A simple and user-friendly protocol for chitosan nanoparticle synthesis

Abstract

Despite the widespread use of chitosan nanoparticles (CNPs), a simple, cost-effective, and reproducible synthesis protocol remains a critical unmet need. Existing protocols for ionic gelation methods are often laborious, requiring overnight stirring, costly filtration, and time-consuming lyophilization. In this study, we present a novel, easy-to-adopt, cost-effective, scalable, and highly reproducible protocol for synthesizing CNPs via ionic gelation, bypassing these common drawbacks. Our method standardizes the use of low molecular weight chitosan (0.1%) stabilized with Tween 80 in 1% acetic acid solution, crosslinked with sodium tripolyphosphate (STPP) in 3:1 volume ratio to form CNPs. The CNPs are efficiently separated using simple centrifugation, eliminating the need for complex and expensive lyophilization. The nanoparticles obtained were systematically characterized for their physicochemical and structural properties, including particle size, zeta potential, polydispersity index, morphology, functional groups, crystallinity, and elemental composition, using a wide range of analytical techniques such as UV–Vis spectroscopy, Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), Energy-Dispersive X-ray Analysis (EDAX), Atomic Force Microscopy (AFM), and High-Resolution Transmission Electron Microscopy (HRTEM). Comprehensive characterization of synthesized CNPs consistently demonstrated the formation of well-defined, spherical amorphous nanoparticles within the nanometer range, exhibiting a positive surface charge, presence of functional groups, and desirable elemental composition. The protocol’s simplicity, low cost, scalability, accessibility, and reproducibility of the synthesized CNPs make it a significant advancement for researchers in various fields. Given their inherent biocompatibility and functional versatility, these CNPs are highly promising for a wide range of applications, including antimicrobial coatings, food preservation, water treatment, drug delivery, and sustainable agriculture.

Introduction

The advancement in nanotechnology has extended the application of nanomaterials in every sectors of the society. Even though, various nanoparticle formulations are available, most of the formulations are of metal nanoparticles and are toxic in nature. Biodegradable polymers such as chitosan offer eco-friendly, biodegradable, non-toxic and renewable alternative. CNPs are the biopolymers derived from chitin, which has emerged as a viable alternative that offers several benefits for sustainable agricultural practices [1]. CNPs are on the forefront and attracted wide interest due to their versatile physicochemical characteristics such as biodegradability, biocompatibility, and non-toxicity, which plays a major role in biological applications [2]. It provides a multifaceted approach to plant growth promotion, operating across various spatial and temporal scales throughout the plant life cycle. The potential of CNPs as a sustainable option in agricultural production has already been reported [3]. The application of CNPs increased the rate of photosynthesis, induced root nodulation, upregulated nutrient uptake, enhanced the rate of germination of seed, and boosted the plant vigour [4]. CNPs have been used as an effective encapsulation matrix for biofertilizers, as they preserve the structure and thereby enhance bioactivity of microbial inoculants. Besides that the amino (–NH₂) and hydroxyl (–OH) groups of the polymer give high reactivity and charge, improving its capacity for encapsulation [5]. When the mechanical and barrier qualities of biodegradable plastic matrixes need to be improved, CNPs could be utilized as a filler material [6]. Rajitha et al. (2016) reported that CNPs has been used for controlled and continuous drug release because of its slow biodegradation [7]. Encapsulation of drugs with CNPs helps to increase drug efficiency, specificity and accessibility to the target site while minimizing toxicity and side effects [8]. CNPs have been used in waste water treatment to enhance the removal of toxic dyes and heavy metals [9]. Esyanti et al. reported that coating of banana with CNPs reduced post-harvest ripening [10]. Some of the applications of CNPs is shown in Fig. 1.

Fig. 1.

Applications of chitosan nanoparticles

There are several physical, chemical, and green synthesis methods for the preparation of CNPs (Fig. 2) [11]. One simple chemical method is ionic gelation [12], in which a negatively charged compound, like tripolyphosphate (TPP) is used as a crosslinking agent to form polymeric networks of CNPs [13]. Ionic gelation offers a simple and highly controllable approach for the fabrication of chitosan nanoparticles, with the major advantage of avoiding extensive use of organic solvents and toxic reagents [14–16]; apart from that, the size and zeta potential of CNPs can be easily controlled by adjusting the concentration of chitosan and TPP, chitosan to TPP ratio, pH, etc [17].

