*3.5. Ionotropic Cross-Linking*

Ionotropic gelation exploits the capability of polysaccharide-based polyelectrolytes to crosslink in the presence of counter ions under specific ranges of concentration and/or pH [107]. The ionic cross-linking of polysaccharide droplets in aqueous solution gives rise to hydrogel particles (or beads) characterized by a microstructure with interconnected nanofibrillar network [108–111]. Hydrogel physicochemical properties depend upon chemical composition of the selected polysaccharide, its concentration as well as the size (i.e., ionic radius) and the valence (i.e., coordination number) of counter ions and eventually, the presence of water of hydration surrounding cross-linking ions [2,112].

Biocompatible and biodegradable alginate, pectin, chitosan are polyelectrolytes having active functional groups, such as carboxylate, sulphate and amine that can be involved in the ionotropic gelation mechanism [113]. Obviously, the type of counter ion and the gelling conditions must be chosen in relation to the specific polysaccharide used for droplet formation. Alginate and pectin, being polyanionic polysaccharides, tend to cross-link in presence of polyvalent cations. In this case, the gelation is induced by the electrostatic interactions establishing between cations and the polymer anion blocks [114]. On the contrary, chitosan with its amine functional group can undergo ionotropic gelation in presence of anionic counter ions such as tripolyphosphate (TPP), sulphate and citrate. The gel network formation is due to the electrostatic interactions established between anionic counter ions and the chitosan cationic blocks [115–117].

### 3.5.1. Alginate Ionic Cross-Linking

Alginate is certainly the most well-known and studied example of polysaccharide that can be cross-linked via a ionotropic mechanism [118]. It is a linear polysaccharide copolymer consisting of <sup>α</sup>-<sup>l</sup>-guluronic acid ( **G**) and β-<sup>d</sup>-mannuronic acid ( **M**) repeating units forming regions of **M**- and **G**-blocks and alternating structure (MG-blocks) [65,119,120] (Figure 7).

**Figure 7.** Chemical structure of alginate monomers: l-guluronic acid and d-mannuronic acid.

Alginate can be obtained from different brown seaweed species or from certain bacterial strains (e.g., *Pseudomonas aeruginosa*) [121]. The alginate source defines the **G**-to-**M** ratio and the molecular weight of the polysaccharide leading to significant differences in the physicochemical and mechanical properties of the resulting gels [119]. Commercially, alginates are available as alginic acid or in the form of sodium, potassium, or ammonium salts. Generally, most of divalent cations (Ca<sup>2</sup>+, Sr2+, Cd2+, Co2+, Cu2+, Mn2+, Ni2+, Pb2+ and Zn2<sup>+</sup>) and some trivalent cations (Fe<sup>3</sup>+, Cr3+, Al3+, Ga3+, Sc3+ and La3<sup>+</sup>) can interact with **G**-blocks regions of alginate in a highly cooperative manner, generating a 3D network according to the so-called "egg-box" model [122]. Alginate affinity towards such polyvalent cations is directly dependent on the amount of G-blocks present in the alginate structure [123,124]. As demonstrated in [125] by numerous competitive inhibition studies, the main involved gelation mechanism is the dimerization of **G** residues. In detail, the addition of polyvalent cations (often Ca2+ ions) to the alginate solution determines the binding of two **G**-blocks on opposite sides; the result is a diamond shaped hole consisting of a hydrophilic cavity that binds the cations by multicoordination using the oxygen atoms from the carboxyl functional groups. This arrangemen<sup>t</sup> causes the formation of a junction zone shaped like an "egg-box" (Figure 8).

**Figure 8.** Egg-box model representing the interactions between alginate **G**-blocks and calcium ions. Reprinted from [113]. Copyright (2019) Martău, Mihai, Vodnar.

Each cation binds with four **G** residues in the egg-box formation to form a 3D hydrogel network of these interconnected regions [15,126]. It has been reported that in the case of Ca2+, the formation of a stable junction requires eight to twenty adjacent **G** unites [65].

