Eco-friendly synthesis of chitosan and its medical application: from chitin extraction to nanoparticle preparation doi: https://doi.org/10.5599/adme t.1999 435 ADMET & DMPK 11(4) (2023) 435-455; doi: https://doi.org/10.5599/admet.1999 Open Access : ISSN : 1848-7718 http://www.pub.iapchem.org/ojs/index.php/admet/index Review Eco-friendly synthesis of chitosan and its medical application: from chitin extraction to nanoparticle preparation Riyona Desvy Pratiwi*, Sjaikhurrizal El Muttaqien, Nunik Gustini, Najla Salsabilla Difa, Gita Syahputra and A’liyatur Rosyidah Research Center for Vaccine and Drug, Organization Research of Health, The National Research and Innovation Agency, Jalan Raya Bogor Km 46 Cibinong, Bogor 16911, West Java, Indonesia *Corresponding Author: E-mail: riyona.desvy.pratiwi@brin.go.id Received: July 26, 2023; Revised: September 5, 2023; Published: September 23, 2023 Abstract Background and Purpose: Chitosan, a chitin deacetylation product, has been applied in nanoparticle or nano-chitosan for medical applications. However, the chitin extraction from crustacean she lls and other natural resources, chitin deacetylation, and crosslinking of the chitosan forming the nano-chitosan mostly involve hazardous chemical and physical processes. The risks of these processes to human health and the environment attract the attention of scientists to develop safer and greener techniques. This review aims to describe the progress of harmless chitosan synthesis. Experimental Approach: All strongly related publications to each section, which were found on scientific search engines (Google Scholar, Scopus, and Pubmed), were studied, selected, and then used as references in writing this revie w. No limitation for the publication year was applied. The publications were searched from April 2022 - June 2023. Key Results: Nano-chitosan could be synthesized in harmless techniques, including the preparation of the chitosan raw materials and crosslinking the chitosan polymer. Enzymatic processes in shell deproteination in the chitin extraction and deacetylation are preferable to reduce the negative effects of conventional chemical -physical processes. Mild alkalines and deep eutectic solvents also provide similar benefits. In the nano-chitosan synthesis, naturally derived compounds (carrageenan, genipin, and valinin) show potency as safer crosslinkers, besides tripolyphosphate, the most common safe crosslinker. Conclusion: A list of eco-friendly and safer processes in the synthesis of nano-chitosan has been reported in recent years. These findings are suggested for the nano-chitosan synthesis on an industrial scale in the near future. ©2023 by the authors. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/). Keywords green synthesis; nano-chitosan; drug delivery Introduction Nanotechnology is a developing field of research for designing tools and systems with specific cellular, atomic, and molecular features involving nanomaterials of size 1-100 nm. Nowadays, the application of nano- technology has been expanded in the field of biomedical research and clinical practice called nanomedicine [1]. Nanomaterials are used in imaging, drug delivery, and diagnostics in nanomedicine. Since they are small, have a high surface area, and are polydisperse, nanomaterials may have different properties from bulk materials. One of the main advantages of nanoparticles is their small size, which enables them to circulate within the body without obstructing blood flow and to avoid being cleared by the complement and renal systems [2]. https://doi.org/10.5599/admet.1999 https://doi.org/10.5599/admet.1999 http://www.pub.iapchem.org/ojs/index.php/admet/index mailto:name@address.domain http://creativecommons.org/licenses/by/4.0/ R. D. Pratiwi et al. ADMET & DMPK 11(4) (2023) 435-455 436 Chitosan is a naturally occurring polysaccharide primarily made up of (1-4)-linked glucosamine units with a smidgen of N-acetylglucosamine units. Chitosan is a promising material for medicine and pharmacy due to its biocompatibility—biodegradability, low toxicity, and mucoadhesiveness [3]. Nano-chitosan exhibits various applications in medicine and health. The system is suitable for mucosal delivery, such as oral application, nasal drug, pulmonary, muco-adhesin, and other mucosal routes [4]. Like other largely polymeric nanoparticles, nano-chitosan is biodegradable, biocompatible, and applicable for a broad range of active pharmaceutical ingredients, either small or large molecules, for instance, proteins. The nano-sized chitosan offers numerous valuable chemical, physical, and biological properties for better therapy outcomes [5]. In terms of environmental issues, experts agreed that the final product of nanomedicine, such as nano- chitosan, is not necessarily considered to contribute to high ecological risk [6]. However, from past experiences, chitosan was prepared through a rough process, including irritative chemicals in high concentrations [7]. Moreover, in the earlier years, the chitosan particles were synthesized by threatening substances, for instance, glutaraldehyde [8]. This article aims to review a list of eco-friendly processes to prepare the nano-chitosan, from the extraction of chitin as the main raw material, chitosan synthesis by the chitin deacetylation, and selecting the greener and safer crosslinkers in order to form the nano-chitosan. Conventional method for chitin and chitosan production Chitin is an abundant polysaccharide in nature after cellulose in the fungal cell wall and exoskeleton of insects, arthropods, crustaceans, and other invertebrates [9,10]. It has been discovered and explored in several periods since the late eighteenth century [10]. The chemical structure of chitin and cellulose is similar, yet hydroxyl at cellulose C-2 is substituted with an acetamide group in chitin [11]. Chitin is a polymorphic compound with α, β, and γ configurations [12]. It is obtained by extraction from those natural sources, but mostly from shrimp and crab, as up to 80 % of these animals are food wastes [13]. In brief, there are several steps of industrial production of chitin: demineralization, deproteination, bleaching/discoloration, and deacetylation to obtain chitosan, as illustrated in Figure 1. The lengthy extraction process usually takes 17 to 72 h, which includes 1 to 24 h of treatment with hydrochloric acid and 16 to 48 h of sodium hydroxide processing, resulting in a high cost, high energy, and non-environmentally friendly process. Figure 1. Reaction step of conventional chitosan production The synthesis is started by grinding the shrimp waste into smaller sizes, followed by the removal of the contained mineral, including calcium carbonate, calcite, and calcium phosphate (demineralization). In this demineralization step, excessive treatment using dilute acidic solutions such as sulfuric, hydrochloric, nitric, acetic, oxalic and formic acids is done, in which the use of hydrochloric acid during the extraction is preferable to obtain a better quality of chitin [14]. After stirring the pulverized dried shells in acidic solution at room temperature, the mineral such as calcium carbonate is transformed into precipitated calcium chloride and ADMET & DMPK 11(4) (2023) 435-455 Eco-friendly synthesis of chitosan doi: https://doi.org/10.5599/adme t.1999 437 carbon dioxide. Following the demineralization step, the reaction mixture should be rinsed with distilled water to neutralize the pH of the mixture. As chitin occurs naturally in association with protein (chitinprotein), a deproteinization process is needed to disrupt the chemical bonds between chitin and proteins. It is done by treating it with aqueous solution of sodium hydroxide (1-10 %) at elevated temperature (65 to 100 °C) for 0.5 to 12 h. In this step, complete removal of the protein from chitin is preferred, particularly for the biomedical application of chitosan, as the remaining shellfish protein may induce allergic reactions in humans. These acid and alkaline treatments resulted in coloured chitin products. The next step is decolorization to remove the red or orange pigment-formed complexes with chitin. The process could be accomplished mainly by treating it with ethanol or acetone and bleaching it with sodium hypochlorite, yielding a white-coloured chitin powder. To convert chitin to chitosan, the resulting chitin should be deacetylated in the concentrated sodium or potassium hydroxide solution [15]. The acetyl group in the chitin structure causes low solubility and reactivity in many solvents; thus, it is not convenient to use [9]. In 1859, C. Rouget found that the solubilization of chitin in acid increased after being reacted with concentrated potassium hydroxide at high temperatures [16]. This process yields a derivate compound with better solubility and reactivity than its native substance because of the presence of primary and secondary hydroxyl groups [16-18]. The deacetylated chitin was then identified as chitosan by Felix Hoppe Seyler in 1894 [16,19]. After deacetylation, the typical characteristic of each chitin polymorph disappears and becomes amorphous chitosan [12]. Chitosan itself is a cationic polymer (one positive charge for each glucosamine residue) derived from partial (at minimum 50 %) or almost complete alkaline deacetylation (reaching 95 %) at C-2 chitin which presents -NH2 primary group (Figure 2) [20,21]. Among many chitosan derivates such as glycosamine, tosyl - and trityl chitin, chitosan is the most widely used in biomedical and nutraceutical fields [22]. Consisting of amine moiety, chitosan has beneficial biological properties such as biodegradable, highly biocompatible, non-toxic, and low allergenic [23]. Moreover, because of the amino groups, chitosan is a weak base with pKa 6.3 to 6.5, which is protonated below physiological pH, leading to application in a pH-responsive system [24]. Because of the D- glucosamine, chitosan is generally insoluble in water and organic solvents and and is soluble in acidic aqueous solutions, such as acetic acid. However, the solubility highly depends on the degree of deacetylation (DD) [24]. Figure 2. Chitosan as a deacetylation product of chitin DD is a parameter to determine the chitosan characteristic and its biological activity. For instance, aqueous soluble chitosan requires at least 50 % DD, while enzyme or lysozyme non-degradable chitosan must be 95 % DD [25,26]. This parameter is calculated as a unit ratio of 2-acetamido-2-deoxy-glucopyranose (GIcNAc) to 2-amino-2-deoxy-D-glucopyranose (GIcN)) which are linked by a β (1-4) glycosidic bond [27]. DD of the chitosan is analysed by various methods, such as hydrogen bromide titrimetry, infrared spectrometry, UV-Vis spectrophotometry, and 1HNMR spectrometry [28-31]. Besides the excessive need for acid and basic chemicals for chitosan production, which affects the quality of the obtained biopolymer, the waste generated from this well-established process should also