The 21st century revival of chitosan in service to bio-organic chemistry

Abstract

The design and synthesis of biopolymer nano- and micro-formulations are a new trend with growing priority in scientific research and development in the fields of biomedicine, bioorganic/medicinal chemistry, pharmaceutics, agrochemistry and food industry. This incorporates a vast variety of improved and newly-developed analytical and physico-chemical techniques for green and efficient synthesis. The scope of the present review was to outline the recent advances in the methods for design of novel chitosan micro- and nano-carriers and transporters. Special emphasis is laid on their functionalities and capacity for encapsulation of natural bioactive compounds and controlled in vitro/in vivo release in various biological/physiological media. Expectations for the application of chitosan formulations and chitosan-based hybrid systems are progressively increasing as the knowledge regarding their physical, chemical and biological properties constantly expands. Thus, this review proposes insights on the objective assessment of the capacity, applicability and versatility of newly-designed chitosan-based hybrid systems. A detailed integrative approach, which incorporates the innovative scientific achievements based on complex novel, precise and reliable analytical procedures and methods for qualitative and quantitative morphological, structural, spectral, chemical and biochemical analyses of the bio-precursors and the designed chitosan-carrier micro/nano-hybrid systems, is applied. Sustainable knowledge on the mechanism and methods of natural bioactive substances encapsulation and in vitro/in vivo release is reviewed and discussed.

Keywords:

• Chitosan-carrier matrices
• natural bioactive compounds
• biochemical properties
• encapsulation
• in vitro/in vivo release

Introduction

Natural products have served as the source and inspiration for a large fraction of the current pharmacopoeia and continue to provide a diverse and unique source of bioactive compounds for drug discovery[1–4].

Chitosan – a cationic natural polymer, first isolated in 1811, has begun its biomedical “rebirth” since the first decade of the twenty first century. Recent prominent scientific studies provide insights into its unique features and remarkable physical, chemical and biological properties such as biocompatibility, biodegradability, antimicrobial, antifungal, analgesic and antitumor activities. These features define its promising current and future applications in human and veterinary medicine, pharmaceutics, agro- and food industry [5]. Being an excellent excipient, chitosan currently has enormous potential for the preparation of pharmaceutical dosage forms due to its polyelectrolyte properties, gel-forming capability and high adsorption capacity [6]. Novel chitosan gels, suspensions, micro- and nano-particles [7–12], spheres, capsules [13–19] and drops have been recently investigated as potential controlled release carriers of synthetic drugs, proteins, enzymes and natural bioactive substances [20].

Concerning these biologically active compounds, it should be emphasized that modern scientific research has been significantly focused on the exploration of various natural polyphenols, flavonoids, saponins, tannins, etc. [21,22], with diverse bioactivities including antioxidant, anti-inflammatory, anti-cancer, anti-microbial, anti-diabetic capabilities [23], as well as on their applicability and efficiency for nutraceutical, therapeutic and pharmacological purposes. Scientific literature outlines that the basic challenges facing the practical application of these bioactive compounds arise from their high sensitivity to a diverse range of conditions and factors (temperature, oxygen, light, pH, oxidative enzymes, moisture content, etc.), associated with difficulties in the preservation of their stability and valuable biomedical properties. Thus, nano- and micro-encapsulation supports have emerged as reliable techniques to avoid the unwanted degradation of natural bioactive compounds [24].

Another scientific area where chitosan formulations have been widely applied is the field of advanced cell technologies and therapeutics. Two-dimensional and three-dimensional chitosan scaffolds with various cell types cultured on them have been applied in cell-based regenerative therapy and tissue engineering. [25,26]. Increasing scientific knowledge and expectations for the unique potential for applicability of chitosan-based systems, however, face a number of constraints related to the lack of long-term stability, low solubility and sometimes unsatisfactory mechanical strength: challenges that have to be overcome and solved by modern science.

This review outlines the recent advances in the design methods of novel chitosan micro- and nano-carriers and transporters, with emphasis on their functionalities and capacity for encapsulation of natural bioactive compounds and controlled in vitro/in vivo release in various biological/physiological media.

Chitosan: molecular structure, nature and properties

Chitin and chitosan are linear polysaccharides consisting of β-(1→4) linked residues of N-acetyl-2-amino-2-deoxy-D-glucose (AcAG) and 2-amino-2-deoxy-D-glucose (AG). Due to the low content of 2-amino-2-deoxy-D-glucose units, chitin is insoluble in acidic aqueous media. The higher fraction of 2-amino-2-deoxy-D-glucose residues in chitosan determines the polymer solubility in acidic aqueous solutions [25]. The deacetylation process of chitin to chitosan, which could be accomplished either by chemical, or enzymatic hydrolyses, generally involves removal of acetyl (CH₃C = O-) groups resulting in the formation of amino (NH₂-) groups, i.e. the formation of copolymers of N-acetylglucosamines and glucosamines (Figure 1) [27–32].

Figure 1. Deacetyleation of chitin to chitosan.

