Supercritical Antisolvent Process for Pharmaceutical Applications: A Review

: The supercritical antisolvent (SAS) technique has been widely employed in the biomedical ﬁeld, including drug delivery, to obtain drug particles or polymer-based systems of nanometric or micrometric size. The primary purpose of producing SAS particles is to improve the treatment of di ﬀ erent pathologies and to better the patient’s compliance. In this context, many active compounds have been micronized to enhance their dissolution rate and bioavailability. Aiming for more e ﬀ ective treatments with reduced side e ﬀ ects caused by drug overdose, the SAS polymer / active principle coprecipitation has mainly been proposed to o ﬀ er an adequate drug release for speciﬁc therapy. The demand for new formulations with reduced side e ﬀ ects on the patient’s health is still growing; in this context, the SAS technique is a promising tool to solve existing issues in the biomedical ﬁeld. This updated review on the use of the SAS process for clinical applications provides useful information about the achievements, the most e ﬀ ective polymeric carriers, and parameters, as well as future perspectives. of active compounds and their coprecipitation with polymeric carriers. The advantages and disadvantages associated with the use of the SAS technique, the most e ﬀ ective polymeric carriers, and operating conditions, as well as the processed active principles and the potential applications, are indicated. This review provides guidelines for future prospects.


Introduction
Nowadays, the focus on novel polymer/drug formulations is continuously growing, in order to improve the therapeutic efficacy of an active compound and to augment the patient's compliance. In this context, the micronization processes provide a robust solution to issues related to the biomedical field, including drug delivery. The use of polymeric carriers to produce composite particles has multiple functions, such as the drug protection against its oxidation and/or deactivation caused by external agents (light, oxygen, temperature, pH) and the masking of unpleasant taste and/or odor. However, the primary motivation is often the modification of the release kinetics of the active principle embedded in the polymeric particles. In the biomedical field, a rapid or a prolonged release of the active compound can be necessary depending on the pathology, which may require a fast or long-term response. Many prescribed active principles have a low solubility in water, resulting in low bioavailability. The particle size reduction, which is achieved by micronizing the active compound, allows improving the dissolution rate of poorly water-soluble drugs [1][2][3][4][5][6][7]. However, drug dissolution can be further enhanced by developing polymer-based formulations. This approach is also useful when the active compound cannot be micronized alone [8][9][10][11][12][13], or when a highly water-soluble drug has to be released slowly. For example, in the case of infections, long-term antibiotic therapies are often prescribed. Unfortunately, many antibiotics are very soluble in water, and are characterized by a very short half-life; in these cases, prolonged-release drug delivery systems are desirable to reduce the number of administration and the unwanted effects, including the antibiotic resistance [14][15][16][17]. Instead, given the existence of many inflammatory conditions, in the case of non-steroidal anti-inflammatory drugs (NSAIDs), both a fast and a prolonged release can be required for the treatment of minor taken, serious side effects may occur, such as nausea, vomiting, gastrointestinal bleeding, and peptic ulcer. All these issues can be solved by controlling the drug release with suitable polymers, which may have mostly hydrophobic or hydrophilic behavior, so producing alternative administration systems.
The typical release kinetics of an active principle from conventional pharmaceutical forms is sketched in Figure 1a, showing how the drug concentration can go below or above the therapeutic range, in an uncontrolled manner. Thus, in this case, high and repeated drug dosages are necessary, leading to serious side effects on the patient's health. In Figure 1b, different release kinetics from alternative drug delivery systems are represented, such as immediate, prolonged, delayed, or pulsed drug releases. In this way, the therapy is targeted for the specific application, and the drug concentration in the plasma is ensured within the therapeutic region, i.e., above the minimum effective concentration (MEC) required to have a therapeutic effect and below the maximum safe concentration (MSC) to avoid toxicities. There are different technologies to micronize active compounds or to produce composite polymer/drug particles, such as spray drying, freeze-drying, jet-milling, solvent evaporation, or coacervation. Despite the advantages of using these conventional techniques, such as the possibility of scaling up the process without particular complications, in the case of the widely employed spray drying, these methods also present significant limitations [7,12,[21][22][23][24][25]. Indeed, multistage processing is often involved to remove the toxic organic solvents used, whose residues are often not negligible in the final product. Moreover, the employed high process temperatures can cause the thermal There are different technologies to micronize active compounds or to produce composite polymer/drug particles, such as spray drying, freeze-drying, jet-milling, solvent evaporation, or coacervation. Despite the advantages of using these conventional techniques, such as the possibility of scaling up the process without particular complications, in the case of the widely employed spray drying, these methods also present significant limitations [7,12,[21][22][23][24][25]. Indeed, multistage processing is often involved to remove the toxic organic solvents used, whose residues are often not negligible in the final product. Moreover, the employed high process temperatures can cause the thermal degradation of the thermosensitive active principles. In addition, using conventional micronization techniques, it is difficult to control the morphology and the dimensions of the produced particles.
On the contrary, these drawbacks can be overcome using processes based on the use of supercritical fluids [7,12,[21][22][23][24][25], among which the supercritical carbon dioxide (scCO 2 ) is the most commonly employed in the industrial field. Considerably reducing the use of organic solvents, the scCO 2 -based micronization techniques permit to produce regular particles of minimal dimensions, which are difficult to obtain with conventional processes.
This goal is possible because, in supercritical conditions, fluids have some gas-like properties (e.g., high diffusivity, low viscosity) and other liquid-like properties (e.g., high density, high solvent power). In particular, the supercritical antisolvent (SAS) technique stands out for the production of nanoparticles and microparticles of one or more compounds in a controlled manner. The first papers on the SAS process date back to the early 90s. For example, Yeo et al. processed para-linked aromatic polyamides obtaining polycrystalline spherulites and microfibrils [26,27], Bertucco et al. obtained hyaluronic acid benzylic ester microspheres that can be used for controlled drug delivery [28], Reverchon et al. obtained nanoparticles and sub-microparticles of different superconductor precursors [29,30].
