manufacturing processes. In contrast, it can be extracted in its nano-

sized form from sweet corn.[114] Although it is advantageous to start with a nanoparticle such

as glycogen that possesses many desirable intrinsic qualities, the bioengineering of glycogen

nanoparticles for use in the complex in vitro and in vivo biological milieu still requires many

21

parameters to be finely controlled. The ideal drug delivery vehicle should meet several criteria

including stability in serum to avoid loss of functionality post-protein corona formation, long

blood circulation lifetime to effectively reach the target tissue, controlled immune system

recognition and activation, extravasation and penetration into tissues, cellular uptake,

endosomal escape, and intracellular targeting and degradation devoid of off-target effects. The

physicochemical and biological properties of glycogens, such as hydrophilicity, colloidal

stability, degradability, toxicity, immunogenicity, drug loading capacity, can be tailored using

the chemical modification and conjugation approaches described in Figure 4.

5.3.1. Glycogen as Vaccine Adjuvants, Immunostimulators, or Immunosuppressive Agents

Nanoparticles can boost the immune response by inducing inflammatory reaction at the

injection site where phagocytic cells are recruited to phagocytize and digest the biodegradable

nanoparticles. In addition, nanoparticles can drain via the lymph to the lymph node, where they

can interact with the resident dendritic cells. Short-term clearance of nanoparticles is desirable

by excretion via the kidney, liver, or intra-cellular degradation. Although it has been reported

that glycogens extracted from scallops and oysters have an immunomodulating activity,[115] to

our knowledge, the immunostimulant activity of different native unmodified glycogens is

unclear to date. In a study by Kakutani et al.,[115] different ESGs with controlled structures were

used to investigate the relationship between the structure and immunostimulant activity of

glycogen. Glycogens with a M of more than 10,000 kDa hardly activated murine macrophage

cell line RAW264.7, whereas glycogens with a M of 5,000–6,500 kDa strongly stimulated

RAW264.7, thus indicating that the macrophage-stimulating activity of glycogen is related to

its molecular weight instead of fine structural properties. Oral administration of ESG at a dose

of 50 mg mL−1 significantly prolonged the survival time of Meth A tumor-bearing mice,[116]

stimulated macrophage-like cells (J774.1), and increased the production of nitric oxide and

tumor necrosis factor-α. The weight-average degree of polymerization (DPw) and the branch

22

linkage (BL) ratio of ESG were 149,000 and 8.1%, respectively. However, the macrophage-

stimulating activity of ESG was lost when BL increased considerably to 20 upon treatment with

α-amylase. These results suggest that the anti-tumor and macrophage-activating activities of α-

glucans are influenced by the polymer size, degree of branching, and tertiary structure. The

anti-tumor activity by oral administration of the immunostimulant agent ESG against Meth A

was likely mediated through intestinal mucosal immunity. Chemically modified phytoglycogen

nanoparticles have been applied as vaccine adjuvants, when combined with different protein

antigens. For example, Lu et al.[117, 118] reported the synthesis of Nano-11, phytoglycogen

nanoparticles that were modified with octenyl succinic anhydride (forming phytoglycogen

octenyl succinate) and (3-chloro-2-hydroxypropyl)-trimethylammonium chloride (Figure 9A).

The octenyl succinate groups confer hydrophobicity and negative charges, whereas the

quaternary ammonium groups provide positive charges to form a complex with negatively

charged ovalbumin, OVA. After phagocytosis into dendritic cells, Nano-11 induced an

increased expression of costimulatory molecules and the secretion of interleukin-1 β IL-1β and

IL-12p40. Mice that were injected intramuscularly with Nano-11/antigen formulations

displayed an enhanced immune response. Examination of the injection site showed numerous

monocytes/macrophages and relatively few neutrophils at day 1 after injection. Recruited Mac-