Physico-chemical and structural analyses of chitosan nano-/micro-formulations

The majority of the literature reports on the physico-chemical properties and the structure of chitosan-based formulations employ various combinations of analytical methods, such as ultraviolet–visible (UV-VIS) spectrophotometry, fluorescence spectroscopy, Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), conductometric and potentiometric titration, differential scanning calorimetry (DSC), average M_w and/or M_w distribution viscosimetry, light scattering, X-ray diffraction, thermogravimetric analysis (TGA), scanning electron microscopy (SEM), electron paramagnetic resonance (EPR), etc [9,10,16,30,32–40]. Hussain et al. [13] determined the degree of deacetylation (DDA) of alkali-modified essential oil-loaded chitosan by FTIR, potentiometric titration and elemental analysis. A direct relationship between the duration of the alkali treatment and DDA was established. SEM analyses indicated that DDA and crosslinking control the surface smoothness of the obtained microcapsules [13]. The particle size, size distribution and zeta potential of biodegradable chitosan nanoformulations for encapsulation of strawberry extract polyphenols were measured by dynamic light scattering technique (DLS), and the morphology and size of the prepared nanoparticles were studied by SEM. UV-spectrophotometry and FTIR analyses were conducted to investigate the bonding between chitosan functional groups and polyphenol molecules [41]. Harris et al. [14] obtained chitosan hydrochloride nanoparticles and microspheres and proved that they could be adequate vehicles for the encapsulation of natural antioxidants due to maintainance of the antioxidant activity of Ilex paraguariensis extract polyphenols. The nanoparticles prepared by ionic gelation and the microspheres prepared by spray-drying, were characterized in terms of morphology, zeta potential analyses and in vitro release studies [14]. The SEM image of a cross-sectional fracture of chitosan microbeads loaded with thyme (Thymus serpyllum L.) polyphenols showed the presence of polyphenol crystals [42]. Porosity measurements confirmed that, upon encapsulation, polyphenols filled cavities of the matrix, therefore causing reduction in microbeads porosity [42]. The complex structural analysis incorporating transmission electron microscope (TEM), atomic force microscopy (AFM), FTIR and thermogravimetric analysis (TGA)-differential scanning calorimetry (DSC) indicated that tea polyphenol-Zn complex was well incorporated into β-chitosan nanoparticles [7]. The formation of epigallocatechin gallate-sulfobutyl ether-β-cyclodextrin sodium inclusion complexes with chitosan hydrochloride nanoparticles was confirmed by steady-state fluorescence, FTIR and NMR spectroscopy [43]. The photomicrographs of chitosan microparticles synthesized by Cabral et al. [8], by means of a spray-drying method, indicated that the microparticles were homogeneously spherical with a wrinkled surface, while the extent of surface smoothness increased directly with the increase of the quantity of the encapsulated jabutikaba peel extract polyphenols [8]. Newly-synthesized water soluble quercetin loaded chitosan hydrochloride and carboxymethyl chitosan nanoparticles were optimized to small particle size, high zeta potential and high encapsulation efficiency [44]. Their morphology was observed by SEM and quercetin encapsulation efficiency was confirmed based on molecular interactions indicated by FTIR and X-ray diffraction analyses [44]. According to SEM analyses heat-denatured zein/carboxymethyl chitosan complex nanoparticles containing propolis appeared spherical and exhibited smooth surfaces; the carboxymethyl chitosan coating reduced the particle size – an observation that corresponded to DLS data [45]. The comparative FTIR analyses confirmed that the complex nanoparticles were formed through hydrogen bonding, electrostatic and hydrophobic interactions [45]. The real-time fabrication of dextran/chitosan multilayer of microcapsules for polyphenol co-delivery was monitored by quartz crystal microbalance with dissipation monitoring, and the morphology of the nanostructured polymeric capsules was characterized by SEM, whereas UV-spectroscopy and HPLC were applied for assessment of the encapsulation efficiency [15]. The conjugation of resveratrol, genistein and curcumin with two types of chitosan was studied at physiological conditions, using fluorescence spectroscopy, FTIR, TEM and docking studies [46]. Structural analysis indicated the presence of hydrophilic, hydrophobic and H-bonding between polyphenols and chitosan, which induced major alterations of chitosan morphology as established by the morphology studies [46].

Chitosan characteristic parameters

The extent of deacetylation determines two basic and significant interdependent characteristics of chitosan, namely the degree of deacetylation (DD) and the degree of acetylation (DA), that govern important physicochemical, biochemical and biological properties of chitosan related to acid–base characteristics, electrostatic interactions, solubility, biodegradability, self-aggregation, sorption properties, chelation capacity, molecular conformation, etc., which are critical for the effectiveness of the biopolymer for various medical, pharmaceutical, biotechnological and agroindustrial applications [13,33]. DD and DA are measures of the fraction of AG or AcAG monomer residues, respectively, in percentile units [33,47]: (1) $$DD,\mathrm{}\mathrm{\%}=\frac{n_{AG}}{n_{AG}+n_{\mathit{AcAG}}}.100$$(1) (2) $$DA,\mathrm{}\mathrm{\%}=100-DD$$(2)