Regarding the pharmaceutical field, using the SAS process, up to now, micronized drug particles and several polymer/active compound composites have been proposed for different biomedical applications [31][32][33][34][35]. Indeed, SAS precipitated particles can be used for drug delivery by oral, parenteral, transdermal, or topical routes [2,[36][37][38][39][40][41][42]. The versatile SAS particles can be used in powder form for the oral administration of composites or the preparation of aerosol formulations, mainly for the treatment of respiratory diseases; they can also be used for the production of medicated patches or active topical gels, by incorporating nanoparticles/microparticles into various types of dressings, supports or pomades, respectively. The dissolution kinetics of many active principles, both of synthetic and natural origin, have been successfully modified by selecting carriers suitable for the required therapy. Different biopolymers, which are biocompatible [11,22,43,44] and sometimes even biodegradable [32,[45][46][47][48][49], have been studied to tune the release of the active compounds. In literature, there are few reviews about the SAS technique [50][51][52][53]. An older overview [53] was focused on the precipitation of single compounds in the form of microparticles and nanoparticles, mainly introducing the fundamentals of the SAS technique. In this study, the micronization of different kinds of materials was considered, without choosing a specific field of application. Abuzar et al. [50] focused their attention to poorly water-soluble drugs, recommending the SAS process as an innovative tool to enhance the bioavailability of these active principles. In another review [51], the SAS process has been indicated as a promising drug encapsulation method, but a limited number of studies compared to the existing ones have been analyzed. Moreover, the crucial role of the polymeric carrier to produce novel formulations for specific biomedical applications and drug dissolution rate is not highlighted. In the paper of Gonzalez-Coloma et al. [52], the attention was instead given to the supercritical antisolvent fractionation (SAF) of natural products. The SAF process is conceptually similar to the SAS one since, in any case, the supercritical fluid acts as an antisolvent; however, the difference is that, in the former technology, a complex mixture consisting of various compounds in a solvent has to be selectively fractionated, precipitating a compound or a series of compounds of interest. It has to be considered that none of the aforementioned reviews report the interesting results obtained in recent years, such as the new carriers identified, whose use has been optimized lately.
Considering the advantages associated with the use of the SAS technique and the goals achieved so far, it is clear that this technology can still make a significant contribution to improving the treatment of several diseases. Therefore, this updated review is focused on the application of the SAS process in the medical/pharmaceutical field, both regarding the micronization of active compounds and their coprecipitation with polymeric carriers. The advantages and disadvantages associated with the use of the SAS technique, the most effective polymeric carriers, and operating conditions, as well as the processed active principles and the potential applications, are indicated. This review provides guidelines for future prospects.

SAS Process: Fundamentals and Test Procedure
The SAS process is based on some main prerequisites. In this technique, the scCO 2 has the role of the antisolvent; therefore, it has to be completely miscible with the selected liquid solvent. On the contrary, the solute/solutes to be processed has/have to be soluble in the liquid solvent but insoluble in the binary mixture formed in the precipitator consisting of the solvent and the antisolvent. Hence, the precipitation/coprecipitation of solute/solutes occurs because of the fast diffusion of scCO 2 into the liquid solvent and the consequent supersaturation of the solute/solutes.
A schematic representation of the SAS process is reported in Figure 2. Briefly, a typical SAS experiment begins by pumping the CO 2 to reach the desired pressure in the precipitator, which is heated up to the selected temperature. Once the operating conditions are stabilized, the pure solvent is delivered to the precipitator, passing through a nozzle. Then, the liquid solution, which consists of the solute/solutes dissolved in the selected solvent, is injected. Due to the supersaturation, the solute/solutes precipitate on a filter, whereas the mixture solvent/antisolvent is recovered and separated downstream the precipitator, where a vessel to collect the liquid solvent is located. Once the solution is injected, the scCO 2 continues to flow to eliminate the solvent residues. At the end of this washing step, the precipitator is depressurized down to atmospheric pressure, and the precipitated powder can be collected. The SAS process is based on some main prerequisites. In this technique, the scCO2 has the role of the antisolvent; therefore, it has to be completely miscible with the selected liquid solvent. On the contrary, the solute/solutes to be processed has/have to be soluble in the liquid solvent but insoluble in the binary mixture formed in the precipitator consisting of the solvent and the antisolvent. Hence, the precipitation/coprecipitation of solute/solutes occurs because of the fast diffusion of scCO2 into the liquid solvent and the consequent supersaturation of the solute/solutes.
A schematic representation of the SAS process is reported in Figure 2. Briefly, a typical SAS experiment begins by pumping the CO2 to reach the desired pressure in the precipitator, which is heated up to the selected temperature. Once the operating conditions are stabilized, the pure solvent is delivered to the precipitator, passing through a nozzle. Then, the liquid solution, which consists of the solute/solutes dissolved in the selected solvent, is injected. Due to the supersaturation, the solute/solutes precipitate on a filter, whereas the mixture solvent/antisolvent is recovered and separated downstream the precipitator, where a vessel to collect the liquid solvent is located. Once the solution is injected, the scCO2 continues to flow to eliminate the solvent residues. At the end of this washing step, the precipitator is depressurized down to atmospheric pressure, and the precipitated powder can be collected. There are also some modified versions of the SAS technique, in which the scCO2 acts equally as an antisolvent, such as the supercritical gas antisolvent (GAS) process and the solution enhanced dispersion by supercritical fluids (SEDS). In the SEDS process, the scCO2 and the liquid solution are both injected through a coaxial nozzle. Instead, in the GAS process, the scCO2 is introduced into the solution consisting of solute/solutes to be precipitated and the liquid solvent. The SAS process was also proposed in the literature with other different acronyms, such as the aerosol solvent extraction system (ASES).