be carefully considered. The wastewater containing diluted proteins and huge amounts of chloride, calcium, and sodium could potentially induce marine and freshwater ecotoxicity as well as human carcinogenic toxicity [32,33]. Numerous proteins, peptides, and chitooligosaccharides were produced during the deproteinization step as https://doi.org/10.5599/admet.1999 R. D. Pratiwi et al. ADMET & DMPK 11(4) (2023) 435-455 438 impurities could not be utilized further due to the high level of alkali solvents contained [34]. Neutralization and detoxification of the discharged wastewater using large amounts of fresh water are necessary to avoid waste disposal problems and reduce the environmental impact, leading to the increased cost of the chitin-purification process. Therefore, more eco-friendly processes of chitosan production are needed as alternative methods. Eco-friendly chitin and chitosan production The eco-friendly method for chitosan production was introduced to resolve the shortcomings of the conventional counterparts. The green extraction technique could be done through numerous approaches, such as microbial fermentation, enzyme-assisted extraction, and microwave-assisted extraction. For example, the use of organic acid and protease-producing microorganisms like lactic acid bacteria could replace those chemicals in demineralization, deproteinization, and deacetylation steps without critically affecting the yields of chitin and chitosan [7,35,36]. The lists of enzymatic-producing bacteria used in the chitosan production method have been comprehensively reviewed [37]. In the demineralization step, the use of organic lactic acid and/or acetic acid could result in a relatively comparable effectivity to that obtained with hydrochloric acid [38]. Another benefit of using this biological extraction approach is that the obtained protein-rich by-product could be utilized for other applications, including feed or fertilizer, due to the absence of corrosive chemicals. The resulting wastes from this approach are less harmful to the environment while preserving the characteristics of the obtained chitin. Also, a strong base for neutralization is not needed in the demineralization step using organic acid, limiting the production of salt that could inhibit the enzymatic activity during the enzyme-based deproteinization process. Such a combination of greener routes of organic acid demineralization using citric acid and enzymatic deproteinization using papain or bromelain is beneficial and was reported recently by Pérez to produce chitosan from shrimp shells [39]. In the enzyme-assisted extraction approach, Younes and coworkers [40] used a non-commercial Bacillus mojavensis A21 crude enzyme for the deproteinization step of shrimp shells. They optimized several operating parameters, such as enzyme/substrate ratio, temperature and incubation time, by response surface methodology (RSM). The optimal conditions were an enzyme/substrate ratio of 7.75 U/mg, a temperature of 60 °C, and an incubation time of 6 h for obtaining a deproteinization degree of 88 ± 5 %. Valdez-Peña et al. explored the potency of commercial enzymes (Alcalase®, Flavorzyme®, Lysozyme, Inovapure 300, Trypsin VI, Papain DSM) on the deproteinization step of shell-shrimp powder [41]. From this research, a high weight loss of 61 % was achieved by using Alcalase®, while using other commercial enzymes resulted in 35 to 38 % weight losses. The use of Streptomyces griseus protease for enzymatic protein removal from a shrimp-shell powder of Litopenaeus vannamei was reported by Hongkulsup and coworkers [42]. This process achieved 91 % deproteinization at 37 °C, 3 h and 1 wt.% of the enzyme. In the deacetylation step, the combination of the enzymatic method with a mild alkaline solution was advantageous for the quality of chitin and ecological compensation [43]. In the earlier, chitin deacetylation was only performed using highly concentrated sodium hydroxide reaching 60 % at high temperatures (65 to >100 °C) and long incubation time [44]. Different times, temperatures, and concentrations of the alkaline solution affect the number of DD and molecular weight [45,46]. Higher temperature and hydroxyl solution concentration resulted in higher DD, while after saturation, prolonging the incubation time did not significantly increase DD [47]. High DD (>90 %) was only reached in high hydroxyl solution at high temperature and long incubation. On the contrary, the extreme deacetylation condition damaged the polysaccharide chain, yielding low molecular weight (MW) chitosan [48]. According to the MW, chitosan is classified into three groups, i.e. low MW (<100 kDa); medium MW (100 to 1,000 kDa), and high MW (>1,000 kDa) [49]. High DD and low MW chitosan show antimicrobial and antioxidant activity, high DD and high MW chitosan are needed for ADMET & DMPK 11(4) (2023) 435-455 Eco-friendly synthesis of chitosan doi: https://doi.org/10.5599/adme t.1999 439 mucoadhesive activity and application in regenerative medicine or drug delivery, meanwhile, medium MW chitosan has anticancer activity [50,51]. In recent years, modified chitin deacetylations were proposed in order to obtain high DD with lower side effects on the polysaccharide chain. As mentioned above, chemical chitin deacetylation, particularly on a manufacturing scale, inflicts environmental pollution [52]. It was reported that to produce one kilogram of 70 % deacetylated chitosan, at least six kilograms of hydroxy chloride, two kilograms of sodium hydroxide, and a large quantity of nitrogen, water, and organic solvents are customized [53]. Thus, biological processes involving deacetylase enzymes are preferable [54]. The first reported chitin deacetylase (CDA) was identified and purified from Mucor rouxii, a dimorphism (yeast-like and filamentous) fungi microbial. The enzyme is a 75 to 80 kDa glycoprotein with high mannose, approximately 30 % carbohydrate, and commonly expressed in the periplasmic space but also secreted to the extracellular medium [55,56]. The enzyme activity was optimum at pH 4.5, 50 °C for glycol chitin model sub- strate and catalysed at least four N-acetylglucosamine [56]. Afterwards, numerous CADs from other fungi were discovered, isolated, and studied on chitosan production [57]. The CADs have typical biochemical properties, including different molecular sizes from 12 to 150 kDa, acidic pI (2.7 to 4.8), and show optimum bioactivity at 30 to 60 °C in pH 4.5 to 12 [58]. The inefficient yield of the isolated CADs initiated the production of recombinant CADs [59]. Hitherto, several recombinant CADs genes from Colletotrichum lindemuthianum, Saccharomyces cerevisiae, and Mucor circinelloides have been expressed in different bacterial or yeast expression systems such as Escherichia coli or Pichia pastoris, respectively, and showed proper biological activity [59-65]. As microwave has a high potential to accelerate chemical reactions and increase the reaction yield, a chitin deacetylation method using microwave energy was also introduced to reduce incubation time in the hydroxyl solution from hours to less than twenty minutes [66]. The microwave exposure reduced the heating temperature as well as shortened the heating time due to its heating efficiency and homogeneous microwave field within the sample, generating uniform heating [67,68]. Also, the obtained chitosan products from chemical and microwave energy-assisted extraction displayed similar structures, morphologies and chemical compositions. The direct and quick energy transfer of the microwave irradiation to the substrate and catalyst may be responsible for increased reaction efficiency [69]. In this approach, various process parameters, including reaction time, solvent concentration, and solid-to-liquid ratio, should be optimized to obtain the designated degree of deacetylation and molecular mass of the obtained chitosan. In addition, the concentration of hydroxyl solution could be decreased by combination with urea [70]. The deep eutectic solvents (DESs) approach proved more sustainable, biocompatible, inexpensive, and more convenient in preparation [71]. DESs are a mixture of at least one hydrogen-bond acceptor (HBA), such as halide salts, and one hydrogen-bond donor (HBD): urea, glycerol, benzamide, and citric acid [72]. The application of the DESs in chitosan production was not only effective in the deacetylation process but also in the chitin extraction, including deproteination and demineralization [73]. Aside from deacetylation products, natural chitosan has also been found in some micro and invertebrate organisms. From M. rouxii, the first natural chitosan was isolated from the cell wall using an acidic solution, hydrochloric acid, and formic acid [74]. Afterwards, the natural chitosan was also discovered from other micro- organisms like bacteria (Serratia sp. and Bacillus sp.), yeast (Candida albicans and S. cerevisiae), and other fungal microorganisms (Rhizopus oryzae, Gongronella butleri, Cunninghamella elegans, and Phycomyces blakesle- eanus [53]. Zhang and colleagues reported that natural chitosan was only found in budding spores of S. cere- visiae, not in the vegetative form [75]. Chitosan extracted from S. cerevisiae and Aspergillus niger had almost similar DD with chitosan processed from chitin's shrimp [76]. Producing natural chitosan from those sources reduces chemical and toxic materials during the process, including chitin extraction and deacetylation [53]. https://doi.org/10.5599/admet.1999 R. D. Pratiwi et al. ADMET & DMPK 11(4) (2023) 435-455 440 Considering the risk and benefit of each process, chemically processed chitin-derivate chitosan is still widely produced on the industrial scale. The enzymatic deacetylation encompasses an eco-friendly method; even in economic value, the enzyme is relatively higher priced than the chemicals [57]. Likewise, the natural chitosan final yield is still relatively low compared with the chitin-derivate chitosan and highly depends on the culture medium and incubation condition [53,76]. Modified chemically processed chitosan has become interesting because of the rational cost and benefit of reducing harm by the conventional chemical method [57]. Applications of chitosan in nanomedicine Chitosan has been used and reported in many fields, for instance, in the food and beverage industry; pharmacy and health, including biotechnology, biomedicine, dentistry, and veterinary; agriculture and aquaculture; textile; pulp and paper industry; chemistry and environmental chemistry [77]. In pharmaceutical and health, chitosan has been applied in drug and gene delivery systems, tissue engineering and artificial implants, bioimaging, protein binding, wound healing, and contact lenses [78]. As the concept of nanotechnology introduced by Richard Feynman in 1959, the application of nano- technology in medicine has been also emerged [79,80]. Nanotechnology in medicine , terminologically defined as nanomedicine, is