Generally, the greater the atomic radius of the cation, the stronger is the cross-linked polymeric matrix. In fact, as reported by [123,127–129], divalent ions of larger ionic radii such as Ba2<sup>+</sup> and Sr2+ are able to produce stronger alginate gel particles than the Ca2+-based ones (Figure 8). By contrast, having a smaller atomic radius, Mg<sup>2</sup>+ is not able to cross-link alginate [112,130]. Overall, alginate affinity to cations increases in the following order: Mn2<sup>+</sup> < Zn2+, Ni2+, Co2+ < Fe3<sup>+</sup> < Ca2+ < Sr2+ < Ba2<sup>+</sup> < Cd2+ < Cu2+ < Pb2+ [123]. For practical applications, the use of highly toxic cations such as Pb2+, Cu2+, and Cd2+ is limited. The use of Sr2+ and Ba2+, which are mildly toxic, has been reported in cell immobilization applications although only at low concentrations [131]. Ca2+ is certainly the divalent cation most used to form ionic alginate gels for its good binding affinity to alginate and lack of toxicity under normal conditions of use [15]. Although it is generally recognized that most divalent and trivalent cations are able to form alginate gels according to the pioneering "egg-box" model (valid mainly for **GG** sequences), it is important to consider that the chemical composition of such polymers and hence the **M**/**G** ratio can significantly vary [132]. This means that binding affinity with cations as well as polymeric conformation can vary too. Some binding studies have shown that Sr2+ is able to bind to G-blocks only, Ca2+ to both **G**- as well as **MG**-blocks, and Ba2<sup>+</sup> to both **G**- and **M**-blocks [65,123]. In general, binding affinity with cations is lower for both **MM** and alternating **MG** blocks, requiring a rather high polyvalent ion concentration to be more efficiently complexed. Compared to **GG** blocks, both **MM** and **MG** ones present a more open geometry that make them more available for interchain aggregations along polymer (self-assembly of alginate chains) causing significant irregularities in the arrangemen<sup>t</sup> of uronic units in alginate chains; this happens for the gels cross-linked by relatively low ion radius cations [125]. Since in the pharmaceutical field the ionotropic gelation is commonly used to entrap an API between the polymer chains, this arrangemen<sup>t</sup> can affect the drug release in a controlled manner.

### 3.5.2. Pectin Ionic Cross-Linking

Pectin is a linear polysaccharide mainly consisting of galacturonic acid units which are connected via α-(1–4) bonds and with a certain degree of methyl esterification of carboxyl groups (Figure 9) (DE, degree of esterification) depending on the polysaccharide quality and source [133]. It is commonly extracted from apple pomace and citrus peels under slightly acidic conditions. There are currently three commercially types of pectins: (1) low methoxyl (LM) pectin, where less than 50% of galacturonic acid groups are esterified with methyl groups; (2) high methoxyl (HM) pectin, where more than 50% of existing galacturonic acid groups are esterified with methyl groups; and (3) amidated pectin (A), where acid groups are partly amidated. Gelling properties highly depend on the ratio of esterified and amidated acid groups [134].

**Figure 9.** Pectin structure.

As reported by Braccini and Perez [114], despite the structural analogy between polyguluronate and polygalacturonate chains, the egg box model valid for alginates (guluronate system) cannot be directly transposed to the pectate gels. In this case, the most favorable antiparallel associations of galacturonate chains may, at best, be considered as "shifted egg boxes". The observed "shift" seems to lead to an efficient association with several van der Waals contacts; it reduces the original large cavity and provides two symmetrical sub-cavities of appropriate size for binding a cation and it creates an efficient periodic intermolecular hydrogen bonding network. Generally, the methyl esterification of carboxyl groups weakens the crosslinker-pectate interaction and might hamper subsequent dimer-dimer. Therefore, ionotropic gelation is more pronounced for lower-methylated pectins and at pH around 3–3.5; increasing the pH leads to deprotonation of acidic groups which prevents aggregation of chains and eventually gelation. Pectin has been also mixed with alginate to form in presence of cross-linking ions an interpenetrated network made up by heterogeneous interactions [135–137].

