Chitosan nanoparticles: Green synthesis, biological activities, and sustainable frontiers in targeted drug delivery and cancer nanomedicine – A comprehensive review

Abstract

Chitosan, a biopolymer derived from chitin—the second most abundant natural polysaccharide found in crustaceans such as crabs, lobsters, and shrimp—has become a cornerstone in biomedical innovation. Its unique properties, including hydrophilicity, biocompatibility, biodegradability, low toxicity, and intrinsic cationic nature, make it an ideal candidate for the development of sustainable and multifunctional nanomaterials. Chitosan nanoparticles (CNPs), distinguished by their nanoscale size and enhanced physicochemical characteristics, offer significant advantages in biomedicine, particularly in diagnostic imaging as targeted delivery systems for drugs, genes, and biomolecules in cancer therapy. The green synthesis of CNPs through methods such as microemulsion, polyelectrolyte complexation, ionic gelation, emulsification-solvent diffusion, and reverse micellization further emphasizes their eco-friendly and sustainable production. Recognized as a Generally Recognized as Safe (GRAS) material by the USA Food and Drug Administration, chitosan is widely accepted for use in biomedical applications. This review comprehensively explores the structural features, environmentally friendly synthesis strategies, and advanced characterization techniques of CNPs. Moreover, it highlights their interdisciplinary biomedical applications, including drug delivery via ocular, oral, pulmonary, vaginal, and nasal routes, as well as their promising role in tissue engineering and cancer therapy. By integrating green chemistry principles with advanced biomedical design, CNPs are reshaping the future of nanomedicine, offering sustainable and targeted therapeutic solutions.

Graphical abstract

1. Introduction

Nanoparticles made from various polymers, typically within the 1–100 nm size range, are commonly used in nanomedicine and biomedical applications [1]. Nanoparticle production can be achieved through top-down techniques, including high-pressure processing, homogenization, sonication, and bottom-up procedures such as reactive precipitation and solvent displacement [1]. Owing to their nanoscale dimensions, nanoparticles possess distinct properties compared to their bulk counterparts, enabling their precise engineering with unique characteristics [2].

Nanoparticles can be classified into two categories: organic and inorganic. Recently, inorganic nanoparticles have gained interest owing to their remarkable stability under demanding conditions [3]. Notable metal oxide nanoparticles, such as silver, zinc, titanium, and magnesium, have garnered attention for their diverse optical properties and strength [4]. Conversely, the distinctive structural, metallurgical, and electrical properties of organic materials, including polymers, lipids, and carbon nanotubes, render them highly adaptable for various applications [5]. Both natural and synthetic polymers are well-suited for producing polymeric nanoparticles due to their inherent stability and versatility in surface modification [67]. Additionally, Biopolymeric nanoparticles offer several advantages, including high biocompatibility, as well as low toxicity and enhanced biodegradability [6,7]. They can be easily derived from natural sources like starch, cellulose, collagen, pectin, chitin, chitosan, and silk fibroin [7].

Chitosan, a chemically degradable biocompatible polymer, is obtained through the deacetylation of chitin and is structurally similar to cellulose, consisting of alternating units of N-acetyl glucosamine and glucosamine linked by 1-4-glycosidic bonds [8,9]. It is a pale, inelastic carbohydrate used for various purposes, such as agriculture, biomedicine, the food sector, water purification, preventing pollution, photography, and paper production [10]. The nanoparticles derived from chitosan, known as chitosan nanoparticles (CNPs), combine the advantageous properties of both chitosan and nanomaterials [11]. The economic feasibility and widespread availability of chitosan make it particularly valuable for medicinal applications, such as enhancing wound healing [12] and the development of drug delivery systems [13]. Moreover, chitosan possesses mucoadhesive characteristics that enable the administration of CNPs through several transmucosal pathways, including the intratracheal, intranasal, intravaginal, intrapulmonary, and intraocular pathways [14].

In the agricultural sector, chitosan is used in fertilizer formulations and as a standard component in food coatings [15]. Moreover, it is incorporated into cosmetic formulations to help combat skin dryness [16]. Chitosan can be chemically modified to create polymers with varied properties, thus broadening its applications [14]. For instance, polymeric CNPs facilitate targeted delivery of medications through multiple injection routes. These nanoparticles, which are characterized by a positive surface charge and mucoadhesive properties, effectively attach to the mucosal surfaces, allowing for the gradual release of their drug payload [17]. Colloidal nanoparticles have the potential to be used in non-invasive drug delivery systems for conditions such as cancer, eye diseases, respiratory ailments, and gastrointestinal disorders [18].

Due to its beneficial properties, chitosan has emerged as a naturally occurring material with considerable promise in nanomedicine [19]. Its versatility facilitates the production of various three-dimensional scaffolds with customized pore networks for bone tissue engineering [19]. Furthermore, chitosan may be incorporated into components such as polymers and ceramics to produce synthetic scaffolds exhibiting exceptional biological and mechanical features [20]. CNPs effectively deliver growth factors or peptides for bone tissue engineering [13]. The synthesis of CNPs may be efficiently achieved via a modified precipitation method employing sodium hydroxide as the precipitating agent [13].

The current review explores the revolutionary potential of CNPs, focusing on their green synthesis, multifunctional design, and diverse applications across disciplines. By elucidating their production processes, unique properties, and transformative capabilities, this review underscores the role of CNPs in advancing biomedicine and fostering sustainable innovation.

2. Structure and physicochemical properties of chitosan

The structural and physicochemical characteristics of chitosan have garnered considerable attention. Henri Braconnot (1811) initially identified the chemical chitin while examining mushrooms. Later, in 1859, Charles Rouget demonstrated that treating chitin with alkali could yield a chemically distinct organic polysaccharide that was acid-soluble, which was named chitosan by Hoppe Seiler [21]. Chitin ranks as the second most abundant natural carbohydrate, constituting a significant component of the exoskeleton of crustaceans, such as crabs, shrimp, and lobsters [22]. It is also a key component of the insect cuticles and cell walls of certain yeasts, fungi, and green algae [23]. Although chitosan is less prevalent than chitin in the environment, it is present in the cell walls of certain fungi [24].

Structurally, chitin is a polymer composed of β-(1–4)-2-acetamido-2-deoxy-D-glucopyranose units, closely resembling that of cellulose except for the replacement of the C(2) hydroxyl group with an acetamido group [25]. Chitin can be classified into three forms: β, α, and γ [21]. γ-chitin is a hybrid of the two types, while β-chitin exhibits intersheet hydrogen bonding between parallel chains. Chitin is the main biosource of chitosan [26], containing at least 60% D-glucosamine units (D Unit) [27], the concentration of which significantly influences chitosan's solubility in acidic aqueous solutions [27].

The deacetylation degree (DD) represents the molar fraction of deacetylated units in chitosan [28]. It plays a crucial role in determining the effectiveness of chitosan in various applications [29]. DD can be assessed through various techniques, including ultraviolet (UV)-visible spectrophotometry, solid-state nuclear magnetic resonance (NMR) spectroscopy, liquid-state NMR spectroscopy, potentiometric titration, and infrared spectroscopy. Although chemically distinct from cellulose, chitin is poorly soluble in solvents typically used to dissolve cellulose, such as cuprammonium hydroxide, cadox, and cupriethylenediamine [30].

The solubility of chitosan is affected by many factors, including pH, DD, temperature, solvent type, and polymer crystallinity. The protonation of amino (–NH₂) groups plays a vital role in solubility in aqueous solutions; chitosan becomes soluble when approximately 50% of the amino groups are protonated [31]. Moreover, chitosan dissolves in acetic acid when its DD is below 28%. Therefore, DD significantly influences chitosan's solubility, influencing the length of the glucosamine units and its crystalline structure, provided all other factors remain constant [31].

The molecular weight and viscosity of chitosan in aqueous solutions critically affect its potential use in biochemistry, nanomedicine, and pharmacology. Other essential factors include moisture content, crystallinity, ash content, crystal size, and levels of heavy metals [32]. Additionally, chitosan extraction is an environmentally friendly process that mitigates the ecological impact of discarded crustacean shells from the seafood industry [21]. The waste produced annually from crab shells poses a challenge for decomposition, contributing to environmental pollution. Transforming these shells into chitin and chitosan effectively reduces contamination, imparting both compounds with many valuable applications [21].

The molecular structure of chitosan comprises three reactive groups: OH groups at C₃–OH and C₆–OH, and –NH₂ group at C₂–NH₂. The C₆-OH group demonstrates a higher chemical reactivity than the C₃–OH group. The glycosidic bond acts as a functional group that enables chemical modification, allowing the polymer to develop new properties and behaviors [31]. Interacting suitable reagents with the amino group produces N-modified chitosan derivatives. Similarly, altering the –OH groups produces O-modified varieties of chitosan with improved physicochemical properties [33].

Although the chemical modification of chitosan may affect its functions, modifying the –OH groups often produces slight variations in its biophysical characteristics [21]. However, replacing the O-amine group with the N-cinnamyl group increases chitosan's hydrophobicity and engenders vigorous antibacterial activity against pathogens such as Pseudomonas aeruginosa, Bacillus cereus, Escherichia coli, and Staphylococcus aureus [34]. C6 N-quaternary ammonium-O-sulfobetaine modified with a chitosan-based glycopolymer, is hydrophilic and can effectively interact with lectins [35]. This modified chitosan exhibits antibacterial properties [36]. Studies have indicated that O-acylated chitosan nanofibers, modified with fatty acids and anhydrous side groups, manifest diverse degrees of hydrophobicity and hydrophilicity linked to the length of the acyl group substitution [37].

Chitosan quaternary ammonium salts display coagulation and flocculation properties that are effective against the cyanobacterium Microcystis aeruginosa [38]. Antibacterial properties of chitosan arise from its cationic nature, allowing the positively charged polymer to bind to bacterial surfaces, thereby disrupting membrane integrity and inhibiting microbial proliferation [39]. Chitosan with a higher degree of deacetylation and under low pH conditions tends to exhibit enhanced antibacterial properties [40]. Lowering the molecular weight may increase the effectiveness against Gram-negative bacteria and reduce it against Gram-positive bacteria [40].

The low toxicity of chitosan to mammalian cells is likely due to its fundamental hydrophilic nature, which facilitates its interaction with microbial cells [12,39]. Consequently, they can efficiently encapsulate enzymes, medications, and nucleic acids with controlled release for various applications across different industries [39].

When combined with Fe₃O₄ nanoparticles, the resulting salts can function as bio-adsorbents for chromium (VI) ions and methyl orange [41]. Moreover, hydrogels created by crosslinking chitosan with terephthalaldehyde, glutaraldehyde, and glyoxal have proven effective in organ transplantation and functional restoration [42]. In addition, radionuclides such as Lu-166, Sm-153, and Ho-166 have been combined with chitosan for focused radiotherapy [43].

3. Synthesis of CNPs

Chitosan produces chitosan microparticles and CNPs [44]. The quality of chitosan is influenced by the source of chitin, the extraction method, and the DD [45]. Chitin and chitosan primarily originate from aquatic and terrestrial species and specific microorganisms [46]. The extraction process of chitosan and chitin from marine creatures like shrimp, lobsters, crabs, and squid generates considerable bio-waste [47]. Seasonal variations, irregular raw material supply, and unpredictable processing conditions impede the commercial manufacture of chitosan from the waste products of these organisms [48,49]. Consequently, terrestrial organisms, including mushrooms, crabs, and insects, have been used for chitosan derivatization [29,45].

Special focus has been given to silkworms and honeybees, as their waste products could serve as valuable resources for the large-scale production of chitosan and chitin [50]. Many microorganisms, including yeasts, molds, ciliates, certain chrysophyte algae, and some bacteria, particularly prosthecate bacterial stalks and streptomycete actinobacteria, contain chitosan and chitin [51]. Using chitosan in conjunction with microorganisms presents a viable approach because it allows for the production of consistent and tailored products [52].

Despite the potential for synthesizing chitosan and chitin from various terrestrial microorganisms, species selection restricts commercial viability [53]. A notable characteristic of chitosan is its minimal tendency to trigger significant inflammation or immune responses. This biopolymer is particularly effective for nanoparticle synthesis because of its cationic properties, low toxicity, excellent biocompatibility, mucoadhesive qualities, absorption-enhancing ability, and environmental degradability [14,54].

The cationic nature of chitosan facilitates ionic crosslinking with highly charged anions; the several unbound –NH₂ groups in its linear polyamine form are essential for crosslinking. These features are vital for producing nanoparticles. CNPs exhibit characteristics that increase their affinity for negatively charged cellular membranes, enabling precise targeting of specific in vivo locations [55]. Given their unique properties and versatility, CNPs are particularly suitable for use in mucosal tissues, including nasal, oral, and ocular applications. The gelation and bead formation of chitosan upon interaction with anions render it appropriate for drug delivery. However, the effectiveness is constrained by the bead size, which generally ranges from 1 to 2 mm [56,57]. There are different approaches for synthesizing CNPs, and the most widely used methods are ionotropic gelation and polyelectrolyte complexation because they are straightforward and do not necessitate high shear pressures or organic solvents [58]. Table 1 shows various synthesis methods for CNPs and their possible uses according to their size. The size distribution of CNPs generated from different production processes is shown in Fig. 1.

Table 1.

Various methods of synthesizing chitosan nanoparticles and their possible uses according to their size.

Synthesis method	Principle/mechanism	Particle size (nm)	Advantages	Disadvantages	Cross-linking agents	Applications	References
Ionic gelation (ionotropic gelation)	Electrostatic interaction between cationic chitosan and anionic crosslinker	50–1000	Simple, mild conditions, aqueous-based, no organic solvents, cost-effective, scalable	Limited control over particle size, potential aggregation, pH dependent	Tri-polyphosphate, alginate, sulfates, phosphates	Drug delivery, gene therapy, vaccine delivery, wound healing	[371]
Polyelectrolyte complexation	Complexation between oppositely charged polyelectrolytes	100–800	Versatile, can use various polyelectrolytes, mild conditions	Complex optimization, potential instability	Hyaluronic acid, heparin, dextran sulfate	Protein delivery, tissue engineering	[372]
Emulsification and cross-linking	Formation of emulsion followed by cross-linking with chemical agents	200–2000	Good encapsulation efficiency, suitable for hydrophobic drugs	Use of organic solvents, potential toxicity, complex purification	Glutaraldehyde, formaldehyde, genipin	Sustained drug release, hydrophobic drug delivery	[373]
Reverse micellar	Formation of reverse micelles in organic phase containing chitosan	10–100	Small particle size, narrow size distribution, high drug loading	Use of organic solvents, surfactant removal required	Aerosol-OT (AOT, sodium bis (2-ethylhexyl) sulfosuccinate, or cetyltrimethylammonium bromide span surfactants	Targeted drug delivery, imaging agents	[81]
Microemulsion	Formation of stable microemulsion system for nanoparticle synthesis	50–500	Controlled particle size, good stability, suitable for lipophilic drugs	Complex formulation, surfactant removal needed	Various surfactants and co-surfactants	Cosmetic applications, topical delivery	[374]
Solvent evaporation	Dissolution of chitosan in solvent followed by evaporation	100–1500	Simple process, good for drug encapsulation	Residual solvents, environmental concerns	Chemical cross-linkers if needed	Pharmaceutical formulations	[375]
Precipitation	Precipitation of chitosan from solution using non-solvent	100–1000	Rapid process, no need for cross-linking agents	Broad size distribution, potential aggregation	Non-solvents (alcohols, acetone)	Research applications, proof-of-concept studies	[376]
Spray drying	Atomization of chitosan solution using spray dryer	166–3500	Continuous process, good for large-scale production	High temperature exposure, potential degradation	Tri-polyphosphate or other ionic cross-linkers	Industrial-scale production, food applications	[377]
Supercritical CO2	Use of supercritical CO2 as antisolvent or processing medium	50–500	Green technology, no toxic solvents, uniform particles	Expensive equipment, limited scalability	None typically required	Pharmaceutical applications, drug delivery	[378]
Self-assembly	Spontaneous assembly of amphiphilic chitosan derivatives	277–731	No external cross-linking required, biocompatible	Limited to specific chitosan derivatives	None (self-cross-linking)	Biomedical applications, smart drug delivery	[379]
Top-down approach (milling)	Mechanical size reduction using milling techniques	500–5000	Simple equipment, suitable for bulk production	High energy consumption, heat generation	Post-processing cross-linking if needed	Industrial applications, bulk production	[380]
High-pressure homogenization	Application of high pressure to achieve size reduction	100–1000	Uniform particle size, high throughput	High energy requirement, expensive equipment	Chemical stabilizers may be added	Pharmaceutical manufacturing	[381]
Ultrasonication	Use of ultrasonic waves for particle size reduction	100–1000	Simple, energy-efficient, can reduce aggregation	Potential degradation, heat generation	Optional chemical cross-linkers	Laboratory-scale synthesis, research	[382]

Fig. 1.

