Recent developments in chitosan based microgels and their hybrids

Abstract

Chitosan based microgels have gained great attention because of their chemical stability, biocompatibility, easy functionalization and potential uses in numerous fields. Production, properties, characterization and applications of chitosan based microgels have been systematically reviewed in this article. Some of these systems exhibit responsive behavior towards external stimuli like pH, light, temperature, glucose, etc. in terms of swelling/deswelling in an aqueous medium depending upon the functionalities present in the network which makes them a potential candidate for various applications in the fields of biomedicine, agriculture, catalysis, sensing and nanotechnology. Current research development and critical overview in this field accompanying by future possibilities is presented. The discussion is concluded with recommended possible future works for further progress in this field.

1. Introduction

Microgels are three-dimensional polymeric network with diameter range of 0.1–10 μm [1]. They are colloidal particles and are usually dispersed in water. Microgels are a class of smart materials because they show variation in size by changing external stimuli like pH, temperature, ionic strength etc. [1,2]. Microgels possess various potential advantages over the macrogels because of their submicron size including better dispersibility, high response kinetics, and micro-range applications [3]. The microgels can be categorized into different types depending upon the morphology, size, crosslinking strength, feed composition and crosslinking density. Usually, microgels starting from hydrophilic pre-polymers are synthesized through physical or chemical crosslinking methods [4,5]. In the physical crosslinking methods, the microgels are synthesized through molecular entanglements, metal ligand coordination, ionic interactions, hydrogen bonding and hydrophobic interactions [6]. Generally, ionic cross-linkers are used to synthesize microgels through physical crosslinking [7,8]. The ionic crosslinked microgels have lower stability than microgels having covalent bond based cross-linking in their network [8]. In chemical crosslinking method, covalent bonds are formed between cross-linker and main polymer chains as a result of polymerization. Chemically crosslinked microgels are acquired by polymerization of monomer/pre-polymer in the presence of suitable crosslinker. Glutaraldehyde (GLA), N,N′-methylenebisacrylamide (MBA), genipin, ethylene glycol diglycidyl ether (EGDGE) and epidichlorohydrin (ECH) are the most popular chemical crosslinking agents for the fabrication of microgels [8–12] while synthetic monomers, low molecular weight pre-polymers and natural polymers like cellulose, chitosan and alginate etc. are extensively used as starting material to synthesize chemically crosslinked microgels [13,14]. The unique properties of natural polymers, such as non-toxicity, good biocompatibility, biodegradability, higher adsorption ability and low price make them special starting material in the preparation of microgels [15–17]. Chitosan (CS) is the most abundant polymer for the preparation of microgel systems [16]. Chitosan is obtained by partial deacetylation of chitin, which is second most abundant polymer in nature and found in exoskeletons of insects, cell walls of algae and fungi [18–21]. Chitosan is made of unsystematically distributed β_1–4 linked N-acetylglucosamine units and glucosamine units [2,22,23]. The backbone of chitosan contains numerous reactive amino and hydroxyl groups that make the structure of chitosan suitable for physical and chemical modifications [5,24]. These methodologies are extremely versatile because of the great diversity of synthetic options and chemical groups offered by the suitable combination of functional monomers and crosslinkers [1,25]. Chitosan based microgels responsive to outer physical or chemical stimuli can be established by combining chitosan with monomer responsible for smart behavior [26–28]. By varying the particular external stimulus, they can undergo variations in size, shape, wettability, optical, mechanical and electrical properties [29,30]. These stimuli-responsive microgel systems have various applications in adsorption, drug delivery, bio-sensing and catalysis [15,31–35]. The selection of feed composition is made on the basis of the requirement of the application of the microgel system associated with responsive behavior towards a specific stimuli [36–40]. For example, temperature responsive microgels can be obtained through free radical precipitation polymerization of N-isopropylacrylamide (NIPAM) or N-isopropylmethacrylamide (NIPMAM) in the presence of chitosan and suitable crosslinker. Similarly, polymerization of acrylic acid (AAc) [41,42] or methacrylic acid (MAAc) [43] in the presence of chitosan gives CS based microgels with pH sensitivity. P(NIPAM) and P(NIPMAM) are well recognized temperature-sensitive microgels and undergo speedy swelling or deswelling near a particular temperature named volume phase transition temperature (VPTT) in aqueous medium [44–47]. The VPTT value of P(NIPAM) microgels in aqueous medium is 30–34 °C whereas that of P(NIPMAM) is 38–44 °C [48–50]. If temperature is lower than the VPTT, the temperature responsive microgels are hydrophilic and swollen in water, while by rising the temperature higher than the VPTT, they become hydrophobic and exist in de-swollen form due to release of water from the network. Therefore, polymerization of NIPAM or NIPMAM with chitosan gives thermoresponsive microgel system suitable for the drug delivery applications [51–55]. Microgels obtained from chitosan and some synthetic monomers have large volume for storage and transportation of small molecules. These microgels can be utilized for the preparation of several types of inorganic nanoparticles (NPs) [9,56]. Metal nanoparticles (MNPs) can be stabilized within microgels because of donor-acceptor interactions among MNPs and functional groups of the polymer system. The dimensions of MNPs can be altered by adjusting crosslinking density of the microgel systems [57,58]. Microgels loaded with inorganic nanoparticles are known as hybrid microgels which have properties of both organic polymeric network and inorganic nanoparticles. Synthesis of chitosan based microgels and their hybrids has become the focus of present interest [59,60] due to their rapid swelling and deswelling in comparison to macrogels/hydrogels. Size and change in size of the microgels can be accurately measured by available techniques like transmission electron microscopy and dynamic light scattering in case of microgels. They have excellent dispersibility in water and may be applied for a variety of applications. Microgels can be used as model systems to understand the behavior of CS based macrogels. Properties of CS based microgels can be more easily altered by using various co-monomers in comparison to bulk gels. Our subject of current interest is to review chitosan based microgels and their hybrids. To the best of our knowledge, synthesis, properties and applications of multi-responsive chitosan based microgels have not been reported in an organized way in the form of a review article formerly.

Here in, we have reported a critical analysis of literature published on methodologies to synthesize chitosan based microgels and hybrid microgels, their properties and applications. A brief introduction of the topic is given in Section 1. Methods to synthesize chitosan based microgels have been discussed in Section 2. Section 3 is dealing with several characterization techniques used to investigate chitosan based microgels. Temperature, pH, glucose, light and magnetic field responsive behavior, influence of crosslinker feed composition on the properties of chitosan based microgels have been discussed in Section 4. In Section 5, applications of chitosan based microgels and their hybrids in environment, catalysis, sensing, agriculture and biomedicine have been described in detail. Conclusion and future guidelines for the development of this particular microgel system are given in Section 6.

2. Synthesis of chitosan based microgels and their hybrids

2.1. Synthesis of chitosan based microgels

2.1.1. Through irradiation polymerization

Chitosan based microgels/nanogels are synthesized by irradiation polymerization using ionizing radiations like gamma rays. Polymerization occurs by irradiating the mixture of monomers, co-monomers and crosslinker for a specific period of time. The irradiation process is favorable over some chemical procedures for the synthesis of particular microgel system. Because chemical crosslinking agents are poisonous and can decrease the number of functional groups of microgel particles by reacting with them and thus reduces their adsorption capability [61,62]. However, microgels/nanogels synthesized through gamma irradiation process, are not monodisperse because of that, their applications are limited. Radwan et al. [63] created a series of chitosan-co-poly(acrylic acid) nanogels by gamma irradiation at room temperature. First, they dissolved chitosan in an aqueous solution of CH₃COOH under stirring for six hours until complete dissolution. Chitosan molecules got protonated in the presence of acetic acid. Then, they mixed the acrylic acid with chitosan solution at a ratio of 1:1 under stirring. When this protonated CS was mixed with dissociated acrylic acid solution, an electrostatic force of attraction between positive and negative charge produced a complex via H-bonding. These solutions were sealed in plastic bags, and irradiated with gamma radiation of different doses of ranging from 10 to 50 kGy at a dose rate of 0.56 Gy/s using ⁶⁰Co gamma cell. The polymerization process was initiated by gamma radiation. Almost all the COOH groups of acrylic acid got ionized and the previously formed complex disrupted. The strong electrostatic forces between polymerized acrylic acid and chitosan chain resulted in the formation of cross-linked polymeric network. The transparent solution in sealed packs turned turbid after the gamma irradiation, which was another indication of polymerization. The prepared nanogels particles depending on their size, were separated by centrifugation. The polymerization yield for 30 kGy dose was maximum i.e. 88 %. However, further increase in radiation dose caused the degradation of polymeric network and a reduction in yield was observed.

2.1.2. Through free radical precipitation polymerization

Free radical precipitation polymerization is the most common process used for the preparation of chitosan based microgels [1,9,30]. In this method, ammonium per sulfate (APS) [64,65] and potassium per sulfate (KPS) [66–68] have been reported as initiators using MBA as crosslinker and sodium dodecyl sulfate (SDS) as emulsifier as shown in Fig. 1. For the preparation of chitosan based microgels, chitosan is dissolved in aqueous CH3COOH (1 % w/v) in a three necked round bottom flask fitted out with condenser, N₂ inlet and temperature probe for 24 h to make sure complete dissolution. After that, the co-monomers (if any) and SDS (emulsifier) are mixed with the reaction mixture under N₂ supply at constant stirring at room temperature. After half an hour of continuous stirring, MBA (crosslinker) is added into the reaction contents and the temperature of the reaction mixture is brought up to 75 ° C. After one hour of heating, the initiator usually solution of APS or KPS is added dropwise to start the polymerization. The change in color from transparent to milky of the reaction mixture indicates the on-set of polymerization. Reaction is kept in progress further for 4–5 h under N₂ purging with continuous stirring at 75 °C. The resulting chitosan based microgels are dialyzed for few days against distilled water to remove unreacted moieties i.e. unreacted monomers, initiator and surfactant. With minor change in methodology, majority of the researchers have reported free radical precipitation polymerization route of preparation of homogenously crosslinked chitosan based microgels [1,69–71].

Fig. 1.

Route of synthesis of chitosan based microgels through free radical precipitation polymerization method.

Caputo et al. [1] prepared various samples of chitosan based microgels using different feed compositions of AAc, AAm, NIPMAM and NIPAM co-monomers in the presence of MBA (crosslinker), SDS (surfactant) and APS (initiator) through free radical precipitation polymerization method as stated above. It was found that the swelling/de-swelling, pH and temperature responsive properties of microgels can be easily adjusted by varying the feed composition of microgel components. It was described that this is the best method to synthesize chitosan based multi-responsive and monodisperse microgels with tunable properties according to the required applications.