### 3.5.3. Chitosan Ionic Cross-Linking

Among several polysaccharides amenable to ionotropic cross-linking, chitosan is another noteworthy example even if by far less investigated than negatively charged polysaccharides such as alginate and pectin [138]. Chitosan is typically obtained from the alkaline deacetylation of the highly abundant, naturally occurring polymer chitin, but other sources can directly provide it as such (e.g., yeasts) [113]. Chitosan is composed of β-1,4-linked glucosamine and *N*-acetylglucosamine residues [139]. Both the degree of acetylation (DA) and the degree of polymerisation (DP) of chitosan are crucial factors determining the structural and functional properties of this family of polymers and their resulting engineered materials [140]. Commercially available chitosans vary in their DA, usually between 5 and 20%, and in their DP or molecular weight, typically ranging between 10 and 500 kDa. Unlike chitin, chitosan is soluble in water under mild acidic conditions thanks to the protonation of its amino groups (pKa = 6.2–7) able to promote the solvation of polymer chains. In these conditions, chitosan behaves as a polycation and, consequently, can form a gel by ionic interaction in presence of multivalent anions. Tripolyphosphate (TPP) is by far the most employed cross-linker to ionically reticulate chitosan due to its high net negative charges (ranging from one to five depending on pH) per monomeric unit and nontoxicity [116,141,142]. TPP has been widely exploited in pharmaceutical field to obtain chitosan microparticles [143–145], nanoparticles [143,146] and nano/micro-gels [147,148] intended for controlled drug delivery [149].

### *3.6. Di*ff*erent Approaches to Hydrogel Formation by Ionotropic Cross-Linking*

Hydrogels can be generated using ionotropic gelation technique by three main methods that differ in the way crosslinking ions are introduced to the polymer, realizing the so-called external, internal or inverse gelation [18,150].

In the external gelation or diffusion controlled method, polysaccharide solution is added dropwise into the gelling bath. The hydrogel matrix is formed through the diffusion of the cross-linking agents from the external continuous phase into the inner structure of polymeric droplets [151]. As expected, at the outermost layer of the hydrogel, gelling kinetics is rapid and gel formation is instantaneous. Then, counter-ions start to diffuse towards the center of the particle creating an inhomogeneous gelation profile in which the interaction between ions and polymer functional groups is maximum at the surface and zero at the core [152,153].

The internal gelation also called in situ gelation is an approach widely used to produce calcium alginate particles [154]. In this case, an insoluble calcium salt (e.g., CaCO3 and CaSO4) is mixed with the polysaccharide solution, and the obtained mixture is then extruded into an acidic gelling bath [155–157]. The acidic environment increases the solubility of calcium salt, allowing its release that leads to the formation of the polysaccharide gel network. This mechanism guarantees a controlled and homogeneous alginate exposure to cations and hence a uniform gel network formation [120]. Despite the good homogeneity, the internal cross-linked matrices results less dense, with larger pore sizes and thus more permeable than those obtained for external gelation, with lower encapsulation efficiencies and faster release rates [157,158]. This happens because matrix permeability is affected by competition between Ca2+ and H<sup>+</sup> ions due to the acid added. It seems that while the acid in the gelling bath liberates Ca2+ from the insoluble salt, it also competes with Ca2+ for interaction with the alginate/polymer. This drawback can be overcome by manipulating the pH of the medium and the amount of calcium salt employed [150].