2+ macrophages efficiently phagocytized the majority of the injected particles at the injection

site, and particles were found in the draining lymph nodes (1 day after injection) with the

majority contained in migratory dendritic cells. There was no evidence of

Figure 9. (A) Schematic describing the preparation of Nano-11 (phytoglycogen-octenyl succinate-(3-chloro-2-hydroxypropyl)-trimethylammonium chloride, PG-OS-CHPTAC) from phytoglycogen. Reproduced with permission.[117] Copyright 2015, Elsevier. (B) Mice were injected in both calf muscles with 200 μg of Alexa Fluor 647-labeled Nano-11 (1 and 2) or non-labeled Nano-11 (C, control) with 10 μg OVA and imaged under a fluorescence imager at different time points; the internal organs imaged on day 21 are shown in lower left. (C) Fluorescence intensity of the injection site over time (expressed as a percentage of the intensity at 0 h). Reproduced with permission.[118] Copyright 2017, NPJ Vaccines. (D) Schematics showing the distribution of hydroxypropyl and octenylsuccinate groups within octenylsuccinate hydroxypropyl-functionalized phytoglycogen (OHPP) and the (E) incorporation and (F) release

23

of active pharmaceutical ingredients (APIs) with OHPP. Dissolution profiles of (G) celecoxib and (H) docetaxel in the presence of OHPP-based solid dispersions. Reproduced with permission.[113] Copyright 2017, Elsevier. uptake of Nano-11 by neutrophils. In addition, Nano-11 was not detected in muscle cells or

fibroblasts at the site of injection. The inflammation nearly completely disappeared within 2

weeks of injection, indicating a transient inflammatory response that resolves quickly. The

clearance mechanism of Nano-11 after administration, and its interaction with immune cells in

vivo and influence on the delivery of co-injected antigen to the draining lymph node were

investigated using an in vivo imaging system. The distribution of Alexa Fluor 647-labeled

Nano-11 after injection over a 3-week period in mice was monitored. A fluorescent signal was

detected at the injection sites only (Figure 9B lower left). After day 7, the fluorescent signal

decreased with time and by day 21, about 20% of the original signal was detected (Figure 9C).

There was no evidence of accumulation of Nano-11 in other organs (including the lung, liver,

and kidney). Amine-functionalized phytoglycogen nanoparticles have shown

immunosuppressive properties, acting as a suppressor of the type 1 interferon-dependent innate

antiviral response in vertebrate cells. The use of this modified phytoglycogen was recently

proposed for suppressing an anti-viral response, for prevention or treatment of autoimmune,

inflammatory disease and sepsis.[119]

5.3.2. Glycogen as a Nanocarrier for Lipophilic Drugs

Poorly water-soluble APIs require solubilizers to enhance their efficacies for desirable

pharmacological responses. An inert solubilizer inhibits nuclei formation and crystal growth of

APIs, reducing the particle size of API crystallites and improving their wettability. To act as

solubilizer, typically phytoglycogen is functionalized with octenyl succinate moieties[110, 111, 113,

120] that endow the resulting particles with a negative charge and hydrophobicity. The resulting

particles, commonly referred as octenylsuccinate hydroxypropyl phytoglycogen (OHPP

particulate), are highly soluble in polar organic solvents and aqueous buffers with pH ≥ 5.0.

24

The solubilizing power of the modified glycogen particles increases with increasing level of

hydrophobicity and strength of the crystal structure of the APIs.[111] It is assumed that

functionalization with octentyl moieties, among others, occurs primarily at the particle surface,

therefore allowing the APIs to physically embed in the surface-functionalized chains (Figure

9D–F).[113] These functionalized particles significantly increased the dissolution of celecoxib

and docetaxel (Figure 9G and H). Xie and Yao[121] used a similar system to increase the aqueous

solubility of niclosamide, an anti-tumor agent—the solubility increased by almost 12,000 times

when compared with that of niclosamide. In another study by Bi et al.,[110] phytoglycogen

nanoparticles were subjected to β-amylosis, followed by functionalization with octenyl

succinate moieties. The resulting nanoparticles were then assessed in prolonging the efficacy

of nisin against the pathogen Listeria monocytogenes. A high degree of β-amylosis and

substitution resulted in a high retention of nisin activity against the pathogen when compared

