*5.4. Core-Shell PbHPs*

Generally, the production of core-shell PbHPs through a generic dripping device can be easily conducted exploiting coacervation. The major driving force for the coacervation method is electrostatic attraction between cationic and anionic water-soluble polysaccharides. The resultant "coacervate" generally forms the particle shell. The power of the interaction between the polysaccharides and the nature of the complex is a ffected by many factors such as pH, concentration, ionic strength, biopolymer type, and the ratio of biopolymers [213]. With this method several core-shell PbHPs have been produced [214–216]. For instance, alginate and chitosan can be used together because of their opposite charges to form alginate particles coated with chitosan. The electrostatic interaction of carboxylic groups of alginate with the amine groups of chitosan results in the formation of a membrane surrounding the surface of the core particles and reduces their porosity [47,217,218]. Ren et al. [219] exploited this method to prepare alginate-chitosan microcapsules for protein delivery via oral route. This study confirmed that such systems are pH sensitive; in acidic solution, due to the ionic bond between the chitosan and alginate as well as the physical barrier provided by the interphasic membrane itself, microcapsules maintained integrity well, and e ffectively prevented the direct exposure of protein to the gastric fluid. In fact, less than 10% of protein (i.e., IgG) was released in SGF and 80% of its activity was preserved; during the first hour of permanence into simulated intestinal fluid, a burst release of IgG was observed.

In general, to increase the performances of such particle systems and make more e fficient the delivery of macromolecules as well as drugs throughout the GIT, the manufacturing phase should provide a major control on shell formation process. To this regard, an important achievement was the design and the development of multiparticulate beads in core-shell configuration using (a) prilling apparatus in its basic configuration followed by an enteric coating process [32,205] and (b) prilling apparatus in coaxial configuration [31,43]. This latter is certainly the most innovative approach; it employs multiple concentric nozzles to produce a smooth coaxial jet comprising polymer annular shell and core material, which are broken up by acoustic excitation into uniform core-shell droplets and gelled into a cross-linking solution. PbHPs in core-shell configuration consisting of zinc-ALM pectinate as core and zinc-alginate as shell were produced. Both NSAID (piroxicam, PRX) and SAID (betamethasone, B) were respectively loaded as model drug within the pectin core. The aim was to combine the pH dependent solubility (gastro-resistance) of zinc alginate and the colon targeted selectivity of zinc-ALM pectinate [220,221] in a unique system to obtain an enteric carrier targeting colon. The most critical process parameter to obtain uniform double-layered particles was identified in the ratio between the nozzle viscosity of the inner and outer polymer solutions. In fact, beads with homogeneous layered, good spherical shape and smooth particle surface were obtained when this ratio was above 6. Moreover, optimization of other process parameters such as selection of the cross-linker, pH of the gelling solution as well as cross-linking time was necessary to obtain well-formed and homogeneously coated microcapsules (see Figure 15, panel a) with strong drug/polymer core showing a tailored control of drug release in the gastrointestinal tract (see Figure 15, panel b).

μ

μ

**Figure 15.** Main in vitro results obtained for core-shell (Pectin/Alginate) beads loaded with piroxicam: (**a**) SEM microphotographs showing the complete and homogeneous alginate shell surrounding the pectin core; (**b**) Release profiles of mono-layered "only core" (Pectin) beads (---) and bi-layered "core-shell" (Pectin/Alginate) beads (--), performed in simulated intestinal fluid by using USP Apparatus 2. Reprinted from [43] with permission from Elsevier. Copyright (2014).

### *5.5. Design of PbHPs in Form of Aerogels*

−− −− −S− The application of biopolymer aerogels as drug delivery systems has gained increased interest during the last decade since these structures have large surface area and accessible pores allowing for, e.g., high drug loadings. Examples of oral, mucosal, and most recently pulmonary drug delivery routes have been highly discussed in the literature. Furthermore, thanks to high pore volume and swelling ability both pristine and drug-loaded polysaccharide aerogel particles have been suggested as superabsorbent and for wound healing applications [44,222]. Being largely mesoporous solids, aerogels can accommodate drugs in the amorphous state suppressing re-crystallization [223]. This feature along with the high specific surface area and rapid pore collapse upon contact with liquid media gives rise to unusually fast drug release.