Another approach is the inverse gelation, based on the dripping of the medium containing the cross-linking agents into the polysaccharide solution. This method is usually applied to emulsions for producing alginate microcapsules with an oily content and soft shell [5,159,160]. In comparison to other cross-linking methods, it exploits low amounts of biopolymer leading to the formation of a soft particle shell. Clearly, as highlighted by Martins at al. [161] in a recent published paper, the properties of the obtained microcapsules (e.g., mechanical resistance and release of bioactive substances) can vary based on the type of emulsion used (W/O or O/W) for the inverse gelation

### **4. Influence of Drying Process on Gel Particle Characteristics**

As discussed above, polysaccharide-based hydrogel particles can be used in the hydrated form for di fferent applications [119,162–166]. However, to avoid their chemical or microbiological degradation a drying step is often required [167,168]. Hydrogel particles can be dried using several techniques such as conventional, dielectric, freeze or supercritical drying, each one with a significant impact on the physicochemical and textural properties of the final dried beads [169]. Phenomena such as modifications of the highly interconnected hydrogel network, solute migration, polymorphism, damages by overheating and many other e ffects can occur [170]. Therefore, the choice of the drying method represents one of the critical step on the pathway to the production of PbHPs. As illustrated in Figure 10, each drying process leads to polymeric material with di fferent inner structure; conventional or dielectric methods allow to obtain "xerogels" whereas freeze and supercritical drying generally allow to achieve "cryogels" and "aerogels", respectively [171–175].

**Figure 10.** Pathway for the production of xerogels, cryogels and aerogels from polysaccharide based hydrogels via several steps including: (**a**) gelation (SOL →GEL), (**b**) solvent exchange pre-treatment, if required (e.g., the replacement of the water contained in the pores of the hydrogel with a suitable organic solvent), (**c**) drying final step with a specific technology. The images of xerogel, cryogel and aerogel beads were here reprinted (with some modifications) from [176] with permission from Elsevier. Copyright (2016).

Each drying method presents specific advantages and disadvantages (Table 1), and the choice must be done based on the desired performances for the final product, system cost-e ffectiveness and realizable scale-up.

In the following paragraphs, production and properties of xerogel, cryogel and aerogel particles are discussed.


**Table 1.** Main characteristics of the drying methods for polysaccharide hydrogels.

### *4.1. Conventional and Dielectric Drying to Produce Xerogels*

Conventional drying (e.g., using ambient air and an oven) as well as dielectric treatments generally cause the collapse of the nanoporous structure of the parent hydrogel due to the high capillary pressure gradient established during the solvent removal. The collapsing of the polymer structure produces a massive volume shrinkage leading to the formation of a highly aggregated and densely packed material without pores; the formed compact structure is known as "xerogel". However, as discussed in a large number of papers [175,177–179], porous texture of the dried material can be tuned by a proper selection of solvent, temperature, carrier gas used during evaporation and, specifically for microwave heating, the irradiating regimen. These parameters alone or in combination can allow to obtain either microporous, micro-mesoporous or micro-macroporous textures of the dried beads. For instance, as reported by [176] the porous structure of cellulose-based wet gels may resist to collapse to a certain limit when alcohol, having low surface tension and low vapor pressure, is used as organic solvent. In addition, an interesting study of [180] showed that degree of substitution (DS) of cellulose can play an important role for the production, via ambient drying, of low density, open porous and hydrophobic cellulose material defined "Xerocellulose". During this research, tritylcellulose with di fferent DS was synthesized in homogeneous conditions and then subjected to dissolution-coagulation-drying producing "Xerocellulose". Results showed that, depending on DS, the chemical modification leads to the development of unusual microstructure due to the di fferent manner of self-assembly of cellulose molecules and lack of hydrogen bonding.

Xerogel porosity may be specifically tuned, as demonstrated by studies of our research group aimed to verify the feasibility of the tandem technique Prilling/Microwave assisted drying for the production of alginate-based beads loaded with non-steroidal anti-inflammatory drugs (NSAIDs) [181,182]. Microwaves at di fferent regimens of irradiation a ffected matrix porosity, solid state of the loaded drug (i.e., ketoprofen or piroxicam) and drug-polymer interaction, leading to beads with significant di fferences in drug release profiles. Interestingly, high MW irradiation level led to dried beads with highly porous and swellable inner matrix able to rapidly release the encapsulated drug in the simulated gastrointestinal fluids. By contrast, low MW irradiation levels produced beads with few pores in the inner matrix acting as NSAID delayed delivery systems.