The DD exhibited by chitosan can be controlled during a relatively aggressive alkaline hydrolysis process applied to chitin, through a combination of exposure duration and temperature [48,49]. Foster et al. [49] studied the variation in the physiochemical, mechanical and biological properties for a range of commercial chitosans within a narrow DD range (72–85%), with a view to chitosan potential in the regeneration of nerve tissue [49]. The lowest chitosan DD values cited in literature are variable but usually within the range of 40–60%. However, the majority of commercial chitosan samples have average DD values (70–90%) [13,30]. The process of preparation of highly deacetylated chitosans (DD > 95%), which are appropriate for specific biological applications, often results in partial depolymerization of the polyssacharide and is associated with increased economic cost [13,50]. The solubility of chitosan is usually investigated in weakly acidic solutions of diluted inorganic (H₃PO₄, H₂SO₄) and organic (HCOOH, CH₃COOH, C₂H₂(OH)₂(COOH)₂ tartaric acid, C₂H₄(OH)(COOH)₃ citric acid) acids [31,50]. Due to the presence of primary –NH₂ groups, chitosan is positively charged. The magnitude of the charge density, however, depends on the pH, DD and ionic strength. In acidic conditions, the –NH₂ groups (pK_a ∼ 6.5) are partially protonated (Figure 2), which in turn provokes repulsion between positively charged polymer chains, thereby allowing diffusion of water molecules and subsequent solvation of the macromolecules [30]. Sogias et al. [51] showed that chemical disruption of chitosan crystallinity by partial re-acetylation or physical disruption caused by the addition of urea and guanidine hydrochloride broadens the pH-solubility range for this biopolymer [51].

Figure 2. Chitosan protonation/deprotonation.

According to Tachaboonyakiat et al. [52], the main cause of the water insolubility of phosphorylated chitosan is inter- and intra-molecular crosslinking. Fu and Xiao [53]developed a facile physical approach to make chitosan soluble in acid-free water by modulating the solution concentration and varying the precipitants.

The capacity of chitosan for systemic bioabsorption, bioadsorption and diffusion is also dependent on its molecular weight (M_w). Scientific studies outlined that oligomers display greater affinity for sorption, whereas larger M_w chitosans were excreted without being sorbed [48,50]. Besides, the cytotoxic effects of these biopolymers were also dependent on the M_w [54,55]. According to the investigations of Chae et al. [54], chitosan oligosaccharides (M_W < 10 kDa) showed negligible cytotoxic effect on Caco-2 cells and were characterized with the highest plasma concentration. Due to the semicrystallinity of chitosan, it displays polymorphism which depends on its physical state [48]. Recent scientific investigations [13,30,33,47–54] outlined strong and complex interdependence between various chitosan functionality parameters and intrinsic/extrinsic factors, which is schematically illustrated in Figure 3.

Figure 3. Interdependence between chitosan functionality parameters and intrinsic/extrinsic factors.

Physico-chemical methods for chitosan modification

The abundant amine and hydroxyl groups present in chitosan monomers offer a unique opportunity for attaching targeting ligands or imaging agents. The scientific literature over the last 5 years abounds with studies on numerous newly-designed derivatives of chitosan tailored to improve the physicochemical properties of various chitosan formulations: size, shape, charge, density, solubility [55], so as to enhance their biological activity in view of biocompatibility, antibacterial, antifungal, antioxidant activity, biodegradability, etc. Chitosan-based biodegradable materials, including micro- and nano-particles, gels, composites and films, have grown to become a key choice in agriculture due to their proven antimicrobial and plant-growth promoting activities [56–59]. Recent research findings are subjected to the diverse biomedical and pharmaceutical applications of chitosan-based formulations for drug-delivery, gene delivery, cell encapsulation, tissue engineering, wound healing, bioimaging, antibacterial food packaging, etc. [60,61]. The variety of modification procedures [28] and modifing agents applied in scientific investigations is highly specific depending on the intended future application of the chitosan composites (Table 1) [32,52,55,62].

Table 1. Modified chitosan derivatives applied in various biomedical, agricultural and environmental fields (recent scientific literature review 2016-2019).

