Sterile or disinfected aerogels and methods for simultaneous preparation and sterilisation thereof

Abstract

The present invention relates to sterile or disinfected aerogels, for example, for use as nanostructured biomaterials for biomedical and food purposes. The present invention further relates to methods for preparing said sterile or disinfected aerogels.

Description

[0001] STERILE OR DISINFECTED AEROGELS AND PROCEDURES FOR THEIR PREPARATION AND SIMULTANEOUS STERILIZATION

[0002] TECHNICAL SECTOR

[0003] This disclosure relates to disinfected or sterile aerogels, e.g., for use as nanostructured biomaterials for biomedical and food purposes. This disclosure also relates to methods for preparing such disinfected or sterile aerogels.

[0004] BACKGROUND OF THE INVENTION

[0005] Aerogels based on natural polymers are of increasing interest in the biomedical and food fields due to their biocompatibility, bioactivity, biodegradability, and, in some cases, biomimicry of the extracellular matrix. The high and open porosity of aerogels, along with their high specific surface areas and thermal insulation properties, are of interest for applications such as drug delivery, wound treatment, regenerative medicine, oncotherapy, and as carriers of functional compounds and fat substitutes in the food industry. Furthermore, recent advances in aerogel formulation have resulted in a high versatility of external morphologies, porous structures, and composition, providing customized biological performance for various routes of administration (oral, nasal, pulmonary, cutaneous).

[0006] However, the requirement for sterility or disinfection is a critical quality attribute that limits the use of aerogels in biomedicine and food. The production of sterile or disinfected aerogels is not straightforward due to their complex nanostructure (high mesoporosity and tortuosity). Some methods used to obtain sterile or disinfected aerogels include (i) aseptic processing of aerogels from sterile precursors, and (ii) post-sterilization or post-disinfection of aerogels. The first option, aseptic processing of aerogels, results in increased manufacturing costs due to the higher costs of purchasing certified sterile raw materials or sterilization pretreatments, along with the higher processing costs associated with operating under aseptic conditions due to its greater complexity (including packaging).The option of subsequent sterilization or disinfection of aerogels introduces an additional processing step, leading to greater complexity and variability in results. Furthermore, conventional sterilization techniques (e.g., the use of ethylene oxide, gamma irradiation, thermal, and UV radiation) are unable to achieve the required sterility levels due to penetration problems in the aerogel's intricate porous structures, or they fail to preserve the aerogel's integrity in terms of physical properties (textural, mechanical) or composition (changes in molecular weight, physicochemical degradation) to varying degrees.

[0007] Therefore, there is a need to develop aerogel preparation methods that allow obtaining aerogels with a satisfactory level of sterility and that, at the same time, maintain their nanostructure and chemical identity.

[0008] GENERAL DESCRIPTION

[0009] In this regard, this disclosure focuses on a method that allows for the in-situ sterilization of aerogels during their production. This document reports, for the first time, the direct production of sterile or disinfected aerogels through the implementation of an integrated and environmentally friendly production technology. This technology utilizes a supercritical fluid, such as supercritical carbon dioxide (SCCO2), in combination with a microbial inactivating agent, employing a combination of dynamic (with CO2 flow) and static (without flow) stages.

[0010] Aerogels are gels made of a porous solid material where the dispersed phase is a gas and they possess a high open mesoporosity. A gel is a non-fluid colloidal or polymeric network that expands throughout its volume through a fluid. Aerogels are usually produced by supercritical drying of gels, for example, in a process assisted by SCCO2. SCCO2 is also recognized as a biocidal agent and has been proposed for the sterilization of heat-sensitive materials such as biopolymers, materials sensitive to degradation by hydrolysis, food products, implantable biological tissues, pharmaceuticals, drug delivery systems and medical devices without impacting the integrity of the material and the material properties after treatment (see for example the patent documents published as US 6,149,864 A, US 2007 / 0003432 A1, EP 1 782 839 A1, US 2009 / 0041620 A1, EP 1 782 839 A1, US 2004 / 0120852, US2014 / 0193552 A1).

[0011] Several documents describe the use of supercritical fluids for the sterilization of porous materials. For example, without addressing aerogels, the international application published as WO 2022 / 084569 A1 describes a sterile implantation system comprising a thermosensitive polymer matrix that modifies its structure in the presence of a compressed gas or a supercritical fluid, resulting in a solid or semi-solid with a porosity greater than 60%. The document also addresses a process for preparing such systems using SCCO2 and hydrogen peroxide. Bento et al. (Bento, CSA; Alarico, S.; Empadinhas, N.; de Sousa, HC; Braga, MEM “Sequential SCCO2 Drying and Sterilization of Alginate-Gelatine Aerogels for Biomedical Applications.” J. Supercrit. Fluids 2022, 184, 105570, doi: 10.1016 / j. supflu.2022.105570) describes an integrated process that uses scCO2 for drying alginate-gelatin alcogels and sterilizing them to provide alginate-gelatin aerogels for biomedical applications. For this purpose, the alginate-gelatin gels were dried and sterilized sequentially with scCO2 in a continuous process and in the same high-pressure vessel, for example, by means of pressurization / depressurization cycles. Santos Rosales et al. (Santos-Rosales, V.; Ardao, Alvarez-Lorenzo, C.; Ribeiro, N.; Oliveira, A.; García-González, C. “Sterile and Dual-Porous Aerogels Scaffolds Obtained through a Multistep Supercritical CO2-Based Approach.” Molecules 2019, 24, 871, doi:10.3390 / molecules24050871) describe the preparation of aerogels using zein, a family of proteins present in tissue engineering materials, as a sacrificial porogen to obtain macroporous starch aerogels and implementing a post-treatment of supercritical CO2 sterilization for these aerogels in the presence of hydrogen peroxide (H2O2).

[0012] It has been shown that the in situ sterilization or disinfection of aerogels during their production using SCCO2 in combination with a microbial inactivating agent, by contacting a lyogel with SCCO2 in a specific sequence of steps, as described in this document, results in an effective treatment to obtain aerogels with a degree of microbial inactivation suitable according to the requirements for biomedical and food applications, while preserving the physicochemical properties of the aerogels.

[0013] In particular, this disclosure relates to a procedure for the preparation and simultaneous sterilization or disinfection of an aerogel, comprising contacting a lyogel with SCCO2 inside an autoclave, wherein contacting comprises five steps in the following order: i) a pressurization step comprising pressurizing the autoclave with CO2 to SCCO2 conditions;

[0014] i) a first dynamic stage comprising maintaining the autoclave under pressure with a flow of CO2; iii) a static stage comprising maintaining the autoclave at a pressure of SCCO2 without flow; iv) a second dynamic stage comprising maintaining the autoclave under pressure with a flow of CO2; and v) a depressurization stage comprising depressurizing the autoclave to atmospheric pressure, obtaining a sterile or disinfected aerogel;wherein during the static stage (iii) a microbial inactivating agent is introduced into the autoclave under SCCO2 pressure, preferably a microbial inactivating agent selected from hydrogen peroxide (H2O2), peracetic acid, acetic acid, trifluoroacetic acid, sodium hypochlorite, chlorine dioxide, boric acid, didecyldimethylammonium chloride (DDAC), formic acid, sodium dichloroisocyanurate, tert-butyl hydroperoxide, formaldehyde, glutaraldehyde, water, methanol, ethanol, ammonium salts, ethylene oxide, as well as phenols, polyphenols, terpenes, essential oils, glucosinolate derivatives, alkaloids and thiols with antimicrobial activity, and combinations thereof in mixtures, most preferably the microbial inactivating agent being H2O2.

[0015] This procedure not only allows for obtaining aerogels with an acceptable level of microbial inactivation, even for the most demanding applications, but also allows for obtaining aerogels without damaging their nanostructure and chemical identity. Furthermore, sterile or disinfected aerogels obtained using the methods described in this disclosure have demonstrated cytocompatibility with cell lines. These methods, therefore, allow the use of aerogels for novel biomedical or food applications, for example, as scaffolds for tissue engineering, as carriers in pharmaceutical formulations, or as carriers of functional compounds and fat substitutes in foods requiring controlled levels of sterilization or disinfection.Therefore, this disclosure also addresses a sterile or disinfected aerogel obtainable by said procedure and a sterile or disinfected aerogel having a controlled degree of microbial inactivation, e.g., SAL-6 as detailed below; and at the same time having acceptable degrees of desirable aerogel properties such as, e.g., porosity, specific surface area, total specific pore volume, specific mesopore volume and / or mean pore diameter, as also detailed below.

[0016] The methods and products described in this report offer a promising and technologically and economically feasible solution that has not been achieved until now, opening new perspectives in aerogel engineering for new biomedical purposes, such as aerogels as scaffolds for tissue engineering or as carriers in pharmaceutical formulations, and food purposes that require controlled levels of sterilization or disinfection.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] To complement the description being made and in order to help a better understanding of the characteristics of the invention, a set of drawings is included as an integral part of said description, in which, for illustrative and non-limiting purposes, the following has been represented:

[0019] Figure 1.- Autoclave including a liogel (e.g., an alcogel) in a procedure for the preparation and simultaneous disinfection or sterilization of an aerogel with SCCO2 by adding hydrogen peroxide (H2O2) in the autoclave under SCCO2 pressure and temperature conditions of 140 bar and 39 °C, respectively.

[0020] Figure 2. Process flow diagram of an example of equipment that can be used in a procedure for the preparation and simultaneous sterilization of an aerogel with SCCO2. The thick black lines allow for the in-situ addition of the microbial inactivating agent (e.g., H2O2) in the pressurized autoclave. Legend: 1. CO2 supply; 2. Pressure gauge (Pl); 3. Chiller; 4. CO2 pump (e.g., twin-piston); 5. Heat exchanger; 6. Valves (e.g., needle valves); 7. Pressure autoclave, e.g., thermally insulated and equipped with, e.g., pressure gauge (Pl), magnetic stirrer (M), heating jacket, and temperature gauge or thermocouple (TI); 8. Backpressure valve pressure controller and indicator (PIC); 9. Separator-collector vessel; 10. Valve (e.g., ball valve); and 11: Equipped container where H2O2 is pre-charged and then introduced.

