Antimicrobial Properties and Application of Polysaccharides and Their Derivatives

With the quick emergence of antibiotic resistance and multi-drug resistant microbes, more and more attention has been paid to the development of new antimicrobial agents that have potential to take the challenge. Polysaccharides, as one of the major classes of biopolymers, were explored for their antimicrobial properties and applications, owing to their easy accessibility, biocompatibility and easy modification. Polysaccharides and their derivatives have variable demonstrations and applications as antimicrobial agents and antimicrobial biomaterials. A variety of polysaccharides, such as chitosan, dextran, hyaluronic acid, cellulose, other plant/animal-derived polysaccharides and their derivatives have been explored for antimicrobial applications. We expect that this review can summarize the important progress of this field and inspire new concepts, which will contribute to the development of novel antimicrobial agents in combating antibiotic resistance and drug-resistant antimicrobial infections.


INTRODUCTION
Microbial infection is a common community and nosocomial acquired disease that has been a global challenge and seriously threatens the health of human life. [1] Although anti-microbial treatment has made great progress in the past century, infectious diseases are still one of the three leading causes of death in the world according to the report of the International Health Organization. [2] In recent years, multidrug resistant microbial infections have been increasingly serious problems, especially with drain of the antibiotic pipeline. [3] As reported, the global death toll caused by drug-resistant bacterial infections will increase from 7×10 5 in 2015 to 1×10 8 in 2050, if without effective action and strategy. [4] Therefore, it is in great need to develop novel antimicrobial agents for variable applications.
There are many glycoconjugates or glycocalix that cover the surfaces of mammalian cells. In the process of microbial infection, pathogens usually use glycoconjugates to recognize and bind to host cells. [5] The changes of glycans over time and space reflect strategies that pathogens use the host surface to evade defense. [6] Therefore, polysaccharides are a class of natural polymers worth exploring for the antimicrobial applications.
Polysaccharides have many important physiological functions such as promoting the production of various cytokines (e.g. interferon, interleukin, etc.), stimulating macrophages and lymphocytes, and promoting antibody production to enhance organismal immunity. [7] In addition, polysaccharides also have antiviral, antibacterial and anti-inflammatory functions to some extent, mainly by inhibiting viral and bacterial reproduction. [8,9] The excellent biocompatibility, wealthy resources and low prices of polysaccharides altogether result in the increasing interest in exploring polysaccharides and their derivative as emerging antimicrobial agents.
Various polysaccharides exist in nature, such as chitosan, dextran, hyaluronic acid, cellulose and other plant/animal-derived polysaccharides, which have been explored for antimicrobial applications. In this review, we attempt to provide an overview on the development of polysaccharide-based antimicrobial agents and their applications, and a prospective on the future of this field (Scheme 1).

CHITOSAN
Chitosan, β-(1→4)-2-amino-2-deoxy-D-glucose, the product of partially or fully deacetylated chitin, is the second most abundant natural polysaccharide after cellulose. [10] Chitin mainly exists in the shells of crustacea, such as crab and shrimp, cell walls of fungi, algae and plants. [11] Chitosan is the only basic polysaccharide in nature, bearing free amino group. Due to its unique biological characteristics, such as biodegradability, biocompatibility and bacteriostasis, chitosan has been widely used in food, textiles, agriculture, environmental protection, cosmetics, biomedicine and other fields. [12,13]

Antimicrobial Mechanism
In 1979, Allan et al. proposed broad-spectrum antibacterial properties of chitosan. [14] Since then, chitosan has been widely studied and applied in various fields. Chitosan can inhibit the growth of bacteria and chitosan-sensitive fungi. [15] In general, according to the different targets of chitosan on cells, its antibacterial mechanism can be divided into two categories ( Fig. 1). Young et al. proposed that chitosan targets bacterial cell membranes. [16] Briefly, under acidic conditions, the amino groups on chitosan are protonated to show positive charges and bind to the surface of negatively charged bacteria. The uniform distribution of charge on the bacterial cell wall is disturbed, which affects the synthesis of the cell wall or even destroys the cell wall. Without cell wall protection, the permeability of cell membrane changes greatly and the cell contents leak out. Another mechanism proposed by Roller suggested that chitosan could penetrate through the porous cell wall of bacterial and enter into the bacteria after chitosan is adsorbed on bacteria. [17] Chitosan may form a stable complex with DNA and interfere with the synthesis of DNA or RNA, thereby inhibiting the reproduction of bacteria. [18] In addition to electrostatic effects, other interactions may also contribute to the antibacterial mode-of-action, [19] such as chitosan-mediated chelation of metal ions. [20]