Modified chitosan	Modification procedure	Properties	Application	Ref.
Carboxymethyl chitosan	Carboxymethyl chloride; the resultant carboxymethyl chitosan (CMCTS) was conjugated to α-cyclodextrin (α-CD) to yield CMCTSCD	Improved water solubility; hydrophobic cavities functioning as crosslinking points by forming an inclusion complex with poly(ethylene glycol)	Injectable hydrogel for bone tissue engineering	[63]
Graphene oxide and amine-modified graphene oxide chitosan	Incorporation of graphene oxide and amine-modified graphene oxide into Chitosan-Gelatin scaffold	Improved mechanical, physico-chemical, and biological properties; absence of cytotoxicity; in vitro biomineralization ability; improved cell viability	Tissue engineering scaffolds	[64]
Methyl methacrylate modified chitosan conjugate	Green method via Michael addition reaction between chitosan and methyl methacrylate in ethanol	Good transfection efficiency, entrapment and drug release efficiency	Potential gene and drug delivery agent	[65]
Quaternized chitosan modified nanofiber membrane	Quaternary amine (i.e., glycidyl trimethyl ammonium chloride, GTMAC)	99.95% antibacterial activity against Escherichia coli	Microfiltration membrane to effectively disinfect against E. coli	[66]
Chitosan beads modified with molybdenum (mocb), iron (ICB), single super phosphate (SSPCB) and mono calcium phosphate (MCPCB)	Molybdenum (MoCB), iron (ICB), single super phosphate (SSPCB) and mono calcium phosphate (MCPCB)	Efficiency in reducing Zn bioavailability and shoot uptake, effectively decreased Zn root uptake	Amendment for remediation of Zn toxicity in contaminated and wasteland soils	[67]
Modified chitosan	Reaction of N-acylation with maleic anhydride	Excellent antibacterial action against Staphylococcus aureus and Escherichia coli, evidencing no activity against the protozoan Leishmania amazonensis; absence of cytotoxicity in mammalian cells	Biomedical applications	[62]
Surface modified chitosan	Crosslinking of chitosan (I) with 2-hydroxy-1-naphthaldehyde. Further modification by grafting with CuCl2 to prepare	Improved interaction with pentachlorophenol (PCP) and enhanced the ability to uptake PCP	Bioadsorbent with enhanced adsorption properties to remove pentachlorophenol (PCP) pesticide	[68]
Diisocyanate-modified chitosan	Cross-linking reaction with chitosan and diphenyl methane diisocyanate	High degree of bacterial growth inhibition against E. coli	Novel biodegradable material with improved antibacterial properties for biomedical applications	[69]
Salicylic acid functionalized chitosan nanoparticle	Modification with salcylic acid	Promoting plant Defense and growth in maize	Biostimulant for plant disease control and higher yield	[39]
Schiff bases modified chitosan-graft-poly(acrylonitrile)	- Modification by grafting with acrylonitrile using potassium persulfate as initiator - modification with Schiff bases	Antibacterial activities against Streptococcus pneumonia, Staphylococcus aureus and Escherichia coli as gram-negative bacteria; antifungal activity against Aspergillus fumigatus, Candida albicansand Geotricum candidum	Antibacterial activity	[70]
Chitosan-based hydrogels modified with silver nanoparticles	Modification with nanosilver particles	Nanosilver slightly reduces sorption ability of modified materials; Antimicrobial activity against Staphylococcus aureus; absence of cytotoxicity to dermis cells; biodegradability; biocompatibility	Biomedical applications	[71]
Photo-crosslinkable hydrogel biomaterial based on trans-3-(4-pyridyl)acrylic acid (PYA) modified chitosan	Photo-crosslinked under UV irradiation through intermolecular [2π + 2π] photo-induced cycloaddition reaction	Optimized hydrogel enzymatic biodegradation; flexibility of the polymeric matrix	Potential for biomedical and pharmaceutical applications	[72]
Gallic acid/chitosan coating		Increased antimicrobial activity; inhibited haemoglobin, lipid and protein oxidation	Practical hurdle technology in preservation of fresh pork to improve food safety and quality	[73]
Microwave-modified chitosan	Grafting and crosslinking with acrylic acid and acrylonitrile by microwave route	Excellent antibacterial activities against E. coli, P. aeruginosa and S. euros	Antibacterial applications	[74]
Diethoxyphosphoryl polyaminoethyl chitosan	Grafting polyaminoethyl and diethoxyphosphoryl groups on chitosan backbone	Enhanced antifungal activity against P. capsici, F. solani and B. cinerea, good water solubility, low cell toxicity	Antimicrobial applications	[75]
Salicylic acid-chitosan microparticles	Modification with salicylic acid, crosslinking with tripolyphosphate	Positively modulate lettuce growth; potential to improve plant defense responses	Activator of plant defense responses	[13]
Lanthanum-modified chitosan oligosaccharide nanoparticles	Ionic cross-linking with citric acid and LaCl3	Growth promotion effects in terms of plant height and fresh weight of treated rice; significant defense response for rice blast	Promising environmentally friendly nanocarrier of pesticides in agricultural scenarios	[76]

Diversity of chitosan formulations applied for encapsulation of natural bioactive compounds

Nano-/micro-particles, nano-/micro-spheres, microbeads, nano-/micro-capsules, hydrogels etc. are basic representatives of the variety of multiple unit chitosan-based formulations recently applied as potential drug carrier systems in the segment of novel drug delivery [17].

The main disadvantages of chitosan-based matrices regarding the release of the incorporated bioactive substances are uncontrollable diffusion profile, passive drug release over time, poor control of external or internal stimuli [77]. Efforts are directed towards overcoming these limitations and improving the physico-chemical, biochemical and biomedicinal properties of chitosan carriers by modification and optimization of existing methodologies and the development of innovative synthesis techniques. In this respect, a number of scientific studies during the last decade outlined that chitosan nano- and micro-formulations tested for different application routes, i.e. oral, ocular, nasal, vaginal, buccal, parenteral and intravesical, possess some unique characteristics over other delivery systems. These advantages include slow and controlled release of biologically active compounds, in situ gelling, mucoadhesion, hydrophilic behavior, enhanced transfection and permeation, improved drug encapsulation efficiency, bioavailability and controlled release of the target compound at specific active sites [17,77–80]. Mohapatra et al. [77] developed a novel drug delivery platform based on biocompatible chitosan microbeads cross-linked with glyoxal and embedded with Fe₃O₄ magnetic nanoparticles, which was responsive to electric stimulation and exhibited statistically significant drug release efficiency . According to Aslan et al. [55], the ionic cross-linking of chitosan is advantageous due to the facility of the method and the possibility for its performance under mild conditions without the use of organic solvents. Chitosan nanoparticles were synthesized by ionotropic gelation with tripolyphosphate, and their applicability for the incorporation of low molecular drugs, proteins, DNA/siRNA was examined. Novel chitosan-based polyelectrolyte nanocapsules were prepared and their improved encapsulation efficiency toward curcumin was established. The oral administration of these formulations induced significant decrease in hyperglycemia after 7 days [81]. Encapsulation in chitosan nanoparticles improved the stability of tea polyphenols by preventing oxidation or degradation in the gastrointestinal tract was [82]. The study of Wang et al. [83] demonstrated the superior benefits of using cellulose nanocrystal as a macro-ion crosslinking agent over sodium tripolyphosphate, for the synthesis of chitosan microcapsules applied for improving the encapsulation efficiency and stability of blueberry anthocyanin extracts. Their results showed that CNC incorporation into chitosan functioned not only as a macro-ion crosslinking agent on the positively charged amino groups in chitosan, but also as a filler for the chitosan matrix, thus generating more rigid and stable microcapsules. The results revealed that the stable rigid complex microcapsules produced by the innovative crosslinking method were characterized with improved encapsulation efficiency and enhanced the stability of the encapsulated anthocyanins [83]. The broad-spectrum imidazolinone herbicide imazaquin was encapsulated in starch and chitosan beads reinforced with alginate [80]. The microformulations characterized with high porosity demonstrated improved matrix strength and prevented leakage of the encapsulated herbicide, which was due to interactions between the biopolymer functional groups and imazaquin molecules [80]. A systematic classification of varous recently developed chitosan nano- and micro-formulations for the encapsulation of natural bioactive substances with emphasis on the preparation procedures is presented in Table 2.

