**1. Introduction**

In the last three decades, there has been a constant development of polysaccharide-based hydrogel particles (PbHPs) as smart tools to release drugs with the right kinetic and target. The encapsulation of an active pharmaceutical ingredient (API) inside these polymeric micro-particles


Several techniques can be used for the preparation of PbHPs. Many methods are based on the preparation of spherical droplets made by mixtures of the API and the polymeric excipients. For the development of such PbHPs is crucial the polysaccharide droplet formation phase (Figure 1) that in

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turn defines the size and the size distribution of the resulting microparticles, the two primary factors affecting drug release [1–3].

**Figure 1.** Illustration of the general way for producing hydrogels in form of particles: transition from polysaccharide solution (SOL phase) to a gel particle (GEL phase) followed by possible drying treatments.

In general, the processes used to prepare monodispersed particles starting from polysaccharidedroplets can be divided into:


For both methodologies, the critical parameters able to determine size and shape of the liquid droplets, are the following: the viscosity of each phase, the surface tension of the polysaccharide solution compared to the surrounding medium (gas/air or liquid) and the dynamic interactions of the droplets with the matrix fluid (laminar or turbulent flow). In case (a), where the liquid is pushed through a nozzle at a constant flow rate, surface tension between droplet and air (liquid-air interface) is essential. In the second case (b), the liquid is broken down in an immiscible fluid system in form of droplets and the interfacial tension between dispersed and continuous phases is usually controlled by surfactants [4].

As shown in Figure 2, the main processes involving droplet formation in gaseous phase can be grouped according to the mechanism of liquid jet break-up in: simple extrusion (conventional dripping, Figure 2a), vibrating nozzle (Figure 2b), electrostatic (Figure 2c) and mechanical cutting method (Figure 2d).

Conventional dripping has been widely used to produce mainly alginate particles able to encapsulate cells, enzymes, probiotics, plant extracts, oils and flavours [5–14]. This method involves the manual extrusion of polymeric droplets from a fluid filled syringe or pipette into a gelation or coagulation bath (Figure 2a). When the polysaccharide solution flows out, a droplet is formed at the orifice. The polymeric droplet grows in size until it detaches from the orifice under the influence of gravity, falling toward the gelling medium. In this method, there is no precise control on the formation of the droplets that, during the falling, have the tendency to become spherical due to the surface tension of the liquid before being gelified. Although extrusion by syringe or pipette is the simplest way to produce polysaccharide gel particles, this method generally leads to large gel particles that are polydisperse and not always spherical in shape [4,15,16]. This happens because gravity is the main driving force to generate the droplet from the orifice. In addition, several scale-up difficulties limit this method to a lab scale setup [4,17,18]. Another limitation is represented by the possibility to process only low viscosity feed solutions due to pumping problems and needle blockage [19].

Considering the other technologies illustrated in Figure 2b–d, the breaking up of the polysaccharide liquid jet into droplets is determined by specific devices that give the possibility to strictly control droplet formation [4]. Among them, vibrating nozzle method, also known as prilling or laminar jet break-up, has been widely reported in literature for its grea<sup>t</sup> versatility, reproducibility and high scalability potential [20–23].

The present review surveys the main results gained in prilling technology addressing: (i) the basic aspects of the droplet formation technique, its possible implementations and the ionic crosslinking as main gelation method of the droplets formed by prilling, (ii) the main polysaccharides suitable for PbHPs production by prilling; (iii) the possible approaches exploitable for the ionic gelation, e.g. external, internal or inverse, (iv) the influence of the applied drying method on polymer matrix characteristics and hence on its properties affecting the release of the entrapped drug.

**Figure 2.** Illustration of dripping devices: (**a**) conventional dripping method influenced by gravity, surface tension and viscosity; breaking up of liquid jets into droplets stimulated by (**b**) vibrating nozzle method, (**c**) electrostatic forces and (**d**) a mechanical cutting device. Reprinted (with some modifications) from [4]. Copyright (2018) Ganesan, Budtova, Ratke, Gurikov, Baudron, Preibisch, Niemeyer, Smirnova, Milow.

