Method for integrating nanobubbles and nanodrops of oxygen into hydrogels for biological and regenerative medicine applications

Abstract

The invention relates to a method for stably incorporating nanobubbles and / or nanodrops of oxygen into hydrogels for biological and regenerative medicine applications, wherein gas micro / nanobubbles (MNBs) and / or micro / nanodrops (MNDs) are formed using compression and expansion cycles in a closed system, by means of cavitation by vacuum degassing before polymerising the hydrogel. The invention also relates to the hydrogel formed and the use thereof: as a biological substrate for tissue regeneration / generation; as a biological matrix as a cell substrate for cell invasion; to supply oxygen and, optionally, active components such as drugs, growth and / or migration factors, antibodies, peptides, vesicles, cellular derivatives and / or oligonucleotides, to tissues.

Description

[0001] METHOD FOR THE INTEGRATION OF NANOBUBBLES AND NANODROPS OF OXYGEN IN HYDROGELS FOR BIOLOGICAL AND REGENERATIVE MEDICINE APPLICATIONS.

[0002] Descriptive Memory

[0003] TECHNICAL FIELD

[0004] The invention relates to the field of hydrogels, specifically hydrogels suitable for biological and regenerative medicine applications. For example, hydrogels that serve as scaffolds for living cells, such as mesenchymal stem cells, can be used in tissue regeneration, for example. The invention allows for the production of porous hydrogels with a supply of gases, such as oxygen, in the form of micro / nanobubbles or micro / nanodroplets within the hydrogel, where the gas can be transferred to the cells contained within said hydrogel or scaffold.

[0005] BACKGROUND OF THE INVENTION

[0006] The replacement of damaged tissues and organs through regenerative medicine and tissue engineering is considered the best treatment for many chronic diseases or critically ill injuries. In the context of the shortage of organ and tissue donors, the creation of new functional tissues or organs is a priority for public health. Tissue engineering has emerged as an ideal technology for this effort. Simply put, tissue engineering provides a matrix or scaffold with healthy cells and inserts them into the tissue to be recovered, allowing these new cells to regenerate the damaged tissue.

[0007] Tissue engineering must consider several transcendental factors for its use in therapy, highlighting the role of scaffold selection and other biological components capable of mimicking the cells' native environment and replacing damaged tissue with the highest fidelity or bioinspiration. In this context, scaffold design must consider both the physical and biological aspects of tissues for appropriate signaling toward cellular maintenance, differentiation, and tissue regeneration. Regarding physical aspects, we highlight porosity and stiffness as aspects that contribute to tissue mechanobiology.Regarding biological factors, biocompatibility, cell infiltration, cell adhesion, encapsulation or binding of growth factors, and the incorporation of helper cells can also be modulated to improve the performance of biomaterials to repair, replace, or induce regeneration of injured tissues and organs.

[0008] Scaffolds are three-dimensional arrays of biomaterials capable of mimicking the native extracellular matrix (ECM) and facilitating cell expansion and / or the patterning or arrangement of different cell types, matrix deposition, and tissue production. In this regard, one of the most promising biomaterials is hydrogels. They are composed of a hydrophilic polymer network capable of absorbing water molecules through hydrogen bonding, polar, and ionic interactions. Hydrogels can mimic the ECM and be used in biomedical applications such as drug delivery and tissue engineering.

[0009] In some tissue engineering applications, hydrogels are formed from a liquid composition of polymers and cells suspended in solution. This pre-composition, or pre-gel, is cross-linked through irreversible mechanisms such as exposure to UV light, generating cross-linking of the matrix through free radical polymerization (FRP). However, the inventors noted that FRP-polymerized hydrogel scaffolds generate an extremely hypoxic environment, close to anoxia. This could compromise cell viability, proliferation, mitochondrial function, and other regenerative functions, in addition to the risk of cell death during the avascular phase of biomaterial implantation, mainly in the central zone of the hydrogel.

