## 4 Production of Nanoparticles by Using CO2 as a Co-Solute
As expected, by decreasing the chitosan/doxorubicin ratio from 4/1 to 1/1 \(w\)/\(w\), the particle size reduced, as a result of a decrease in viscosity and surface tension of the solution due to the lower molecular weight of the chemotherapy drug compared to the polymer. Dissolution tests conducted in liquid media at different pH values revealed that the doxorubicin-loaded nanoparticles were highly pH-sensitive. In particular, the drug release rate is greatly enhanced by decreasing the medium pH. The in vitro cytotoxicity profiles of the chitosan/doxorubicin nanoparticles were similar to the unprocessed drug, showing that the drug's activity was preserved during the SAA-HCM process. The SAILA technique consists in the formation of an expanded liquid solution constituted by scCO\({}_{2}\) and an organic solvent in which the solute of interest is dissolved. The expanded ternary solution formed inside a high-pressure saturator is depressurized into a receiving water phase (in which the solute is insoluble) through a nozzle. The water phase is continuously pumped inside the vessel, and the nanoparticle suspension is continuously recovered through a regulation valve [146; 147]. The SAILA process is based on two prerequisites: (1) the solute has to be soluble in the organic solvent and insoluble in water; (2) water and the organic solvent have to be miscible. In this process, carbon dioxide has the role of a co-solute, whereas water has the role of the antisolvent. SAILA technique has been successfully applied for the micro/nanonization of pure compounds [148; 149; 150; 151; 152] or for the attainment of polymer/drug coprecipitates [146; 153; 154]. The organic solvents commonly used for this process are acetone and ethanol; however, in some cases, isopropanol has also been used. The parameters mostly affecting morphology, mean size, and PSD are GLR and temperature. Indeed, in correspondence with different GLR, expanded liquid mixtures with different CO\({}_{2}\) molar fractions are obtained. Particle size decreases with the increase in GLR and, consequently, the increase in X\({}_{\text{CO}2}\). Regarding the effect of temperature, it is possible to observe that, when increasing the temperature, the surface tension of the liquid mixture containing scCO\({}_{2}\) decreases and, as a consequence, a reduction in nanoparticle diameter is generally obtained. In summary, although the particles produced mainly through the SAA process are characterized by at least sub-micrometric dimensions, PSDs are narrower than those obtained with the PGSS method, in which difficult control of the shape/size of the precipitates is encountered. Undoubtedly, among the techniques based on the use of scCO\({}_{2}\) as a co-solute, the smaller particles with a narrower and less polydisperse PSD are those obtained through the SAILA technique. From a commercial point of view, some limitations are linked to the scale-up of the technologies assisted by the use of scCO\({}_{2}\) as a co-solute, such as the possible nozzle blockage and any troubles related to the collection of ultrafine particles, among others. On the other hand, good adaptability to the spray-drying facilities still existing in the pharmaceutical industries can be encountered. However, Thar (USA), Cerbios-Pharma (Switzerland), and Feyecon (the Netherlands) are some of the companies with pilot/industrial facilities. A list of the pharmaceutical nanoparticles produced by using scCO\({}_{2}\) as a co-solute is reported in Table 3, indicating the solvent, the operating pressure, and temperature employed in the mixer or the precipitator. The morphology of the precipitated powder and the particle size are also specified.