SAS Micronization of Active Compounds
Different factors can influence the oral bioavailability of an active compound, including the firstpass metabolism, the drug permeability, and its solubility and dissolution rate in water [54]. In particular, poor solubility and slow dissolution rates are the leading causes of the low bioavailability for most of the active principles [55][56][57]. Consequently, a low drug amount, often at an insufficient concentration, is available at the specific site of action. There are also some modified versions of the SAS technique, in which the scCO 2 acts equally as an antisolvent, such as the supercritical gas antisolvent (GAS) process and the solution enhanced dispersion by supercritical fluids (SEDS). In the SEDS process, the scCO 2 and the liquid solution are both injected through a coaxial nozzle. Instead, in the GAS process, the scCO 2 is introduced into the solution consisting of solute/solutes to be precipitated and the liquid solvent. The SAS process was also proposed in the literature with other different acronyms, such as the aerosol solvent extraction system (ASES).

SAS Micronization of Active Compounds
Different factors can influence the oral bioavailability of an active compound, including the first-pass metabolism, the drug permeability, and its solubility and dissolution rate in water [54]. In particular, poor solubility and slow dissolution rates are the leading causes of the low bioavailability for most of the active principles [55][56][57]. Consequently, a low drug amount, often at an insufficient concentration, is available at the specific site of action.
In the last decades, drug particles with nanometric or micrometric size have been applied to treat or prevent different pathologies or medical conditions. As previously mentioned, the micronization of an active compound revealed to be a possible approach to enhance its dissolution rate, since the particle size reduction leads to an increase of the specific surface area in contact with the aqueous solution.
Most of nonsteroidal anti-inflammatory drugs (NSAIDs) are a striking example of poorly-water soluble drugs. The micronization of active principles is also a valid solution to reach a target part of the body. Aerosol drug administration or anticancer therapies are striking applications. The drug micronization is also a valid solution when a specific range of particles' size is required for a certain disease; for example, in the case of pulmonary diseases, drug particles (e.g., antibiotics) with mean diameters lower than 1 µm and a controlled particle size distribution (PSD) are desirable.
As in the case of the other supercritical fluids assisted technologies, SAS micronization allows overcoming the main drawbacks of conventional micronization techniques, such as spray drying, freeze-drying, jet-milling, solvent evaporation, and grinding [7,25]. For example, Rogers et al. [73] focused their review on a comparison between scCO 2 -based processes, including SAS, and cryogenic spray-freezing technologies in the micronization of active principles. Both approaches were presented as potential solutions for the production of particles of active compounds that cannot be processed by conventional micronization techniques, such as spray drying and jet-milling. However, the authors highlighted the advantage of using an environmentally-friendly antisolvent as the scCO 2 (in the specific case of the SAS technique) compared to the organic and toxic antisolvents conventionally employed. Moreover, the gas-like and liquid-like properties of scCO 2 , such as the high diffusivity and the high solvent power, allow obtaining dry particles without solvent residues in one step. In reverse, lyophilization or liquid antisolvent extraction are needed to dry powders prepared by the cryogenic micronization technologies. The study of Park et al. [25] demonstrated the superiority of the SAS process with respect to spray drying in the micronization of cefdinir (a model drug). Indeed, microparticles with a mean size equal to 2.32 ± 1.76 µm were obtained by spray drying, whereas nanoparticles with a narrow PSD (0.15 ± 0.07 µm) were produced by the SAS process. Moreover, the specific surface area of SAS-processed particles (55.79 ± 0.06 m 2 /g) was higher than that of spray-dried particles (35.01 ± 0.63 m 2 /g). The increase in the surface area led to a better contact with the water molecules, so the dissolution of the SAS-processed cefdinir was faster than the spray-dried drug particles. According to the results obtained by Ha et al. [7], SAS micronization also proved to be more effective than milling techniques. Indeed, SAS nanoparticles and sub-microparticles (size in the range: 0.15-0.50 µm) allowed to speed up the dissolution of resveratrol, with respect to crystals (18.75 µm) or crystals/irregular coalescent particles (1.94 µm) produced by Fritz milling and jet-milling, respectively. Therefore, it is ascertained that SAS micronization provides benefits in the final pharmaceutical product both in terms of morphology and dimensions of the particles that constitute it.
Similarly, in other works [1][2][3][4][5][6][7]38,66,72,[74][75][76][77], the SAS micronization led to an enhancement of the drug dissolution rate; in some cases, a slight improvement was already observed even when smaller crystals and no regular particles were obtained by SAS with respect to the unprocessed drug. The SAS technique offers the possibility of obtaining both crystalline and amorphous materials by modulating the operating conditions of pressure and temperature. For example, at a fixed temperature, crystals mostly precipitate working at pressures below the mixture critical point (MCP) of the drug/solvent/scCO 2 system, i.e., in the biphasic region. The possibility of producing an amorphous material increases by increasing the pressure, up to a value much higher than the critical pressure, i.e., at fully-developed supercritical conditions. The attainment of amorphous powders involves numerous benefits, including the enhancement of the drug dissolution rate in an aqueous environment, due to a higher contact between the water molecules and the active principle with an increased specific surface area.