[0021] Figure 3.- Pressure-time process profiles with: (i) a pressurization stage with SCCO2; (ii) a first dynamic stage with a CO2 flow; (iii) a static stage without flow; (iv) a second dynamic stage with CO2 flow; and (v) a depressurization stage. Figure 3A illustrates a procedure for the preparation of aerogels, and Figure 3B illustrates a procedure for the preparation of aerogels with simultaneous sterilization (in situ sterilization during aerogel preparation with the drying process) to obtain sterile or disinfected aerogels with the addition of H2O2 to the autoclave in step (iii), e.g., under SCCO2 pressure, and with the presence of F^C^ in the equipment with a microbial inactivating effect also in the subsequent steps (iv) and (v), although its presence in these subsequent steps is reduced.

[0022] Figure 4.- SEM images of alginate aerogels (Figure 4A) and starch aerogels (Figure 4B) obtained for the blank, an example without microbial inactivating agent (without H2O2) prepared under the conditions of comparative example 2 (BS_1_0), and an example with microbial inactivating agent (with H2O2) prepared under the conditions of example 3e (BS_1).

[0023] Figure 5.- Effect of aerogel preparation with simultaneous sterilization in the presence of a microbial inactivating agent (e.g., H2O2 at an amount of 3300 ppm) on the textural properties of starch aerogels, e.g., starch cylinders (the three solid bars on the left) and alginate aerogels, e.g., alginate pearls (the three striped bars on the right) obtained for the blank (Blank_3), the example without a microbial inactivating agent (without H2O2, prepared under the conditions of comparative example 2, BS_1_0), the example with a microbial inactivating agent (with H2O2, prepared under the conditions of example 3e, BS_1) and using water as the microbial agent according to the conditions of comparative example 3 (BS_1_water). Total porosity (E) (Figure 5A), specific surface area (ABET) (Figure 5B), and specific total pore volume (V) are represented. v) (Figure 5C), represented as the sum of the specific volume of macropores (VP, macro) (light color) and the specific volume of mesopores (VP, meso) (dark color) and the mean pore diameter (DP) (Figure 5D). Bars grouped with the same letter (a, boc) denote statistically homogeneous groups.

[0024] Figure 6.- Effect of exposure time on the textural properties of sterile or disinfected starch aerogels, e.g. starch cylinders (the three solid bars on the left) and alginate aerogels, e.g. alginate beads (the three striped bars on the right), prepared with a static stage (iii) duration of 1 hour (1 h stage (iii) according to the conditions of example 3e, BS_1), 2 hours (2 h stage (iii) according to the conditions of example 3d, BS_2) and 3 hours (3 h stage (iii) according to the conditions of example 3f, BS_3) including porosity (E) (Figure 6A), specific surface area (ABET) (Figure 6B), total specific pore volume (V v ) (Figure 6C), represented as the sum of the specific volumes of macropores (VP, macro) (light color) and mesopores (VP, meso) (dark color), and the mean pore diameter (DP) (Figure 6D). Bars grouped with the same letter (a, boc) denote statistically homogeneous groups.

[0025] Figure 7.- Effect of agitation time on the textural properties of sterile or disinfected starch aerogels, e.g. starch cylinders (the three solid bars on the left) and alginate aerogels, e.g. alginate beads (the three striped bars on the right), including an example without agitation (prepared according to the conditions of example 3k, N_1), an example with agitation in step (iii) (prepared according to the conditions of example 3h, B_1) and an example with agitation in steps (iii) and (iv) (prepared according to the conditions of example 3e BS_1), said properties including porosity (E) (Figure 7A), specific surface area (ABET) (Figure 7B), total specific pore volume (V v) (Figure 7C), represented as the sum of the specific volumes of macropores (VP, macro) (light color) and mesopores (VP, meso) (dark color), and the mean pore diameter (DP) (Figure 7D). Bars grouped with the same letter (a, boc) denote statistically homogeneous groups.

[0026] Figure 8. Effect of other sterilization techniques on the textural properties of sterile or disinfected starch aerogels, e.g., starch cylinders (the three solid bars on the left), and alginate aerogels, e.g., alginate beads (the three striped bars on the right). Included are an example sterilized by ethylene oxide (OE, middle bars), another by gamma ray irradiation (Y-rays, right bars), and, for comparison, an example of the materials sterilized according to the invention by agitation in steps (iii) and (iv) prepared under the conditions of example 3e (BS_1, left bars). The properties compared are porosity (E) (Figure 8A), specific surface area (ABET) (Figure 8B), and total pore specific volume (V). v) (Figure 8C), represented as the sum of the specific volumes of macropores (VP, macro) (light color) and mesopores (VP, meso) (dark color), and the mean pore diameter (DP) (Figure 8D). Bars grouped with the same letter (a, boc) denote statistically homogeneous groups.

[0027] Figure 9.- Cell viability studies, expressed as a percentage, determined after 24 and 48 h of contact with starch aerogel formulations, e.g., starch cylinders (solid bars, Figure 9A) and alginate aerogels, e.g., alginate pearls (striped bars, Figure 9B), prepared with comparative example 2 (BS_1_0, left bars for each time) and example 3e (BS_1, right bars for each time).

[0028] Figure 10. Effect of aerogel preparation with simultaneous sterilization in the presence of a microbial inactivating agent (e.g., H2O2 at 3300 ppm) on the textural properties of methylcellulose aerogels with morphology obtained by 3D printing, obtained for the blank (Blank_1), and the example with a microbial inactivating agent (with H2O2, prepared under the conditions of example 3e, BS_1). The properties compared are porosity (E) (Figure 10A), specific surface area (ABET) (Figure 10B), and total specific pore volume (V v ) (Figure 10C), represented as the sum of the specific volumes of macropores (VP, macro) (light color) and mesopores (VP, meso) (dark color), and the mean pore diameter (DP) (Figure 10D). Bars grouped with ns denote values ​​with no significant difference.

[0029] DETAILED DESCRIPTION

[0030] In particular, this disclosure relates to a process for the preparation and simultaneous sterilization or disinfection of an aerogel, comprising contacting a lyogel with scCÜ2 inside an autoclave, wherein contacting comprises five steps in the following order: i) a pressurization step comprising pressurizing the autoclave with CO2 to SCCO2 conditions;

[0031] i) a first dynamic stage comprising maintaining the autoclave under pressure with a flow of CO2; iii) a static stage comprising maintaining the autoclave at a pressure of SCCO2 without flow; iv) a second dynamic stage comprising maintaining the autoclave under pressure with a flow of CO2; and v) a depressurization stage comprising depressurizing the autoclave to atmospheric pressure, obtaining a sterile or disinfected aerogel;wherein during the static stage (iii) a microbial inactivating agent is introduced into the autoclave under SCCO2 pressure, preferably a microbial inactivating agent selected from H2O2, peracetic acid, acetic acid, trifluoroacetic acid, sodium hypochlorite, chlorine dioxide, boric acid, DDAC, formic acid, sodium dichloroisocyanurate, tert-butyl hydroperoxide, formaldehyde, glutaraldehyde, water, methanol, ethanol, ammonium salts, ethylene oxide, as well as phenols, polyphenols, terpenes, essential oils, glucosinolate derivatives, alkaloids and thiols with antimicrobial activity, and combinations thereof in mixtures, most preferably the microbial inactivating agent being H2O2.;

[0032] The preparation and simultaneous sterilization or disinfection of an aerogel is understood as the in situ sterilization or disinfection of the aerogel during its preparation.

[0033] In this document, disinfection is defined as a process that reduces the number of microorganisms present in an aerogel, resulting in a disinfected aerogel, and sterilization is defined as a process that eliminates all microorganisms from an aerogel, resulting in a sterile aerogel. Specifically, the level of disinfection or sterility of an aerogel is described in detail below.

[0034] It has been observed that simultaneous sterilization or disinfection during the preparation of an aerogel, as described in this document, advantageously reduces the steps required to obtain a sterile or disinfected aerogel and at the same time allows obtaining aerogels with a high level of sterility without significantly damaging the nanostructure and chemical identity of the aerogel.

[0035] In particular, a gel is understood here, as in the state of the art, as a non-fluid colloidal network or polymer network that expands throughout its volume by a fluid.

[0036] An aerogel, as described above, is understood to be a gel, preferably one with high open mesoporosity, in which the fluid is a gas. The gas present in the aerogel, as described herein, may be air, CO2, a controlled atmosphere, or an inert gas.

[0037] A lyogel is understood here, as in the state of the art, as a gel where the fluid is a liquid. The liquid may be a solvent, for example, selected from ethanol, acetone, methanol, dimethyl sulfoxide, diethyl ether, ethyl methyl ketone, isopropanol, water, and mixtures thereof, most preferably the solvent being ethanol or water, still more preferably being ethanol.

[0038] An alcogel is understood here, as in the state of the art, as a lyogel in which the liquid is a solvent comprising predominantly or may consist of an alcohol or a mixture of alcohols. The alcohol may preferably be selected from, for example, ethanol, methanol, and isopropanol; more preferably, the alcohol may be ethanol.

[0039] When the solvent is water, the liogel can be referred to as a hydrogel.

[0040] In various embodiments, a gel, for example, a lyogel (such as an alkogel) or an aerogel, comprises a polymeric material. The polymeric material may be selected, for example, from organic polymers, inorganic polymers, and combinations thereof, optionally with additions. A polymeric material with additions refers to, for example, a polymeric material with additions of particles and / or fibers of a ceramic, polymeric, metallic, or biological nature, or a composite polymeric material such as, for example, a sandwich type.

[0041] In various embodiments, the organic polymers can be selected from polysaccharides, resorcinol-formaldehyde; polyurethane; proteins; polynucleotides; and mixtures thereof.

[0042] In various embodiments, inorganic polymers can be selected from silica, carbon, and mixtures thereof.

