**1. Introduction**

Recently, one of the main thrusts of the micro and nano technologies application in the biomedical and clinical field has certainly been observed in the pharmaceutical drug delivery technologies optimization. Whether it is based on active or passive drug delivery, the way in which drugs are delivered substantially impact their e fficacy and toxicity a ffecting their biocompatibility, pharmacokinetics, and pharmacodynamics. Drugs and active molecules can be introduced into the body via a number of administration routes such as buccal/sublingual, nasal, ocular, oral, pulmonary, anal/vaginal, transdermal and parenteral drug delivery [1–3]. Since a high percentage of the active pharmaceutical ingredients settled by the pharmaceutical production are precluded for a classical

administration route, due to their low bioavailability [4], novel technologies assist modern drug delivery. As a result, an increase is observed in the effectiveness and reduction of side effects of the formulations in relation to patient compliance and costs reduction. In the past years, many drug delivery systems as organic and inorganic micro- and nanoparticulated systems as nanoparticles, micelles, liposomes, extracellular vesicles, nanotubes, metal–organic frameworks (MOF) and hydrogels have been used to deliver drugs at their therapeutic concentration to specific cell types and tissues [5–8]. Both material and design should be taken into account when optimizing a drug delivery carrier able to guarantee tuneable release (sustained, controlled, or pulsed), to act as a temporary reservoir, to increase the solubility of hydrophobic formulations, to float in the gastrointestinal tract and to protect the biological cargo from degradation [9].

Porous carriers have been successfully used as drug delivery matrices for their surface properties, high surface area and tuneable pore dimensions [10,11]. According to their pore sizes, porous materials are classified into three different categories, namely microporous, mesoporous, and macroporous [9,12]. Microporous materials such as MOFs and zeolites, are characterized by a well-interconnected network of pores less than 2 nm in size and high thermal stability and catalytic activity [13]. In macroporous materials, pores dimension ranges between 50 and 1000 nm [14] while in mesoporous materials pore size is between 2 to 50 nm [15]. In more details, mesoporous materials with a narrow pore dimension distribution and high surface area can be considered valuable candidates in drug delivery applications [16,17]. In the wide category of mesoporous, many materials are included such as mesoporous silica, hydroxyapatite and carbon, hydrogel and nanogel, metal and metal-doped nanoparticles. These materials have grea<sup>t</sup> versatility since their actions can be regulated by tuning the chemical environment optimizing the loading and consequent release of the chosen drug [18–20]. The drug incorporation into a mesoporous material is usually carried out by embedding the matrix in a concentrated solution of the drug and by a successive drying step. The size of the absorbable molecule (from small active molecules to proteins) is related to the dimension of the pore, and generally, a pore/drug size ratio >1 allows the adsorption of active molecules inside the pores. By using polymeric structure-directing agents, varying the chain length of surfactant or solubilizing supplementary substances into micelles, mesopores sizes can be adjusted from some nanometres to several tens of nanometers [21].

Recent advancement in micro/nano-fabrication techniques, materials science, chemistry and pharmacology has allowed the development of a number of mesoporous materials for drug delivery application characterized by evident structural advancement such as tuneable pore sizes, different grade of skeleton rigidity and two/three dimensional (2D–3D) architectures arrangemen<sup>t</sup> [22–26].

Hard (nanocasting) or soft templating approaches are applied to produce ordered mesostructured porous materials. The templated synthesis usually requires three successive steps: template preparation, template-directed synthesis and template removal. Hard templating leads to very robust structures containing several constituents as carbon, and metals (oxides, nitrides and sulphides) [27,28]. It is a synthetic method based on the deposition of the targeted materials into the narrowed spaces of the template, resulting in a reversed copy of the mold. The pores of these templates are soaked with a precursor of the looked-for product (e.g., a metal salt for metal oxides) which is in situ thermally transformed to the final product. When the template is removed, mesoporous material remains as the negative replica of the hard template [29].

Soft-templating techniques allow direct synthesis of porous materials through block copolymers including blocks of ionic and non-ionic oligomers, amphiphilic surfactants employed as structure-directing agents (SDAs) and through the addition of precursors as metal salts for metal oxide nanomaterials and organosilanes or triethoxysilane for SiO2-based nanomaterials. Soft-templating techniques are those in which small sub-units self-assemble to define the final structure, which is an aggregate of these starting units, which are not embedded in other matrices or removed as in the techniques described above. Upon self-assembly in a solvent, a micellar structure is realized by the fact that the hydrophobic sides of the molecules of the amphiphilic surfactants point inward and

the hydrophilic ones outward in case the solvent is polar, while the opposite occurs if the solvent is non-polar. After this step, micelles are functionalized on their external corona structure using functional groups, frequently polymeric oligomers. Finally, it is the cross-linking of these external terminations which assemble the micelles in a mesoporous superstructure [30].