Fig. 2.

Some important physical and chemical synthesis methods of chitosan nanoparticles

The protocols for synthesis of CNPs are many but none of which gave a good yield. Despite its advantages, optimizing the synthesis of CNPs remains one of the challenges. Synthesis methods are many, however reproducible, scalable, simple, and low cost protocols are lacking in literature. Existing protocols on ionic gelation methods are often laborious, requiring overnight stirring, costly filtration, and time-consuming lyophilization. Bekmukhametova et al. [18] stated that, it is still challenging to produce a reliable protocol for the fabrication of CNPs. Hence, a study was undertaken to standardize a simple protocol for CNPs synthesis by ionic gelation method which is cheap, less time consuming, user-friendly, reproducible, and would give comparatively higher yield.

Methods

Synthesis of chitosan nanoparticles

CNPs were synthesized by ionic gelation method (Fig. 3), the complexation of polyelectrolyte between positive charge of chitosan and negative charge of sodium tripolyphosphate (STPP) [19], by following modification in procedure described by Des Bouillons-Gamboa et al. [20]. Low molecular weight chitosan of 300 mg (Sigma-Aldrich) was dissolved in 300 mL of 1% acetic acid solution (3 mL of glacial acetic acid in 300 mL of distilled water) to obtain 0.1% chitosan solution. The pH of the chitosan solution was 2 initially which was increased to 5.5 by using 10 N sodium hydroxide solution followed by homogeneous mixing for 30 min at 40 °C using a magnetic stirrer (1000 rpm).

Fig. 3.

Flowchart illustrating the synthesis of chitosan nanoparticles using the ionic gelation method

The temperature of the magnetic stirrer was then reduced to 25 °C, and 30 µL of Tween 80 was added as a stabilizing agent, followed by 10 min of stirring. Subsequently, 1% STPP solution (1 g of STPP in 100 mL of distilled water) was added drop wise to the chitosan solution in 3:1 ratio (3 parts of chitosan solution to 1 part of STPP solution) while being stirred on the magnetic stirrer. The mixture was stirred for one hour to obtain CNPs suspension. Change in colour of suspension from colourless to off-white indicates the formation of CNPs.

The nanoparticle suspension was kept undisturbed for 30–60 min for settling of the nanoparticles. After incubation at room temperature, the suspension was centrifuged at 10,000 rpm for 10 min to collect the pellets of nanoparticles formed. The pellet was washed twice with distilled water in centrifuge at 10,000 rpm for 5 min to remove any impurity. The washed pellet was spread in a Petri dish using stainless steel spatula and dried in a hot air oven at 60 °C for 24–48 h. The dried pellets were ground to fine powder using a mortar and pestle. The CNPs formed were stored at 4 °C for further studies.

Characterization of chitosan nanoparticles

1. UV visible spectroscopy

The CNPs were dispersed in distilled water and sonicated using ULTRA probe sonicator for 30 min followed by UV spectroscopy characterization. UV–visible spectra of CNPs were recorded using Shimadzu UV–visible 1800 spectrophotometer at Nano cell, Dept. of Plant Pathology, College of Agriculture, Vellanikkara, Thrissur for the confirmation of chitosan nanoparticles, as their formation was verified primarily through hydrodynamic diameter measurements. The CNPs suspension was scanned in the range of 200–600 nm range with distilled water as reference [2].

• b.Dynamic light scattering (DLS)

2 mg of CNPs was dispersed in 5 ml of distilled water followed by sonication using Sonics vibra cell sonicator (bath sonicator) for 30 min at 25 °C [2]. Particle size, zeta potential and polydispersity index of CNPs was performed using Zetasizer (Malvern Instruments, U.K) at Agri Business Incubator, Dept. of Agriculture Engineering, College of Agriculture, Vellanikkara, Thrissur with distilled water as reference.

• c.Scanning electron microscopy (SEM)

The size and the morphology (shape) of oven dried CNPs were examined under Carl Zeiss (EVO 18) SEM [21] at CSIR-NIIST, Thiruvananthapuram. A small amount of CNPs samples were kept on an SEM stub using double-sided adhesive tape at 50 mA current for 6 min through sputter. Sputter coating was done with Au-Pd alloy in sputter coater, initially for 150 s followed with 120 s and 12 mA current. Afterwards, the stub containing the sample was placed in the SEM Chamber. The photomicrograph was taken at an acceleration voltage of 20 KV.