with that of free nisin. Nisin is a model amphiphilic antimicrobial peptide that is positively

charged at physiological pH, allowing for strong electrostatic interactions with the negatively

charged glycogen particles. Furthermore, nisin contains several hydrophobic residues,

including isoleucine, leucine, alanine, methionine, and valine, contributing further to the

hydrophobic interactions with the octentyl moieties of the phytoglycogen.[110, 122] Sarkar et

al.[122] demonstrated substantially enhanced nisin adsorption on phytoglycogen octenyl

succinate-based oil-in-water emulsions. Such an enhancement enabled prolonged activity of

nisin against food pathogens.

5.3.3. Exploiting the Specific Bio-Interactions of Glycogen Nanoparticles

There is growing interest in developing glycosylated nanoparticles for targeting carbohydrate-

binding proteins on mammalian cells for imaging and therapeutic applications, where

multivalent receptor–ligand presentation in biological systems can be exploited to compensate

for weak and low affinity binding between proteins and carbohydrates.[123] Glyconanoparticles,

25

through specific carbohydrate–lectin interactions, can potentially interfere with a wide range of

cellular activities, including cell-to-cell communication, immune response, cell clustering, and

bacterial and viral infections.[93, 124] Glycogen should, in its native form, interact with α-D-

glucosyl-specific lectins. For example, when concanavalin-A (Con-A), a tetrameric α-D-

glucosyl- and α-D-mannosyl-specific lectin, is added to solutions of glycogen, rapid

precipitation of the particle–protein complex occurs, which is dependent on the average chain

length within the glycogen particles.[125] Different glycogens can be differentiated from one

another based on their capacity to react with Con-A. Cifonelli et al.[126] found that when the

outer branches of glycogen are removed by treatment with β-amylase, an increase in the

magnitude of the precipitation with Con-A results, implying that the lectin–glycogen complex

primarily occurs on inner branches. These same specific lectin–carbohydrate interactions have

been exploited for producing noncovalent, yet strong, films composed of glycogen and Con-A

on different substrates, including quartz slides,[127] quartz crystal microbalance gold

electrodes,[128] and mesoporous silica nanoparticles.[129] For example, films have been deposited

in a layer-by-layer approach with targeting motifs on the penultimate layer that disassembled

at endosomal pH, thus allowing for the release of entrapped cargo such as doxorubicin.[130]

Similar approaches were used to construct chitosan, Con-A, and glycogen layer-by-layer films

(Figure 10A–C) that can respond (Figure 10D) and disassemble to enzymatic and electrical

cues.[131] Zhang et al.[129] developed systems of mesoporous silica nanoparticles that were

sequentially coated by Con-A and glycogen, and with galactomannan as the final coating

(Figure 10E), which allowed for enhanced interaction with liver cells. Such systems enable the

use of noncovalent lectin–carbohydrate interactions for fabricating multifunctional coatings.

Con-A exhibits an intrinsic affinity for glucose over various other monosaccharides, which has

been used as a basis for glucose-sensitive glycogen-based films.[132] However, such networks

can be too strong (i.e., not able to readily disassembly) at physiological pH[133] to enable

adequate glucose sensitivity for application in diabetes treatment, for example.

26

Figure 10. (A) Schematic of the layer-by-layer (LbL) self-assembly of Con A/glycogen films. (B) Fluorescence images of films fabricated with fluorescein isothiocyanate-Con A indicate that the electrodeposited chitosan serves as a template to enable spatially controllable LbL self-assembly. (C) Electrochemical quartz crystal microbalance (E-QCM) measurements demonstrate the electrodeposition of chitosan (Chit, inset) and LbL self-assembly of Con-A, glycogen, and glucose oxidase (GOx). (D) Nyquist plot indicates increasing impedance with the assembly of each added glycogen-Con A layer and the final GOx layer. Reproduced with permission.[131] Copyright 2019, American Chemical Society. (E) Schematic describing the self-assembly of nanoparticle multilayer coatings on silica particles. Reproduced with permission.[129] Copyright 2015, Wiley-VCH.