Khan et al. [72] synthesized chitosan-poly(MAAc) microgels with different feed compositions of the microgel system via free radical copolymerization in aqueous medium. It was reported that various physiochemical properties (electrical properties, volume phase transitions, colloidal stability, swelling/deswelling) of these microgels were highly dependent on the composition of the microgel system and can be easily tuned by this method. Free radical precipitation polymerization is very convenient and easy technique to synthesize different microgels of desired characteristics [2,28,30,73].

2.1.3. Through ion-imprinting technique

Chitosan based imprinted microgels are also synthesized by ion imprinting technique [74,75]. In this method, the complex formation occurs with the metal ions in the presence of ligand. During preparation, the microgel particles are imprinted with specific metal ions. Afterward, these metal ions are removed using eluting agent and the synthesized imprinted microgel particles are used to extract corresponding metal ions from aqueous solution. Zhang et al. [39] synthesized ion-imprinted chitosan microgel beads through glutaraldehyde (crosslinker) and impregnation of Ag⁺ ions as a template for selective elimination of Ag⁺ ions from water solution of Ag⁺ and Cu²⁺ having equal concentration. The Ag⁺-imprinted chitosan microgel beads were synthesized by shaking the aqueous solution of silver nitrate and chitosan beads for 48 h at 150 rpm. The resulting Ag⁺-imprinted chitosan beads were purified by deionized water and acetone. The template was removed from chitosan beads using aqueous solution of H₂SO₄ and thiourea. It was reported that the ion-imprinted chitosan beads have greater selectivity towards Ag⁺ ions as compared to Cu²⁺ ions.

2.1.4. Through microfluidic devices

Microfluidic technology provides an easy approach for the synthesis of monodisperse microgels with required sizes in the range from 10 μm to 100 μm [76]. Microfluidic technology processes and accurately manipulate the minute quantity of fluids ranging from 1 × 10⁻⁹ L to 1 × 10⁻¹⁸ L with channels of 10–100 μm in size. Because of very small size, these microchannels provide greater mass transfer, large specific surface area and heat transfer performance. So, microfluidic technology has many advantages like small sample or reagent consumption, quick response, accurate control of chemical and physical properties of fluid and greater mixing efficiency overcome the disadvantages observed in conventional microgels synthesis methods. Compared with the other methods, the microfluidics technology exhibits exceptional advantages in the synthesis of chitosan based microgels with controlled size distribution, shape and morphology [77]. Generally, microfluidic method for creating droplets consists of the injection of one liquid phase (called inner phase) into another immiscible liquid phase (called outer phase) through a specifically designed device called microfluidic device. The droplets are sheared off at a single point called junction where the two liquids meet each other. Many researchers reported the synthesis of chitosan based microgels through microfluidic technology for various applications [78–81].

Zamora-Mora et al. [6] reported the synthesis of chitosan microgels by on-chip crosslinking of CS with sodium tripolyphosphate (TPP) through microfluidic technique. Four-inlet flow focusing geometry was formed in which a dispersion medium containing mineral oil + span 80 (3 % w/v) and three dispersed phases, CS (0.25 % w/v), TPP (0.05 % w/v) and CH₃COOH (1 % v/v) were employed. The flow rate of dispersion medium was changed from 6.7 to 11.7 μL/min to obtain the CS microgels with mean diameter ranging from 68.0 to 42.0 μm. Microfluidic synthesis is not only limited to the fabrication of microgel systems. Small particles or biological units may be loaded within the microgel system using microfluidic devices. For example, Mora-Boza et al. [82] synthesized chitosan microgels for encapsulation of human cell mesenchymal stem cells by using microfluidic device. The chitosan lactate microgels with and without encapsulating human mesenchymal stem cells were obtained by using flow focusing devices with three independent flow inlets having chitosan lactate, crosslinker and dispersion medium. Initially, the chitosan lactate (1 wt% in phosphate buffer saline) and crosslinker phase were mixed at a T-junction to enable polymer and crosslinker interaction within the device. After that, the reaction mixture was focused towards the dispersion medium to form w/o emulsion and droplet formation. The resulting homogeneous microgels were collected through the outlet tubing and centrifuged to remove surfactant, oil and crosslinker traces. Synthesis of islet loaded CS based microgels with diameter of 302 μm using microfluidic devices via Michael addition reaction between thiol grafted CS and maleimide grafted CS has been reported by Li et al. [83]. In the next step, a zwitterionic layer around the CS microgels was made by covalent bonding between −SH (thiol) groups of thiol modified cellulose and maleimide groups of CS microgels. The system was found highly stable with excellent biocompatibility and low immunogenicity for regulation of glucose level in vivo.

2.1.5. Through spray drying approach

Spray drying technique comprises the use of a spray dryer generally comprising of a drying chamber and an atomizer. Solutions/suspensions containing feed composition of the CS based microgels to be formed is injected into the drying chamber to generate fine droplets by atomizer. In the drying chamber, the stream of hot air induces rapid evaporation of solvent from the droplets, resulting in the fabrication of microgels or microspheres with a diameter ranging from 1 to 10 μm [84].

Kang et al. [85] reported the synthesis of chitosan based microgels through spray drying method. The glutaraldehyde was used as a cross-linker for the synthesis of microgels. Chitosan was dissolved in acetic acid and glutaraldehyde (crosslinking agent) was added into the chitosan dispersion. After stirring the mixtures, the microgels were synthesized by spray drying method described above. The droplets were generated by using inlet temperature of 160 °C, 4 mL/min feed rate of flow of 4.0 mL/min and air flow rate of 6.90 L/min. The synthesized microgels were washed for 5 h with distilled water and centrifuged to remove the unreacted chitosan and glutaraldehyde. Many other scientists reported the fabrication of chitosan based microgels through spray drying technique [86–90].

2.1.6. Physical crosslinking method

The efficient way to synthesize microgels/nanogels without using toxic chemical agents is physical crosslinking method. The main benefit of this method is not using the toxic crosslinking agents which decreases the biocompatibility of microgels. They can be achieved either by hydrophobic associations, hydrogen bonding, through polyanions and association with small ionic species.

Cao et al. [91] prepared micro beads made of chitosan and hydrolyzed polyacrylamide (HPAM) through electrostatic interactions between −COO⁻, Al³⁺ and $-\mathrm{N}{\mathrm{H}}_3^{+}$ in a two stage crosslinking process. HPAM was mixed with solution of AlCl₃ and stirred for 2 h at laboratory temperature. After 2 h, the synthesized HPAM-Al micro beads were washed with purified water three times and dipped in a 20 mL chitosan solution for 24 h. These chitosan-HPAM micro beads were washed with purified water three times and dried for 48 h at 50 °C. Huang et al. [92] have prepared pH-sensitive chitosan-based microgels by physical cross-linking of polysaccharide chitosan and sodium tripolyphosphate (STP) via ultrasound treatment. Multiple cross-linked polymeric networks were obtained by varying the mass ratios of CS and STP under different mechanical stirring speeds.

2.2. Synthesis of chitosan based hybrid microgels

Chitosan based microgels having some inorganic material within the network are termed as chitosan based hybrid microgels. Inorganic materials loaded into chitosan based microgels may include nanoparticles of metals, metal oxides or SiO₂. Chitosan based microgels containing metal nanoparticles can be produced by in situ reduction of metal ions inside the network of microgels by using an appropriate reducing substance. For this purpose, the prepared microgels suspension is taken in a simple reactor equipped with magnetic stirrer, nitrogen source and condenser. Then the metal salt solution is added into the reactor under continuous stirring and N₂ gas purging. The metal ions incorporated into the cavities of microgels due to electrostatic attraction between negatively charged functionalities of polymeric network of the microgels and positively charged metal ions or donor-acceptor interaction between polymer functionalities and metal ions. Freshly prepared solution of reducing agent usually NaBH₄ is added dropwise into the reaction flask. Then these metal ions are reduced to metal atoms which combine to produce metal nanoparticles in voids of polymeric systems. The color of dispersion is changed due to the formation of metal nanoparticles. The prepared hybrid microgels are dialyzed against distilled water to remove unreacted species. Ahmad et al. [9] synthesized poly(chitosan-N-isopropylmethacrylamide-acrylic acid) P(CS-NIPMAM-AAc) microgels and fabricated silver nanoparticles (AgNPs) within the network. For this purpose, the microgel suspension was diluted with deionized water in a three necked round bottom flask having condenser, N₂ inlet and a magnetic stirrer. After adding AgNO₃ into reaction flask, the reaction contents were stirred for 30 min under N₂ purging. After adjusting the pH value at 6, freshly prepared NaBH₄ solution was added dropwise into reaction mixture and the color of the dispersion was changed to greenish brown due to the creation of AgNPs. The dispersion was stirred for 2 h under N₂ supply and the prepared hybrid microgels were dialyzed for 20 min. Zhang et al. [93] synthesized Ag-carboxymethylchitosan-P (NIPAM) hybrid microgels by using the approach as described above. For this purpose, the AgNO₃ solution was added into the aqueous dispersion of carboxymethylchitosan-P(NIPAM) microgels at pH of 8.8. After stirring for 4 h in the dark, the solution was dialyzed for 2 d against distilled water to remove metal ions present outside the network. The Ag⁺ ions were reduced into Ag nanoparticles by adding freshly prepared NaBH₄ solution into the reaction mixture. After 1 h of continuous stirring, the hybrid microgel dispersion was dialyzed against distilled water. NaBH₄ can be replaced by some environmentally benign reducing agent for the fabrication of silver nanoparticles within the CS based microgels. George et al. [94] reduced Ag⁺ ions loaded into P(CS-AAc) microgels using Euphorbia Maculate leaf extract as reducing agent in aqueous medium to obtain highly stable Ag- P(CS-AAc) hybrid microgels for H₂O₂ sensing applications. Fabrication of CS based hybrid microgels can also be achieved by simple addition of already prepared metal nanoparticles into the dispersion of chitosan based microgels. Mutharani et al. [95] prepared palladium metal nanoparticles (PdNPs) by reduction of PdCl₂ in the presence of trisodium citrate using NaBH4 as reducing agent. Then PdNPs were added into the aqueous dispersion of P(NIPAMCS) microgels under stirring. PdNPs were not found in the interior of microgels. However, the stable microgels decorated with PdNPs were successfully obtained. CS based hybrid microgels can also be obtained by carrying out the free radical precipitation polymerization in the presence of already prepared inorganic nanoparticles. This methodology is also useful to get chitosan based microgels containing metal oxide nanoparticles. Echeverria et al. [96] obtained Magnetic P(NIPAM-CS) hybrid microgels by carrying out free-radical precipitation polymerization of NIPAM in the presence of CS and iron oxide nanoparticles using MBA as crosslinker and APS as initiator under N₂ atmosphere in aqueous medium. Preparation of Fe₃O₄-P(CS-NIPAM) core shell microgels with Fe₃O₄ core and P(CS-NIPAM) shell following above two step synthesis methodology has been reported by Li and co-workers [97]. In the first step, oleic acid modified Fe₃O₄ nanoparticles with mean diameter of 13 nm were synthesized using FeCl₂·4H₂O and FeCl₃·6H₂O chemical co-precipitation method under N₂ in aqueous medium in the presence of oleic acid. In the next step, P(CS-NIPAM) shell was built around oleic acid modified Fe₃O₄ nanoparticles by carrying out the emulsion polymerization of chitosan and NIPAM using MBA as a crosslinker and APS as initiator in water phase under N₂ in the presence of Na₂SO₄.