However, as emerged from several papers, the selection of the proper liquid solvent is crucial. Indeed, the success of SAS precipitation strongly depends on the affinity between the solvent and the supercritical antisolvent, i.e., the solubility of the liquid solvent in the supercritical CO 2 and the fast gas-like diffusion of the supercritical CO 2 in the solvent. These fundamental aspects assure the attainment of small particles. Many studies showed that dimethylsulfoxide (DMSO) is one of the best solvents for SAS micronization [1,3,6,17,46,[58][59][60][61][62]65,67,71], as it often allows to produce particles with good morphology and small size, in addition to being the most used solvent. Respecting the basic prerequisites of SAS precipitation, up to now, DMSO allowed micronizing a wide variety of active principles in the form of nanometric and micrometric particles. Ethanol [5,7,63,64,69,78] and acetone [2,68] also emerged as good solvents for the SAS micronization of active compounds. In addition, some active principles were effectively micronized using methanol [7,25,72] and N-methyl-2-pyrrolidone (NMP) [38,58,60]. Moreover, acetone belongs to the category of solvents that have a narrow pressure range to switch from the two-phase to the single-phase mixing with scCO 2 ; using this kind of solvent, nanoparticles are generally obtained [79]. Instead, microparticles usually precipitate with DMSO, EtOH, and NMP, which are solvents that present a broad transition zone from the two-phase to the single-phase mixing with scCO 2 . For the latter category of solvents, high operating pressures are necessary to produce nanoparticles. In some papers, SAS precipitation was also attempted by solubilizing drugs into solvent mixtures, which consisted of the solvents above, but satisfying results were reached only in few cases [1,7,13,59,66,80,81].
Another critical parameter for a successful micronization is the molar fraction of CO 2 , which should allow the complete miscibility between the solvent and the antisolvent at the selected pressure and temperature. For this purpose, in most of the reported papers, CO 2 molar fractions higher than 0.95 were used. In addition, the operating pressure and the concentration of the active compound in the liquid solution also influence the morphology and the size of the precipitated powders. In particular, when spherical particles are produced, a decrease of the pressure or an increase of the drug concentration often results in an increase of particles' dimensions [5,38,58,61,62,69,72]. However, it is possible that large crystals significantly precipitate, increasing drug concentration. This result can be explained considering that the presence of the active compound can modify the high-pressure vapor-liquid equilibria (VLEs) of the solvent/antisolvent binary system.
Consequently, the MCP of the drug/solvent/scCO 2 ternary system shifts towards higher pressures with respect to the MCP of the solvent/scCO 2 binary system [82,83]. Therefore, the operating point could lie below the MCP, i.e., in the biphasic region. This effect is accentuated by increasing the drug concentration in the liquid solvent.
The morphology and the dimensions of SAS-processed particles obviously will influence the dissolution of the active compound. Indeed, as highlighted in various papers [7,38,66,72], a smaller particle size, which means an increased surface area, led to an increase in the drug dissolution rate.
However, some active compounds are not processable by the SAS technique. Indeed, in many cases, SAS micronization substantially fails since crystals precipitate [11][12][13]74,87,90,93] or, even, the active compound can be extracted by the solvent/scCO 2 mixture [8,9] not respecting the fundamental prerequisites of the SAS process.
In Table 1, a summary of the active compounds processed by SAS micronization is reported. The active principle to be micronized, the morphology and size of precipitated powders, as well as the applications are indicated. The operating conditions that mainly influence the morphology of particles are also specified, i.e., the liquid solvent used, the pressure (P), the temperature (T), and the drug concentration in the injected solution (C).
As occurs for the drug micronization, the coprecipitation of a polymer and an active principle via the SAS process offers many advantages if compared with conventional techniques [12,[21][22][23][24]. The study of Wu et al. [12] proved the superiority of the SAS process with respect to spray drying in the attainment of polyvinylpyrrolidone (PVP)/piroxicam microparticles. Indeed, the PSD obtained with the SAS technique was narrower than that of the spray-dried particles. Moreover, the dissolution rate of piroxicam released from SAS particles is approximately twice as fast than that of the drug released from the spray-dried particles. In particular, about 2% of pure piroxicam dissolved in 5 min, whereas the percentage of piroxicam released from SAS-processed microparticles and the spray-dried microparticles was about 100% and 55%, respectively [12].
Similarly, Lee et al. [22] prepared inclusion complexes both by the freeze-drying method and the SAS technique. By coprecipitating cetirizine dihydrochloride (an antihistamine drug) with β-cyclodextrin (β-CD), large and irregular crystals were produced by the freeze-drying method, whereas spherical particles were obtained by the SAS technique. In the work of Won et al. [23], the SAS process also showed better performance with respect to the conventional solvent evaporation in the preparation of felodipine-loaded particles based on hydroxypropylmethyl cellulose (HPMC) and poloxamers. In particular, SAS particles proved to be more stable over time, in addition to exhibit a higher drug dissolution rate.
As previously mentioned, polymer/drug composites are produced for various purposes, but the main challenge is to suitably modify the dissolution kinetics of the active principles, aiming for excellent therapies. Depending on the medical application, different drug release kinetics can be required. The choice of the right polymeric carrier is strategic to release the drug at the desired time/speed and/or to a specific site of action. In this context, it was proven that the use of different carriers for SAS coprecipitation leads to different drug releases [46,65]. In particular, when a hydrophilic polymer is used as the carrier, the dissolution of the active principle contained in the SAS composite particles is enhanced. PVP belongs to this category of polymers; indeed, its use allowed to increase the dissolution rate of various poorly-water soluble active compounds [8,9,11,31,33,65,70,81,93,94,97,[115][116][117]. In reverse, the drug release is prolonged using a polymer with a hydrophobic behavior, as occurs, for example, by selecting zein [17,[46][47][48][49]. Similarly, a sustained or prolonged release of the active compound was observed from particles based on polylactic acid (PLA) and poly (L-lactic acid) (PLLA) [32,67,98,99,111,112,[118][119][120][121], which, until now, has been mostly employed to deliver anticancer drugs.
