**1. Introduction**

Zinc oxide (ZnO) is a multifunctional material possessing unique physical and chemical properties, such as high chemical stability, a high electrochemical coupling coefficient, a broad range of radiation absorption and high photostability [1]. For these reasons it is largely used in many applications, ranging from electronics, optoelectronics, sensors and photocatalysis [2,3].

ZnO is also widely used in topical formulations to address several skin conditions, like burns, scars, and irritations, thanks to its non-toxicity, biocompatibility and antimicrobial activity [4].

ZnO exhibits three crystal structures named wurtzite, zinc-blend, and an occasionally noticed rock-salt [5], which allow it to be employed as a nanostructured material for different nanotechnology applications in many industrial areas, such as gas sensors, biosensors, semiconductors, piezoelectric devices, field emission displays and photocatalytic degradation of pollutants [6].

Due to the wide spread of nanotechnology, cosmetics and pharmaceuticals have also been revolutionized. Among all the materials, ZnO has been developed in different nanostructures to enhance its interaction with the skin and to improve the existing products. A promising application consists of the addition of ZnO in wound dressing materials. Nanocomposites represent a good example [7]: they consist of the addition of ZnO nanostructures to polymeric matrices [8–11] in order to impart novel functionalities, such as antibacterial activity. It is well-established in the literature that ZnO displays significant bactericidal properties over a broad range of bacteria [12,13]. This occurs due to several mechanisms, such as generation of reactive oxygen species (ROS), zinc ion release, membrane dysfunction, and nanoparticle penetration. Moreover the physico–chemical parameters of the nanomaterial, such as size, morphology, and specific surface area, remarkably affect the antibacterial properties of ZnO [14,15]. It has also been demonstrated that zinc ion release can improve wound healing [16,17], since zinc is an essential trace element in the human body and acts as a cofactor in zinc-dependent matrix metalloproteinases that augmen<sup>t</sup> auto debridement and keratinocyte migration during wound repair.

The treatment of painful wounds is another important issue in biomedicine. It has been demonstrated that painful wounds can take more time to heal, leading to a lack of compliance by the patients. Several research works [18] have addressed the development of innovative wound dressings, able to deliver small doses of anti-inflammatory analgesic drugs to the wound [19]. In this context the use of ZnO as a drug carrier to be included in the wound dressing device could be of particular interest thanks to its outstanding biocompatibility, even though the application of this material as a drug delivery system has not been investigated widely in the literature [20] and may be considered at its nascent stage.

The use of organic solvents in pharmaceutical technologies is another challenging issue since it leads both to health concerns, which are related to the toxicity of residual solvents in the final products, and to a negative environmental impact. In the last decade, supercritical fluid technology has been emerging as a green drug impregnation method [21]. Supercritical carbon dioxide (scCO2) is the most used supercritical solvent because it is readily available, cheap, non-toxic, non-flammable, and recyclable. At the end of the scCO2–mediated drug impregnation process a simple depressurization step allows a ready-to-use organic-solvent-free drug loaded material to be obtained. Furthermore, it offers the possibility to tailor the operating parameters of the impregnation process, such as temperature, pressure, and time, on the basis of the selected drug/carrier system [22]. This permits a better drug/carrier interaction to be obtained, with the drug in an amorphous state, which improves its dissolution profile and, consequently, its bioavailability [23].

Notwithstanding the above reported remarkable advantages, some drawbacks in the use of this technology have emerged, such as the scarce ability of scCO2 to dissolve polar and ionic species, since it is a linear molecule with no net dipole moment. Furthermore, the elevated pressure required and high maintenance cost can represent a limitation in the use of this technology [21].

Even though the incorporation of active pharmaceutical ingredients (APIs) in organic and inorganic carriers through scCO2 has been proposed in different research areas [22,24,25], the loading of drugs on ZnO carriers has not been investigated ye<sup>t</sup> [23] and, to the best of our knowledge, the clotrimazole incorporation described in our previous work [26] represents the very first study about the loading of an API on nanostructured ZnO by means of scCO2.

The fundamental idea of this research project is to combine the physico–chemical and biological properties of ZnO with the eco-friendly features of the scCO2–mediated drug impregnation process to develop a green multifunctional device for treating painful wounds. This consists of the combination of antibacterial and anti-inflammatory/analgesic action in a single delivery system. ZnO is particularly suitable for this role, because its nanostructure can be tailored to host drug molecules and because it can offer intrinsic antimicrobial activity [15]. Ibuprofen (IBU) has been selected as the drug to be

hosted in the ZnO nanostructures, since it is one of the most commonly used and most frequently prescribed non-steroidal anti-inflammatory drug (NSAID) for oral and topical administration due to its prominent analgesic role [27,28], and it has already been employed to prepare innovative pain-reducing wound dressings [18,19]. Furthermore IBU has also been widely used in many scCO2–mediated drug impregnation processes [23].

This work is a feasibility study aiming at investigating the possibility of loading IBU on different ZnO carriers by means of scCO2 and checking the antimicrobial activity as well as the capability of the obtained system to release Zn2+ ions and the drug, which are essential requirements for the development of a multifunctional device for wound healing applications. Two nanostructured ZnO (NsZnO) powders with different morphologies and physico–chemical parameters were synthetized through wet organic-solvent-free processes [26] and characterized by means of powder X-ray diffraction, FESEM images, and nitrogen adsorption analysis. The samples were also characterized from a biological point of view; particularly, their antimicrobial activity against different microbial strains and the in vitro Zn2+ release profiles from the NsZnO matrices were evaluated. IBU was loaded on the NsZnO carriers with a scCO2–mediated drug impregnation process and in vitro dissolution studies of the loaded drug were performed.

### **2. Materials and Methods**