The average particle size (nm) of chitosan nanoparticles formed from various synthesis techniques.

CNPs exhibit exceptional chemical, physical, and morphological characteristics, which are determined by their composition and the methods used for their synthesis [54]. Although chitosan is not soluble in water, it can dissolve in acidic solutions, such as tartaric, citric, and acetic acids [59,60]. The molecular weight of chitosan can vary widely, ranging from 3,800 to 20,000 Da. The DD and molecular weight are critical in influencing the properties of the nanoparticles. Chitosan-based polymeric carriers have proven effective in delivering various therapeutic agents, such as anticancer drugs [61], peptides [13], antimicrobials [13,54], growth factors [62], and anti-inflammatory medications [63]. The large surface area of CNPs enhances their ability to encapsulate, dissolve, and bond with bioactive compounds, facilitating their adsorption onto the nanoparticle matrix. Additionally, their nanoscale dimensions enhance their ability to penetrate epithelial cells. Colloidal CNPs can deliver diverse drugs, proteins, and DNA of varying molecular weights while possessing a negative charge that targets specific tissues, cells, and organs [55].

Several different synthesis techniques for CNPs are described in Table 1 and in the sections described below (3.1–3.9). Furthermore, Table 2 presents the most recent green synthesis technologies that have been developed for CNPs.

Table 2.

Recent green synthesis technologies for chitosan nanoparticles.

Method	Key Features	Advantages	References
Enzyme-catalyzed	Uses chitinases/chitosanases under mild conditions	Clean, mild, precise	[101]
Microbial fermentation	Utilizes microbial and plant pathways for nanoparticle fabrication. Example: fermentation with Eucalyptus citriodora	Sustainable, tunable, scalable	[395]
Plant extract bioreduction	Plant extract serves as a reducing agent in green synthesis. Example: utilizing lavender essential oils	Non-toxic, cost-effective	[396]
Nanocomposite synthesis	Combine chitosan with other green-synthesized nanoparticles. Example: chitosan-zinc oxide	Synergistic bioactivity, stability	[108]

3.1. Ionotropic gelation: a green and efficient approach

Chitosan can undergo physical and chemical crosslinking to form nanoparticles because its macromolecular structure contains –NH₂ groups that can be protonated into the NH₃⁺ form under acidic conditions [64]. Physical crosslinking has gained considerable interest because it avoids toxic substances, minimizes side effects, and enhances biocompatibility. Furthermore, this method is straightforward and mild, enabling effective crosslinking [64]. Interactions between the positive charge of chitosan and the negative charge of multivalent ions like sodium tri-polyphosphate (TPP), citrate, and sulfate are critical for physicochemical crosslinking [65].

Ionic gelation occurs when chitosan interacts with small anionic molecules like citrate, sulfate, or phosphate, facilitating the formation of a polyelectrolyte complex [65]. This technique leverages the electrostatic attraction between negatively charged polyanions, such as sodium TPP, and the –NH₂ groups of chitosan [66].

Chitosan is typically dissolved in 2% acetic acid or combined with a poloxamer as a stabilizing agent, followed by the addition of sodium TPP under intense agitation; the inclusion of anionic particles aids in crosslinking, resulting in nanoparticles ranging from 200 to 1000 nm [67]. CNPs are obtained through several centrifugation and rinsing steps, followed by heat or freeze-drying. The method allows altering the nanoparticles' surface charge and size by varying the chitosan-to-stabilizer ratio [67,68]. An increased ratio of chitosan to polyanion typically leads to larger particle sizes [69]. Moreover, nanoparticles dispersed in a saline solution exhibited improved stability, and the addition of a monovalent salt like sodium chloride reduced the repulsion among chitosan's –NH₂ groups, resulting in enhanced flexibility of the polymer chains and improved stability [70].

Combining ionotropic gelation with radical polymerization leads to the gelation of chitosan, whereas acrylic or methacrylic acid undergoes polymerization [71]. The ionotropic gelation technique represents the most straightforward and affordable approach for moving from laboratory studies to industrial applications. This method uses simple and cost-effective materials and equipment that are readily available in standard research settings. In addition, this approach utilizes electrical interactions instead of chemical ones, thus eliminating the need for organic solvents and reducing the harmful effects of toxic compounds [72].

3.2. Emulsion-based methods: balancing efficiency and sustainability

In this method, a pharmaceutical compound and an aqueous chitosan solution are rapidly mixed with liquid paraffin and a stabilizer like Span™ 83 to form a stable water-in-oil emulsion. The interaction and merging of droplets from both emulsions during this process lead to the precipitation of chitosan droplets, resulting in the formation of nanoparticles approximately 452 nm in size [73,74]. An organic solvent such as methylene chloride or acetone is introduced into an aqueous chitosan solution, and a hydrophilic drug is mixed with a stabilizer like lecithin or poloxamer to form an oil-in-water emulsion [75]. This combination is then vigorously mixed and subjected to high-pressure homogenization to eliminate the methylene chloride. As the organic solvent diffuses into the water, the solubility of chitosan is reduced by the transfer of acetone into the water phase, leading to polymer precipitation [75].

The nanoparticles produced by this technique have an average size of 100–500 nm. A large volume of water was used to ensure that the acetone diffused completely and that the nanoparticles were effectively separated by centrifugation. This novel method may aid in reducing the size and distribution of chemically synthesized CNPs [76]. Although the emulsification technique assists in regulating the particle size, it often requires potent crosslinking agents, which may impede the thorough removal of residual agents [77].

Using this emulsion fluid dispersion approach with lipophilic and lipophobic medicines is possible. A double emulsion containing multiple water/oil/water layers was created for hydrophilic drugs, in which the pharmaceutical components dissolved in the water [78]. However, this approach has two significant drawbacks: it necessitates high shear pressure and requires the use of organic solvents in nanoparticle fabrication [78].

3.3. Reverse micellar technique: precision in green synthesis

The reverse micellar technique generates nanoparticles of the polymeric material with a narrow-size distribution [79]. Drug-delivering micelles may be created using various polymers. A reverse micelle is a thermodynamically stable combination of oil, surfactant, and water. This technique produces nanoparticles with a fine-size distribution and exhibits more dynamic behavior compared to conventional emulsion polymerization methods [79,80]. The process involves the use of reverse micelles formed when a surfactant dissolves in an organic solvent. The micellar droplets undergo random displacement due to Brownian motion, dividing the droplets into two micelles through water content exchange [81].

A hydrophilic surfactant, such as sodium bis-(2-ethylhexyl) sulfosuccinate, is combined with an organic solvent, like n-hexane (water-in-oil emulsion), to formulate the organic case. This solution is prepared by mixing the drug with chitosan in water and then continuously stirring. Following the addition of a crosslinking agent, the mixture is stirred overnight to facilitate crosslinking. Removal of the organic solvent results in the formation of a dry product in the water [67,81].

The surfactant is then removed by adding salt, and the CNPs are separated via centrifugation. Three primary steps are involved in separating the nanoparticles: precipitating the surfactant using calcium chloride, dialysis to eliminate unreacted components, and freeze-drying [76,82]. This process may produce small and consistent particle sizes. Colloidal CNPs loaded with bovine serum albumin (size, 80–180 nm) can be created using the reverse micellar method [76,83,84].

3.4. Desolvation technique: low-energy, high-yield synthesis

The desolvation technique can create nanometer-scale carriers [85]. Synthesizing CNPs often utilizes a modified approach using sodium sulfate, which helps disperse DNA and proteins [86]. Key principles, such as phase separation and coacervation, are integral to the desolvation process. Nanoparticles precipitate upon the addition of desolvation agents like acetone or ethanol. A crosslinking agent is used to enhance the stability of the nanoparticles [86,87].

This technique involves treating an aqueous solution of chitosan, which is combined with a stabilizer (e.g., Tween 20) and a precipitating agent (like sodium sulfate). The saline solution of chitosan effectively removes water from the chitosan layers, and the insolubility of chitosan results in precipitation [87]. Glutaraldehyde is subsequently used to increase the firmness of the nanoparticles, with an average diameter of 373 ± 71 nm [87]. The primary advantage of this method lies in its ability to produce nanoparticles using a single process with economic efficiency, low energy consumption, and minimal frequency [88].

3.5. Nanoprecipitation: a rapid and scalable green method

The solvent displacement method, or nanoprecipitation, offers several advantages over other techniques [89]. This manufacturing method is based on the Marangoni effect principle. Nanoprecipitation involves the production of nanoparticles from a colloidal suspension by gradually adding the oil phase to the aqueous phase while stirring at a moderate speed [90]. The formation of nanoparticles occurs almost instantly, requiring only one step for a quick and simple process [90].

Several critical factors significantly influence the nanoprecipitation process, including the rate of the organic phase injection, the stirring speed of the aqueous phase, and the ratio of the oil phase to the aqueous phase [90,91]. This technique enables the synthesis of particles with a narrow size distribution due to the absence of shear stress [91]. Although it can incorporate hydrophilic drugs, it is primarily suited for encapsulating hydrophobic pharmaceuticals. Polymers and drugs are dissolved in organic solvents such as methanol or acetone that mixes well with water. This solution is then added dropwise to a water-based solution containing the surfactant [92]. The rapid diffusion of the solvent leads to instant nanoparticle formation, following which the solvents are separated using a reduced vacuum. The peristaltic pump, running at a velocity of 0.8 mL/min, injects the diffusion component of the dissolved chitosan into the dispersed phases (methanol) [92].

Tween 80 is added to the dispersion phase to create the nanoparticles [92]. Smaller particles improve the available contact area, which is crucial for applications in adsorption and desorption systems; therefore, the nanoprecipitation process may yield nanoparticles with a size ranging from 50 to 300 nm [92,93]. This technology is notably effective in producing particles (as small as 170 nm), thus expanding its potential applications [93].

3.6. Spray-drying: continuous and ecofriendly synthesis

This process utilizes a nano-spray dryer because chitosan is first dissolved in glacial acetic acid and water, and then left to sit overnight [94]. The resulting solution is atomized with an atomizer, creating droplets that are exposed to a drying gas that evaporates the liquid phase, producing CNPs [95]. With a flow rate of 2 mL/min, the diameter of the spray dryer nozzle typically ranged from 4.0 to 7.0 μm. The drying gas had an output temperature of 80°C and an intake temperature of 120°C, with a flow rate of 1.3 m³/min. Factors such as the initial feed and operational parameters—including the flow rate, nozzle size, and inlet and outlet temperatures—impact the characteristics and production yield of CNPs during the spray-drying process [76,95].

Spray-drying is commonly used in the pharmaceutical sector to produce microencapsulated antibiotics, including vancomycin, ampicillin, and amoxicillin [96]. This single-step method is straightforward, continuous, and minimally affected by the dissolved state of the drugs and matrix. The method applies to hydrophilic and hydrophobic polymers and heat-resistant, heat-sensitive, water-insoluble, and soluble medicines [97].

The spray-drying method is an effective and straightforward technique for producing protein-loaded CNPs [98]. Öztürk and Kiyan [99] created CNPs with dexketoprofen via a spray dryer and assessed their anti-inflammatory properties; the dexketoprofen trometamol-loaded CNPs showed promise as a low-dose, high-efficacy, oral extended-release drug delivery system.

3.7. Enzyme-catalyzed green synthesis

Recent studies on the green synthesis of CNPs have focused on eco-friendly, sustainable, and non-toxic approaches, with enzyme-catalyzed synthesis and microbial fermentation emerging as prominent technologies [100,101]. Enzyme-catalyzed methods use biological catalysts (such as chitinases, chitosanases, and other relevant enzymes) to facilitate the controlled conversion of chitosan into nanoparticles [101]. These approaches offer several advantages over traditional chemical methods, including improved control over particle size and distribution due to the selectivity of enzyme action [102]. Cleaner reaction conditions, as enzymes work efficiently under mild temperatures and pH, reduce energy costs and chemical waste, and enhance stability, as enzymatic modification can be paired with surface coatings (such as chitosan itself) to improve the dispersion and thermal properties of the CNPs [101,102].

A recent study developed chitosan-enzyme-magnetic nanoparticle bioconjugates for recyclable biocatalyst systems, significantly enhancing the colloidal stability and thermal resilience of the nanoparticles. The immobilization of enzymes on chitosan-modified nanoparticles enables the production of nanoparticles that are not only eco-friendly but also highly functional for downstream applications in biocatalysis and drug delivery [101,102].

3.8. Microbial fermentation approaches

Microbial fermentation leverages the metabolic pathways of specific microorganisms (such as fungi or bacteria) to convert chitin or chitosan into nanoparticles, often as part of their natural metabolic processes [103]. This approach is gaining attention for several reasons, including the use of renewable bioresources and standard fermentation technology [104]. The process can be fine-tuned by adjusting fermentation conditions (pH, temperature, chitosan concentration, and incubation time) to optimize yield and nanoparticle characteristics [[103], [104], [105], [106]].

Recent studies have demonstrated the use of plant extracts and microbial fermentation for producing chitosan nanoparticles with potent antimicrobial and antibiofilm activities, showing consistency in nanoparticle size and bioactivity [107,108]. Optimization studies (using experimental designs such as Box–Behnken) have outlined factors controlling nanoparticle yield and size, with maximum production achieved at specific pH, temperature, and incubation times [100].

3.9. Combination and hybrid technologies

These green approaches are often combined with other sustainable methods, such as biosynthesizing hybrid nanoparticles (e.g., chitosan-coated zinc oxide or copper nanoparticles), utilizing plant extracts, or employing enzymatic crosslinking [108,109]. The synergy of bio-capping agents (from natural extracts) and microbial or enzymatic processes results in uniform, stable nanoparticles endowed with enhanced biological activity, confirmed through physicochemical characterization and bioassays [109].

4. Characterization techniques for multifunctional CNPs

A comprehensive understanding of the synthesis and the physical, chemical, and therapeutic properties of CNPs necessitates thorough characterization. Numerous studies have examined the characterization of CNPs to clarify their physicochemical properties [[110], [111], [112]]. This section provides a brief overview of the current characterization techniques used to assess the physicochemical properties of CNPs.

The size of CNPs is essential for determining their drug-binding capacity, crystallographic structure, and overall electrical charge [12]. Dynamic light scattering (DLS) or transmission electron microscopy (TEM) is used to quantify the size of CNPs. However, accurately determining the size with DLS can be challenging due to the high levels of polydispersity in CNPs [110,111].

Various complementary techniques, such as Raman spectroscopy, atomic force microscopy (AFM), X-ray diffraction (XRD), and scanning electron microscopy (SEM), are available to analyze the optical, morphological, and structural properties of CNPs [[110], [111], [112]].

4.1. XRD

XRD, the primary analytical method for identifying the crystalline phases of materials, can be used to determine the crystallinity of CNPs [113]. In addition to assessing structural features, XRD can be used to measure the average size of particles [114]. The Scherrer equation may be used to compute the average crystallite size in powdered materials subjected to XRD analysis while considering peak broadening caused by lattice defects, strain, and instrumental factors. According to Nasiri et al. [115], the Scherrer equation assumes that the average size of a nanoparticle is equivalent to the crystallite size and connects the absolute and full widths at half maximum of a particular XRD peak of a crystalline phase (with the previously mentioned adjustments) to the average nanoparticle size [115].

The XRD study of chitosan by Ali et al. [116] showed two different peaks at 2δ ≈ 11° and 19.6°, which disappeared when CNPs were formed due to their crosslinking with triglyceride polyol. The XRD pattern of the CNPs lacked clearly defined peaks, indicating an amorphous structure. The reduced crystallinity of CNPs may disrupt the complex network produced by long-chain polymers by infiltrating dense counter ions from dimethyl pyrophosphate into CNPs [117,118].

4.2. Raman spectroscopy

Raman spectroscopy is a sensitive tool for detecting structural changes in macromolecular complexes using Raman-active vibrational modes [119]. Light scattering plays a part in the Raman effect; this includes inelastic scattering at a wavelength altered by molecular vibrations and elastic scattering at the same wavelength as incoming light [119].

Raman spectroscopy was used to investigate possible structural alterations in proteins resulting from the adsorption of ovalbumin (OVA) onto CNPs [43]. The Raman spectrum of OVA-loaded CNPs displayed notable variations compared to the spectra of the individual components or the spectrum produced from a straightforward physical combination of both components [43].

This suggests a conformational shift in the OVA protein throughout the loading procedure, which is explained by the interactions between ions or dipoles and hydrogen bonds [120]. Such conformational shifts could lead to various effects in the Raman spectra, including frequency shifts, intensity variations, and broadening of relevant Raman bands [120]. Empirical evidence suggests that alterations in the vaccine structure can directly affect the recipients’ immunological response [121].

4.3. TEM

TEM is used to analyze nanoparticle size, structure, and crystallinity using an intense stream of high-energy electrons focused on the sample [122]. It allows an imaging system, such as a screen with phosphors placed across the electron beam, to record the resultant picture. The image was created from the interference between the transmitted electrons and those scattered by the sample. High-resolution imaging in TEM can achieve an effective resolution of approximately 2 Å, but samples must be thin for clear images, typically approximately 500 Å or less [123].