Antimicrobial Effect and Influencing Factors
The antibacterial effect of chitosan is affected by different factors. Chitosan from different sources and in purity was used and reported in literatures, therefore, it is difficult to make a direct comparison. Besides the molecular weight and types of bacteria, the antibacterial properties of chitosan are largely affected by the degree of deacetylation, resulting in different densities of protonable amines. Other conditions such as the type and concentration of the solvent, pH value, and the metal ion strength of the environment will also affect the antibacterial properties of chitosan. The influencing factors have been elaborated in detail by Park et al. [21] To improve the solubility and antibacterial effect of chitosan, many chitosan derivatives have been developed. Due to the reactivity differences of the primary amine and primary alcohols within chitosan, it is easy to obtain chitosan derivatives through modification.

Antimicrobial polysaccharides
Other plant/animal-derived polysaccharides A q u a c u lt u r e T e x t i l e A n t ib a c te ri al ad ditives in pa p e rm a k in g  Quaternized chitosan shows not only good water solubility, [22] but also moderate to strong antibacterial properties against bacteria and fungi. Mohamed et al. reporated the minimum inhibitory concentration (MIC) of a series of quaternized chitosan against Escherichia coli (E. coli) to be 6−300 μg/mL. [23] In order to improve the antibacterial activity, Chen et al. synthesized N-quaternary ammonium-O-sulfobetainechitosan. The quaternary ammonium group and sulfobetaine were introduced to improve antibacterial activity and biocompatibility of chitosan, respectively. [24] Tang et al. incorporated quaternised chitosan derivatives (hydroxypropyltrimethyl ammonium chloride chitosan, HACC) in poly(methyl methacrylate) (PMMA) bone cement and showed that 26% HACC-loaded PMMA prevented biofilm formation of staphylococcus. [25] Sajomsang et al. synthesized methylated N-(4-N,Ndimethylaminocinnamyl) chitosan chloride, and proved its antibacterial activity against Staphylococcus aureus (S. aureus) and E. coli at the neutral pH. [26] Másson et al. investigated the antibacterial structure-activity relationship of chitosan derivatives with different degrees of substitution on the 2-amino group of chitosan, N,N,N-trimethylamine, N-acetyl and N-stearoyl, and identified the best substitution range. [27] Though quaternization is an effective strategy to increase the antibacterial activities of chitosan, the cell toxicity of quaternized chitosan remains a serious obstacle for biological applications such as antimicrobial biomaterials. Ma and Guo et al. synthesized quaternized chitosan grafted polyaniline (QCS-g-polyaniline) using oxidized dextran as the crosslinker, and developed a series of in situ forming antibacterial, conductive and degradable hydrogels called QCS-g-polyaniline hydrogels (Fig. 2). The results showed that the introduction of polyaniline into QCS could reduce the cytotoxicity of QCS and improve the antibacterial activity QCS. The antibacterial activities of the hydrogels with 3 wt% polyaniline were 95% and 90 % against E. coli and S. aureus, respectively. [28] Some other derivatives were also explored, such as carboxy-methyl chitosan, [29] N-alkyl chitosan, N-benzyl chitosan, [30] chitosan-sulfonamide derivatives, [31] chitosan-g-aminoanthracene derivatives [32] and urea-functionalized chitosan derivatives [33] with enhanced antibacterial activity. Kritchenkov et al. developed N-(3-azido-2-hydroxypropyl)chitosan that showed higher antibacterial activity than that of ampicillin and gentamicin. The finding implied potential application of chitosan in food coatings. [34] Alkylated chitosan was also prepared under basic ionic liquid conditions and was found to show excellent antibacterial activity against P. aeruginosa. [35] In addition, nanoparticles of chitosan or chitosan derivatives showed high antibacterial activity, which may be attributed to the high positive charge density on the surface. Junginger et al. synthesized N-trimethyl chitosan nanoparticles and showed a high growth inhibitory on S. aureus. [36] Marangon et al. developed antimicrobial nanoparticles combining chitosan with rhamnolipid (Fig. 3a). With the addition of rhamnolipid, chitosan/rhamnolipid nanoparticles showed decreased size and dispersity index, a higher density of positive charge and a better antimicrobial activity against S. aureus than that of either single rhamnolipid or chitosan for both planktonic bacteria and biofilms. [37] Omidi and Kakanejadifard developed new chitosan nanoparticles by grafting pyridinium salts on the surface (Fig. 3b) and these new nanoparticles showed good antibacterial activity against two types of Gram-positive bacteria: S. aureus and Bacillus cereus (B. cereus). [38]