In addition, it is also possible to promote a targeted drug release by producing SAS particles based on pH-sensible polymers, i.e., polymers that dissolve at specific pH values. The Eudragit polymers are a striking example [43,44,98,99,104,122].
Two significant examples of improvement of the drug release kinetics by SAS-produced particles are reported in Figure 3. In Figure 3a, the dissolution of a model NSAID (i.e., ketoprofen) was speeded up by selecting PVP as a hydrophilic carrier to ensure a rapid relief against inflammations, such as headache or toothache. In Figure 3b, the release of a model antibiotic (i.e., ampicillin) was prolonged by producing particles based on zein as a hydrophobic carrier, thus reducing the frequency of administration and the side effects due to antibiotics overuse. However, it is necessary to identify and to use carriers that can be processed by the SAS technique. Up to now, satisfactory results have been reached only with a reduced number of polymers, which are therefore identified as effective carriers for the SAS coprecipitation. PVP is currently the most used polymer and is considered one of the best carriers [8,9,11,31,33,35,65,70,80,81,93,94,97,103,[115][116][117]123] since it is often allowed to obtain regular and spherical composite particles. PVP is followed by PLA [45,108,124] and PLLA [41,67,98,99,[111][112][113][118][119][120][121][125][126][127] that are also suitable carriers often employed. Recently, zein [17,46] is also established as a good polymeric carrier for SAS coprecipitation; moreover, β-CD [22,128] seems to be a promising polymer, but its use has to be further investigated given the very few existing papers. Accurately selecting the solvent and the operating conditions, Eudragit L100-55 also led to the formation of regular composite particles. Therefore, it can be considered a novel carrier more suitable for SAS coprecipitation with respect to the previously tested Eudragits, including Eudragit RS100 and Eudragit RL100 [43,98,99,122]. Eudragit L100-55 is soluble at a pH higher than 5.5, corresponding to the first intestinal tract (duodenum), thus the active compound is protected against the acid gastric fluid; in the meanwhile, the side effects on the gastric tract are also avoided. Eudragit L100-55 allowed reaching a controlled release of NSAIDs, antibiotics, and bronchodilator drugs from SAS microspheres [44,104]. Until now, the SAS coprecipitation was attempted using other kinds of polymers, such as polyethylene glycol (PEG) [35,106,129,130], poly(lactide-co-glycolide) (PLGA) [ [95,96,[131][132][133][134][135]. However, the use of these carriers must be further investigated, by changing the selected solvents or/and the operating conditions, because the morphology of the polymer/drug precipitated powder is not yet satisfactory.
Indeed, in addition to respecting the essential prerequisites of the SAS technique, it has to be considered that the SAS coprecipitation seems to be effective when composite microspheres are obtained, i.e., microparticles consisting of a polymeric matrix in which the drug is homogeneously dispersed [46]. In this case, in the meanwhile that the liquid solution is fed to the precipitator, the liquid jet break-up and the subsequent atomization quickly occur and prevail with respect to the surface tension vanishing. As a consequence, the polymer and the active compound are entrapped in However, it is necessary to identify and to use carriers that can be processed by the SAS technique. Up to now, satisfactory results have been reached only with a reduced number of polymers, which are therefore identified as effective carriers for the SAS coprecipitation. PVP is currently the most used polymer and is considered one of the best carriers [8,9,11,31,33,35,65,70,80,81,93,94,97,103,[115][116][117]123] since it is often allowed to obtain regular and spherical composite particles. PVP is followed by PLA [45,108,124] and PLLA [41,67,98,99,[111][112][113][118][119][120][121][125][126][127] that are also suitable carriers often employed. Recently, zein [17,46] is also established as a good polymeric carrier for SAS coprecipitation; moreover, β-CD [22,128] seems to be a promising polymer, but its use has to be further investigated given the very few existing papers. Accurately selecting the solvent and the operating conditions, Eudragit L100-55 also led to the formation of regular composite particles. Therefore, it can be considered a novel carrier more suitable for SAS coprecipitation with respect to the previously tested Eudragits, including Eudragit RS100 and Eudragit RL100 [43,98,99,122]. Eudragit L100-55 is soluble at a pH higher than 5.5, corresponding to the first intestinal tract (duodenum), thus the active compound is protected against the acid gastric fluid; in the meanwhile, the side effects on the gastric tract are also avoided. Eudragit L100-55 allowed reaching a controlled release of NSAIDs, antibiotics, and bronchodilator drugs from SAS microspheres [44,104]. Until now, the SAS coprecipitation was attempted using other kinds of polymers, such as polyethylene glycol (PEG) [35,106,129,130], poly(lactide-co-glycolide) (PLGA) [36,37,109], ethyl cellulose (EC) [91,[100][101][102]105], HPMC [34,39,42,70,107], and poly(hydroxybutyrate-co-hydroxyvalerate (PHBV) [95,96,[131][132][133][134][135]. However, the use of these carriers must be further investigated, by changing the selected solvents or/and the operating conditions, because the morphology of the polymer/drug precipitated powder is not yet satisfactory.
