One of the main biomedical development lines consists on the study of implants that would not cause a rejection reaction from the patients’ organisms. It is well known that Spain is one of the leading countries in this type of interventions, with a record number of 5,500 transplants completed in 2019. Despite this accomplishment, the situation is still far from optimal. According to the Spanish National Transplant Organization, the number of patients who are registered to receive a liver transplant each year, for example, is 25% greater than the number of transplants that are actually performed. In addition to this, rejection and/or other implant related disorders may reach up to 50%.

In response to these challenges, efforts have been made for decades to develop an alternative for a better future, namely tissue engineering, which is based on promoting the self- regeneration of the damaged organ by the patient’s organism itself. While the biological mechanisms that allow a bone fracture or other minor injuries to regenerate are being investigated, research efforts are focusing on the use of biocompatible materials; that is, materials that do not produce an adverse reaction by the body and serve as a protective and supportive medium for the cells in the vicinity, providing them with an environment that favors the regeneration of the new tissue. Consequently, and in order to ensure a complete acceptance of the new tissue, implants must exhibit similar permeability and mechanical stability with respect to the new tissue [1]. In this sense, some studies have already proven that, when cells are grown in 3D structures, their behavior and response is similar to that registered in in vivo essays [2]. When, additionally, a greater control on the growth of the new tissue is desired, such porous 3D devices may act as pharmacological biomaterials by incorporating slow-releasing drugs, such as bioactive molecules with anesthetic, anti- inflammatory, antibiotic properties or even with the growth factors that are associated to the type of tissue to be repaired [3].

A good alternative for the production of biomaterials are the so-called conjugated polymers. Conjugated polymers are composite materials that are formed by an oxidized polymer skeleton and a certain amount of negatively charged species (dopants) that balance the total charge. The combination of these two elements allows a rapid electronic movement through the polymer structure that makes them highly conductive [4]. Their main disadvantage with regard to its biomedical usage is their poor mechanical properties and their difficulty to be handled and processed, since they are usually fragile, rigid and not very durable because of their propensity to delaminate.
In order to avoid such negative effects, the combination of a conjugated polymer with other non-conductive and more malleable polymers has recently been studied so as to benefit from the favorable characteristics of both materials [5,6]. The conjugated polymers that are most commonly used in the biomedical field are PPy, PANI and PEDOT [1] and the most often used non-conductive malleable polymers are PLA, PLGA and PCL. Another option under study consists on the hydrothermal treatment of these polymers, since it has been proven to effectively modify the structure and properties of polymers [7]. Rapid heating/cooling procedures and short reaction times [8] may improve the performance of the polymers.

And a good alternative for polymer impregnation is the supercritical impregnation [3]. Supercritical impregnation exploits the density and diffusivity properties of CO2-sc. On the one hand, CO2-sc has a good solvation ability due to its relatively high density that allows the solubilization of a large variety of compounds. On the other hand, its high diffusivity facilitates the penetration of the solvent into a wide range of polymer matrices. Supercritical impregnation can be briefly described in three steps: 1. Dissolution of the solute in the CO2; 2. Contact between the solution and the polymer, for the diffusion and impregnation of the former into the polymeric matrix; 3. Depressurization to remove the CO2 while the solute compounds are held within the polymer. During the last step, the temperature of depressurization speed must be controlled, since they have a considerable influence on the mechanical properties of the matrix that is being impregnated.

The versatility of this technique lies not only with the advantages of CO2-sc as a substance carrier, but also with the effect that it has on the polymers under supercritical conditions, since the modification of their structural characteristics can give rise to devices that would be suitable for different applications, mainly depending on whether or not a process known as foaming has taken place during such depressurizing phase.

Foaming is a process that arises when a polymer is brought into contact with CO2-sc: the CO2- sc is introduced between the polymer chains and turns the polymer into a paste by lowering its vitreous transition temperature. This system reaches a supersaturated state that causes phase separation and the formation of pores within the polymer matrix. This technique is mainly applied to amorphous polymers, with the exception of polymers with a high crystallinity or vitreous transition temperature [9].