Chitosan and Chitosan Derivatives Grafted with Peptides and Polypeptides
Inspired by peptidoglycan, a component of microbial cell wall, polysaccharides linked with peptides were proposed for antibacterial application.  Fig. 2 The formation of QCS-g-polyaniline hydrogel (Reproduced with permission from Ref. [28]; Copyright (2015) Elsevier). materials. Fan et al. synthesized complexes of nisin, a natural antimicrobial peptide, [39] and chitosan derivatives by combining nisin and chitosan via electrostatic action, and found the complex showed some antimicrobial effect. [40] Subsequently, they grafted nisin onto hydroxypropyl chitosan (HPCS) to improve antimicrobial activity in a weakly acidic environment (Fig. 4a). [41] Quaternary ammonium chitosan (QCS) with grafted nisin showed antibacterial activity and strong antioxidant activity as a wound dressing (Fig. 4b). [42] Másson et al. grafted anoplin, a peptide isolated from the venom of the solitary wasp Anoplius Samariensis, to chitosan polymer (Fig. 4c) and found their antibacterial activity was affected by differences in Nterminal or C-terminal binding sites of alkyne groups and graft density of anoplin. Nevertheless, the chitosan complex maintained high activity against Gram-negative bacteria. [43] Chitosan grafted with polypeptides ε-Poly-L-lysine (ε-PL) is a naturally occurring antimicrobial poly-peptide consisting of the carbonyl group at the α-position of lysine and the amino group at the ε-position. Li et al. grafted ε-PL on chitosan with different molecular weights using a copper-free thiol-ene 'click' reaction ( Fig. 5a) and found chitosan complexes displayed increased antimicrobial activity and 20 times higher selectivity than that of ε-PL. The effectiveness of the antimicrobial chitosan complexes was further verified by in vivo studies on animals. [44] Du et al. explored the antimicrobial properties of self-assembled peptidopolysaccharides that were synthesized by grafting antimicrobial polypeptides to chitosan (Fig. 5c). The hollow structure of the nano-micelle assemblies could easily wrap drugs. [45] Chan-Park et al. explored synthetic polypeptides and chitosan complex, a copolymer of chitosan and polylysine (CS-g-K16) (Fig. 5d), to mimic peptidoglycan components and found excellent antimicrobial activity and high selectivity. [

Chitosan grafted with short peptides
The scheme of C-NPs and C/RL-NPs synthesis (Reproduced with permission from Ref. [37]; Copyright (2020) American Chemical Society). (b) The synthesis of modified chitosan nanoparticle (Reproduced with permission from Ref. [38]; Copyright (2019) Elsevier).
Albicans (C. albicans) and Fusarium solani (F. solani) after microbes were treated with CS-g-K16. Short chitosan grafted with oligolysine chloride salt could self-assemble to nanoparticles and effectively inhibit MRSA growth by four orders of magnitude in a mouse excision wound model (Fig. 5b). [47]