Reverchon et al. [208,209] produced alginate-based aerogels as carriers for the fast delivery of slightly soluble NSAIDs in the upper gastrointestinal tract by prilling of drug/alginate feed solutions followed by cross-linking in ethanol or aqueous CaCl2 solutions, water replacement and, supercritical-CO2-assisted drying. The selected techniques allowed to successfully produce spherical aerogels (sphericity coefficient 0.97–0.99) in narrow size distribution with reduced particle shrinkage and smooth surface (surface roughness 1.10–1.13); the internal porous texture of the parent hydrogels was preserved and appeared as a network of nanopores with diameters around 200 nm (see Figure 16a).

**Figure 16.** Main in vitro results obtained for alginate-based aerogel beads loaded with ketoprofen: (**a**) SEM microphotographs showing the inner nanoporous structure; (**b**) Release profiles of beads dried by both supercritical-CO2 and conventional drying, performed in simulated gastro-intestinal fluids by using USP Apparatus 2. Reprinted from [208] with permission from Elsevier. Copyright (2012).

Recently, we verified the influence of the alginate molecular weight, the solvent used in the gelation solution on porosity, textural properties and stability of the alginate aerogel beads produced via prilling/ionotropic gelation/SC-CO2 drying route [169]. Gelation in ethanolic media promoted the formation of aerogels with higher textural properties compared to the aerogels derived from particle crosslinked in aqueous media. As expected, the textural properties of aerogels were far higher than those obtained from cryogels and xerogels obtained by freeze-drying and oven drying, respectively. This study also highlighted that the use of medium molecular weight alginate led to aerogels with reduced shrinkage and enhanced porosity. By contrast, the use of high molecular weight alginate promoted the formation of aerogels with higher surface area. Finally, stability studies showed non-significant variations in aerogels weight and specific surface area after 3 months of storage, especially, in the case of aerogels produced with medium molecular weight alginate. Overall, this study allowed to highlight the suitability of such materials for wound dressing applications. In fact, thanks to their high surface area, aerogels can rapidly absorb the exudate once applied on a wound and at the same time, they can promote a controlled release of the active substance eventually embedded within the polymer network.

Several other researchers developed aerogel-based formulations for the managemen<sup>t</sup> of chronic wounds. For instance, with this aim <sup>L</sup>ópez-Iglesias and coworkers [224] produced vancomycin-loaded chitosan aerogel beads, starting from the simple dripping of a chitosan solution into a basic NaOH 0.1 M solution. In this sol-gel method, the gelation took place immediately after contact with the medium. After that, the productive process continues with the solvent exchange carried out using absolute EtOH, and it ends with the SC drying. The dried particles showed a fibrous structure characterized by a high porosity (>96%) and large surface area (>200 m<sup>2</sup>/g); they preserved their initial spherical structure, with an overall volume shrinkage of 57.0 ± 4.5% attributable to the flexibility of the polymeric chains of chitosan that are brought closer after the extraction of the solvent.

A significant number of investigations also focused on the possibility to produce layered aerogels. Veronovsky et al. [225] prepared multilayer amidated LM pectin aerogel particles via ionotropic gelation by dripping 2% Wt pectin solutions through a needle into calcium chloride solution. The obtained hydrogels were then dripped into a 1% Wt pectin solution for obtaining membranes around the core particles, and again were crosslinked in CaCl2 solution. Three-membrane hydrogel particles were produced by repeating the process. After solvent exchange, particles were dried with supercritical CO2. To verify the ability of such materials to act as carriers for drug delivery, two model drugs that is theophylline and nicotinic acid were loaded within the core. The selected operative conditions allowed obtaining multilayer pectin aerogel particles with diameter of 8.0 and 9.8 mm, depending on the source of pectin (apple and citrus, respectively). Specific surface area varied from 469 to 593 m<sup>2</sup>/g based on pectin source and its concentration. The release of both loaded drugs turned out to be controlled by swelling and dissolution of pectin matrix. Aerogels from citrus pectin showed more controlled release behavior than those from apple. Moreover, core-shell aerogels have been designed to delivery antibiotics. De Cicco et al. [44] combined amidated LM pectin with alginate to produce core-shell aerogels loaded with doxycycline hyclate, by means of prilling technique in coaxial configuration. Ionotropic gelation was conducted via di ffusion method (external gelation) in an ethanolic CaCl2 solution. The obtained aerogels showed spherical shape, smooth surface and an apparent density of around 0.3 g/cm3.