[0043] In various embodiments, the polymeric material may preferably be an organic polymer and more preferably a polysaccharide. In various particular embodiments, a polysaccharide may be selected from alginate, starch, chitosan, and / or cellulosic derivatives, such as methylcellulose, even more preferably from alginate, starch, and methylcellulose, and still more preferably the polymeric material is alginate and / or starch.

[0044] The liogel can be any shape suitable for the final application of the resulting aerogel. For example, the liogel (e.g., alcogel) can be in the form of cylinders (also referred to as monoliths) and / or beads, such as starch cylinders or alginate beads.

[0045] In a method described in this dissertation, an aerogel is obtained from a lyogel and more particularly from an alcogel.

[0046] A lyogel (e.g., an alcogel) may be commercially available or prepared by methods known in the art. Briefly, a lyogel, for example, an alcogel, can be prepared by dissolving a polymeric material in a solvent, for example, comprising an alcohol, as detailed above. Alternatively, an alcogel can be obtained from another lyogel by dissolving a polymeric material in a first solvent, for example, water, acetone, ethanol, acetonithlo, or aqueous saline solutions (such as potassium hydroxide and thiourea in water) or dimethyl sulfoxide (such as lithium bromide in DMSO), preferably water, to provide a first gel where the fluid is said first solvent (e.g., a hydrogel where the fluid is water). Optionally, intermediate aging steps can be carried out in intermediate solutions containing salts or chemical crosslinking agents.Subsequently, once the lyogel has been obtained, or optionally after such intermediate steps, the first solvent can be exchanged for a second solvent. The second solvent can comprise any solvent (different from the first solvent) to provide a second gel that is a lyogel, or more particularly, it can comprise, for example, an alcohol to provide a second gel that is an alcogel. The solvent exchange can be carried out by immersing the first gel in the second solvent, for example, sequentially with intermediate mixtures of both solvents or directly in the second solvent, and separating the gel from the solvent. To obtain the lyogel, e.g., the alcogel, if necessary, the process of immersing the gel in the second solvent and separating it from the solvent can be repeated one or more times, preferably twice. For the preparation of an alcogel, the solvent comprising an alcohol (e.g., the second solvent) may comprise an alcohol or consist of an alcohol or a mixture of alcohols, as described above for the alcogel.

[0047] Lyogel can also be obtained by 3D printing in the form of, for example, a hydrogel or alkogel. The solvent in the 3D-printed lyogel can also be changed by solvent exchange, as described above. For example, if the lyogel is obtained by 3D printing as a hydrogel, the corresponding alkogel can be obtained by such a solvent exchange (e.g., by replacing water with ethanol).

[0048] For example, hydrogels can be prepared from polymers dissolved in a solvent, e.g., water; these solutions are also referred to as aqueous inks. Suitable polymers include methylcellulose, carboxymethylcellulose, alginate, pectin, collagen, and gelatin, with methylcellulose being a good choice. In particular, an aqueous solution of methylcellulose can be used (e.g., 5–20% w / v, more specifically 10–15% w / v), using a printer (e.g., a commercial bioprinter such as Cellink BIOX (Boston, MA, USA)). For example, the printer can use an extrusion print head under the recommended working conditions (e.g., 40°C, 40 kPa, and 3 mm / s, a 3 mL syringe, and a 600 µm nozzle). 3D printing allows for the creation of various structures. For example, hydrogels obtained by 3D printing can have a grid pattern. A hydrogel of one or more layers, e.g., three layers, can be obtained by 3D printing.

[0049] A method described in this document comprises contacting a liogel with scCC>2 inside an autoclave, which comprises the liogel.

[0050] An autoclave suitable for the method described herein is preferably a vessel in which operations are carried out at pressures above atmospheric pressure and which is adapted to maintain a fluid under pressure and to modify and maintain the temperature within the autoclave. Examples of suitable autoclaves include, for instance, a stainless steel or Hastelloy pressure vessel with a metal or polymer seal compatible with high pressure and the presence of CO2, solvents, and microbial inactivating agents. Such an autoclave must have at least one port for the inlet and outlet of CO2.

[0051] A method described herein may comprise, before contacting the lyogel with the SCCO2, introducing the lyogel (e.g., an alcogel) into the autoclave or preparing the lyogel inside the autoclave. Preferably, the lyogel may be placed inside a sterilization bag.

[0052] In various embodiments, the liogel to be in contact with the scCÜ2 can be placed inside an autoclave. Preferably, the liogel can be placed inside a sterilization bag. For example, the liogel can be placed in a sterilization bag, which can then be heat-sealed or self-sealing. Suitable sterilization bags for a method as described herein can be commercially available. By way of example, heat-sealable and self-sealing sterilization bags made of paper (such as medical-grade paper) and / or polymeric material (such as polyester polypropylene, PET, or shutterless polypropylene (CPP), among others) can be used, which may be commercially available, e.g., from MEDIPACK AG, Sendal SI, or Soplahl Hispania. The liogel can be commercially obtained or previously prepared (e.g.,(as described above) and can be introduced into an autoclave, for example, through an inlet or with the autoclave open, preferably with the gels separated and not stacked. In particular, the lyogel can be prepared, preferably by dissolving a polymeric material in a solvent or by 3D printing from an ink composed of a polymeric material dispersed or dissolved in a liquid solvent, e.g., as described in more detail above.

[0053] In various embodiments, the lyogel can be prepared inside the autoclave. For example, the lyogel can be prepared inside the autoclave from a pre-existing lyogel via solvent exchange, or from a polymeric material and a solvent, as described above. In this way, the lyogel is already present in the autoclave for subsequent contact with SCCO2, thus avoiding the step of preparing the lyogel separately and placing it in the autoclave. In this case, it is necessary to maintain a sterile environment when opening the autoclave and when packaging the sterile material.

[0054] One method described herein involves contacting the liogel with SCCO2. SCCO2 refers to CO2 at a temperature and pressure above its critical point, also known as supercritical conditions, where the fluid, in this case CO2, exhibits properties between those of a gas and a liquid. The critical point of CO2 is at a pressure of 7.39 MPa (critical pressure) and a temperature of 31.1 °C (critical temperature). Therefore, the supercritical conditions for CO2 are a pressure above 7.39 MPa and a temperature above 31.1 °C.

[0055] In a procedure described herein, the autoclave temperature can be from 31.1 to 70 °C, preferably from 33 to 60 °C, and more preferably from 35 to 50 °C. In various embodiments, the autoclave temperature is from 37 to 41 °C, and more preferably around 39 °C. In a procedure described herein, the autoclave pressure can be at least 74 bar (i.e., 7.4 MPa), in particular at least 85 bar (i.e., 8.5 MPa), and more particularly at least 110 bar (i.e., 11 MPa).

[0056] Contacting the liogel with scCC>2 is generally carried out within the autoclave containing the liogel. In some embodiments, the autoclave may contain excess solvent (preferably the same solvent as that of the liogel) at the bottom of the autoclave. The presence of excess solvent can prevent, for example, premature evaporation of the solvent from the liogel.

[0057] Contacting the liogel with scCC>2 comprises five steps in a specific order.

[0058] The first step is a pressurization step (i) comprising pressurizing the autoclave with CO2 to supercritical CO2 conditions. Pressurizing the autoclave can be accomplished by adding CO2 into the autoclave, for example, through an inlet pipe, and if necessary, heating the autoclave until supercritical conditions for CO2 or the supercritical mixture of CO2 with the lyogel solvent are achieved (in particular, a pressure above 7.39 MPa and a temperature above 31.1 °C), thus pressurizing the autoclave with supercritical CO2. For pressurization, the inlet flow rate of CO2 can be from 0.25 to 25 g / min per 100 mL of autoclave volume, preferably from 0.5 to 20 g / min, more preferably from 1 to 15 g / min, even more preferably from 2 to 10 g / min, and still more preferably from 5 to 7 g / min.

[0059] In specific applications, the autoclave can be pressurized to a predetermined pressure. For example, the autoclave can be pressurized to a pressure of 80 to 180 bar (i.e., 10–18 MPa), preferably 110 to 150 bar (i.e., 11–15 MPa), and even more preferably around 120 bar (i.e., 12 MPa).

[0060] The autoclave can be kept under pressure with or without CO2 flow.

[0061] For example, when the autoclave has a certain pressure, that pressure can be maintained with a flow of CO2 by allowing CO2 to enter the autoclave, for example, through an inlet channel, and CO2 to exit the autoclave, for example, through an outlet channel, particularly so that the inlet flow of CO2 and the outlet flow of SCCO2 are the same.

[0062] In a method described herein, the CO2 entering the autoclave may enter under non-supercritical conditions and reach supercritical conditions inside the autoclave. Therefore, although the incoming CO2 may also be under supercritical conditions in some embodiments, it is generally referred to as an incoming CO2 flow. Conversely, the CO2 inside the autoclave that exits as an outgoing flow will generally be under supercritical conditions and is therefore generally referred to herein as the outgoing CO2 flow. Furthermore, the terms "with" and "without" flow generally refer to a flow of CO2, without specifying whether that CO2 is supercritical.

[0063] On the other hand, the autoclave can be kept under pressure without flow, for example, by closing the inlet and outlet flows of the autoclave.

[0064] After pressurizing the autoclave, contacting the liogel (e.g., alcogel) with SCCO2 can be done by alternating a first dynamic stage (i) with CO2 flow; a static stage

[0065] (iii) without flow; and a second dynamic stage (iv) with CO2 flow. In particular,

[0066] i) a first dynamic stage comprising maintaining the autoclave under pressure with a flow of CO2; iii) a static stage comprising maintaining the autoclave under pressure with a flow of CO2; and iv) a second dynamic stage comprising maintaining the autoclave under pressure with a flow of CO2.

[0067] In some specific embodiments, the static stage (iii) and / or the second dynamic stage (iv) can be carried out under stirring; preferably, both stages can be carried out under stirring. The stirring can be imparted, for example, using stirring media such as a magnetic stirrer, for example, a commercial magnetic stirrer such as the IKA RCT basic S000. The level of stirring applied can be from 100 to 1500 rpm, particularly from 200 to 1200 rpm, more particularly from 400 to 900 rpm, and still more particularly from 500 to 800 rpm, and preferably around 700 rpm.

