**2. Preparation procedures**

#### **2.1. Ionotropic gelation**

Ionotropic gelation, that is by far the most used technique for preparation of nanoparticles from chitosan derivatives, was first reported in 1997 by Calvo et al. [13]. The basic concept is that a polycationic polymer in aqueous solution passes, in appropriate conditions, from sol to dispersed gel following electrostatic crosslinking with an adequate anionic substance. This technique has been used with several quaternized chitosans carrying fixed, pH-independent positive charges, the most known of which is *N*-trimethyl chitosan (TMC). Sodium tripoly‐ phosphate (TPP) has widely been employed as the ionotropic crosslinker [14-19].

The nanoparticles prepared by ionotropic gelation of quaternized chitosans with TPP were generally 200-300 nm in size, i.e., smaller than those obtained by the same method starting from plain chitosan which, by the way, showed lesser stability and tended to re-dissolve after some time from formation. The zeta potential was always positive, in the 10-20 mV range. The solution of the chitosan derivative into which the TPP solution was dripped would often contain a surfactant, usually Tween 80, to hinder nanoparticle aggregation and facilitate their re-dispersion after centrifugation. In fact, centrifugation was necessary to clear the particles of non-encapsulated drug.

The technique under discussion has also been used to prepare nanoparticles from thiolated derivatives of chitosan [20-23].

These polymers have shown mucoadhesive properties due to the ability of their thiol groups to form covalent disulfide bridges by reacting with the thiol residues present on the glyco‐ proteins of mucus. For this reason the nanoparticles derived from these chitosan derivatives were themselves endowed with mucoadhesivity. The thiolated nanoparticles formed by ionotropic gelation with TPP were stabilized via oxidation of thiols with H2O2 which formed interchain disulfide bonds. These would bestow gastroresistance on the particles, which would be particularly appropriate in case of oral administration of the nanoparticle formulation. However the presence of some non-oxidized thiols on the nanoparticle surface was needed to confer enhanced mucoadhesivity on such a surface. This goal was actually achieved by Bernkop-Schnurch et al. [21]. These authors also studied the crosslinker effect on nanoparticle size. Under similar preparation conditions, sizes in the 200-300 nm range were obtained with TPP as the crosslinker, whereas sizes beyond the micron resulted using Na2SO4.

#### **2.2. Gelation from polyelectrolyte complex (PEC) formation**

This method involved ionotropic gelation, just as described in the preceding section, only in the present case the crosslinker was a polyanionic polymer with charges opposite to those of the chitosan derivative, with which it formed a PEC. To this purpose *N*-carboxymethyl chitosan, poly(γ-glutamic acid), poly(aspartic acid) and hyaluronic acid were used as polyan‐ ions, while TMC and glycidyl trimethyl ammonium chitosan were the polycations [24-27]. In a case both the polyanion (hyaluronic acid) and the polycation (TMC) were thiolated and the nanoparticles were stabilized by the formation of interchain disulfide bonds [28].

#### **2.3. Polymer-drug complexes**

**2. Preparation procedures**

244 Advances in Biomaterials Science and Biomedical Applications

Ionotropic gelation, that is by far the most used technique for preparation of nanoparticles from chitosan derivatives, was first reported in 1997 by Calvo et al. [13]. The basic concept is that a polycationic polymer in aqueous solution passes, in appropriate conditions, from sol to dispersed gel following electrostatic crosslinking with an adequate anionic substance. This technique has been used with several quaternized chitosans carrying fixed, pH-independent positive charges, the most known of which is *N*-trimethyl chitosan (TMC). Sodium tripoly‐

The nanoparticles prepared by ionotropic gelation of quaternized chitosans with TPP were generally 200-300 nm in size, i.e., smaller than those obtained by the same method starting from plain chitosan which, by the way, showed lesser stability and tended to re-dissolve after some time from formation. The zeta potential was always positive, in the 10-20 mV range. The solution of the chitosan derivative into which the TPP solution was dripped would often contain a surfactant, usually Tween 80, to hinder nanoparticle aggregation and facilitate their re-dispersion after centrifugation. In fact, centrifugation was necessary to clear the particles of

The technique under discussion has also been used to prepare nanoparticles from thiolated