nano-scaled medicine obtained from an engineering process resulting in different physical, chemical, and biological features than its original material for diagnosis, monitoring, control, prevention, and treatment of diseases [81,82]. However, the exact definition of nanomedicine is still debated among scientists, regulatory agencies, and enterprisers related to size, organic/inorganic material, pharmacodynamic profile, pathology, and route of administration [83]. In general, nanomaterial should be between 1 to 100 nm in size. Still, for nanomedicine, the material, particularly organic nanomedicine such as liposome or micelle, which is more than 100 nm (i.e., 400 nm), is also considered as 'nano,' as long it shows significantly improved properties (i.e., higher solubility and bioavailability) than the bulk material [83]. On the other hand, iron oxide only exhibits altered characteristics below 20 nm [83]. In another literature, nanomedicine is determined in the 1 to 1000 nm range [84], but in the context of pathology and route of administration ( i.e., enhanced permeability and retention for cytotoxic therapy), the effect could not be observed from >200 nm particles [85]. According to the types of material, nanomedicine is classified into seven groups: oxide/metal nanoparticles; polymer-based nanoparticles; lipid-based nanoparticles; micelles, liposomes, protein-based nanoparticles, and unspecified others [86,87]. Chitosan-based nanoparticle or nano-chitosan is categorized in polymer-based nanoparticles [88]. In the field of medicine, nano-chitosan has been explored for small and large molecule delivery carrier, wound healing, and tissue regeneration, besides the potential as antimicrobial/anticancer/antioxidant given from the chitosan itself [88]. Eco-friendly crosslinkers for nano-chitosan preparations Nano-chitosan (and micro-chitosan) is formed by several methods, i.e., ionic gelation crosslinking, reverse micelles, emulsification, precipitation, radical polymerization, and top-down method [8]. The nano-chitosan is characterized differently by each method [89]. Emulsification, reverse micelles, and precipitation involve aqueous chitosan, stabilizer, and organic phases. On the other hand, the organic solvent-free method is much preferred to those methods in which the usage of organic solvent is required. Thus, to date, ionic gelation is the most favourable method for nano-chitosan production. The particle is produced by ionic interaction between positively charged amine groups of chitosan with polyanions crosslinker [8,89]. At the beginning of chitosan development, glutaraldehyde was used as the crosslinker [8]. The crosslinking is formed via imine bonds by the interaction of amine groups of the chitosan and an aldehydic group of the glutaraldehyde. The amount of glutaraldehyde affects the particle size as the higher concentration of ADMET & DMPK 11(4) (2023) 435-455 Eco-friendly synthesis of chitosan doi: https://doi.org/10.5599/adme t.1999 441 glutaraldehyde reduces particle size [90]. In addition, the reaction of glutaraldehyde-chitosan depends on the pH solution, which determines the protonation of the amine groups (Figure 3) [91]. Glutaraldehyde is known as a stable crosslinker for chitosan. However, because of its high toxicity to human health, for instance, skin and eye irritation, respiratory tract problems, and environmental issues, it has been substituted with other safer crosslinkers, such as tripolyphosphate (TPP) [92,93]. As described in Table 1, TPP has been the most commonly used crosslinker for nano-chitosan production. Besides the TPP, some crosslinkers, which are natural-derived compounds, have been reported to exhibit remarkable advantages as harmless crosslinker in the nano-chitosan synthesis (Table 2). Table 1. Application of nano-chitosan in medicine Type Characteristic Ref. Therapeutic protein /peptide carrier Salivary protein (histatins) delivery, tripolyphosphate (TPP)-crosslinked chitosan [94] Oral delivery of insulin, 3-APBA & L-valine crosslinked carboxymethyl chitosan [95] Oral delivery of insulin, chitosan with SAR6EW (a cell-penetrating peptide) [96] Buccal delivery of bioactive peptide, TPP-crosslinked chitosan [97] Intranasal delivery of recombinant interleukin-17 receptor for asthma, TPP-crosslinked chitosan [98] Delivery of recombinant human bone morphogenetic protein-2, alginate crosslinked chitosan [99] Small molecule drug carrier Oral delivery of rifampicin, TPP-crosslinked chitosan [100] Oral delivery of ciprofloxacin, cyanocobalamin-crosslinked chitosan [101] Targeted delivery of methotrexate for HeLa cells, folic thiolated chitosan [102] Oral delivery of repaglinide and diltiazem HCl, TPP-crosslinked chitosan [103] Delivery of triclosan and flurbiprofen for periodontitis, chitosan nanogels [104] Brain delivery of rotigotine for Parkinson’s disease, TPP-crosslinked chitosan [105] Vaccine drug carrier Cancer vaccine delivery of whole tumor cell lysate, mannose-alginate coated chitosan [106] Avian infectious bronchitis virus (IBV) delivery, TPP-crosslinked chitosan [107] Hepatitis A vaccine delivery, alginate-coated, sodium sulfate-crosslinked chitosan [108] Recombinant hepatitis B surface antigen delivery, TPP-crosslinked, mannosylated chitosan [109] Curdlan intranasal vaccine delivery, quartenized chitosan [110] Vo protein for meningitis vaccine, polylactic glycolic acid - chitosan combination [111] DNA (pDNA-E7mut) vaccine, TPP-crosslinked chitosan [112] Delivery of vaccine for diarrhea (LSC-chimeric recombinant protein), TPP-crosslinked chitosan [113] Chitosan-metal nanoparticle complex Chitosan-polyethylene oxide (PEO) nanofibers combined with silver and zinc oxide nanoparticles for antibacterial wound healing [114] Chitosan-gold nanoparticles for DNA carrier [115] Anti-infective wound healing HemCon®, FDA approved for hemorrhage in 2003 [116] Composite sponge of chitosan and hydroxybutyl chitosan [117] Electrospun chitosan nanofibers [118] Microchannelled alkylated chitosan sponge [119] Chitosan microneedle array, antiangiogenic and antibacterial [120] Chitosan-poly vinyl alcohol patch [121] Chitosan-bentonite nanocomposite [122] Chitosan-glucan-collagen complex with aloevera [123] Hypericum perforatum oil incorporated chitosan cryogel [124] Chitosan-iron (III) sulfate-levofloxacin dressing [125] Tissue regeneration Nerve tissue regeneration, chitosan-collagen composite scaffold for Schwann cells [126] Skin tissue regeneration, keratinocyte loaded chitosan-polyvinyl alcohol-silk electrospun system [127] Bone tissue regeneration, gelatin-chitosan scaffold for hydroxyapatite (HAp), β‑tricalcium phosphate (β-TCP) and 58s bioactive glass [128] Bone tissue regeneration, composite of chitosan, chondroitin sulfate, and nano-bioglass [129] Cartilage tissue regeneration, chitosan-poly(L-lactide)-peptin composite [130] TPP, also available in sodium TPP (Na5P3O10), reacts with the amine groups of the chitosan through the nega- tively charged P3O10 5- (Figure 4). Characteristics of nano-chitosan from the chitosan polymer and TPP depend on several experimental factors, such as the ratio of chitosan and TPP, pH solution, ionic strength, stirring type, and rate [131]. TPP was reviewed as the smallest and strongest nano-chitosan's crosslinker among the others crosslinkers [132]. To overcome the environmental issues, a certain amount of innovative natural-based crosslinker for nano-chitosan has been explored, including carrageenan, genipin, citric acid, and vanillin [129]. https://doi.org/10.5599/admet.1999 R. D. Pratiwi et al. ADMET & DMPK 11(4) (2023) 435-455 442 Table 2. Eco-friendly crosslinkers in nano-chitosan production Crosslinker Application Materials Ref Tripoly- phosphate (TPP) Nanocomposite film of gelatin-coated chitosan-sodium TPP nanoparticles for wound healing 0.5 % chitosan in 2 % acetic acid pH 4.5; 10 mL sodium TPP solution (0.7 mg/mL) [133] Nanocomplex of cashew gum, chitosan, and TPP for insulin loading 0.2% chitosan in 0.1 % lactic acid pH 3.4; 13.5 mM TPP pH 9.1; cashew gum 0.5 % pH 6.2 [134] Carrageenan Slow-release nanoparticles/polyelectrolytes complexes of chitosan and κ-carrageenan 100 mg chitosan and 100 mg κ-carrageenan in 0.2 M sodium acetate buffer (pH 3-6) [135] Polyelectrolytes complexes of chitosan and κ- carrageenan for protein delivery 1.5 g chitosan in 400 mL acetic acid 1 %; 1.5 g κ- carrageenan in 100 mL deionized water enriched with 25 g sodium chloride [136] Genipin Biocompatibility study of genipin-chitosan hydrogels for vaccine delivery 1.5 % chitosan in 1 % acetic acid. Ratio chitosan: genipin = 1: 0.1 to 0.3 [137] Genipin crosslinked caseinate-chitosan for curcumin delivery as an anticancer drug 1 mg/mL chitosan in 0.1 M acetic acid; 20 mg/mL genipin in DMSO [138] Vanillin Vanillin-chitosan particles for zinc sustained release system Molar ratio of vanillin: chitosan (0.1 to 2.0) in 3 mL glacial acetic acid; loaded with 1g/L zinc acetate ethanol solution pH 6 [139] Cytotoxicity drug-loaded vanillin-chitosan nanoparticles 2 % chitosan in 1 % acetic acid (10 mL); 25 mg/mL vanillin (50 mL); 50 mg 5-fluorouracil [140] Figure 3. Glutaraldehyde crosslinked nano-chitosan Figure 4. TPP crosslinked nano-chitosan Carrageenan is a sulphated galactosan extracted from the Rhodophyceae family containing main sugars (o-galactose) and 3,6-anhydro-o-galactose; the main substituent (sulphate); carbohydrate residues (xylose, glucose, and uronic acids), and other substituents (methyl esters) [141]. It is an anionic polymer because of the high component of the ester-sulphate. In addition, because of variations in the sulphate content, carrageenan is classified into six types (κ, λ, ι, θ, β, ν, and μ carrageenan). Each type shows different physio- chemical properties (such as solubility and gelling mechanism) and bioactivity [142]. ADMET & DMPK 11(4) (2023) 435-455 Eco-friendly synthesis of chitosan doi: https://doi.org/10.5599/adme t.1999 443 As a negatively charged polymer, carrageenan is suitable as a crosslinker in the nano-chitosan preparation. Carrageenan (κ-carrageenan) and chitosan were used to form polyelectrolyte complexes (PEC) (Figure 5). Not only does it function as a drug delivery system, tissue regeneration, cell cultivation, and enzyme immobilization, but the PEC also exhibits biological activity as an anti-inflammatory via histamine activation [143]. Figure 5. Carrageenan crosslinked nano-chitosan Khaliq and colleagues reported that cefotaxime sodium loaded κ-carrageenan - chitosan hydrogel was applicable to dress diabetic wounds by releasing the drug in 24 h for effectively inhibiting bacterial propagation [144]. The drug release profile depends on pKa or pI and the extrinsic pH. For instance, diflunisal (pKa = 2.94) was released from a κ-carrageenan - chitosan nanocapsule in intestinal fluid and blood distribution (pH 7.4) in which the drug was found in anionic and highly soluble form. Otherwise, in lower pH, such as in acidic gastric