Indeed, in addition to respecting the essential prerequisites of the SAS technique, it has to be considered that the SAS coprecipitation seems to be effective when composite microspheres are obtained, i.e., microparticles consisting of a polymeric matrix in which the drug is homogeneously dispersed [46]. In this case, in the meanwhile that the liquid solution is fed to the precipitator, the liquid jet break-up and the subsequent atomization quickly occur and prevail with respect to the surface tension vanishing. As a consequence, the polymer and the active compound are entrapped in the same droplet, which behaves as an isolated reactor. The droplet drying by scCO 2 leads to the attainment of the composite microsphere, as represented in Figure 4.  On the contrary, when nanoparticles precipitate, the coprecipitation fails at least in part. In particular, nanoparticles are formed when the surface tension vanishing is very fast, and the gas mixing prevails on the breakage of the liquid jet. Consequently, no droplets are formed and the precipitation of nanoparticles occurs by gas-to-particles nucleation and growth. In this case, a sort of physical mixture at the nanometric level is obtained, i.e., polymer nanoparticles mixed with nanoparticles of the active compound. In addition, in the case of the sub-microparticles, a specific portion of the recovered powder consists of the polymer and the drug precipitated together, but with an irregular distribution of the two materials. Hence, the achievement of an effective polymer/active compound coprecipitation seems to be strongly influenced by the size and the morphology of the particles produced, as well as by their shape. Indeed, the coprecipitation fails also in the case of the attainment of crystals, which occurs by operating in the miscibility gap between the solvent and the antisolvent, where the two compounds tend to precipitate separately, resulting in polymer crystals and drug crystals.
This correlation between a successful coprecipitation and the morphology/size of particles was proved in the study of Franco et al. [46] employing the dissolution tests. In particular, using zein as the carrier and diclofenac sodium as the model drug, it was observed that, increasing the polymer/drug ratio, the particles' diameter increased, whereas the release of the active compound was further slowed down. Moreover, as the particle size decreased, the burst-like effect, i.e., the dissolution of the drug near/on the particles' surface, which, therefore, dissolve as quickly as the unprocessed drug increased.
Several papers showed that the selection of a proper carrier could force even the morphology of active principles that cannot be processed alone by the SAS technique [5,[8][9][10][11][12][13]44]. Moreover, the entrapment of the active compound into the amorphous polymeric matrix can also favor the inhibition of the drug recrystallization [136].
Nevertheless, it has to be taken into account that the presence of the active compound can modify the polymer processability even if, under the selected process conditions, the carrier can be micronized alone in well-defined particles [44]. This influence may be a consequence both of an interaction between the polymer and the drug, but usually, it is due to the presence of solutes On the contrary, when nanoparticles precipitate, the coprecipitation fails at least in part. In particular, nanoparticles are formed when the surface tension vanishing is very fast, and the gas mixing prevails on the breakage of the liquid jet. Consequently, no droplets are formed and the precipitation of nanoparticles occurs by gas-to-particles nucleation and growth. In this case, a sort of physical mixture at the nanometric level is obtained, i.e., polymer nanoparticles mixed with nanoparticles of the active compound. In addition, in the case of the sub-microparticles, a specific portion of the recovered powder consists of the polymer and the drug precipitated together, but with an irregular distribution of the two materials. Hence, the achievement of an effective polymer/active compound coprecipitation seems to be strongly influenced by the size and the morphology of the particles produced, as well as by their shape. Indeed, the coprecipitation fails also in the case of the attainment of crystals, which occurs by operating in the miscibility gap between the solvent and the antisolvent, where the two compounds tend to precipitate separately, resulting in polymer crystals and drug crystals.
This correlation between a successful coprecipitation and the morphology/size of particles was proved in the study of Franco et al. [46] employing the dissolution tests. In particular, using zein as the carrier and diclofenac sodium as the model drug, it was observed that, increasing the polymer/drug ratio, the particles' diameter increased, whereas the release of the active compound was further slowed down. Moreover, as the particle size decreased, the burst-like effect, i.e., the dissolution of the drug near/on the particles' surface, which, therefore, dissolve as quickly as the unprocessed drug increased.
Several papers showed that the selection of a proper carrier could force even the morphology of active principles that cannot be processed alone by the SAS technique [5,[8][9][10][11][12][13]44]. Moreover, the entrapment of the active compound into the amorphous polymeric matrix can also favor the inhibition of the drug recrystallization [136].
Nevertheless, it has to be taken into account that the presence of the active compound can modify the polymer processability even if, under the selected process conditions, the carrier can be micronized alone in well-defined particles [44]. This influence may be a consequence both of an interaction between the polymer and the drug, but usually, it is due to the presence of solutes (polymer and/or active principle) that can alter the high-pressure VLEs. Therefore, the critical point of the solvent/polymer/drug/antisolvent quaternary mixture can shift with respect to that of the solvent/scCO 2 binary mixture [82,83]. Each compound (i.e., carrier and drug) has a different influence on VLEs, which might be negligible or not; thus, the morphology of the precipitated composites also depends on this factor.
In the case of the coprecipitation, it is evident that the polymer/drug ratio plays a crucial role in the attainment of composite particles. Moreover, the polymer content strongly affects the dissolution rate; in general, the release of the active compound is mostly modified by increasing the polymer/drug ratio [23,41,46,99,110,117,119].
Aiming to obtain spherical microspheres that guarantee an effective coprecipitation, in some cases, it is not possible to reduce the amount of polymer in the composite particles beyond a specific value [97,123]. In particular, a high polymer content in the pharmaceutical formulations can be a limitation of the SAS coprecipitation when some carriers are employed. Among these, up to now, the use of Eudragit L100-55 allowed producing well-defined microparticles only at polymer/drug ratios equal to 20/1 and 10/1 w/w [44,104]. Similarly, in the studies of Chhouk et al. [123] and Ozkan et al. [97], polymer/active compound ratios equal to 30/1 and 20/1 w/w were indicated as optimal for the production of PVP/curcumin and PVP/quercetin particles, respectively. However, this drawback was found only in these few cases using PVP as the carrier. Alternatively, a high amount of polymer in the SAS coprecipitated powders can be avoided by selecting β-CD.