Table 2. Multiplicity of chitosan nano- and micro-formulations applied for the encapsulation of natural biologically active compounds.

Chitosan matrix type	Preparation procedure	Natural biologically active compound/Biological activity		Ref.
Nanoparticles
Chitosan nanoparticles	Ionic gelation of tripolyphosphate pentasodium (TPP) and chitosan hydrochloride	Natural polyphenols extracted from Ilex paraguariensis (Yerba mate)		[14]
β-Chitosan nanoparticles	Ionic gelation between CS and sodium tripolyphosphate (TPP). The β-CS was depolymerized to molecular mass (MM) < 40 kDa for obtaining β-CS NPs at < 50 nm.	Tea polyphenol-Zn complex		[7]
Chitosan/sulfobutylether-β-cyclodextrin nanoparticles	Cross-linking chitosan hydrochloride (CSH) and sulfobutyl ether-β-cyclodextrin sodium (SBE-β-CD)	Tea polyphenol		[43]
Chitosan hydrochloride and carboxymethyl chitosan nanoparticles	Modified ionic gelation method	Quercetin		[44]
Carboxymethyl chitosan nanoparticles	Acid-induced aggregation and calcium cross-linking	Propolis		[45]
Chitosan nanoparticles	Commercial chitosan-15 and chitosan-100 kDa nanoparticles	Antioxidant polyphenols resveratrol, genistein and curcumin		[46]
Microparticles
Zein-chitosan nanoparticles and microparticles	Electrospraying process	Curcumin and piperine		[9]
Chitosan microparticles	Spray-drying process	Rosmarinic acid		[36]
Chitosan microparticles	Spray drying of medium molecular weight chitosan	Antioxidant compounds from Plinia cauliflora (jabuticaba) fruit peel extract		[8]
Microcapsules, microbeads
Alginate–chitosan microcapsules enhanced with ascorbic acid	Electrostatic extrusion, cross-linking solution 2% (w/v) calcium chloride, 0.5% (w/v) of chitosan dissolved in the previously prepared plant extract containing 2% (w/v) ascorbic acid (pH = 2.65)	Raspberry leaf, hawthorn, ground ivy, yarrow, nettle and olive leaf extracts		[84]
Chitosan/dextran multilayer microcapsules	Layer-by-layer self-assembly technique with chitosan. dextran sulfate and calcium carbonate sacrificial microparticles, obtained by mixing calcium chloride and sodium carbonate solutions	Tyrosol, caffeic acid, vanillic acid, p-coumaric acid		[15]
Chitosan microbeads	Emulsion-crosslinking technique: GA -crosslinking agent. itaconic acid solution (7.5% (w/v)); Tween 80 - emulsifier	Thyme (Thymus serpyllum L.) polyphenols		[42]
Microspheres
Chitosan microspheres	Spray-drying with 0.5% and 0.0% HCS solution, 0.1% and 0.2% TPP	Polyphenols extracted from Ilex paraguariensis (Yerba mate)		[14]
Chitosan microspheres	Imine formation (Schiff base reaction) method and microwave irradiation	Curcumin		[16]
Double shells, hydrogel-based scaffolds
Ferritin-chitosan double shells	Recombinant soybean seed H-2 ferritin - the protein shell material, reversible assembly of ferritin cage; electrostatic interactions	Epigallocatechin gallate		[34]
Chitosan hydrogels	Injectable hydrogels for bone regeneration consisting of chitosan, sodium beta-glycerophosphate (Na-β-GP) and alkaline phosphatase	Phloroglucinol and gallic acid		[35]
Nanocellulose reinforced chitosan hydrogel	Chemical crosslinking method and Tween 20 incorporation to the hydrogel via in situ loading method	Curcumin extracted from dried rhizomes of Curcuma longa		[85]
Membranes, films
Polylactic acid-tea polyphenol-chitosan composite membranes	Stretch film methodwith polylactic acid, tea polyphenol and chitosan	Tea polyphenols decrease the rotting rate and mass loss rate significantly, postpone the consumption of soluble solids and vitamin C, maintain the quality of the cherries, and extend the shelf life, thus proving its potential for application in food packaging.		[37]
Ultrasound treated polyvinyl alcohol/tea polyphenol composite films	Polyvinyl alcohol - a film-forming substrate added to extracted tea polyphenols; ultrasonication by tape-casting method	Excellent bacteriostatic properties against Staphylococcus aureus and Escherichia coli		[38]
Multi stimuli responsive particles
Magnetic stimulus responsive chitosan microbeads	Chitosan microbeads cross-linked with polyethylene glycol dimethacrylate (PEGDMA) and embedded with magnetic nanoparticles	Enable on-demand dosage optimization to actual therapeutic requirements; Drug boost at later time points, which increase drug elution to therapeutic levels Increased efficacy and reduced risk of drug-resistant bacterial strains		[79]
Stimuli-responsive implantable biocompatible chitosan microbeads	Glyoxal cross-linked chitosan microbeads containing embedded biocompatible Fe3O4	Promising technique for in vivo implantation		[86]
Multi-responsive chitosan biomaterials doped with biocompatible ammonium ionic liquids	Chitosan and two ammonium-based ionic liquids: choline chloride and choline dihydrogen phosphate	- Application as improved electrical and pH-sensitive drug delivery systems; - Development of biocompatible and biodegradable drug delivery responsive systems for several biomedical/pharmaceutical applications such as iontophoretic devices.		[87]
Multi-stimuli responsive smart chitosan- based microcapsules	- Folic acid functionalized thiolated chitosan, sonochemical method; - Folic acid and red fluorescent dye (Rhodamine B isothiocyanate, RITC) onto the shells of microcapsules; oleic acidmodified Fe3O4 magnetic nanoparticles, a model hydrophobic drug, green fluorescent dye (Coumarin 6, C6), were encapsulated into the microcapsules	- Excellent magnetic responsive ability; - Favorable selectively folate-receptor-mediated targeting functionality to HeLa cells; - Improved reduction-responsive release ability for hydrophobic drugs - Multi-stimuli responsive, biocompatible and non-immunogenic smart carrier for targeted delivery and triggered release of hydrophobic drugs		[19]