[0068] In some embodiments, the CO2 flow rate in the first dynamic stage (i) and / or the second dynamic stage (iv) can be 0.25 to 25 g / min per 100 mL of autoclave volume, preferably 0.5 to 20 g / min, more preferably 1 to 15 g / min, even more preferably 2 to 10 g / min, and still more preferably 5 to 7 g / min. These CO2 flow rates allow for the extraction of a large portion of the solvent from the gel during dynamic stage (i) and the remaining solvent and the microbial inactivating additive during dynamic stage (iv). The flow rates are sufficient to ensure that the CO2 is renewed at least three times in the autoclave.

[0069] In various executions, in the first dynamic stage (i) and / or the second dynamic stage

[0070] (iv) the autoclave can be maintained at a pressure of, for example, 80 to 180 bar (i.e. 10 - 18 MPa), preferably 110 to 130 bar (i.e. 11 - 13 MPa), and more preferably around 120 bar (i.e. 12 MPa).

[0071] In some embodiments, the static stage (iii), which comprises maintaining the autoclave under pressure without CO2 flow, may comprise maintaining the autoclave at a scCÜ2 pressure of 80 to 180 bar, preferably 130 to 150 bar, more preferably above 135 bar, and even more preferably around 140 bar. In some embodiments, the autoclave pressure in the first dynamic stage (i) and in the second dynamic stage (iv) may be lower than the autoclave pressure in the static stage (iii). For example, in some particular embodiments, the pressure in the first dynamic stage (i) and in the second dynamic stage (iv) may be around 120 bar, and the pressure in the static stage may be around 140 bar.

[0072] In other implementations, the pressure may be the same in the static stage (iii) and in both dynamic stages (i) and (iv). For example, the pressure in all stages (i), (iii) and (iv) may be around 140 bar.

[0073] As indicated above, the autoclave pressure in the static stage (iii) may be higher than the autoclave pressure in the first dynamic stage (i). In some embodiments, the static stage (iii) may involve increasing the autoclave pressure, for example, by adding CO2 to the autoclave with no outflow or at an outflow rate lower than the inflow rate, and then transitioning to the absence of both inflow and outflow, thus maintaining the pressure in the autoclave. The inflow of CO2 without CO2 outflow or with an outflow rate lower than the inflow rate results in an increase in autoclave pressure.

[0074] As also indicated above, the autoclave pressure in the static stage (iii) may be higher than the autoclave pressure in the second dynamic stage (iv). In some embodiments, the second dynamic stage (iv) may involve lowering the autoclave pressure, for example, by releasing a flow of SCCO2 from the autoclave without any inflow, or with an inflow of CO2 into the autoclave that is less than the outflow. The release of SCCO2 without CO2 inflow, or with an outflow of SCCO2 greater than the inflow of CO2, results in a decrease in autoclave pressure.

[0075] Maintaining the autoclave under pressure with a flow of CO2, in the first dynamic stage (i) and / or in the second dynamic stage (iv), can be achieved once the autoclave has the desired pressure by, for example, introducing into the autoclave an inlet flow of CO2 equal to the outlet flow of SCCO2.

[0076] In a method described in this document, during the static stage (iii) a microbial inactivating agent is introduced into the autoclave under SCCO2 pressure.

[0077] Preferably, a microbial inactivating agent can be selected from H2O2, peracetic acid, acetic acid, thiofluoroacetic acid, sodium hypochlorite, chlorine dioxide, boric acid, DDAC, formic acid, sodium dichloroisocyanurate, tert-butyl hydroperoxide, formaldehyde, glutaraldehyde, water, methanol, ethanol, ammonium salts, ethylene oxide, as well as phenols, polyphenols, terpenes, essential oils, glucosinolate derivatives, alkaloids, and thiols with antimicrobial activity, and combinations thereof in mixtures, most preferably the microbial inactivating agent being H2O2. It has been observed that the use of such microbial inactivating agents, and of H2O2 in particular, advantageously results in aerogels with good microbial inactivation properties and nanostructure and chemical identity.Furthermore, it has been observed that the use of these microbial inactivating agents, and H2O2 in particular, results in aerogels that do not exhibit cytotoxicity and, therefore, good biocompatibility.

[0078] The microbial inactivating agent can be added in an amount of 10 to 6000 ppm relative to the autoclave volume, specifically milligrams (mg) of microbial inactivating agent per liter (L) of autoclave volume, preferably from 250 to 5750 ppm, and more preferably from 600 to 5500 ppm. These amounts have been observed to contribute to producing sterile or disinfected aerogels without adversely affecting the cytotoxicity, nanostructure, or chemical identity of the resulting aerogels.

[0079] In some embodiments, in the static stage (iii), the microbial inactivating agent, in solution or solid form, can be added along with a stream of CO2 or another pressurized fluid at the bottom of the autoclave, preferably with CO2, via a feed tube that has its outlet at the bottom of the autoclave. An example of an autoclave adapted for the introduction of the microbial inactivating agent (e.g., H2O2) at the bottom of the autoclave can be seen in Figure 1. In certain specific embodiments, this feed tube may have a nozzle of known diameter at its outlet for spraying the microbial inactivating agent. Other configurations for adding the microbial inactivating agent are possible, preferably avoiding direct physical contact of the agent's inlet with the gels present in the autoclave.

[0080] In some embodiments, the first dynamic stage (i) can last from 0.25 to 5 hours, preferably from 0.5 to 4 hours, more preferably from 1 to 3 hours, and even more preferably around 2 hours. A stage duration within these ranges has been observed. These ranges favor the correct formation of porous nanostructures depending on the inlet CO2 flow rate used and the size of the sterile or disinfected aerogel to be prepared.

[0081] In some embodiments, the static step (iii) can last from 0.25 to 5 hours, preferably from 0.5 to 4 hours, and more preferably from 1 to 3 hours. A step duration within these ranges has been observed. These ranges contribute to obtaining nanostructures and a level of microbial inactivation acceptable for most applications of interest. The exact duration may vary depending on the nature and dimensions of the aerogel to be prepared and the nature and quantity of the microbial inactivating agent used.

[0082] In some embodiments, the second dynamic stage (iv) can last from 0.25 to 4 hours, preferably from 0.5 to 3 hours, more preferably from 1 to 2 hours, and still more preferably around 1.5 hours. It has been observed that a stage duration within these ranges contributes to obtaining porous nanostructures and levels of microbial inactivation acceptable for most applications of interest, as well as eliminating the microbial inactivating agent. The exact times may vary depending on the specific scC₂ outlet flow rate used.

[0083] After the second dynamic stage (iv), a procedure described in this document comprises a depressurization stage (v) which involves depressurizing the autoclave to atmospheric pressure, obtaining a sterile or disinfected aerogel.

[0084] Atmospheric pressure refers to the ambient pressure at the location of the autoclave. For example, at sea level, atmospheric pressure might be around 1 bar (i.e., around 0.1 MPa).

[0085] The depressurization stage (v) can be carried out at a depressurization rate of, for example, 0.5 to 5 bar / min (i.e., 0.05 to 0.5 MPa / min), preferably 0.75 to 4 bar / min (i.e., 0.075 to 0.4 MPa / min), and 1 to 3 bar / min (i.e., 0.1 to 0.3 MPa / min). The autoclave pressure at the end of the depressurization stage corresponds to atmospheric pressure (e.g., around 1 bar, i.e., 0.1 MPa).

[0086] At the end of the procedure, the sterile or disinfected aerogel may contain CO2 as a fluid. The sterile or disinfected aerogel may be exposed to air and / or stored in, for example, a controlled atmosphere, such as an inert atmosphere. Storing the sterile or disinfected aerogel in an inert atmosphere can be achieved, for example, by placing the aerogel in a container under pressure from an inert gas such as nitrogen. Depending on the duration and type of atmosphere with which the aerogel is exposed, the aerogel fluid may change from CO2 to other gases such as air or nitrogen.

[0087] It has been observed that an aerogel obtainable by a procedure described herein has a degree of sterilization or disinfection that is acceptable for most applications. In particular, the preparation procedure can be adjusted to obtain aerogels suitable for applications requiring a high degree of disinfection, for example, requiring specific levels of sterilization, or to obtain aerogels suitable for applications that do not require sterilization, for example, for which certain levels of disinfection are sufficient.

[0088] For example, a sterile product can be defined (as established in the Spanish standard UNE 556, which corresponds to the European standard EN 556) as a product where the probability of survival of a microorganism is no greater than one in a million (less than 1 *10' 6) and this expression is what is internationally known as the SAL Level (for its acronym in English “Security Assurance Level”) of 10' 6 or -6, also referred to as SAL-6 Level. Higher SAL levels, i.e., still lower survival levels, for example, SAL-7 (less than 1 x10' 7 ), SAL-8 (less than 1 x10' 8 ), etc., also denote sterility.

[0089] The level of sterilization or disinfection can be determined against different organisms. Depending on the sterilization or disinfection method used, some microorganisms are more resistant than others, making it more difficult to achieve the same SAL level (e.g., SAL-6). For example, the level of sterilization or disinfection obtained with a procedure as described in this document can be evaluated against spores of a microorganism selected from Bacillus stearothermophilus, Bacillus atrophaeus, and / or Bacillus pumilus.

[0090] As an example, it is known that the bacterium B. pumilus is the standard for radiation sterilization (see, for example, ISO 11137-1:2006) and is the microorganism most resistant to sterilization treatment using a supercritical sterilization method such as the one described herein. Therefore, B. pumilus can be used as a bioindicator (Bl) for sterilization under supercritical conditions, and a sterilization level of SAL-6 against B. pumilus can be considered the highest verified sterilization level achievable with procedures of the type described herein. B. stearothermophilus is known as the standard Bl for steam sterilization (see, for example, ISO 17665-1:2006), and B. atrophaeus as the standard Bl for ethylene oxide sterilization (see, for example, ISO 11135:2014).

[0091] Therefore, a SAL-6 level, or higher, against one or more of these microorganisms denotes a good level of sterilization.