### *5.6. Design of Experiments (DoE) and Artificial Intelligence (AI) for the Productionof PbHP by Inverse Gelation Technique*

Recently, our research group exploited the possibility to apply the Design of Experiment for the development of wet alginate soft-capsules with a hydrophilic core through inverse gelation [199]. Soft-capsules, designed for topical administration, were produced through a vibrating nozzle device, dripping a thickened calcium chloride solution into an alginate bath. The experimental design applied revealed the e ffect of several critical process parameters such as the solution's composition, frequency and flow-rate onto critical quality attributes of the produced soft-capsules as e.g., drug content, encapsulation efficiency, size, shape and mechanical strength. The overall knowledge gained through the DoE exercise showed that the inverse gelation process was capable of producing PbHPs as soft-capsules with the desired attributes, resistant enough to allow handling during storage, but also very easy to break when applied onto the skin.

Process optimization may be gained by integration of several methods into the process of the formulation design of PbHPs. Artificial intelligence (AI) is one of the possibilities to predict the optimal process conditions, reproducibility and grea<sup>t</sup> precision and accuracy in data analysis. In the last years, we proposed inverse gelation for the production of PbHPs as wet core-shell microcapsules using AI tools for the process optimization [200]. A w/o emulsion containing aqueous calcium chloride solution in sunflower oil pumped through the inner nozzle of a prilling encapsulator apparatus gave the core while an aqueous alginate solution, coming out from the annular nozzle, produced the particle shell (see Figure 17). The numerous operative conditions such as w/o constituents, polymer concentrations, flow rates and frequency of vibration were optimized by two commercial software, FormRules® and INForm®, which implement neurofuzzy logic and artificial neural networks together with genetic algorithms, respectively. The optimized parameters by AI tools allowed to manufacturing of spherical core-shell beads (sphericity coefficient about 0.98) with diameter about 1.1 mm and a narrow size distribution containing the oily droplet wrapped by a thin and regular alginate layer (about 95 μm). This technique certainly represents an innovative approach to produce PbHPs in form of core-shell particles with different hardness loaded with oil or drugs dispersed into lipids.

**Figure 17.** Schematic illustration of the method employed for the production of core-shell microparticles by inverse gelation optimized with artificial intelligent tools. Reprinted from [200] with permission from Elsevier. Copyright (2018).

### **6. PbHPs: Safety by Design and Clinical Translations**

Despite the largely evolving knowledge and techniques for the development of PbHPs as controlled DDS able to improve biopharmaceutical properties of various bioactive molecules (e.g., small molecules, protein, oligonucleotides), their clinical study remains very limited [2]. As it always happens, opportunities are accompanied by challenges, and for new process technologies in pharmaceutical field, the main issue is the insecurity concerning their safe implementation. The main difficulty to translate such systems into clinical studies is due to a general lack of:


both aspects falling in the field of safety. In particular, it is important to consider that natural-based polysaccharides are not a single discrete chemical system, as they vary in number and distribution of repeating building blocks along the backbone [2]. Since polymer molecular weight and composition can significantly vary, their main physicochemical properties such as solubility, chain flexibility, intraand intermolecular forces, carrier size/shape, loading capacity, surface charge, and degradation profile can vary, and consequently also the in vivo performances. These aspects must be taken into account by regulatory authorities during control phases for a successful translation of PbHPs from bench to bedside.