Recently, Besford et al.[93] reported the functionalization of glycogen nanoparticles with

terminal β-galactoside moieties (lactose), achieved through copper(I)-catalyzed “click”

chemistry (Figure 11A), for targeted interaction with β-galactoside-specific lectins. The

particles were functionalized with alkyne moieties in a polar organic solvent (dimethyl

sulfoxide), which likely resulted in surface-constrained functionalization. The lactosylated

glycogen nanoparticles exhibited strong, multivalent interactions with peanut agglutinin, a β-

galactoside-specific lectin, in vitro, with an affinity constant of 3.4 × 105 M−1, which could not

be competitively displaced by free lactose.

When incubated with prostate cancer cells (PC3) in vitro, the lactosylated glycogen

nanoparticles exhibited high affinity for the cell membrane (Figure 11B) and induced

aggregation between cells (as observed from the bright-field microscopy analysis in Figure

11B), indicating to specific extracellular galectin-1 targeting. β-Galactoside residues are known

to be galectin 1-targeting moieties markedly upregulated on PC3 cells.[134] This highlights the

potential of extracellular Gal-1 as a target for drug delivery to inhibit cell proliferation and

survival of prostate cancer cells by using β-galactoside-functionalized glycogen nanoparticles.

Importantly, the functionalization strategy retains the biodegradability of the particles and

“click” chemistry can be used to endow further functionality or incorporate therapeutic drugs

into the particles. Detection and quantification of endogenous or exogeneous glycogen

nanoparticles in cells and tissues by immunostaining with anti-glycogen antibodies is important

27

for studying their intracellular trafficking and metabolism in mammalian cells. Quantification

of glycogens is challenging and involves measuring the glucose content after hydrolyzing in

hydrochloric acid. Anti-glycogen antibodies are a powerful tool for quantification of different

glycogen nanoparticles in a biological context. An anti-glycogen monoclonal antibody

(IV58B6) was previously constructed by Baba et al.[135] to detect intracellular glycogens;

however, cross-reactivity with other molecules could not be excluded. A recyclable probe to

specifically detect glycogen in cultured cells or tissues was recently bioengineered using a

recombinant protein.[136] The probe contained the carbohydrate-binding module from human

starch-binding domain containing protein 1 (Stbd1) fused to glutathione S-transferase and c-

myc tags. The resulting probe bound to glycogen in vitro and was used for immunofluorescent

detection of glycogen in mammalian cells and individual muscle fibers from mouse models of

Lafora and Pompe diseases. Another anti-glycogen monoclonal antibody using enzymatically

synthesized glycogen B (EGS-B), as the antigen, and designated as ESG1A9- mAb was

constructed.[137]

Figure 11. (A) The chemical modification of bovine liver glycogen (BLG) to produce galacto-glycogen (GG). (B) FITC-filtered and bright-field merged confocal microscopy images of control (absence of glycogen), FITC-labeled BLG, and FITC-labeled GG incubated with PC3 cells for 24 h. Optical microscopy images of untreated PC3 cells, and PC3 cells treated with BLG or lactosylated glycogen at a concentration of 0.5 mg mL−1. The red circles indicate regions of intercell contact, indicating aggregation. Reproduced with permission.[93] Copyright 2017, American Chemical Society.

5.3.4. Glycogen as a Nanocarrier in Gene Therapy

Cationic glycogen derivatives can be complexed with plasmid deoxyribonucleic acid (pDNA)

or siRNA for use as gene delivery vectors.[138] Liang et al.[90] developed cationic oyster

glycogen/pDNA constructs that were about 100–250 nm in size. The constructs exhibited high

transfection efficiency, good blood compatibility and low cytotoxicity. The positively charged

complexes were formed at a pDNA-to-oyster glycogen weight ratio of 2 and functionalized

with 3-(dimethylamino)-1-propylamine moieties and 1-(2-aminoethyl)piperazine via

28

carbonyldiimidazole activation method.[139] Liu et al.[140] investigated the use of oyster