### **2. Prilling Technique to Produce Polymeric Droplets**

### *Prilling or Laminar Jet Break-Up*

Prilling process is based on the mechanical dispersion of the feed solution through pressurecontrolled injection in a specific gelation or coagulation medium after breaking apart into mono-sized drops by means of a vibrating nozzle device [23,24]. The technology has been shown especially suitable to immobilize microorganisms or entrap bioactive substances in polymeric beads; these are formed by fall of a mixed host-polymer liquid formulation into an appropriate polymer gelling solution [25–28]. Recently, many pharmaceutical applications of such beads have been developed in order to control the drug release in orally administered formulations [29,30] or the colon targeting [31,32].

In the vibrating nozzle method (Figure 3), the monodispersed droplets are formed from a laminar liquid jet by applying superimposed vibrations with an optimal frequency either on the nozzle or on the liquid that is approaching the nozzle. The vibrations can be generated using sound waves (ultrasound) [4,33]; the acoustic jet excitation process involved in prilling was patented to produce uniform microspheres of alginate [34], collagen [35] and PLGA [36]. Practically, polymeric feed solution is pressurized using a pump or gas through a nozzle in order to generate the liquid jet. The superimposed vibrations destabilize the liquid jet (Rayleigh instability) and the jet is disintegrated into monodispersed liquid droplets [3].

Several variables, such as density, dynamic viscosity and flow rate of the feed solution, nozzle geometry and diameter, frequency of vibration as well as falling distance, can affect shape, size and size distribution of the droplets and consequently of the resulting hydrogel particles [16,37–40].

**Figure 3.** Schematic illustration of prilling technology with the indication of the main process variables.

Viscosity is certainly one of the most important variable of this technique; prilling is able to process only solutions with viscosity values lower than few hundreds of mPa·<sup>s</sup> [21] and it is essential to study the so-called nozzle viscosity (dynamic viscosity) using appropriate theoretical model [23].

As regards to the droplet size, it is estimated to be at least twice the nozzle inner diameter and can be varied by changing the flow rate of the liquid and nozzle diameter [40]. In addition, particle sphericity can be highly influenced by the distance between the vibrating nozzle and the gelling bath. In fact, when the droplets hit the surface of the gelation medium, their spherical shape can be deformed if the droplet viscosity and surface tension forces are unable to overcome the surface tension exerted by the gelling solution [17,41]. Different papers have demonstrated that the liquid droplets are generally able to overcome the impact forming spherical gel particles when the falling distance is greater than 10 cm [15,16]. Uniformity of the polymeric gel particles can also be improved by reducing the surface tension of the gelling bath by the addition of surfactants [15,42]. Moreover, smaller nozzle diameters and higher frequencies increase the possibility of coalescence [24]. For this reason, frequency is usually kept as low as possible in order to avoid the formation of satellite droplets leading to a broader size distribution [29].

Prilling technology can be also used in the co-axial configuration (Figure 4) to obtain droplets with multiple layers formed by different polysaccharides, in a single manufacturing step [43,44]. Core-shell beads can be easily fabricated by prilling in co-axial configuration designing formulations able to obtain a drug controlled release and to diminish the effect of the GI environment. The appropriate combination of two or more polysaccharides may be, for example, an effective way to produce a polymer-drug core (e.g., pectin) enveloped by gastroresistant shell (e.g., alginate) able to prevent the early release of the drug in the upper part of the gastro-intestinal tract (GIT) [45–48]. An enteric shell may release the drug in a specific district of the organism (intestinal/colonic tract); moreover, from the use of bioadhesive polymers may increase the gastric retention time and, therefore, improve the localized action in GI tract or even delay the release in a precise moment of the day as required, for example, for the treatment of severe chronic mucosal inflammations such as Inflammatory bowel disease IBD [31].

**Figure 4.** Schematic reproduction of prilling process in co-axial configuration. Reprinted from [43] with permission from Elsevier. Copyright (2014).

Particle manufacturing through the vibrating nozzle device is easily to scale up e.g., by using a multi-nozzle system (Figure 5) without changing other process parameters such as flow rate and the vibration frequency [21,26]. The most important element is about the arrangemen<sup>t</sup> of the nozzles which must ensure equal jet formation and equal pressure drops between the nozzles [26]. The pilot apparatus using this technique is now being sold by some companies such as Brace GmbH (Karlstein am Main, Bavaria, Germany), Nisco Inc. (Zurich, Switzerland), EncapBioSystems AG (Greifensee, Switzerland) [24,49].