### *4.2. Freeze-Drying to Produce Cryogels*

During the freeze drying, the liquid entrapped into the hydrogel body is frozen and sublimed under regulated vacuum [118,183] reducing the volume shrinkage of the beads to 40%–50%. Unless special precautions are often taken to prevent the growth of ice crystals, freezing may destroy the pore structure and damage the nanostructured gel matrix as freezing always implies the growth of crystals [184]. The increase of the solvent volume upon crystallization induces the formation of a dendritic network of the crystalline solvent phase. The dendrites are, depending on the cooling rate, typically in the range of few up to a few tens of micrometers' size; they push the walls of the network at the crystal boundaries destroying the morphology/inner structure [185–189]. The resulting material is open porous product with a pore size in the range of several micrometers termed "cryogels".

In certain cases, modifying the freeze-drying conditions it is possible to modulate the ultrastructure of the porous matrix, moving from nanofibrillar to sheet-like skeletons with hierarchical micro- and nanoscale morphology. For instance, by increasing the cooling rate of the hydrogel precursors with e.g., liquid propane, or by using the spraying freeze drying approach, it is possible to avoid the macroporous 2D-sheet morphology of cellulose cryogels, producing aerogel-like dried systems with intermediate textural properties (BET-specific surface areas of 70–100 m<sup>2</sup>/g) [190]. Interestingly, the freeze drying of alcogels from resorcinol-formaldehyde using t-butanol as solvent resulted in a significant improvement of the mesoporosity of the resulting dried gels if compared to the freeze drying of hydrogel counterparts [187].

### *4.3. Supercritical Assisted Drying to Produce Aerogels*

Supercritical drying is the best method to preserve the porous texture and structural properties of the wet gel network in a dry form without cracks as well as without substantial volume reduction or packed network structure due to the intrinsic absence of surface tension in the pores of the gel. Supercritical drying produces nanostructured materials with low-density (typically <0.2 g cm<sup>−</sup>3), high-porosity (>96% *v*/*v*) in the mesoporous range, with full pore interconnectivity and large surface area (>250 m<sup>2</sup>/g), commonly called "aerogels". Aerogels have been designed in a several morphologies (e.g., cylinders, beads, microparticles) and configurations (e.g., only core, core-shell, coated particles) with an attractive processing versatility [191–194].

During supercritical drying, the organic solvent in the hydrogel pores (deriving from the pre-treatment named solvent exchange procedure, see Figure 10), is removed under supercritical conditions. As well known, a fluid reaches its supercritical state when it is compressed and heated above its critical point. Supercritical fluids have liquid-like densities and gas-like viscosities [195]. Supercritical carbon dioxide is the most commonly used fluid for supercritical drying due to its mild critical point conditions (304 K, 7.4 MPa), nontoxicity (it is considered to be Generally Recognized as Safe or GRAS), environmental friendliness, widely availability and cheapness/cost-e ffectiveness [191,196,197]. Generally, prior to the supercritical drying, the solvent exchange (that is the replacement of the water contained in the pores of the hydrogel with a suitable organic solvent) is needed due to the low a ffinity of water to supercritical carbon dioxide (SC-CO2) [196,198]. The presence of even small amounts of water in the pores of the wet gel can cause a dramatic change in the initially highly porous polysaccharide network upon supercritical drying. The usual approach in SC-CO2 drying procedure is the displacement of the water using a solvent with high solubility in CO2, commonly alcohol or acetone [191] and then the immersion of the gel in SC-CO2. The extraction time depends mainly on the thickness of the gel samples. Therefore, it can be still reduced from the several hours needed for thick monoliths to only few minutes for polysaccharide particles of millimeter size.

