Hydrogel manufacturing method and uses thereof

Abstract

The invention relates to a method for manufacturing a hydrogel from at least one crosslinkable polymer, the method comprising the following steps: a) providing a solution comprising at least one crosslinkable polymer and an oxidase, b) crosslinking the at least one polymer with the oxidase by adding at least one oxygenated species in order to obtain a hydrogel (10'), wherein said at least one oxygenated species is added during step b) by a device (30) for generating a cold plasma (31).

Description

[0001] Description

[0002] Title of the invention: Method for manufacturing hydrogel and its uses The invention relates to the field of manufacturing bioactive hydrogels and the use of the latter in particular as a substrate for the development of bio-ink usable in 3D bio-printing (3D printing technique) and / or production of hydrogel patches or as a therapeutic tool (injectable hydrogel in situ / in vitro), in particular in the context of cancer treatment.

[0003] A hydrogel typically consists of a matrix of polymer chains that are initially water-soluble but become insoluble after crosslinking by absorbing a large amount of water. Hydrogels can be made from either natural polymers, such as polysaccharides (alginate, chitosan, hyaluronic acid, polyglucuronic acid, etc.), proteins (gelatin, collagen, etc.), or synthetic polymers, such as ethylene oxide, vinyl alcohol, acrylic acid, etc.

[0004] Hydrogels possess a high degree of flexibility (elasticity, compressibility, etc.), comparable to that of living tissues. Furthermore, due to their predominantly water content, they are biocompatible with such tissues. Consequently, hydrogels have numerous applications in the biomedical field, including tissue engineering (bone, cartilage, tendons, muscles, blood vessels, etc.), regenerative medicine, drug, protein, and cell delivery / transfer, personalized medicine, and more.

[0005] The porous structure of hydrogels offers the advantage of being able to trap active compounds and / or cells for various medical applications.

[0006] It is known to employ 3D bioprinting technology, which uses additive manufacturing processes to artificially produce biological tissues by layering hydrogel, some of which can be loaded, for example, with living cells. In this case, it is referred to as 3D printing "bioink".

[0007] Nevertheless, there is still a need to improve 3D bioprinting techniques. In particular, there is a need for the rapid fabrication of a hydrogel that exhibits structural reliability and sterility for use in numerous medical applications.

[0008] To this end, the invention relates to a method for manufacturing a hydrogel from at least one crosslinkable polymer, the method comprising the following steps: a) providing a solution comprising at least one crosslinkable polymer and an oxidase, b) crosslinking said at least one crosslinkable polymer (11) by the oxidase (13) using the addition of at least one oxygenated species in order to obtain a hydrogel (10'),

[0009] where the addition of said at least one oxygenated species during step b) is provided by a cold plasma generation device.

[0010] The process according to the invention advantageously allows for the rapid production of hydrogels with a reliable and homogeneous structure. The manufacturing process of the invention makes it possible to obtain all types of 3D biomodels, such as mono- or multilayer biomaterials (hydrogel film patches, 3D constructions), tissues, complex organs, etc. The process according to the invention has the advantage of applying a plasma treatment during the crosslinking step, thus directly providing the oxygenated species necessary for the controlled crosslinking of the polymers in the presence of an oxidase-type enzyme, without any other external input.

[0011] The hydrogel obtained by this process can be used in numerous applications, from cell culture to therapy, particularly for cancer treatment. Indeed, the parameters of step b) can be adjusted according to the desired end use of the hydrogel, as will be discussed in detail later.

[0012] Plasma is defined as the fourth state of known matter. It has long been used in many medical applications such as sterilization, cauterization (stopping bleeding), and tissue ablation.

[0013] Cold plasma, also known as atmospheric pressure plasma, differs from hot plasma in its composition: a gas weakly ionized by electrons from the current supplied by a high-voltage power source. Unlike hot plasma, where electrons and heavy particles are in thermal equilibrium, cold plasma is characterized by thermal disequilibrium; the electrons are hot (10 to 20 kK) and the heavy particles are cold (atoms, molecules, and ions are at a few tens of degrees Celsius). Indeed, at atmospheric pressure, the temperature of heavy particles in cold plasma varies between 25°C and 45°C, thus allowing treatments on organisms without significantly damaging the structure of the targeted surface.

[0014] The cold plasma in the process of the invention is applied at atmospheric pressure and ambient temperature. For the purposes of this invention, "ambient temperature" means a temperature between 20°C and 30°C, in particular 25°C. The cold plasma produces reactive oxygen and nitrogen species (ROS), including, but not limited to, atomic oxygen and singlet oxygen ( 1µ2), ozone (O3), hydroxyl radicals (*OH), hydrogen peroxide (H2O2), nitric oxide (NO), peroxynitrite (ONOO), nitrogen dioxide (*NO2), and dinitrogen trioxide (N2O3). It is therefore possible to vary the amount of RONS present within the hydrogel depending on the desired application. The presence of RONS is particularly useful for cell culture, as it provides an antiseptic effect. A high concentration of RONS, on the other hand, allows for antitumor applications to trigger apoptosis in tumor cells. Because the RONS are incorporated during the polymer crosslinking process, they are present more homogeneously and reproducibly in the hydrogel than if plasma treatment were applied after the polymers had already been crosslinked.