• d.Fourier transfer infrared (FTIR) spectroscopy

FTIR analysis of CNPs sample was performed [2] with PerkinElmer Spectrum Two FT-IR Spectrophotometer at Radio Tracer Laboratory, KAU, Thrissur. Sample spectra were recorded in the middle infrared range from 4000 cm⁻¹ to 400 cm⁻¹ with a resolution of 4 cm⁻¹ in the transmittance mode for 10 scans at room temperature to obtain an overview of the chemical composition of the prepared CNPs. FTIR spectra of CNPs were obtained by placing 1 mg of samples (chitosan, STPP and CNPs respectively) on the sensor of the instrument. The spectrum generated for the CNPs was subsequently compared with those of chitosan and STPP.

• e.X-ray diffraction

X-ray diffraction (XRD) analysis of chitosan and CNPs were conducted [22] using a Malvern Panalytical Aeris diffractometer with Cu-Kα radiation (K-Alpha1 wavelength − 1.540598 Å, K-Alpha2 wavelength − 1.544426 Å, and Ratio K-Alpha2/K-Alpha1–0.5) at 40 kV and 15 mA at Department of Physics, St. Thomas College, Thrissur. Finely powdered samples were smeared over a low background sample holder (amorphous silica holder) and fixed on the sample stage on the goniometer. The relative intensities were recorded within the range of 5⁰–80⁰ (2θ) at a scanning rate of 5° min⁻¹.

• f.Energy dispersive (EDAX) X-ray analysis

Elemental analysis of chitosan, STPP and CNPs were done [23] with the Joel 6390LA/ OXFORD XMXN, equipped with Oxford X-Max N Detector from Sophisticated Test and Instrumentation Centre, Cochin University of Science and Technology, Kochi. 1–2 mg of dry samples was smeared over multisided adhesive carbon tape fixed on specimen stubs. The stub was then placed into SEM sample holder. Elemental analysis of the samples were obtained using Aztec software (EDAX acquisition settings - EDAX resolution 136 eV, EDAX detector area 30 mm², accelerating voltage – 20 kV).

• g.Atomic force microscopy (AFM)

High resolution imaging and structural analysis of chitosan and CNPs were done using MultiMode 8-HR AFM [20] from CSIR-NIIST, Thiruvananthapuram. The samples were dispersed in deionized water to an appropriate concentration and ultrasonicated for 15–30 min. 10 µL of the suspension was spread evenly on a clean, ethanol washed, air dried glass piece (1 m x 1 m) by drop casting followed by drying in dessicator. The prepared glass piece was mounted on AFM stage followed by loading of Antimony (n) doped silicon cantilever. The imaging of the samples was done followed by analysis using Nanoscope analysis software.

• h.High-resolution transmission electron microscopy (HRTEM)

The chitosan and CNPs were imaged using JM-2100 transmission electron microscope (JEOL Ltd.) [24] at 200 kV from Sophisticated Test and Instrumentation Centre, Cochin University of Science and Technology, Kochi. Samples were made into slightly turbid suspension with minimal amount of sample in water/ethanol followed by ultra-sonication to evenly distribute the sample in solvent. A drop of suspension (approx. 5 µl) was then drop-casted using a pipette onto carbon-coated copper grids of 300 mesh and dried at room temperature. The grid was then fixed on the sample holder inserted into Jeol JM 2100 instrument followed by capturing the image using the Gatan camera.