3. Characterization of chitosan based microgels

Various characterization techniques like Transmission electron microscopy (TEM) [98], Scanning electron microscopy (SEM) [30], Atomic force microscopy (AFM) [27], Dynamic light scattering (DLS) [29], Nuclear magnetic resonance spectroscopy (NMR) [99], Fourier transform infrared spectroscopy (FTIR) [28], UV–visible spectroscopy (UV–Vis) [100], Energy dispersive X-ray spectroscopy (EDX) [34], Differential scanning calorimetry (DSC) [71], Thermogravimetric analysis (TGA) [101], Differential mechanical analysis (DMA), X-ray diffraction (XRD) [102], Atomic absorption spectroscopy [103] and Raman spectroscopy (RS) have been used to characterize chitosan based microgels and their hybrids.

The surface morphologies of chitosan based microgels and their hybrids are explored using spectroscopic techniques like SEM, AFM and TEM [27,99,104]. TEM is also used to investigate the size and shape of microgels and nanoparticles loaded into chitosan based microgels. DLS/particle size analyzer are used to measure the size distribution and hydrodynamic diameter of microgels and their hybrids. This method is also utilized to study the pH and temperature sensitive behavior of chitosan based microgels [72]. FTIR, NMR and RS are extensively used techniques for identification of functionalities of synthesized microgel particles [21,28]. DSC, DMA and TGA are widely used for determination of thermal decomposition and thermal stability of chitosan based microgels and their composites. TGA is also helpful for investigation of metal content in chitosan based microgels loaded with MNPs. UV–Vis spectroscopy is used to study the VPTT of microgels and their hybrids [53]. This technique is also useful for confirmation of formation and stabilization of plasmonic nanoparticles inside the microgels. UV–Vis spectrophotometry may also be used to observe the advancement of reaction catalyzed by MNPs loaded chitosan based microgels [9]. Flame atomic absorption spectroscopy is used to determine the amount of heavy metal ions (Cr⁶⁺, Pb²⁺, Cd²⁺, Cu²⁺, Ni²⁺, Co²⁺, Zn²⁺) adsorbed on chitosan based microgels [103].

Viscoelastic behavior of hydrogels can be studied by performing rheological measurements [65,107]. XRD and EDX techniques are employed to study the crystalline properties of CS microgels and their hybrids [105]. These methods are also useful for confirmation of metallic nature of inorganic material loaded within microgels [56]. Cytotoxicity and drug release studies of chitosan based microgels and their hybrids are performed through MTTS method and quantitative analysis of drug release is investigated through high performance liquid chromatography (HPLC) technique [1,16]. Chitosan based microgels characterized by various techniques are illustrated in Table 1.

Table 1.

Chitosan based microgels/nanogels characterized by different techniques.

Composition of microgels	Characterization techniques	References
P(CS-NIPMAM-AAc), P(CS-NIPAM-AAc), P(CS-NIPMAM-MAAc), P(CS-NIPAM-MAAc)	DLS, AFM, UV–Vis spectroscopy, HPLC, MTTS	[1]
Chitosan-P(NIPAM-co-MAAc)	FESEM, FTIR, 13C NMR	[2]
Poly(CS-NIPMAM-AAc)	TEM, FTIR, UV–Vis spectroscopy	[9]
Chitosan-SiO2-VTS	DLS, SEM, UV–Vis spectroscopy, MTTS	[16]
Poly(Chitosan)	FTIR, CLSM, AFM, Pendant drop tensiometer	[27]
Chitosan-g-PNIPAM	SEM, FTIR, 1H NMR, UV–Vis spectroscopy, Particle analyzer	[28]
CS-PdNPs, CS-GO-PdNPs, CS-CNTs-PdNPs, CS-LDHs-PdNPs	FTIR, SEM, TEM, XRD, UV–Vis spectroscopy	[56]
PNIPAM-CS-PAAc	FTIR, DLS, UV–Vis spectroscopy, Ostwald viscometer	[64]
Chitosan-PNIPAM	FTIR, DLS, Rheometer	[65]
Chitosan loaded Cu2+ and Zn2+	FTIR, SEM, MTTS	[104]
Chitosan-alginate	UV–vis spectroscopy, DLS, FTIR, XRD, HPLC, TEM	[105]
Chitosan-gellan	DLS, Optical microscopy, CSLM	[106]

4. Properties of chitosan based microgels

Chitosan based microgels exhibit responsive behavior to external stimuli like pH, temperature, magnetic field, glucose, light etc. due to different moieties present in the network are summarized in Table 2.

Table 2.

Stimuli-responsive behavior of chitosan based microgels.

Composition of microgels	Stimulus	References
Chitosan-carrageenan	Temperature, pH	[21]
Chitosan-P(MAAc-co-NIPAM)	Temperature, pH	[51]
Chitosan-PNIPAM	Temperature, pH	[118]
Chitosan	pH	[27]
Chitosan-g-PNIPAM	Temperature, pH	[28]
Chitosan-PNIPAM	Temperature, pH	[52]
ZnO-(PNIPAM-Chitosan-PAAc)	Temperature, pH	[30]
Chitosan-iron	pH	[119]
Chitosan-PNIPAM	Temperature, pH	[53]
PNIPAM-chitosan-PAAc	Temperature, pH	[64]
Chitosan-PNIPAM	Temperature, pH	[65]
Chitosan-PMAAc	Temperature, pH	[71]
Chitosan-PNIPAM-Fe3O4	Light, Magnetic	[109]
Carboxymethyl chitosan functionalized rGO-aldehyde functionalized PEG	Light, pH	[120]
Chitosan crosslinked β-GP	Light, pH	[111]
Chitosan-Fe3O4 NPs	Magnetic, pH	[121]
Chitosan-polypyrrole	Light, pH	[112]
Chitosan-PVA	Light	[110]
Chitosan-polyethylene glycol dimethacrylate	Magnetic	[122]
Chitosan-GMACoFe2O4	Magnetic	[19]
Chitosan-Con A	Glucose	[123]
Chitosan-4-formylphenylboronic acid	Glucose, pH	[124]

4.1. pH responsive behavior

Microgels which exhibit instant change in their hydrodynamic radius with change in pH of the medium are called pH responsive microgels. Highly pH sensitive chitosan based microgels can be obtained by copolymerization of chitosan with ionic monomers. Chitosan based microgels show pH responsive behavior due to the presence of −NH₂ groups and −COOH groups (coming from copolymerization of CS with some ionic monomer). At pH ≥ pKa of ionic co-monomer, the −COOH groups get deprotonated and swelling of microgels takes place due to the electrostatic repulsion among negatively charged −COO⁻ groups. When pH < pKa, the microgels remain in de-swollen state due the absence of such repulsions. At pH < pKa of chitosan (pKa ≈ 6.5), the protonation of −NH₂ groups to $-\mathrm{N}{\mathrm{H}}_3^{+}$ groups takes place which results in the swelling of microgels due to electrostatic repulsion between $-\mathrm{N}{\mathrm{H}}_3^{+}$ groups present within the polymer network. So pH of the medium adjusts the swelling/de-swelling properties of chitosan based microgel particles. Khan and coworkers reported pH sensitive behavior of P(CS-MAAc) microgels in aqueous medium using DLS measurements as shown in Fig. 2 [108]. It was revealed that the value of hydrodynamic diameter (D_h) of P(CS-MAAc) microgels increases with rise of pH of the medium and reaches its maximum value at pH 7.5. The swelling of microgels was attributed to the repulsion between the −COO⁻ groups at pH 7.5. A decrease in the Dh value of the polymer particles when the pH of the medium was in the range of 6–4.5 because of the electrostatic attractions between positively charged $-\mathrm{N}{\mathrm{H}}_3^{+}$ groups of chitosan and negatively charged −COO⁻ groups of PMAAc. At pH < 4, all the −COO⁻ groups become protonated to −COOH and −NH₂ groups of chitosan become protonated to $-\mathrm{N}{\mathrm{H}}_3^{+}$ groups. The swelling of polymer particles was occurred due to the electrostatic repulsion between positively charged $-\mathrm{N}{\mathrm{H}}_3^{+}$ groups of chitosan [72]. Rasib et al. [51] studied the similar pH responsive behavior of chitosan-P(MAAc-co-NIPAM) microgels in aqueous medium. The analogous pH responsive behavior of chitosan-P(NIPAM) [65], chitosan-carrageenan (CS-CRG) composite microgels [21], ZnO-P(NIPAM-CS-AAc) [30] microgels have been published in literature. The pH responsive behavior of chitosan based microgels having −NH₂ and −COOH functionalities in their network is shown in Fig. 2.

Fig. 2.

pH responsive behavior of chitosan based microgels.

