Recent Advancement of Molecular Structure and Biomaterial Function of Chitosan from Marine Organisms for Pharmaceutical and Nutraceutical Application

activities of chitosan with a molecular weight of 3 K, 65 K and 600 kda and the zero-valent selenium (Se ) nanoparticles stabilized by oligosaccharides. High molecular weight chitosan stabilized nanoparticles are easier to release selenium than low molecular weight chitosan, and to be absorbed by HepG2 cells through electrostatic action. Additionally, they are more effective in inhibiting the activity of HepG2 cells. These nanoparticles could produce highly toxic Se from the less toxic Se and release selenium upon high ROS production by cancer cells. This high toxic Se causes apoptosis and mitochondrial dysfunction via consuming antioxidant enzymes. Chitosan and its derivatives can regulate the immune system through molecular mediation, enhance the body’s resistance to various pathogenic microorganisms, and show antitumor activity. The antitumor activity of chitosan varies with the molecular weight and the substituted functional groups. Additionally, there are more negative charges on the surface of tumor cells, and chitosan and its derivatives are polycationic electrolytes, which are easy to adsorb to the surface of cancer cells and neutralize the charges, which can inhibit the growth and metastasis of tumor cells and even kill cancer cells. The research on the characteristics and application of chitosan in biomedicine has developed rapidly and become one of the hot research fields. Compared with α-chitosan, β-chitosan has weaker binding force, better solubility and biological activity. However, there are a few kinds of research on it at present. Therefore, exploring the economic and environmental protection of β-chitosan production process and modification research may become one of the research hotspots of potential biomedical materials in the future. With the rapid development of biomaterials, higher requirements and challenges have been put forward for scaffold materials and drug carries. However, chitosan and its derivatives limit their application to some extent due to their defects, which need further study. At the same time, it is also necessary to strengthen the research of composite with other biomaterials to form new functional materials of marine organisms with multiple advantages, which should be one of the research hotspots of biomaterials in tissue engineering.

. Overview of production and structure of chitosan.
In 1894, the German scientist Hopper Seyler [6] used potassium hydroxide solution to boil chitin for modification and obtained deacetylated chitin, which was named chitosan. Chitosan is a high molecular weight compound with a deacetylation degree higher than 55% of chitin, the deacetylation degree or degree of acetylation (DA) is derived from the amount of the acetamido-2-deoxy-dglucopyranose monomeric unit that exists in the polymer chain. The deacetylated chitin (chitosan) is the only natural basic polysaccharide, soluble in aqueous solutions of inorganic or organic acids, with more than 90% glucosamine content, which is found in large quantities in the biological world.
Chitosan is an innate cationic biological polysaccharide polymer, naturally obtained from chitin deacetylation, that possesses broad-spectrum properties such as antibacterial, biodegradability, biocompatibility, non-toxic, non-immunogenicity, and so on. Chitosan can be easily modified owing to its molecular chain that contains abundant active amino and hydroxyl groups, through various modifications.
Not only does it possess excellent properties but it also greatly accelerates its solubility and endows it with additional special properties. It can be developed into bioactive materials with innovative properties, functions, and multiple uses, especially in the biomedical fields. In this paper, the unique properties and the relationship between the molecular structure of chitosan and its derivatives are emphasized, an overview of various excellent biomedical properties of chitosan and its current progress in the pharmaceutical and nutraceutical field have prospected, to provide the theoretical basis for better development and utilization of new biomedical materials of chitosan and its derivatives.

Introduction and history
Chitosan is structurally similar to mucopolysaccharide, which is widely distributed in tissues, and is one of the organic components of cell membranes. It possesses excellent biological activities, such as biocompatibility, biodegradability, film-forming, bacteriostaticity and non-toxicity, and can resist inflammation and bacteria, promote wound healing and has acid resistance, is antiulcer, reduces lipids and reduces the cholesterol effect [7,8]. Furthermore, chitosan demonstrates anticancer activity via activating the immune system and promotes it when applied to combine with existing anticancer drugs. It has become a research hotspot in the field of biomaterials in recent years owing to containing safe and reliable natural bioactive activities [9,10,11].