### **5. Case-Studies: Polysaccharide-Based Hydrogel Particles Produced by Prilling**/**Ionotropic Gelation and Their Application as Drug Delivery Systems (DDS)**

Polysaccharide-based hydrogel particles can be used as drug carriers, which application in pharmaceutics depends on the characteristics of the polysaccharides and the drugs, the particle configuration, as well as on the inner structure, namely xerogels, cryogels and aerogels. Table 2 summarizes the di fferent kinds of PbHPs produced via prilling/ionotropic gelation, particle configuration (only core or core-shell), their main physico-chemical and technological properties, and the potential pharmaceutical applications.

As shown in Table 2, hydrogels can be obtained as simple monolayered (only core) or multi-layered (core-shell) systems, in the hydrated or in the dried form. Based on the specific drying treatment, hydrogel beads can be transformed into xerogel, cryogel or aerogel form. The choice of a specific polysaccharide and particle system must be driven by the final desired application and performance requirements. Prior to any process design, it is necessary to study physico-chemical characteristics of the polymeric materials, like viscosities, densities, gelation and, physico-chemical and biopharmaceutical properties of the carried drug.


*Molecules*

 **2020**, *25*, 3156

### *5.1. Design of PbHPs in Form of Xerogels*

As reported in Table 2, successful outcomes have been achieved through a formulation design based on prilling in tandem with conventional drying that provide e fficient drug delivery of both steroidal (e.g., prednisolone and betamethasone) and non-steroidal (e.g., ketoprofen, ketoprofen lysine salt and piroxicam) anti-inflammatory drugs, targeting chronic inflammation and early morning pathologies.

The accurate selection of biopolymer, the opportune set-up of process parameters and gelling conditions allowed to produce interesting delivery systems in the xerogel form with controlled drug release both for low soluble and highly soluble NSAIDs. Zn2<sup>+</sup> as external cross-linking agen<sup>t</sup> for alginate/ketoprofen (K) solutions gave PbHPs with good technological properties such as drug loading, particle size, morphology, hardness of cross-linked matrix [202]. In vitro and in vivo release behavior resulted to be strongly influenced by the amount of NSAID loaded inside the polymer; the loading of high amount of drug into feed solutions promotes, during the gelling phase, the formation of a compact gel polymeric network via intermolecular interactions, as hydrophobic or hydrogen bonding, which stabilize the well-known alginate "egg-box" structure. This phenomenon leads to tough polymer beads (Figure 11a), reducing the leaching of the drug from the drops into the gelling medium. Accordingly, the formulation obtained with the highest drug content (F20, K/alginate ratio 1:5) showed the highest entrapment of the drug within the matrix (encapsulation e fficiency, 53%) and a delayed release of the drug in simulated intestinal fluid (see Figure 11b). This in vitro release pattern was clearly reflected in the in vivo prolonged anti-inflammatory e ffect evaluated using a modified carrageenan-induced acute edema assay in rat paw. F20, administered 3 h before edema induction, showed a significant anti-inflammatory activity, reducing maximum paw volume in response to carrageenan injection, whereas no response was observed for pure ketoprofen (see Figure 11c).

**Figure 11.** Main in vitro and in vivo results obtained for Zn-alginate-based xerogel beads loaded with ketoprofen: (**a**) SEM microphotographs showing the compact inner matrix; (**b**) relese profile performed by USP Apparatus 4 and, (**c**) edema volume reduction in rats (\*\* *p*-value ≤ 0.01, \*\*\* *p*-value ≤ 0.001 compared with control). These images were here reprinted (with some modifications) from [202] with permission from Elsevier. Copyright (2015).

Zn2<sup>+</sup> as external cross-linking agen<sup>t</sup> for pectin solutions was able to produce PbHPs containing ketoprofen lysine salt (KL), a highly soluble NSAID [204]. In this case, the best results were obtained using amidated low methoxyl pectin (esterification degree 24% and amidation degree 23%) producing beads with good morphological properties and size, high drug content and encapsulation e fficiency (93.5%), and interesting KL sustained release profiles.