fluid (pH 2-3), diflunisal was neutral and insoluble in an aqueous solution [145]. Along with TPP, the combination of these two marine polymers was also used for the delivery system of α - mangostin and showed cytotoxicity in the MCF-7 cell line and antibacterial activity [146,147]. Genipin, another naturally derived crosslinker in nano-chitosan preparation, is found in Genipa americana fruit. It was soluble in aqueous solution, alcohol, and propylene glycol, less toxic than other synthetic crosslinkers (showed 0.01 % cytotoxicity compared with glutaraldehyde), and reported for several pharma- cological activities [148]. This compound actively reacts with primary amine groups of chitosan by substituting oxygen at the dihydropyran ring and the aldehyde group, which depends on environmental pH and oxidation level (Figure 6) [149,150]. Most of the recent applications of genipin-crosslinker chitosan were proposed for tissue regeneration and wound healing. Heimbuck and team described the capabilities of genipin-crosslinked chitosan hydrogel to hamper bacterial growth and to neutralize environmental pH as a wound dressing [151]. A list of genipin-crosslinked chitosan for skeletal regeneration has been reviewed by Wang et al. [152]. Figure 6. Genipin crosslinked nano-chitosan https://doi.org/10.5599/admet.1999 R. D. Pratiwi et al. ADMET & DMPK 11(4) (2023) 435-455 444 Another fascinating natural chitosan crosslinker is vanillin or 4-hydroxy-3-methoxy benzaldehyde, an aromatic compound extracted from Vanilla sp. beans and commonly used in food and beverage industries. The exorbitant cost of the natural vanilla extraction and harvest limitation caused the idea to chemically synthesize the vanillin compound for a more rational commercial value [153]. 4-hydroxybenzaldehyde was used to produce this artificial vanillin. However, the precursor was then reported to harm the environment and human health [153,154]. Therefore, biotechnological-derived approaches, such as plant-based, enzyme-based, and microorganism- based methods, have been applied in vanillin production to obtain lower costs with decreased side effects [153]. The aldehyde group of the vanillin reacts with the chitosan's primary amine, yielding an imine group (Schiff base). The reaction is reversible by the presence of a para-hydroxyl group (of the imine group) that forms a hydrogen bond with the adjacent hydroxyl of the amino group (Figure 7). Because the reversible hydrogen bond exists, the interaction of vanillin and chitosan relies on pH and temperature. The hydrogen bonds showed high stability at low temperatures [155]. Figure 7. Vanillin crosslinked nano-chitosan Discussion and conclusion The idea of an eco-friendly process, previously called green chemistry, appeared in 1991 to avoid excessive exposure to harmful chemicals to the environment and human health. The harmful chemicals are classified into three following groups: physical problem (explosive and flammable substances); toxicity to human health (causing cytotoxicity, mortality); environmental danger (causing climate change; ozone layer depletion; over-consumption of energy; or uncontrollable waste) [156]. Pharmaceutical industries were reported to contribute significantly to pollutant and waste by-products among chemistry industries. In the last decade, the pharmaceutical industries have applied green chemistry principles as reviewed by Mishra et al., including reducing waste and preventing pollution, using safer chemical or renewable materials, and increasing energy efficiency [157]. Production of nanomaterial for nanomedicine applications, such as nano-chitosan, is also known to involve hazardous substances and waste for the environment and health. Therefore, eco-friendly or green synthesis is requested not only during the nanoparticle assembly but must be applied during the raw materials preparations. In short, two crucial eco-friendly concepts in the nano-chitosan preparation, which are using safer or reducing dangerous chemicals in the chitosan synthesis (including chitin extraction and deacetylation) and determining harmless crosslinkers for the nano-chitosan preparation, have been discussed. Biotechnology processes, particularly enzymatic and microbiological methods, have also been applied in this field, even so, up to now, in terms of industrial capacity and cost, it gives less benefit than the other green methods, for instance, by using sustainable reagents such as DESs. Above all, the natural ADMET & DMPK 11(4) (2023) 435-455 Eco-friendly synthesis of chitosan doi: https://doi.org/10.5599/adme t.1999 445 properties of chitin, chitosan, or nano-chitosan (hydrophilicity, biocompatibility, no organic solvent needed) are the main key to the appropriateness of the eco-friendly or green synthesis concept [158]. Acknowledgements: We would like to thank to Vaccine and Drug Funding Program 2023, Organization of Health, The National Innovation and Research Agency, Republic of Indonesia. Conflict of interest: The authors declare no conflicts of interest. Contributions: RDP contributed to conceptualization, writing and revising the manuscript; SEM wrote and improved the manuscript in the section on chitin and chitosan production; NG wrote the abstract; NSD & GS contributed in writing technical process; AR contributed to conceptualization and corrected the whole first manuscript. All authors read, corrected, and approved the final manuscript. References [1] M.K. Teli, S. Mutalik, G.K. Rajanikant. Nanotechnology and Nanomedicine: Going Small Means Aiming Big; Current Pharmaceutical Design 16 (2010) 1882-1892. https://doi.org/10.2174/138161210791208992 [2] R. Foulkes, E. Man, J. Thind, S. Yeung, A. Joy, C. Hoskins. The Regulation of Nanomaterials and Nanomedicines for Clinical Application: Current and Future Perspectives. Biomaterial Science 8 (2020) 4653-4664. https://doi.org/10.1039/d0bm00558d [3] H. Peniche, C. Peniche. Chitosan Nanoparticles: A Contribution to Nanomedicine. Polymer International 60 (2011) 883-889. https://doi.org/10.1002/pi.3056 [4] M.A. Mohammed, J.T.M. Syeda, K.M. Wasan, E.K. Wasan. An Overview of Chitosan Nanoparticles and Its Application in Non-Parenteral Drug Delivery. Pharmaceutics 9 (2017) 53. https://doi.org/10.3390/pharmaceutics9040053 [5] D. Chenthamara, S. Subramaniam, S.G. Ramakrishnan, S. Krishnaswamy, M.M. Essa, F.H. Lin, M.W. Qoronfleh. Therapeutic Efficacy of Nanoparticles and Routes of Administration. Biomaterial Research 23 (2019) 20. https://doi.org/10.1186/s40824-019-0166-x [6] I. Mahapatra, J.R.A. Clark, P.J. Dobson, R. Owen, I. Lynch, J.R. Lead. Expert Perspectives on Potential Environmental Risks from Nanomedicines and Adequacy of the Current Guideline on Environmental Risk Assessment. Environmental Science Nano 5 (2018) 1873-1889. https://doi.org/10.1039/c8en00053k [7] P. Beaney, J. Lizardi-Mendoza, M. Healy. Comparison of Chitins Produced by Chemical and Bioprocessing Methods. Journal of Chemical Technology and Biotechnology 80 (2005) 145-150. https://doi.org/10.1002/jctb.1164 [8] M. Yanat, K. Schroën. Preparation Methods and Applications of Chitosan Nanoparticles; with an Outlook toward Reinforcement of Biodegradable Packaging. Reactive and Functional Polymer 161 (2021) 104849. https://doi.org/10.1016/J.REACTFUNCTPOLYM.2021.104849 [9] B.T. Iber, N.A. Kasan, D. Torsabo, J.W. Omuwa. A Review of Various Sources of Chitin and Chitosan in Nature. Journal of Renewable Materials 10 (2022) 1097-1123. https://doi.org/10.32604/JRM.2022.018142 [10] G. Crini. Historical Review on Chitin and Chitosan Biopolymers. Environmental Chemistry Letter 17 (2019) 1623-1643. https://doi.org/10.1007/s10311-019-00901-0 [11] S.P. Chawla, S.R. Kanatt, A.K. Sharma. Chitosan, in Polysaccharides, K. Ramawat, J.M. Mérillon (Eds)., Springer Cham, 2015, 219-246. https://doi.org/10.1007/978-3-319-16298-0_13 [12] M.V. Tsurkan, A. Voronkina, Y. Khrunyk, M. Wysokowski, M, I. Petrenko, H. Ehrlich. Progress in Chitin Analytics. Carbohydrate Polymers 252 (2021) 117204. https://doi.org/10.1016/J.CARBPOL.2020.117204 [13] M.A. Hossin, N.H.K. Al Shaqsi, S.J. Al Touby, M.A. Al Sibani. Review of Polymeric Chitin Extraction, Characterization, and Applications. Arabian Journal of Geosciences 14 (2021) 1870. https://doi.org/10.1007/s12517-021-08239-0 https://doi.org/10.5599/admet.1999 https://doi.org/10.2174/138161210791208992 https://doi.org/10.1039/d0bm00558d https://doi.org/10.1002/pi.3056 https://doi.org/10.3390/pharmaceutics9040053 https://doi.org/10.1186/s40824-019-0166-x https://doi.org/10.1039/c8en00053k https://doi.org/10.1002/jctb.1164 https://doi.org/10.1016/J.REACTFUNCTPOLYM.2021.104849 https://doi.org/10.32604/JRM.2022.018142 https://doi.org/10.1007/s10311-019-00901-0 https://doi.org/10.1007/978-3-319-16298-0_13 https://doi.org/10.1016/J.CARBPOL.2020.117204 https://doi.org/10.1007/s12517-021-08239-0 R. D. Pratiwi et al. ADMET & DMPK 11(4) (2023) 435-455 446 [14] I. Younes, M. Rinaudo. Chitin and chitosan preparation from marines sources. Structure, properties and applications. Marine Drugs 13 (2015) 1133-1174. https://doi.org/10.3390/md13031133 [15] M.N.R. Kumar. A review of chitin and chitosan applications. Reactive and Functional Polymers 46 (2000) 1-27. https://doi.org/10.1016/S1381-5148(00)00038-9 [16] M.H. Periayah, A.S. Halim, A.Z.M. Saad. Chitosan: A Promising Marine Polysaccharide for Biomedical Research. Pharmacognosy Review 10 (2016) 39-42. https://doi.org/10.4103/0973-7847.176545 [17] S. Islam, M.A.R. Bhuiyan, M.N. Islam. Chitin and Chitosan: Structure, Properties and Applications in Biomedical Engineering. Journal of Polymers and The Environment 25 (2017) 854-866. https://doi.org/10.1007/s10924-016-0865-5 [18] R. Ravindra, K.R. Krovvidi, A.A. Khan. Solubility Parameter of Chitin and Chitosan. Carbohydrate Polymers 36 (1998) 121-127. https://doi.org/10.1016/S0144-8617(98)00020-4 [19] S.B. Nimbeni, B.S. Nimbeni, D.D. Divakar. Role of Chitosan in Remineralization of Enamel and Dentin: A Systematic Review. International Journal of Clinical Pediatric Dentistry 14 (2021) 562-568. https://doi.org/10.5005/jp-journals-10005-1971 [20] F. Gao, B.S. Zhang, J.H. Zhao, J.F. Huang, P.S. Jia, S. Wang, J. Zhang, J.M. Zhou, H.S. Guo. Deacetylation of Chitin Oligomers Increases Virulence in Soil-Borne Fungal Pathogens. Nature Plants 5 (2019) 1167-1176. https://doi.org/10.1038/s41477-019-0527-4 [21] L. Pérez-Álvarez, L. Ruiz-Rubio, J.L. Vilas-Vilela. Determining the Deacetylation Degree of Chitosan: Opportunities to Learn Instrumental Techniques. Journal of Chemical Education 95 (2018) 1022- 1028. https://doi.org/10.1021/acs.jchemed.7b00902 [22] O.U. Akakuru, H. Louis, P.I. Amos, O.C. Akakuru, E.I. Nosike, E.F. Ogulewe. The Chemistry of Chitin and Chitosan Justifying Their Nanomedical Utilities. Biochemistry and Pharmacology 7 (2018) 241. https://doi.org/10.4172/2167-0501.1000241 [23] J. Forsythe, G. Rassu, P. Giunchedi, R. James Gilbert, J.C. Mateos-Díaz, D.D. Ojeda-Hernández, D.; A.A. Canales-Aguirre, J. Matias-Guiu, U. Gomez-Pinedo. Potential