From the biomedical point of view, foaming has some advantages and disadvantages depending on the objective that is being pursued. In the case of tissue regeneration, the device must be highly porous, as it is the case with scaffolds. Foaming is highly interesting in this case, since it allows a number of homogeneous and interconnected pores to be generated, which would favors cell growth and the acceptance of the biomedical implant by the patient’s body. However, for other devices, such as intraocular lenses [10] or stents [11], an excessive porosity of the polymer would have a negative impact on the final product, either because of the increased turbidity or because of the excessive cell proliferation that it might cause. Therefore, supercritical impregnation processes using CO2-sc require a strict control on some of the operating conditions if such phenomenon is to be avoided.

Supercritical technology has already been used to produce scaffolds through different methods. One of such methods consists on the placing of the polymer in a cell and then introducing the CO2 that would remain in contact with the polymer for as long as necessary to reach the equilibrium of the CO2 dissolution inside the polymer. The system is then depressurized to remove the CO2 while giving place to the foaming of the polymer. Scaffolds made of PCL [12] or polyvinylidene fluoride copolymerized with hexafluoropropylene [13] have been produced using this method. Another method to produce scaffolds using CO2-sc consists on the phase inversion or immersion precipitation. In this case, the polymer, which has been previously dissolved in an organic solvent, is introduced into a bath with CO2-sc where the polymer is not soluble. The contact between the solvent and the CO2-sc promotes phase separation, the organic solvent is extracted and the scaffold is precipitated because of the anti- solvent effect. By means of this method, scaffolds have been prepared based on chitosan [14], PLA, PMMA, natural polymers [15-19] and PCL [20]. A third method that has been developed consists on the formation of a polymeric gel that is subsequently dried out. This technique has been used to obtain PLA scaffolds [21].

Apart from generating scaffolds, supercritical technology has also been used to impregnate them. In the literature we can find different studies on the impregnation of collagen scaffolds with nanoparticles from natural propolis extract [22]; hybrid PCL/PLA scaffolds impregnated with black seed extracts from Nigella sativa [23]; PCL and hydroxyapatite containing two chitosan and collagen growth factors, BMP2 and VEGF [24], plus ibuprofen and silver particles for the treatment of wound infections by transdermal administration [25]; alginates impregnated

with ibuprofen and calcium, to be used in the treatment of wounds and burns [26]; chitosan with dexamethasone [27]; PMMA with a protein [28]; PCL with nimesulide [29]; mixtures of PLA/PLGA with indomethacin [30]; LCP impregnated with natural compounds extracted from lichen of the genus Usnea [31]; PLGA impregnated with gemcitabine [32]; mixtures of PLA/PLGA with 5-fluorocyl [33] and LCP containing 5-fluorouracil, nicotinamide and triflusal [34].

As far as stents are concerned, although the trend that is currently being studied involves the use of pharmacoactive stents made up of absorbable polymers, i.e., drug releasing agents that, once their objective of opening the body’s duct has been achieved, are absorbed by the patient’s organism, hardly any references have been found in the literature on the use of supercritical technology for this purpose. Nevertheless, the studies by Barros et al. analyze the supercritical impregnation of biodegradable ureteral stents based on alginate and gelatin gum [11].

This project proposes the study of the foaming process and the functionalization of combined polymers obtained through supercritical impregnation with natural extracts from mango leaves that exhibit pharmacological properties. This extract has been studied previously by the research group [35]. Mango extract has shown excellent nutraceutical and pharmacological properties and is considered a promising agent in the treatment of various degenerative diseases such as cancer [36] and Alzheimer’s [37].

The project that we are applying for represents the extension of a previous project entitled Impregnation of extracts and functionalization of antioxidant nanoparticles obtained from mango leaves through high pressure processes and their application in biomedicine (CTQ2017-86661-R. IP: Clara Pereyra. 2018-2020) and arises as a logical continuation based on the results obtained by some of the research team members in previous experiences related to the use of polymers and the impregnation of porous matrices.

In the last decade, the main researchers in the present project have developed their work in two lines of research financed by the calls for projects under the National Programme for Research Aimed at the Challenges of Society. On the one hand, the development of nanoparticles and nanocapsules of interest in the pharmaceutical sector, led by Dr. Clara Mª Pereyra (CTQ2010-19368: Co-precipitation of non-steroidal anti-inflammatories and polymers with supercritical carbon dioxide using the RESS technique – IP Clara Mª Pereyra 2011-2013; CTQ2013-47058-R: Impregnation of silica particles with natural antioxidant nanocapsules using supercritical technology. IP Clara Mª Pereyra. 2014-2016); and on the other hand, the extraction of active compounds and their application to the food industry, led by Dr. Casimiro Mantell (CTQ2011-22974: Fractionation and purification of antioxidant compounds from agricultural by-products using high-pressure separation techniques. IP Casimiro Mantell. 2012-2015; CTQ2014-52427-R: Supercritical impregnation of natural extracts in the preservation of perishable foods. IP Casimiro Mantell. 2015-2018).