4.2. Thermosensitive behavior

Microgels which exhibit an abrupt variation in their hydrodynamic diameter or radius by changing the temperature of the system are called thermo-responsive microgels. Thermo-sensitivity can be induced in chitosan based microgels/nanogels by copolymerization with some thermosensitive monomers. By changing the temperature, some polymeric systems quickly undergo volume phase transition at a specific value of temperature which is known as the lowest critical solution temperature (LCST). Above the LCST, polymer chains go from coil conformation to globule conformation due to increase in hydrophobic interactions between nonpolar groups of the polymer chains. Below the LCST, polymer chains exist in coil conformation due to hydrogen bonding between polymer functionalities and water molecules. Microgels obtained from the monomers responsible for temperature sensitivity can change their size with change in temperature because of the same reason and are known as thermoresponsive microgels. The value of temperature at which significant decline in particle size of microgels occurs is termed as volume phase transition temperature (VPTT). Wang et al. [28] investigated the temperature responsive behavior of P(CS-NIPAM) microgels using particle size analyzer in the temperature range of 20–40 °C. It was noted that below 32 °C (VPPT), microgels are hydrophilic and remain in swollen state due to strong polymer-solvent interaction. Above 32 ° C, the interaction between polymer particles and water molecules becomes weak and polymer-polymer interactions increases due to increase in hydrophobic character of P(NIPAM). Consequently, the microgels shrink due to dehydration as shown in Fig. 3. It was found that the presence of chitosan does not only affect the value of hydrodynamic diameter but also has a prominent effect on VPTT of P(CS-NIPAM) microgels. The increase in weight %age of chitosan in feed composition of the P(CS-NIPAM) microgels decreases thermo-sensitivity and increase the value of VPTT.

Fig. 3.

Thermo-sensitivity of chitosan based microgels.

Khan et al. [64] studied the thermo-sensitivity of [PNIPAM-CS-PAAc] microgels in terms of change in hydrodynamic radius at constant pH using DLS measurements. It was observed that by increasing the temperature from 15 °C to 38 °C, the size of microgels increases and show maximum swelling. AAc is hydrophilic unit and its presence in microgels may increase the value of VPTT. Above 38 ° C, the size of microgels start decreasing because above this temperature, the hydrophobic character of P(NIPAM) and CS increases which results in greater polymer-polymer interaction in comparison to polymer-water interaction. Hence, the microgels start to shrink due to dehydration. This decrease in size of microgels is less prominent above 45 °C. It means that no significant dehydration occurs after this temperature. Many other researchers have also reported the temperature responsive behavior of chitosan based microgels [29,65,71]. In addition to the content of CS, the temperature responsive behavior of chitosan-P(NIPAM) microgels can be adjusted by altering the crosslinker feed contents. Khan et al. [65] reported the influence of crosslinker feed content on the temperature-responsive behavior of chitosan-P(NIPAM) microgels. It was found that the size of microgels increases with increasing the feed content of crosslinker up to 20 mg/100 mL of reaction mixture, whereas further increase of crosslinker feed content reduces the size of microgels. Microgel particles with higher crosslinker content are less swollen due to compact/tight structure. Hence microgels with small crosslinker feeding are flexible and more thermo-sensitive in contrast to microgels with higher crosslinker content.

4.3. Light responsive behavior

Chitosan based microgels having photosensitive groups, plasmonic MNPs and photoactive molecules show light responsive behavior by absorbing photon energy. Microgels having photosensitive groups show phase transition behavior by absorbing certain energy photons. The microgels having photoactive molecules create ions and change the osmotic pressure which result in swelling of the polymeric network. Chitosan based hybrid microgels loaded with plasmonic MNPs exhibit light-responsive behavior because the light absorbed by plasmonic MNPs is transformed into heat which causes shrinkage in the network. Pourjavadi et al. [109] synthesized the magnetic field responsive and light-responsive chitosan-P(NIPAM) nanogels loaded with Fe₃O₄ NPs and AuNPs for drug release through light as an external stimulus. When the hybrid system was irradiated with green light, the in-situ fabricated AuNPs on the surface of chitosan units absorb light at particular wavelength called surface Plasmon resonance wavelength (λ_SRP) and create local heat. Due to the presence of thermosensitive NIPAM units in the system goes from swollen state to shrunken state to release the drug. Various light-responsive chitosan based hybrid systems have been documented for drug delivery applications [110–112].

4.4. Magnetic field sensitivity

The microgels can be furnished with magnetism by combining the magnetic nanoparticles and crosslinked polymeric systems. These inorganic NPs are actual responsible for magnetic field responsive behavior of the hybrid microgels. Alternating magnetic field produces heating effect by conversion of electromagnetic energy into heat which increases the temperature of the medium. The increase in temperature causes deswelling of hybrid microgels made of magnetic nanoparticles and negative temperature sensitive microgels. A wide variety of magnetic hybrid microgels has been reviewed recently in literature [113] and is beyond the scope of this review. A few reports on chitosan based magnetic hybrid microgels for their applications in adsorption of dyes [114] and heavy metals [115], magnetic imaging [116] and drug delivery [117] are available in literature. Li et al. [97] reported pH, temperature and alternating magnetic field responsive behavior of Fe₃O₄-P(CS-NIPAM) core shell microgels in water. The value of mean diameter of the core shell microgels was decreased on applying alternating magnetic field due to heating caused by conversion of electromagnetic energy into heat. Moreover, the value of temperature as a function of time in alternating magnetic field was also investigated for determination of the value of the specific absorption rate (R_sp) from the product of specific heat of water and the slope of temperature vs time curve which is an important parameter for treatment of cancer via magnetic induction hyperthermia. The value of R_sp was found in the range of 10–15 W/g which is considered as optimum value for hyperthermia treatment. Pellá et al. [19] synthesized magnetic chitosan microgels loaded with GMA modified CoFe₂O₄ NPs. The magnetic field-responsive behavior of the hybrid microgels was used to investigate the release of drug from the polymeric network in the presence and absence of external magnetic field employing vitamin-B12 as an exemplary drug. In the absence of magnetic field, a rapid release of drug was noticed at pH 7.4 and the equilibrium was achieved after 30 min. While in the presence of magnetic field, a two-step drug release was found at pH 7.4, in first 30 min a greater release rate of drug was observed, followed by a more controlled sluggish release step that continued 50 min. The slower release of drug in second step was due to the presence of magnetic field which promoted the high degree organization of chains of microgels, because ^GMACoFe₂O₄ NPs affect the swelling kinetics of microgels by aligning itself in the direction of magnetic field. This kind of release is significant for gastric wounds treatment.

4.5. Glucose-responsive behavior

The glucose sensitive microgels change their size in the presence of glucose molecules in the outer environment. Glucose oxidase, phenylboronic acid and bean protein A are the main substances used to synthesize glucose-responsive chitosan based microgels. Mechanism of glucose responsiveness of chitosan based microgels loaded with glucose oxidase (GO_x) for the self-regulating release of drug is shown in Fig. 4.

Fig. 4.

Glucose responsive chitosan based microgels containing glucose oxidase for drug release. Glucose (C₆H₁₂O₆) reacts with oxygen (O₂) to form gluconic acid which decreases the pH of the medium. Protonation of −NH₂ groups of chitosan takes place which causes electrostatic repulsion between the positively charged $-\mathrm{N}{\mathrm{H}}_3^{+}$ groups. The increase in $-\mathrm{N}{\mathrm{H}}_3^{+}$ groups in the network increases the hydrophilicity of the network. So water molecules diffuse from bulk region to the interior of the network which causes swelling in the polymeric network. The increase in diameter of microgels with increase of glucose concentration in the medium is known as glucose sensitivity.

Therefore, such kind of chitosan based microgels can be used as glucose responsive drug delivery system (Fig. 4) as reported by Kim et al. [125]. H₂O₂ produced in oxidation of glucose catalyzed by GO_x has severe toxic effects. H₂O₂ is converted into harmless O₂ and water in the presence of catalase (CAT). The detail of the process will be given in next sections.

5. Applications of chitosan based microgels

5.1. Environmental applications

5.1.1. Removal of dyes from water

The main source of water pollution is industrial waste which contains dyes, heavy metals and other toxic chemicals, which cause serious health problems. Removal of these toxic chemicals from waste water is the worldwide concern. Adsorption is the most efficient technique to remove these toxic pollutants from water and waste water. Chitosan based microgels have outstanding ability to adsorb toxic heavy metals and dyes from water, because the functional groups of crosslinked polymeric systems have the attraction towards adsorbate molecules. In acidic solution, the $-\mathrm{N}{\mathrm{H}}_3^{+}$ groups of chitosan have affinity to attract anionic dyes. Ramalingam et al. [126] synthesized chitosan based magnetic core shell hybrid microgels decorated with silver nanoparticles for adsorptive removal of both anionic and cationic dyes from aqueous medium under various pH values of the medium. The adsorption of anionic dyes was ideal (99 % removal at 50 mg/mL of starting concentration) under acidic pH conditions (from 2.0 to 4.0), because in acidic medium, all the −NH₂ groups of chitosan get protonated to $-\mathrm{N}{\mathrm{H}}_3^{+}$, which attracts the negatively charged dyes, enhancing the adsorption power of the hybrid system while slightly basic pH (pH > 8.0) was found appropriate for removal of cationic dyes from aqueous medium which definitely may be attributed to conversion of $-\mathrm{N}{\mathrm{H}}_3^{+}$ groups to −NH₂ groups of chitosan of Fe₃O₄-CS core shell microgels decorated with Ag nanoparticles. This hybrid microgel system has three major advantages. Both anionic and cationic dyes can be selectively removed from water by changing the pH of the medium. Secondly, Fe³O₄ core of Fe₃O₄-CS-Ag hybrid system makes it magnetically separable after extracting dyes from aqueous medium. Moreover, the presence of silver nanoparticles in the outer region of the CS shell make it a potential antibacterial system.

Zhang et al. [127] have reported the adsorptive removal of orange II, a widely used dye in textile, pulp and paper industries and dangerous to the environment, from an aqueous medium using zirconium-based chitosan (Zr-CS) micro-composites. They claimed that the prepared system has an excellent adsorptive capacity of 926 mg/g and followed the pseudo-second-order kinetics and the Langmuir isotherm model. The adsorption of orange II dye under different conditions of temperature, pH and concentration of dye was studied. It was stated that with an increase in temperature from 277 to 313 K, the adsorption capacity was also increased from 534 mg/g to 955 mg/g, because of higher kinetic energy, moreover, the process of adsorption was endothermic. The maximum adsorption was observed at pH = 2 because under acidic condition, amide groups of CS were in protonated form. Increased adsorption capacity at lower pH was due to the electrostatic interaction between negatively charged dye and positively charged $-\mathrm{N}{\mathrm{H}}_3^{+}$ groups of Zr-CS composites. A decrease in percentage removal from 100 % to 63 % was observed with an increase in dye concentration from 50 mgL⁻¹ to 300 mgL⁻¹ because of saturation at binding sites.