of Chitosan and Its Derivatives for Biomedical Applications in the Central Nervous System. Frontiers in Bioengineering and Biotechnology 8 (2020) 389. https://doi.org/10.3389/fbioe.2020.00389 [24] V.K.Thakur, M.K. Thakur. Recent Advances in Graft Copolymerization and Applications of Chitosan: A Review. ACS Sustainable Chemistry and Engingeering 2 (2014) 2637-2652. https://doi.org/10.1021/sc500634p [25] T. Feng, Y. Du, J. Li, Y. Wei, Y.; Yao, P. Antioxidant Activity of Half N-Acetylated Water-Soluble Chitosan in Vitro. European Food Research and Technology 225 (2007) 133-138. https://doi.org/10.1007/s00217-006-0391-0 [26] Q.Y. Deng, C.R. Zhou, B.H. Luo. Preparation and Characterization of Chitosan Nanoparticles Containing Lysozyme. Pharmceutical Biology 44 (2006) 336-342. https://doi.org/10.1080/13880200600746246 [27] K. Sweidan, J. Abdel-Motalleb, N.D. Al-Jbour, R.M. Obaidat Jordan. Further investigation on the degree of deacetylation of chitosan determined by potentiometric titration. Journal of Excipients and Food Chemicals 2 (2011) 16-25. https://doaj.org/article/ad90534b4543471f998602bde4a1bf7f [28] T.A. Khan, K.K. Peh, S.H. Ch’ng. Reporting Degree of Deacetylation Values of Chitosan: The Influence of Analytical Methods. Journal of Pharmacy and Pharmaceutical Science 5 (2002) 205-212. https://pubmed.ncbi.nlm.nih.gov/12553887/ [29] R. Czechowska-Biskup, D. Jarosińska, B. Rokita, P. Ulański, J.M. Rosiak. Determination of Degree of Deacetylation of Chitosan - Comparison of Methods. Progress on Chemistry and Application of Chitin and Its Derivatives 17 (2012) 5-20. https://www.researchgate.net/publication/288104933_ Determination_of_degree_of_deacetylation_of_chitosan_-_Comparision_of_methods [30] Y. Jiang, C. Fu, S. Wu, G.Liu, J. Guo, Z. Su. Determination of the Deacetylation Degree of Chitooligosaccharides. Marine Drugs 15 (2017) 332. https://doi.org/10.3390/md15110332 [31] M. Lavertu, Z. Xia, A.N. Serreqi, M. Berrada, A. Rodrigues, D. Wang, M.D. Buschmann, A. Gupta. A Validated 1H NMR Method for the Determination of the Degree of Deacetylation of Chitosan. https://doi.org/10.3390/md13031133 https://doi.org/10.1016/S1381-5148(00)00038-9 https://doi.org/10.4103/0973-7847.176545 https://doi.org/10.1007/s10924-016-0865-5 https://doi.org/10.1016/S0144-8617(98)00020-4 https://doi.org/10.5005/jp-journals-10005-1971 https://doi.org/10.1038/s41477-019-0527-4 https://doi.org/10.1021/acs.jchemed.7b00902 https://doi.org/10.4172/2167-0501.1000241 https://doi.org/10.3389/fbioe.2020.00389 https://doi.org/10.1021/sc500634p https://doi.org/10.1007/s00217-006-0391-0 https://doi.org/10.1080/13880200600746246 https://doaj.org/article/ad90534b4543471f998602bde4a1bf7f https://pubmed.ncbi.nlm.nih.gov/12553887/ https://www.researchgate.net/publication/288104933_%20Determination_of_degree_of_deacetylation_of_chitosan_-_Comparision_of_methods https://www.researchgate.net/publication/288104933_%20Determination_of_degree_of_deacetylation_of_chitosan_-_Comparision_of_methods https://doi.org/10.3390/md15110332 ADMET & DMPK 11(4) (2023) 435-455 Eco-friendly synthesis of chitosan doi: https://doi.org/10.5599/adme t.1999 447 Journal of Pharmaceutical and Biomedical Analysis 32 (2003) 1149-1158. https://doi.org/10.1016/S0731-7085(03)00155-9 [32] A. Riofrio, T. Alcivar, H. Baykara. Environmental and economic viability of chitosan production in Guayas-Ecuador: a robust investment and life cycle analysis. ACS Omega 6 (2021) 23038-23051. https://doi.org/10.1021/acsomega.1c01672 [33] W. Wang, Y. Du, Y. Qiu, X. Wang, Y. Hu, J. Yang, et al. A new green technology for direct production of low molecular weight chitosan. Carbohydrate polymers 74 (2008) 127-132. https://doi.org/10.1016/j.carbpol.2008.01.25 [34] I. Younes, S. Hajj, V. Frachet, M. Rinaudo, K. Jellouli, M. Nasri. Chitin extraction from shrimp shell using enzymatic treatment. Antitumor, antioxidant and antimicrobial activities of chitosan. International Journal of Biological Macromolecules 69 (2014) 489-498. https://doi.org/10.1016/j.ijbiomac.2014.06.013 [35] Y. Yu, X. Liu, J. Miao, K. Leng. Chitin from Antarctic Krill shell: eco-preparation, detection, and characterization. International Journal of Biological Macromolecules 164 (2020) 4125-4137. https://doi.org/10.1016/j.ijbiomac.2020.08.244 [36] H. Zhang, S. Yun, L. Song, Y. Zhang, Y. Zhao. The preparation and characterization of chitin and chitosan under large-scale submerged fermentation level using shrimp by-products as substrate. International Journal of Biological Macromolecules 96 (2017) 334-339. https://doi.org/10.1016/j.ijbiomac.2016.12.017 [37] K. Mohan, A.R. Ganesan, P.N. Ezhilarasi, K.K. Kondamareddy, D.K. Rajan, P. Sathishkumar, J. Rajarajeswaran, L. Conterno. Green and Eco-Friendly Approaches for the Extraction of Chitin and Chitosan: A Review. Carbohydrate Polymers 287 (2022) 119349. https://doi.org/10.1016/j.carbpol.2022.119349 [38] N.S. Mahmoud, A.E. Ghaly, F. Arab. Unconventional approach for demineralization of deproinated crustacean shells for chitin production. American Journal of Biochemistry and Biotechnology 3(1) (2007) 1-9. https://doi.org/10.3844/ajbbsp.2007.1.9 [39] A.O. Ameh, M.T. Isa, D. Abutu, A. Danlami. Kinetic modelling of the demineralization of shrimp exoskeleton using citric acid. Leonardo Electron Journal Practice and Technology 25 (2014) 99-108. http://lejpt.academicdirect.org/A25/099_108.pdf [40] I. Younes, O. Ghorbel-Bellaj, R. Nasri, M. Chaabouni, M. Rinaudo, M. Nasri. Chitin and chitosan preparation from shrimp shells using optimized enzymatic deproteinization. Process Biochemistry 47 (2012) 2032-2039. https://doi.org/10.1016/j.procbio.2012.07.017 [41] A.U. Valdez-Peña, J.D. Espinoza-Perez, G.C. Sandoval-Fabian, N. Balagurusamy, A. Hernandez- Rivera, I.M. De-la-Garza-Rodriguez, J.C. Contreras-Esquivel, Screening of industrial enzymes for deproitenization of shrimp head for chitin recovery. Food Science and Biotechnology 19 (2010) 553- 557. https://doi.org/10.1007/s10068-010-0077-z [42] C. Hongkulsup, V.V. Khutoryanskiy, K. Niranjan. Enzyme assisted extraction of chitin from shrimp shells (Litopenaeus vannamei). Journal of Chemical Technology & Biotechnology 91 (2016) 1250- 1256. https://doi.org/10.1002/jctb.4714 [43] M.N. Marzieh, F. Zahra, E. Tahereh, K.N. Sara. Comparison of the physicochemical and structural characteristics of enzymatic produced chitin and commercial chitin. International Journal of Biological Macromolecules 139 (2019) 270-276. https://doi.org/10.1016/j.ijbiomac.2019.07.217 [44] M. Hossain, A. Iqbal. Production and Characterization of Chitosan from Shrimp Waste. Journal of the Bangladesh Agricultural University 12 (2014) 153-160. https://doi.org/10.3329/jbau.v12i1.21405 [45] C.T.G.V.M.T. Pires, J.A.P Vilela, C. Airoldi. The Effect of Chitin Alkaline Deacetylation at Different Condition on Particle Properties. Procedia Chemistry 9 (2014) 220-225. https://doi.org/10.1016/j.proche.2014.05.026 https://doi.org/10.5599/admet.1999 https://doi.org/10.1016/S0731-7085(03)00155-9 https://doi.org/10.1021/acsomega.1c01672 https://doi.org/10.1016/j.carbpol.2008.01.25 https://doi.org/10.1016/j.ijbiomac.2014.06.013 https://doi.org/10.1016/j.ijbiomac.2020.08.244 https://doi.org/10.1016/j.ijbiomac.2016.12.017 https://doi.org/10.1016/j.carbpol.2022.119349 https://doi.org/10.3844/ajbbsp.2007.1.9 http://lejpt.academicdirect.org/A25/099_108.pdf https://doi.org/10.1016/j.procbio.2012.07.017 https://doi.org/10.1007/s10068-010-0077-z https://doi.org/10.1002/jctb.4714 https://doi.org/10.1016/j.ijbiomac.2019.07.217 https://doi.org/10.3329/jbau.v12i1.21405 https://doi.org/10.1016/j.proche.2014.05.026 R. D. Pratiwi et al. ADMET & DMPK 11(4) (2023) 435-455 448 [46] R.F. Weska, J.M. Moura, L.M. Batista, J. Rizzi, L.A.A. Pinto. Optimization of Deacetylation in the Production of Chitosan from Shrimp Wastes: Use of Response Surface Methodology. Journal of Food Engineering 80 (2007) 749-753. https://doi.org/10.1016/J.JFOODENG.2006.02.006 [47] G. Galed, E. Diaz, F.M. Goycoolea, A. Heras. Influence of N-Deacetylation Conditions on Chitosan Production from α-Chitin. Natural Product Communications 3 (2008) 543-550. https://doi.org/10.1177/1934578X0800300414 [48] V.Y. Novikov, I.N. Konovalova, N. Dolgopyatova, N. The Mechanisms of Chitin and Chitosan Deacetylation During Long-term Alkaline Treatment. Applied Biochemsitry and Microbiology 58 (2022) 273-279. https://doi.org/10.1134/S0003683822030097 [49] J. Santoso, K.C. Adiputra, L.C. Soerdirga, K. Tarman. Effect of Acetic Acid Hydrolysis on the Characteristics of Water-Soluble Chitosan. In Proceedings of the IOP Conference Series: Earth and Environmental Science 414 (2020) 012021. https://doi.org/10.1088/1755-1315/414/1/012021 [50] C. Casadidio, D.V. Peregrina, M.R. Gigliobianco, S. Deng, R. Censi, P. di Martino. Chitin and Chitosans: Characteristics, Eco-Friendly Processes, and Applications in Cosmetic Science. Marine Drugs 17 (2019) 369. https://doi.org/10.3390/md17060369 [51] M.K. Rasweefali, S. Sabu, K.V. Sunooj, A. Sasidharan, K.A.M. Xavier. Consequences of Chemical Deacetylation on Physicochemical, Structural and Functional Characteristics of Chitosan Extracted from Deep-Sea Mud Shrimp. Carbohydrate Polymer Technologies and Applications 2 (2021) 100032. https://doi.org/10.1016/j.carpta.2020.100032 [52] G.M. Pawaskar, S. Pangannaya, K. Raval, D.R. Trivedi, R. Raval. Screening of Chitin Deacetylase Producing Microbes from Marine Source Using a Novel Receptor on Agar Plate. International Journal of Biology Macromolecular 131 (2019) 716-720. https://doi.org/10.1016/j.ijbiomac.2019.03.118 [53] J. Sebastian, T. Rouissi, S.K. Brar. Fungal Chitosan: Prospects and Challenges. Handbook of Chitin and Chitosan Preparation and Properties 1 (2020) 419-452. https://doi.org/10.1016/B978-0-12- 817970-3.00014-6 [54] I.A. Hoell, G. Vaaje-Kolstad, V.G.H. Eijsink. Structure and Function of Enzymes Acting on Chitin and Chitosan. Biotechnology Genetic Engineering Review 27 (2010) 331-366. https://doi.org/10.1080/02648725.2010.10648156 [55] S. Bartnicki-Garcia’, W.J. Nickerson. Nutrition, Growth, and Morphogenesis of Mucor rouxii. Journal of Bacteriology 84 (1962) 841-867. https://doi.org/10.1128/jb.84.4.841-858.1962 [56] D. Kafetzopoulos, A. Martinou, V. Bouriotis. Bioconversion of Chitin to Chitosan: Purification and Characterization of Chitin Deacetylase from Mucor rouxii. Proceedings of the National Academy of Sciences of the United States of America 90 (1993) 2564-2568. https://doi.org/10.1073/pnas.90.7.2564 [57] M.B. Kaczmarek, K. Struszczyk-Swita, X. Li, M. Szczęsna-Antczak, Daroch, M. Enzymatic Modifications of Chitin, Chitosan, and Chitooligosaccharides. Frontiers Bioengineering and Biotechnolology 7 (2019) 243. https://doi.org/10.3389/fbioe.2019.00243 [58] L. Grifoll-Romero, S. Pascual, H. Aragunde, X. Biarnés, A. Planas. Chitin Deacetylases: Structures, Specificities, and Biotech Applications. Polymers 10 (2018) 