[0015] According to a preferred embodiment of the invention, step b) is carried out under agitation. Such agitation allows, on the one hand, for better homogenization of the hydrogel and, on the other hand, prevents the formation of a passive surface layer, which can limit the penetration of RONS into the hydrogel and therefore of oxygenated species for the crosslinking reactions.

[0016] According to a preferred embodiment of the invention, the cold plasma is generated from an air stream with a relative humidity of 80% or higher, preferably saturated with water vapor, and / or from an air stream containing dihydrogen. In this respect, the process according to the invention has the advantage of using air as the gas instead of another gas such as helium, thus avoiding supply and cost problems. A high relative humidity (greater than 80%) and / or the presence of dihydrogen in the air stream also allows for the production of large quantities of oxygenated species, and in particular hLCh, which is useful both for efficient crosslinking and for greater controlled integration of RONS into the hydrogel, particularly for therapeutic purposes (treatment of cancer cells / tumors).

[0017] Specifically, the humidity level is greater than or equal to 85%, for example greater than or equal to 90%. By "vapor-saturated air" we mean air with a humidity level of 95% to 100%.

[0018] According to a preferred embodiment of the invention, during step a) said at least one crosslinkable polymer is present in the solution from 0.1 to 6%, preferably from 0.2 to 1% by weight by volume of said solution.

[0019] According to a preferred embodiment of the invention, step b) lasts from 1 to 30 minutes and preferably from 1 to 20 minutes. The duration of step b) notably influences the degree of polymer crosslinking and the amount of trapped RONS. According to a preferred embodiment of the invention, the crosslinkable polymer or at least one of the crosslinkable polymers is a polysaccharide, alone or in combination with one or more thermosensitive crosslinkable polymers. Preferably, in combination with one or more thermosensitive crosslinkable polymers at body temperature, particularly in the context of an in situ / in vitro injectable hydrogel application.

[0020] According to a preferred embodiment of the invention, the oxidase is selected from the group consisting of an oxidoreductase, a peroxidase, a catalase, a laccase, a tyrosinase, a monosaccharide oxidase, and a mixture thereof. Preferably, the oxidase is a peroxidase or a laccase, in particular horseradish peroxidase (HRP). According to a preferred embodiment, the manufacturing process according to the invention is a computer-aided 3D printing process. By "computer-aided," it is understood that the steps followed by the process are established according to instructions provided by a computer program stored on a tangible, non-transient, computer-readable storage medium.

[0021] The invention also relates to a hydrogel obtained by the process as defined above. The process according to the invention makes it possible to obtain a wide range of hydrogels exhibiting different properties adapted to each desired application. The hydrogel according to the invention can thus be active and suitable for in situ / in vitro injection in therapeutic treatments, but can also be a patch or film suitable for molding methods, or even a bio-ink suitable for use in 3D printing.

[0022] In particular, hydrogel is an activated solution.

[0023] The invention also relates to a bioactive hydrogel as defined above for use in a method of treating a disease. Preferably, said disease is cancer.

[0024] Brief description of the figures

[0025] The invention will be better understood in light of the following description, which is purely illustrative and not limiting, and is made with reference to the accompanying drawings in which:

[0026] Figure 1 represents an installation enabling the implementation of an embodiment of the process according to the invention.

[0027] Figure 2 shows the appearance of several hydrogels obtained by an embodiment of the manufacturing process according to the invention. Figure 2A shows a phenolized polyglucuronic acid hydrogel after 8 minutes of crosslinking under cold plasma. Figure 2B shows a phenolized alginate hydrogel after 10 minutes of crosslinking under cold plasma. Figure 2C shows a phenolized chitosan hydrogel after 10 minutes of crosslinking under cold plasma.

[0028] Figure 3 shows the viscosity (in Pa·s) as a function of the shear rate (1 / s) of phenolic polyglucuronic acid (PGU-ph) hydrogels obtained by a manufacturing process according to the invention. The light gray solid curve represents a PGU-ph hydrogel at 25°C obtained after 8 minutes of cold plasma treatment. The dark gray solid curve represents a PGU-ph hydrogel at 4°C obtained after 4 minutes of cold plasma treatment. The light gray dashed curve represents a PGU-ph hydrogel at 25°C obtained after 4 minutes of cold plasma treatment. The dark gray dashed curve represents a PGU-ph hydrogel at 37°C obtained after 4 minutes of cold plasma treatment.

[0029] Figure 4 shows the viscosity (in Pa·s) as a function of the shear rate (1 / s) of phenolized polyglucuronic acid hydrogels obtained by a comparative manufacturing process where H₂O₂ is directly added at a concentration of 50 pM, without the application of cold plasma. The dark gray solid curve represents a PGU-ph hydrogel at 4°C. The light gray dashed curve represents a PGU-ph hydrogel at 37°C obtained after 4 minutes of cold plasma treatment. The dark gray dashed curve represents a PGU-ph hydrogel at 25°C obtained after 4 minutes of cold plasma treatment.