Since 1970, the use of this natural polymer has been accelerated in many countries. Several international symposiums have been held and several academic monographs on chitosan have been published. Besides, the number of papers and patents published each year also shows a significant growth trend. This fully reflects that people are more and more interested in the application value of chitosan with abundant resources. Biomaterials are natural or synthetic special functional materials used to contact and interact with living systems to diagnose, repair, replace or induce regeneration of cells, tissues and organs. It usually consists of living cells or biological tissues combined with inanimate materials to form a single, composite or hybrid material. Biomaterials interact with biological organisms, and the direct combination is a characteristic of their therapeutic method [12]. Compared with nonpharmaceutical natural polymers, the biocompatibility and biodegradability of synthetic polymers are limited. Nevertheless, the reactivity and capacity for further application of extensive materials found in nature are limited. Due to the similarity between natural polymers and biomolecules, natural polymers are easy to be recognized by the biological environment and thus easy to be metabolized into non-toxic residues for natural elimination, which has attracted wide attention [13]. In this regard, aments of scientific literature showed that natural and abundant chitosan with biocompatibility, biodegradability and non-immunogenic properties could be the smart polymers for numerous biomedical purposes such as drugs and gene delivery vehicles, permeable membranes, tissue engineering scaffolds and so on [14].
Chitosan can be biodegraded in the human body, so it is not accumulated in the body, and there is no toxicity, irritation and antigen immunity. It is generally considered to have good biocompatibility and plays a very important role in human physiological activities.
Although chitosan has superior biological activity, biocompatibility, biodegradability, antibacterial, antiseptic, hemostatic and wound healing and other special functions, its application is limited to a certain extent by its poor water solubility and mechanical properties [15]. In a practical application, due to the molecular chain of chitosan containing a large amount of amino and hydroxyl groups, the chemical modification of chitosan, such as acylation, carboxylation and etherification, especially graft copolymerization and blending modification, can change the molecular structure of chitosan, generating a series of chitosan derivatives, which can improve its water solubility, biological activity and mechanical properties, and endow on it some special properties, extending the application of chitosan in various fields.
A variety of polymer materials can be grafted on the primary, secondary hydroxyl and amino groups of chitosan. The graft copolymer of chitosan not only has the original biocompatibility and degradability of chitosan but also improves its solubility and endows it with other special properties. However, due to the small reactivity difference between the three functional groups, it is difficult to introduce a side chain at a fixed point and quantitatively, and it is difficult to separate and purify the intermediate product or the final product of the reaction, which limits the study on the chitosan grafting reaction to some extent. The research focuses on finding the appropriate reaction reagent, mild experimental conditions and efficient separation methods. At present, the application of the chitosan graft copolymer in medicine is still under continuous exploration. With the development of the research, the application of the chitosan graft copolymer in pharmacy will become more extensive. Blending modification is a common method of polymer modification. It is easy to give full play to the advantages of two or more kinds of polymers and effectively expand the application range of polymer materials. When the compatibility of components is good, a thermodynamic stable system can be formed to achieve the synergetic effect. Poor compatibility results in the separation of components. To avoid this disadvantage, a proper amount of cross-linking agents can be added to make the components cross-linked or the blends crosslinked through a hydrogen bond. The blend of chitosan with other natural or synthetic polymer materials can synthesize the excellent properties of each component to produce functional materials suitable for various fields.
According to the Pharmacopoeia of the people's Republic of China (Volume 4), chitosan is used for pharmaceutical excipients, disintegrants, thickeners, etc. [89]. Chitosan has a variety of biomedical properties and is widely used in wound dressings, orthopedics, dentistry, antitumor therapy, vascular repair and other fields [90,91]. Aments of studies have confirmed that the unique biomedical properties of chitosan and its derivatives are closely related to its structure.