352. https://doi.org/10.3390/polym10040352 [59] K. Tokuyasu, S. Kaneko, K. Hayashi, Y. Mori. Production of a Recombinant Chitin Deacetylase in the Culture Medium of Escherichia coli Cells. FEBS Letters 458 (1999) 23-26. https://doi.org/10.1016/S0014-5793(99)01113-8 [60] P. Bhat, G.M. Pawaskar, R. Raval, S. Cord-Landwehr, B. Moerschbacher, B.Raval, K. Expression of Bacillus Licheniformis Chitin Deacetylase in E. coli PLysS: Sustainable Production, Purification and Characterisation. International Journal of Biolology Macromolecule 131 (2019) 1008-1013. https://doi.org/10.1016/j.ijbiomac.2019.03.144 [61] S. Naqvi, S. Cord-Landwehr, R. Singh, F. Bernard, S. Kolkenbrock, N.E. Gueddari, B.M. Moerschbacher. A Recombinant Fungal Chitin Deacetylase Produces Fully Defined Chitosan https://doi.org/10.1016/J.JFOODENG.2006.02.006 https://doi.org/10.1177/1934578X0800300414 https://doi.org/10.1134/S0003683822030097 https://doi.org/10.1088/1755-1315/414/1/012021 https://doi.org/10.3390/md17060369 https://doi.org/10.1016/j.carpta.2020.100032 https://doi.org/10.1016/j.ijbiomac.2019.03.118 https://doi.org/10.1016/B978-0-12-817970-3.00014-6 https://doi.org/10.1016/B978-0-12-817970-3.00014-6 https://doi.org/10.1080/02648725.2010.10648156 https://doi.org/10.1128/jb.84.4.841-858.1962 https://doi.org/10.1073/pnas.90.7.2564 https://doi.org/10.3389/fbioe.2019.00243 https://doi.org/10.3390/polym10040352 https://doi.org/10.1016/S0014-5793(99)01113-8 https://doi.org/10.1016/j.ijbiomac.2019.03.144 ADMET & DMPK 11(4) (2023) 435-455 Eco-friendly synthesis of chitosan doi: https://doi.org/10.5599/adme t.1999 449 Oligomers with Novel Patterns of Acetylation. Applied Environmental Microbiology 82 (2016) 6645- 6655. https://doi.org/10.1128/AEM.01961-16 [62] B. Shrestha, K. Blondeau, W.F. Stevens, F.L. Hegarat. Expression of Chitin Deacetylase from Colletotrichum Lindemuthianum in Pichia pastoris: Purification and Characterization. Protein Expression and Purification 38 (2004) 196-204. https://doi.org/10.1016/J.PEP.2004.08.012 [63] C. Mishra, C.E. Semino, K.J. Mccreath, H. de La Vega, B.J. Jones, C.A. Specht, P.W. Robbins. Cloning and Expression of Two Chitin Deacetylase Genes of Saccharomyces cerevisiae. Yeast 13 (1997) 327- 336. https://doi.org/10.1002/(SICI)1097-0061 [64] M.B. Kaczmarek, K. Struszczyk-Swita, M. Xiao, M. Szczęsna-Antczak, T. Antczak, M. Gierszewska, A. Steinbüchel, M. Daroch. Polycistronic Expression System for Pichia pastoris Composed of Chitino- and Chitosanolytic Enzymes. Frontiers Bioengineering and Biotechnolology 9 (2021) 710922. https://doi.org/10.3389/fbioe.2021.710922. [65] A. Martinou, D. Koutsioulis, V. Bouriotis. Cloning and Expression of a Chitin Deacetylase Gene (CDA2) from Saccharomyces cerevisiae in Escherichia coli: Purification and Characterization of the Cobalt-Dependent Recombinant Enzyme. Enzyme Microbiology Technology 32 (2003) 757-763. https://doi.org/10.1016/S0141-0229(03)00048-6 [66] H. el Knidri, J. Dahmani, A. Addaou, A. Laajeb, A. Lahsini. Rapid and Efficient Extraction of Chitin and Chitosan for Scale-up Production: Effect of Process Parameters on Deacetylation Degree and Molecular Weight. International Journal of Biological Macromolecules 139 (2019) 1092-1102. https://doi.org/10.1016/j.ijbiomac.2019.08.079 [67] A. Zaeni, E. Safitri, B. Fuadah, I.N. Sudiana. Microwave-assisted hydrolysis of chitosan from shrimp shell waster for glucosamine hydrochlorid production. The 5th International Conference on Theoretical and Applied Physics, IOP Publishing of Physics Conference Series. 846 (2015) 23-25. https://doi.org/10.1088/1742-6596/846/1/012011 [68] J. Zhang, M. Feng, X. Lu, C. Shi, X. Li, J. Xin, et al. Base-free preparation of low molecular weight chitin from crab shell. Carbohydrate Polymers 190 (2018) 148-155. https://doi.org/10.1016/j.carbpol.2018.02.019 [69] S. Tsubaki, J.I. Azuma. Total fractional of green tea residue by microwave-assisted alkaline pretreatment and enzymatic hydrolysis. Biosensor Technology. 131 (2013) 485-491. https://doi.org/10.1016/j.biortech.2013.01.001 [70] F. Li, X. You, Q. Li, D. Qin, M. Wang, S. Yuan, X. Chen, S. Bi. Homogeneous Deacetylation and Degradation of Chitin in NaOH/Urea Dissolution System. International Journal of Biological Macromolecules 189 (2021) 391-397. https://doi.org/10.1016/J.IJBIOMAC.2021.08.126 [71] F.A. Vicente, M. Huš, B. Likozar, U. Novak. Chitin Deacetylation Using Deep Eutectic Solvents: Ab Initio-Supported Process Optimization. ACS Sustainable Chemical Engineering 9 (2021) 3874-3886, https://doi.org/10.1021/acssuschemeng.0c08976 [72] E.L. Smith, A.P. Abbott, K.S. Ryder. Deep Eutectic Solvents (DESs) and Their Applications. Chemical Reviews 114 (2014) 11060-11082. https://doi.org/10.1021/cr300162p [73] D. Zhao, W.C. Huang, N. Guo, S. Zhang, C. Xue, X. Mao. Two-Step Separation of Chitin from Shrimp Shells Using Citric Acid and Deep Eutectic Solvents with the Assistance of Microwave. Polymers 11 (2019) 409. https://doi.org/10.3390/polym11030409 [74] S.A. White, R. Farina-Peter, I. Fulton. Production and Isolation of Chitosan from Mucor rouxii. Applied and Environmental Microbiology 38 (1979) 323-326. https://doi.org/10.1128/aem.38.2.323-328.1979 [75] H. Zhang, H. Tachikawa, X.D. Gao, H. Nakanishi. Applied Usage of Yeast Spores as Chitosan Beads. Applied and Environmental Microbiology 80 (2014) 5098-5105. https://doi.org/10.1128/AEM.00677-14 [76] M. Afroz, M. Nayeem, H. Kashem, M. Masirul Afroz, N.H. Kashem, K.M. Prottoy, S. Piash, N. Islam. Saccharomyces Cerevisiae as an Untapped Source of Fungal Chitosan for Antimicrobial Action. https://doi.org/10.5599/admet.1999 https://doi.org/10.1128/AEM.01961-16 https://doi.org/10.1016/J.PEP.2004.08.012 https://doi.org/10.1002/(SICI)1097-0061 https://doi.org/10.3389/fbioe.2021.710922 https://doi.org/10.1016/S0141-0229(03)00048-6 https://doi.org/10.1016/j.ijbiomac.2019.08.079 https://doi.org/10.1088/1742-6596/846/1/012011 https://doi.org/10.1016/j.carbpol.2018.02.019 https://doi.org/10.1016/j.biortech.2013.01.001 https://doi.org/10.1016/J.IJBIOMAC.2021.08.126 https://doi.org/10.1021/acssuschemeng.0c08976 https://doi.org/10.1021/cr300162p https://doi.org/10.3390/polym11030409 https://doi.org/10.1128/aem.38.2.323-328.1979 https://doi.org/10.1128/AEM.00677-14 R. D. Pratiwi et al. ADMET & DMPK 11(4) (2023) 435-455 450 Applied Biochemistry and Biotechnology 193 (2021) 3765–3786. https://doi.org/10.21203/rs.3.rs- 490748/v1 [77] N. Morin-Crini, E. Lichtfouse, G. Torri, G. Crini. Applications of Chitosan in Food, Pharmaceuticals, Medicine, Cosmetics, Agriculture, Textiles, Pulp and Paper, Biotechnology, and Environmental Chemistry. Environmental Chemistry Letters 17 (2019) 1667-1692. https://doi.org/10.1007/s10311- 019-00904-x [78] Z. Shariatinia. Pharmaceutical Applications of Chitosan. Advanced Colloid Interface Science 263 (2019) 131-194. https://doi.org/10.1016/J.CIS.2018.11.008 [79] S. Bayda, M. Adeel, T. Tuccinardi, M. Cordani, F. Rizzolio, A. Baeza. Molecules The History of Nanoscience and Nanotechnology: From Chemical-Physical Applications to Nanomedicine. Molecules 25 (2020) 112. https://doi.org/10.3390/molecules25010112 [80] R.A. Freitas. What Is Nanomedicine? Nanomedicine 1 (2005) 2-9. https://doi.org/10.1016/J.NANO.2004.11.003 [81] B. Pelaz, C. Alexiou, R.A. Alvarez-Puebla, F. Alves, A.M. Andrews, Φ.S. Ashraf, L.P. Balogh, L. Ballerini, A. Bestetti, C. Brendel. Diverse Applications of Nanomedicine Nano Focus. ACS Nano 11 (2017) 2313-2381. https://doi.org/10.1021/acsnano.6b06040 [82] J. Morais Catita, B. Salas Valdez, R. Jorge, C. Vitorino, S. Soares, J. Sousa, A. Pais. Nanomedicine: Principles, Properties, and Regulatory Issues. Frontiers in Chemistry 1 (2018) 360. https://doi.org/10.3389/fchem.2018.00360 [83] P. Satalkar, B.S. Elger, D.M.Shaw. Defining Nano, Nanotechnology and Nanomedicine: Why Should It Matter? Science Engineering Ethics 22 (2016) 1255-1276. https://doi.org/10.1007/s11948-015- 9705-6 [84] D. Astruc, Introduction to Nanomedicine. Molecules 21(1) (2016) 4. https://doi.org/10.3390/molecules21010004 [85] H. Kobayashi, R. Watanabe, P.L. Choyke. Improving Conventional Enhanced Permeability and Retention (EPR) Effects; What Is the Appropriate Target? Theranostics 4 (2014) 81-89. https://doi.org/10.7150/thno.7193 [86] M. Germain, F. Caputo, S. Metcalfe, G. Tosi, K. Spring, A.K.O. Åslund, A. Pottier, R. Schiffelers, A. Ceccaldi, R. Schmid. Delivering the Power of Nanomedicine to Patients Today. Journal of Controlled Release 326 (2020) 164-171. https://doi.org/10.1016/J.JCONREL.2020.07.007 [87] P. Ray, N. Haideri, I. Haque, O. Mohammed, S. Chakraborty, S. Banerjee, M. Quadir, A.E. Brinker, S.K. Banerjee. The Impact of Nanoparticles on the Immune System: A Gray Zone of Nanomedicine. The Journal of Immunological Science 5 (2021) 19-33. https://doi.org/10.29245/2578- 3009/2021/1.1206 [88] D. Zhao, S. Yu, B. Sun, S. Gao, S. Guo, K. Zhao. Biomedical Applications of Chitosan and Its Derivative Nanoparticles. Polymers (Basel) 10 (2018) 462. https://doi.org/10.3390/polym10040462 [89] E.F. Ribeiro, T.T. de Barros-Alexandrino, O.B.G. Assis, A.C. Junior, A. Quiles, I. Hernando, V.R. Nicoletti. Chitosan and Crosslinked Chitosan Nanoparticles: Synthesis, Characterization and Their Role as Pickering Emulsifiers. Carbohydrate Polymers 250 (2020) 116878. https://doi.org/10.1016/J.CARBPOL.2020.116878 [90] O.A.C. Monteiro, C. Airoldi. Some Studies of Crosslinking Chitosan-Glutaraldehyde Interaction in a Homogeneous System. International Journal of Biological Macromolecules 26 (1999) 119-128. https://doi.org/10.1016/S0141-8130(99)00068-9 [91] N.R. Kildeeva, P.A. Perminov, L. Vladimirov, V. Novikov, S.N. Mikhailov. About Mechanism of Chitosan Cross-Linking with Glutaraldehyde. Russian Journal of Bioorganic Chemsitry 35 (2009) 360- 369. https://doi.org/10.1134/S106816200903011X [92] K. Song, H. Xu, B. Mu, K. Xie, Y. Yang. Non-Toxic and Clean Crosslinking System for Protein Materials: Effect of Extenders on Crosslinking Performance. Journal of Cleaner Production 150 (2017) 214-223. https://doi.org/10.1016/j.jclepro.2017.03.025 https://doi.org/10.21203/rs.3.rs-490748/v1 https://doi.org/10.21203/rs.3.rs-490748/v1 https://doi.org/10.1007/s10311-019-00904-x https://doi.org/10.1007/s10311-019-00904-x https://doi.org/10.1016/J.CIS.2018.11.008 https://doi.org/10.3390/molecules25010112 https://doi.org/10.1016/J.NANO.2004.11.003 https://doi.org/10.1021/acsnano.6b06040 https://doi.org/10.3389/fchem.2018.00360 https://doi.org/10.1007/s11948-015-9705-6 https://doi.org/10.1007/s11948-015-9705-6 https://doi.org/10.3390/molecules21010004 https://doi.org/10.7150/thno.7193 https://doi.org/10.1016/J.JCONREL.2020.07.007 https://doi.org/10.29245/2578-3009/2021/1.1206 https://doi.org/10.29245/2578-3009/2021/1.1206 https://doi.org/10.3390/polym10040462 https://doi.org/10.1016/J.CARBPOL.2020.116878 https://doi.org/10.1016/S0141-8130(99)00068-9 https://doi.org/10.1134/S106816200903011X https://doi.org/10.1016/j.jclepro.2017.03.025 ADMET & DMPK 11(4) (2023) 435-455 Eco-friendly synthesis of chitosan doi: https://doi.org/10.5599/adme t.1999 451 [93] T. Takigawa, Y. Endo. Effects of Glutaraldehyde Exposure on Human Health. Journal of Occupational Health 48 (2006) 75-87. https://doi.org/10.1539/joh.48.75 [94] Y. Zhu, L.M. Marin, Y. Xiao, E.R. Gillies, W.L. Siqueira. pH-Sensitive Chitosan Nanoparticles for Salivary Protein Delivery. Nanomaterials 11 (2021) 1028. https://doi.org/10.3390/nano11041028 [95] L. Li, G. Jiang, W. Yu, D. Liu, H. Chen, Y. Liu, Z. Tong, X. Kong, J. Yao. Preparation of Chitosan-Based Multifunctional Nanocarriers Overcoming Multiple Barriers for Oral Delivery of Insulin. Materials Science and Engineering: C 70 (2017) 278-286. https://doi.org/10.1016/J.MSEC.2016.08.083 [96] L. Li, L. Yang, M. Li, L. Zhang. A Cell-Penetrating Peptide Mediated Chitosan Nanocarriers for Improving Intestinal Insulin Delivery. Carbohydrate Polymers 174 (2017) 182-189. https://doi.org/10.1016/J.CARBPOL.2017.06.061 [97] P. Batista, P. Castro, A.R. Madureira, B. Sarmento, M. Pintado. Development and Characterization of Chitosan Microparticles-in-Films for Buccal Delivery of Bioactive Peptides. Pharmaceutics 12 (2019) 32. https://doi.org/10.3390/ph12010032 [98] Y. Lv, J. Zhang, C. Wang. Self-Assembled Chitosan Nanoparticles for Intranasal Delivery of Recombinant Protein Interleukin-17 Receptor C (IL-17RC): Preparation and Evaluation in Asthma Mice. Bioengineered 12 (2021) 3029-3039. https://doi.org/10.1080/21655979.2021.1940622 [99] M. Zohri, H.A. Javar, T. Gazori, M.R. Khoshayand, S. Hamid Aghaee-Bakhtiari, M.H. Ghahremani. Response Surface Methodology for Statistical Optimization of Chitosan/Alginate Nanoparticles as a Vehicle for Recombinant Human Bone Morphogenetic Protein-2 Delivery. International Journal of Nanomedicine 15 (2020) 8345-8356. https://doi.org/10.2147/IJN.S250630 [100] R. Ghosh, A.B. Susmita Mondal, D. Mukherjee, A. Adhikari, S.A. Ahmed, C.D.I. Reem Alsantali, A.S. Khder, C.F.M. Hatem Altass, Z. Moussa, R. Das, et al. Oral Drug Delivery Using a Polymeric Nanocarrier: Chitosan Nanoparticles in the Delivery of Rifampicin. Materials Advances 3 (2022), 4622-4628. https://doi.org/10.1039/d2ma00295g [101] N. Hosseini-Ashtiani, A. Tadjarodi, R. Zare-Dorabei. Low Molecular Weight Chitosan- Cyanocobalamin Nanoparticles for Controlled Delivery of Ciprofloxacin: Preparation and Evaluation. International Journal of Biological Macromolecules 176 (2021) 459-467. https://doi.org/10.1016/j.ijbiomac.2021.02.093 [102] E. Mazzotta, S. de Benedittis, A. Qualtieri, R. Muzzalupo. Pharmaceutics Actively Targeted and Redox Responsive Delivery of Anticancer Drug by Chitosan Nanoparticles. Pharmaceutics 12 (2020) 26. https://doi.org/10.3390/pharmaceutics12010026 [103] G. Khairnar, V. Mokale, A. Mujumdar, J. Naik. Development of Nanoparticulate Sustained Release Oral Drug Delivery System for the Antihyperglycemic with Antihypertensive Drug. Materials Technology 34 (2019) 880-888. https://doi.org/10.1080/10667857.2019.1639019 [104] N. Aminu, S.Y. Chan, M.F. Yam, S.M. Toh. A Dual-Action Chitosan-Based Nanogel System of Triclosan and Flurbiprofen for Localised Treatment of Periodontitis. International Journal of Pharmacy 570 (2019) 118659. https://doi.org/10.1016/j.ijpharm.2019.118659 [105] A.S. Tzeyung, S.K. Bhattamisra, T. Madheswaran, N.A. Alhakamy, H.M. Aldawsari, A.K. Radhakrishnan. Pharmaceutics Fabrication, Optimization, and Evaluation of Rotigotine -Loaded Chitosan Nanoparticles for Nose-To-Brain Delivery. Pharmaceutics 11 (2019) 26. https://doi.org/10.3390/pharmaceutics11010026 [106] G.N. Shi, C.N Zhang, R. Xu, J.F. Niu, H.J. Song, X.Y. Zhang, W.W. Wang,Y.M. Wang, C. Li, X.Q. Wei. et al. Enhanced Antitumor Immunity by Targeting Dendritic Cells with Tumor Cell Lysate-Loaded Chitosan Nanoparticles Vaccine. Biomaterials 113 (2017) 191-202. https://doi.org/10.1016/J.BIOMATERIALS.2016.10.047 [107] P.D. Lopes, C.H. Okino, F.S. Fernando, C. Pavani, V.M. Casagrande, R.F.V. Lopez, M. Montassier, H.J. Montassier. Inactivated Infectious Bronchitis Virus Vaccine Encapsulated in Chitosan Nanoparticles Induces Mucosal Immune Responses and Effective Protection against Challenge. Vaccine 36 (2018) 2630-2636. https://doi.org/10.1016/J.VACCINE.2018.03.065 https://doi.org/10.5599/admet.1999 https://doi.org/10.1539/joh.48.75 https://doi.org/10.3390/nano11041028 https://doi.org/10.1016/J.MSEC.2016.08.083 https://doi.org/10.1016/J.CARBPOL.2017.06.061 https://doi.org/10.3390/ph12010032 https://doi.org/10.1080/21655979.2021.1940622 https://doi.org/10.2147/IJN.S250630 https://doi.org/10.1039/d2ma00295g https://doi.org/10.1016/j.ijbiomac.2021.02.093 https://doi.org/10.3390/pharmaceutics12010026 https://doi.org/10.1080/10667857.2019.1639019 https://doi.org/10.1016/j.ijpharm.2019.118659 https://doi.org/10.3390/pharmaceutics11010026 https://doi.org/10.1016/J.BIOMATERIALS.2016.10.047 https://doi.org/10.1016/J.VACCINE.2018.03.065 R. D. Pratiwi et al. ADMET & DMPK 11(4) (2023) 435-455 452 [108] N.H. AbdelAllah, Y. Gaber, M.E. Rashed, A.F. Azmy, H.A. Abou-Taleb, S. AbdelGhani. Alginate-Coated Chitosan Nanoparticles Act as Effective Adjuvant for Hepatitis A Vaccine in Mice. International Journal of Biological Macromolecules 152 (2020) 904-912. https://doi.org/10.1016/J.IJBIOMAC.2020.02.287 [109] M. Mehrabi, N.M. Dounighi, S.M.R. Sorkhabadi, D. Doroud, A. Amani, M. Khoobi, S. Ajdary, Y. Pilehvar-Soltanahmadi. Development and Physicochemical, Toxicity and Immunogenicity Assessments of Recombinant Hepatitis B Surface Antigen (RHBsAg) Entrapped in Chitosan and Mannosylated Chitosan Nanoparticles: As a Novel Vaccine Delivery System and Adjuvant. Artificial Cells Nanomedine and Biotechnology 46 (2018) 230-240. https://doi.org/10.1080/21691401.2017.1417868 [110] S. Zhang, S. Huang, L. Lu, X. Song, P. Li, F. Wang. Curdlan Sulfate-O-Linked Quaternized Chitosan Nanoparticles: Potential Adjuvants to Improve the Immunogenicity of Exogenous Antigens via Intranasal Vaccination. International Journal of Nanomedicine 13 (2018) 2377-2394. https://doi.org/10.2147/IJN.S158536. 13 2377-2394 [111] J. Zhang, H. Sun, C. Gao, Y. Wang, X. Cheng, Y. Yang, Q. Gou, L. Lei, Y. Chen, X. Wang, et al. Development of a Chitosan‐modified PLGA Nanoparticle Vaccine for Protection against Escherichia coli K1 Caused Meningitis in Mice. Journal of Nanobiotechnology 19 (2021) 69. https://doi.org/10.1186/s12951-021-00812-9 [112] C. Rodolfo, D. Eusébio, C. Ventura, R. Nunes, H.F. Florindo, D. Costa, A. Sousa, J. Valente, A. Panitch. Pharmaceutics Design of Experiments to Achieve an Efficient Chitosan-Based DNA Vaccine Delivery System. Pharmaceuitics 13 (2021) 1369. https://doi.org/10.3390/pharmaceutics13091369 [113] B.H.G. Marandi, M.R. Zolfaghari, R. Kazemi, M.J. Motamedi, J. Amani. Immunization against Vibrio Cholerae, ETEC, and EHEC with Chitosan Nanoparticle Containing LSC Chimeric Protein. Microbial Pathogenesis 134 (2019) 103600. https://doi.org/10.1016/J.MICPATH.2019.103600 [114] M. Bagheri, M. Validi, A. Gholipour, P. Makvandi, E. Sharifi. Chitosan Nanofiber Biocomposites for Potential Wound Healing Applications: Antioxidant Activity with Synergic Antibacterial Effect. Bioengineering and Translational Medicine 7 (2021) 10254. https://doi.org/10.1002/btm2.10254 [115] P. Abrica-González, A. Zamora-Justo, A. Sotelo-López, G.R. Vázquez-Martínez, J.A. Balderas-López, A. Muñoz-Diosdado, M. Ibáñez-Hernández. Gold Nanoparticles with Chitosan, N-Acylated Chitosan, and Chitosan Oligosaccharide as DNA Carriers. Nanoscale Research Letters 14 (2019) 258. https://doi.org/10.1186/s11671-019-3083-y [116] Y.J. Zhang, B. Gao, X.W. Liu. Topical and Effective Hemostatic Medicines in the Battlefield. International Journal of Clinical Experimental Medicine 8 (2015) 10-19. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4358424/ [117] S. Hu, S. Bi, D. Yan, Z. Zhou, G. Sun, X. Cheng, X. Chen. Preparation of Composite Hydroxybutyl Chitosan Sponge and Its Role in Promoting Wound Healing. Carbohydrate Polymers 184 (2018) 154- 163. https://doi.org/10.1016/J.CARBPOL.2017.12.033 [118] C. Cui, S. Sun, S. Wu, S. Chen, J. Ma, F. Zhou. Electrospun Chitosan Nanofibers for Wound Healing Application. Engineered Regeneration 2 (2021) 82-90. https://doi.org/10.1016/J.ENGREG.2021.08.001 [119] X. Du, L. Wu, H. Yan, Z. Jiang, S. Li, W. Li, Y. Bai, Wang, Z. Cheng, D. Kong, et al. Microchannelled Alkylated Chitosan Sponge to Treat Noncompressible Hemorrhages and Facilitate Wound Healing. Nature Communication 12 (2021) 4733. https://doi.org/10.1038/s41467-021-24972-2 [120] J. Chi, X. Zhang, C. Chen, C. Shao, Y. Zhao, Y. Wang. Antibacterial and Angiogenic Chitosan Microneedle Array Patch for Promoting Wound Healing. Bioactive Materials 5 (2020) 253-259, https://doi.org/10.1016/J.BIOACTMAT.2020.02.004 [121] K.S. Venkataprasanna, J. Prakash, S. Vignesh, G. Bharath, M. Venkatesan, F. Banat, S. Sahabudeen, S. Ramachandran, G.D. Venkatasubbu. Fabrication of Chitosan/PVA/GO/CuO Patch for Potential Wound Healing Application. International Journal of Biological Macromolecules 143 (2020) 744-762. https://doi.org/10.1016/J.IJBIOMAC.2019.10.029 https://doi.org/10.1016/J.IJBIOMAC.2020.02.287 https://doi.org/10.1080/21691401.2017.1417868 https://doi.org/10.2147/IJN.S158536.%2013%202377–2394 https://doi.org/10.1186/s12951-021-00812-9 https://doi.org/10.3390/pharmaceutics13091369 https://doi.org/10.1016/J.MICPATH.2019.103600 https://doi.org/10.1002/btm2.10254 https://doi.org/10.1186/s11671-019-3083-y https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4358424/ https://doi.org/10.1016/J.CARBPOL.2017.12.033 https://doi.org/10.1016/J.ENGREG.2021.08.001 https://doi.org/10.1038/s41467-021-24972-2 https://doi.org/10.1016/J.BIOACTMAT.2020.02.004 https://doi.org/10.1016/J.IJBIOMAC.2019.10.029 ADMET & DMPK 11(4) (2023) 435-455 Eco-friendly synthesis of chitosan doi: https://doi.org/10.5599/adme t.1999 453 [122] N. Devi, J. Dutta. Preparation and Characterization of Chitosan-Bentonite Nanocomposite Films for Wound Healing Application. International Journal of Biological Macromolecules 104 (2017) 1897- 1904. https://doi.org/10.1016/J.IJBIOMAC.2017.02.080 [123] A.M. Abdel-Mohsen, J. Frankova, R.M. Abdel-Rahman, A.A. Salem, N.M. Sahffie, I. Kubena, J. Jancar. Chitosan-Glucan Complex Hollow Fibers Reinforced Collagen Wound Dressing Embedded with Aloe Vera. II. Multifunctional Properties to Promote Cutaneous Wound Healing. International Journal of Pharmacy 582 (2020) 119349. https://doi.org/10.1016/J.IJPHARM.2020.119349 [124] N. Bölgen, D. Demir, M.S. Yalçın, S. Özdemir. Development of Hypericum Perforatum Oil Incorporated Antimicrobial and Antioxidant Chitosan Cryogel as a Wound Dressing Material. International Journal of Biological Macromolecules 161 (2020) 1581-1590. https://doi.org/10.1016/J.IJBIOMAC.2020.08.056 [125] I. Koumentakou, Z. Terzopoulou, A. Michopoulou, I. Kalafatakis, K. Theodorakis, D. Tzetzis, D. Bikiaris. Chitosan Dressings Containing Inorganic Additives and