A negative staining agent like uranyl acetate can be used to prepare specimens for TEM analysis, or a liquid suspension of the sample can be placed directly onto a carbon-coated copper grid [124]. TEM was used to geometrically analyze the shape and size of magnetic CNPs, revealing that the Fe₃O₄ particles were successfully coated with chitosan; the CNPs measured 10–80 nm in diameter [124].

4.4. AFM

AFM enables the observation of nanomaterials at the atomic scale, the acquisition of a three-dimensional surface profile, and the measurement of the force exerted by the sample's surface on the AFM tip at the nano-Newton level [125]. AFM can be used to investigate the surface structure of nanocomposite chitosan-maghemite films with thin layers and potentially utilize them for plasmon resonance-based optical detection of Hg²⁺ ions [126]. Surface morphology can be evaluated using the root-mean-square (RMS) ruggedness value, computed based on the cross-section or two-dimensional shape of the sample [126].

In one study, the RMS smoothness coefficients for chitosan, γ-Fe₂O₃, and chitosan/γ-Fe₂O₃ were reported as 1.40, 47.00, and 37.30 nm, respectively, and surface diffusion was responsible for the smoothing impact of the chitosan treatment on the maghemite surface [43]. The surface structure of the nanostructured chitosan-maghemite composite thin film improved the detection of Hg²⁺ ions [43].

4.5. SEM

In SEM, a centered high-energy electron beam can be used to image the surface of a specimen [127]. Typically, samples are examined after they have dried, except when using environmental SEM. The effective imaging of nonconductive samples requires a thin carbon or metal coating. Analyzing the X-ray emission spectra can determine a material's elemental composition. SEM allows for direct viewing of the size and surface appearance of nanoparticles, particularly when combined with energy-dispersive spectroscopy [128].

Ionic gelation was used to produce copper-doped CNPs (Cu-CNPs), CNPs, and chitosan–saponin nanoparticles and to evaluate their distinct antifungal characteristics [129]. SEM, which was used to validate the nanoscale architecture of the CNPs, demonstrated that the chitosan–saponin nanoparticles had a spherical morphology, while the Cu-CNPs presented a compact polyhedral configuration. In addition, SEM was used to analyze the morphology of the carbon nanotubes of CNPs crosslinked with TPP and combined with salicylic acid and gentamicin; the colloidal nanoparticles exhibited a roughly spherical shape with an average diameter of approximately 200 nm [129].

4.6. DLS

DLS measures the fluctuations of particles caused by Brownian motion in a solution by assessing the intensity of the scattered light [130]. The illumination source is often monochromatic and originates from a laser. The particles in the solution scatter light, producing dynamic diffraction patterns called speckle profiles. The Stokes–Einstein equation can be used to determine the average particle size within a sample. Furthermore, DLS was used to measure the polydispersity of the nanoparticles, known as the polydispersity index (PDI), along with their Zeta potential [131]. Accurate nanoparticle-size measurements using DLS often involve observing intensity fluctuations at multiple detector angles and across various polarizations of the resulting lighting [132].

In the research conducted by Fan [133], DLS was used to assess copper-coated CNPs, CNPs, and saponin-coated CNPs at a laser-scattering angle of 90°. The mean particle size and PDI values were approximately 192 nm and 0.6 for the CNPs, 196 nm and 0.5 for the copper-loaded CNPs, and 374 nm and 1.0 for the saponin-loaded CNPs, respectively. The reduced PDI values indicate that CNPs and Cu-CNPs have much smaller size distributions than saponin-CNPs [133].

Zeta potential studies revealed that the CNP and Cu-CNP samples had elevated values of +45.33 and + 88.00 mV, respectively, whereas the saponin-CNPs recorded a Zeta potential of +31.00 mV. The Zeta potential quantitatively assesses the static stability of the nanoparticles in a fluid by measuring the electric potential between the fluid adjacent to the nanoparticle surface and the bulk fluid [133]. Thus, the higher Zeta potential values of the CNPs and Cu-CNPs indicate better stability than that of the saponin-CNPs in aqueous environments [133].

Kiselova-Kaneva et al. [134] developed DNA-loaded CNPs grafted with polyethylenimine (PEI) for gene therapy purposes in osteoarthritis. Using DLS, they showed that increasing the weight-to-weight ratio of CNP to DNA decreased the particle size and increased the surface charge, resulting in higher Zeta potential values [134].

5. Applications of CNPs

The various applications of CNPs in different fields are illustrated in Fig. 2 and Table 3. Furthermore, Fig. 3 illustrates the tree map depicting the many applications of chitosan nanoparticles based on their commercial availability.

Fig. 2.

Diverse applications of chitosan nanoparticles (CNPs) in vitro and in vivo.

Table 3.

Field applications of chitosan nanoparticles (CNPs).

Application Field	Specific applications	Commercial availability	Key benefits	Regulatory status	References
Drug delivery	Oral, nasal, pulmonary, ocular, vaginal drug delivery, and controlled release systems	Available	Biocompatibility, controlled release, and targeting capability	Approved (some formulations) by the USA Food and Drug Administration	[383,384]
Biomedical applications	Medical implants, biosensors, diagnostic tools, and medical device coatings	Limited	Non-toxic, biodegradable, versatile, and surface modification	Under evaluation	[385]
Wound healing and tissue engineering	Wound dressings, skin grafts, bone regeneration, cartilage repair, and scaffolds	Emerging	Antimicrobial, hemostatic, promotes healing, and scaffolding	Approved (wound dressings) by the USA Food and Drug Administration	[386]
Food industry and packaging	Food preservation, active packaging, edible films, and shelf-life extension	Available	Natural preservative, biodegradable, and barrier properties	Generally recognized as safe (GRAS) in many countries	[387]
Agriculture and plant protection	Biopesticides, nano-fertilizers, plant growth promotion, and pathogen control	Emerging	Eco-friendly, slow-release, and plant compatibility	Approved in several countries	[388]
Environmental remediation	Heavy metal removal, water purification, air filtration, and soil remediation	Limited	High adsorption capacity, renewable, and cost-effective	Environmentally approved	[389]
Cosmetics and personal care	Skincare formulations, hair care products, UV protection, and anti-aging	Available	Natural origin, moisturizing, and antimicrobial	Cosmetic grade approved	[390]
Gene and vaccine delivery	DNA delivery, RNA interference, vaccine adjuvants, and genetic engineering	Research phase	High transfection efficiency, low toxicity, and stability	Research phase/clinical trials	[391]
Antimicrobial applications	Antibacterial coatings, antifungal treatments, and disinfectants	Available	Broad spectrum activity, natural, and safe	Generally recognized as safe	[392]
Cancer therapy	Targeted chemotherapy, immunotherapy, and combination treatments	Clinical trials	Targeted delivery, reduced side effects, and enhanced efficacy	Clinical trial phase	[281]
Aquaculture	Fish health management, water quality improvement, and feed supplements	Limited	Disease prevention, growth promotion, and environmentally safe	Approved for aquaculture use	[393]
Water treatment	Wastewater treatment, industrial effluent cleaning, and membrane separation	Emerging	Effective pollutant removal, reusable, and sustainable	Industrial use approved	[394]

Fig. 3.

Tree map illustrating the diverse applications of chitosan nanoparticles according to their commercial availability, with higher percentages indicating greater applicability and availability, and lower percentages signifying reduced applicability and restricted availability.

5.1. Biological activities of CNPs

CNPs exhibit a wide range of biological activities, including antimicrobial, antioxidant, anti-inflammatory, antidiabetic, anticancer, and wound healing properties. They are biocompatible, biodegradable, and non-toxic, making them suitable for various biomedical applications. CNPs can also be used in drug delivery, enhancing drug solubility, stability, and efficacy (Fig. 2, Fig. 3).

5.1.1. Antimicrobial properties of CNPs

Microorganisms play pivotal roles in ecosystems, human health, and industrial processes [135]. The rising concern over microbial resistance to conventional antibiotics and disinfectants has spurred interest in alternative antimicrobial agents. Chitosan has also emerged as a promising candidate due to its biocompatibility, biodegradability, and broad-spectrum antimicrobial activity [136]. The inherent positive charge of CNPs enables them to interact with negatively charged bacterial and fungal cell membranes, disrupting their structure and preventing their growth, thus rendering CNPs particularly beneficial in managing common skin conditions such as acne, eczema, and fungal infections (e.g., athlete's foot or ringworm) [137].

The nanoparticles target Propionibacterium acnes, which is responsible for developing pimples and inflammation [138]. In fungal infections, the nanoparticles can penetrate the affected skin layers more effectively than traditional topical treatments, providing a higher concentration of active compounds directly at the site of infection [139]. The small size of the nanoparticles allows them to bypass the skin's outermost barrier, facilitating deeper penetration and enhanced drug delivery. Furthermore, their ability to gradually release therapeutic agents over time ensures prolonged antimicrobial activity, leading to more effective treatment and reduced recurrence of infections [139]. In addition to these direct effects, CNPs can promote healing by reducing the inflammatory response often associated with bacterial or fungal skin infections, further improving overall health and appearance [140].

5.1.1.1. Antibacterial activity of CNPs

Bacteria are ubiquitous, flourishing in all environments on Earth, including soil, water, extreme habitats such as hot springs and deep-sea vents, as well as within the human body [141]. Bacteria play essential roles in various ecological processes, such as nutrient cycling, and are integral to ecosystem functioning. In the human body, beneficial bacteria are crucial for processes like digestion, in which they help break down complex carbohydrates and synthesize vitamins [142]. These beneficial bacteria form part of the microbiota that maintains gut health and supports the immune system. However, some can be pathogenic, causing mild infections and severe illnesses [143].

CNPs have emerged as potent antimicrobial agents against bacterial infections by killing bacterial cells primarily through direct interaction with the bacterial cell membrane. Numerous theories have been proposed to explain this framework [144]. The mechanism begins with positively charged CNPs interacting with negatively charged components of the bacterial cell wall, such as lipopolysaccharides in Gram-negative bacteria and teichoic acids in Gram-positive bacteria [144]. This electrostatic interaction disrupts the structural integrity of the cell membrane, leading to altered membrane permeability, leaking of essential intracellular contents, and finally, cell death. Furthermore, CNPs may alter the electron transport chain in bacteria [145].

CNPs can penetrate bacterial cells and interact with intracellular components [144]. Once inside, they can bind to bacterial DNA, inhibiting the DNA replication and transcription processes essential for protein synthesis. This dual mechanism (disruption of the cell membrane and interference with genetic material) ensures that CNPs are highly effective in killing bacterial cells and preventing the proliferation of bacterial infections [144]. Additionally, this multifaceted approach reduces the likelihood of bacteria developing resistance to chitosan, making it a promising alternative to traditional antibiotics [144,145].

Another possible mechanism for antimicrobial action is the capacity of chitosan to form chelates with metal ions, thereby promoting the synthesis of toxins while obstructing bacterial life [146]. In acidic environments, the metal ion chelating capacity of chitosan is greatly enhanced (e.g., Fe²⁺, Mg²⁺, Ni²⁺, Co²⁺, Cu²⁺, and Zn²⁺), a property that contributes to its potent antibacterial activity because metal ions play a key role in the stabilization and normalization of bacterial cell wall components [146]. This mechanism mainly functions at elevated pH levels because chitosan sequesters positive ions owing to the presence of unprotonated NH₂ groups and the availability of electron pairs for donation to the metal ions on the amine nitrogen [146].

Inevitably, the metallic complex is encased by chitosan molecules, which prevent the essential flow of nutrients and cause cellular death. Consequently, the effective handling of CNPs depends on several customizable factors [147]. Hipalaswins et al. [148] evaluated the antibacterial activity of manufactured CNPs against various therapeutically important bacterial species, such as Enterobacter aerogenes, Pseudomonas fluorescens, Proteus mirabilis, E. coli, Klebsiella pneumoniae, and S. aureus. E. coli had the highest susceptibility, followed by K. pneumoniae, E. coli, P. mirabilis, and P. fluorescens. The CNPs exhibited considerably reduced toxicity against S. aureus [148]. Furthermore, the antibacterial efficacy against four specific bacteria (Listeria monocytogenes, S. aureus, E. coli, and Shigella dysenteriae) was observed by combining CNPs with lime essential oil; S. dysenteriae showed considerable sensitivity to CNPs, indicating the most significant susceptibility [149].

Curcumin-loaded CNPs have proven effective in drug delivery and the targeted activation of antibacterial mechanisms, as indicated by their suppression of S. aureus and P. aeruginosa infections in murine models [150]. Fig. 4 illustrates the mechanisms by which CNPs combat bacterial infections. Furthermore, Table 4 demonstrates the antibacterial efficacy of CNPs.

Fig. 4.

The antibacterial mechanism of chitosan nanoparticles (CNPs) in combating bacterial infections.

Table 4.

Antibacterial activity of chitosan nanoparticles (CNPs).

Experimental studies	CNPs variant	Target microorganisms	Methodologies	Results and observations	References
Antibacterial efficacy of pure CNPs against food-borne pathogens	CNPs	Foodborne pathogens such as Salmonella enterica and Listeria monocytogenes	Broth dilution and scanning electron microscopy imaging	Effective membrane disruption and inhibition at low concentrations	[397,398]
Antibacterial efficacy of CNPs loaded with tetracycline against periodontal pathogens	CNPs loaded with tetracycline	Porphyromonas gingivalis (periodontal pathogen)	Agar diffusion, minimum inhibitory concentration assay and minimum bactericidal concentration assay	Zone of inhibition, Effective in inhibiting growth in vitro	[399]
Silver-coated CNPs against multidrug-resistant strains	CNPs with silver nanoparticles	multidrug-resistant strains of Escherichia coli and Pseudomonas aeruginosa	Minimum inhibitory concentration assay and biofilm inhibition assay	Superior efficacy against biofilms and planktonic cells due to silver synergism	[400,401]
CNPs loaded with plant extracts against bacteria	CNPs infused with neem extract	Bacillus cereus and Staphylococcus aureus	Minimum inhibitory concentration assay and growth curve analysis	Enhanced efficacy due to the combined antibacterial properties of chitosan and neem	[402]
CNPs-Copper oxide-NPs for biofilm management	Copper oxide-incorporated CNPs	Streptococcus pyogenes	Disk diffusion assay and fluorescence microscopy	Potent activity against biofilms and resistant bacterial strains	[403]
Pure CNPs against agricultural pathogens	CNPs	Xanthomonas campestris and Erwinia amylovora	Zone of inhibition assay and greenhouse studies	Promising reduction of bacterial growth and disease symptoms in plants	[404,405]
Zinc-oxide-loaded CNPs against bacteria	Zinc oxide- CNPs	Klebsiella pneumoniae and Acinetobacter baumannii	Time-kill assays, scanning electron microscope and transmission electron microscopy imaging	Synergistic antibacterial effects and cell wall disintegration	[406,407]

5.1.1.2. Antiviral activity of CNPs

To multiply and spread, viruses need to penetrate host cells, where they take over the host's metabolism and produce new viral particles [151]. This parasitic nature underlies their ability to cause a wide range of diseases across humans, animals, and plants [152]. CNPs offer a promising antiviral strategy targeting several stages of the viral life cycle. A primary mechanism by which CNPs inhibit viral infections is by blocking the initial binding and entry of viruses into host cells [153]. The positively charged CNPs can bind to the negatively charged viral particles, preventing them from interacting with the host cell receptors [153].

This binding not only inhibits the attachment of viruses to the host cell surface but also prevents subsequent penetration and entry, effectively halting the infection process at its earliest stage [154]. In addition, the immunomodulatory properties of chitosan can enhance the host's immune response by stimulating the activation of immune cells and the production of cytokines. CNPs can help the host's immune system recognize and clear viral infections more efficiently [154].

Furthermore, CNPs can be tailored to target specific viruses by modifying the surface properties or functionalizing the nanoparticles with specific ligands that recognize viral components [155]. This targeted approach increases the efficacy of CNPs and minimizes potential side effects, making them a highly attractive option for developing antiviral therapies [155]. Fig. 5 illustrates the antiviral mechanism of CNPs in inhibiting viral infections.

Fig. 5.

The antiviral mechanism of chitosan nanoparticles (CNPs) in the inhibition of viral infections.

5.1.1.3. Antifungal activity of CNPs

Fungi provide essential functions in ecosystems, especially in the decomposition of organic materials and the cycling of nutrients [156,157]. However, some fungi are pathogenic to humans, causing various infections [158]. These infections can range from superficial to systemic and life-threatening, particularly in immunocompromised individuals [158].