Results and discussion

Nanotechnology can be applied to natural polymers such as chitosan by changing to nanoparticle size so that it will increase absorption which will expand the surface of the chitosan [25]. Chitosan is a natural polysaccharide, produced by the alkaline deacetylation of chitin, which possesses excellent characteristics, including low toxicity, low cost, biodegradability, biocompatibility, environmental non-toxicity, and adsorption abilities [26, 27]. There are several physical, chemical, and green synthesis methods for the preparation of CNPs, among them ionic gelation method is reported as the best. CNPs synthesized by high-intensity ultrasonication induced considerable damage on the CNPs and were not spherical, instead an irregular shape with branching arms were formed which could affect its overall function [28]. Zhang et al. prepared CNPs using ultrafine milling method. However, they were not able to reduce the size of particles below 350 nm [29]. Even though chitosan nanofibers are prepared using electrospinning, the method has some drawbacks, such as the high electrical potential, which can impact directly on scale-up limitation [30]. The microemulsion method can generate CNPs with a narrow size distribution, but large quantities of organic solvent must be used [31]. The major disadvantages of CNPs synthesized by emulsification solvent diffussion include the high shear forces required during nanoparticle preparation and the use of large quantities of organic solvents [32]. Compared to other emulsion-based methods, the reverse micellar method has the advantage of producing ultrafine nanoparticles of around 100 nm or even less, with a narrower size range. Nevertheless, disadvantages such as the difficult isolation of nanoparticles and the need for larger amounts of solvent, have been reported [33]. CNPs by covalent crosslinking method involve the formation of covalent bonds between chitosan or its derivatives and the functional cross-linking agent such as glutaraldehyde which is known to be toxic [34]. The main limitations of green synthesis of CNPs are the lack of size uniformity, low stability, and poor reproducibility due to variations in biological precursors [35]. Currently the most widely used synthesis of CNPs is through ionic gelation, a bottom-up process whereby an anionic crosslinker such as sodium tripolyphosphate (TPP), is leading to the self-assembly of chitosan and TPP into CNPs. Although several anionic crosslinkers like glutaraldehyde (which is toxic) can be used, the favourable properties of TPP, including biocompatibility and biodegradability, make it a better crosslinker [36]. Ionic gelation, which crosslinks polymer units via electrostatic interactions with an oppositely charged molecule, is a widely used method for the manufacture of CNPs [37]. STPP is a safe material approved by the Food and Drug Administration (FDA), commonly used in the synthesis of nanoparticles by the ionic gelation method as a crosslinking agent [38]. According to Chandrasekaran et al., the ionic gelation method is superior for CNPs synthesis because it avoids toxic crosslinking agents and yields well-defined nano-sized CNPs [39]. Koukaras et al. reported that the CNPs formation by ionic gelation method is due attraction of positive and negative charges [40]. Typically, cations and polyanions are released when chitosan and STPP are dissolved in acetic acid and distilled water, respectively. When STPP is added dropwise to the chitosan solution, the polyanions (negatively charged) interact electrostatically with the amino groups (positively charged) of chitosan, inducing gel ionization and leading to the formation of CNPs (Fig. 4) [41]. The primary interactions in ionic crosslinking configuration are H-link and T-link. The H-link is interaction of O⁻ and NH₃⁺ in the same plane, while the T-link is interaction of nonbrinding oxygen atom and NH₃⁺ in different plane (Fig. 5) [40]. Rochima et al. also reported that the chitosan has a positive charge which will bind with negatively charged STPP, then break the size of the chitosan into smaller ones without changing the functional group of the chitosan itself [42].

Fig. 4.

Schematic representation of ionic crosslinking of chitosan with tripolyphosphate [43]

Fig. 5.

Illustration of ionotropic gelation depicting electrostatic interactions between chitosan and TPP, highlighting the H-link configuration (same-plane interaction) and the T-link configuration (cross-plane interaction) [44]