[0092] The level of disinfection or sterilization of an aerogel can be determined using methods known to the art. For example, the SAL level can be determined by measuring turbidity and / or the number of colony-forming units (CFUs). In particular, dried spore strips of a bioindicator can be placed in the autoclave along with a lyogel and subjected to the same conditions used for aerogel preparation and sterilization or disinfection. At the end of the process, to determine the level of microbial inactivation with respect to the tested bioindicator, bacterial growth can be evaluated from the spore strips subjected to the procedure and compared to the growth level of strips that have not been subjected to the procedure (positive control) and to the growth level of a growth medium without spores (negative control).For example, if after incubating the spore strips subjected to the procedure (and the positive and negative controls) at a temperature suitable for the growth of the evaluated bioindicator, for a few days, for example, seven days, the survival level of the bioindicator is no greater than one in a million (less than 1 xi o. -6 The procedure can be considered to have achieved a SAL-6 level against this microorganism, and the aerogels obtained with it can also be considered to have achieved this level. As indicated above, the survival level can be determined by measuring turbidity, for example, by optical density, and / or by measuring the number of colony-forming units (CFLIs), for example, by plating on culture medium.

[0093] Accordingly, this specification also applies to a sterile aerogel, for example, obtainable by a process described herein. Such sterile aerogel may have a SAL-6 or higher grade against B. stearothermophilus, B. atrophaeus, and / or B. pumilus, preferably at least against B. stearothermophilus, more preferably also against B. atrophaeus, and even more preferably also against B. pumilus.

[0094] This specification also addresses a disinfected aerogel, for example, obtainable by a process described herein. Such a disinfected aerogel can typically have a SAL grade lower than SAL-6 for one or more of B. stearothermophilus, B. atrophaeus, and B. pumilus. For example, a disinfected aerogel can have at least a SAL-1 grade, for example, from SAL-1 to SAL-5, in particular at least a SAL-2, SAL-3, or SAL-4 grade, against B. stearothermophilus, B. atrophaeus, and / or B. pumilus, preferably at least against B. stearothermophilus, preferably also against B. atrophaeus, and more preferably also against B. pumilus.

[0095] It was also observed that sterile or disinfected aerogels obtained using the procedure described herein can have a low level of cytotoxicity, implying good cytocompatibility. Therefore, such sterile or disinfected aerogels may be particularly useful for applications where cytocompatibility is important, such as in biomedical applications.

[0096] In particular, it was observed that these sterile or disinfected aerogels can have a cell viability level of at least 70%, according to the UNE-EN ISO 10993-5:2009 standard, preferably greater than 90%. The cell viability level can be measured by methods known in the art. For example, samples of these sterile or disinfected aerogels can be incubated with cells of interest. The incubation temperature can be, for example, 30 to 45 °C, particularly 36 to 39 °C, and more specifically around 37 °C, which is physiological temperature. The incubation time can be, for example, 6 to 96 h, particularly 12 to 72 h, and more specifically 24 to 48 h. The cells used may be cells that may be found in contact with the aerogels in their final application or representative cells of them that can be expected to react in the same or a similar way.In some examples, fibroblasts can be used, such as cells from cell lines NIH-3T3, NCTC clone 929, BALB / 3T3 clone A31, MRC-5, WI-38, Vero Cells, BHK-21, or V-79-379A. After incubation with aerogels, cell viability can be determined using, for example, the resazurin assay, which measures mitochondrial function in metabolically active cells, as described, e.g., by Iglesias-Mejuto et al. (Iglesias-Mejuto, A., Magariños, B., Ferreira-Gonzáíez, T., Starbird-Pérez, R., Álvarez-Lorenzo, C., Pinto-Reis, C., Ardao, I., García-Gonzáíez, CA “Vancomycin-loaded methylcellulose aerogel scaffolds for advanced bone tissue engineering.” Carbohydrate Polymers 2024,324, 121536 doi: 10.1016 / j.carbpol.2023.121536).

[0097] Without being tied to any specific theory, it is proposed that the sequence of steps (i) to (v) of a procedure as described herein can contribute to obtaining an aerogel with a low level of toxic residues, such as residues of microbial inactivating agent. For example, the amount of microbial inactivating agent present in a sterile or disinfected aerogel obtained by a procedure described herein can be below 500 ppm, relative to the total weight of the aerogel, specifically below 100 ppm, more specifically below 25 ppm, and even more specifically below 10 ppm.Therefore, this specification also applies to a sterile or disinfected aerogel, for example, obtainable by a procedure described herein, which further contains an amount of microbial inactivating agent below 500 ppm, in particular below 100 ppm, more particularly below 25 ppm, and still more particularly below 10 ppm.

[0098] It has been observed that a procedure as described herein results in sterile or disinfected aerogels with a suitable nanostructure. A suitable nanostructure is understood to be a nanostructure equivalent to that of a non-sterile or disinfected aerogel, in particular one that has not been subjected to a sterilization or disinfection process, i.e., prepared under similar conditions, for example, by supercritical drying, without being subjected to sterilization conditions.By way of example, a sterile or disinfected aerogel obtained by a process as described herein (including steps (i)-(v)) may have a nanostructure equivalent to an aerogel obtained by a process that includes steps equivalent to (i)-(v) but under conditions that do not result in a sterile or disinfected aerogel, for example without using a microbial inactivating agent in the static step (iii), or a process that instead of having two dynamic steps (i) and (iv) and one static step (iii) comprises simply a single dynamic step.

[0099] In various embodiments, a sterile or disinfected aerogel, for example, obtainable by a procedure as described herein, may have characteristics of, for example, porosity, specific surface area, total specific pore volume, and / or mean pore diameter similar or equivalent to those of an aerogel that has not been subjected to a sterilization process. The porosity of an aerogel can be represented by the Greek letter epsilon (ε) and expressed as the fraction of the total specific pore volume (V) v ) relative to the total apparent volume of a sample (VT) and can be calculated, usually in %, using equation 1:

[0100] E(%) = 100 (1)

[0101] W77

[0102] The V v The volume of an aerogel sample can be determined from the difference between the total volume of the aerogel solid (V s ) and the VT of the sample.

[0103] The total specific pore volume (V v ) and porosity (E) can also be determined from the apparent density or envelope density of the aerogel (p¡) and the actual density of the aerogel (p eS q). The term apparent density (p¡ = p app ) can be used, for example, referring to aerogel cylinders, such as starch cylinders, or 3D printed structures and the term enveloping density (p¡ = p env ) can be used, for example, when referring to aerogel beads. The apparent or shell density (ρi) can be determined by measuring the dimensions and weight of the aerogels. The true density (ρ esq ) can be determined, for example, using a pycnometer such as a helium pycnometer under temperature and pressure conditions of, for example, 25 °C and 1.01 bar.

[0104] Porosity can be calculated in % using equation 2:

[0105] A sterile or disinfected aerogel, for example, obtainable by a procedure as described herein, may have a porosity (E) of more than 65%, preferably more than 75%, more preferably more than 78%, and still more preferably more than 80%. The porosity may be determined by helium pycnometry and weight and volume measurements, in particular, for example, from the apparent or envelope densities (ρi) and the true densities (ρ). esq ) of the aerogel with equation 2.

[0106] The total specific pore volume (V v ) can be expressed in cm 3 / gy can be calculated using equation 3:

[0107] V v (cm 3 / g) = ( - -) (3)

[0108] Pi Pesq /

[0109] In the absence of microporosity in the aerogel, the total specific pore volume corresponds to the sum of the specific volumes of the mesopores and macropores of the aerogel. As is known in the field, macropores are pores larger than 50 nm, mesopores are pores between 2 and 50 nm in size, and micropores are pores smaller than 2 nm. The specific volume of mesopores (VP, meso), which can be determined, for example, by the Barrett-Joyner-Halenda (BJH) method in the pore range between 2 and 50 nm, and the specific volume of macropores (VP, macro), which can be calculated, for example, by the difference between V v and V P , meso.

[0110] A sterile or disinfected aerogel, for example, obtainable by a procedure as described in this document, may have a specific mesopore volume (VP, meso) of more than 0.3 cm³ 3 / g, preferably larger than 0.6 cm 3 / g, which can be determined by nitrogen adsorption-desorption analysis using the Barrett-Joyner-Halenda (BJH) method.

[0111] A sterile or disinfected aerogel, for example, obtainable by a procedure as described herein, may have a total specific pore volume (V v ) of more than 2 cm 3 / g. It could be the V v determined, for example, from the apparent or envelope densities (p¡) and real (p esq ) of the aerogel with equation 3.

[0112] The specific surface area of ​​an aerogel can be defined as the solid surface area per unit mass and can be determined by methods known as the Brunauer-Emmett-Teller (BET) method and expressed as the BET Area (ABET) in units of m² 2 / g, as described, e.g., by Sing et al. (Sing, KS H / ., Everett, DH, Haul, RAW, Moscou, L, Pierotti, RA, Rouquérol, J., Siemieniewska, T. “Reporting physisorption data for gas / solid systems with special reference to the determination of surface area and porosity” Pure and Applied Chemistry 1985, 57, 4, doi: 10.1351 / pac198557040603). Briefly, ABET is obtained from the adsorption branch of the nitrogen adsorption-desorption isotherm, in the relative pressure range between 0.05 and 0.30, using the BET equation. The vacuum degassing time prior to sample analysis, as well as the temperature at which the degassing is carried out, can influence the measurement. For example, the degassing temperature used for starch aerogels might be 60°C for, say, at least 18 hours, while alginate aerogels might be degassed for the same amount of time at, say, 40°C.A sterile or disinfected aerogel, for example, obtainable by a procedure as described in this document, can have a specific surface area of ​​more than 50 m². 2 / g determined by the BET method, preferably over 80 m 2 / g.