Producing porous hierarchical materials by integrating macropores in mesoporous tools manifestly increases their practical drug delivery applicability since macropores increase mass transport decreasing diffusion restraints characterizing purely mesoporous materials, while the mesopores empower grea<sup>t</sup> surface area [31,32].

Many methodologies have been optimized to engineer the hierarchically structured mesoporous solutions. The dual-templating synthesis method, applying colloidal crystal (opal) hard-templating and soft-templating techniques, is employed for realizing, as schematized in Figure 1, 3D macro/mesoporous materials for a wide range of applications, including the drug delivery ones [33,34].

**Figure 1.** Schematic representation of 3D macro/mesoporous materials preparation reproduced with permission from [34], Chemistry of Materials, 2018.

## **2. 3D Printed (3DP) Mesoporous Sca**ff**olds Fabrication Technique**

The idea of realizing a macroscopic object via a bottom-up approach has been attractive for a long time but recently, the advancement of both the materials to be used and the techniques to be exploited have made possible the fabrication of 3D printers able to produce any shape in many different natural [35], synthetic, plastic and metallic materials, at variable size scales and with potentially very high accuracy in positioning [36–38]. This has pushed some researchers towards the idea to explore the possibility to use these techniques to realize solutions with different designs, characterized by being made of different types of mesoporous materials [21]. 3D porous substrates, used with or without further functionalization or engineering, are used more and more frequently in in vitro and in vivo drug delivery studies to assist cell growth or tissue regeneration ensuring the right degree of asepticity and differentiation [39–41].

2D and 3D printing tools are appealing for drug delivery applications since state-of-the-art equipment allows the deposition of liquid, gel, and solid constituents enclosing a wide range of pharmaceutics according to predefined schemes. The layer-by-layer assembling mode to print scaffold allows exact control of the design and of the geometry of the internal pores system, which consequently leads to tune the strength of the final products [42,43].

3DP technology can successfully assist engineers, pharmacologists and clinicians in the design and realization of 3D mesoporous scaffolds to be used for different medical applications such as tissue engineering and regenerative medicine implants characterized by the adjustable loading and unloading activity of pharmacologically active substances such as, antibiotics, growth and differentiation factors *(*Figure 2) [44].

**Figure 2.** Schematic layout summarizing pre-loading and direct loading 3DP porous substrate fabrication for in vitro and in vivo drug delivery applications.

These active substances can be incorporated inside the mesoporous 3D structures in two different main steps: during the manufacturing process (pre-loading, PL) by mixing the substances with the printable material and then proceeding with the 3DP technique in mild conditions (i.e., electrospinning or inkjet printing), or at the end of the printing step (direct loading, DL), by soaking the 3D-printed scaffold in a solution of the molecule to be loaded as reported for bone morphogenetic protein-2 (BMP-2) mesoporous calcium silicate (MesoCS) 3D-printed scaffold [45]. PL methods are usually applied for the production of scaffolds able to locally deliver antibiotiotics [46], but unfortunately, antibiotics such as those of the cephalosporin family have significantly reduced efficiency when exposed to heat and, consequently, the DL method is definitely applied to sensitive molecules when the 3DP process is carried out at high temperatures or pressures [47].

There are many 3DP strategies available to the scientific community that allow the realization of mesoporous scaffolds under computer aids combining different processes and materials like carbon nanotubes, nanoparticles, nanofibers, polymers with active biomolecules with or without live cells. These 3DP fabrication techniques, as summarized in Figure 3, include fused deposition modeling (FDM), material jetting as inkjet printing (IP), electron beam melting (EBM), selective laser sintering (SLS), stereolithography (SLA) and digital light processing, electrospinning, and two-photon polymerization (TPP).

## *2.1. Fused Deposition Modeling*

FDM is one of the most inexpensive nozzle-based deposition systems that allows direct printing of 3D CAD designed layer by layer objects. Thermoplastic degradable (polylactic acid, PLA, poly(ε-caprolactone), PCL, polyvinyl alcohol, PVA) and non-degradable (acrylo-nitrile butadiene styrene, ABS, ethylene vinyl acetate, EVA, poly methyl methacrylate, PMMA) polymer filament are pushed into the heater block to melt before extruding from a high-temperature nozzle solidifying onto the previous layer on the build plate [48].

The easiest method of loading target drugs into the thermoplastic polymer filament is the impregnation obtained leaving the just printed device in a concentrated drug solution (mostly ethanol or methanol) followed by a drying step [49,50].