Synthesis parameters including homogenization time and speed must be carefully tuned, while parameters such as pH and ratio of chitosan to TPP may need to be considered to avoid aggregation and excess growth of the particles [45, 46]. The first demonstration of CNPs synthesis was performed by Calvo et al. [47], focused on the ratio of chitosan: TPP as a control of particle size and dispersity. They reported that the formation of CNPs was possible only for some specific concentrations of chitosan and STPP. We used low molecular weight chitosan (0.1%) crosslinked with STPP in 3:1 volume ratio resulted in effective CNPs formation, suggesting that this specific ratio is appropriate for CNPs synthesis. Chitosan converted to CNPs tends to stick together and form agglomerates; therefore, a surfactant must be added to disperse the CNPs and prevent agglomeration. Sektiaji et al. reported the use of Tween 80 as a surfactant to prevent agglomeration of CNPs [48]. During the incubation, the CNPs formed precipitated at the bottom of the container leaving a clear supernatant. In the study we derived a protocol in which the overnight stirring of chitosan solution [2, 49, 50] is reduced to just 1 h stirring. Placing of chitosan solution in ice before addition of STPP [11] is excluded in our study. Sonication of the chitosan solution and usage of deionized water in protocol explained in Ali et al. [22] is excluded in our protocol. Only stirring of the chitosan solution using magnetic stirrer was adopted in this study. Instead of deionized water which is costly and difficult to obtain we have used double distilled water which was easily available in most of the labs. Lyophilization is a widely used technique for removing moisture and preserving nanoparticles for extended periods, thereby improving their physicochemical stability. However, the high susceptibility of CNPs to environmental factors and process conditions, such as freezing, can impose stress on its structure and cause polymer degradation [51]. In some cases, it may even be difficult to achieve complete redispersion after lyophilization due to aggregation or irreversible melting of the nanoparticles [52]. Thus, the addition of a suitable cryoprotectant at optimal concentrations prior to freezing is necessary, which is formulation specific for polyelectrolytes [53]. The lyophilization step for separation of CNPs from CNPs-based suspension [2, 20, 54–56] is costly as well as time consuming and is substituted with oven drying in our study which is comparatively cheap. The filteration of solutions [2, 49, 57, 58] is not followed in the study. None of the solutions are filtered in this study. The particle size is also affected by the reaction time. On one hour stirring, the chitosan-based solution changed color from colorless to off-white colloidal suspension, indicating the formation of CNPs. The smallest CNPs size were reported to be obtained at 1 h but larger particles were obtained when the reaction time exceeded 60 min [59] which is supporting our protocol. Different pH levels were used but the CNPs formation occurred only when the pH of chitosan solution reached 5.5. Studies suggest that changing the pH can effectively prevent aggregation and reduce particle development [46]. Han et al. reported that the addition of STPP to chitosan solution near pH 5.0 results in the formation of nanosized CNPs, which is in accordance with our results [60]. We further noted that when the amount of Tween 80 was increased beyond 30 µL, the strength of CNPs pellets increased and was very difficult to grind to powder form. It is in agreement with Akdasci et al. [61], they stated that through changing the proportion of chitosan to stabilizing agent, it is possible to alter the nanoparticles size. There is effect on CNPs on storage temperature, at elevated storage temperature, the particle size was reported to decrease due to polymer degradation and mass loss [62]. No significant change in particle size was observed when stored at 4 °C upto 12 months [52]. So we have stored CNPs at 4 °C for further studies.

However, this widely used method is prone to uncontrollable intra and intermolecular crosslinking between chitosan and STPP, resulting in aggregation, excess growth, and high polydispersity of CNPs [52, 63]. The main disadvantage of CNPs synthesised by ionic gelation method tend to agglomerate soon [64]. This process of swelling and aggregation over time can be attributed to Brownian motion and osmosis due to the presence of STPP [65]. Lopez-Leon et al. reported that CNPs obtained by the ionic gelation method tend to lose integrity if they are kept in solution for long as it is unstable in acidic condition [66]. As soon as the precipitation of CNPs were obtained the suspension was taken for centrifugation inorder to prevent losing of integrity of CNPs. Upon oven drying nanoparticles pellet at 60 °C, flakes of CNPs were formed, which is then grounded to form CNPs (Fig. 6). A high yield of 300 ± 0.02 mg CNPs were obtained in this study. Van Bavel (2023) reported an yield of about 1.05 mg in their study, which was reported as high yield [43] .

Fig. 6.

Chitosan nanoparticles obtained

UV absorbance peak was observed at 236 nm (Fig. 7). Agarwal et al. reported the formation of absorbance peak of CNPs between 225 and 240 nm [2], which is aligning well with our findings. The UV absorbance peak gives only a preliminary idea that CNPs may have formed. So, we have carried out further characterization techniques to confirm CNPs formation.

Fig. 7.