[0030] Detailed description

[0031] According to a first object, the invention relates to a method for manufacturing a hydrogel, comprising the following steps:

[0032] a) provide a solution comprising at least one crosslinkable polymer and an oxidase, b) crosslink said at least one polymer with the oxidase by means of the addition of at least one oxygenated species in order to obtain a hydrogel,

[0033] where the addition of at least one oxygenated species during step b) is provided by a cold plasma generation device.

[0034] In the context of the invention, the term "hydrogel" refers to a gel in which water is used as the dispersion medium, and which is formed by introducing a portion of a hydrophobic group and a hydrophilic residue into a water-soluble, crosslinkable polymer having a network-linked structure. The crosslinking of the crosslinkable polymer may be partial or complete. In particular, when partial, the hydrogel may correspond to an activated solution that retains flow rate and viscosity properties that allow it, in particular, to be injected through a nozzle (for example, for a 3D printing application) or a catheter (for example, for a therapeutic application). Partial crosslinking may be achieved, in particular, by prematurely interrupting step b) or by selecting the various parameters involved in crosslinking to obtain partial crosslinking.When the hydrogel is an activated solution, almost total, or even total, crosslinking of the crosslinkable polymer can subsequently be achieved either by terminating step b) or by repeating this step, or by other means of crosslinking such as the application of heat or pressure.

[0035] Figure 1 shows an installation 1 for a process of manufacturing a hydrogel 10' according to the invention. In particular, a solution 10 is deposited in a receptacle 20. The solution 10 comprises water 12 in which at least one crosslinkable polymer 11 and an oxidase 13 are dissolved. Figure 1 shows a crosslinking step b) in progress, such that the solution 10 and the hydrogel 10' are mixed.

[0036] Step a)

[0037] Any type of hydrophilic crosslinkable polymer 11 known to those skilled in the art is usable within the scope of the present invention. This crosslinkable polymer(s) 11 is / are selected from the group consisting of synthetic crosslinkable polymers, crosslinkable biopolymers, and mixtures thereof. "Mixture" means (i) a mixture of at least two synthetic crosslinkable polymers, (ii) a mixture of at least two crosslinkable biopolymers, and (iii) a mixture of at least one synthetic crosslinkable polymer and at least one crosslinkable biopolymer. In a mixture, at least two different crosslinkable polymers may be unlinked to each other or linked to each other via covalent and / or non-covalent bonds.

[0038] The crosslinkable polymer(s) 11 included in the solution 10 used in the invention is / are typically biodegradable. Indeed, when the hydrogel 10' is intended to be implanted in a patient's body, it is essential that the polymer(s), once crosslinked, can / are easily degraded without generating toxic residue.

[0039] Typically the synthetic polymers used in the invention are chosen from the group consisting of thermosensitive polymers, polyethylene glycols, polyacrylic acids (PAA), polyvinyl alcohols (PVA), pluronic acids, methylcelluloses, their mixtures and a salt of these.

[0040] By "biopolymer" we mean a polymer produced by a living organism or a derivative thereof, or is a synthetic version of that polymer produced by abiotic chemical pathways.

[0041] Typically, the crosslinkable biopolymers implemented in the invention include, but are not limited to, proteins, polyphenolic compounds and / or polysaccharides. In particular, the crosslinkable biopolymers used in the invention are selected from the group consisting of starches (including amylose and / or amylopectin), chitosans, hemicelluloses, glycans, lignins, tannins, lignosulfonates, celluloses, chitins, alginates, dextran, pullan, polyhydroxyalkanoates, fibrins, cyclodextrins, collagen, gelatin, silk fibroins, pectin, glycosaminoglycans, polylactic acids, polyuronic acids, natural or synthetic polyglucuronic acids, a salt of these, a substituted form of these, a salt of the latter, and mixtures thereof.

[0042] More specifically, the or at least one of the crosslinkable polymers 11 is a polysaccharide, notably chosen from the group consisting of alginates, chitosans and polyglucuronic acids.

[0043] According to one embodiment, the crosslinkable polymer(s) 11, or at least one of them, is a polysaccharide, alone or in combination with one or more thermosensitive crosslinkable polymers 11, in particular those that are thermosensitive to body temperature. This aspect of the invention makes it possible to obtain hydrogels suitable for implantation in a patient and which harden only upon contact with the patient.

[0044] In solution 10, the crosslinkable polymer or each of the crosslinkable polymers 11 is present in particular in small quantities, in order to avoid gelation problems during step b). Thus, according to one embodiment of the invention, the crosslinkable polymer or each of the crosslinkable polymers 11 is present in solution 10 from 0.1 to 6% by weight per volume of solution (w / v). In particular, the crosslinkable polymer or each of the crosslinkable polymers 11 is present in the solution 10 from 0.1 to 4% w / v, particularly from 0.1 to 3% w / v, particularly from 0.2 to 4% w / v, particularly again from 0.2 to 3% w / v, particularly from 0.2 to 2% w / v, particularly from 0.2 to 1% w / v, particularly from 0.2 to 1% w / v, particularly again from 0.2 to 0.8% w / v, particularly from 0.3 to 0.7% w / v, for example 0.5% w / v.