These polymers have shown mucoadhesive properties due to the ability of their thiol groups to form covalent disulfide bridges by reacting with the thiol residues present on the glyco‐ proteins of mucus. For this reason the nanoparticles derived from these chitosan derivatives were themselves endowed with mucoadhesivity. The thiolated nanoparticles formed by ionotropic gelation with TPP were stabilized via oxidation of thiols with H2O2 which formed interchain disulfide bonds. These would bestow gastroresistance on the particles, which would be particularly appropriate in case of oral administration of the nanoparticle formulation. However the presence of some non-oxidized thiols on the nanoparticle surface was needed to confer enhanced mucoadhesivity on such a surface. This goal was actually achieved by Bernkop-Schnurch et al. [21]. These authors also studied the crosslinker effect on nanoparticle size. Under similar preparation conditions, sizes in the 200-300 nm range were obtained with

TPP as the crosslinker, whereas sizes beyond the micron resulted using Na2SO4.

nanoparticles were stabilized by the formation of interchain disulfide bonds [28].

This method involved ionotropic gelation, just as described in the preceding section, only in the present case the crosslinker was a polyanionic polymer with charges opposite to those of the chitosan derivative, with which it formed a PEC. To this purpose *N*-carboxymethyl chitosan, poly(γ-glutamic acid), poly(aspartic acid) and hyaluronic acid were used as polyan‐ ions, while TMC and glycidyl trimethyl ammonium chitosan were the polycations [24-27]. In a case both the polyanion (hyaluronic acid) and the polycation (TMC) were thiolated and the

**2.2. Gelation from polyelectrolyte complex (PEC) formation**

phosphate (TPP) has widely been employed as the ionotropic crosslinker [14-19].

**2.1. Ionotropic gelation**

non-encapsulated drug.

derivatives of chitosan [20-23].

Some negatively charged active principles, such as insulin or gene drugs, when mixed with cationic chitosan derivatives in adequate proportions spontaneously formed nanoparticulate dispersions of insoluble complexes [29-34]. TMC nanoparticles obtained by ionotropic gelation with TPP in the presence of insulin were compared with nanoparticles obtained by PEC formation between TMC and insulin. In the latter instance higher encapsulation efficiency and zeta potential (positive), and smaller particle size were observed, which is particularly appropriate for particle internalization into cells. In addition, a higher stability in simulated intestinal fluid (pH 6.8) of the nanocomplex compared to the nanoparticles prepared with TPP resulted [31,32].

### **2.4. Self-assembly**

Amphiphilic derivatives of chitosan in aqueous solution were found, at a critical aggregation concentration (CAC), to spontaneously arrange into nanoparticles of sizes in between 100-400 nm. Such derivatives were prepared by connecting hydrophobic structures to the chitosan or glycol chitosan backbone via the amino group of the chitosan repeating unit. Examples of the above amphiphilic derivatives are the following: glycol chitosan-5β-cholanic acid conjugate [35-40]; palmitoyl chitosan [41]; palmitoyl glycol chitosan [42]; oleoyl chitosan [43]. Other amphiphiles were prepared from chitosans bearing fixed positive charges, in the case of quaternary ammonium palmitoyl glycol chitosan [42], or negative fixed charges, as in the case of linoleic acid-modified *O*-carboxymethyl chitosan [44], or deoxycholic acid-modified *N,O*carboxymethyl chitosan [45]. Usually, after suspending the polymer in an aqueous medium, probe sonication was applied to limit particle size. The formation of nanoparticles was monitored and the CAC determined fluorometrically, or through UV absorption spectra, or measuring the enthalpy change by a microcalorimeter [42,44,45].

The CAC for the hydrophobically modified chitosan derivatives is usually in the μM range, whereas the CMC of small-molecular weight surfactants is in the mM range. This is one of the most important characteristics of amphiphilic polymers, pointing to stability of the selfaggregates in dilute conditions, such as those the nanoparticles are supposed to encounter after administration to the organism. The CAC values of these polymers have been found to decrease with increasing hydrophobic content of derivatives [44]. In fact, the nanoparticles formed from these chitosan derivatives are characterized by a core-shell structure, i.e., a hydrophobic core in a hydrophilic shell. The drug incapsulation method was chosen on the basis of the hydrophilic or hydrophobic nature of the drug. With hydrophobic drugs the solution of polymer and drug in a water-miscible organic solvent was mixed with an aqueous medium and the organic solvent was cleared away by dialysis or evaporation [36,39,40]. Hydrophobic drugs having a fair water solubility and polar drugs have been loaded into nanoparticles via direct addition to the aqueous polymer dispersion [41,42,44,45]. The nonencapsulated drug has been separated by ultracentrifugation, filtration or dialysis.