In general, cyclodextrins (CDs) are characterized by a hydrophilic external surface, which makes them very soluble in water, and a hydrophobic internal cavity, in which various molecules can be incorporated to form guest/host inclusion complexes by non-covalent interactions. The production of CDs-based inclusion complexes by the SAS technique has been exploited to mask the bitter taste and/or to increase the dissolution of various active compounds, such as antioxidants [114,128,137,138], antihistamine drugs [22], antibiotics [139], and others [140]. In these papers, hydroxypropyl-β-cyclodextrin (HP-β-CD) [114,[137][138][139][140] is mainly employed, followed by β-CD [22,128]. In particular, from the few available studies, β-CD seems to be a more suitable carrier for the SAS process with respect to H-β-CD, since composite particles of regular morphology and size were successfully produced only with β-CD. In addition, inclusion complexes are generally formed by using a low polymer content, i.e., from molar ratio polymer/drug equal to 3/1 up to 1/2 mol/mol. Therefore, the application of β-CD may be a useful tool to reduce the amount of polymeric carrier in the SAS-prepared composite particles significantly, when a rapid drug release is desired.
In addition to the polymer/active compound ratio, other parameters influence the morphology and size of composite particles, mainly the pressure and the overall concentration of solutes in the liquid solution [8,9,44,65,80,81,93,94,97,110,117]. If the pressure is too high, the disappearance of the surface tension prevails over the breakage of the liquid jet, and the coprecipitation partly fails with the formation of nanoparticles, as previously mentioned. This failure also occurs if the pressure is too low and below the MCP, hence the operating point lies in the biphasic region, resulting in the precipitation of crystals or aggregates; even, the liquid can be recovered in the precipitator. Regarding the total concentration, undoubtedly, the upper limit is represented by the solubility of the solutes in the selected solvent. Nanoparticles or coalescent particles often precipitate when the concentration of solutes is too low, because of the fast vanishing of the surface tension and the low viscosity of the injected solution, not allowing the generation of well-defined droplets. Moreover, in the case of a low concentration in the solution, the content of the polymeric carrier, which acts as a microsphere-forming agent, is not enough to force the morphology of the composite system and to trap the active compound into the polymeric matrix. On the contrary, if the total concentration is too high, aggregates or even crystals can be obtained due to the influence that solutes may have on VLEs, as explained above.
In some cases, the variation of the temperature can favor the attainment of well-separated microparticles, as occurs with zein [17,46]. As demonstrated in the study of Franco et al. [17], by coprecipitating ampicillin (a model antibiotic) and zein at a fixed pressure (90 bar) and total concentration (50 mg/mL), polymer/drug 20/1 w/w sub-microparticles (mean size: 0.36 ± 0.30 µm) were produced working at 40 • C. Instead, spherical microparticles with a mean size in the range 19-9 µm were obtained at 50 • C, also managing to reduce the ampicillin/zein ratio up to 5/1 w/w. The morphology and shape of zein-based particles prepared by the SAS process are also definitely better than aggregates and irregular or collapsed particles obtained up to now with the techniques commonly used in the pharmaceutical field, namely spray freeze-drying and spray-drying [141][142][143].
A similar trend to that shown in the study of Franco et al. [17] was also reported in the paper of Montes et al. [99] since the dimensions of ibuprofen-loaded particles produced using PLLA or Eudragit L100 increased by increasing the temperature. Instead, Kalogiannis et al. [59] and Patomchaiviwat et al. [119] observed a rise in the degree of coalescence of the PLLA-based particles by increasing the temperature up to 50 • C, probably due to the PLLA plasticization. Indeed, it is well known that the glass transition temperature (T g ) of a polymer can be depressed in the presence of the scCO 2 , because of its absorption into the polymeric matrix that leads to a weakening of the intramolecular and intermolecular attractions between the polymeric segments [144][145][146]. Therefore, the polymer T g decreases in proportion to the amount of scCO 2 that the polymer is able to absorb. The CO 2 absorption and, consequently, its plasticizing effect in the presence of semicrystalline polymers also depends on the operating temperature, which influences the scCO 2 properties.
A glaring gap emerged from the SAS literature: Up to now, the use of polymeric blends has been employed to produce drug delivery systems in very few papers [23,37,40,41]. Polymer blends can be exploited to tune the drug release as desired, by modulating the ratio between the hydrophilic and hydrophobic polymers that constitute the microparticles. In addition, they are useful to improve other features of the final product, such as mechanical properties and moisture absorption. For example, the very hydrophilic PVP can induce a hygroscopic effect, which could lead to low stability over time and high water uptake in the produced composite powders. This critical issue can be overcome by introducing hydrophobic polymers in the formulation, thus increasing the particles' resistance to humidity. Lee et al. [37] selected PLGA and PLLA to produce microspheres loaded with bupivacaine HCl, a local anesthetic drug generally administered by parental route for the regional anaesthesia, i.e., the local pain control. Various PLGA/PLLA mass ratios were investigated, as well as the use of PLLA at two different molecular weights (MW 2000 or 50,000). It was proved that the release of bupivacaine HCl from particles obtained by SAS depends on the PLGA/PLLA ratio and on the molecular weight of PLLA. In particular, by increasing the PLGA content, the drug dissolution rate increased, until having a rapid release (in about 4 h) when only PLGA was used.
