## 5 Fabrication Techniques for Chitosan-Based Nanoparticles  
The fabrication techniques for the development of chitosan nanoparticles depend upon the choosen target for delivery and desired nanoparticle characteristics. Figure 3 summarizes the techniques heavily explored in the literature for the development of chitosan nanoparticles. 1. IONIC GELATION: At the nanoscale level, the physical, optical and mechanical properties of the polymer may differ as compared to the macroscopic level. The fabrication of chitosan-based nanoparticles by ionic gelation method was first reported by professor [PERSON] and his group, and is by far the most widely used technique [35]. In short, the method is based on the spontaneous interactions between the cationic chitosan (CS) polymer in the presence of an anionic crosslinking agent. Sodium tripolyphosphate (TPP) is commonly used anionic crosslinker which initiates the formation of a polyelectrolyte complex named TPP/CS. Under appropriate conditions, the complex is stabilized due to the electrostatic interactions created between the CS-NH\({}_{3}\)\({}^{+}\) and TPP-O- groups. This results in a three-dimensional entanglement from sol to dispersed gel-like nanoparticles. The drugs or genes added during the process are encapsulated to form nanoparticles. The process results in nanoparticle formation in the size range of 200-300 nm [36];
2. POLYELECTROLYTE COMPLEX FORMATION: This method is based on similar lines to ionotropic gelation, with the difference that it employs a polyanionic polymer. The most widely used polymers include poly (\(\gamma\)-glutamic acid), poly (asparatic acid) and hyaluronic acid which \begin{table}
\begin{tabular}{c c} \hline
**Variables** & **Desired Characteristics** \\ \hline
**Organoleptic properties** & White or almost white fine powder No taste, no odor \\
**Degree of deacetylation** & \(>\)80\% \\
**Molecular weight** & Low molecular weight or oligomers \\
**Viscosity (1\% in 1\% AcOH, 20 \({}^{\circ}\)C)** & \(<\)5 cps \\
**Moisture content** & 5–15\% \\
**Ash content** & \(<\)1\% \\
**Protein content** & \(<\)1–0.2\% \\
**Insolubility** & \(<\)0.5–0.1\% \\ \hline \end{tabular}
\end{table} 
Table 1: Summary of Chitosan characteristics for medical use. have opposite charges to those on chitosan derivatives. Polycationic chitosan derivatives like trimethyl chitosan (TMC), glycidyl trimethyl chitosan interact with the aforementioned polyanions, resulting in nanoparticles. As compared to the conventional polyelectrolyte complex formation, thiolated trimethyl chitosan (TMC-SH) and thiolated hyaluronic acid (HA-SH) demonstrated better stabilization due to the intermolecular covalent disulfide bonds [37];
3. COMPLEX COACERVATION: The origin of the term coacervation derives from Latin root \"coacervare\", meaning to crowd or pile. The basic concept of complex coacervation involves liquid-liquid phase separation which is due to the mixing of two oppositely charged macroions. It is one of the easily applied techniques for nanoparticle formation which depends upon electrostatic interactions between two oppositely charged biopolymers. This method can be employed for the packaging of different plasmids [38]. Recently, zein-chitosan nanoparticles formed by complex coacervation were examined to encapsulate curcumin, which is known for its antioxidant properties. The nanoparticles that were formed possessed greater encapsulation efficiency and were affected with multiple factors like pH and zein to chitosan ratio [39]. The nanoparticles reported having improvised stability, controlled release of bioactive compounds, and biodegradability;
4. POLYMER-DRUG COMPLEX FORMATION: When active principles like insulin or genes consisting of an inherent negative charge are added in adequate proportions to cationic chitosan, they undergo nanoparticle formation. These insoluble complexes have higher encapsulation efficiency, stability, and a smaller particle size for better cell internalization.