Methods and mechanism of bioactive compounds encapsulation

There are a number of techniques recently used for the encapsulation of natural and synthetic biologically active compounds on chitosan carriers, e.g. ionotropic gelation, emulsion phase separation, cross-linking with various agents, emulsion solvent diffusion, coacervation, etc. (Figure 4) [55,77,82,88,89].

Figure 4. Generalized procedure for chitosan nano-/micro-particles synthesis and encapsulation. Adapted from [18,78,88,90–96].

Other methods of chitosan micro- and nano-particles production are based on the coalescence phenomenon, collision of dispersed emulsion phases, formation of reversed micelles, opposite charged poly-ions complexation, etc. [12,14,50] (Reviewed in [77]). The design of new bioactive compound carriers together with the accurate mathematical modeling of the mass transport phenomena [97] allows prediction of their behavior during shelf-life, product preparation or consumption, as well as controlling the release of the bioactive compounds [91]. A systematic summary of the recently applied methods for encapsulation of natural bioactive compounds by various chitosan carriers, with emphasis on the analytical methodologies, the most significant functional characteristics of the microformulations and the mechanism of encapsulation/release, is presented in Table 3.

Table 3. Recent methods for bioactive compounds encapsulation on chitosan formulations.

System	Encapsulation method	Analytical methods	Formulation characteristics	Encapsulation/release mechanism	Ref.
Vitamin B12/chitosan microparticles	Spray drying	SEM; laser granulometry analysis (LGA); UV–VIS spectrophotometry	Regular spherical shape; Smooth surface; Average dimeter 3 - 8 μm; Tendency to aggregate	Matrix type with the encapsulated substance distributed in the encapsulating agent; Release mechanisms controlled by solvents action and diffusion	[11]
Tannic acid/chitosan/NaOH/fly ash composites	Adsorption	Wavelength Dispersive X-Ray Fluorescence (WD-XRF);Energy Dispersive X-Ray Analysis (EDX) coupled with Field Emission Scanning Electron Microscopy (FE-SEM); FTIR; UV–VIS spectrophotometry	The surface of zeolitic crystal covered with an organic layer; The crystalline depositions completely disappeared; Pore size: 20 Å (1 Å=0.1 nm) - 500 Å; Surface area of pores: 23.75 m^2/g; pHPZC 7.3	Electrostatic attraction; Hydrogen bonding; Organic partitioning	[98]
Peppermint and green tea essential oils/chitosan nanoparticles	Emulsification-ionic gelation	Differential Light Scattering (DLS); TEM analysis;FTIR; UV–VIS spectrophotometry Thermogravimetric analysis (TGA)	Positive zeta potential values; Stable particles; No aggregation observed; Spherical particles; 20–60 nm (TEM analyses) - on dry matter with restricted particle mobility; 217.2 – 256.3 nm (DLS analyses) - in liquid suspension, the sizes are directly related to the hydrodynamic diameter of the sample in the solvated state	The release follows a Fickian behavior; Release caused by diffusion and tiny swelling	[99]
Oregano essential oil/ chitosan nanoparticles	Oil-in-water (o/w) emulsification, and ionic gelation	UV–VIS spectrophotometry; FTIR; TGA; XRD; SEM; AFM; Laser Light Scattering (LLS)	Regular distribution; Spherical shape; Stable over the steps of the preparation process; Average diameter: 281.5 nm; Chitosan loaded particles diameter 309.8–402.2 nm	In vitro release profile - a two-step biphasic process, i.e. an initial burst release followed by subsequent slower release; The diffusion of toregano oil dispersed into the polymer matrix as the dominant mechanism; The incorporation of oregano oil results in a change in the chitosan–TPP packing structure	[10]
Vitamin C/chitosan nanoparticles	Ionic gelation method	SEM; FTIR; TG/DTG; XRD; UV/VIS spectrophotometry	Particle diameter: 6.45 nm from XRD; Particle diameter: 10 nm from SEM; Spherical, uniform particles	Intense electrostatic interaction between positively charged amine groups of CS and negatively charged TPP; Release of vitamin C depends on swelling and diffusion control mechanism	[100]
Gallic acid/chitosan films with grape seed extract and carvacrol	Solvent casting technique;chitosan films, formulated with grape seed extract and carvacrol	HPLC	Film thikness 0.042 − 0.062 nm	Constant effective diffusion coefficient; Release dependent on temperature, described by an Arrhenius-type equation, becoming faster at higher temperatures	[90]
Extracts of Argentinean medicinal plants (Larrea divaricata, L. cuneifolia, L. nitida, Zuccagnia punctata and Tetraglochin andina)/chitosan microcapsules	Electrospraying	Folin–Ciocalteau method; HPLC-ESI-MS/MS; SEM; FTIR; UV/VIS spectrophotometry	Variation in capsule diameter: 0.1–1.5 µm; Low homogeneity of the microcapsules	Interactions between the chitosan matrix and the polyphenolic extracts, mediated by H-bonding or hydrophobic interactions; Stabilization of carbohydrate matrices bypolyphenols described and attributed to the presence of physical interactions between both components; Increased solubility and release in aqueous medium; 100% release after 24 h; Unaffected biological properties of the extracts	[88]