CNPs exhibit potent antifungal activity and are particularly useful for combating fungal infections. The primary mechanism involves the disruption of the fungal cell wall and membrane [159]. The cell wall of fungi is a complex structure composed of chitin, glucans, and proteins, which maintain the cell's shape and integrity. The positively charged CNPs interact with the negatively charged components of the fungal cell wall, such as glucans and chitin, causing structural damage to the cell wall, compromising its integrity, and increasing its permeability [159]. The fungal cell membrane exhibits increased permeability, impacting vital internal components such as ions, proteins, and nucleic acids. This leakage results in the loss of cellular function and ultimately cell death [159].

Furthermore, CNPs may prevent chitin production, a crucial component of the fungal cell wall, hence affecting cell wall development and further weakening the structural integrity of fungal cells. In addition, CNPs can penetrate fungal cells and interfere with intracellular processes [160]. They can bind to DNA and RNA, inhibit nucleic acid synthesis, and affect vital cellular functions, such as replication and transcription [161].

Furthermore, the biodegradability and biocompatibility of chitosan enhance its appeal as an antifungal agent, minimizing potential side effects and environmental impact [162]. Thus, CNPs may be considered a promising alternative to conventional antifungal drugs, particularly in the face of rising antifungal resistance [162].

5.1.1.4. Antiprotozoal activity of CNPs

Protozoa are single-celled eukaryotic organisms that thrive in various habitats and exhibit various behaviors. Protozoa play significant roles in ecosystems, contributing to the decomposition of organic matter and serving as a food source for larger organisms [163]. Nevertheless, some protozoa are pathogenic and can cause severe diseases in humans and animals [164]. These diseases can range from mild gastrointestinal discomfort to severe, life-threatening conditions, particularly in regions with limited access to medical care [164].

CNPs offer a mechanism of action against protozoa similar to bacteria and fungi, primarily involving the disruption of the protozoan cell membrane. The positively charged CNPs interact with the negatively charged components of the protozoan cell membrane, causing structural damage and increased permeability [165]. This interaction leads to the formation of pores in the membrane, resulting in the leakage of essential intracellular contents, which compromises cellular function and leads to cell lysis and death [165].

In addition to causing membrane disruption, CNPs can interfere with the metabolic activities of protozoa by penetrating the cell and disrupting various intracellular processes, including DNA replication, transcription, and protein synthesis [166]. CNPs bind to nucleic acids and inhibit the synthesis of vital macromolecules, thereby impairing the growth and reproduction of protozoan cells. This multifaceted mode of action ensures a comprehensive approach to protozoan control, reducing the likelihood of resistance development [166].

Moreover, the ability of CNPs to enhance the host's immune response further contributes to their effectiveness against protozoan infections. By stimulating the activity of immune cells and promoting the production of cytokines, these nanoparticles help the host's immune system recognize and eliminate protozoan pathogens more efficiently [167]. The biocompatibility and biodegradability of CNPs make them an attractive alternative to traditional antiparasitic drugs, which are often accompanied by significant side effects and the risk of developing resistance [167].

As research continues to explore the full potential of CNPs, their application in treating protozoan infections holds great promise for improving public health, particularly in areas where protozoan diseases are endemic [167].

5.1.1.5. Antialgae activity of CNPs

Algae are predominantly found in aquatic environments, including freshwater and marine ecosystems, which are crucial for maintaining the ecological balance [168]. Algae contribute significantly to global oxygen production and are essential for the survival of many aquatic organisms [169]. In addition to their ecological importance, algae have substantial economic value [170,171]. However, excessive algal growth, often called algal blooms, can lead to environmental problems in water bodies, killing fish and disrupting aquatic ecosystems [172].

CNPs inhibit algal growth by binding to the algal cell surfaces. The positively charged CNPs interact with the negatively charged components of the algal cell membranes, causing structural damage [173]. This interaction disrupts the integrity of the cell membrane, leading to increased permeability and leakage of essential cellular contents, cell dysfunction, and death [173].

Furthermore, CNPs can interfere with photosynthesis in algae by blocking light absorption, which prevents algae from effectively capturing light energy, which is crucial for converting carbon dioxide and water into glucose and oxygen [174]. This mechanism inhibits the proliferation of algae and reduces the risk of resistance development. The potential of CNPs to address algal overgrowth and its associated environmental impacts is increasingly evident [174].

5.1.1.6. Antiarchaea activity of CNPs

Archaea are a group of single-celled prokaryotes that are genetically distinct from eubacteria despite their similar size and structure [175]. Archaea can thrive in extreme environments, such as hot springs, salt lakes, deep ocean vents, and even acidic or alkaline habitats [176]. Archaea have unique metabolic pathways, enabling them to utilize unconventional energy sources like methane and hydrogen gas, further distinguishing them from other microorganisms [177]. Despite their adaptability, some archaeal species act as pathogens, causing infections in humans and animals, although such occurrences are relatively rare compared to bacterial or fungal infections [178]. Researchers have shown interest in exploring the potential for targeting archaea for therapeutic purposes. However, the antimicrobial mechanisms that can disrupt these organisms remain poorly understood [179,180].

The primary proposed mechanism by which CNPs act against archaea is by disrupting the archaeal cell membrane [180]. Like bacteria, the positively charged CNPs interact with the negatively charged components of the archaeal cell membrane, leading to structural damage, increased membrane permeability, leakage of intracellular contents, cell dysfunction, and death [180]. Additionally, CNPs may target the unique metabolic pathways of archaea. Given the distinct biochemical processes of archaea, CNPs could interfere with essential metabolic functions, such as energy production and nutrient processing [181], thereby inhibiting the growth and survival of archaeal cells [181].

Further research is needed to fully understand the extent of the antimicrobial effects of CNPs on archaea and to develop targeted strategies for their use in archaea-related diseases or environmental management.

5.1.2. Anti-inflammatory properties of CNPs

CNPs possess significant anti-inflammatory properties and are highly beneficial for individuals with various inflammatory skin conditions, such as psoriasis, rosacea, and dermatitis [182]. Chronic inflammation is a hallmark of these conditions, often leading to skin irritation, redness, swelling, and discomfort. The nanoparticles work by modulating the body's inflammatory response and reducing the production of pro-inflammatory cytokines and mediators contributing to the irritation and flare-ups seen in these conditions [183].

By inhibiting these inflammatory pathways, CNPs help calm the skin, reduce the redness and swelling, and alleviate the discomfort associated with such disorders [184]. In addition to lowering general inflammation, CNPs can help restore the skin's barrier function, which is often compromised in conditions like eczema and psoriasis [184].

For people with sensitive or reactive skin, these nanoparticles offer long-term comfort by strengthening the skin's natural defences against irritation and lowering the chance of further flare-ups [185]. Moreover, the ability of CNPs to penetrate deeper layers of the skin ensures that they can deliver anti-inflammatory effects directly to the affected areas, in which traditional topical treatments may be less effective [185].

This targeted approach enhances the skin's immediate calming and contributes to the long-term management of these conditions. Furthermore, the gentle nature of CNPs means that they can be used on delicate or inflamed skin without causing additional irritation, making them suitable for those with conditions like rosacea, in which skin sensitivity is a concern [185]. CNPs offer a promising solution for managing inflammation in various dermatological conditions, promoting healthier skin, and preventing recurrent flare-ups [184,185].

5.1.3. Antioxidant properties of CNPs

CNPs are renowned for their potent antioxidant properties, which are crucial in protecting the skin from the harmful effects of oxidative stress caused by free radicals [186]. Free radicals are unstable molecules that can damage healthy skin cells, accelerate the aging process, and contribute to the development of various skin concerns, including wrinkles, fine lines, and skin sagging [187]. The antioxidant capabilities of CNPs help neutralize these free radicals, preventing cellular damage and reducing the visible signs of aging [186]. By preventing oxidative stress, the nanoparticles shield the skin from external and internal factors that cause premature aging, like pollution, smoking, and UV radiation [188]. CNPs also aid in improving the skin's general tone and texture by lowering oxidative stress and boosting its firmness and elasticity [188].

Thus, CNPs are valuable in anti-aging skincare because they support collagen production and maintain skin hydration, thereby visibly reducing the appearance of fine lines and wrinkles [189]. Moreover, CNPs provide long-lasting protection against future oxidative damage, offering a preventive approach to skin aging [189]. Additionally, the antioxidant properties of CNPs can benefit individuals with acne. By neutralizing free radicals, these nanoparticles help calm irritated skin and promote faster healing of acne lesions [189].

Antioxidants protect cells against oxidative stress by neutralizing harmful free radicals and reactive oxygen species (ROS), which are implicated in various diseases and aging processes. The unique chemical structure of chitosan is particularly influenced by its –NH₂ and –OH groups, which effectively scavenge free radicals, chelate metal ions, and inhibit lipid peroxidation [190]. Consequently, CNPs are used in diverse fields, such as food preservation, pharmaceutical drug delivery, and cosmetic formulations, in which their antioxidant potential enhances product stability, efficacy, and longevity [191].

5.1.3.1. Free radical scavenging

CNPs exhibit a strong ability to neutralize various free radicals, including highly reactive species like OH⁻ radicals, superoxide anions (O₂⁻), and peroxyl radicals (ROO•) [192]. These radicals are mainly implicated in oxidative stress and the damage of cellular components, such as lipids, proteins, and DNA [192]. The unique chemical structure of chitosan, particularly its functional groups, plays a critical role in its antioxidant activity [193]. The –NH₂ and OH⁻ groups in CNPs are particularly effective in interacting with and neutralizing these radicals [193].

The –NH₂ groups can donate a hydrogen atom or an electron to stabilize the free radical, converting it into a less reactive species. Likewise, OH⁻ groups, with their ability to form hydrogen bonds, can also trap and neutralize radicals through similar electron-donating mechanisms [194]. Both electron donation and hydrogen atom transfer enhance the scavenging capacity of CNPs, making them particularly effective in mitigating oxidative damage [194]. The smaller particle size and the bigger surface area of the nanoparticles can influence their radical-scavenging efficiency, making CNPs a promising candidate for reducing oxidative stress in biological systems and extending the shelf life of products sensitive to oxidation [194].

5.1.3.2. Chelation of metal ions

CNPs exhibit significant chelating properties, particularly for metal ions such as iron (Fe²⁺, Fe³⁺) and copper (Cu²⁺), which are key players in the generation of ROS via Fenton and Haber–Weiss reactions [195]. In these reactions, metal ions convert hydrogen peroxide (H₂O₂) into highly reactive –OH radicals, leading to oxidative damage. The ability of CNPs to bind and sequester these metal ions effectively prevents the metal-catalyzed formation of ROS, thereby reducing oxidative stress [196].

The chelation process occurs through the interaction of the functional groups in chitosan, particularly the –NH₂ and –OH groups, with the metal ions [197]. These groups have a strong affinity for metal cations, allowing the nanoparticles to form stable complexes with the ions, rendering them unavailable for catalyzing oxidative reactions [197]. CNPs help shield cells from oxidative damage that may otherwise result in inflammation, aging, and several chronic diseases by blocking metal-mediated ROS generation [197].

Moreover, the chelating activity of CNPs extends beyond just iron and copper; they can also interact with other divalent and trivalent metal ions, further enhancing their potential as antioxidants [197]. The chelation property of CNPs makes it particularly valuable in applications wherein metal-induced oxidative damage is a concern, such as in food preservation, cosmetic formulations, and pharmaceutical products [196,197].

5.1.3.3. Inhibition of lipid peroxidation

Lipid peroxidation is a critical process during oxidative stress, wherein ROSs attack and degrade polyunsaturated fatty acids in cellular membranes [197]. This process leads to the formation of lipid peroxides, which disrupt the integrity of cell membranes, impair cellular function, and contribute to various pathological conditions, such as neurodegenerative diseases, cancer, and cardiovascular disorders. CNPs effectively inhibit lipid peroxidation and protect against this form of cellular damage [198].

The antioxidant activity of the nanoparticles, including their ability to scavenge free radicals and chelate metal ions, directly impacts the inhibition of lipid oxidation [199]. CNPs contribute to preserving the structural integrity of cell membranes, necessary for appropriate cellular communication, nutrition transport, and general cell viability, by inhibiting the production of lipid peroxides [199].

Furthermore, the ability of the nanoparticles to interact with lipid bilayers and stabilize them may enhance the fluidity and flexibility of the membranes, contributing to better cellular function [200]. This protective effect on lipids also helps prevent the cascade of damage that often results from lipid peroxidation, such as the activation of pro-inflammatory pathways and the release of cytotoxic compounds [200].

The ability of CNPs to inhibit lipid peroxidation is particularly beneficial in developing therapeutic and cosmetic products designed to combat aging, inflammation, and oxidative damage, as well as in food preservation, wherein the stability of fat-rich ingredients is a concern [200].

5.1.3.4. Enzyme modulation

In addition to directly scavenging free radicals and chelating metal ions, CNPs influence the activity of endogenous antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, which play vital roles in the body's defense against oxidative stress [201]. SOD catalyzes the conversion of superoxide anions (O₂⁻) into hydrogen peroxide (H₂O₂), while catalase further decomposes H₂O₂ into water and oxygen, preventing the accumulation of reactive species that can damage cellular components [202]. CNPs may enhance the expression or activity of these enzymes by directly interacting with the cells to promote enzyme synthesis or by modulating cellular signaling pathways involved in the antioxidant response [202].

Nanoparticles help strengthen the body's natural defense system by boosting the activities of SOD and catalase, thus allowing them to neutralize ROS and mitigate oxidative damage more efficiently [202]. This enzyme modulation helps reduce oxidative stress and protects cellular structures, including lipids, proteins, and DNA, from free radical-induced damage. Additionally, CNPs may exert a more sustained, long-term protective effect than conventional antioxidants by stimulating the body's antioxidant defense mechanisms [201,202]. These characteristics make them particularly promising for applications that enhance the body's resilience against oxidative stress in various contexts, including aging, inflammatory diseases, and chronic conditions associated with oxidative damage [201,202].

5.1.3.5. Synergy with other antioxidants

CNPs enhance the antioxidant activity of other natural antioxidants, such as polyphenols, vitamins, and flavonoids, via synergistic interactions [203]. Polyphenols are known for their free radical-scavenging properties, whereas vitamins like vitamin C and E significantly neutralize reactive species [203]. Combining CNPs with these antioxidants can lead to a complementary effect, wherein the CNPs may help improve the bioavailability, stability, and controlled release of the antioxidants, thereby increasing their effectiveness [204].

Additionally, the nanoparticles may enhance the solubility of lipophilic antioxidants, allowing them to act more efficiently in both aqueous and lipid environments, which is particularly important in complex systems like human cells or food matrices [205]. CNPs may also provide a structural scaffold that facilitates the sustained release of these antioxidants, prolonging their activity and reducing the rapid degradation often encountered in antioxidant compounds [206].

This synergistic effect can be particularly beneficial in various applications, such as functional foods, skincare products, and pharmaceuticals, wherein a robust, multifaceted antioxidant defense is desired [206]. The collaborative action between CNPs and other antioxidants enhances their collective ability to protect cells, tissues, and products from the damaging effects of oxidative stress, offering a more assertive approach to combat aging, inflammation, and oxidative-related diseases [206,207].

5.2. Interdisciplinary applications of CNPs in biomedicine

Polymeric nanoparticles play a vital role in the biomedical field, serving as essential tools for disease identification and management [208]. Their ability to encapsulate various therapeutic agents allows them to act as effective drug delivery systems, enhancing the efficiency of medication administration [208]. Moreover, bioactive compounds can covalently interact with the surfaces of these nanoparticles. Their ability to selectively interact with molecules that target specific cell membrane receptors and effectively infiltrate cells opens innovative avenues for drug delivery and gene therapy [209]. The surfaces of hydrophilic polymeric nanoparticles restrict nonspecific protein adsorption, making them suitable for use as carriers [209].

Chitosan is also recognized for its mucoadhesive properties, which enable innovative drug delivery systems through mucosal pathways and enhance the absorption of substances with limited mucus affinity [98]. These properties aid in the permeation of drugs through epithelial layers by facilitating the opening of tight junctions [210]. Chitosan's interaction with platelets and surface amino acid chains has led to extensive use in wound healing studies [211]. The hemostatic properties of chitosan are valuable in wound treatment, as it can activate neutrophils and macrophages, accelerate the development of tissue granulation, promote re-epithelialization, reduce scarring and contraction, and enhance hemostasis [212]. In addition, it offers features such as chemoattraction, pain relief, and intrinsic antibacterial properties when used as a wound dressing [212].

Research indicates that chitosan and its derivatives can effectively scavenge free radicals, with low-molecular-weight chitosan demonstrating distinct advantages in radical elimination over its higher molecular-weight counterpart [213]. According to Zhao et al. [214], the carboxyl and amino groups in chitosan may enhance its antioxidant properties by stabilizing free radicals. The superior biodegradability, modifiability, and biocompatibility of CNPs make them ideal candidates for therapeutic delivery [215]. The degradation products N-acetylglucosamine and glucosamine are known to be tolerated by humans, and the resulting intermediates are unlikely to elicit allergic reactions [215].