The adsorptive removal of ionic dyes [methyl orange (MO) and methylene blue (MB)] was studied using magnetic chitosan-polyethyleneimine embedded hydrophobic sodium alginate micro-composites by Zeng and co-workers [128]. With an increase in pH of the medium from 2 to 7, the adsorption of MO decreases 2.11 times. Because the protonated functional groups on the surface of micro-composite possessed a strong electrostatic attraction with the sulfonic acid groups on MO leading to higher adsorption at lower pH value. However, in case of MB dye, the adsorption increased 2.47 time with an increase in pH from 2 to 12. At higher pH, functional groups got deprotonated and had a strong electrostatic attraction with the quaternary ammonium groups on MB, resulting in higher adsorption. By changing the pH of the medium both dyes were removed from the aqueous medium.

Adsorption potential and experimental data of chitosan based microgels and their hybrids is given in Table 3.

Table 3.

Chitosan based microgels along with experimental data for dye and heavy metals removal.

Microgels	Adsorbate	Adsorption capacity (mg/g)	Kinetic model	Isotherm	References
Chitosan hydrogel crosslinked with glutaraldehyde	Food Blue 2	112.4
Chitosan hydrogel crosslinked with glutaraldehyde		155.1
			Pseudo first and second order		[10]
	Food Red 17	92.9
		133.9
Nano-TiO2-chitosan-PNIPAM	Acid fuchsin	45.4	Pseudo second order	Langmuir	[129]
Nano-ZnO-chitosan	Reactive Black 5	189.4	–	Langmuir	[130]
Chitosan-alunite composite	Acid Red 1	596	Pseudo first order	Langmuir	[131]
	Reactive Red 2	423.3
Chitosan-PVA	Hg2+	950.6	–	Langmuir	[132]
Ag-chitosan/clay	Cu2+	181.5	–	–	[133]
Chitosan	Cd(II)	125	Pseudo second order	Langmuir	[134]
Chitosan/Cu(OH)2	As5+	39.1	Pseudo second order	Langmuir	[135]
Chitosan/CuO		28.1

5.1.2. Removal of heavy metals from water

The extensive distribution of heavy metal ions in environment due to several agricultural, domestic, medical and industrial applications, increasing worry over their possible effects on environment and human health. They are highly toxic that can damage multiple organ, even at minor exposure and categorized as human carcinogens. Current concern is the removal of these toxic heavy metals based cations like Cr⁶⁺, Cd²⁺, Cu²⁺ and Pb²⁺ from water or waste water using chitosan based microgels as an adsorbents. Du et al. [92] designed chitosan based microgels via spray drying method using formaldehyde as crosslinker and used them as adsorbents for removal of Cu(II) ions from aqueous medium. Adsorption process was carried out under various conditions of pH of the medium ranging from 2.0 to 7.0. It was noted that extent of adsorption of Cu(II) ions increases with increase in pH up to pH value of 6 because protonated amino groups of chitosan prevent Cu(II) from reaching the microgel particles because of electrostatic repulsion and then started to decrease due to precipitation of Cu(II) ions in the form of copper hydroxide. The maximum adsorption capacity of the microgel system was 144.93 mg/g at pH = 6.0. Adsorption data was the best fitted in Langmuir adsorption isotherm for determination of values of Langmuir parameters. Chen et al. [136] also found that Langmuir adsorption isotherm is the best suited model for the sorptive extraction of Cu(II), Zn (II), Ni(II) and Pb(II) ions from aqueous medium by the chitosan system crosslinked by epichlorohydrin. Moreover, the adsorption capacity of the microgel system can be increased by using metal ions imprinting concept. For this purpose, microgels were synthesized in the presence of each metal ions separately and then template (metal ions) was removed and the resulting templated microgel system was used as sorbent for the removal of same metal ions from aqueous medium. It was noted that adsorption capacity significantly increased in case of metal ions templated microgel system in comparison to microgel synthesized without metal ions template. This idea was also found useful for the selective removal of specific metal ions from aqueous medium. The microgel system was found to regenerate for the re-use.

Lalita et al. [137] synthesized CS-g-PNIPAM, CS-g-(PNIPAM-co-AAc) and CS-g-(PNIPAM-co-AAm) microgels and studied the removal of three metal ions (Cu²⁺, Fe²⁺, Cr⁶⁺) from their water solution at different values of pH and temperature. It was reported that all the microgel samples show maximum adsorption of these three metal ions at lower temperature and higher pH values. Because at higher temperature, the interaction between active groups of microgels and metal ions become weak. At higher pH values, the swelling of CS-g-(PNIPAM-co-AAc) samples was observed due to formation of –COO⁻ groups. The negatively charged carboxyl groups repel each other and cause swelling in polymeric network. Due to swelling and greater electrostatic attraction between positively charged metal ions and negatively charged carboxylic groups, the adsorption of metal ions increased. The percent uptake of metal ions was observed >95 % for Cu²⁺, 90 % for Fe²⁺ and 75 % for Cr⁶⁺ by chitosan grafted with binary monomers. Because grafting of chitosan with PNIPAM-co-AAc and PNIPAM-co-AAm enhanced the binding capacity of metal ions and revealed best results for sorption of all three metal ions. So adsorption potential of CS based microgels can be enhanced by copolymerization of CS with suitable monomers and by changing the conditions of medium. CS microgels having no co-monomers have ability to extract metal ions and cationic dyes from aqueous medium under high pH conditions and can release the adsorbate (metal ions and cationic dyes) under low pH conditions (Fig. 5). After removing metal ions or cationic dyes, the same CS microgel system can be used again as adsorbent in the next cycle.

Fig. 5.

Uptake and release of cationic dyes and heavy metal ions using CS based microgels as adsorbents under different pH conditions.

Vilela et al. [31] synthesized chitosan-PAAc and PAAc microgels and investigated the adsorption of Cd (II) ions from water. It was reported that, in an acidic medium, the adsorption capacity of chitosan based hydrogel decreases due to protonation of −NH₂ groups to $-\mathrm{N}{\mathrm{H}}_3^{+}$, which increases the electrostatic repulsion between $-\mathrm{N}{\mathrm{H}}_3^{+}$ groups of adsorbent and metal cations. At higher pH value, due to deprotonation of $-\mathrm{N}{\mathrm{H}}_3^{+}$ to −NH₂ and formation of –COO⁻ groups of AAc, the electrostatic attraction between –COO⁻ groups of AAc and Cd²⁺ ions which enhances the adsorption capacity of the system. It was observed that, the chitosan-PAAc system has higher adsorption efficiency than PAAc based hydrogel due to greater number of available active sites. Cho et al. [138] prepared hydrous zirconium oxide impregnated chitosan microgels for the removal of Pb²⁺ and F⁻ from aqueous solution. This composite system proved to be a potential adsorbent for the simultaneous removal of Pb²⁺ and F⁻ from water, because chitosan shows binding affinity with cations while zirconium oxide with anions. This system was found efficient to adsorb 89 % of Pb²⁺ within a short time period (2.5 h). Many researchers have reported the removal of heavy metal ions using chitosan microgels as adsorbents. Their findings are summarized in Table 3.

5.2. CS based microgels as carriers of inorganic nanoparticles

Chitosan based polymer microgels have three dimensional network which have swelling/deswelling ability under external stimuli (pH, temperature, light etc.). Inorganic nanoparticles can be easily synthesized and stabilized within polymeric network of microgels through insitu reduction method. Due to donor-acceptor interaction of nanoparticles with functional groups of polymeric structure of microgels, the inorganic nanoparticles are highly stable within the network of microgels as shown in Fig. 6. Ahmad et al. [9] fabricated AgNPs within chitosan based microgels [P(CS-NIPMAM-AAc)] by in situ reduction of AgNO₃ using NaBH₄ as a reducing agent. AgNPs were fabricated within the polymeric network of microgels through reduction of Ag⁺ ions. The aqueous solution of AgNO₃ was stirred with the aqueous suspension of synthesized microgels. At higher pH of microgels suspension, the –COOH groups of the microgel system get deprotonated. Due to the presence of –COO⁻ groups in the network of microgels, the Ag⁺ ions were electrostatically attracted towards polymeric network of microgels. The reduction of Ag⁺ ions to Ag^o was achieved in the presence of reducing agent. Many researchers have reported the fabrication of metal nanoparticles within chitosan based microgels in literature [24,57,69,139–141]. Fabrication of metal nanoparticles within chitosan based microgels along with their use in catalysis is summarized in Table 4.

Fig. 6.

Fabrication of MNPs within chitosan based microgels and effect of temperature and pH on rate of catalysis in the existence of Ag-P(CS-NIPMAM-AAc) hybrid microgels.

Table 4.

Summary of fabrication and catalytic applications of chitosan based microgels.

Microgels	Nanoparticles	Catalytic applications	References
Poly(chitosan-NIPMAM-AAc)	Ag	Reduction of 4-NP, 2-NP, 4-NA, 2-NA	[9]
Chitosan-g-PAAc	Ag	Reduction of MB and CR dyes	[34]
Chitosan	Ag	Reduction of 2-NP and acridine orange	[100]
Chitosan-rGO, Chitosan-CNTs, Chitosan-LDHs	Pd	Reduction of 4-NP	[56]
Chitosan-genipin	Cu	Azide-alkyne [3 + 2]-cycloaddition	[58]
Chitosan	ZnO	Degradation of BG and RB dye	[141]
Chitosan-PMAAc	Au	–	[139]
Chitosan	Ag	Reduction of nitroarenes	[35]
Chitosan-[P(NIPAM-co-NVP)]	Ag	–	[142]
Silica/chitosan	Cu	Phenylacetylene and n-butylamine	[140]
ZnO-chitosan-P (NIPAM-AAc)	ZnO	Reduction of 4-NP	[69]
Chitosan-PNIPAM	Ag	Reduction of 4-NP	[93]

5.3. Chitosan based hybrid microgels in catalysis

Chitosan based hybrid microgels loaded with MNPs are widely used as a catalyst and exhibit excellent catalytic activity in various reactions because of high ratio of surface to volume of fabricated MNPs within the network of microgels. Crosslinked network of microgels provides a physical hurdle against the amalgamation of NPs and the NPs remains fixed inside the network because of donor-acceptor type interaction of metal with functional groups of polymer microgels to give long term stability to the catalytic system. The swelling/deswelling behavior of pH and temperature responsive CS based microgels under external stimuli can control the catalytic activity of fabricated NPs as shown in Fig. 6. CS based hybrid microgels remain in dispersed form in aqueous medium and reacting species can easily access metal nanoparticles via diffusion through open polymeric network. Catalysis by CS based hybrid microgels have features of both homogeneous (dispersibility) and heterogeneous (recyclability) catalysis. Ahmad et al. [9] investigated the catalytic activity of prepared Ag-P(CS-NIPMAM-AAc) hybrid microgel catalyst by reducing various nitroarenes (2-nitrophenol, 4-nitrophenol, 2-nitroaniline and 4-nitroaniline) using NaBH₄ as a reducing agent in water. This catalytic system was proved to be an outstanding catalyst for the reduction of multiple nitroarenes simultaneously. The values of rate constants for individual reduction of nitroarenes were higher than simultaneous reduction of nitroarenes.