Epigallocatechin gallate/chitosan/β-lactoglobulin nanoparticles	Ionic complexation Ionic gelation	FTIR	Particle size: 100 − 500 nm; Zeta potential: 10 - 35mV	High number of H-bonding sites in the nanoparticles; Epigallocatechin released slowly over time by diffusion of the outer layer of β-lactoglobulin degraded in the intestinal fluid;	[101]
Quercetin/ crosslinked chitosan Quercetin/ glucosamine-modified chitosan microcapsules	Glucosamine-modification of chitosan by Maillard reaction; Spray-drying	UV/VIS spectrophotometry; SEM	Spherical shape; Average size: 2.0 ± 1.5 μm	Higher release rate under gastric conditions than intestinal conditions; pH-dependent behavior associated with full protonation of the glucosamine moiety; The highest solubility and swelling at very acidic conditions	[102]

In vitro/in vivo release mechanisms from chitosan nano-/micro-formulations

Drug release from biopolymer carriers is influenced by a number of factors including the composition and specific characteristics of the biologically active compound, the polymer, and additives, their ratio, physical and/or chemical interactions among the components, the synthesis and encapsulation methods. According to the mechanism by which a drug “escapes” a carrier, in vitro/in vivo drug release can be conditionally classified into five categories: diffusion-, degradation-, chemical bonds cleavage-, solvent- and stimuli-controlled release [103]. The basic release mechanism principles and their advantageous characteristics described in recent scientific studies are summarized in Table 4.

Table 4. Mechanisms of release of natural bioactive compounds from chitosan formulations.

Release mechanism	Release process principle	Characteristics	References
Diffusion-controlled release	Diffusion-controlled release from biopolymer systems implies drug molecules movement driven by concentration gradient. In degradable systems, the drug release rate is controlled by diffusion through a network of pores and simultaneous or sequential matrix degradation. In non-degradable biopolymer systems, the main driving force is the diffusion of drug molecules out of the matrix.	The release rate is: - constant, - not influenced by concentration gradients; - dependent on the properties of the biopolymeric matrix.	[104]
Chitosan matrix degradation	Biodegradable biopolymer-carriers could release the encapsulated biologically active substances through: - enzymatic decomposition due to degradation of ester, amide bonds, or hydrolysis; - surface erosion from the exterior towards the interior implying matrix degradation starting at the surface, followed by slow reduction of the microparticle size; - bulk erosion associated with water penetration within the biopolymer matrix, followed by homogeneous degradation of the entire matrix.	- Rate of erosion > rate of water penetration in the biopolymer matrix; - Erosion kinetics and drug release are controllable and reproducible; - Due to the low water permeation rate, water-vulnerable drugs are protected; - Rate of water penetration in the biopolymer matrix > rate of erosion - less predictable; - Does not protect drugs from the environment.	[104–108]
Cleavage of chitosan–biologically active molecule linkage	This mechanism is based on drug molecule release inside a cellular compartment or other media through hydrolytic cleavage and detachment of the active molecules from the biopolymer carrier. The cleavable linkers can be placed in the side-chains or in the backbone of the polymer matrix.	Typical release mechanism from various stimuli-sensitive chitosan formulations	[17,109]
Solvent-controlled release	The solvent-controlled release (dissolution release) includes: - Osmosis-controlled release from biopolymer formulations covered with a semi-permeable polymeric membrane, through which water molecules move from externalmedia, with low biologically active compounds concentration, toward the drug-loaded core. As a result of the generated osmotic pressure, the encapsulated molecules are released. - Biopolymer dissolution, which implies drug-release from the biopolymer matrix into a thermodynamically compatible medium or appropriate solvent.	Biologically-active compound release rate could be controlled by: - solute diffusion; - biopolymer dissolution.	[103,104,109,110]
Multistimuli-sensitive destruction	Newly-designed smart chitosan-based drug delivery systems possess the ability to transform some of their physicochemical properties in response to various extra/intracellular stimuli and external accelerating conditions (e.g., pH, redox gradient, temperature, sonication, UV-irradiation, magnetic field, etc.). Such functional sensitivities improve the payload capacity, stimulate the release and regulate the delivery of the encapsulated biologically active substances at target locations, thus achieving improved drug delivery efficiency.	- Short synthesis periods of microcapsules with multi stimuli responsiveness; - High-dose, direct loading of hydrophobic drugs into the microcapsules.	[19,79,109]