UV absorbance of the chitosan nanoparticles

The size distribution profile of CNPs (Fig. 8a) under DLS indicated a mean hydrodynamic diameter of 310.70 nm. The hydrodynamic diameter of CNPs ranges from 150.1 to 682 nm depending upon the concentration and the ratio by which chitosan and NaTPP used [2, 57]. In the present study hydrodynamic diameter falls within this range, indicating successful CNPs formation. Zeta potential implies stability of CNPs. In the present study, a zeta potential of 1.87 was obtained (Fig. 8b) for the CNPs. Studies have reported that the zeta potential of CNPs typically ranges from −28 to 42 mV [36, 67, 68], which is similar with the zeta potential value obtained in our study. A zeta potential of ± 30 mV generally indicates stability in the particles, while lower values suggest reduced stability and a higher tendency for aggregation [69]. In DLS, a polydispersity index of 0.12 was obtained for the CNPs. Agarwal et al. reported that according to dynamic light scattering (DLS) guidelines, PDI (poly-dispersity index) value < 0.5 is favourable for CNPs to have monodisperse particles [2]. The polydispersity index obtained in the study is < 0.5, which implies that CNPs are highly monodisperse particles which are mostly of uniform sizes.

Fig. 8.

Dynamic light scattering analysis of the chitosan nanoparticles: a particle size distribution, b zeta potential

SEM analysis showed that most of the CNPs were in the 5–40 nm range, with a few extending up to 80–100 nm (Fig. 9b), homogeneous, and spherical in shape (Fig. 9a). Chitosan is said to be nano-sized, if the chitosan particles are in range of 1–1000 nm in size [70]. Agarwal et al. also reported the shape of CNPs were obtained as spherical under SEM analysis [2].

Fig. 9.

Scanning electron microscopy of the chitosan nanoparticles: a scanning electron micrograph of chitosan nanoparticles, b histogram showing their size distribution

FTIR analysis was performed to confirm the formation of CNPs. Chitosan was characterized with some specific peaks located at 1658, 896 and 3578 cm^–1 which relate to amide (CONH₂), anhydro glucosidic ring and primary amine (NH₂), respectively (Fig. 10). Kumari et al.. (2016) reported that the peak noticed in chitosan at 1555 cm⁻¹ corresponds to N–H bending of the secondary amide II band of CONH– whereas, the amide I band is generally observed at 1658 cm⁻¹ [71]. In the present case both amide I and amide II bands are present for the CNPs formed. In the case of CNPs, the observed peaks 1658 and 3578 cm⁻¹ got shifted from higher wave number region to lower wave number region as 1653 and 3277 cm⁻¹. The reduction in stretching frequency could be attributed to the TPP interaction with ammonium group of chitosan and more hydrogen bonding in chitosan–Na-TPP complex [57]. We hypothesize that the tripolyphosphate groups of Na-TPP electrostatically bind to the protonated ammonium groups of chitosan, enhancing inter- and intramolecular crosslinking in the CNPs.

Fig. 10.

FTIR spectra of chitosan, sodium tripolyphosphate, and the chitosan nanoparticles

A sharp peak around 20° (2θ value) was observed for chitosan in the XRD analysis (Fig. 11), reflecting the characteristic crystalline nature of chitosan. The X-ray diffractogram of the CNPs showed no distinct peaks, indicating their amorphous nature and confirming that the crystallinity of chitosan is lost upon nanoparticle formation. Sathiyabama et al. also reported that in X-Ray diffractogram no peak was obtained for CNPs [72], stating its amorphous nature, which augment the sorption properties of the materials.

Fig. 11.

X-Ray diffractogram of the chitosan nanoparticles

EDAX analysis confirmed the presence of carbon (42.96%) and nitrogen (0.9%) in CNPs (Fig. 12c), suggesting the presence of chitosan in CNPs formed. The presence of Na (0.84%) and P (8.12%) in EDAX spectrum of CNPs comes from the Na-TPP used for CNPs preparation. The presence of 0.81% Si in EDAX spectra of CNPs might have come from the Petri dish used for grinding the CNPs pellet. The observed elemental composition of CNPs (C—42.96%, N—0.9%, O—46.36%, Na—0.84%, P—8.12%) was comparable to the values reported by Nair et al. [73].

Fig. 12.

EDAX spectrum: a chitosan, b sodium tripolyphosphate, and c chitosan nanoparticles

Morphology of CNPs was examined by AFM. The AFM image of CNPs revealed that the CNPs are of spherical shape with rough surface texture (Fig. 13). The spherical shape was obtained for the CNPs under SEM analysis which was consistent with the AFM results. Joseph et al. also reported the rough nature of CNPs under AFM analysis [74].

Fig. 13.