[0045] Oxidase 13 is notably selected from the group consisting of an oxidoreductase, a peroxidase, a catalase, a laccase, a tyrosinase, a monosaccharide oxidase, and a mixture thereof. In particular, oxidase 13 is horseradish peroxidase (HRP).

[0046] The quantity of oxidase per volume of solution 10 is in particular from 1 to 100 U / ml, more particularly 10 to 100 U / ml, in particular from 10 to 50 U / ml, particularly from 10 to 30 U / ml, for example from 20 U / ml.

[0047] In one particular embodiment, step a) includes a substep of phenolizing one or more of the crosslinkable polymers 11 or the provision of one or more phenolized crosslinkable polymers 11. Phenolization notably allows for improved adhesion between the crosslinked polymers 11. This phenolization is notably carried out by radical polymerization or by selective chemical or enzymatic grafting of a phenolic group onto a carboxylic group (particularly for polyuronic acids such as pectin, alginate, and polyglucuronic acid) and an amine group (for example, for chitosan) of one or more of the crosslinkable polymers 11. In particular, the phenolic group consists of an X-(Y-amino-Z-hydroxyalkyl)-n-hydroxylphenol or one of its derived salts.The said X-(Y-amino-Z-hydroxyalkyl)-n-hydroxylphenol or one of its derived salts is notably chosen from the group consisting of tyramine, dopamine, and octopamine, and is preferably tyramine. In the case of the phenolization of chitosans, the phenolic group may be provided by a derivative of 3-(4-hydroxyphenyl) propionic acid and / or any form of phenolic acid (ferulic, caffeic, gallic, tannic, etc.) or one of its derived salts.

[0048] Thus, according to a preferred embodiment of the invention, said solution 10 comprises at least one or more phenolized polysaccharides 11, alone or in combination with one or more non-phenolized crosslinkable polymers 11 such as thermosensitive polymers, in particular thermosensitive to body temperature.

[0049] According to a preferred embodiment of the invention, said at least one crosslinkable polymer 11 is chosen to provide an injectable hydrogel, notably via a syringe. This aspect of the invention allows for various applications, from 3D printing (crosslinkable bioink for tissue engineering applications) to therapeutic applications for placement in a treatment area (cancerous tumors).

[0050] According to a preferred embodiment of the invention, said at least one crosslinkable polymer 11 is selected to achieve gelation by thixotropic action and / or thermosensitivity, in particular thermosensitivity to body heat. This aspect of the invention is particularly advantageous when the hydrogel formed is an activated solution. Indeed, this solution can thus be injected into the area to be treated in a patient through a catheter, thanks to its flow rate and viscosity properties, and then the crosslinking of the crosslinkable polymer can be continued, or even completed, upon contact with the area to be treated following one or more actions to induce its gelation (for example, by pressure and / or application of heat).

[0051] Step b)

[0052] Step b) concerns the crosslinking of the crosslinkable polymer(s) 11 to form the hydrogel 10'.

[0053] This crosslinking is achieved using a cold plasma generation device 30 31 arranged opposite the solution 10, as shown in Figure 1. The generation device 30 comprises a cold plasma formation chamber 32 31 supplied by a gas injection system 40 comprising a main supply system 42 delivering a gas flow.

[0054] The gas used to generate the cold plasma 31 can be of any type, and in particular a noble gas, such as helium or argon, or even air. The air, injected into the generation device 30 to form the cold plasma 31 and / or through which the cold plasma 31 passes before reaching the solution 10, will be ionized by the latter and provide oxygenated species necessary for the crosslinking of said at least one crosslinkable polymer 11. This ionization also makes it possible to provide other species among the RONS, for various applications.

[0055] According to one embodiment, oxygenated species are selected from the group consisting of O2, 1 U2, H2O2, O3, O2*' and OH. The method according to the invention advantageously allows obtaining all of these species, due to the passage of the cold plasma 31 through the air present between the generation device 30 and the crosslinkable polymer 11, and increases their presence if the gas used to generate the cold plasma 31 is also air. Thus, there is no restriction as to the oxidase that can be used since its oxygenated substrate will certainly be present. Thus, in the case of a laccase or a tyrosinase, the 1 O2 and O2 will be present; in the context of a peroxidase and a catalase, H2O2 will be present. The invention thus simplifies the choice of the oxidase to obtain the best desired properties of the hydrogel, and allows, with the same process, the modification of the oxidase to obtain different properties.

[0056] According to one embodiment of the invention, the gas injected into the generation device 30 is air with a humidity level greater than or equal to 80%, for example greater than or equal to 90%, preferably the air is saturated with water vapor.

[0057] According to an alternative or complementary embodiment, the gas injected into the generation device 30 is air containing dihydrogen.