Zhang et al. [93] studied the reduction of 4-NP catalyzed by the AgNPs loaded in pH and temperature responsive microgels obtained from carboxymethyl chitosan and NIPAM. This hybrid microgel system was employed as a catalyst for the reduction of 4-NP into 4-AP in the presence of sodium borohydride as a reducing agent in water. UV–Vis spectrophotometric measurement was used to monitor the conversion of 4-NP into 4-AP in the presence of AgNPs loaded chitosan based microgels. The higher values of rate constant representing the high catalytic activity of AgNPs which was attributed to the small but highly stable silver nanoparticles. The presence of NIPAM made the CS based microgel system temperature responsive. Therefore, the temperature dependence of rate constant was also examined. It was found that by increasing the temperature (from 20 to 34 °C), the value of apparent rate constant decreased due to deswelling of microgels because in de-swollen state, diffusion of 4-NP from external medium to the surface of AgNPs becomes difficult. So, rate of AgNPs catalyzed reaction was decreased, thus value of apparent rate constant decreased to minimum value at temperature near VPTT. Khan et al. [69] reported stabilization of ZnO nanoparticles in CS-P(NIPAM-AAc) microgels for the catalytic reduction of 4-NP into 4-AP in the presence of NaBH4 in aqueous medium.

Zheng et al. [34] synthesized AgNPs loaded CS-g-PAAc hybrid microgels and the catalytic activity was investigated by reducing Congo red and methylene blue dyes using sodium borohydride as a reducing agent. The complete reduction of methylene blue and Congo red having initial concentration 20 mg/L was achieved within 30 min. Moreover, the Ag-entrapped CS-g-PAAc hydrogel exhibit outstanding reusability and no significant reduction in catalytic efficiency was observed even after ten successive recycles. Sargin et al. [35] have reduced a very large number of nitroarenes into their corresponding aryl amines using NaBH4 as reductant in aqueous medium in the presence of Ag-CS hybrid microgels. Catalytic applications of chitosan based microgels are compiled in Table 4.

5.4. Electrochemical/biosensors

A device used to detect and measure the analyte is called sensor. Electrochemical sensors are widely used to detect analyte in a solution. Variation in concentration, pH and temperature of the medium can also be detected using sensors. Sensors are also used for determination of concentration of the analyte in a complex system. The effect of reduction/oxidation reaction of analyte occurring on electrode-electrolyte interface are converted into electrical signals through transducers by applying potentiometry, amperometry, conductometry and voltammetry in electrochemical sensors. Chitosan based microgels and their hybrids are used to construct a variety of electrochemical sensors.

Thirumalraj et al. [143] fabricated ZnO nano needles on chitosan microgels and used the composite materials to construct chitosan-ZnO nano needles modified carbon electrode for the detection of toxic pollutant, 4-NP in aqueous solution. ZnO nano needles have porous structure and greater surface area. The differential pulse voltammetry showed that the chitosan-ZnO nano needles modified electrode detect 4-NP with a detection limit of 0.23 μM and the sensor revealed a wide linear response range (0.5 to 400.6 μM), even in the presence of interfering species. Moreover, the analytical performance of sensor was excellent and it was successfully utilized for the detection of 4-NP in aqueous solution. Chandrasakaran et al. [144] prepared electrochemical sensor to detect hazardous chloroform by electrochemical depositing chitosan microgel in the form of thin film on the surface of copper electrode. Electrical testing was performed to observe the chitosan sensor’s response towards different concentrations of chloroform. It was observed that the output voltage of electrochemical sensor increased linearly with increasing chloroform concentration. Chitosan microgel sensor proved to be reliable sensor to detect chloroform in air and has additional properties including stability, recoverability and repeatability. Wen et al. [145] constructed a current generating biosensor for the detection of flammable toxic pollutant nitromethane by depositing chitosan-reduced graphene oxide microgels in the form of film on glassy electrode and immobilized the hemoglobin on the film. The prepared biosensor displayed excellent performance for the detection of nitromethane over a wide linear range from 5 μM to 1.46 mM. This biosensor had good reproducibility, selectivity and stability with a low detection limit (1.5 μM) of nitromethane. Applications and technical specification of chitosan based microgel composites in electrochemical/biosensors are illustrated in Table 5.

Table 5.

Electrochemical/biosensors applications of chitosan based microgels.

Microgel	Pollutant	Transduction method	References
Chitosan film	Chloroform	Electrochemical/voltammetry	[144]
Chitosan crafted ZnO nanoneedles	4-NP	Electrochemical/Differential pulse voltammetry	[143]
Chitosan-graphene oxide immobilized hemoglobin	CH3NO2	Electrochemical reduction/Amperometry	[145]
Plant esterase-chitosan/AuNPs-graphene nanosheets	Methyl parathion and Malathion	Electrochemical/Cyclic Voltammetry	[146]
Chitosan-graphene oxide	Sulfamethoxazole	Chemiluminescence	[147]
Chitosan capped AgNPs as fluorophore	Malathion	Fluorescence and absorption	[148]
Chitosan-graphene-CNTs	Methyl Parathion	Electrochemical/Cyclic Voltammetry	[149]

5.5. Agricultural applications

Agriculture is a vital part of the economy. Fertilizer and water are necessary for plant growth, but a lot of fertilizer and water are lost to surroundings and are not used fully for useful purpose, which is not environment friendly and economical. In arid and desert environment, the chitosan based microgels have gained significant attention due to slow release of fertilizer. The leaching is avoided by controlled release formulations of agricultural chemicals and the crop acquires nutrients slowly from soil leading to lower toxicity and little consumption of chemicals. Dhiman et al. [150] synthesized biodegradable CS based microgels by the combination of 2,3-dihydroxybenzoic acid, –SH functionalized chitosan and stearic acid via disulfide crosslinking for controlled release of insecticides and adsorption of heavy metal ions. The system had excellent loading capacity of insecticide (imidacloprid) which was found to release slowly from the polymeric network due to redox responsiveness of the microgel system. The microgel system was found as an excellent adsorbent for the extraction of heavy metal ions from the soil. Good binding ability of the system towards Fe⁺³ ions and excellent foliar adhesion and non-toxicity were additional features of the microgel system for their applications in agriculture.

In the past, large-scale famines and economic displacement have been caused by infections by pathogenic microorganisms, and these infections continue to cause large losses to agricultural crops. Brunel et al. [151] have solved this problem by using complexation of copper (II) with chitosan nanogels. The antifungal activity of Cu(II) loaded CS microgels was investigated using F. graminearum as a model system. The lowest concentration that will completely stop the microorganism’s visible growth after one week of incubation is known as the minimum inhibitory concentration (MIC), of a compound. It was observed that the MIC of Cu(II) ions loaded nanogel was 17 μg (mL)⁻¹ which was quite lower than Cu(II) ions solution i.e. 250 μg (mL)⁻¹. The exact phenomenon of antifungal activity of Cu(II) has not been explored yet. The physical and chemical structure of microgels (amino groups) provided an excellent stability to the Cu(II). Moreover, the Cu(II) ions unload from the polymeric network under acidic conditions because of the protonation of amino groups. Hence, the acidification caused by the pathogens resulted in the unloading of Cu(II) ions. The reported system has multiple advantages including, simpler handling, reduced viscosity, even dispersion on the leaves, and prolonged release of copper in the soil or on the leaves without compromising its antifungal qualities.

5.6. Biomedical applications

Chitosan based microgels and their hybrids are widely used in biomedical field due to their biocompatibility, external stimuli responsive behavior, controllable biodegradability, low cost, excellent biological and physiochemical properties. Biomedical applications of these microgels are summarized in Table 6.

Table 6.

Chitosan based microgels and their hybrids for various biomedical applications.

Microgel system	Drug	Properties	References
Poly(glycol chitosan)	Tannic acid	Drug loading capacity of microgels: 323.89 mg/g, and total amount of drug release was 42.56 ± 2 mg/g within 57 h	[15]
Chitosan-poly(aniline)	Vancomycin	Oral drug delivery, drug loading efficiency 91.3 % and collective drug release was about 76.9 %	[166]
Chitosan loaded SiO2 NPs	Vitamin-B12	Oral drug delivery system with controlled drug release in acidic medium	[16]
Chitosan-gelatin/PEG	Folic acid	Exhibit strong affinity with hydrophobic drug and controlled release at different Ph	[170]
Chitosan-alginate	Jughans regia L. polyphenols	Oral drug delivery: Showed excellent sustained release under gastrointestinal digestion	[105]
Chitosan-dextran	Vancomycin hydrochloride	Colon-specific drug release microgels showed loading capacity of drug 93.67 %	[168]
GMAChitosan-GMACoFe2O4	Vitamin-B12	Magnet-responsive microgels, exhibit controlled release of drug lasted for 50 min for the gastric wound treatment	[19]
Pectin coated-chitosan	5-fluorouracil	100 % encapsulation capacity, controlled release of drug in acidic medium for mucosal and topical treatment	[33]
Zn2+-Carboxymethyl chitosan-hyaluronic acid	Bovine serum albumin	Pulmonary drug delivery, sustained in vitro release in 24 h, 36 h retention time	[4]
Chitosan-genipin	SDF-1α TGFβ-3	Tissue engineering, exhibit in vivo complete degradation in 28 days	[171]
Chitosan-genipin	VEGF	Tissue engineering, endothelial cell proliferation	[172]
HTCC-microgels	Methotrexate disodium	93 % of drug was released in 24 h at pH =7.4	[158]
Chitosan-based microgels	Nifedipine	Drug dissolution and diffusion from microgels was well validated by applying mathematical models	[173]
SiO2 containing GOx and CeNPs @CS based microgels	Insulin and protein	Insulin was released in response to the change in concentration of glucose, the encapsulation efficiency of drug in the microgel was 60.5 ± 1.6 %.	[125]
Chitosan-acrylamide-g-poly(vinyl alcohol)	Cefadroxil	Drug release depends on the concentration of monomers and cross-linker	[174]