Levofloxacin as Potential Wound Care Products with Enhanced Hemostatic Properties. International Journal of Biological Macromolecules 162 (2020) 693-703. https://doi.org/10.1016/J.IJBIOMAC.2020.06.187 [126] J. Si, Y. Yang, X. Xing, F. Yang, P. Shan. Controlled Degradable Chitosan/Collagen Composite Scaffolds for Application in Nerve Tissue Regeneration. Polymer Degradation and Stability 166 (2019) 73-85. https://doi.org/10.1016/J.POLYMDEGRADSTAB.2019.05.023 [127] A. Fathi, M. Khanmohammadi, A. Goodarzi, L. Foroutani, Z.T. Mobarakeh, J. Saremi, Z. Arabpour, J. Ai. Fabrication of Chitosan-Polyvinyl Alcohol and Silk Electrospun Fiber Seeded with Differentiated Keratinocyte for Skin Tissue Regeneration in Animal Wound Model. Journal of Biological Engineering 14 (2020) 27. https://doi.org/10.1186/s13036-020-00249-y [128] S. Dasgupta, K. Maji, S.K. Nandi. Investigating the Mechanical, Physiochemical and Osteogenic Properties in Gelatin-Chitosan-Bioactive Nanoceramic Composite Scaffolds for Bone Tissue Regeneration: In Vitro and in Vivo. Materials Science and Engineering C 94 (2019) 713-728. https://doi.org/10.1016/J.MSEC.2018.10.022 [129] B.N. Singh, V. Veeresh, S.P. Mallick, Y. Jain, S. Sinha, A. Rastogi, P. Srivastava. Design and Evaluation of Chitosan/Chondroitin Sulfate/Nano-Bioglass Based Composite Scaffold for Bone Tissue Engineering. International Journal of Biological Macromolecules 133 (2019) 817-830. https://doi.org/10.1016/J.IJBIOMAC.2019.04.107 [130] S.P. Mallick, B.N. Singh, A. Rastogi, P. Srivastava. Design and Evaluation of Chitosan/Poly(l- Lactide)/Pectin Based Composite Scaffolds for Cartilage Tissue Regeneration. International Journal Biological Macromolecules 112 (2018) 909-920. https://doi.org/10.1016/J.IJBIOMAC.2018.02.049 [131] A.M. dos Santos, S.G. Carvalho, L.M.B. Ferreira, M. Chorilli, M.P.D. Gremião. Understanding the Role of Electrostatic Interactions on the Association of 5-Fluorouracil to Chitosan-TPP Nanoparticles. Colloids Surface A Physicochemical Engingeering Aspects 640 (2022) 128417. https://doi.org/10.1016/j.colsurfa.2022.128417 [132] M.F. Warsito, F.A. Agustiani. A Review on Factors Affecting Chitosan Nanoparticles Formation. International Symposium on Applied Chemistry, IOP Conference Series: Materials Science and Engineering, Indonesia, 2020, 012027. https://doi.org/10.1088/1757-899X/1011/1/012027 [133] S. Hajji, N. Ktari, R. Ben Salah, S. Boufi, F. Debeaufort, M. Nasri. Development of Nanocomposite Films Based on Chitosan and Gelatin Loaded with Chitosan-Tripolyphosphate Nanoparticles: Antioxidant Potentials and Applications in Wound Healing. Journal of Polymers and the Environment 30 (2022) 833-854. https://doi.org/10.1007/s10924-021-02239-7 [134] J.M.N.A. Bezerra, A.C.J. Oliveira, E.C. Silva-Filho, P. Severino, S.B. Souto, E.B. Souto, M.F.L.R Soares, J.L. Soares-Sobrinho. The Potential Role of Polyelectrolyte Complex Nanoparticles Based on Cashew Gum, Tripolyphosphate and Chitosan for the Loading of Insulin. Diabetology 2 (2021) 107-116. https://doi.org/10.3390/diabetology2020009 [135] H.C. Yew, M. Misran. Preparation and Characterization of pH Dependent κ-Carrageenan-Chitosan Nanoparticle as Potential Slow Released Delivery Carrier. Iranian Polymer Journal (English Edition) 25 (2016) 1037-1046. https://doi.org/10.1007/s13726-016-0489-6 https://doi.org/10.5599/admet.1999 https://doi.org/10.1016/J.IJBIOMAC.2017.02.080 https://doi.org/10.1016/J.IJPHARM.2020.119349 https://doi.org/10.1016/J.IJBIOMAC.2020.08.056 https://doi.org/10.1016/J.IJBIOMAC.2020.06.187 https://doi.org/10.1016/J.POLYMDEGRADSTAB.2019.05.023 https://doi.org/10.1186/s13036-020-00249-y https://doi.org/10.1016/J.MSEC.2018.10.022 https://doi.org/10.1016/J.IJBIOMAC.2019.04.107 https://doi.org/10.1016/J.IJBIOMAC.2018.02.049 https://doi.org/10.1016/j.colsurfa.2022.128417 https://doi.org/10.1088/1757-899X/1011/1/012027 https://doi.org/10.1007/s10924-021-02239-7 https://doi.org/10.3390/diabetology2020009 https://doi.org/10.1007/s13726-016-0489-6 R. D. Pratiwi et al. ADMET & DMPK 11(4) (2023) 435-455 454 [136] C. Li, S. Hein, K. Wang. Chitosan-Carrageenan Polyelectrolyte Complex for the Delivery of Protein Drugs. ISRN Biomaterials 2013 (2013) 629807. https://doi.org/10.5402/2013/629807 [137] N.T.N. Vo, L. Huang, H. Lemos, A.L. Mellor, K. Novakovic. Genipin-Crosslinked Chitosan Hydrogels: Preliminary Evaluation of the in Vitro Biocompatibility and Biodegradation. Journal of Applied Polymer Science 138 (2021) 50848. https://doi.org/10.1002/app.50848 [138] M.A. Razi, R. Wakabayashi, Y. Tahara, M. Goto, N. Kamiya. Genipin-Stabilized Caseinatehitosan Nanoparticles for Enhanced Stability and Anti-Cancer Activity of Curcumin. Colloids Surface B Biointerfaces 164 (2018) 308-315. https://doi.org/10.1016/j.colsurfb.2018.01.041 [139] C. Huang, H. Liao, X. Liu, M. Xiao, S. Liao, S. Gong, F. Yang, X. Shu, X. Zhou. Preparation and Characterization of Vanillin-Chitosan Schiff Base Zinc Complex for a Novel Zn2+ Sustained Released System. International Journal of Biological Macromolecules 194 (2022) 611-618. https://doi.org/10.1016/j.ijbiomac.2021.11.104 [140] P.W. Li, G. Wang, Z.M. Yang, W. Duan, Z. Peng, L.X. Kong, Q.H. Wang. Development of Drug-Loaded Chitosan-Vanillin Nanoparticles and Its Cytotoxicity against HT-29 Cells. Drug Delivery 23 (2016) 30- 35. https://doi.org/10.3109/10717544.2014.900590 [141] G.A. De Ruiter, Rudolph Brian. Carrageenan Biotechnology. Trends in Food Science & Technology 8 (1997) 389-395. https://doi.org/10.1016/S0924-2244(97)01091-1 [142] E.M. Pacheco-Quito, R. Ruiz-Caro, M.D. Veiga. Carrageenan: Drug Delivery Systems and Other Biomedical Applications. Marine Drugs 18 (2020) 583. https://doi.org/10.3390/md18110583 [143] V.N. Davydova, I.V. Sorokina, A.V. Volod’ko, E.V. Sokolova, M.S. Borisova, I.M. Yermak. The Comparative Immunotropic Activity of Carrageenan, Chitosan and Their Complexes. Marine Drugs 18 (2020) 458. https://doi.org/10.3390/md18090458 [144] T. Khaliq, M. Sohail, M.U. Minhas, S.A. Shah, N. Jabeen, S. Khan, Z. Hussain, A. Mahmood, M. Kousar, H. Rashid. Self-Crosslinked Chitosan/κ-Carrageenan-Based Biomimetic Membranes to Combat Diabetic Burn Wound Infections. International Journal of Biological Macromolecules 197 (2022) 157-168. https://doi.org/10.1016/j.ijbiomac.2021.12.100 [145] S. Rochín-Wong, A. Rosas-Durazo, P. Zavala-Rivera, A. Maldonado, M.E. Martínez-Barbosa, I. Vélaz,J. Tánori. Drug Release Properties of Diflunisal from Layer-by- Layer Self-Assembled k- Carrageenan/Chitosan Nanocapsules: Effect of Deposited Layers. Polymers (Basel) 10 (2018) 760. https://doi.org/10.3390/polym10070760 [146] T.H. Nguyen, T.C. Nguyen, T.M.T. Nguyen, D.H. Hoang, D.M.T. Tran, D.T. Tran, P.T. Hoang, V.T. Le; T.K.N. Tran, H. Thai. Characteristics and Bioactivities of Carrageenan/Chitosan Microparticles Loading α-Mangostin. Journal of Polymers and the Environmental 30 (2022) 631-643. https://doi.org/10.1007/s10924-021-02230-2 [147] N. Wathoni, L. Meylina, A. Rusdin, A.F.A. Mohammed, D. Tirtamie, Y. Herdiana, K. Motoyama, C. Panatarani, I.M. Joni, R. Lesmana, et al. The Potential Cytotoxic Activity Enhancement of α- Mangostin in Chitosan-Kappa Carrageenan-Loaded Nanoparticle against Mcf-7 Cell Line. Polymers (Basel) 13 (2021) 1681. https://doi.org/10.3390/polym13111681 [148] A.M. Ramos-de-la-Peña, C.M.G.C. Renard, J. Montañez, M. de la Luz Reyes-Vega, J.C. Contreras- Esquivel. A Review through Recovery, Purification and Identification of Genipin. Phytochemistry Reviews 15 (2016) 37-49. https://doi.org/10.1007/s11101-014-9383-z [149] C. Pizzolitto, M. Cok, F. Asaro, F. Scognamiglio, E. Marsich, F. Lopez, I. Donati, P. Sacco. On the Mechanism of Genipin Binding to Primary Amines in Lactose-Modified Chitosan at Neutral pH. International Journal of Molecular Science 21 (2020) 6831. https://doi.org/10.3390/ijms21186831 [150] R.A.A. Muzzarelli. Genipin-Crosslinked Chitosan Hydrogels as Biomedical and Pharmaceutical Aids. Carbohydrate Polymers 77 (2009) 1-9. https://doi.org/10.1016/j.carbpol.2009.01.016 [151] A.M. Heimbuck, T.R. Priddy-Arrington, M.L. Padgett, C.B. Llamas, H.H. Barnett, B.A. Bun nell, M.E. Caldorera-Moore. Development of Responsive Chitosan-Genipin Hydrogels for the Treatment of Wounds. ACS Applied Bio Materials 2 (2019) 2879-2888. https://doi.org/10.1021/acsabm.9b00266 https://doi.org/10.5402/2013/629807 https://doi.org/10.1002/app.50848 https://doi.org/10.1016/j.colsurfb.2018.01.041 https://doi.org/10.1016/j.ijbiomac.2021.11.104 https://doi.org/10.3109/10717544.2014.900590 https://doi.org/10.1016/S0924-2244(97)01091-1 https://doi.org/10.3390/md18110583 https://doi.org/10.3390/md18090458 https://doi.org/10.1016/j.ijbiomac.2021.12.100 https://doi.org/10.3390/polym10070760 https://doi.org/10.1007/s10924-021-02230-2 https://doi.org/10.3390/polym13111681 https://doi.org/10.1007/s11101-014-9383-z https://doi.org/10.3390/ijms21186831 https://doi.org/10.1016/j.carbpol.2009.01.016 https://doi.org/10.1021/acsabm.9b00266 ADMET & DMPK 11(4) (2023) 435-455 Eco-friendly synthesis of chitosan doi: https://doi.org/10.5599/adme t.1999 455 [152] Z. Wang, H. Liu, W. Luo, T. Cai, Z. Li, Y. Liu, W. Gao, Q. Wan, et al. Regeneration of Skeletal System with Genipin Crosslinked Biomaterials. Journal of Tissue Engineering 11 (2020) 1-24. https://doi.org/10.1177/2041731420974861 [153] V. Paul, D.C. Rai, R.L. Ramyaa, S.K. Srivastava, A.D. Tripathi. A Comprehensive Review on Vanillin: Its Microbial Synthesis, Isolation and Recovery. Food Biotechnology 35 (2021) 22-49. https://doi.org/10.1080/08905436.2020.1869039 [154] D.F. Taber, S. Patel, T.M. Hambleton, E.E. Winkel. Vanillin Synthesis from 4-Hydroxybenzaldehyde. Journal of Chemical Education 84 (2007) 1158. https://doi.org/10.1021/ed084p1158 [155] A.C. Alavarse, E.C.G. Frachini, R.L.C.G. da Silva, V.H. Lima, A. Shavandi, D.F.S. Petri. Crosslinkers for Polysaccharides and Proteins: Synthesis Conditions, Mechanisms, and Crosslinking Efficiency, a Review. International Journal of Biological Macromolecules 202 (2022) 558-596. https://doi.org/10.1016/j.ijbiomac.2022.01.029 [156] O.V. Kharissova, B.I. Kharisov, C.M.O. González, Y.P. Méndez, I. López. Greener Synthesis of Chemical Compounds and Materials. Royal Society Open Science 6 (2019) 191378. https://doi.org/10.1098/rsos.191378 [157] M. Mishra, M. Sharma, R. Dubey, P. Kumari, V. Ranjan, J. Pandey. Green Synthesis Interventions of Pharmaceutical Industries for Sustainable Development. Current Research in Green and Sustainable Chemistry 4 (2021) 100174. https://doi.org/10.1016/j.crgsc.2021.100174 [158] P. Bradu, A. Biswas, C. Nair, S. Sreevalsakumar, M. Patil, S. Kannampuzha, A.G. Mukherjee, U.R. Wanjari, K. Renu, B. Vellingiri. Recent Advances in Green Technology and Industrial Revolution 4.0 for a Sustainable Future. Environmental Science and Pollution Research (2022). https://doi.org/10.1007/s11356-022-20024-4 ©2023 by the authors; licensee IAPC, Zagreb, Croatia. 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