−−

−−

−S−

### *5.2. Investigation on the E*ff*ect of Di*ff*erent Cations on Gelation Process*

Many researchers evaluated the influence of di fferent divalent cations on PbHP properties. For instance, Chan et al. [210] studied the ability of calcium chloride and zinc sulphate to cross-link alginate microspheres prepared by emulsification.

In this study, the aqueous phase, consisting of 2.5% *w*/*w* sodium alginate and 1% *w*/*w* sulphaguanidine was dispersed in isooctane with the aid of surfactants and a mechanical stirrer. The fine globules of sodium alginate produced were gelified by addition of calcium chloride and zinc sulphate, alone or in combination. The microspheres formed were collected by filtration, washed and oven dried at 40 ◦C. The results of characterization studies showed that the simultaneous use of these two salts led to di fferent particle morphology and slower drug release compared to particles cross-linked by the calcium salt alone. These e ffects were attributed to a greater extent of interaction between zinc cations and the alginate molecules able to produce a less permeable alginate matrix. Cerciello et al. also investigated the specific e ffect of these two divalent cations, i.e., Ca2+ and Zn2+, used alone or blended in di fferent ratios (Ca<sup>2</sup><sup>+</sup>:Zn2+, ratio 1:1, 1:4 or 4:1), on the properties of alginate beads obtained via prilling/external gelation [201]. The synergistic e ffect of the two cations, when used in the gelling bath in the ratio Ca2<sup>+</sup>:Zn2<sup>+</sup> 1:4; positively a ffected particle morphology, size, inner structure, ability to encapsulate the model drug (SAID, prednisolone, P) and to control its release from the polymer matrix. Figure 12 shows SEM and SEM-EDS microphotographs of cryofractured blank beads obtained using the di fferent ratios Ca<sup>2</sup>+/Zn2<sup>+</sup> in the gelling solution. As showed, formulations gelified using Ca2<sup>+</sup>:Zn2<sup>+</sup> in the ratios 1:1 and 4:1 exhibited an internal structure enriched in Ca2+, due to the higher di ffusivity of this cation, compared to Zn2+. Only with a ratio Ca<sup>2</sup>+/Zn2<sup>+</sup> 1:4 was possible to observe an equilibrium between the two cations quantities into the polymeric matrix. This specific ratio, in fact, exploited the Ca2+ ability to establish quicker electrostatic interactions with **G** groups of alginate and the Zn2<sup>+</sup> ability to establish covalent-like bonds with both **M** and **G** blocks of alginate. Drug release profiles clearly reflected the advantages deriving from the simultaneous use of both cations. Their proper mixing allowed to produce a polymeric matrix tougher and more resistant compared to those obtained with a single cation (zinc or calcium) and with an interesting P prolonged release.

**Figure 12.** SEM and SEM-EDS microphotographs of cryofractured blank xerogel beads produced with di fferent Ca<sup>2</sup>+/Zn2<sup>+</sup> ratios: (**a**) 1:1, (**b**) 1:4 and (**c**) 4:1. Reprinted from [201] with permission from Elsevier. Copyright (2017).

### *5.3. Prilling to Obtain Floating PbHPs*

A significant number of studies was conducted to develop floating PbHPs, mainly alginate based particles, using gas-forming agents such as CaCO3 or NaHCO3. In the most of them, calcium has been used as external cross-linker for the gelation phase [157,211,212]. More recently, an important milestone was achieved with the simultaneous use of two different divalent cations to produce floating and prolonged release alginate PbHPs for the oral administration of prednisolone, P [207]. Critical parameters were established: prilling/ionotropic gelation was used as microencapsulation technique, zinc acetate in the gelling solution as the alginate external crosslinker, and calcium carbonate in the feed acting as the internal crosslinking agen<sup>t</sup> able to generate gas when in contact with the acidic zinc acetate solution. The double gelation process (internal- and external) promoted by Ca2+ and Zn2<sup>+</sup> ions gave alginate beads with extremely high encapsulation efficiency values (up to 94%) and a very porous inner matrix conferring buoyancy in vitro in simulated gastric fluid up to 5 h. Particularly, the best formulation F4 (P/Alginate ratio 1:5; Alg/CaCO3 ratio 1:0.50) was able to control the drug release in acidic medium for the entire time corresponding to the floating period. Although porous, the tougher matrix obtained thanks to the double gelation process is able to reduce swelling and erosion processes in simulated gastric fluid (SGF). F4 was also able to prolong the in vivo anti-inflammatory effect up to 15 h compared with raw prednisolone. Therefore, this alginate-based system has been proposed as a new technological platform able to extend the anti-inflammatory efficacy of SAID such as prednisolone (characterized by high efficacy and high tolerability, but short half-life) for many hours and successfully treat patient suffering from chronic inflammatory diseases, also reducing the frequency of the oral administration.