5.6.1. Drug delivery

Chitosan based microgels and their hybrids have gained a lot of attention in drug delivery due to their responsive behavior towards external stimuli (temperature, pH, magnetic field, light depending upon the microgel components), biodegradability, low cost, good mechanical strength and biocompatibility. A wide variety of drugs has been loaded into different chitosan based microgel systems and their release has been investigated in literature. Chemistry of the process of drug delivery using chitosan microgels is different in different cases. Therefore, this section has been further divided into various subsections:

5.6.1.1. Insulin delivery.

Chitosan based microgel systems may be used to load and release insulin for the treatment of diabetes (both type-I and type-II). Gu et al. [152] loaded insulin into pH responsive chitosan microgels containing glucose oxidase (GOx) and catalase (CAT) and investigated its self-regulating release from the microgel system. GOx present in the microgel system is an enzyme which acts as a catalyst for the oxidation of glucose into gluconic acid and H₂O₂. The formation of gluconic acid decreases the pH of the medium due to increase of concentration of H⁺ ions in the medium. The protonation of −NH₂ groups of CS (formation of positively charged $-\mathrm{N}{\mathrm{H}}_3^{+}$ in the microgel network) causes swelling in microgel system because of electrostatic repulsion between positively charged $-\mathrm{N}{\mathrm{H}}_3^{+}$ of the microgels. As a result, the insulin is released rapidly. Oxidation of glucose in the presence of GOx produces H₂O₂ which is toxic in nature. It deactivates GOx. Activity of the glucose oxidase is significantly affected by the H₂O_2. So, conversion of H₂O₂ into harmless product is mandatory. Catalase (CAT) is an enzyme which has ability to rapidly convert H₂O₂ into O₂ and H₂O. Water is harmless while the O₂ is needed for the oxidation of glucose. Catalase does not have a good stability. Therefore, rate of conversion of H₂O₂ into O₂ is low. Moreover, decline in catalytic activity under variation in the environment, difficulties in recovery and reusability and expensiveness are major drawbacks of the natural enzymes. However, the rate of conversion of H₂O₂ into water and O₂ can be increased by replacing the conventional catalase by artificial inorganic catalyst with high surface to volume ratio. Kim et al. [125] synthesized cerium oxide (ceria) nanoparticles to be utilized as an artificial inorganic catalyst as a replacement of catalase for rapid conversion of H₂O₂ into water and O₂ (Fig. 7). Mesoporous SiO₂ containing GOx and ceria nanoparticles were integrated into CS based microgels for insulin as well as protein delivery. The rate of release of insulin from the microgel system was regulated according to concentration of glucose.

Fig. 7.

Mechanism of insulin/protein release from chitosan based microgels integrated with mesoporous Silica (MCF) containing glucose oxidase (GOx) and Ceria nanoparticles (artificial inorganic catalase) via glucose induced pH responsiveness of the hybrid system.

(Reproduced with permission from ref. [121]. Copyright © 2017 American Chemical Society.)

Yin et al. [153] designed glucose sensitive self-regulating insulin delivery system based on microgels of the derivative of CS (glucosyloxyethyl acrylated chitosan) and concanavalin A crosslinked by genipin. Chitosan derivative acts as ligand for concanavalin A and can immobilize concanavalin A without any modification. Rate of in vitro release of insulin from the microgel system was found dependent on glucose concentration. No destruction in the structure of insulin was observed during the loading and release process. The system has no cytotoxicity and may be a suitable one for self-regulating insulin delivery. Some other CS microgels based insulin delivery systems may be found in literature [154–157].

5.6.1.2. Cancer therapeutics.

CS based microgels have gained attention in cancer therapeutics due to control over their size, bioconjugation ability, biodegradability, biocompatibility, low toxicity, stimuli responsiveness and controlled drug release. These features of the CS based microgels make them suitable for targeted drug delivery with minimum side effects and enhanced efficiency. Zhang et al. [158] obtained highly monodisperse CS based microgels with diameter of 180 nm from N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC) using Sodium tripolyphosphate (TPP) as crosslinker via ionic association between HTCC and TPP (Fig. 8).

Fig. 8.

(a–b) Bioconjugation of CS based microgels, (b–c) Drug (MTX) loading into Bioconjugated microgels and (c–d) pH triggered drug release from Bioconjugated microgels.

(Adopted from ref. [158] Copyright © 2006 American Chemical Society.)

Bioconjugated recognition molecules of apo-transferrin were attached with the surface of the microgels by stirring of aqueous solution of apo-transferrin and dispersion of microgels in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) in phosphate buffered saline (PBS) for 2 h under physiological pH conditions (pH = 7.4). Microgel particles were found stable at pH = 7.40. An anticancer drug, methotrexate disodium (MTX) was loaded into the HTCC microgels via electrostatic attraction between negatively charged drug and positively charged HTCC microgel system by simple mixing of dispersion of drug at microgel dispersion in PBS at pH = 7.4 under various concentrations of the drug. The loading capacity was found to increase with the increase in MTX concentration due to increase in difference of concentration of drug in the bulk and in the interior of the polymeric network (concentration gradient) which is responsible for mass transport by diffusion. The in vitro cumulative release of MTX from the microgel system as function of time under physiological temperature at two different values of pH (7.4 and 5.0) in PBS buffer solution was investigated. 66 % of drug was released from HTCC system within one day and about 30 % of MTX was not released from the microgel system due to strong ionic attraction and hydrogen bonding among the drug and polymer functionalities. But rate of release of drug from the microgel system was much higher at pH = 5.0 in comparison to that at pH = 7.4 and about 93 % of MTX was released from the microgel system within 24 h while 95 % release was achieved in three days because at pH = 5.0, drug gets partly protonated and has positive charge. As a result, interaction of MTX with positively charged polymer functionalities becomes weaker. Moreover, swelling of polymeric network due to electrostatic repulsion between positively charged amino groups (Fig. 8) facilitates the diffusion of drug from interior of the microgel to the bulk region. Microgels are localized in the lysosome or late endosomes via receptor mediated endocytosis after entering the cells because the pH values of these organelles lie in the range of 5–6. Sorafenib is a low molecular weight anticancer oral drug for treatment of cancer but it is poorly soluble in water in the pH range of 1.2–7.4 which reduces its bioavailability and therapeutic efficacy. Caputo et al. [159] synthesized a variety of microgels using different combinations of monomers and chitosan for loading and release of sorafenib. The microgel system obtained from chitosan, N-isopropylmethacrylamide, acrylamide and BIS were found more appropriate for successful loading and release of the drug. The protocol for incorporation of the drug into the microgels was optimized. About 40 % loading efficiency was achieved using aforementioned microgels as carriers. >10 % of the drug entrapped in the microgel system was released within 16 h. Puga et al. [33] prepared chitosan microgels loaded with 5-fluorouracil by using super hydrophobic surface-based encapsulation technology for optical chemotherapy and oral drug delivery. The encapsulation efficiency of microgels with diameter range from 200 μm to 600 μm for 5-fluorouracil an antitumor drug was found to be 100 %. The rate of drug release was higher at pH 7.4 than at acidic pH, exhibiting pH dependent rate of drug release. Pectin coating of microgels on the super hydrophobic surfaces enhanced the controlled-release of 5-fluorouracil in lower pH and premature leakage of the drug was also prevented and potentially useful for colorectal tumors and skin treatment.

All studies related to loading and release of anticancer drug using CS based microgels as carriers cannot be covered in this short report. Therefore, readers are referred to the relevant literature [160–162].

5.6.1.3. Antibiotic drug delivery.

CS based microgels can also be used for site-specific delivery of antibiotic drugs. Vancomycin hydrochloride (VMC) is an antibiotic drug which is widely used for the treatment of bacterial infection in colonic region of intestine. Loading of VMC into the CS based microgels and its release from the microgel system has become the subject of interest [163–165] due to easy monitoring of release of VMC by UV–Vis spectrophotometry as the value of wavelength of maximum absorption is 280 nm.

Li et al. [166] synthesized chitosan-poly(aniline) microgels using glutaraldehyde as a crosslinker. The prepared microgel system was treated with NaCl for the charge reversal (from positive to negative) to load cationic vancomycin via electrostatic attraction. The reversal of charge was achieved by attachment of Cl⁻ ions coming from ionization of NaCl with polyaniline segments of the microgels. The lysozyme-triggered vancomycin release from vancomycin-loaded N-chitosan-poly(aniline) microgels for the treatment of inflammatory bowel disease was investigated. It has been reported that the inflamed intestine has specific micro environment containing lysozymes with pH = 6.8 [167] which is suitable for enzymatic hydrolysis of the lysozyme-cleavage of 1,4-β-glycosidic bonds of the CS backbone. Therefore, degradation of the microgel system loaded with VMC in the presence of lysozymes at pH = 6.8 was confirmed by taking the TEM images at different times and by measuring the hydrodynamic diameter of the microgels under said conditions. The microgel system was completely decomposed within two hours. The cumulative rapid release (76.9 %) of vancomycin in the simulated inflammatory intestinal microenvironment was observed. Extraordinarily, the microgels loaded with VMC do not only avoid the early leakage of the drug in the gastrointestinal region but also show resistant to severe gastric acidity. Li et al. [168] synthesized a series of pH responsive biocompatible and biodegradable azide-modified dextran and alkyne-modified chitosan microgels in the presence of Cu(II) as crosslinker. At various pH values, the microgels show polyampholyte behavior and can carry negative or positive charges in aqueous solution. Moreover, the microgels revealed high encapsulation capability about 93.67 % of an antibiotic vancomycin hydrochloride through electrostatic interactions under negatively charged state of the microgel systems. The microgels were degradable in pH >9.0 due to hydrolysis of carbonate esters. They were also degradable in the presence of Dextranase due to cleavage of 1,6-α-glucosidic linkages of dextran segments of the network for maximum release of VMC from the system at pH = 6.8. The degradation of the polymeric network under micro environment of colon region of intestine as a function of time was confirmed by DLS, FTIR, proton NMR and TEM analysis. US Food and Drug Administration (FDA) has approved tannic acid (TA), a polyphenolic substance as food additive due to its antibacterial, antiviral, anti-oxidative and anticancer activities [169]. Sahiner et al. [15] loaded TA into glycol chitosan (TA@P(GCS)) microgels crosslinked by divinyl sulfone. The loading and releasing behavior of tannic acid was studied. The tannic acid loading capacity of poly(glycol chitosan) microgels was found to be 323.89 mg/g. Within 9 h, the release of TA from P(GCS) microgels was linear and the total quantity of tannic acid (42.56 ± 2 mg/g) was released within 57 h.