[0058] The fact that the airflow used to form the cold plasma 31 has a high humidity level (greater than or equal to 80%) and / or contains dihydrogen significantly increases the quantity and diversity of rons generated compared to other gases, particularly noble gases. Indeed, when a noble gas is used, only the air through which the plasma passes allows the formation of rons, which de facto reduces the quantity that can be produced. A high humidity level compared to air with low water saturation (humidity level below 80%) provides an overabundance of water molecules, which allows for better production of H2O2 molecules. Similarly, the presence of dihydrogen molecules also allows for better production of H2O2 molecules.Furthermore, a high humidity level helps to at least partially compensate for the water loss that can occur when the cold plasma 31 is applied to the solution 10, preventing at least partial drying of the hydrogel 10' and better preserving the desired viscoelastic properties. This embodiment is particularly useful for manufacturing hydrogel 10' for use as injectable therapeutic active hydrogels, especially in cancer treatment.

[0059] To achieve saturation and a high humidity level, the injection system 40 further includes a supplementary supply system 41 delivering a water flow into which the air flow supplied by the main supply system 42 is injected. Thus, the air flow delivered by the main supply system 42 is projected against the water flow delivered by the supplementary supply system 41, thereby achieving a high humidity level in the air. For this purpose, the flow rate of the supplementary supply system 41 is lower than the flow rate of the main supply system 42. In this context, the flow rate supplied by the supplementary supply system 41 can be from 1 to 10 ml / hour, in particular from 1 to 5 ml / hour, and especially from 2 to 4 ml / hour, for example, 2 or 3 ml / hour. The flow rate supplied by the main supply system 42 can be from 2 to 15 l / min, in particular from 3 to 12 l / min, in particular from 5 to 10 l / min, for example from 7 l / min.The airflow with a high humidity level injected by the injection system 40 is, in particular, at ambient temperature. In order to obtain an airflow containing dihydrogen, the supplementary supply system 41 can, in particular, deliver a dihydrogen flow into which the airflow supplied by the main supply system 42 is injected. Thus, the airflow delivered by the main supply system 42 is projected against the dihydrogen flow delivered by the supplementary supply system 41. In particular, the flow rate of the dihydrogen supply system 41 is lower than the flow rate of the main supply system 42. In particular, the flow rate of the main supply system 42 is 2 to 10 times greater than that of the flow rate of the supplementary supply system 41, in particular 3 to 7 times, in particular 4 to 6 times, for example 5 times.The flow rate supplied by the main supply system 42 can be from 2 to 15 l / min, in particular from 3 to 12 l / min, in particular from 5 to 10 l / min, for example from 7 l / min. The airflow containing dihydrogen injected by the injection system 40 is, in particular, at ambient temperature.

[0060] According to an embodiment not shown, the injection system 40 may include several supplementary feed systems 41, each delivering a water flow or a dihydrogen flow, in order to obtain a combination of high humidity and the presence of dihydrogen in the airflow delivered to the cold plasma generation device 31. The supplementary feed system(s) 41 and the main feed system 42 each include a pumping system. This pumping system may include any type of pump capable of providing a constant flow rate. In particular, the pumping system may include a pneumatic diaphragm pump, a diaphragm pump, a piston pump, a centrifugal pump, a bellows pump, an electromagnetic pump, or a peristaltic pump.

[0061] The chamber 32 of the cold plasma generator is in particular made in an insulating tube, for example of quartz, and includes in particular a gas-flow dielectric barrier discharge (DBD) 33. Any type of gas-flow dielectric barrier discharge 33 can be used, in particular a single-electrode configuration or a two-electrode configuration, and in particular a plasma gun DBD, a Tesla plasma jet DBD or a DBD with planar electrodes.

[0062] Figure 1 shows a DBD 33 with a plasma gun. This DBD 33 includes two electrodes 34, 35.

[0063] The dielectric barrier discharge is connected to a high voltage AC electric generator 50.

[0064] According to one embodiment, the electric generator 50 can supply a voltage from 10 kV to 30 kV, in particular from 11 kV to 25 kV, in particular from 13 to 20 kV, more particularly from 15 to 19 kV, for example 18 kV.

[0065] According to one embodiment of the invention, the frequency of the electric generator 50 can in particular be from 10 to 30 kHz, in particular from 15 to 25 kHz, in particular from 17 to 22 kHz, for example 20 kHz.

[0066] The generation device 30 can be arranged so that the visible tip of the cold plasma 31 is positioned at a distance from the solution 10, in order to avoid damaging the forming hydrogel 10'. In particular, the generation device 30 is arranged so that the visible tip of the cold plasma 31 is positioned at a distance of 0.5 to 10 cm from the solution 10, specifically 1 to 5 cm.

[0067] To ensure better homogenization of the crosslinking and improved trapping of RONs within the hydrogel, step b) can be carried out under agitation. Indeed, due to the positioning of the cold plasma 31 opposite the solution 10, a surface layer will initially receive the RONs (including H2O2), triggering the crosslinking of the polymers 11 present at this level. This crosslinking of the surface layer can act as a shield, preventing the RONs from penetrating the solution 10. Therefore, agitation helps prevent the formation of such a barrier by renewing the polymers 11 exposed to the cold plasma 31.