An interesting production innovation was obtained designing floating PbHPs with controlled release properties without using any gas-generating agent. Our research group designed a hollow multipolymer matrix made up of alginate, ALM-pectin and HPMC. Results showed that particle shape and sphericity can be correlated to nozzle viscosity of the feed solutions; the higher the nozzle viscosity, the slower the break-up of the polymeric laminar-jet and, thus, droplet formation. The high entanglement existing between the chains of three different polymers makes the polymeric jet highly cohesive (viscoelastic stresses dominate) delaying drops detachment from the nozzle. At the lowest feed concentration (4.75 *w*/*w*), corresponding to a nozzle viscosity of 24.4 mPa·s, polymer chains are relaxed and surface tension dominates, allowing the formation of droplets that, falling in the gelation bath, give rise to spherical particles. Optimized formulation F4 (Drug/Polymers ratio 1:15; Pol1/Pol2/Pol3 ratio 1.25:3:0.5) showed beads spherical in shape with a sphericity coefficient mean value of 0.94 and a mean diameter around 2200 μm. This formulation acts as a floating-system able to release the encapsulated model drug (piroxicam, PRX) in a controlled and delayed manner.

Floating properties of F4 are due both to the swelling of the hydrocolloid particles and to hollow inner structure (Figure 13). The hydration of the hydrocolloid particle surface in SGF results in an increased bulk volume and, at the same time, the presence of internal pores make beads able to entrap air. As a result beads had bulk density <1, and, therefore, remained buoyant on the acidic medium. In addition, the inner cross-linked multi-polysaccharide matrix acts as reservoir for slow and sustained PRX. Drug release was controlled by a diffusion mechanism process through the swollen polymers gel layer, as shown by in vitro release assay. Figure 14 shows the presence of pores and air bubbles entrapped within the gel barrier after 60 min of floating in acidic medium.

**Figure 13.** SEM microphotographs showing the hollow inner matrix of polysaccharide-based floating beads produced with different Pol1/Pol2/Pol3 ratios: (**<sup>a</sup>**,**b**) 1.75:4:1, (**<sup>c</sup>**,**d**) 1.75:3:0.5 and (**<sup>e</sup>**,**f**) 1.25:3:0.5. Reprinted from [206] with permission from Elsevier. Copyright (2018).

**Figure 14.** Microphotographs obtained using bright-field (**<sup>a</sup>**,**b**) and fluorescent microscopy (**<sup>c</sup>**,**d**) showing the presence of pores and air bubbles entrapped within the gel matrix of hydrated beads, able to confer their floating ability in SGF. Reprinted from [206] with permission from Elsevier. Copyright (2018).

As expected by morphology and results from in vitro assays, a promising application of floating PbHPs is the treatment of chronic inflammatory-diseases in elderly patients needing a rapid onset of drug action followed by a maintenance dose. In this regard, the in vivo anti-inflammatory activity of this type of new floating PbHPs, evaluated using the modified protocol of carrageenan-induced acute edema in rat paw previously developed [202], showed an incredible extension, up to 48 h, of the anti-inflammatory e ffect compared to standard PRX, as e ffect of both floating and sustaining release abilities.