glycogen-mediated complexation of siRNA to target NF-κB p65 in human retinal pigment

epithelial cells. siRNA was fully complexed by 3-(dimethylamino)-1-propylamine (DMAPA)-

glycogen at a w/w ratio of 5. However, at this ratio the system had the tendency to form large

aggregates (~400 nm). Conversely, at higher glycogen-to-siRNA ratios (w/w > 10), particles

ranging from 70 to 120 nm and with a positive surface potential were formed. Significant

downregulation of protein expression (90%), assessed by reverse transcription polymerase

chain reaction and western blot, was achieved over an incubation period of 72 h. Cationic

derivatization of ESG was performed by modifying the hydroxyl group at C6 position with

glycidyltrimethylammonium chloride, resulting in the introduction of quaternary ammonium

groups via an epoxide ring-opening reaction.[138] Effective complexation of siRNA molecules

with cationic ESG was achieved at siRNA-to-glycogen molar ratio of 200. A limited

knockdown (35%) of manganese superoxide dismutase (SOD2) protein in ES-2 ovarian clear

carcinoma cells was achieved. Nevertheless, the construct showed sustained siRNA delivery

for up to 6 days, while exhibiting less toxicity than a common transfection agent lipofectamine

RNAiMAX. Wojnilowicz et al.[28] investigated the use of glycogen nanoparticles from different

bio-sources and with different structural features to form constructs with siRNA. The

cytotoxicity of bovine liver (BG), rabbit liver (RG), and oyster glycogen (OG) nanoparticles

was evaluated on four different cell lines: human prostate, breast and lung cancer cells (PC3,

MDA-MB-231, and A549), and murine macrophages (Raw 264.7). Neither of the glycogen

nanoparticles studied impacted on cell viability over the wide range of concentrations studied

(0.5-0.15 mg mL-1), indicating that the particles were not cytotoxic. Amine groups were

introduced on glycogen nanoparticle by reductive amination using ethylenediamine (EDA). In

addition, methoxy-poly(ethylene glycol) (PEG) chains (2 kDa) (Figure 12A) were conjugated

to BG. To efficiently deliver nucleic acids, the size of the carrier and surface charge must be

finely tuned. A size ranging from 50 to 150 nm and a neutral or slightly negative ζ-potential are

29

considered suitable properties.[141] Optimum control over the complex size was achieved using

bovine liver glycogen (BG)-EDA/siRNA and BG-EDA-PEG/siRNA at weight ratios of 4:1 and

6:1, respectively, which afforded constructs of about 150 nm in diameter with a slight negative

ζ-potential (Figure 12E and F). Conversely, the binding of siRNA to RG-EDA or OG-EDA

resulted in aggregates of approximately 300–400 nm in diameter, which are not suitable for

complexation of nucleic acids. Super-resolution microscopy analysis of the BG-EDA-siRNA

and BG-EDA-PEG-siRNA constructs after incubation with serum protein indicated a slight

increase in size to 133 ± 28 and 186 ± 37 nm, respectively. Importantly, the complexes mediated

the silencing of luciferase and survivin proteins in both monolayer cell cultures and 3D

multicellular spheroids (Figure 12B–D), highlighting the ability of the glycol complexes to both

protect and transport siRNA, as well as penetrate the complex tumor microenvironment. The

ability of these complexes to penetrate multicellular tumor spheroids (293T and PC3 cells),

were confirmed by confocal microscopy imaging. BG-EDA-siRNA could penetrate the

spheroid core of both cell types (Figure 12A), whereas the PEGylated complexes mainly

associated on the outer layer of the spheroids, which can be ascribed to the slightly larger size

of these complexes.