Chitosan and its derivatives act as wound healing materials with a broad application prospect, which is attributed to their advantages of promoting wound healing, unique bactericidal and biodegradable properties and providing certain nutrients for cell growth. In the process of wound healing, chitosan can regulate the function of macrophages and the secretion of cytokines such as interleukin and tumor necrosis factor. Aamna et al. [92] synthesized silver nanoparticles in-situ in chitosan sericin composite to prepare chitosan sericin silver nanocomposite film by solvent casting technology and studied its antibacterial activity. The composite films were tested in Sprague Dawley male rats burn wound model for 7 days and confirmed a remarkable wound-healing ability with complete neovascularization, fibrosis, epidermal regeneration and collagen reorganization. Ouyang [93] constructed a new composite sponge by using chitosan/tilapia peptides microspheres as fillers and chitosan as a matrix and proposed for hemorrhage regulation. The findings indicated that by absorbing a high amount of water, the composite scaffolds accelerate platelet adhesion, speed up blood clotting, and stimulate the fibrin formation from fibrinogen. The bleeding volume was significantly reduced by decreasing bleeding time. Chitosan can accelerate the regeneration of epithelial cells, promote wound healing through cell proliferation and attract a large number of polymorphonuclear cells and macrophages through mild acute inflammatory response to remove tissue debris and blood clots. Chitosan can promote the chemotaxis, migration and activation of stromal fibroblasts and accelerate cell proliferation and tissue remodeling. It can also promote the formation of granulation and epithelial tissue, reduce the contraction of the wound surface, thus reducing scar formation.
Chitosan and its derivatives are also widely used in tissue engineering, especially as biological scaffolds of skin and bone, due to its

Application of Chitosan Biomaterials
ability of no expansion in water, high porosity and water absorption, interconnecting pores and uniform pore size, which are suitable for material exchange and growth metabolism of cells [94,95,96,97,98] [105,106]. In skin tissue engineering, the rigid structure of chitosan fibers can enhance the mechanical resistance of the dermal matrix and prolong the degradation of the dermal matrix by wound cell collagenase [107]. In cartilage tissue engineering, chitosan sustained-release microspheres have good drug loading and drug-releasing properties. Microsphere scaffolds can well maintain the phenotype of chondrocytes, promote their adhesion and proliferation, and have a good application prospect in the construction of cartilage and repair of cartilage damage as a carrier of chondrocytes. Chitosan and its derivatives have been extensively applied in the study of artificial nerves because of their excellent biodegradability and biocompatibility. During the construction of artificial nerves, the function of normal peripheral nerves was not affected, which could promote nerve regeneration and provide conditions for the attachment, migration and proliferation of Schwann cells to play their normal functions. Gels, nanoparticles, films, compressed tablets, beads and microspheres are currently used as potential drug delivery systems [108,109,110]. Chitosan has excellent biological activities as mentioned before and has been widely used in the study of the drug carrier systems as drug conjugates, hydrogel systems and biodegradable release systems [111,112,113]. It is mainly used in gene therapy, biological imaging, delivery of proteins/peptides, anti-inflammatory drugs, growth factors, antibiotics and vaccines. Drug delivery routes include oral administration, nose, eye and percutaneous administration [114,115]. The ionic interaction between the negatively charged sialic acid substructure in the mucus and the positively charged primary amino group of chitosan polymer could offer adhesion and permeability properties of chitosan. Self-assembled nanospheres were prepared by chemically-linked active amino groups on the chitosan backbone, which can circulate in the blood for a long time without being engulfed and can be transplanted to the target ligand, which is easy to deposit in the designated lesion site for treatment. Kim [116] used carbodiimide to connect the bile acid to the glycolytic chitosan skeleton so that the chitosan had strong hydrophilicity, and the nanomicrospheres circulated in the blood for a long time and could be loaded with doxorubicin, paclitaxel, doxycycline and other anticancer drugs to effectively treat tumors.