**Figure 5.** (**a**) Schematic illustration representing a multi-nozzle encapsulator (1, double piston pump; 2, sterile barrier; 3, damper; 4, vibrator; 5, membrane of pulsation chamber; 6, concentric split; 7, pulsation chamber; 8, nozzle plate; 9, bypass system; 10, reaction vessel; 11, stirrer; and 12, input hardening solution). Reprinted from [26] with permission from Elsevier. Copyright (1998). (**b**) Picture showing the equipment supplied by Brace GmbH (https://www.brace.de).

Once the droplets are formed, the SOL-GEL transition in hydrogels (gel network formation) must take place as soon as possible to prevent either the aggregation of polymer droplets or the undesired leakage of encapsulated drugs. The chemical nature of the droplets (dispersed phase) determines

the subsequent consolidation step, in which the droplets are transformed into solid particles known as gel-beads, involving: (i) non-solvent induced phase separation (NIPS), (ii) temperature or pH modifications, (iii) chemical reactions or ionic cross-linking for water soluble polymers or solvent evaporation/extraction for oil soluble polymers [24]. During the hardening process, the droplets can shrink. The shrinkage is influenced by the type of polysaccharide, its concentration and nature of the hardening medium.

### **3. Methods for the Gelation of Polymeric Droplets to Produce Gel-Particles**

Figure 6 shows the main mechanisms involved in the gelation of polysaccharide droplets to produce gel particles.

**Figure 6.** Illustration of the main mechanisms to induce SOL-GEL transition of polysaccharide droplets: (**a**) non-solvent approach to produce a non-solvent filled gel network, (**b**) pH-induced gelation, (**c**) temperature-induced (thermotropic) gelation in which the polysaccharides undergo structural transition from coil to helix and then to double helix, (**d**) covalent crosslinking approach in which the polysaccharide chains are covalently crosslinked to form gel network and (**e**) ions-induced (ionotropic) gelation in which the polysaccharide molecules are crosslinked by ions. Reprinted (with some modifications) from [4]. Copyright (2018) Ganesan, Budtova, Ratke, Gurikov, Baudron, Preibisch, Niemeyer, Smirnova, Milow.

### *3.1. Non-Solvent Induced Phase Separation*

Non-solvent induced phase separation (NIPS) is also known as coagulation or immersion precipitation. In this case, the polymer is dissolved in a specific solvent and when this solution is extruded into the coagulation bath containing the non-solvent, there is a rapid decrease of polymer solubility leading to phase separation. Polymer chains self-associate and form a 3D self-standing network with the non-solvent in the pores (see Figure 6a). Generally, polysaccharide macromolecules shrink upon the addition of non-solvent, but not completely collapse if polymer concentration is above the overlap concentration. The NIPS process has been applied from several authors to a diverse set of polysaccharides, such as cellulose [50,51], alginate [52,53], pectin [54] and chitin [55]. In these publications, different liquids were exploited as non-solvents to induce phase separation. Pérez-Madrigal et al. studied the ability of aqueous sodium alginates to gelify upon mixing with dimethyl sulfoxide (DMSO) and other organic solvents such as dimethylformamide, methanol, ethanol etc. Gel formation was shown to depend on nature of the non-solvent, solution viscosity (which is correlate to polymer molecular weight and concentration) and gelation time. Similar results were obtained by Tkalec et al. [56,57]. Chitin and chitin-graft-poly(4-vinyl pyridine) were coagulated in ethanol [58,59]; in this case the gelation occurs for the increase of the hydrophobic interactions between the polysaccharide chains, an effect depending on the polymer type. The obtained gel particles are usually defined "alcogels", an attractive opportunity for aerogels processing by supercritical drying [57,60] as we will discuss in the next paragraphs.

The non-solvent properties of ethanol have been also utilized for hardening of alginate and pectin hydrogel microparticles prepared with other techniques such as emulsion gelation [61,62] and ionic cross-linking [61–64] with the aim to further stabilize the polymeric gel network by a combination of hydrogen bonds and hydrophobic interactions [53].