Figure 12. (A) Glycogen-nucleic acid constructs for gene silencing in multicellular tumor spheroids. Glycogen from bovine liver (BG) is modified with ethylenediamine (EDA) and poly(ethylene glycol) (PEG) and mixed with siRNA to form glycoplexes. Reproduced with permission.[17] Copyright 2018, American Chemical Society. (B) Luciferase (luc) gene knockdown in 293T-Luc cells after transfection for 48 and 72 h. (C) Survivin gene knockdown in PC3 cells after transfection for 48 and 72 h. (D) Luciferase and survivin knockdown in 293T and PC3 spheroids 72 h post-transfection. (E) Hydrodynamic diameter and (F) ζ-potential of BG-EDA-, BG-EDA-PEG-, RG-EDA-, and OG-EDA-siRNA constructs at increasing w/w ratios. RG, rabbit liver; OG, oyster glycogen. (G) Confocal laser scanning microscopy images of sections of 293T spheroids treated with BG-EDA-siRNA (left) and BG-EDA-PEG-siRNA (right) constructs for 24 h taken with a 20× and a 60× magnification objective (inset) at increasing spheroid depth. Blue, nucleus; Red, particles. Reproduced with permission.[28] Copyright 2018, Elsevier.

Similar cationic glycogen/siRNA complexes have recently been used to probe the mechanism

of endosomal escape upon cellular internalization as observed by stochastic optical

30

reconstruction microscopy (STORM) imaging.[17] The use of STORM enabled the observation

of individual polyplexes released in the cytosol upon rupture of the endosomes of PC3 prostate

cancer cells. Multicolor STORM super-resolution images of glycoplexes and early/late

endosome compartments cells, at different incubation times (Figure 13A–D) enabled

visualization of glycoplexes colocalized with endosomes (yellows dots, Figure 13A4),

glycoplexes approaching bursting endosomes (red and green signal, Figure 13A5), non-

colocalized glycoplexes (green objects, Figure 13A6), and non-colocalized endosomes (red

objects). The statistical analysis of these signals (Figure 13E–H) suggested that most

glycoplexes were confined within endosomal compartments after incubation for 2 h, whereas a

significant decrease in colocalization of glycoplexes with both early and late

endosomes/lysosomes was observed after incubation for 4 h. Notably, the highly branched

cationic glycogen was effective in inducing osmotic swelling and ultimately endosome rupture.

STORM images depicting ruptured early endosomes and glycoplexes protruding throughout

the lipid membrane were acquired to demonstrate the endosomal escape mechanism (Figure

13I). Overall, this study gave an insight into the cellular uptake and intracellular trafficking of

glycoplexes. The rate-limiting step for intracellular delivery of siRNA mediated by cationic

glycogen was suggested to be the cytosolic disassembly of the complex.

Figure 13. STORM super-resolution images of glycoplexes and early endosome compartments. Intracellular trafficking of (A,C) BG-EDA and (B,D) BG-EDA-PEG glycoplexes and early endosome compartments after incubation with PC3 cells for 2 h (A,B) and 4 h (C,D). (E–H) Statistical analyses of STORM images (A–D respectively) to evaluate the extent of colocalization of glycoplexes with endosomes as a function of incubation time. (I) STORM images depicting ruptured early endosomes and glycoplexes protruding throughout the lipid membrane. Reproduced with permission.[17] Copyright 2018, American Chemical Society.

5.4. In Vivo Properties of Glycogen-Based Platforms

Oyster glycogen has been examined as a nanoprobe for in vivo imaging.[112] By modifying

glycogen with MRI and fluorescent probes such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-

tetraacetic acid gadolinium (III) complex (DOTA-Gd), and Dyomics 615 (λex 610 nm; λem 631

31

nm)[142] respectively, the in vivo fate of the modified particles after intravenous injection into

healthy mice could be tracked. The nanoparticles were mainly excreted in the urine after 6 h,

with no toxic side effects. There is an uncertainty as to whether these nanoparticles were

degraded in the blood plasma. However, fluorescence lifetime correlation spectroscopy

measurements showed that the modified glycogen nanoparticles were stable over a 60 min

mixing period with blood plasma.[142] In vivo studies in tumour bearing mice models showed

that glycogen functionalized with DOTA-Gd is a good contrast agent that can be used for MRI

imaging of tumours and the contrast enhancement in tumors was comparable with Omniscan,

a contrast typically used in biomedical imaging. Other in vivo studies by Wojnilowicz et al.[28]

investigated the circulation properties and biodistribution of bovine glycogen-, BG-EDA-, and

BG-EDA-PEG-siRNA constructs approximately 150–200 nm in diameter and with a slight

negatively charged surface.