5.2.1. CNPs-assisted delivery systems for advanced therapies

The application of nanomaterials has emerged as a pivotal strategy for developing refined treatment methodologies, particularly for pulmonary and autoimmune disorders [216,217]. This approach leverages nanomaterial-assisted gene editing tools and synthetic biology techniques to promote more precise and efficient therapies [218].

5.2.1.1. Polymer nanoparticles for pulmonary delivery

Polymer nanoparticles, a significant category of nanomaterials, are solid colloidal particles made from natural, synthetic, or semi-synthetic biodegradable polymers [219]. Their diverse chemical and physical attributes are highly customizable for specific applications [220]. Optimizing these systems requires meticulous consideration of properties such as size, shape, porosity, surface charge, and crystallinity [221].

These nanoparticles typically form through the self-assembly of amphiphilic molecules and can encapsulate therapeutic agents as nano-capsules (a liquid core surrounded by a solid shell) or nanospheres (solid matrices) [222]. Polymer micelles, formed by the aggregation of hydrophobic polymer segments into a core shielded by hydrophilic segments, are particularly effective at transporting hydrophobic drugs in aqueous environments [223].

Polymers are categorized as natural (e.g., chitosan, sodium alginate, gelatin) or synthetic. Natural polymers are often preferred for their superior cytocompatibility and biodegradability, with chitosan and alginate known to enhance nebulization for pulmonary delivery [224,225]. However, they present limitations, such as potential safety concerns with chronic lung exposure to chitosan and the rapid drug release profile of alginate [226]. In contrast, synthetic polymers offer enhanced control over delivery profiles and release kinetics, enabling more efficient and sustained drug release [226]. A notable example is the development of pH-responsive amphiphilic polymer nanoparticles (mPEG-PC7A) that efficiently co-deliver Cas9 ribonucleoprotein and single-strand oligonucleotides to the lungs for cystic fibrosis gene therapy via endocytosis [218].

The advantages of polymers in pulmonary drug delivery include manipulable surface properties, protection of drugs from degradation, and the facilitation of sustained release [227]. For instance, the uniform three-dimensional structure of dendrimers allows for the incorporation of diverse bioactive agents, enabling efficient conveyance of drugs with varying solubilities and the treatment of inflammatory conditions like asthma [228,229].

Furthermore, polymers like poly(lactic-co-glycolic acid) (PLGA) are extensively explored as carriers for various pulmonary medications and are even food and drug Administration (FDA)-approved for gene transfer [230]. Despite these accomplishments, challenges persist, including the dose-dependent toxicity of dendritic macromolecules and the mechanical deficiencies of some natural polymers [230].

5.2.1.2. Nanomaterial strategies for autoimmune diseases

Autoimmune diseases (ADs) are chronic, relapsing inflammatory conditions that pose a significant burden. Conventional immunosuppressive therapies often reduce systemic immune function, potentially leading to opportunistic infections. Biomaterial-based drug delivery systems (DDSs) offer significant advancements through improved bioavailability, specific targeting, and precise controlled release of drugs [217]. Their inherent immunomodulatory functions and ability to target immune cells can also provide novel therapeutic strategies to induce immune tolerance [231].

Many traditional AD medications can be reformulated into nanomaterial-based DDSs to enhance bioavailability and reduce adverse effects. For instance, the lipophilic immunosuppressant Cyclosporin A (CsA) has poor solubility and unstable oral bioavailability. Liposomes can effectively solubilize CsA and enhance its oral absorption [231]. Similarly, chitosan-coated microspheres can prolong their residence time in the gastrointestinal tract (GIT), prevent enzymatic degradation, and improve absorption [232,233]. An optimized lipid particle system (∼40 nm) was also developed for the lipophilic drug Tacrolimus, significantly improving its lymphatic delivery and oral bioavailability compared to commercial capsules [234].

Proteins and peptides pose a greater challenge for oral delivery due to their susceptibility to denaturation in the GIT. Insulin, essential for Type 1 diabetes mellitus (T1DM), is a primary focus for oral formulation development to replace subcutaneous injections [235]. Strategies include using hyaluronic acid-coated chitosan nanoparticles (HCPs) to promote enzymatic stabilization, control release, and enhance cellular uptake [235].

For inflammatory bowel disease (IBD), an AD of the GIT, oral delivery is the preferred method. Research focuses on creating stimuli-responsive DDSs that target the inflamed colon using triggers like the GIT's pH gradient, transit time, and bacterial enzymes [236]. For example, a pH-sensitive composite hydrogel of hyaluronic acid and gelatin (HA/GE) combined with carboxymethyl chitosan microspheres was developed to extend drug release at the colonic site, remaining stable at pH 7.4 but accelerating release at the inflamed site's pH of 6.8 [237].

Enzyme-responsive CNPs designed to be degraded by specific colonic enzymes have also shown effective colon-targeting, reducing inflammatory markers in colitis mice [238]. Another innovative concept uses fermentation by intestinal microorganisms as a release trigger. One design used alginate hydrogel microspheres containing Bifidobacterium bifidum and drug-modified nanoscale dietary fiber [239]. The fermentation in the colon facilitated drug release and acted as a probiotic to modulate intestinal microbiota and alleviate inflammation [239].

5.2.1.3. CRISPR/Cas9 delivery and challenge

The high specificity and efficiency of CRISPR/Cas9 gene editing technology make it a powerful tool for treating human diseases, especially those involving multiple genetic modifications [240]. A major clinical challenge, however, is the development of optimized delivery vectors. Biomaterials and nanomaterials are considered the best choice for this role due to their tunability, biocompatibility, and efficiency, fueling hope for their clinical application in oncology [240].

Chitosan is a natural polymer with good biocompatibility but relatively low transfection efficiency. This can be improved by modifying it with compounds like 5β-bile acid or stearic acid to enhance its interaction with cell surfaces [241,242]. Zhang et al. [243] demonstrated this by using lactate-targeted CNPs (CLPV nanoparticles) to co-deliver a sgVEGFR2/Cas9 plasmid and paclitaxel (PTX) for hepatocellular carcinoma therapy. This system showed sustained release, good cellular uptake, a synergistic therapeutic effect, and a 38% genome editing efficiency, inhibiting tumor progression by more than 70% in a mouse model [243]. A significant limitation of chitosan is its poor solubility, caused by extensive hydrogen bonding, which can restrict its interaction with guide RNA (sgRNA) [244].

5.2.1.4. Advanced models for drug screening

The development of accurate disease models and effective drug screening is a key area of modern medical research. Traditional methods often fail to replicate the complex architecture of human tissues [245]. Advances in biomaterial-assisted organoid technology are revolutionizing biomedical research by enabling the cultivation of 3D cellular structures in vitro that closely emulate organ function. The evolution of biomaterials is pivotal in supporting organoid culture, thereby facilitating more accurate disease modeling and rigorous evaluation of drug efficacy and safety [245].

5.2.1.4.1. Ocular pharmaceutical administration

Due to their mucoadhesive properties, CNPs are beneficial for controlled drug delivery through mucosal membranes [246]. When exposed to nearly neutral aqueous solutions, CNPs form a gel layer on their surface, which prolongs their retention time on the mucosal surfaces and improves drug delivery to the ocular tissues [246].

Sulfobutylether-β-cyclodextrin (SBE-β-CD) crosslinked CNPs were investigated for their potential to deliver the antifungal drug econazole nitrate (ECO) to the eye of albino rabbits [43]. The loading percentage of the ECO drug ranged from 13% to 45%. In vivo tests indicated that CNPs loaded with ECO were more effective in treating ocular fungal infections than ECO solution, demonstrating their significant potential as pharmaceutical carriers for antifungal ocular therapies [43].

Furthermore, Santhi et al. [247] produced fluconazole-loaded CNPs with an average particle size of 152.85 ± 13.7 nm using the emulsification preparation and demonstrated an ideal drug-loading capacity of more than 50%, as confirmed by comparing their antifungal efficiency with that of fluconazole eye drops using the cup-plate technique [247]. Their findings emphasized the advantageous characteristics of CNPs, such as substantial drug-loading capacity, potent antifungal efficacy, and sustained drug release for fluconazole treatment [247].

5.2.1.4.2. Oral drug delivery

The popularity of oral drug delivery systems can be attributed to several factors, such as ease of administration, controlled release, low production costs, and improved patient compliance [248]. However, traditional oral drug delivery methods face challenges, such as solubility issues in acidic gastric fluid, degradation, and reduced efficacy due to enzymatic action, and poor membrane permeability [248]. Nanomedicine offers promising solutions to overcome these challenges in oral drug distribution [249].

CNPs are excellent candidates for improving the administration of oral medications owing to their physicochemical characteristics, which include biocompatibility, mucoadhesion, flexibility in drug conjugation, and high surface-to-volume ratios [250]. In diabetic rats, the oral administration of insulin attached to positively charged CNPs, ranging in size from 250 to 400 nm, improved intestinal insulin absorption and normalized glucose levels for extended periods by adjusting the insulin dosage within the CNPs [250].

Although the specific mechanism remains uncertain, CNPs stabilize insulin in the GIT, facilitating its absorption [250]. CNPs were orally administered to dogs to assess the bioavailability enhancement of lipophilic drugs like cyclosporine [43]. The concentration of the medicine was measured by collecting blood samples at different time points after administration. The average diameter and Zeta potential of the chitosan hydrochloride nanoparticles were 148 nm and +31 mV, respectively. Using the commercial microemulsion Neoral®, the study demonstrated that CNP encapsulation enhanced cyclosporine bioavailability by 73% compared with oral administration [43].

According to Grewal and Saral [251], positively charged CNPs interact better than neutral or negatively charged carriers with negatively charged GIT epithelial cells, improving permeability and medication absorption [251]. CNPs may enhance lipid metabolism impairment, which is closely linked to the gene expressions involved in lipogenesis and oxidative stress [252]. Furthermore, incorporating 2% CN into camel yogurt resulted in satisfactory sensory attributes and microbiological quality [253].

5.2.1.4.3. Pulmonary drug delivery

The lungs have many physiological characteristics that facilitate drug transport, such as a thin absorption barrier for drug uptake, a substantial surface area, and significant vascularization [254]. Analogous to oral medication delivery, pulmonary drug delivery is believed to leverage the physicochemical characteristics of CNPs, which include high encapsulation efficiency, mucosal membrane adhesion, antibacterial capabilities, positive charge, and prolonged drug release [254].

Numerous key studies have explored the effectiveness of controlled-release polymers of CNPs in lung drug delivery [255]. For instance, conjugated nanoparticles encapsulating elcatonin were used to develop a drug that lowered blood calcium levels using PLGA copolymer, which was then aerosolized for pulmonary administration [255]. CNP-PLGAs loaded with elcatonin facilitated a prolonged release of the medication for up to one day, indicating effective delivery [255]. The positive charge of PLGA-modified CNPs facilitates the opening of tight junctions in lung epithelial cells, thereby enhancing drug uptake [255]. Rawal et al. [255] also synthesized CNPs containing rifampicin for pulmonary injection in rats to evaluate medication delivery for tuberculosis. A key advantage of pulmonary delivery is its potential to minimize severe side effects associated with oral medication. The laboratory results demonstrated prolonged drug release for 24 h with minimal toxicity [255].

5.2.1.4.4. Nasal drug delivery

The nasal route offers an efficient, non-invasive platform for delivering vaccines, nucleic acids, peptides, and other therapeutic agents encapsulated in nanocarriers [256]. This delivery pathway is particularly advantageous due to its potential to stimulate a robust immune response [256]. However, nasal drug absorption faces significant physiological barriers, including the presence of mucus gel, mucociliary clearance, and limited permeability of the nasal epithelium to lipophilic compounds [256].

CNPs have emerged as promising carriers for nasal drug delivery owing to their mucoadhesive properties, biocompatibility, and low toxicity [167]. One study explored the use of thiolated CNPs loaded with leuprolide—a peptide drug used to treat uterine fibroids, prostate cancer, and endometriosis—to enhance bioavailability via nasal administration [257]. The results demonstrated significantly improved leuprolide absorption in rats when delivered intranasally through thiolated carbon nanoparticles compared to the administration of a leuprolide solution alone [257].

A major limitation of conventional oral administration of antiepileptic drugs is the restricted passage across the blood-brain barrier, which can contribute to drug resistance. To address this, Liu and Ho [258] investigated the intranasally delivery of carbamazepine—an anticonvulsant—encapsulated in carboxymethyl CNPs. The synthesized CNPs exhibited an average size of 219 nm and a drug entrapment efficiency of approximately 80% [258]. In vivo studies in mice revealed that intranasal administration of these nanoparticles significantly enhanced both the bioavailability and brain-targeting efficiency of carbamazepine compared to the intranasal delivery of the drug solution [258]. However, the authors noted that the limited volume capacity of the nasal cavity remains a challenge that may constrain the overall therapeutic efficacy of this approach [258].

5.2.1.4.5. Buccal drug delivery

Buccal drug delivery represents a valuable alternative for administering therapeutics, especially macromolecules unsuitable for oral delivery [259]. This transmucosal route bypasses gastrointestinal degradation and first-pass hepatic metabolism, potentially reducing the required drug dose while enhancing systemic bioavailability [260].

A notable example involves the use of curcumin-loaded CNPs for the treatment of periodontal disease. These nanoparticles were incorporated into buccal films and coated with polycaprolactone to ensure stability and controlled release [260]. AFM and SEM confirmed the uniform dispersion of CNPs throughout the film matrix. In vitro studies conducted in simulated saliva demonstrated that the films achieved optimal swelling at approximately 80% moisture content, facilitating sustained curcumin release—an essential feature for effective periodontal therapy [260].

5.2.1.4.6. Vaginal pharmacological administration

The vaginal mucosa represents a viable alternative route for drug delivery, supplementing other mucosal pathways such as buccal administration. Vaginal delivery serves two primary purposes: localized treatment of vaginal conditions and systemic drug absorption through the highly vascularized mucosa [261,262]. Due to their mucoadhesive and bioconjugative properties, CNPs have emerged as promising carriers for vaginal distribution that aim to produce systemic effects [261]. Due to their mucoadhesive properties, CNPs have emerged as promising carriers for vaginal drug delivery, particularly when systemic effects are desired [262].

However, effective drug delivery via the vaginal route faces several physiological challenges, including an acidic pH (3.8–4.5), high mucosal fluid secretion, and the complex folding of the epithelial tissue. To address these barriers, Martínez-Pérez et al. [263] developed a formulation combining CNPs with PLGA and infused it with clotrimazole for enhanced vaginal antifungal therapy. In vitro results demonstrated improved antifungal activity with the CNP-PLGAs formulation compared to conventional treatments lacking CNPs [263,264].

In a related study, a hydrogel system comprising chitosan and hydroxypropyl methylcellulose (HPMC) was developed for treating fungal infections caused by Candida albicans and non-albicans strains [264]. The system incorporated either CNPs or monomolecular chitosan in combination with metronidazole. In vitro evaluations and mucoadhesion tests confirmed that hydrogels containing CNPs or monomolecular chitosan exhibited superior antifungal activity and enhanced mucoadhesive properties, making them effective candidates for vaginal drug delivery systems [264].

5.3. Cancer treatment: targeted and multifunctional approaches

Cancer encompasses a diverse group of diseases marked by the uncontrolled proliferation and invasion of abnormal cells across various tissues. Traditional treatments, including chemotherapy and radiotherapy, although effective, are often associated with systemic toxicity and limited specificity for tumor cells [265].

Recent advances in nanotechnology, particularly the use of CNPs, have introduced more targeted and multifunctional strategies for cancer therapy [266]. Derived from chitosan, a naturally occurring biopolymer, CNPs possess unique characteristics that make them highly suitable for oncological applications [207]. These include their ability to deliver chemotherapeutic agents or genetic material to tumor sites selectively, induce oxidative stress in cancer cells, serve as immunoadjuvants in cancer vaccines, and promote apoptosis [207]. Such multifunctionality positions CNPs as promising tools in the development of next-generation cancer therapies with enhanced efficacy and reduced systemic toxicity [207].

The anti-cancer impact of chitosan nanoparticles is demonstrated in Table 5. As illustrated in Fig. 6, CNPs exhibit an anti-carcinogenic mechanism that encompasses several different signaling pathways.

Table 5.

Anticancer effect of chitosan nanoparticles.