In this contest, the safety-by-design (SbD) approach acquires a significant relevance. In general, SbD concepts foresee the risk identification and reduction as well as uncertainties regarding human health and environmental safety during the early stages of product development, by altering its design and by ensuring safety along its lifecycle. The SbD concept is therefore di fferent from conventional risk assessment approaches, which only consider safety when the product is already fully developed. As well known, while the concept of quality-by-design (QbD) is widely used by pharmaceutical industry and its implementation is foreseen by the pharmaceutical development guidelines [226,227], that of SbD is new, and it is not ye<sup>t</sup> included in ICH, EMA, or FDA guidelines. This means that even if safety is taken into account during the pharmaceutical development, there is still no systematic SbD approach in place, yet. As the QbD requires for its application the definition of the critical quality attributes (CQA) that will lead to the achievement of a product with proven e ffectiveness, in the same way the SbD has to establish CQA leading to a product with low safety concerns. Few papers focus on this intriguing and complex topic involving multidisciplinary knowledge (i.e., life science, clinical medicine, material science, chemistry, and engineering) as well as active collaboration among regulatory authorities, pharmaceutical companies, academics, and governments.

Recently, Schmutz et al. [228] elaborated a useful methodological SbD approach (referred as GoNanoBioMat SbD approach) focusing on polymeric biomaterials widely used to prepare nanoparticles and microparticles for drug delivery. Such approach allows identifying and addressing the relevant safety aspects to face with when developing biopolymeric-based DDS during design, characterization, assessment of human health and environmental risk, manufacturing and handling. As shown in Figure 18, the pillars of such approach are the following:


For the Human Health Risks step, the route of administration/exposure, the dosage, the duration and frequency should be determined as safety of polymeric biomaterials depends on the route of administration/exposure and the resulting respective pharmacokinetic profile. If one final candidate has been selected, the developer of such materials should go to the Manufacturing and Control step to ensure product safety and quality. The goal of this step is to scale-up the production by applying Good Manufacturing Practices (GMPs), preventing contamination and ensuring uniformity between the batches. In this step, CQAs of nanobiomaterials must be identified as well as Critical Process Parameters. These are defined as the "process parameters that influence CQAs and therefore should be monitored or controlled to ensure the process produces the desired quality" (ICH Q8 (R2), 2009). The goal of the final step is guarantee safe storage and transport.

**Figure 18.** Safety-by-Design approach. Blue arrows correspond to the flow of polymeric nanobiomaterials as drug delivery systems from design to storage and transport, red arrows are feedback loops uses whenever the nanobiomaterial product is unsafe, inefficient or has unwanted side effects, and bullet points represent the methods/tools or endpoints at each step. Reprinted from [228]. Copyright (2020) Schmutz, Borges, Jesus, Borchard, Perale, Zinn, Sips, Soeteman-Hernandez, Wick and Som.

A recent paper by Poel and Robay [229] highlights the usefulness to "design for the responsibility for safety", rather than directly for safety, and propose some heuristics to use in deciding how to share and distribute responsibility for safety through design; in summary, designers should think where the responsibility for safety is best situated and design technologies, accordingly. The solution to safety issues is not to be sought in transferring all responsibility to the users of a technology (or to other stakeholders) but rather in a model of responsibility shared among the various actors involved, such as operators and users. The authors state that it is better to accept indeterminacy and to use it as potential source for safety rather to design it out in an attempt to achieve absolute safety, which is unattainable; in many real-world situations, human actions should be considered not a source of risks but of safety. Reason argues that the human possibility to improvise may be crucial to react to (unexpected) risks and is, therefore a source of safety [230]. Based on these considerations, indeterminacy is not only a liability but also an asset as it opens the possibility to use the expertise and insights of the actors in the value chain to identify risks unknown during the design phase.

To date, there are still many open questions on safety and conformity assessment. From this point of view, an early and deep dialogue between experts from academic community, industry and regulatory authorities is of utmost importance to "anticipate" quality and safety requirements of PbHPs. In general, regulatory requirements as well as regulatory/scientific guidance on new technologies and nanomaterials applied in medicinal products as well as medical devices are emerging. A new regulatory framework for medical devices was recently published in Europe [231]. The new regulation contains several provisions for nanomaterials, including a definition, specific attention for safety of nanomaterials and classification rules leading to di fferent routes for conformity assessment. For the implementation of such aspects, more guidance is needed.