Figure 14. (A) In vivo fluorescence imaging of Cy7.5-labelled BG-EDA-siRNA and BG-EDA-PEG-siRNA constructs in male Balb/C mice at 24 h after intravenous injection of 100 μL (1 mg mL−1). (B) Fluorescence images of organs harvested 24 h after injection of Cy7.5-labelled BG-EDA-siRNA and BG-EDA-PEG-siRNA constructs. (C) Liver accumulation profiles of BG-EDA-siRNA (red) and BG-EDA-PEG-siRNA (black) constructs over time. (D) The level of liver-related enzymes, alanine aminotransferase (ALT) and aminotransferase (AST), in serum after 8 and 24 h. Reproduced with permission.[28] Copyright 2018, Elsevier.

Soon after injection of the constructs in the mice tail, the mice were imaged at different time

intervals from 5 min to 24 h (Figure 14A) and individual organs harvested after 24 h were

analyzed (Figure 14B). The visceral fluorescence signal for BG-EDA-siRNA construct (Figure

14C, red line) increased in the first 2 h, followed by a progressive decrease in the next 22 h.

Conversely, the BG-EDA-PEG-siRNA construct showed signal in the liver only after 2 h

circulation (Figure 14C, black line), with a peak observed after 10 h. Overall, this study suggests

that PEGylation can prolong blood circulation of glycogen-siRNA constructs. Both constructs

displayed high hepatic uptake, poor accumulation in the spleen and absence of significant lung-

associated activity (Figure 14B). The hepatobiliary excretion of the nanoparticles (Figure 14B)

32

was indicated by the fluorescence signal in the feces. The nanoparticles are too large for kidney

clearance into the urine and they are not disassembled in the kidney. Both BG-EDA-siRNA and

BG-EDA-PEG-siRNA constructs showed good biocompatibility as the levels of alanine

aminotransferase and aminotransferase in serum after 8 and 24 h (Figure 14D) together with

H&E staining of the liver sections (Figure 14E) indicated lack of inflammation and minimal

damage to the liver.

6. Conclusion and Future Outlook

We have reviewed the recent literature on developing functional materials from glycogen,

highlighting their promising application as therapeutic and diagnostic agents. Glycogen is

readily available from a variety of sources, spanning mammalian tissues to plant-based sources,

and may also be synthesized from sucrose. This availability places glycogen on a similar

standing to other polysaccharides in terms of commercial viability. The native particles have a

high water solubility, which may be tuned by simple modification approaches. Importantly,

glycogen is biodegradable and nontoxic. However, after chemical modification with functional

moieties, the possible cytotoxic and mutagenic response induced by glycogen nanoparticles

requires carefully evaluation.

The progress thus far on developing glycogen as a functional advanced material is promising

but several key challenges lay ahead. Most pertinently, studies on the in vivo fate of glycogen

particles of different size, molecular weight, and surface functionalization, particularly with

regards to immune system stimulation, are needed. Of emerging importance is the role of the

protein corona on determining the in vivo fate of glycogen nanoparticles, and how this may, or

may not, contribute to immune system recognition,[143, 144] and in vivo biodistribution. In this

context, we are investigating the use of hydrophilic peptides to improve the pharmacokinetics

and biodistribution of functionalized glycogen nanoparticles. To our knowledge, there are only

very limited studies on the adsorption of serum proteins, other than lectins, onto glycogen.[28,

33

93] Furthermore, glycogen particles in vivo are associated with a population of proteins that

mediate their biological function.[145] Given that the “hard” protein corona can only be removed

from particles under extreme alkaline conditions,[144] it stands to reason that there remain

associated proteins on commercially available glycogen, particularly embedded proteins such

as glycogenin. This can give rise to fluorescence of the nanoparticles[99] and may provide an

anchor point to further modify the nanoparticles through polymerization.[146]