Cancer Type	CNP modification/formulation	Loaded drug/agent	Mechanism/target	Model used	Key findings	Reference
Breast cancer	Folic acid-conjugated CNPs	Doxorubicin	Targeting folate receptor overexpressed on tumor cells, receptor-mediated endocytosis	MCF-7 (in vitro), murine model (in vivo)	Enhanced selective uptake; decreased systemic toxicity	[408]
Lung cancer	PEGylated CNPs	Paclitaxel	Prolonged circulation; improved tumor penetration and induction of apoptosis via microtubule stabilization	A549 cells (in vitro)	Enhanced cytotoxicity; improved stability and solubility of the drug	[409]
Neuroblastoma	pH-sensitive CNPs	Curcumin	This formulation evokes prolonged oxidative stress, stabilizing HIF-1α, and inducing caspase-dependent apoptosis; ROS-mediated apoptosis and G2/M arrest	Human childhood neuroblastoma (MYCN-amplified cells)	Superior antitumor activity in an acidic environment	[410]
Liver cancer	Galactosylated CNPs	5-Fluorouracil (5-FU)	ASGPR-targeted delivery to hepatocytes; inhibition of DNA synthesis	HepG2 (in vitro), rat model	Liver-specific accumulation; enhanced therapeutic index	[411]
Colorectal cancer	Chitosan-coated PLGA and PCL NPs	Docetaxel	Lowering the rate of DTX, in vitro release was achieved within 48 h by using chitosan-coated NPs. Furthermore, a tremendous increase in DTX cytotoxicity was observed by chitosan-decorated PLGA NPs compared to all other NPs, including DTX-free-NPs and pure DTX	HT29 colorectal cancer (in vitro), In vivo	The in vivo study revealed a significant enhancement in DTX bioavailability from chitosan-decorated PLGA NPs, with a more than 4-fold increase in AUC compared to DTX solution.	[412]
Lung cancer	Thiolated CNPs	Cisplatin	Enhanced mucoadhesion; improved transport across mucosal barriers; DNA cross-linking	NSCLC (in vitro)	Increased permeability and drug retention; reduced IC50	[413]
Glioblastoma	Transferrin-conjugated CNPs	Temozolomide	Blood–brain barrier penetration; transferrin receptor-mediated targeting	U87MG (in vitro), mouse model	Enhanced brain delivery and apoptosis induction	[414]
Melanoma	Mannose-modified CNPs	siRNA against BRAF V600E	Dendritic cell targeting; RNA interference mechanism	B16-F10 (in vitro and in vivo)	Gene silencing; tumor regression observed in mice	[415]
Pancreatic cancer	Lipid-coated CNPs	Gemcitabine	Enhances cell membrane penetration and drug stability	PANC-1 (in vitro)	Improved bioavailability and cytotoxicity	[416]
Cervical cancer	Antibody-conjugated CNPs (anti-EGFR)	Doxorubicin	Active targeting via EGFR recognition; increased intracellular delivery	HeLa (in vitro)	Stronger tumor specificity; lower off-target effects	[417]
Breast cancer	Chitosan/alginate NPs loaded with α-Mangostin	α-Mangostin	Oral doses (20 mg) controlled body weight and shrank tumor volume nearly as effectively as tamoxifen; histopathology showed tissue repair and apoptosis	DMBA-induced breast tumors (Wistar rats, in vivo)	Reduced tumor volume and body weight loss similar to tamoxifen	[418]
Colon cancer	Chitosan hydrogel + gold NP–paclitaxel	Paclitaxel	Induced apoptosis via BAX/BAD upregulation and BCL2 downregulation; showed potent cytotoxicity	LS174T colon cancer cells (in vitro)	Induced BAX/BAD expression and reduced BCL2; enhanced apoptosis	[419]
Esophageal cancer	Plain chitosan NPs	–	Inhibited cancer-associated fibroblast proliferation and motility; downregulated CXCR4, CXCR7, CCR5, SDF-1α markers linked to metastasis	Human CAFs (in vitro)	Suppressed CXCR4/7 and SDF-1α; reduced proliferation/motility	[420]
Lung (NSCLC)	HA-modified chitosan NPs with anti-BCL2 siRNA	siRNA	Improved CD44-mediated targeting, efficient BCL2 silencing, and inhibited tumor growth in vivo	A549 cells and in vivo NSCLC model	Efficient BCL2 silencing; CD44-targeting; tumor regression	[421,422]
Pancreatic cancer	CNPs with AMTB hydrochloride	AMTB	Enhanced anti-cancer activity over free drug, triggered apoptosis and ferroptosis pathways	Pancreatic cancer cells (in vitro)	Triggered apoptosis and ferroptosis	[423]
Oral squamous cell carcinoma	Cisplatin-gold-chitosan nanocomposite	Cisplatin and gold NPs	Increased ROS, DNA damage, cell cycle arrest with selective tumor cytotoxicity	Patient-derived cultures (in vitro)	Trigger apoptosis	[424,425]
Lung cancer	Chitosan/hyaluronic acid hydrogel (pH-responsive) with CDDP and doxorubicin	Cisplatin and doxorubicin	Dual release enhanced cytotoxicity versus a single-loaded system	A549 cells (in vitro)	Cytotoxicity studies performed in A549 lung cancer cells confirmed the enhanced activity of the material as a dual drug carrier compared with the single-loaded system	[426]
Colon cancer	Chitosan hydrogel/gold NP/Paclitaxel complex	Paclitaxel	Upregulated BAX/BAD, downregulated BCL2; potent apoptosis	LS174T cells (in vitro)	Trigger apoptosis	[419]
Breast and liver cancer	Anthocyanin + cisplatin/chitosan NPs	Anthocyanin + Cisplatin	Mitochondrial apoptosis, reduced migration/angiogenesis markers	MCF-7 and HepG2 cells (in vitro)	Trigger mitochondrial apoptosis	[427]
Breast cancer	Zinc oxide/Chitosan conjugate and Cisplatin	Cisplatin	Enhanced selectivity and apoptosis; minimal necrosis	MCF-7 cells (in vitro)	Trigger apoptosis	[428]
Cervical cancer	Cisplatin-loaded, chitosan-coated solid lipid NPL	Cisplatin	Lower IC50 (0.61 μg/mL); sustained release; higher apoptosis	HeLa cells (in vitro), and animal model	Trigger apoptosis	[428]
Breast Cancer	Doxorubicin/loaded GO–Chitosan/β-GP hydrogel	Doxorubicin	Controlled release; enhanced uptake and cytotoxicity	MCF-7, MDB-231, FaDu (in vitro)	Trigger cytotoxicity	[429]

PLGA, poly(lactic-co-glycolic acid); NPs, nanoparticles; CNPs, chitosan nanoparticles; ROS, reactive oxygen species; DTX, docetaxel; PCL, polycaprolactone; IC₅₀, half maximal inhibitory concentration; EGFR, epidermal growth factor receptor; NCSLC, non-small-cell lung cancer; AUC, area under curve.

Fig. 6.

The anti-carcinogenic mechanism of chitosan nanoparticles (CNPs), including detailed signaling pathways. ROS, reactive oxygen species; ER, endoplasmic reticulum.

5.3.1. CNPs for cancer gene therapy and immunotherapy

CNPs possess a unique combination of attributes—biodegradability, biocompatibility, excellent membrane permeability, high drug-loading efficiency, pH-responsive release, multifunctionality, and prolonged circulation time—that make them highly attractive for cancer chemotherapy and diagnostic applications [54]. To modulate specific cancer-related genes, exogenous nucleic acids are introduced into tumor cells or their microenvironment [267]. However, challenges such as inefficient targeting, charge repulsion at the cell membrane, and limited endosomal escape hinder the efficacy of nucleic acid-based therapies. Thus, the development of efficient gene delivery systems is critical [267].

Gene delivery typically relies on viral or non-viral vectors. While viral vectors offer high transfection efficiency, their use in cancer gene therapy is limited due to safety concerns, including mutagenicity and carcinogenicity [268]. Conversely, non-viral vectors—such as liposomes, diverse nanoparticles, and polymer-based carriers—are increasingly recognized as safer alternatives [268]. Among these, liposomes are well-studied but suffer from drawbacks like low encapsulation efficiency, non-specific toxicity, limited stability, and short shelf life [269].

Cationic polymers have emerged as promising gene carriers due to their ability to encapsulate genetic material effectively and their proton sponge effect, which facilitates endosomal escape [270]. Chitosan stands out among these polymers for its low toxicity and favorable physicochemical properties. It readily forms complexes, microspheres, or nanoparticles with nucleic acids through electrostatic interactions, making it particularly suitable for gene delivery applications [271].

Chitosan's structural versatility has encouraged its widespread exploration in cancer gene therapy. The presence of abundant free amine groups enables the conjugation of chemotherapeutic agents. For instance, water-soluble doxorubicin (DOX) has been successfully linked to chitosan via a succinic anhydride spacer [272] facilitates carboxylic formation and subsequent conjugation using carbodiimide chemistry [273]. The resulting chitosan-DOX complex self-assembles into nanoparticles under mild aqueous conditions. Interestingly, increasing DOX concentrations reduced conjugation efficiency, highlighting the importance of dose optimization [273].

Further functionalization with targeting moieties enhances the specificity of CNPs. For example, trastuzumab, a monoclonal antibody targeting the human epidermal growth factor receptor 2 (Her2+), was covalently attached to chitosan-DOX nanoparticles through thiolated lysine residues [273]. This targeted formulation demonstrated superior selectivity and uptake in Her2+ cancer cells compared to non-targeted CNPs or free drugs, underscoring its potential in receptor-specific drug delivery [273].

To overcome challenges associated with poorly water-soluble drugs, CNPs have been engineered to encapsulate hydrophobic compounds [272]. Glyceryl monooleate-chitosan core-shell nanoparticles, synthesized via the emulsification-evaporation method, significantly enhanced the anticancer efficacy of paclitaxel in MDA-MB-231 breast cancer cells, reducing the IC₅₀ value by 1000-fold—an effect that could potentially minimize off-target toxicity [272].

Amphiphilic CNPs have also been developed for paclitaxel delivery by modifying glycol chitosan with 5β-cholanic acid or incorporating human chorionic gonadotrophin (HCG) nanoparticles [274]. These formulations achieved encapsulation efficiencies of up to 80%, surpassing the conventional Cremophor EL-based system and showing reduced cytotoxicity [272]. Moreover, tumor-specific ligands can be conjugated to CNPs for receptor-mediated targeting. Such ligand-functionalized CNPs can be internalized by cancer cells through receptor–ligand interactions and can release their payload in the acidic endo-lysosomal environment via pH-sensitive linkers [272].

Chitosan is also gaining recognition as a potent immunoadjuvant in cancer vaccine development [275]. As adjuvants, chitosan plays a critical role in enhancing immune responses; researchers are investigating polysaccharide-based alternatives for safer and more effective vaccination strategies [275]. Chitosan's cationic nature, biocompatibility, and antigen-carrying capacity make it ideal for this purpose [276]. Although its immunostimulatory potential has been known for over two decades, only recently has chitosan been seriously considered as a non-toxic adjuvant for cancer and infectious disease vaccines [277,278].

CNPs have garnered attention for their immune-regulatory and anti-fibrotic effects, owing to their unique physicochemical properties and capacity for cellular interaction [279]. Mechanistically, CNPs exert pronounced immunomodulatory effects by acting on pivotal immune cells such as dendritic cells and macrophages. Chitosan facilitates dendritic cells activation and maturation, enhancing antigen presentation and downstream T-cell activation, which are indispensable for robust innate and adaptive immune responses [279,280].

Intriguingly, these effects can occur via TLR4-independent pathways, in which cGAS–STING and interferon signaling play key roles, and are accompanied by the upregulation of costimulatory molecules, such as CD80 and CD86, as well as the selective modulation of cytokines-elevating IFN-γ, TNF-α, and IL-1β while limiting IL-12 production [280]. The immunostimulatory impact of chitosan also extends to its molecular weight: low-molecular-weight CNPs more potently increase macrophage pinocytic activity and stimulate secretion of pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL-6, as well as inducible nitric oxide synthase (iNOS), suggesting a bias toward M1 macrophage polarization [280,281]. These responses may be traced to GlcNAc units within the chitosan backbone, rather than GlcN residues, thus elucidating further structural specificity in their immune activity [[279], [280], [281]].

Beyond direct immunostimulation, CNPs can act as vaccine adjuvants or delivery vehicles for antigens, amplifying the desired immune protection by targeting dendritic cells and promoting both systemic and mucosal immunity [282,282]. Notably, in plant systems, CNPs have demonstrated the ability to enhance innate immunity by upregulating defense enzymes, antioxidant genes, and increasing nitric oxide as a signaling molecule [282,283]. This induction is often accompanied by increased phenolic content and heightened resistance to stress, highlighting the conserved potential of chitosan nanostructures for immune potentiation across biological kingdoms and possibly offering templates for biomedicine [[281], [282], [283]].

With respect to anti-fibrosis, CNPs show clear molecular and physiological impacts in diverse models [284]. Fibrosis, characterized by excessive extracellular matrix (ECM) deposition and altered tissue architecture, often involves heightened TGF-β1 signaling, a driver of collagen and fibronectin synthesis, as well as the inhibition of ECM-degrading proteases [285,286]. Experimental data showed that CNPs, either alone or co-delivered with agents such as curcumin, significantly attenuate muscle fibrosis in injury models [280]. This occurs via the downregulation of TGF-β1 and TNF-α, which limits the fibroproliferative environment and supports muscle regeneration, as indicated by enhanced myotube diameter and more robust regenerative tissue [280]. Notably, these effects are dose-dependent and are demonstrated in both the short-term and healing phases, suggesting a durable modulation of fibrotic pathways [280].

Moreover, CNPs have been shown to modulate key cellular processes underlying tissue scarring, such as autophagy and oxidative stress [281]. For instance, CNPs have been reported to upregulate Beclin-1 and LC3-II, proteins crucial for autophagic turnover, thereby contributing to decreased ECM accumulation and tissue repair, as well as reducing toxic beta-amyloid aggregates in neurodegenerative models [287]. These effects are complemented by the antioxidant property of chitosan, which limits ROS-driven secondary fibrotic changes. Mechanistically, the nanoparticles' positive charge enhances their affinity for negatively charged cell surfaces and ECM components, promoting cellular uptake and facilitating interaction with the fibrotic microenvironment [281,287].

In clinical translational terms, by mitigating inflammation, regulating immune function, and remodeling tissue architecture, CNPs offer a highly versatile platform for disease-modifying interventions in immune-related and fibrotic disorders [287]. Ongoing research continues to unravel their signaling pathways and optimize nanoparticle design for maximal therapeutic gain [287].

5.3.2. Treatment of carcinoma cells

Carcinomas are malignant tumors originating from epithelial cells, which line various organs and tissues throughout the body, including the skin, lungs, liver, intestines, and others [288]. They present the most prevalent type of cancer and encompass several subtypes with distinct histological and biological characteristics [288]. Adenocarcinomas arise from glandular epithelial tissues and are commonly found in the lungs, colon, breast, and prostate [288]. Squamous cell carcinomas originate from flat, scale-like epithelial cells, present in the skin, lungs, and mucous membranes [288]. Basal cell carcinoma, the most prevalent form of skin cancer, originates from basal epidermal cells and, while infrequently metastatic, can result in considerable local tissue damage [288].

CNPs have emerged as a promising nanocarrier system for the targeted treatment of carcinoma. Owing to their positive charge, CNPs exhibit strong electrostatic interactions with the negatively charged membranes of carcinoma cells, enhancing cellular uptake via endocytosis [272]. Once internalized, CNPs can release therapeutic agents—such as chemotherapeutic drugs or genetic material—in response to specific stimuli in the tumor microenvironment, including low pH and overexpressed enzymes [289]. This stimuli-responsive release facilitates precise drug delivery, minimizing off-target toxicity and preserving surrounding healthy tissues [289].

Moreover, CNPs have demonstrated intrinsic anticancer activity by inducing apoptosis in carcinoma cells [290]. They promote the generation of ROS, leading to oxidative damage to cellular macromoleculaes such as DNA, proteins, and lipids. This oxidative stress impairs mitochondrial function, a key trigger for intrinsic apoptotic pathways [291]. Through these mechanisms, CNPs not only inhibit tumor proliferation but also prevent metastasis. The combined effects of targeted drug delivery, ROS-mediated cytotoxicity, and apoptosis induction position CNPs as a potent and versatile platform for carcinoma therapy, offering the potential for more effective and less toxic treatment options [291].

5.3.3. Treatment of sarcoma cells

Sarcomas are malignant tumors arising from mesenchymal tissues, including bone, cartilage, fat, muscle, or blood vessels [292]. Although less common than carcinomas, sarcomas are typically aggressive and can affect individuals across all age groups, including children and young adults [293]. They are classified based on tissue of origin: osteosarcoma (bone), chondrosarcoma (cartilage), and rhabdomyosarcoma (skeletal muscle) [293]. Despite their diversity, all sarcomas share the potential for invasion and distant metastasis [293].

CNPs offer a compelling strategy for sarcoma treatment due to their ability to exploit the unique features of tumor microenvironment. Functionalization of CNPs with targeting ligands or antibodies enables selective binding to surface markers overexpressed on sarcoma cells or associated vasculature, allowing for precise drug delivery [272]. Upon binding, CNPs are internalized by cancer cells and release their payload—commonly chemotherapeutic agents like DOX—directly into the cytoplasm, thereby enhancing cytotoxic efficacy while minimizing systemic side effects [294].