[0068] Agitation can be achieved by any means. In particular, agitation is carried out using a magnetic or mechanical device. Specifically, agitation is achieved using a magnetic bar 61 set in motion by a magnetization system 60. The magnetic bar 61 can be rotated at a speed of 500 to 10,000 rpm, in particular from 1,000 to 5,000 rpm, in particular from 1,200 to 2,000 rpm, for example, 1,500 rpm. Mechanical agitation can also be achieved using stirring blades, a vortex mixer, or a homogenizer at the same speeds as those mentioned for the magnetic bar. The duration of step b) will influence the degree of crosslinking of the hydrogel and the amount of RONs trapped within it. This duration is adjusted according to the type of crosslinkable polymer(s) 11 present in the solution 10 and the desired application of the hydrogel 10'.According to one embodiment of the invention, step b) lasts from 1 to 30 minutes, in particular from 1 to 20 minutes, in particular from 2 to 15 minutes, in particular from 3 to 10 minutes, in particular from 4 to 8 minutes.

[0069] According to a preferred embodiment of the invention, the process is a 3D printing process.

[0070] According to a preferred embodiment of the invention, the crosslinking of at least one crosslinkable polymer 11 following step b) is not complete. In particular, the crosslinking of at least one crosslinkable polymer 11 is adapted to obtain an activated solution. This aspect of the invention makes it possible to obtain a handleable hydrogel within a conduit, the crosslinking of which of at least one crosslinkable polymer 11 can be continued, and in particular completed, at another time and in particular at another location, depending on the desired application of the hydrogel.

[0071] According to one embodiment of the invention, the process comprises the successive addition of glucose oxidase and glucose. This aspect makes it possible to intrinsically increase the amount of hhCh present in the hydrogel 10', a product of the reaction of glucose oxidase with glucose, and also to obtain improved rigidity of the hydrogel, depending on the requirements. To this end, step a) may include the addition of glucose to solution 10 and step b) may subsequently include the addition of glucose oxidase after the use of cold plasma. Conversely, step a) may include the addition of glucose oxidase to solution 10 and step b) may subsequently include the addition of glucose after the use of cold plasma. The invention also relates to a hydrogel 10' obtained by the process as defined above. Said hydrogel 10' comprises within its structure oxygenated species originating from the application of cold plasma, in particular selected from O2, 1 C>2, H2O2, O3, O2*' and OH.

[0072] These oxygenated species are in particular distributed homogeneously in the hydrogel.

[0073] In particular, hydrogel is an activated solution.

[0074] The hydrogel includes in particular 0.2 to 6% of crosslinked polymers by weight per volume of solution (w / v), in particular 0.1 to 4%, in particular 0.1 to 3%, in particular 0.2 to 4%, in particular 0.2 to 3%, in particular 0.2 to 2%, in particular 0.2 to 1%, in particular 0.2 to 1%, in particular 0.2 to 0.8% w / v, in particular 0.3 to 0.7% of crosslinked polymers w / v, for example 0.5% of crosslinked polymers w / v.

[0075] In particular, the hydrogel exhibits viscoelastic properties enabling it to be injected using an injection device, notably a device of the pneumatic extrusion injection type or a laser-assisted injection type. The hydrogel suitable for this aspect of the invention is in particular an activated solution.

[0076] Hydrogel can be used in many applications, such as tissue engineering (bone, cartilage, tendons, muscles, vessels, skin, etc.), regenerative medicine (organ, etc.), drug transport (vectorization of therapeutic assets), proteins and cells, and therapy.

[0077] The hydrogel according to the invention, due to the quantity of RONS that can be incorporated into it, finds a particular application in therapeutic treatment. Indeed, RONS are known to generate significant DNA breaks in cancer cells, triggering their apoptosis.

[0078] Thus, the invention also relates to a hydrogel as defined above for its use in treating a disease in a subject, the hydrogel being administered to the subject in an effective quantity.

[0079] In the context of the invention, a "subject" preferably refers to a mammal, and more particularly to a human being.

[0080] The term "disease" refers to changes in cells, tissues, organs, or organisms compared to normal (healthy) cells, tissues, organs, or organisms. In some cases, physiological functions related to the natural functioning of organs, homeostasis, aging, or regeneration may be altered, such as abnormal organ development, inflammatory diseases, autoimmune diseases, chronic diseases, infectious diseases, or cancer. An "effective amount" or "effective dose," as used here, is an amount that produces the desired effect. For therapeutic purposes, an effective amount is an amount sufficient to achieve a beneficial or desired clinical outcome.The preferred effective amount for a given application can be readily determined by a person skilled in the art by taking into account, for example, the size, age, and weight of the subject, the type of disease or disorder to be prevented or treated, and the time elapsed since the onset of the disease or disorder. In the context of the present invention, an effective amount is an amount sufficient to induce apoptosis of the cells.