On the contrary, both molecular weight PLLA prolonged the bupivacaine dissolution, but a slower drug release with a lower burst-like effect was achieved when the lower molecular weight PLLA (MW 2000) was employed. These results are partly also since the particles' morphology improved as the PLGA content decreased. Amphiphilic block copolymers, obtained by combining hydrophilic and hydrophobic polymeric segments, can also be employed to deliver active compounds. For example, in the paper of Jung et al. [41], PLLA, methoxy poly-(ethylene glycol)-b-poly(L-lactide) (mPEG-PLLA) block copolymers or PLLA/PEG blends were chosen as carriers for controlled delivery of leuprolide acetate. The last-mentioned one is a non-steroidal antiandrogen drug used to treat prostate and breast cancers and endometriosis, which is usually administered via the parenteral route, being not orally bioavailable. However, leuprolide-based therapies require long-term daily injections and frequent administrations because this drug is very water-soluble and it has a short half-life when administrated via the parenteral route. The mPEG-PLLA diblock copolymers were synthesized by ring opening polymerization of L-lactide in the presence of PEG chain lengths. The effect of the mPEG block length, the PEG/PLLA blending, and the polymer/drug ratio on the leuprolide acetate release were investigated. A dichloromethane/methanol 1/1 v/v mixture was used for the SAS coprecipitation, and in all the investigated conditions, spherical microparticles were produced. It was observed that the mean diameter of PLLA-based microparticles was higher than the one of microparticles prepared using mPEG-PLLA.
Moreover, an increase in the mean particle size was noted as the PEG block length increased in the mPEG-PLLA diblock copolymer. The drug entrapment efficiency was not significantly influenced by the mPEG block length in mPEG-PLLA, the blending of PLLA and PEG, as well as the polymer/drug ratio. However, these factors influence the release of leuprolide acetate from SAS microparticles. Indeed, the drug dissolution rate increased by increasing the PEG block length in the mPEG-PLLA. Microparticles based on PEG/PLLA blends showed a faster drug release with respect to the mPEG-PLLA copolymer containing an equal mass ratio PEG/PLLA. Concerning the effect of the polymer/drug ratio, a higher amount of mPEG-PLLA allowed a slower drug release rate.
Recently, some changes to the SAS process have been suggested [13,47]. In particular, Matos et al. [13] proposed a single-step coprecipitation and coating by using the supercritical antisolvent. A SAS coprecipitation of PVP/curcumin particles was attempted to coat the surface of different polymers, namely microcrystalline cellulose (MCC) (size of crystals/irregular particles: 175 µm), corn starch (size: 15 µm), or lactose (size: <5 µm). Briefly, before starting the experiment, irregular particles/crystals of MCC, starch, or lactose were located into the precipitator; then, the solution containing PVP and curcumin is injected and, because of the antisolvent effect of the scCO 2 , composite particles precipitated onto the different polymers used as support. During the single-step coprecipitation and coating, the stirring into the precipitator ensured a homogeneous coating onto the polymer surface. However, a proper morphology of the final product was not guaranteed, since it strongly depends on the shape of the polymeric supports initially charged into the precipitator. Moreover, from the release tests, it can be noted that only about 10% of the unprocessed curcumin dissolved in 60 min. In contrast, the drug released from PVP-based particles precipitated by a conventional SAS coprecipitation (i.e., without other polymeric supports) already reached more than 95% of dissolution in 5 min. The dissolution of curcumin did not show a further improvement when PVP/curcumin particles were precipitated onto the polymeric supports. Instead, Liu et al. [47] attempted to incorporate an anticancer compound, namely 10-hydroxycamptothecin (HCPT), into zein-based particles. By coprecipitating the polymer and the drug through the SAS technique, rods-like crystals and drug nanoparticles were obtained. These results suggest that coprecipitation did not occur because the polymer and the drug precipitated separately and not in the form of composite microspheres. An unsatisfactory morphology was also achieved using the built-in ultrasonic dialysis process (BUDP), which couples the ultrasonic emulsification with the dialysis technologies [147]. Then, the authors combined the supercritical process with the BUDP by dispersing the SAS coprecipitated zein/HCPT powders into an ethanol/water mixture, which was used as the dialysis solution for BUDP. In this way, well-defined zein/HCPT microspheres were produced, also prolonging the drug release. However, nowadays, aiming at the process intensification, the use of a multistage-procedure is not the best option, both in terms of costs and of process times.
A summary of the polymer/active compound composites produced by the SAS technique for medical purposes is reported in Table 2. The polymeric carriers and the active principles processed were indicated, as well as the main process parameters, including the liquid solvent used, the pressure (P), the temperature (T), and the overall concentration of solutes (polymer + drug) in the liquid solution (C tot ). The morphology and the dimensions of SAS-obtained samples are also specified. In particular, the literature results are listed in Table 2, firstly reporting the polymers that revealed to be the most used and the most suitable for the SAS technique.

Conclusions
This review is focused on the application of the SAS technique in the biomedical field. In this context, the micronization and coprecipitation by the supercritical antisolvent have been proposed for several purposes. Regarding the production of composite particles, the opportunity of modulating the drug release by choosing a carrier based on the required therapy is particularly impressive. In this way, it is possible to release the active principle to a specific site of action and/or at the desired dissolution rate, therefore reducing the side effects caused by drug overdoses and improving the patient's compliance. However, to date, few polymers can be defined as valid for a successful SAS coprecipitation in the form of microspheres, including PVP, PLA, PLLA, zein, and Eudragit L100-55. From the very few conducted studies, β-CD also seems a promising SAS carrier, but its use has yet to be deeply investigated. The main challenge is, therefore, to identify new polymeric carriers suitable for the SAS coprecipitation, in order to develop novel polymer/active compound systems and overcome the main issues still existing in the biomedical field. A key role may be played by the use of polymeric blends, which is still practically unexplored with regard to the SAS technique. Maintaining the microparticles' morphology and varying the content of hydrophobic and hydrophilic polymers, the release can be tuned according to the specific application. Anyway, the use of the SAS technique in the biomedical field is strategic to produce composite systems without residues of solvents that are toxic for human health and to produce particles in a controlled manner with a narrow PSD.