Similar to their mechanism in carcinoma, CNPs induce oxidative stress in sarcoma cells, resulting in mitochondrial dysfunction and subsequent activation of apoptotic cascades [295]. In addition, CNPs interfere with cellular processes such as cell cycle progression, a hallmark of sarcoma cell proliferation [296]. By arresting the cell cycle and promoting apoptosis, CNPs can effectively suppress tumor growth and proliferation [296].

Overall, the multifaceted therapeutic actions of CNPs—including targeted delivery, ROS generation, apoptosis induction, and disruption of cell cycle regulation—highlight their potential as a powerful nanotherapeutic approach for sarcoma treatment [297]. Their ability to selectively target tumor cells while reducing systemic toxicity underscores their promise for future clinical applications in oncology [297].

5.3.4. Treatment of leukemia cells

Leukemia is a malignancy of the blood and bone marrow characterized by the uncontrolled proliferation of dysfunctional white blood cells, which compromises normal hematopoiesis and immune function [298]. The disease is categorized into subtypes based on the rate of progression and the type of affected white blood cells. Acute lymphocytic leukemia (ALL), a rapidly progressing cancer of lymphoid cells, primarily affects children, whereas chronic lymphocytic leukemia (CLL) progresses more slowly and is commonly diagnosed in older adults [299]. Despite their clinical differences, all leukemia subtypes share a hallmark feature: excessive production of immature or dysfunctional white blood cells that leads to bone marrow failure and immunosuppression [300].

CNPs have emerged as promising nanocarriers for targeted leukemia therapy. These positively charged nanoparticles facilitate enhanced cellular uptake via endocytosis due to their electrostatic interaction with the negatively charged membranes of leukemia cells [301]. CNPs can encapsulate chemotherapeutic agents such as methotrexate and paclitaxel, allowing for controlled and sustained drug release in response to the tumor microenvironment [272]. This targeted delivery system enhances drug accumulation within leukemia cells, disrupts cell cycle progression, and impairs DNA replication and repair, ultimately inhibiting rapid cell division [272].

Beyond traditional drug delivery, CNPs can be engineered to transport small interfering RNA (siRNA) or microRNA (miRNA), which selectively silence genes responsible for chemotherapy resistance [18]. This gene-silencing capability can significantly sensitize leukemia cells to treatment, addressing one of the major hurdles in leukemia therapy [302]. In addition, CNPs have been shown to induce apoptosis by promoting mitochondrial dysfunction and oxidative stress, effectively triggering programmed cell death and reducing leukemic cell proliferation [302].

5.3.5. Treatment of lymphoma cells

Lymphoma is a hematologic malignancy that originates in the lymphatic system, which includes the lymph nodes, spleen, tonsils, and bone marrow—critical components of the immune system [303]. The disease results from the abnormal proliferation of lymphocytes, leading to compromised immune responses and symptoms such as lymphadenopathy, fever, weight loss, and night sweats [303].

CNPs provide a versatile platform for delivering chemotherapy agents directly to lymphoma cells [302]. Their ability to selectively accumulate in tumor tissues enhances the therapeutic index while minimizing systemic toxicity. In addition to their drug delivery function, CNPs can activate innate immune cells such as macrophages and dendritic cells, thereby enhancing anti-tumor immunity—a particularly valuable feature when treating lymphomas, which inherently involve the immune system [272].

Moreover, CNPs can be functionalized to block immune checkpoints, regulatory molecules that tumor cells exploit to evade immune detection [304]. By inhibiting these pathways, CNPs restore immune surveillance and support the immune system's capacity to target and destroy lymphoma cells [304]. Furthermore, CNPs can downregulate anti-apoptotic proteins that are often overexpressed in lymphoma cells, thereby promoting apoptosis and inhibiting growth and dissemination [305].

5.3.6. Treatment of myeloma cells

Myeloma is a type of cancer that originates in plasma cells, a subset of white blood cells responsible for producing antibodies to help combat infections [306]. In multiple myeloma—the most common and severe form—these plasma cells become malignant, proliferating uncontrollably and disrupting the production of healthy blood cells [306]. This malignancy primarily affects bone marrow, leading to complications such as bone lesions, anemia, kidney impairment, and immune dysfunction. Typically diagnosed in advanced stages and more common in older adults, multiple myeloma remains a challenging disease to manage [306].

CNPs offer promising therapeutic potential in the targeted treatment of multiple myeloma. These biodegradable and biocompatible carriers can encapsulate and deliver chemotherapeutic agents such as bortezomib and thalidomide, both widely used in clinical practice [272]. Bortezomib functions as a proteasome inhibitor, leading to the accumulation of misfolded proteins within myeloma cells and inducing apoptosis [307]. Thalidomide, on the other hand, exhibits multiple mechanisms of action, including immunomodulation and inhibition of angiogenesis [307].

By incorporating these drugs into CNPs, targeted delivery to malignant plasma cells is achieved, enhancing therapeutic efficacy while reducing systemic toxicity. One of the significant advantages of CNPs in treating multiple myeloma lies in their ability to penetrate the bone marrow microenvironment and selectively attack cancerous cells without damaging surrounding healthy tissues [272]. This precision-targeted approach not only improves drug bioavailability and therapeutic index but also minimizes the adverse effects commonly associated with conventional chemotherapy [272].

5.3.7. Treatment of melanoma cells

Melanoma is an aggressive form of skin cancer that arises from melanocytes—the pigment-producing cells responsible for skin coloration [308]. While primarily affecting the skin, melanoma can also develop in the eyes or mucous membranes. Its progression is often linked with excessive ultraviolet radiation exposure from sunlight or tanning beds, which induces DNA mutations in melanocytes [308]. If not detected early, melanoma can metastasize to distant organs, making it a life-threatening malignancy. Timely diagnosis and effective treatment are therefore critical for improving patient survival [308].

CNPs are particularly well-suited for melanoma therapy due to their capacity to penetrate skin tissues, allowing for localized, topical delivery of anticancer agents [309]. This targeted application enhances drug concentration at the tumor site while reducing systemic exposure. Once delivered, CNPs can induce ROS generation, resulting in oxidative damage to vital cellular components such as DNA, proteins, and lipids [310]. This oxidative stress disrupts melanoma cell function and activates apoptotic pathways, leading to selective cancer cell death [310].

Moreover, CNPs can be functionalized with targeting ligands—such as antibodies or peptides—that bind specifically to melanoma-associated antigens [311]. This targeted strategy improves drug delivery precision, enhances therapeutic outcomes, and minimizes off-target effects. The combination of localized delivery, ROS-mediated cytotoxicity, and molecular targeting makes CNPs a powerful tool in the emerging field of nanomedicine for melanoma treatment [311].

5.3.8. Treatment of brain and spinal cord tumors

Brain and spinal cord tumors are malignancies originating in the central nervous system, which comprises the brain and spinal cord [312]. These tumors can arise from various cell types, including neurons, glial cells, and other supportive or structural tissues [312]. Common tumor types include gliomas (originating from glial cells), meningiomas (from the meninges), and pituitary tumors arising in the pituitary gland at the brain's base [313]. Treating central nervous system tumors is particularly challenging due to the critical and delicate nature of the brain and spinal cord, where even minor damage can have severe consequences [313].

CNPs have emerged as innovative vehicles for the treatment of neurodegenerative diseases due to their unique biocompatibility, biodegradability, and ability to be tailored for targeted drug delivery across the blood-brain barrier, a significant obstacle in effectively managing neurological disorders such as Alzheimer's, Parkinson's, and other neurodegenerative conditions [314,315].

The use of CNPs has shown several therapeutic advantages, including enhanced drug absorption, prolonged retention in the central nervous system, and protective effects against degradation of encapsulated agents [315,316]. Notably, surface modifications enable CNPs to carry targeting ligands, thereby enhancing their ability to deliver drugs directly to affected neural tissues, which is crucial for diseases characterized by selective neuronal loss and chronic neuroinflammation [[314], [315], [316], [317]].

Recent studies have demonstrated that CNPs can deliver a variety of therapeutic agents, including small molecule neuroprotectants, anti-amyloid compounds, microRNAs, and anti-inflammatory drugs, often resulting in improved outcomes in animal models of neurodegeneration [318]. For instance, chrysin-loaded CNPs have been shown to effectively reduce amyloid-beta aggregation, production of ROS, and neuronal death, ultimately improving cognition and synaptic function in Alzheimer's disease models [319]. Through such delivery, both the CNPs matrix and its cargo can act synergistically. Chitosan itself possesses mild neuroprotective and anti-inflammatory properties, yielding approaches that are multimechanistic and highly relevant for multifactorial neurological pathologies [314,318,319].

The integration of CNPs with advanced therapeutic modalities, such as phototherapy and immunotherapy, has become a particularly promising strategy [320]. In phototherapy, CNPs serve as nanocarriers for photosensitizers, such as methylene blue, which, upon light activation, generate singlet oxygen to inhibit amyloid-beta fibrillogenesis—a central pathological hallmark of Alzheimer's disease [321]. Encapsulation of the photosensitizer within CNPs not only stabilizes the compound, protecting it from enzymatic reduction and premature deactivation, but also enhances its accumulation in brain tissue [320]. While traditional phototherapy faces the challenge of delivering light into deep brain tissues, advances such as optogenetic tools and wireless microLED implants offer potential solutions, enabling more effective and targeted photo-activation of drugs in situ [320,321].

CNPs are also being explored as adjuvants or carriers in immunotherapeutic approaches for neurodegenerative diseases [314]. Their immunomodulatory properties, ability to serve as antigen or cytokine carriers, and pronounced effects on microglia activation state add new capability to immune-based treatments aimed at reducing neuroinflammation and promoting neurorepair [321]. Coupling CNPs with immunotherapies can facilitate the delivery and sustained release of immunomodulatory agents or vaccines, potentially improving outcomes in conditions where inflammation or autoimmunity contributes to disease progression [314,321].

Intranasal administration of CNPs has recently garnered attention as a non-invasive route for direct nose-to-brain delivery, bypassing the blood-brain barrier and reducing systemic side effects [321]. Clinical translational challenges remain primarily concerning scalable production, regulatory approval, and long-term safety [314]. However, ongoing innovation in chitosan nanoparticle design, functionalization, and combined application with phototherapy or immunotherapy provides a robust foundation for their future as multitargeted, precision nanomedicines for neurodegenerative disorders [314,321].

5.3.9. Treatment of neuroendocrine tumors

Neuroendocrine tumors (NETs) are a rare and heterogeneous group of malignancies derived from neuroendocrine cells—specialized cells with both neuronal and endocrine functions [322]. These cells are distributed throughout the body and are involved in hormone production and nervous system regulation [322]. NETs can develop in multiple organs, with common types including pancreatic neuroendocrine tumors (PNETs) and small cell lung cancer (SCLC). Many NETs are hormonally active, secreting excess hormones that result in clinical symptoms such as flushing, diarrhea, and other endocrine-related disturbances [322].

Due to their hormonal activity and diverse anatomical origins, NETs present complex diagnostic and therapeutic challenges. CNPs have emerged as a versatile platform for delivering anti-cancer agents such as temozolomide or everolimus directly to NET cells [323]. Their utility lies in their ability to be engineered for active targeting, exploiting specific surface markers and receptors that are often overexpressed on NET cells. This allows for precise drug delivery, minimizing off-target effects on healthy tissues [323].

Beyond chemotherapy, CNPs can also deliver hormone-modulating therapies, which are crucial in managing hormonally active NETs [324]. For example, CNPs can encapsulate somatostatin analogs, which help regulate hormone secretion and control tumor-associated symptoms [324]. By combining chemotherapeutic and hormone-based treatments within a single delivery platform, CNPs offers a highly adaptable and practical approach for NET management [324].

5.4. Tissue engineering and CNPs

Tissue engineering is interested in the special qualities and increasingly focuses on the unique properties of naturally derived bioactive materials, which offer chemical and morphological similarities to native human tissues [272]. These natural materials have gained attention due to their lower toxicity, superior biocompatibility, and potential to mimic the extracellular matrix (ECM), surpassing many synthetic polymers in biomedical applications [272]. A major challenge in tissue engineering is the development of biomaterials capable of effectively restoring damaged or necrotic tissues, particularly under physiological stress [325]. Traditionally used natural and synthetic materials—such as chitosan, collagen, alginate, and hydroxyapatite—exhibit promising biological compatibility but are often limited by uncontrolled degradation, poor mechanical strength, infection risk, and challenges in storage and processing [326].

To address these limitations, advanced hybrid biocomposites have been developed by combioactivity, and functionality [327]. Chitosan, in particular, has emerged as a leading candidate in tissue engineering owing to its abundance of functional groups—hydroxyl, amino, and carboxyl—that enable facile chemical modification and composite formation with other materials [272]. These attributes, alongside its excellent biocompatibility and biodegradability, make chitosan especially suitable for applications such as skin tissue regeneration and chronic wound healing [328].

According to Entekhabi et al. [329], chitosan modulates the healing process at multiple stages, promoting neutrophil migration and enhancing IL-8 production to N-acetylation. [328]. In addition, micro- and nanoscale chitosan particles have demonstrated immunomodulatory properties by stimulating inflammasome formation in macrophages [63,330]. Interestingly, while nanostructured chitosan triggers IL-1 production of IL-1 and inflammasome activation, macro-sized chitosan scaffolds have shown inhibitory effects, reducing inflammatory responses in vitro [331].

Chitosan further influences wound healing by modulating growth factor expression through electrostatic interactions with anionic biomolecules [332]. It promotes dermal fibroblast proliferation, supports fibrous tissue formation, and enhances epithelial cell regeneration [333]. Innovative formulations, such as a freeze-dried chitosan-cordycepin hydrogel, leverage the electrostatic attachment of negatively charged cordycepin to positively charged chitosan without crosslinkers [269]. Similarly, post-thermal treatment of a chitosan-coated polyethylene terephthalate textile significantly extended chlorhexidine release while improving mechanical durability [334].

Chitosan is often employed as a base layer in asymmetric wound dressings and can be used alone or with other natural polymers [335]. Incorporating nanoparticles into chitosan-based hydrogels is a promising strategy for developing multifunctional synthetic biomaterials [336]. For example, a silver-chitosan-sericin nanocomposite film loaded with loxifloxacin demonstrated strong antibacterial efficacy against methicillin-resistant S. aureus (MRSA) and promoted wound healing in rats, performing comparably to standard clinical dressings [337]. Composite films combining chitosan and collagen have also shown intrinsic regenerative properties. A UV-crosslinked human keratin–chitosan membrane has been introduced as a novel wound dressing with enhanced healing capabilities [338].

Polyelectrolyte complexes comprising chitosan and chondroitin possess excellent antibacterial properties and cytocompatibility, making them ideal for wound healing applications [339]. Chitosan's positive charge facilitates the incorporation and controlled release of growth factors and cytokines, enhancing regenerative outcomes [339]. For instance, CNPs produced via ionotropic gelation with tripolyphosphate were used to deliver granulocyte-macrophage colony-stimulating factor (GM-CSF) in a nanocrystalline cellulose-hyaluronic acid scaffold [340]. In vivo studies revealed that GM-CSF-loaded CNPs significantly accelerated wound healing compared to GM-CSF-free composites, achieving a loading efficiency of 97.40% with sustained release over two days [214].

Further developments include chitosan macromers linked to the peptide Ser-Ile-Lys-Val-Ala-Val, which enhanced collagen deposition, angiogenesis, and TGF-β1 expression while downregulating pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in murine wound models [341]. The synthesis of heparin-like polysaccharides, such as 2-N, 6-O-sulfated chitosan, has further improved chitosan's affinity for vascular endothelial growth factor (VEGF), surpassing even that of heparin due to its higher sulfation degree [341].

Tissue engineering for central nervous system disorders, including neurodegenerative diseases and traumatic injuries to the brain and spinal cord, remains particularly challenging due to the limited degenerative capacity of the central nervous system [342,343]. CNPs serve as biocompatible, biodegradable scaffolds promoting cell proliferation and differentiation [344], especially when integrated with conductive polymers that mimic the brain's native electrical properties. Electrically conductive scaffolds derived from chitosan composites are being explored for regenerating neuronal, muscular, and cardiac tissues [272]. To support these functions, biomaterials must exhibit appropriate mechanical properties and porous structures to facilitate nutrient diffusion and cell migration [272].

Due to the complexity of tissue requirements, single-component polymers rarely fulfill all desired criteria. Hence, hybrid materials are increasingly explored for hard tissue engineering [331,332]. While colloidal nanoparticles show promise in bone regeneration, they often suffer from poor mechanical stability in aqueous environments [76]. Chitosan's affinity for calcium and phosphate has been leveraged to enhance biomineralization, especially in hybrid hydrogels combining chitosan with polyethylene glycol diacrylate [334]. Hydroxyapatite, resembling bone's inorganic matrix, is widely used to improve the mechanical strength of chitosan scaffolds [335]. Additional hybrids, such as strontium-modified chitosan/montmorillonite and nano-calcium zirconate/chitosan composites, have shown enhanced mechanical and biological performance [337].