[0113] The average pore diameter (D PThe mean pore diameter (MPD) of an aerogel can be defined as the effective diameter of the pore body or its neck and can be expressed as the average pore diameter of the pores present in a sample in nanometers (nm). The mean pore diameter can range from 1 to 400 nm, preferably from 2 to 50 nm. The mean pore diameter can be measured by methods such as the Barrett-Joyner-Halenda (BJH) method of the desorption isotherm branch of nitrogen adsorption-desorption analysis, as described, for example, by Sing et al. The vacuum degassing time prior to sample analysis, as well as the temperature at which this degassing is carried out, can influence the measurement. This may be due to the presence of gases and vapors adsorbed onto the material prior to testing or to thermal degradation of the material if an excessive temperature is used (Sing et al.).For example, the degassing temperature used for starch aerogels can be 60°C for at least, e.g., 18h, while alginate aerogels can be degassed for the same amount of time at, e.g., 40°C.

[0114] A sterile or disinfected aerogel, for example, obtainable by a procedure such as described in this document, may have a mean pore diameter (PD) of more than 4 nm.

[0115] In various particular embodiments, a sterile or disinfected aerogel, for example, obtainable by a procedure as described in this document, may have:

[0116] - a porosity (E) of more than 65%, preferably more than 75%, more preferably more than 78%, still more preferably more than 80%;

[0117] - a specific surface area (ABET) of more than 50 m 2 / g, preferably over 80 m 2 / g; and / or

[0118] - a specific mesopore volume (VP, meso) of more than 0.3 cm³ 3 / g, preferably larger than 0.6 cm 3 / g.

[0119] As indicated above, the degree of sterilization of such sterile aerogel has at least a SAL-6 grade against B. stearothermophilus, preferably against B. atrophaeus and most preferably against B. pumilus.

[0120] In view of the foregoing, this specification is also addressed, in particular, to a sterile aerogel that may comprise alginate, methylcellulose and / or starch that may have a degree of sterilization of SAL-6 against B. stearothermophilus, preferably against B. atrophaeus and more preferably against B. pumilus and at least one of the following characteristics:

[0121] - a porosity (E) of more than 65%, preferably more than 75%, more preferably more than 78%, still more preferably more than 80%;

[0122] - a specific surface area (ABET) of more than 50 m 2 / g, preferably over 80 m 2 / g; and / or

[0123] - a specific mesopore volume (VP, meso) of more than 0.3 cm³ 3 / g, preferably larger than 0.6 cm 3 / g.

[0124] Said sterile aerogel can also be obtained by a procedure as described in this document.

[0125] Sterile or disinfected aerogels, such as those described herein, obtainable using the procedures outlined herein, and possessing the controlled absence of microorganisms and the nanostructure detailed above, are particularly well-suited for applications requiring the highest levels of microbial inactivation. Therefore, this document also addresses the use of such sterile or disinfected aerogels in biomedical and / or food applications, such as in tissue engineering, as carriers in pharmaceutical, cosmetic, or food formulations, and as fat substitutes in the food industry.

[0126] In this text, the words "comprises", "includes" and their variants (such as "comprising", "including", etc.) should not be interpreted in an exclusionary way, that is, they do not exclude the possibility that what is described includes other elements, steps, etc.

[0127] Furthermore, the invention is not limited to the specific embodiments described but also encompasses, for example, those that can be carried out by the average person skilled in the art (for example, regarding the choice of materials, dimensions, components, configuration, etc.), within the scope of the claims.

[0128] Specific realizations of procedures and products as described in this report are detailed in the following examples, without being limited to or including them.

[0129] EXAMPLES Materials

[0130] A sodium salt of alginic acid from brown algae (with a guluronic / mannuronic acid ratio of 70 / 30 and a molecular weight (MW) of 403 kDa) was supplied by Sigma Life Science (Irvine, UK). Native maize starch (with an amylose content of 52.6%) was supplied by Roquette Frères SA (Lestrem, France). Anhydrous calcium chloride (CaCh, >99% purity) and absolute ethanol (EtOH, >99.9% purity) were supplied by Scharlab (Barcelona, ​​Spain) and VWR (Radnor, PA, USA), respectively. CO2 (99.8% purity) was supplied by Nippon Gases (Madrid, Spain) and 30% (v / v) hydrogen peroxide (H2O2) by Sigma-Aldrich (Madrid, Spain). Methylcellulose (viscosity 15 cps, MW 14 kDa) was supplied by Sigma-Aldhch (Steinheim, Germany). Water was purified by reverse osmosis (resistivity > 18 MΩ cm, Milli-Q, Millipore®, Madrid, Spain). Biological sterility indicators: B. spore strips.pumilus (ATCC 27142) (10. 6 spores / strip) and B. stearothermophilus spore strips (ATCC 7953) (10 6 spores / strip) were purchased from Sigma-Aldrich (Madrid, Spain), while the B. atrophaeus spore strips (cell line 9372) (2.4 x 10 6 Spores / strips) were obtained from Crosstex International (Rush, NY, USA). Thypticase soy broth (TSB) and thiocasein soy agar (TSA) medium were purchased from BIOKAR Diagnosis (Pantin, France). Ultrapure nitrogen (N2 > 99% purity) supplied by Nippon Gases (Madrid, Spain) was used for adsorption-desorption textural analyses.

[0131] Example 1: Preparation of alcogels. Alginate hydrogel beads were prepared by external ionic gelation using the dropper method, as described by Remuiñán-Pose, et al. (Remuiñán-Pose, P.; López-Iglesias, C.; Iglesias-Mejuto, A.; Mano, JF; García-González, CA; Rial-Hermida, Ml. Preparation of Vancomycin-Loaded Aerogels Implementing Inkjet Printing and Superhydrophobic Surfaces. Gels 2022, 8, 417, doi:10.3390 / gels8070417). Briefly, alginate powder was dissolved in distilled water with magnetic stirring (300 rpm) overnight to obtain a final concentration of 2.0% (w / v) based on the weight of alginate and the total volume of water.Next, 20 mL of the alginate solution were transferred to a syringe with a 2 mm nozzle diameter for alginate bead formation by dropping it into a 50 mL bath of 150 mM CaCh solution from a height of 20 cm at a constant flow rate of 1.4 mL / min using a syringe pump (AL-1000, World Precision Instruments, Sarasota, FL, USA). After 2 h of aging in the CaCh solution, the gel particles were immersed in EtOH for solvent exchange.

[0132] Starch hydrogel cylinders were obtained by thermal gelation in molds, as described by Santos-Rosales et al. (see previous reference). Briefly, aqueous starch dispersions at 8% (w / v) were prepared based on the weight of starch and the total volume of water and subjected to heat treatment at 121 °C and 1.1 bar for 20 min to dissolve the starch. After dissolving the starch solutions into cylindrical molds (6 cm high and 12 mm in diameter), the starch was gelled. The starch gels were stored at 4 °C for 48 h for retrogradation and then immersed in EtOH and cut into 3.5 mm long cylinders.

[0133] Methylcellulose hydrogels were prepared from aqueous inks containing 12% (w / v) methylcellulose. The hydrogels were produced using a Cellink BIOX bioprinter (Boston, MA, USA) with an extrusion printhead at 40 °C, 40 kPa, and 3 mm / s, a 3 mL syringe, and a 600 pm nozzle. The resulting hydrogels had dimensions of 20 x 20 x 1 mm, a grid pattern, and three layers.

[0134] In all three cases, two consecutive direct solvent exchange steps were performed every 24 h to replace water with EtOH in the gel structure.

[0135] Example 2 (comparative): Production of non-sterile aerogels by supercritical drying

[0136] Starch and alginate alcogels prepared according to Example 1 were subjected to supercritical drying to obtain aerogels. Briefly, EtOH-soaked alcogels were placed in a 100 mL autoclave (TharSFC, Pittsburg, PA, USA; represented illustratively as item 7 in Figure 2), excess EtOH was added to the bottom of the autoclave, and the alcogels were subsequently contacted with SCCO2. The gel samples were previously enclosed in sterilization bags and heat-sealed, as preliminary tests (not shown) confirmed that drying in such bags did not impact the internal porous structure of the aerogels. Heat-sealable autoclave sterilization bags (Soplaril Hispania, Barcelona, ​​Spain) were filled with the alginate and starch alcogels and then heat-sealed.A supercritical drying process (without microbial inactivating agent) was then carried out at 39 °C in five consecutive stages of different regimes as shown, for illustrative purposes, in Figure 3A (Blank_1 test): a stage (i) of pre-suction with CO2 up to SCCO2 conditions of 120 bar; a stage (ii) with a first dynamic regime (or a first dynamic stage) at 120 bar with a CO2 flow of 7 g / min, for 2 h; a stage (iii) with a static regime at 135 bar in the presence of SCCO2, without CO2 flow, for 1 h; a stage (iv) with a second dynamic regime (or a second dynamic stage) at 120 bar with a CO2 flow of 5 g / min, for 1.5 h; and a final depressurization stage (v) in which the autoclave was depressurized at a rate of 1 bar / min until atmospheric pressure was reached.

[0137] The effect of increasing the depressurization rate was compared between the Blank_1 and Blank_3 trials in which the autoclave was depressurized at 1 bar / min and 3 bar / min, respectively.

[0138] Example 3: In situ sterilization during aerogel production

[0139] For the preparation and simultaneous sterilization of an aerogel, also referred to as in situ sterilization of the aerogel during its production or preparation, the process described for Example 2 was followed with the five stages described, but with modifications to the depressurization rate, the conditions of the static stage (iii), and the addition of a microbial inactivating agent (H2O2). This process is illustrated in Figure 3B. The depressurization rate was increased from 1 bar / min to 3 bar / min. In the static stage (iii), the pressure was increased by 5 bar, to 140 bar, and the duration of stage (iii) varied in different tests, ranging from 1 to 3 hours depending on the test. Furthermore, at the beginning of this stage (i¡¡) H2O2 was added in different concentrations (1700-5000 ppm, in particular, milligrams (mg) of microbial inactivating agent per liter (I) of autoclave volume).The additive was injected through a new line implemented in the system (thick black lines in Figure 1), which allowed the addition of the microbial inactivating agent once the autoclave was already under pressure (CO2). To improve the homogenization of the H2O2, the effect of agitation at 700 rpm was evaluated either only during step (i) (test B_1) or during steps (i) and (iv) (test BS_1). A test (test BS140_1) was performed in which the pressure was kept constant at 140 bar throughout the test, with all other conditions similar to those in test BS_1. On the other hand, a comparative example (Ex. Comp. 3: BS_1_water) was carried out with the same conditions as the BS_1 test but injecting 3300 ppm of water in stage (iii) instead of H2O2, to test and evaluate the effect of adding H2O2 on the textural properties of the aerogels.While it was observed that the aerogel had similar textural properties with water (BS_1_water test) or with H2 <D2 (BS_1), los aerogeles obtenidos con agua resultaron ser no estériles.