Antimicrobial Application of Chitosan and Its Derivatives
Chitosan and its derivatives can be used as antibacterial agents and preservatives in the food industry. Savvaidis et al. reported chitosan can enhance the freshness of fish during storage. [48]   Chitosan-based films can be used as food packaging materials. Rhim et al. used chitosan and sulfur nanoparticles to prepare antibacterial composite films, taking advantage of sulfur nanoparticles' antibacterial function. This composite membrane has a good antibacterial effect on E. coli and Listeria monocytogenes (L. monocytogenes) and can be used for food packaging. [49] Siripatrawan et al. prepared nanocomposite of nanosized titanium dioxide and chitosan, with the ability of ethylene photocatalytic degradation and antibacterial effect on bacteria and fungi (Fig. 6a), [50] which has potential as active packaging materials for post-harvest application. Lin et al. fixed nisin-loaded poly-γ-glutamic acid/chitosan nanoparticles on PEO nanofibers as potential antibacterial packaging materials for food preservation, and tested the antibacterial effect on L. monocytogenes. [51] Chitosan and its derivatives are also widely studied as medical materials. Ji et al. constructed an antibacterial film via layer-by-layer assembly of heparin and chitosan, [52] which showed the potential in surface modification of medical devices. Wang et al. developed a novel chitosan-based antibacterial hydrogel adhesive by integrating hydrocaffeic acidmodified chitosan (CS-HA) with hydrophobically modified chitosan lactate (hmCS lactate) (Fig. 7). The study suggested chitosan-based hydrogels as promising sutureless materials in surgery. [53] Chitosan is also used in antibacterial surface materials, which has been discussed by Yu et al. [54,55] Chitosan is applied to wound dressings in various forms such as hydrogels, fibers, membranes and sponges. Tariq and Hasan et al. used chitosan-PEG-silver nitrate to prepare silver nanoparticle-based hydrogels that showed excellent antibacterial activity (Fig. 6c). Experiments have shown that this hydrogel can continuously release silver nanoparticles for at least seven days, which accelerates the healing of diabetic wounds. [56] Guo et al. designed multifunctional injectable hydrogel dressing by mixing the N-carboxyethyl chitosan and oxidized hyaluronic acid-graft aniline tetramer (OHA-AT) polymer under physiological conditions. It can be used as a potential bioactive wound dressing for skin wound healing. [57] Guo et al. synthesized quaternized chitosan-g-polyaniline and mixed with poly(ethylene glycol)-co-poly(glycerol sebacate) functionalized benzaldehyde group to obtain injectable hydrogel for wound dressings. [58] Combining the favorable hemostatic effect and biocompatibility of chitosan, Guo and Ma et al. prepared an injectable shape memory hemostatic dressing, using carbon nanotubes and glycidyl methacrylate functionalized quaternized chitosan (QCSG/CNT) to possess antibacterial activity and blood absorptive capacity. [59] Chan-Park et al. reported an antimicrobial hydrogel based on highly quaternized dimethyldecylammonium chitosan grafting with poly(ethylene glycol) methacrylate and poly(ethylene glycol) diacrylate, and proposed an "anionic sponges" antimicrobial mechanism, which attracts anionic phospholipids from bacterial cell membranes to the gel pores and eventually destroys the cell membrane to kill bacteria (Fig. 6b). [60] Mandracchia et al. prepared chitosan-based hydrogels by crosslinking glycol chitosan with diepoxy PEG, and the obtained hydrogel displayed antibacterial activity against S. aureus and a pronounced pro-angiogenic activity, indicating potential applica-  Fig. 6 Antimicrobial application of chitosan and its derivatives.   tion as wound dressing materials. [61] In addition to hydrogels, electrospun fiber mats can be used as wound dressings due to the interconnected network, high surface area and designable porosity. Khorasani et al. used electrospinning to prepare PVA/chitosan/starch nanofiber mats and verified their mechanical properties, cell compatibility and antibacterial properties (Fig. 6d). These nanofiber mats can be used as a wound dressing to protect wounds from bacterial infections and effectively accelerate wound healing. [62] Chen et al. develop a novel surface fluidswellable chitosan fiber with better water absorption capacity and stronger antibacterial activities than chitosan fiber, which had great potential to be used as wound dressings. [63] Membrane is another important form for wound dressing. Bacterial cellulose chitosan membrane was prepared by immersing bacterial cellulose in chitosan, which can be used to treat wounds and has obvious growth inhibition effect on E. coli and S. aureus. [64] Ren and Qiu et al. developed quaternary ammonium N-halamine chitosan (CSENDMH) based nanofibrous membranes, which offered antibacterial activity and hemostasis capability as potential wound dressings. [65] Chitosan and its derivatives are also applied to textile industry. Cotton fabric treated with carboxymethyl chitosan at 0.1% concentration showed good antimicrobial activity against E. coli and S. aureus. [66] Novel core-shell particles consisting of poly(n-butyl acrylate) cores and chitosan shells were prepared and designed as an antibacterial coating for textiles. The cotton fabric treated with the particles confers excellent antibacterial property. [67] Chitosan was added into the main chain polyurethanes which can improve antibacterial activity of polyurethanes. This synthesized chitosan-polyurethanes can be used as an antibacterial finishing with potential application in polyester/cotton textiles. [68] DEXTRAN Dextran, a polysaccharide composed of glucose as a monosaccharide, with glucose units connected by α-(1→6) glycosidic bonds, widely exists in microorganism, plant and animals. Dextran has attracted attentions owing to its advantages, such as water solubility, biocompatibility, biodegradability, immunomodulation and easy chemical modification. [69] O'Connor et al. prepared dextran-polyallylamine (DexPAA) hydrogel which showed antibacterial activity. [70] Nichifor et al. designed a series of cationic amphiphilic dextran derivatives, with a hydrophobic alkyl chain at the reduction end and a quaternary ammonium group on the main chain (Fig. 8). These researchers studied the effects of dextran molar mass, terminal alkyl chain length, and side chain quaternary ammonium salt structure on antimicrobial activity, and found that a proper balance between the hydrophobic and hydrophilic groups within the polymer has great influence on antibacterial activity. [71] Aminlari et al. prepared lysozyme-dextran conjugate using Maillard reaction and found the conjugate reduced the number of E. coli in cheese curd by 3 log after 40 days storage. [72] Dextran methyacrylate (Dex-MA), a photo-crosslinking derivative of dextran, can mitigate bacterial biofilm. Hence, Haldar et al. synthesized antibacterial hydrogel by mixing the cationic biocide and Dex-MA to kill bacteria. The gel can remove the formed bacterial biofilm in vitro and in superficial skin infection. [73] Li et al. grafted polypeptide to thiolated dextran via thiol-ene 'click' reaction and demonstrated effective therapeutic effects in a mouse model of sepsis. [74] HYALURONIC ACID Hyaluronic acid (HA), a polysaccharide consisting of D-glucuronic acid and N-acetylglucosamine, is an important component of extracellular matrix. HA has many important functions in the body, such as lubricating joints, regulating the permeability of blood vessel wall, promoting wound healing and moisturizing. [75] Though HA has some activiy of anti-adhesion and antibiofilm of bacteria, [76] further modifications are necessary to expand the application in antibacterial field. Xia and Zhang prepared antibacterial wound dressing by conjugating antimicrobial peptide Tet213 onto the substrates of alginate, hyaluronic acid, and collagen, to exhibit antimicrobial activity (Fig. 9a). [77] Thebault et al. coupled nisin to HA and achieved antimicrobial activity against Gram-positive bacteria. [78] To improve the mechanical strength and anti-infection ability of HA hydrogel, polydopamine (PDA) and sulfurized hyaluronic acid (HA-SH) were used (Fig. 9b). [79] PDA endows HA hydrogel with good tissue adhesion, efficient free-radical scavenging and antibacterial ability for wound dressing applicatoin. Francolini and Piozzi fabricated chitosan (CS)-HA matrices as wound dressing, with HA incorporated into CS matrix at over 5% to reduce S. epidermidis fouling on CS matrix. [