The application of mathematical models to glycogen structure has offered more information on

branching structure and density, thus providing greater insight into the solution behavior of the

particles. The recent development of force fields for molecular dynamics (MD) simulations of

polysaccharides in aqueous systems,[147] along with tools for building polysaccharide

structures,[148] offer a new strategy for studying the surface behavior of glycosidic chains on

glycogen. This has the potential to allow for MD studies on the interaction of glycogen surface

chains with solutes, such as proteins and enzymes,[149] as well as a direct probe of the hydration

layer structure.[150] This may prove to be a powerful complement to in vivo and in vitro

experiments for studying the interaction of glycogen particles with biologically relevant

proteins.

Acknowledgements

This work was conducted and funded by the Australian Research Council (ARC) Centre of

Excellence in Convergent Bio-Nano Science and Technology (project no. CE140100036). This

research was also supported by the ARC under the Future Fellowship scheme (F. Cavalieri,

FT140100873). F. Caruso acknowledges the award of a National Health and Medical Research

Council Senior Principal Research Fellowship (GNT1135806). We gratefully acknowledge

Marco Savioli, Agata Glab and Pietro Pacchin Tomanin for assistance with illustrations and

helpful discussions.

34

Received: ((will be filled in by the editorial staff)) Revised: ((will be filled in by the editorial staff))

Published online: ((will be filled in by the editorial staff))

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Quinn A. Besford received his PhD in 2016 from The University of Melbourne in the field of statistical mechanics and then was a postdoctoral fellow with Dr. Francesca Cavalieri and Prof. Frank Caruso at The University of Melbourne (2016–2019). He currently is a Ludwig Leichhardt Memorial Fellow of the Alexander von Humboldt Foundation and an Honorary Fellow of The University of Melbourne (Australia). His research focuses on developing responsive polymer systems and on advancing glycogen toward material applications.

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Francesca Cavalieri received her Laurea degree, ‘maxima cum laude’, in Industrial Chemistry in 1995 from the University of Rome La Sapienza. She completed her PhD in 2014 at The University of Melbourne. She was employed as researcher at ENEA from 1998 to 2002. Since 2002, she has been a tenured Assistant Professor at the University of Rome ‘Tor Vergata’. She has received many research fellowships including ARC Future and RMIT VC Senior Research fellowships. Her research focuses on the engineering of nanostructured biomaterials and supramolecular assembly of biopolymers for drug/gene delivery.

Frank Caruso received his PhD in 1994 from The University of Melbourne and thereafter conducted postdoctoral research at CSIRO Division of Chemicals and Polymers. He was an Alexander von Humboldt Research Fellow and a group leader at the Max Planck Institute of Colloids and Interfaces from 1997 to 2002. Since 2002, he has been a professor and is currently an NHMRC Senior Principal Research Fellow at The University of Melbourne. He is also Deputy Director of the ARC Centre of Excellence in Convergent Bio-Nano Science and Technology. His research focuses on developing advanced nano- and biomaterials for biotechnology and medicine.

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Glycogen is one of nature’s unique biological polysaccharide nanoparticles that holds promise

as a material for biomedical, including therapeutic, applications. Herein, the recent literature on

advancing glycogen as a building block for advanced biofunctional materials is reported.

Keywords: glycogen, nanoparticles, polysaccharides, therapeutic materials, biodegradable

materials

Q. A. Besford, F. Cavalieri,* F. Caruso*

Glycogen as a Building Block for Advanced Biological Materials

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s:

Besford, QA; Cavalieri, F; Caruso, F

Title:

Glycogen as a Building Block for Advanced Biological Materials

Date:

2020-05-07

Citation:

Besford, Q. A., Cavalieri, F. & Caruso, F. (2020). Glycogen as a Building Block for

Advanced Biological Materials. Advanced Materials, 32 (18),

https://doi.org/10.1002/adma.201904625.

Persistent Link:

http://hdl.handle.net/11343/230737

File Description:

Accepted version