[0140] The effect of increasing the depressurization rate and agitation was compared between trials Blanco_1 and BS_1_0, in which the autoclave was depressurized to 1 bar / min and 3 bar / min, respectively, and in the second, agitation was used during steps (iii) and (iv). Additionally, a trial, Blanco_H2C>2, was conducted in which the microbial inactivating additive was added to the autoclave at the same time as the lyogels, at the beginning of the process, and the process was run under the same conditions as Example 2, to verify the effect of introducing the additive in step (iii).

[0141] Table 1 shows the conditions for the different sterilization tests performed, numbered as Examples 3a-3k and Comparative Examples 2 and 3, and further denoted by three characters: X_Y_C, where X is the agitation applied in the experiment: samples that are not agitated are designated N, those agitated only in step (iii) are designated B, and those agitated in steps (iii) and (iv) are designated BS; the second character, Y, is the duration of step (iii) in hours; and the last character, C, is the amount (in ppm) of H2O2 added in the test. The notation for tests performed with 3300 ppm of H2O2 was shortened to X_Y.

[0142] Example 4: Microbiological evaluation of the effectiveness of sterilization treatment

[0143] The efficacy of in situ sterilization treatments was evaluated using the bioindicators (Bl): B. stearothermophilus (standard Bl for steam sterilization), B. atrophaeus (standard Bl for ethylene oxide sterilization), and B. pumilus (standard Bl for radiation sterilization). In each experiment, dried spore strips of all three Bls were placed in the autoclave along with the alcoholgels and subjected to the same sterilization tests (see Example 3). After processing, the spore strips were collected and inoculated onto Tryptic Soy Broth (TSB) medium. B. atrophaeus and B. pumilus were then incubated at 37 °C and B. stearothermophilus at 55 °C for 7 days. Bacterial growth was assessed by optical measurement of turbidity in the tubes and confirmed by plating on Tryptic Soy Agar (TSA) plates daily for 7 days.The results obtained for the different conditions of the preparation tests of examples 2 and 3 and detailed in table 1, are presented in table 2 indicating a YES when a SAL-6 sterility level was achieved for the Bl in question (when there was no bacterial growth after 7 days of incubation for said Bl) and a NO when a SAL-6 sterilization level was not achieved (when there was growth after seven days of incubation for said Bl).

[0144] TSB medium (negative microbiological control) and untreated spore strips (positive microbiological control) inoculated onto TSB were incubated under the same conditions as the process controls. Table 1

[0145] *3300 ppm of water instead of H2O2 Table 2

[0146] As can be seen in Table 2, the blanks and comparative examples 2 and 3 failed to sterilize the sample against any of the tested bioindicators. The remaining examples did achieve a SAL-6 sterilization level against at least one or two of the bioindicators, although most conditions achieved a SAL-6 sterilization level against all three tested bioindicators.

[0147] Example 5: Sterilization of aerogels by other techniques Comparatively, aerogels were subjected to standardized sterilization treatments with ethylene oxide (EO) and gamma radiation (y rays). Starch and alginate aerogels were introduced into heat-sealed sterilization bags before being subjected to the sterilization treatments.

[0148] The process was carried out in a SUOD142483 sterilizer (Suphatec SL, Bigues i Riells, Spain) by Esterilización SL (Barcelona, ​​Spain), following ISO 11135:2014. The samples were treated at 55 °C and a minimum relative humidity of 40% for 4 hours. To eliminate any remaining toxic traces of oxygen in the samples, sequential aeration steps were performed. First, the samples were subjected to a vacuum process for 21 minutes, followed by a flow of fresh nitrogen for 8 minutes at 600 mbar. A second vacuum period of 12 minutes was then applied, followed by air aeration for 8 minutes at 600 mbar. Finally, the samples were aerated with fresh air at 500 mbar for 34 minutes and could be collected 48 hours later.

[0149] The samples were treated with X-rays at a first-class radioactive facility, IR / B-02 / 69 (Aragogamma SL, Les Franqueses del Vallés, Spain). The treatment was performed using a source of 6O Co, reaching a dose of 30.6 kGy and exposing the material for 7.53 hours, in accordance with ISO 11137-A2:2020.

[0150] Example 6: Physicochemical characterization of aerogels

[0151] The texture of the aerogels was studied by scanning electron microscopy (SEM) using an UltraPlus FESEM microscope (Zeiss, Jena, Germany). The aerogels were previously coated with a 10 nm iridium layer to enhance contrast (Q150 TS / E / ES instrument, Quorum Technologies, Lewes, UK). Figure 4 shows SEM images of alginate (Figure 4A) and starch (Figure 4B) aerogels obtained for the blank, for the example without a microbial inactivating agent (without H2O2) prepared under the conditions of comparative example 2 (BS_1_0), and for the example with a microbial inactivating agent (with H2O2) prepared under the conditions of example 3e (BS_1).

[0152] The apparent density of starch aerogel cylinders and methylcellulose aerogel 3D structures (p app ) and the enveloping density of the alginate aerogel beads (p env) were determined by measuring the dimensions and weight of the aerogels. The actual density of the aerogels (p esq The porosity (E) was determined using a helium pycnometer (Quantachrome, Boynton Beach, FL, USA) at room temperature (25 °C) and 1.01 bar pressure. Values ​​were obtained from five replicates (standard deviation < 5%). The resulting overall porosity (E) was calculated according to Equation 2, defined above: where p¡ is the apparent density of starch aerogel cylinders or methylcellulose aerogel 3D structures, or the envelope density of alginate aerogel beads, respectively.

[0153] The textural properties of the aerogels were determined by nitrogen adsorption-desorption analysis (ASAP 2000 Micromeritics Inc., Norcross, GA, USA). Prior to analysis, the samples were degassed under vacuum (<1 mPa) for 24 h at degassing temperatures of 40 °C, 60 °C, and room temperature for alginate, starch, and methylcellulose aerogels, respectively. The specific surface area (ABET) of the aerogels was determined by the Brunauer-Emmett-Teller (BET) method, while the Barrett-Joyner-Halenda (BJH) method was used to determine the mean pore diameter (DP) of the desorption branch of the isotherm. The same BJH method was used to determine the specific volume of mesopores (VP, meso). The total specific pore volume (V v ) was calculated from equation (3), defined above:

[0154] The specific volume of macropores (VP, macro) was calculated by the difference between Vv and VP, meso.

[0155] The results obtained for aerogels prepared under different preparation and sterilization conditions from Table 1 are shown in Figures 5, 6, and 7.

[0156] In particular, Figure 5 shows the effect of preparing aerogels from starch cylinders (the three solid bars on the left) and alginate beads (the three striped bars on the right) obtained for the blank (Blank_3), for the example without a microbial inactivating agent (without H2O2, prepared under the conditions of Comparative Example 2, BS_1_0), for the example with H2O2 as the microbial inactivating agent (prepared under the conditions of Example 3e, BS_1), and for the example where water is used as the microbial inactivating agent, replacing H2O2 (prepared under the conditions of Comparative Example 3, BS_1_water). Porosity (E) (Figure 5A), specific surface area (ABET) (Figure 5B), and total specific pore volume (V) are shown. v(Figure 5C), represented as the sum of the specific volume of macropores (light color) and the specific volume of mesopores (dark color) and the mean pore diameter (DP) (Figure 5D). Bars grouped with the same letter (a, boc) denote statistically homogeneous groups.

[0157] Figure 6 shows the effect of exposure time on the textural properties of sterile or disinfected aerogels of starch cylinders (the three solid bars on the left) and alginate beads (the three striped bars on the right), prepared with a static stage (iii) duration of 1 hour (1 h stage (iii) according to the conditions of example 3e, BS_1), 2 hours (2 h stage (iii) according to the conditions of example 3d, BS_2), and 3 hours (3 h stage (iii) according to the conditions of example 3f, BS_3), including porosity (Figure 6A), specific surface area (Figure 6B), total specific pore volume (Figure 6C), represented as the sum of the specific volumes of macropores (light color) and mesopores (dark color), and mean pore diameter (Figure 6D). Bars grouped with the same letter (a, boc) denote statistically homogeneous groups.

[0158] Figure 7 shows the effect of agitation time on the textural properties of sterile or disinfected starch aerogels, e.g. starch cylinders (the three solid bars on the left) and alginate aerogels, e.g. alginate beads (the three striped bars on the right), including the example without agitation (prepared according to the conditions of example 3k, N_1), the example with agitation at step (i) (prepared according to the conditions of example 3h, B_1) and the example with agitation at steps (i) and (i) (prepared according to the conditions of example 3e, BS_1), including porosity (Figure 7A), specific surface area (Figure 7B), total specific pore volume (Figure 7C), represented as the sum of the specific volumes of macropores (light color) and mesopores (dark color), and mean pore diameter (Figure 7D). Bars grouped with the same letter (a, boc) denote statistically homogeneous groups.

[0159] Figure 8 shows the effect of various sterilization techniques on the textural properties of sterile or disinfected starch aerogels, e.g., starch cylinders (the three solid bars), and alginate aerogels, e.g., alginate beads (the three striped bars), including an example of the materials sterilized according to the invention by agitation in steps (iii) and (iv) (prepared according to the conditions of Example 3e BS_1, left bars), an example sterilized by ethylene oxide (EO, middle bars), and by gamma ray irradiation (γ rays, right bars). The properties studied comprise porosity (E) (Figure 8A), specific surface area (ABET) (Figure 8B), and total pore specific volume (V). v) (Figure 8C), represented as the sum of the specific volumes of macropores (VP, macro) (light color) and mesopores (VP, meso) (dark color), and the mean pore diameter (DP) (Figure 8D). Bars grouped with the same letter (a, boc) denote statistically homogeneous groups.