[0081] In particular, the hydrogel according to the invention is used in a curative treatment setting by triggering apoptosis, notably to combat cancer. The term "cancer," as used here, refers to a malignant neoplasm. More specifically, the term "cancer" here refers to any member of a class of diseases or disorders characterized by uncontrolled cell division and the ability of these cells to invade other tissues, either by direct growth in adjacent tissues (invasion) or by implantation in distant sites (metastasis). Metastasis is defined as the stage at which cancer cells are transported by the bloodstream or the lymphatic system.The term "cancer" preferably refers to solid cancer and includes, among others, esophageal cancer, stomach cancer, duodenal cancer, small bowel cancer, appendix cancer, large bowel cancer, colon cancer, rectal cancer, colorectal cancer, anal cancer, pancreatic cancer, liver cancer, gallbladder cancer, spleen cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, endometrial cancer, ovarian cancer, vaginal cancer, vulvar cancer, breast cancer, lung cancer, thyroid cancer, thymus cancer, brain cancer, nervous system cancer, gliomas, oral cavity cancer, skin cancer, blood cancer, lymphomas, eye cancer, bone cancer, muscle cancer, etc. In particular, the cancer is prostate cancer.

[0082] Materials and Methods

[0083] Solutions

[0084] Three polymer solutions were tested:

[0085] an alginate solution (ALG) (Sigma Aldrich (W201501)),

[0086] a chitosan (CS) solution (chitosan CS5 (GBS022021401) supplied by Chibio Biotech)), and a polyglucuronic acid (PGU) solution (PGU-Biopi supplied by BIOPI laboratory, UPJV, Amiens).

[0087] Phenolization of polymers

[0088] The success of the synthesis of a phenolized polymer was evaluated by UV-visible absorption spectrophotometer (JASCO V-630) and by FTIR infrared spectroscopy (Thermo Ficher, Nicolet iS5 EN 60825-1).

[0089] To phenolize 5 g of ALG polymer and PGU polymer, a 4-morpholinoethanesulfonic acid (MES) buffer (Sigma Aldrich, M3671) was prepared in 500 mL by dissolving 10.65 g of MES. Once fully dissolved, the pH of the buffer was adjusted to 6 using 1 M sodium hydroxide (NaOH). The 5 g of polymer were then dissolved in the MES buffer under stirring at 350 rpm and at room temperature until complete dissolution of the polymer. Next, 1.4185 g of carbodiimide (Sigma Aldrich, E7750) was added, followed after 5 minutes by 426.5 mg of NHS (N-Hydroxysuccinimide) (Sigma Aldrich, 130672), and after another 5 minutes, 3 g of tyramine (Sigma Aldrich, T2879) to initiate the phenolization reaction under stirring at 350 rpm at room temperature for 20 to 24 hours. After 24 hours, the pH was stable around 6, and the reaction was then stopped by adjusting the pH to 8.6 using 5 M or 10 M NaOH.The phenolized polymer solution was then precipitated in 1500 mL of acetone (three times the volume of the solution) and stirred for approximately 10 minutes at room temperature to begin washing. The precipitate was then filtered. Next, the precipitate was washed in 300 mL of 80% ethanol (EtOH) with stirring for 10 minutes, and then filtered again. This step was repeated four times. At the end of the EtOH wash, the product was washed a final time in 250 mL of acetone with stirring for 15 minutes at room temperature, and then filtered. The product was then oven-dried overnight at 50°C and subsequently ground into a powder.

[0090] The phenolization process for CS was similar to that used with ALG and PGU, except that tyramine was replaced by 3-(4-hydroxyphenyl)propionic acid (H PP) (Sigma Aldrich, H52406) because CS was phenolized at its amine (NH2) group. During the washing step, filtration was replaced by a centrifugation step at 10,000 g at 4°C due to the low molar mass of CS, which results in the formation of small precipitates dispersed in solution.

[0091] Enzymatic polymerization by HRP

[0092] Polymer crosslinking was performed using horseradish peroxidase (HRP) (Sigma Aldrich®), added to the starting solutions at a concentration of 20 U / ml of solution. Cold plasma generation device

[0093] Cold plasma was produced by a plasma gun-type plasma generator. The plasma gun was equipped with an internal electrode (1 mm diameter) subjected to a high, quasi-sinusoidal alternating voltage (RLC Electronic®, NanoGen®) of a few kilovolts, directly in contact with the airflow, and an external annular electrode connected to ground. These two electrodes were separated by a 1 cm diameter quartz insulating tube. The circulating gas was an airflow supplied by an air pump (HAILEA model ACO-208). A low-flow water supply system was connected to the airflow system. The electrical power supplied to the plasma generator was calculated from current (Stangenes 0.5-1.0 probe) and voltage (TESTEC HVP-2739 probe) measurements displayed on an oscilloscope (Tektronix 2 series MSO (200 MHz)).