CELLULOSE
Cellulose is the most widely distributed polysaccharide in nature. While cellulose itself has no antimicrobial activity, many researches have been done to enhance cellulose's antimicrobial activity and application. Zhang et al. synthesized cellulose-based Schiff base through condensation of dialdehyde cellulose (DAC) with lysine (Fig. 10a), which showed higher antibacterial activity than DAC and potential application in papermaking. [81] Anionic poly(ionic liquids) (PILs) were antimicrobial agents formed by choline and amino acids (AAs) and they were incorporated into the bacterial cellulose to fabricate composite membranes (Fig. 10b), which exhibited efficient antibacterial and antifungal activity, and good biocompatibility as a promising antibacterial wound dressing. [82] Zheng et al. prepared aminoalkyl-grafted bacterial cellulose membranes to have antibacterial pro perties (Fig. 10c). [83] Liu et al. grafted nisin to 2,3-dialdehyde cellulose by Schiff base reaction, and the modified cellulose can be used as a food packaging material to extend shelf life of fresh pork owing to the packing material's antimicrobial activity. [84]

POLYSACCHARIDES FROM OTHER PLANTS OR ANIMALS
In addition to chitosan, dextran, HA, cellulose and their derivatives, there are other polysaccharides showing antibacterial activity as summarized in Table 1 mainly from medicinal plants. These polysaccharides could be explored as potential natural bacteriostatic agents in the food and pharmaceutical industries. However, the antibacterial mechanism of most these polysaccharides remains unclear.

CONCLUSIONS AND PERSPECTIVE
Multidrug-resistant microbial infection is emerging quickly all over the world, which has become a formidable challenge for public health globally. The drain of new antibiotic pipeline indicates the arrival of the era of post-antibiotics. Therefore, it is urgent to develop new antimicrobial agents with highefficiency, favorable biocompatibility and low susceptibility for microorganisms to develop resistance. Polysaccharides exist widely in plants, animals and microorganisms and regulate various biological functions, including resistance to bacterial invasion and immune regulation. Therefore, polysaccharides and their derivatives have received extensive attention as potential antibacterial agents and have shown antibacterial properties. However, most researches are still in the conceptual and model study stage, and further exploration is necessary for both fundamental and practical studies. Polysaccharides  Step I Step II Step III