[0160] Example 7: In vitro compatibility studies

[0161] The cytocompatibility of the aerogels was tested by incubating them with the NIH-3T3 cell line (ATCC: CRL-1658). Briefly, the cells were seeded into 24-well plates containing 500 pL of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% bovine serum and 1% penicillin-streptomycin (12,000 cells per cm²). 2 for 24 h and 8000 cells per cm 2Cells were inoculated with aerogels (for 48 h) and incubated at 37°C in a humidified atmosphere supplemented with 5% CO2. To measure cell viability in contact with the aerogels, after 24 h of cell incubation, the aerogels were placed in triplicate in 24-well culture inserts (etched PET membrane with a 0.4 µm pore size), and 100 pL of medium was added to the well plate containing the seeded cells. Cell viability was measured after 24 h of incubation. To ensure that any residual H2O2 dissolved in the cell medium, viability was also measured after 48 h of incubation with the cells. Negative controls consisting of cells without aerogel were incubated under the same conditions and in triplicate for all formulations.To evaluate the cytotoxicity of the blank samples (aerogels not sterilized by SCCO2), these aerogel samples were first UV sterilized for 30 min and then placed in the cell wells using culture inserts.

[0162] Cell viability was assessed using the resazurin assay, which measures mitochondrial function in metabolically active cells. The aerogel was removed from the cells after 24 and 48 h of incubation, the culture medium was aspirated, and 300 pL of 44 pM resazurin was added to each well. After 3 h of incubation under identical conditions, fluorescence was measured at an excitation wavelength of 544 nm and an emission wavelength of 259 to 590 nm using a microplate reader (Infinite® M200, Tecan Group Ltd., Manneddorf, Switzerland).

[0163] The results obtained are presented in Figure 9, which shows cell viability expressed as a percentage after 24 and 48 h of contact with starch cylinder formulations (solid bars, Figure 9A) and alginate beads (striped bars, Figure 9B), prepared using comparative example 2 (BS_1_0, left bars for each time point) and example 3e (BS_1, right bars for each time point). No statistically significant differences were found between the samples.

[0164] Figure 10 shows the effect of preparing methylcellulose aerogels. The bars on the left represent the results obtained for the blank (Blank_1), and the bars on the right represent the results obtained for the example with H2O2 as the microbial inactivating agent (prepared under the conditions of Example 3e, BS_1). Porosity (E) (Figure 10A), specific surface area (ABET) (Figure 10B), and total specific pore volume (V) are represented. v ) (Figure 10C), represented as the sum of the specific volume of macropores (light color) and the specific volume of mesopores (dark color) and the mean pore diameter (DP) (Figure 10D). Bars grouped with ns denote values ​​with no significant difference.

[0165] Statistical analysis

[0166] Results are expressed as mean ± standard deviation. Statistical analyses of textural properties and cell viability were performed using a 2-way ANOVA test, followed by a post-hoc Tukey HDS multiple comparison test using GraphPad Prism v.8.0.2 (GraphPad Software, Boston, MA, USA).

Claims

1. CLAIMS 1. A method for preparing and simultaneously sterilizing or disinfecting an aerogel, comprising contacting a lyogel with supercritical carbon dioxide (SCCO2) inside an autoclave, wherein contacting comprises five steps in the following order: i) a pressurization step comprising pressurizing the autoclave with CO2 to SCCO2 conditions; i) a first dynamic stage comprising maintaining the autoclave under pressure with a flow of SCCO2; iii) a static stage comprising maintaining the autoclave under pressure of scCO2 without flow; iv) a second dynamic stage comprising maintaining the autoclave under pressure with a flow of SCCO2; and v) a depressurization stage comprising depressurizing the autoclave to atmospheric pressure, obtaining a sterile or disinfected aerogel;wherein during the static stage (iii) a microbial inactivating agent is introduced into the autoclave under SCCO2 pressure, preferably a microbial inactivating agent selected from H2O2, peracetic acid, acetic acid, trifluoroacetic acid, sodium hypochlorite, chlorine dioxide, boric acid, DDAC, formic acid, sodium dichloroisocyanurate, tert-butyl hydroperoxide, formaldehyde, glutaraldehyde, water, methanol, ethanol, ammonium salts, ethylene oxide, as well as phenols, polyphenols, terpenes, essential oils, glucosinolate derivatives, alkaloids and thiols with antimicrobial activity, and combinations thereof in mixtures, most preferably the microbial inactivating agent being H2O2.; 2. The method of claim 1, comprising, before contacting the liogel with SCCO2: introducing the liogel into the autoclave, preferably with the liogel located inside a sterilization bag, and, optionally, before introducing the liogel into the autoclave: • Prepare the lyogel, preferably by dissolving a polymeric material in a solvent or 3D printing from an ink composed of polymeric material dispersed or dissolved in a liquid solvent, and / or • where the lyogel liquid is a solvent, perform a lyogel solvent exchange; or Prepare the liogel inside the autoclave from a first liogel by means of a solvent exchange or from a polymeric material and a solvent.

3. The process of claim 1 or 2 wherein in the pressurization step (i) the autoclave is pressurized with scCÜ2 to a pressure of 80 to 180 bar, preferably 110 to 150 bar, and more preferably around 120 bar.

4. The method of any of claims 1 to 3 wherein the flow of scCÜ2 in the first dynamic stage (i) and / or in the second dynamic stage (iv) is 0.25 to 25 g / min per 100 mL of autoclave volume, preferably 0.5 to 20 g / min, more preferably 1 to 15 g / min, still more preferably 2 to 10 g / min, and still more preferably 5 to 7 g / min.

5. The procedure of any of claims 1 to 4 wherein - in the static stage (iii) the autoclave is maintained under a pressure of scCÜ2 of 80 to 180 bar, preferably from 130 to 150 bar, more preferably above 135 bar and even more preferably around 140 bar; and / or - in the first dynamic stage (i) and / or in the second dynamic stage (iv) the autoclave is kept under a scCÜ2 pressure of 80 to 180 bar, preferably 110 to 130 bar, more preferably around 120 bar.

6. The method of any of claims 1 to 5 wherein in the static step (iii) the microbial inactivating agent is introduced into the autoclave under SCCO2 pressure, preferably at the bottom of the autoclave, preferably through a feeder tube having its outlet at the bottom of the autoclave.

7. The procedure of any of claims 1 to 6 wherein: - the first dynamic stage (i) lasts from 0.25 to 5 hours, preferably from 0.5 to 4 hours, more preferably from 1 to 3 hours, and still more preferably around 2 hours; - the static stage (iii) lasts from 0.25 to 5 hours, preferably from 0.5 to 4 hours, and more preferably from 1 to 3 hours; and / or - the second dynamic stage (iv) lasts from 0.25 to 4 hours, preferably from 0.5 to 3 hours, more preferably from 1 to 2 hours, and even more preferably around 1.5 hours.

8. The method of any of claims 1 to 7 wherein the autoclave temperature is from 31.1 to 70 °C, preferably from 33 to 60 °C, and more preferably from 35 to 50 °C.

9. The process of any of claims 1 to 8 wherein the liogel comprises: - a polymeric material, preferably a polymeric material selected from organic polymers, inorganic polymers and combinations thereof, optionally with additions of particles and / or fibers of ceramic, polymeric, metallic or biological nature, or a composite polymeric material of sandwich type; and - a solvent, preferably a solvent selected from ethanol, acetone, methanol, dimethyl sulfoxide, diethyl ether, ethyl methyl ketone, isopropanol, and mixtures thereof, most preferably the solvent being ethanol.

10. The procedure of claim 9 wherein - Organic polymers are selected from polysaccharides, resorcinol-formaldehyde; polyurethane; proteins; polynucleotides; and mixtures thereof; and / or - Inorganic polymers are selected from silica, carbon, and mixtures thereof.

11. The process of claim 9 or 10 wherein the polymeric material is an organic polymer, preferably a polysaccharide, more preferably a polysaccharide selected from alginate, starch, chitosan and / or cellulosic derivatives, in particular methylcellulose, even more preferably from alginate, starch and methylcellulose, and still more preferably the polymeric material is alginate and / or starch.

12. The method of any of claims 1 to 11 wherein the microbial inactivating agent is introduced in an amount of 10 to 6000 ppm with respect to the volume of the autoclave, preferably 250 to 5750 ppm, more preferably 600 to 5500 ppm.

13. A sterile or disinfected aerogel obtainable by the process of any of claims 1 to 12 wherein - the sterile aerogel has a SAL-6 grade or higher; and - the disinfected aerogel has at least a SAL-1 grade, in particular from a SAL-1 grade to a SAL-5 grade, preferably at least a SAL-2, SAL-3 or SAL-4 grade; against B. stearothermophilus, B. atrophaeus and / or B. pumilus, preferably at least against B. stearothermophilus, more preferably also against B. atrophaeus and even more preferably also against B. pumilus.

14. The sterile or disinfected aerogel of claim 13 having: - a porosity (E) of more than 65%, preferably more than 75%, more preferably more than 78%, still more preferably more than 80%; - a specific surface area (ABET) of more than 50 m 2 / g, preferably over 80 m 2 / g; and / or - a specific mesopore volume (VP, meso) of more than 0.3 cm³ 3 / g, preferably larger than 0.6 cm 3 / g.

15. A sterile aerogel comprising alginate and / or starch having a degree of sterilization of SAL-6 against at least B. stearothermophilus, preferably also against B. atrophaeus and more preferably also against B. pumilus and at least one of the following characteristics: - a porosity (E) of more than 65%, preferably more than 75%, more preferably more than 78%, still more preferably more than 80%; - a specific surface area (ABET) of more than 50 m 2 / g, preferably over 80 m 2 / g; and / or - a specific mesopore volume (VP, meso) of more than 0.3 cm³ 3 / g, preferably larger than 0.6 cm 3 / g.

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