[0094] Cold plasma treatment

[0095] 500 µl of different phenolic polymer solutions (ALG-ph, PGU-ph, and CS-ph) at 0.5% w / v were treated with air plasma for 4, 8, and 10 minutes on a 24-well microplate with magnetic stirring at 1500 rpm at 25 °C. The tip of the plasma device was positioned 1 cm above the microplate. The air plasma generation parameters included a voltage of 18 kV, a frequency of 20 kHz, an air flow rate of 7 L / min, and a water flow rate of 3 mL / hour.

[0096] Rheological analysis

[0097] The viscosity of the different hydrogels was analyzed as a function of the shear rate and the time required for the hydrogel to reach complete gelation. The hydrogel was subjected to a 10% shear stress and a frequency of 0.1 Hz using the AR G2 rheometer (TA Instruments). For a hydrogel containing a given w / v percentage of a polymer, its viscosity varies with the H₂O₂ concentration and therefore with the percentage of bonds formed in the gel. The time-scanning test was used to determine the structural changes of the hydrogel over a specific period. The gelation time is defined as the point at which the storage moduli (G) cross over. 1and loss (G"). Rheological analyses were performed at 4°C, 25°C, and 37°C. These analyses were conducted after a 30-minute waiting period following the discontinuation of cold plasma treatment. During this waiting period, the hydrogels were placed in 5 mL Eppendorf tubes to limit water evaporation and thus avoid altering their behavior.

[0098] Statistical analysis

[0099] Statistical analyses were performed using analysis of variance (ANOVA) with 95% confidence, and Tukey's post-test using the online tool "Statistics Kingdom". Results

[0100] Cold plasma treatment enabled crosslinking of the three different polymers tested (Figure 2). PGU-ph was the polymer that achieved the fastest crosslinking, at just 4 minutes. The other two polymers required a crosslinking time of 10 minutes.

[0101] The viscosity of PGU-ph hydrogels treated for 4 and 8 minutes was analyzed using a rheometer. The PGU-ph hydrogel treated for 8 minutes exhibited a yellowish color compared to the one treated for 4 minutes, which was lighter in color. This color change is due to a greater amount of ROS stored in the hydrogel. The results of the rheological analyses are shown in Figure 3. Figure 4 shows the results obtained for a comparative PGU-ph produced by a cold plasma-free manufacturing process where H₂O₂ was directly added to the solution at a concentration of 50 pM. The results in these figures show that the viscosity of the hydrogels is the same as that obtained with the comparative hydrogels, with a value of 100 (Pa / s) that decreases with increasing shear rate.However, the viscosity is higher for the hydrogel according to the invention treated for 8 minutes, demonstrating that the high amount of ROS present in the latter makes its structure more rigid.

[0102] The pseudo-plastic behavior of PGU-ph hydrogels according to the invention has been confirmed by the Ostwald de Waele mathematical model (Table 1, n = flow behavior index and k = consistency index).

[0103] Table 1

[0104]

[0105] The characteristics of the hydrogel obtained at 37°C appear suitable for application in therapeutic treatment.

Claims

Demands 1. A process for manufacturing a hydrogel (10') from at least one crosslinkable polymer (11), the process comprising the following steps: a) provide a solution (10) comprising at least one crosslinkable polymer (11) and an oxidase (13), b) crosslink said at least one crosslinkable polymer (11) by the oxidase (13) using the addition of at least one oxygenated species to obtain a hydrogel (10'), wherein the addition of said at least one oxygenated species in step b) is provided by a cold plasma (31) generation device (30).

2. Method for manufacturing a hydrogel (10') according to claim 1, wherein step b) is carried out under agitation.

3. Method of manufacturing a hydrogel (10') according to claim 1 or 2, in which the cold plasma (31) is generated from an air stream having a humidity level greater than or equal to 80% and / or from an air stream comprising dihydrogen.

4. A method for manufacturing a hydrogel (10') according to any one of claims 1 to 3, wherein the oxygenated species is selected from the group consisting of O2, 1 O2, H2O2, O3, O2-- and -OH.

5. Method of manufacturing a hydrogel (10') according to any one of claims 1 to 4, wherein the or at least one of the crosslinkable polymers (11) is a polysaccharide, alone or in combination with one or more thermosensitive crosslinkable polymers (11), preferably in combination with one or more thermosensitive crosslinkable polymers (11) at body temperature.

6. A method for manufacturing a hydrogel (10') according to any one of claims 1 to 5, wherein the oxidase (13) is selected from the group consisting of an oxidoreductase, a peroxidase, a catalase, a laccase, a tyrosinase, a monosaccharide oxidase and a mixture thereof, preferably the oxidase (13) is horseradish peroxidase.

7. Method for manufacturing a hydrogel (10') according to any one of claims 1 to 6, said method being a 3D printing method.

8. Bioactive hydrogel (10') obtained by the process according to any one of claims 1 to 7.

9. Hydrogel according to claim 8, said hydrogel being an activated solution.

10. Hydrogel (10') according to claim 8 or 9 for its use in a method of therapeutic treatment of a disease, preferably said disease being cancer.

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