Since both lines have been increasingly growing closer towards a common topic, i.e. supercritical impregnation, when applying for the call under the National Programme for Research Aimed at the Challenges of Society 2018, they jointly applied for the project “Impregnation of extracts and functionalization of antioxidant nanoparticles obtained from mango leaves by means of high-pressure processes and their application in biomedicine” (CTQ2017-86661-R). IP: Clara Pereyra. 2018-2020). This project has analyzed, as part of its objectives, the impregnation of porous matrices with natural extracts from mango leaves and other anonaceae, and has started the study of the foaming phenomenon with excellent results, as can be seen in the following publications:
• García-Casas, I., Montes, A., Valor, D., Pereyra, C., & Martínez de la Ossa, E. J. (2018). Impregnation of mesoporous silica with mangiferin using supercritical CO2. Journal of Supercritical Fluids, 140, 129-136.
• García-Casas, I., Crampon, C., Montes, A., Pereyra, C., Martínez de la Ossa, E. J., Badens, E. (2019). Supercritical CO2 impregnation of silica microparticles with quercetin. Journal of Supercritical Fluids, 143, 157-161.
• García-Casas, I.; Montes, A.; Valor, D.; Pereyra, C.; de la Ossa, E.J.M. Foaming of polycaprolactone and its impregnation with quercetin using supercritical CO2. Polymers. 2019, 11, 1390.
• García-Casas, I., Montes, A., Valor, D., Pereyra, C., Martínez de la Ossa, E. J. (2020). Deposition of CAP/antioxidants systems on silica particles using the supercritical antisolvent process. Applied Sciences, 10(13).
• Montes, A.; Pereyra, C.; de la Ossa, E.J.M. Foaming + Impregnation One-Step Process Using Supercritical CO2, en Advanced Supercritical Fluids Technologies. 2020. Ed. IntechOpen. ISBN: 978-1-83880-709-2, 2020.
• Fernández, M.T., Gómez, E., Cejudo, C.; Casas, L.; Montes, A.; Mantell, C.; Martínez de la Ossa, E. J.; Pereyra, C. 2020.
• Development of functionalized alginate dressing with mango polyphenols by supercritical technique to be employed as an antidiabetic transdermal system. Journal of Supercritical Fluids (under review).
• D. Valor, A. Montes, I. García-Casas, C. Pereyra, E. J. Martínez de la Ossa. Supercritical solvent impregnation of alginate wound dressings with mango leaves extract. Journal of Supercritical Fluids (under review).

The project we are applying for in this call, which arises from the experience gained with the use of polymers — 25 articles (1 on foaming) — and with the application of supercritical impregnation in porous matrices — 10 articles — would consolidate the merging of the two lines by applying supercritical impregnation to biodegradable polymers and by pursuing certain objectives that would give continuity to the project CTQ2017-86661-R as it proposes a more specific application of supercritical fluids to one of its edging lines of development: namely, the study of the functionalization process of polymers through their supercritical impregnation with natural extracts with pharmacological properties for their use in biomedicine.

Besides the members of the research team from the previous project under the Challenges Call, this project has incorporated three new researchers who are experts in hydrothermal processes. A technique that could effectively modify the structure of the polymers under study and increase their effectiveness for the desired purposes. Such researchers also belong to the TEP128 Group, so that in this project, and for the first time since its creation, all the Group’s researchers would be involved in a single project. Furthermore, two experts in molecular biology will join the research team to verify the effectiveness of the products obtained by analyzing their functionality, in vitro cell adhesion, morphology, cell viability and cell proliferation. A clinical researcher from the cardiology area and specialized in the use of biodegradable polymeric scaffolds and stents will be in charge of analyzing the biomedical viability of the products that are generated and determining their potential clinical use.

Given the extensive experience of the members of the research team, and considering the currently available experimental equipment, infrastructure and other resources, we believe that the competence and aptitude of the applicant group to achieve the proposed project objectives should be deemed as solidly supported.

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