The main goals of the design of innovative chitosan-based formulations are achievement of two types of controlled bioactive substance release: temporal and distribution control. The temporal control ensures delivery over an extended or specific time period, whereas distribution control ensures targeted release of the bioalogically active compound to precise active sites [110]. Besides, the search and need for a new generation of biopolymer hybrid systems has been provoked by the necessity of achieving active substance release subjected to desired kinetic model/s, eliminating the so-called undesirable “burst effect” and providing prolonged and controlled release [78]. To accomplish these aims and to overcome the obstacles associated with bioactive compounds encapsulation and release, the basic initial stages required during the development of novel chitosan-based carriers are the investigations and assessment of the encapsulation efficiency, loading and release efficiency of the substance/carrier matrices. Figure 5 shows detailed comparative analyses of the values of these parameters for a number of new hybrid systems comprising of nano-/micro-sized chitosan formulations and natural bioactive substances.

Figure 5. Encapsulation and release efficiency of chitosan micro/nano-formulations loaded with natural biologically active compounds. Graphically presented data adapted from [7–10,14,15,34,39,41,42,44–46,84,85,89,99,100,111–118].

International/European regulatory aspects and future perspectives

Although recent studies indicated that a number of newly-developed chitosan formulations meet the criteria for good biomaterials characterized with unique antibacterial activity, non-allergic response, oxygen permeability, biodegradation to relatively neutral oligomers, unique ability to accelerate healing, functional flexibility, etc. [118], chitosan-based carriers have not been approved yet by the American Food and Drug Association (FDA) in the field of medical applications [48]. In this respect, The American Society of Testing Materials (ASTM F04 division IV) is making efforts to establish standard guidelines for chitosan salts suitable for use in biomedical or pharmaceutical applications, or both, including, but not limited to, tissue-engineered medical products (TEMPS). This guide addresses key parameters relevant for the functionality, characterization and purity of chitosan salts [48,119]. A derivative of chitosan, chitosan hydrochloride, was included in the European Pharmacopoeia (EP) in 2002. The corresponding EP monograph includes tests for heavy metal contamination but does not address bioburden, sterility and bacterial endotoxins (Reviewed in [120]). Taking into account that purity, which is quantified as the remaining ashes, proteins, insolubles and also the bio-burden (microbes, yeasts and moulds, endotoxins, etc.), is vital particularly for high value products, a more detailed characterization is needed [48]. Currently, chitosan has been approved for wound dressing applications and cartilage repairing formulation. However, chitosan approval by the FDA is not a general definition as GRAS (generally recognized as safe), but FDA and other regulatory agencies evaluate and approve materials with respect to their specific applications [27]. Chitosan has been approved as functional food in some Asian countries [48], recognized as safe (GRAS) and approved for dietary use in Italy and Finland. In 2008 a specific monograph was introduced in the European Pharmacopeia and in 2011 in the US National formulary [27]. Based on these facts and regulatory aspects, it could be concluded that the application of chitosan-based carriers for the development of drug formulations is still limited [27]. Thus, the innovative and future studies subjected to the biopolymers biomedical and pharmaceutical applicability have to include detailed and precise clinical investigations assessing probable health risks, side effects, biocompatibility and health benefits associated with the application of chitosan formulations.

With approximately 10¹¹ tons of chitin produced per year from waste crustacean carapaces, chitosan is a cost effective biomaterial [49,50]. Despite the fact that chitosan-based formulations sustain immense biological activities in plants, these materials have not yet been widely adopted as sustainable agricultural remedies due to the lack of reliable knowledge and research on their bioactivity, modes of action towards pathogenic microorganism, plant protection and growth [59].

Conclusions

To satisfy the anticipated claims of chitosan-based formulations and their applicability in the field of bioorganic chemistry, encompassing medical (human and veterinary), pharmaceutical and agricultural scientific research, it is imperative to line up all the possible advantages and strengths, as well as to overcome the established and expected disadvantages and weaknesses associated with the scope of achieving sustainable knowledge, research and development.

Authors’ contributions

Z.Y. developed the review conception and design, performed the acquisition, critical analysis and interpretation of data, prepared the manuscript and revised it critically for important intellectual content; D.I., M.T. and N.N. contributed specific information to the manuscript. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Bulgarian Ministry of Education and Science under the National Research Programme “Healthy Foods for a Strong Bio-Economy and Quality of Life” approved by DCM # 577/17.08.2018” and by Scientific Project 13/2018, FVM, Trakia University, Bulgaria (granted to Z. Y.).