A novel polyelectrolyte scaffold composed of chitosan, chondroitin, and nano-bioglass has demonstrated increased bioactivity, type-1 collagen formation by MG63 osteoblast-like cells, and in vivo osteointegration. Chitosan's mineralization potential can be further amplified by integrating polymers such as fucoidan [339] or bioglass [340], creating bioinspired platforms for osteochondral regeneration [340].

Cartilage tissue engineering remains a focal point due to its avascular nature and limited self-repair capacity. Techniques such as microfracture, mosaicplasty, autologous chondrocyte implantation, and biomaterial scaffolding are commonly employed [345]. Chitosan-based biomaterials present a viable alternative for promoting cartilage regeneration in hypoxic, non-vascular environments, aligning with the primary objective of tissue engineering: the development of regenerative scaffolds that function independently of vascular support [345].

5.5. Diabetes mellitus treatment

Diabetes mellitus is a chronic metabolic disorder characterized by elevated blood glucose levels due to impaired insulin secretion, action, or both [346,347]. It represents a significant health burden, with rising prevalence and serious complications including cardiovascular disease, neuropathy, nephropathy, and retinopathy [181]. Traditional treatments, such as oral hypoglycemic agents and insulin therapy, are often associated with side effects and limited efficacy [348].

Recent advancements in nanotechnology have introduced novel approaches for diabetes management, with CNPs emerging as a promising therapeutic agent due to their multifaceted anti-diabetic properties [348]. These properties include improved insulin sensitivity, inhibition of intestinal glucose absorption, regulation of lipid metabolism, antioxidant and anti-inflammatory activity, and protection and regeneration of pancreatic beta-cells [348,349].

The insulin signaling pathway is initiated when insulin binds to its receptor on target cells such as muscle, liver, and adipose tissue [349]. This activates the receptor's intrinsic tyrosine kinase activity, leading to autophosphorylation and subsequent phosphorylation of downstream substrates, particularly insulin receptor substrate-1 (IRS-1) [350]. Phosphorylated IRS-1 recruits and activates phosphoinositide 3-kinase (PI3K), triggering downstream signaling pathways such as the AKT, which mediate cellular responses like glucose uptake, glycogen synthesis, and lipid metabolism [350].

CNPs have been shown to enhance tyrosine phosphorylation of IRS-1, a key step in promoting insulin sensitivity and facilitating glucose uptake [351]. By improving insulin signaling, CNPs contribute to better glycemic control and reduced insulin resistance—hallmarks of type 2 diabetes. In diabetic conditions, IRS-1 is often inappropriately phosphorylated at serine residues due to elevated levels of inflammatory cytokines and free fatty acids, impairing its activity [272]. The anti-inflammatory properties of CNPs help suppress cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) [352]. By decreasing inflammation, CNPs reduce the serine phosphorylation of IRS-1, thereby preserving IRS-1 functionality and enhancing insulin signaling [352].

Another critical advantage of CNPs lies in their mucoadhesive nature, allowing them to form a gel-like matrix in the acidic environment of the stomach [353]. This gel delays gastric emptying and acts as a physical barrier in the intestines, slowing down glucose absorption and blunting postprandial blood glucose spikes [354]. By reducing the rate of carbohydrate breakdown and glucose transport across the intestinal wall, CNPs help maintain stable postprandial glucose levels—a crucial aspect of diabetes management [354]. In addition, delayed gastric emptying prolongs satiety, helping reduce caloric intake. This appetite-suppressing effect is particularly beneficial for people with type 2 diabetes who struggle with obesity, supporting weight loss and improving metabolic health [355].

Beyond their metabolic effects, CNPs offer significant potential in protecting and regenerating pancreatic beta-cells [356]. Chronic hyperglycemia induces oxidative stress and inflammation, leading to beta-cell apoptosis and decreased insulin production [357]. CNPs exhibit potent antioxidant activity, neutralizing ROS and reducing oxidative damage to beta-cells [358]. Their anti-inflammatory action also attenuates the production of pro-inflammatory cytokines like TNF-α and interleukin-1 beta (IL-1β), further safeguarding beta-cell viability [359].

Moreover, CNPs enhance the pancreatic microenvironment [360], by modulating key survival pathways such as PI3K/Akt, which promotes cell survival and inhibits apoptotic signals [361]. They also stimulate the expression of growth factors like insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF), both of which are vital for beta-cell proliferation and regeneration [362]. In addition, CNPs may facilitate differentiation of pancreatic progenitor cells into functional beta-cells by influencing extracellular matrix components and activating regenerative signaling pathways [272]. This regenerative capacity holds promise for restoring endogenous insulin production and long-term glycemic stability [272].

In summary, chitosan nanoparticles offer a comprehensive approach to diabetes management by targeting multiple pathological mechanisms, including insulin resistance, postprandial hyperglycemia, inflammation, oxidative stress, and beta-cell dysfunction. Their multifunctional properties position them as a compelling nanomedicine strategy for enhancing glycemic control and mitigating diabetes-associated complications. The anti-diabetic mechanism of CNPs is illustrated in Fig. 7.

Fig. 7.

The anti-diabetic mechanism of chitosan nanoparticles (CNPs). IRS-1: insulin receptor substrate 1, PI3K: phosphoinositide 3-Kinase, PDK: phosphoinositide-dependent kinase, AKT: protein kinase B, and GSK-3β: glycogen synthase kinase-3 Beta.

5.6. Treatment of skin disorders

CNPs have garnered significant attention in recent years for their potential in treating various skin disorders [363]. Their intrinsic biocompatibility, biodegradability, and antimicrobial activity make them highly suitable for dermatological applications [333]. Due to their small size and favorable surface properties, CNPs can effectively penetrate the skin, delivering active compounds directly to targeted sites and enhancing therapeutic outcomes [333]. This versatility has led to their application in multiple skincare interventions, including wound healing, infection control, anti-inflammatory therapies, and anti-aging treatments [333].

5.6.1. Wound healing

CNPs have demonstrated remarkable efficacy in accelerating wound healing by promoting key biological processes critical to tissue repair [13]. One of their primary mechanisms involves stimulating cell proliferation, which supports the regeneration of new skin cells at the injury site—particularly valuable for chronic or non-healing wounds [272]. In addition, CNPs enhance collagen synthesis, contributing to the formation of a strong and resilient skin barrier [364]. This not only facilitates wound closure but also improves the structural integrity of the regenerated tissue, minimizing scarring and yielding better cosmetic results [364].

CNPs further promote tissue regeneration by encouraging the migration of fibroblasts and keratinocytes to the wound site, expediting the healing process [140]. In more severe cases, such as burns or ulcers, where both epidermal and dermal layers are compromised, CNPs assist in restoring skin functionality and appearance by supporting comprehensive tissue regeneration [365]. Another unique property of CNPs is their ability to form a gel-like matrix upon skin contact. This matrix maintains a moist wound environment while serving as a protective barrier against microbial invasion [365]. The antimicrobial properties of CNPs further safeguard the healing tissue from infections, thereby reducing complications and ensuring faster recovery [366].

5.6.2. Moisturizing and skin repair

CNPs are particularly beneficial in treating dry skin disorders such as eczema and atopic dermatitis, largely due to their exceptional moisture-retaining capacity [272]. By attracting and binding water molecules, they help restore the skin's hydration balance and maintain long-lasting moisture—essential for individuals with dry, flaky, or irritated skin. Beyond hydration, CNPs enhance the skin's barrier function by reinforcing the lipid layer, preventing transepidermal water loss, and shielding the skin from external irritants, allergens, and pollutants [272]. This fortified barrier reduces the risk of flare-ups and improves overall skin comfort [272].

Moreover, CNPs support skin repair by promoting cell regeneration and turnover, which is particularly beneficial for damaged and compromised skin [272]. By stimulating the renewal of the skin's outermost layers, CNPs help restore elasticity and resilience, making the skin softer, smoother, and more supple [367]. For individuals suffering from chronic dryness or inflammatory conditions like atopic dermatitis, CNPs offer a gentle yet practical approach to enhance hydration, reinforce the skin barrier, and facilitate natural healing [272,367]. These properties position CNPs as a promising nanomedicine platform for long-term skin health and repair [367]. Fig. 8 illustrates the healing mechanism of skin repair utilizing CNPs.

Fig. 8.

The healing mechanism of skin repair using chitosan nanoparticles (CNPs).

5.6.3. Targeted drug delivery

One of the most compelling advantages of CNPs in skincare is their role as efficient carriers for active compounds, enabling targeted delivery of drugs, vitamins, antioxidants, and other bioactive compounds directly to the specific skin layers [368]. The unique physicochemical properties of CNPs—such as their nanoscale size, biocompatibility, and biodegradability—facilitate the encapsulation and controlled release of therapeutic agents, thereby enhancing their stability and bioavailability [369].

CNPs protect encapsulated substances from premature oxidation or degradation, preserving their potency throughout the application process [272]. This function is especially beneficial for addressing localized dermatological issues such as acne, hyperpigmentation, or aging symptoms, where accuracy is essential for treatment effectiveness. In addition, CNPs enable sustained release of their payload over time, which not only prolongs the therapeutic effect but also reduces the risk of irritation associated with higher concentrations of active ingredients [272].

By penetrating the deeper layers of the skin, CNPs allow for more effective treatment of inflammation, oxidative stress, and age-related skin damage [370]. For example, encapsulating antioxidants like vitamin C or E within CNPs enhances dermal absorption, boosts collagen production, and provides robust protection against free radicals—contributing to improved skin firmness and radiance. Furthermore, the enhanced permeability and retention characteristics of CNPs increase the bioavailability of active ingredients, outperforming many conventional formulations that struggle to traverse the skin barrier [188].

Overall, CNP-based delivery systems not only improve the efficacy of skincare treatments but also enable personalized, controlled, and non-invasive therapeutic regimes. Their multifunctionality positions CNPs as a promising platform in both cosmetic and clinical dermatology [54].

6. Limitations of CNPs in biomedical applications, potential solutions, and future studies

CNPs hold great promise in biomedical applications due to their biocompatibility, biodegradability, and multifunctional therapeutic potential. However, several restrictions and challenges hinder their widespread clinical translation. One major limitation is toxicity concerns, particularly when surface modifications or chemical cross-linkers are used.

Although chitosan is generally well-tolerated, variations in molecular weight, DD, and residual chemicals can lead to cytotoxicity or inflammatory responses. From a regulatory perspective, CNPs face strict oversight due to their nanoscale properties and complex behavior in biological systems, with many formulations still lacking comprehensive safety and efficacy data, causing delays in approvals. Stability issues present another obstacle, as CNPs can undergo aggregation, chemical degradation, or premature drug leakage under physiological conditions, reducing their therapeutic efficacy and reproducibility.

Furthermore, challenges in predicting and controlling biodistribution and pharmacokinetics, such as rapid clearance by the reticuloendothelial system or off-target accumulation, impact the precision and safety of CNP-mediated drug delivery. Finally, large-scale production remains a significant bottleneck due to batch-to-batch variability, complex manufacturing processes, and high production costs, which complicate scaling up laboratory methods to industrial levels while maintaining consistent quality and functionality. Addressing these hurdles through advanced formulation strategies, robust characterization, and standardized manufacturing protocols is crucial for realizing the full biomedical potential of CNPs.

To overcome the current limitations of CNPs in biomedical applications, several possible solutions and future research directions have been proposed. To address toxicity concerns, future studies should focus on optimizing the molecular weight and DD of chitosan, as well as employing green synthesis methods and biocompatible cross-linkers to minimize cytotoxic effects and inflammatory responses. Regarding regulatory barriers, the standardization of characterization techniques and the development of comprehensive safety and efficacy databases are essential.

Collaborative efforts among researchers, industry, and regulatory agencies can facilitate the establishment of clear guidelines specifically tailored for nanomedicines, such as CNPs. To improve stability, innovative surface modifications, such as PEGylation or the incorporation of stabilizing agents, can be explored to prevent aggregation and degradation. Advanced delivery systems that respond to specific physiological stimuli (e.g., pH, enzymes) can also enhance the controlled release of encapsulated drugs.

In terms of biodistribution and pharmacokinetics, engineering CNPs with targeting ligands and stealth coatings can enhance selective accumulation at disease sites while reducing off-target effects and rapid clearance. Future investigations using in vivo imaging and pharmacodynamic modeling will be invaluable for optimizing these properties. To overcome obstacles in large-scale production, the implementation of scalable manufacturing methods, including microfluidics, spray drying, and continuous flow synthesis, can guarantee batch-to-batch uniformity and cost-effectiveness.

7. Future studies

Future studies on CNPs should also focus on in-depth toxicological assessments across diverse biological models to comprehensively understand long-term safety profiles, especially for repeated or chronic administration. The development of standardized protocols for nanoparticle characterization, including size distribution, surface chemistry, and batch reproducibility, will be critical to ensure regulatory compliance and clinical success.

Further research should explore novel surface engineering techniques, such as bioinspired coatings and stimuli-responsive materials, to finely tune pharmacokinetics and targeted delivery in complex disease microenvironments. Advanced in vivo imaging and tracking technologies could provide real-time insights into CNP biodistribution, cellular uptake, and clearance, informing design improvements. In terms of production, future work could investigate continuous manufacturing processes coupled with quality-by-design (QbD) approaches for scalable, cost-effective, and reproducible nanoparticle synthesis. Exploring the integration of machine learning and artificial intelligence for process optimization and predictive modeling of biological interactions may significantly accelerate the formulation development process.

Lastly, interdisciplinary collaborations between material scientists, pharmacologists, clinicians, and regulatory agencies are essential for designing CNPs tailored to clinical needs and paving the way for regulatory approval and commercialization. These focused research efforts will collectively address current challenges and unlock the full therapeutic potential of CNPs in the field of biomedicine.

8. Conclusion

Chitosan has emerged as one of the most extensively studied bio-based polymers, recognized as “Generally Recognized as Safe” (GRAS) by the USA FDA. Its inherent biocompatibility, biodegradability, and non-toxic nature have led to its widespread application across medical, pharmaceutical, agricultural, and environmental domains. In nanomedicine, chitosan and its derivatives serve as ideal platforms for drug delivery systems due to their positive surface charge and mucoadhesive properties, which facilitate prolonged retention and sustained release.

CNPs are particularly advantageous for non-parenteral administration routes, including nasal, pulmonary, ocular, and gastrointestinal delivery, and have shown promise in managing respiratory diseases, gastrointestinal disorders, cancer, and ocular infections. Moreover, chemically modified chitosan can significantly enhance transfection efficiency, while unmodified CNPs may have limited stability under variable pH conditions.

The chitosan matrix is versatile, capable of encapsulating a broad range of therapeutic agents—including proteins, peptides, nucleic acids, antiviral drugs, and even inactivated viruses—enabling targeted delivery and improved cellular uptake at disease sites, such as those affected by viral infection or cancer.

List of abbreviations

Abbreviation	Definition
CNPs	Chitosan nanoparticles
NPs	Nanoparticles
PLGA	Poly (lactic-co-glycolic acid)
PEI	Polyethylenimine
FDA	Food and drug administration
ROS	Reactive oxygen species
siRNA	Small interfering RNA
miRNA	Micro RNA
ECM	Extracellular matrix
TPP	Tripolyphosphate
NMR	Nuclear magnetic resonance
UV	Ultraviolet
TEM	Transmission electron microscopy
SEM	Scanning electron microscopy
AFM	Atomic force microscopy
DLS	Dynamic light scattering
EE	Encapsulation efficiency
IC50	Half maximal inhibitory concentration
DDS	Drug delivery systems
RNA	Ribonucleic acid
DNA	Deoxyribonucleic acid
ICU	Intensive care unit
PLA	Polylactic acid
MRI	Magnetic resonance imaging
GO	Graphene oxide

CRediT authorship contribution statement

Mohamed T. El-Saadony: Writing – review & editing, Writing – original draft, Validation, Supervision. Ahmed M. Saad: Writing – original draft, Resources, Formal analysis. Mahmoud Sitohy: Writing – review & editing, Software, Formal analysis. Samar Sami Alkafaas: Writing – original draft, Methodology, Formal analysis. Mthokozisi Dladla: Software, Methodology, Formal analysis. Soumya Ghosh: Writing – review & editing, Formal analysis, Data curation. Dina Mostafa Mohammed: Writing – original draft, Validation, Formal analysis. Tarek N. Soliman: Writing – original draft, Software, Methodology, Formal analysis. Essam H. Ibrahim: Writing – review & editing, Project administration, Formal analysis. Mohamed A. Fahmy: Software, Resources, Data curation. Juwan S. AbuQamar: Resources, Methodology, Data curation. Khaled A. El- Tarabily: Writing – review & editing, Writing – original draft, Supervision, Software.

Declaration of competing interest

Authors declare no conflict of interests.

Data availability

Data will be made available on request.