Chitosan can attach nucleic acids via electrostatic bonding and also could be used to create non-viral gene delivery vectors, which enter into the cells without alienation of the DNA-chitosan complex [117,118,119,120]. It shows the main part in both lysosomal escape and membrane adhesion of the encapsulated DNA for effective cell transfection. Garcia et al. [121] prepared siRNA/folate poly-chitosan lactate nanoparticles by ionic gelation, showing the potential of effective gene therapy for ovarian cancer.
Song [122] investigated the antitumor activities of chitosan with a molecular weight of 3 K, 65 K and 600 kda and the zero-valent selenium (Se ) nanoparticles stabilized by oligosaccharides. High molecular weight chitosan stabilized nanoparticles are easier to release selenium than low molecular weight chitosan, and to be absorbed by HepG2 cells through electrostatic action. Additionally, they are more effective in inhibiting the activity of HepG2 cells. These nanoparticles could produce highly toxic Se from the less toxic Se and release selenium upon high ROS production by cancer cells. This high toxic Se causes apoptosis and mitochondrial dysfunction via consuming antioxidant enzymes. Chitosan and its derivatives can regulate the immune system through molecular mediation, enhance the body's resistance to various pathogenic microorganisms, and show antitumor activity. The antitumor activity of chitosan varies with the molecular weight and the substituted functional groups. Additionally, there are more negative charges on the surface of tumor cells, and chitosan and its derivatives are polycationic electrolytes, which are easy to adsorb to the surface of cancer cells and neutralize the charges, which can inhibit the growth and metastasis of tumor cells and even kill cancer cells.
In a word, the research and application of chitosan and its derivatives are the important direction of biomedical materials research in recent years, which deliver new materials for the development of biomedicine. At present, the research on chitosan is far more than the above-mentioned applications. With the continuous update of science and technology, chitosan and its derivatives in biological medicine are reported quite more every year, including anticancer, antiviral drugs, wound healing promoting materials, implants or blood components, substitutes of tissue components and applications in biotechnology as carriers of biological separators, fermentation industry, biomacromolecules and biosensors. This explains its importance in various fields, especially as biomedical materials. Although chitosan and its derivatives have a significant effect in biomedicine, due to the shortcomings of poor solubility and mechanical properties of chitosan, which limits the development of pure chitosan in the medical field. Additionally, there are still some key scientific problems to be solved, such as the uneven particle size, the deactivation of entrapped drugs, the inability to entrap hydrophobic drugs, and the difficult regulation of release. Therefore, the modification of chitosan, grafting with other materials and strengthening the development of drug loading system, design and construction of safe and efficient granules for protein-peptide sustained release, antitumor drug targeting, intraocular drug delivery and therapeutic vaccine adjuvant are the research hotspot of chitosan as biomedical materials.
Chitosan has good histocompatibility, biodegradability and excellent biomedical properties such as improving immune activity, antitumor, antibacterial, hemostasis and promoting wound healing. These properties are influenced by the degree of deacetylation, molecular weight and groups, especially amino groups of chitosan. To find out the relationship between these properties and its molecular structure has become the focus of many researchers, which provides a theoretical basis for the better development of new materials of chitosan and its derivatives, and makes it have a better application prospect in the field of biomedicine.

Conclusions and Perspectives
The research on the characteristics and application of chitosan in biomedicine has developed rapidly and become one of the hot research fields. Compared with α-chitosan, β-chitosan has weaker binding force, better solubility and biological activity. However, there are a few kinds of research on it at present. Therefore, exploring the economic and environmental protection of β-chitosan production process and modification research may become one of the research hotspots of potential biomedical materials in the future.
With the rapid development of biomaterials, higher requirements and challenges have been put forward for scaffold materials and drug carries. However, chitosan and its derivatives limit their application to some extent due to their defects, which need further study. At the same time, it is also necessary to strengthen the research of composite with other biomaterials to form new functional materials of marine organisms with multiple advantages, which should be one of the research hotspots of biomaterials in tissue engineering.