Atomic force micrograph of the chitosan nanoparticles

The morphology of the particles were observed by TEM and HR-TEM to get direct image of atomic level details. A size of 200 nm for chitosan and 10 nm for CNPs were recorded (Fig. 14). Keawchaoon and Yoksan also reported that the CNPs exhibited spherical shape with an average diameter of 40–80 nm [67].

Fig. 14.

HR-TEM image: a chitosan, b chitosan nanoparticles

CNPs alone or incorporated with metallic, bimetallic, and metal oxide to form nanocomposites possess various applications. CNPs efficiently interact with the skin, enhancing adhesion and prolonging the residence time of active compounds on its surface, making it a suitable component for cosmetics [75]. Abdelraouf et al. (2023) reported that CNPs-encapsulated Pseudomonas fluorescens enhanced soil and plant defense enzyme activities, thereby improved plant growth and mitigated Fusarium wilt in tomato plants [76]. Mannose-modified CNPs target specific receptors on nasal epithelial cells, enhancing drug uptake and bioavailability for intranasal delivery [77]. Zinc deficient/ alkaline soils leads to poor plant growth promotion higher disease incidence, and reduced yields. Choudhary et al. (2019) addressed these challenges with Zn-CNPs (0.01–0.16%) via seed priming and foliar application in maize, and reported a strong in vitro antifungal and seedling growth-promoting activities [78]. Throughout history, wound healing has been a crucial challenge facing all wound care researchers in the medical field. Green-synthesized chitosan–copper oxide nanocomposites (Cs–CuO-NPs), prepared using pomegranate peel extract, exhibited superior antimicrobial activity and showed dual hematological effects by acting as a procoagulant in healthy and diabetic blood while serving as an anticoagulant in hypercholesterolemic samples [79]. Muddin et al. (2024) reported that fabricating magnetic chitosan CNPs (M-Ch-NPs) with surface modification of CNPs using magnetite enhanced the mechanical strength and reusability, acting as a potential biosorbent for the Cr(VI) from contaminated wastewater. Fungal pathogens are the main cause of significant economic loss during the growth of the crop and postharvest handling of fruits [80]. Al-Dhabaan et al. (2017) reported that chitosan–zinc–copper nanocomposites exhibited potent antifungal activity in against Botrytis cinerea, thereby enhancing seedling survival and reducing fruit rot [81].

Although the limitations of this study exist like difficulty in forming uniform size CNPs, the instability of CNPs formed in acidic conditions, and presence of impurities in CNPs introduced during grinding and centrifugation, these donot overshadow their advantages. CNPs produced can be used to synthesize metallic, bimetallic, and metal oxide nanocomposites by various methods, which further enhances its application. This standardized protocol for CNPs synthesis is simple, cost-effective, scalable, and highly reproducible, making it a reliable approach for CNPs synthesis by ionic gelation method. Toxicological studies of the CNPs need to be conducted to check its toxicity level.

Conclusions

In the present study, we successfully developed a simple, cost-effective, reproducible, and user-friendly protocol for the synthesis of CNPs using ionic gelation method. Comprehensive characterization confirmed that synthesized CNPs were spherical and within nanometer size range. Structural analysis using XRD confirmed the amorphous nature of CNPs, while FTIR verified the presence of functional group, confirming successful synthesis and elemental composition. EDAX further confirmed the elemental composition, supporting that the final product was CNPs. This work provides a significant contribution to the field of research by offering a high-yield protocol that is both accessible, simple, and efficient. The well characterized CNPs produced by this method hold potential for various applications like antimicrobial coatings, food preservation, pesticide delivery, plant growth promotion, water treatment, drug delivery etc. Future research will focus on exploring these potential applications to exploit the full potential of these synthesized nanoparticles.

Author contributions

Anju A. B. conducted the experiments, data analysis and wrote the manuscript. Dr. Surendra Gopal K. conceptualized and supervised the study. Dr. Surendra Gopal K. and Dr. Panchami P. S. reviewed the manuscript. Dr. Panchami P. S. and Dr. Reshmy Vijayaraghavan arranged the resources. All authors read and approved the final manuscript for publication.

Funding

This work was financially supported by Department of Science and Technology, Government of India, New Delhi-110016 (under Inspire Programme (DST/INSPIRE Fellowship/2023/IF230155) and Kerala Agricultural University, Vellanikkara, Thrissur, Kerala-680656.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval

Ethical approval is not applicable since no human or animal studies are involved.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.