5.6.1.4. Oral drug delivery.

CS is a natural polysaccharide with minimum toxic effects. So, CS based microgels and their hybrids can be used as carriers for oral drug delivery. Pellá et al. [19] synthesized magnetic chitosan microgels loaded with GMA modified CoFe₂O₄ NPs. The drug release behavior of prepared hybrid microgels in the presence and absence of external magnetic field was investigated using vitamin-B12 as a model oral drug. In the absence of magnetic field, a fast release of drug was observed at pH 7.4 and the equilibrium was achieved after 30 min. While in the presence of magnetic field, a two-step drug release was observed at pH 7.4 in first 30 min a higher release rate of drug was observed, accompanied by a more controlled slower release step that continued for 50 min. The slower release of drug in second step was due to the presence of magnetic field which promoted the high degree organization of chains of microgels because of GMA modified magnetic CoFe₂O₄ NPs which affect the swelling dynamic of microgels by aligning itself in the direction of magnetic field. So, in the presence of magnetic field, the controlled drug release behavior of these hybrid microgels make them a potential candidate for treatment of stomach and intestine ulcers. This kind of release is significant for gastric wounds treatment, which is enhanced by the existence of folic acid in the microgels.

5.6.2. Tissue engineering

The objective of tissue engineering is regeneration of unhealthy or injured tissues by linking bioactive molecules and cells spread into a scaffold or support material. The usage of chitosan based microgels in tissue engineering is beneficial because of three-dimensional arrangement of cells, bearing required mechanical integrity to support nutrient diffusion and developing new tissues to encapsulated cells. Shen et al. [175] synthesized hydroxyapatite-coated chitosan microspheres for osteogenesis promoting microgels. These microspheres are injectable and have the ability to controll degradation. Coating of hydroxyapatite increased cellular proliferation and promoted the differentiation, proliferation and attachment of pre-osteoblastic MC3T3 cells. Li et al. [176] modified chitosan with N-methacryloyl to form UV crosslinkable and soluble in neutral pH aqueous environment. The pre-osteoblastic MC3T3 was encapsulated into modified chitosan based microgels using photolithography techniques and 90 % cell viability was maintained. Mice retaliated with insignificant inflammation, in vivo. Riederer et al. [172] prepared chitosan-genipin microgels by emulsion crosslinking method. The aggregation of microgels was observed at pH 7.4, the microgel could encapsulate the both negatively and positively charged VEGF and stimulated the endothelial cell proliferation. Jin et al. [177] synthesized microgel consisting carboxymethyl chitosan, collagen and sodium alginate. The prepared microgel system was utilized as a hemostatic agent because of their biocompatibility, stability, biodegradability and injectability. It was observed that the microgels improved healing efficiency and stop bleeding by increasing the clotting and wound closure rate without any side effects.

5.6.3. Antibacterial activity enhancement

It has been observed that CS itself has antibacterial activity. However, the mechanism of its antibacterial activity is not clear. But CS based microgels can be used for loading and controlled release of antibiotic drugs. Moreover, CS based microgels can also enhance the process of antibacterial activity. Gong et al. [165] has designed CS based pH-sensitive microgel system with photo-thermal therapy (PPT) assisted antibacterial activity. Oxidative polymerization method was used to obtain co-polymeric system made of CS and polyaniline (PANI) which was further used as backbone in inverse emulsion method for synthesis of CS-PANI microgels crosslinked by glutaraldehyde. Negatively charged CS-PANI microgels were obtained by treating CS-PANI microgels with Na₂SO₄ solution for high loading of cationic antibiotic (vancomycin). Negatively charged CS-PANI microgels loaded with vancomycin was efficiently released from the microgel system in the presence of lysozyme. It was observed that antibacterial activity of the system against drug resistant E-Coli can be significantly enhanced with PPT assistance by rupturing the bacterial membrane using laser light of wavelength of 808 nm. The antibiotic can easily penetrate through broken membrane to kill the bacteria. The same route of antibacterial activity using chitosan based microgel system has been also reported by Li et al. [178]. Chitosan based microgels cannot only be used for the enhancement of antibacterial activity of the antibiotic loaded into the microgel system but some metal nanoparticles known for their antibacterial activity can also loaded into the CS based microgel system for investigation of their catalytic activity. Fan et al. 179 loaded silver ions and chitosan into the sulfonated chitosan via electrostatic attraction and then silver ions were reduced into silver nanoparticles using NaBH4 as reducing agent to obtain hybrid microgel system with excellent bacterial activity. The pH dependent slow release of silver ions from the hybrid system was responsible for long lasting antibacterial activity. Ramalingam et al. [126] have also reported CS based core shell microgel with Fe₃O₄ magnetic core and CS shell containing silver nanoparticles for bacterial decontamination of aqueous medium. The hybrid system had potential to eliminate bacterial contamination without production of any harmful chemicals which are generally produced during the ordinary wastewater treatments (chlorination or ozone treatment). George et al. [94] used agar well diffusion method to investigate the antibacterial activity of Ag-P(CS-AAc) hybrid microgels against waterborne S. aureus (Gram-positive) and E. coli (Gram negative) bacteria. The activity of Ag-P(CS-AAc) hybrid microgels against S. aureus was much higher than that against E. coli because membrane of S. aureus is thinner than that of E. coli. Ag⁺ ions released from Ag nanoparticles penetrate into the bacterial cell through membrane to inactivate the bacterial enzymes.

5.7. Emulsion stabilization

An emulsion is a liquid in a liquid colloidal system. Emulsion is a mixture of two immiscible liquids that is created by dispersing liquid droplets in another liquid in the presence of an emulsifier. Emulsions, whether in the form of oil-in-water (O/W) or water-in-oil (W/O), are crucial to the petrochemical, pharmaceutical, food, and personal care industries [180]. Conventional emulsions are usually stabilized using surfactants; however, this had a number of negative effects, such as recovery difficulties and environmental issues. The particle-stabilized emulsion system is termed as Pickering emulsion. Therefore, solid particles have garnered significant attention recently as potential replacements for conventional emulsifiers due to their exceptional resistance against coalescence, low toxicity, and low cost [181]. Among these biocompatible Pickering emulsifiers, microgels have gained a lot of attention because of their smart behavior and eco-friendly nature [182,183]. Chitosan has emulsifying ability because of the amino and acetyl groups on the polymer backbone. These groups can partially adsorb onto the oil-water interface, creating steric and electrostatic barriers that prevent droplet coalescence. Many researchers have reported CS-based microgels for the stabilization of emulsions, with a slight modification [92,184–186]. Wang et al. [187] have prepared pH and thermo-sensitive chitosan grafted poly(N-isopropylacrylamide) (CS-g-PNIPAM) microgel particles and utilized them for the stabilization of emulsion, the resulting emulsion was also sensitive to the change in pH and temperature of the medium. A biphasic system was prepared by mixing the solution of isooctane and water. This biphasic solution turned into a Pickering emulsion (O/W) upon the addition of CS-g-PNIPAM particles. It was noted that in the absence of microgels, two phases immediately recovered after mixing. The effect of concentration of microgels on the stability and nature of emulsion was also investigated. The average diameter of droplets of emulsion decreased from 32 μm to 11 μm with an increase in the microgel concentration from 0.5 to 3.0 wt%. The emulsion was stable under acidic conditions, due to the hydrophilic interaction of protonated CS moieties. Under alkaline conditions, pH > 7.0, microgels quickly began to aggregate from the water phase, leading to a poor emulsion fraction and macroscopic phase separation. The droplet size became oversized, uneven, and deformed. The effect of temperature on the stability of emulsion at pH = 6 was also studied. At 25 ° C, below the VPTT of NIPAM, the emulsion was stable. With an increase in temperature (T > VPTT of NIPAM), the emulsion started to deform. At 40 °C, two separate phases were obtained due to the shrinkage and disruption of microgel particles. The Pickering emulsion was further used as a biocatalyst for hydrolysis of fish oil and the esterification of oleic acid with n-octanol at oil/water interface. Charisis and co-worker [188] have reported chitosan-coated alginate microgels and their abilities for emulsion stabilization. The emulsion of sunflower-oil in water (O/W) was successfully obtained using CS based microgels as emulsifiers. With an increase in pH from 3 to 7, the size of droplets increased, however emulsion remained stable for 7 days at temperature 25 ± 1 °C.

Chitosan-carrageenan (CS-CG) microgels having lipophilicity have been reported as a suitable candidate for the stabilization of W/O Pickering emulsion [189]. The reported system was responsive to both the change in pH and/or temperature of the medium. A series of CS-CG microgels having different mass ratio of monomers was prepared. All the microgels were claimed as effective stabilizers for emulsion for 24 h of observation. The emulsion was stable under acidic medium (pH = 4.5, 6.5) at room temperature. Under alkaline medium, even at a mild basic pH = 7.5, 8.5, the stability of emulsion was drastically decreased. Two separate phases were observed after 3 h because of disturbed balance of the hydrophilicity and hydrophobicity of CS-CG microgels. Also, when the temperature was increased from 30 to 40 °C, destabilization in the emulsion was observed due to the synergistic effect of heat-induced deprotonation of CS and coil-to-helix transformation of CR at T ≥ 40 °C.

6. Conclusion and future perspective

Chitosan based microgels and their hybrids have obtained considerable attention due to their biocompatibility, low cost, biodegradability, responsive behavior towards external environmental stimuli like pH, temperature, glucose, light and magnetic field according to their feed compositions. Chitosan has ability to be modified and can be combined with a wide variety of materials (both inorganic and organic) by either physical or chemical crosslinking to develop multi-responsive chitosan based microgels for numerous applications like stabilization of inorganic nanoparticles, catalysis, adsorption of heavy metal ions/dyes, slow release of fertilizers, drug delivery and tissue engineering. Precipitation polymerization, spray drying, microfluidic fabrication are extensively used techniques for the preparation of chitosan based microgels. Chitosan based hybrid microgels fabricated with metal nanoparticles are generally synthesized by reduction of metal ions within dispersion of microgels using appropriate reducing agent.

We are hopeful that chitosan based microgels will attain more and more consideration in future because of their special properties including biodegradability, biocompatibility, low toxicity, diverse physical and chemical interaction with other materials, bio-inspired functionality, high mechanical strength, non-toxicity of its degradation products and pH responsiveness. Control over the CS microgel size and its distribution for efficient applications is still challenging and may be key part of the future studies in this fascinating area of research. Metal nanoparticles fabricated in CS based microgels reported in literature were not found perfectly spherical. The techniques to achieve narrow size distribution of inorganic nanoparticles may be discovered in future work. The fabrication of different metal nanoparticles within chitosan based microgels and their catalytic applications have not been explored in detail in literature and may be the subject of future works.