[0010] Delivering oxygen effectively in tissue engineering can be challenging due to limitations of the scaffold and 5) biodegradation rates. Studies have shown that oxygen delivery occurs through passive diffusion from the surrounding environment, leading to insufficient oxygen supply into deeper tissue layers, resulting in cell death and compromised tissue functionality. Furthermore, oxygen diffusion within implanted tissues is limited to approximately ~200 pm [Jain, R et al: Nat Biotechnol 2005, 23, 821-823], resulting in severe oxygen deficiencies in the core of implanted tissues. Therefore, vascularization of the implant is essential to overcome limitations in oxygen diffusion, but this is a slow process, with only 5 mm of vascular penetration in 25 days for implanted porous scaffolds [Mikos, A. G et al: Biotechnology and Bioengineering 1993, 42(6), 716-723], during which period implant viability must be maintained.Cell survival during the early stages of implantation, especially during the prevascular stage, is a major challenge in translational applications of tissue engineering (TE) and 3D bioprinted tissues. Therefore, supplementing oxygen (O2) to transplanted cells through an O2 delivery source such as micro / nanobubbles (MNBs) and / or micro / nanodroplets (MNDs), capable of acting as a reservoir of therapeutic agents and oxygen molecules, is a promising approach to overcome low oxygen levels, mitigate hypoxia-related cell death, and improve cell viability.

[0011] In recent years, several materials have been developed that offer gradual and constant release of oxygen over time [Oh, S. H et al: Biomaterials 2009, 30(5), 757-762.; Gholipourmalekabadi, M., et al: Trends in biotechnology 2016, 34(12), 1010-1021], Although oxygen-releasing biomaterials (OLBs) can help in improving oxygen availability, developing efficient and long-lasting oxygen-releasing materials remains a complex task. Finding the right balance between sustained oxygen release and biocompatibility is a significant challenge.

[0012] To solve this technical problem, the present invention combines the use of micro / nanobubbles and micro / nanodroplets in synthetic or natural biopolymers, preferably in hydrogels generated by crosslinking the matrix through free radical polymerization, such as Methacrylated Salmon Gelatin (sGelMA) hydrogels. In this way, a new BLO formulation is provided capable of mitigating hypoxic stress in cells immobilized in hydrogels, an aspect that has not been sufficiently evaluated until now and currently constitutes a technical limitation in tissue engineering. This problem is especially magnified in hydrogels formed by free radical polymerization, since, as indicated, periods of hypoxia / anoxia are generated when the hydrogel is exposed to UV light and the polymerization process is activated.

[0013] This novel integration of biomaterials with oxygen micro / nanobubbles and micro / nanodroplets allows polymers to be provided with a hybrid composition for oxygen delivery and pore generation in constructs (hydrogels). DESCRIPTION OF THE FIGURES

[0014] FIG. 1 shows the result of the formation of MNBs in culture medium in the presence VC_L1 and absence VC_L0 of surfactant, as explained in example 1. In the Normoxia condition, the amount of nanobubbles per mL, and the Z potential of both compositions according to the invention are graphed. In the Hypoxia condition, a diagram is seen showing the characterization of the composition of the invention (ViaCox_Liq uid ) according to ISO / TC 281 regulations and its oxygen levels during 4 hours of hypoxia. Significance p < 0.05 (One-way Anova) Three independent experiments in triplicate n = 3. VC_L0 (medium + oxygen MNBs). VC_L1 (medium + oxygen MNBs-i- surfactant).

[0015] FIG. 2 is a diagram showing dissolved oxygen levels in UV-polymerized free radical hydrogels and the ability of the inventive composition to mitigate or eliminate anoxia in these hydrogels after polymerization. Ctrl Gel (control hydrogel), VC_Gell (Formulation with MNBs + Surfactant). VC_Gel2 (Formulation with MNBs + Surfactant + Oxygen MNDs), 02 Sat Gel (oxygen-saturated control hydrogel).

[0016] FIG. 3 is a diagram showing the oxygen transfer coefficients in hydrogels developed with the composition of the present invention, showing greater release and rapid transfer of oxygen compared to the control. Significance p<0.05 (unpaired t-test, One-way Anova). Three independent experiments in triplicate n=3. Control_Gel (Control Hydrogel). ViaCox_Gell (formulation with oxygen MNBs + surfactant).

[0017] FIG. 4 is a diagram showing the oxygen transfer coefficients in hydrogels developed with the composition of the present invention, showing differential coefficients in oxygen transfer (hydrogels with rapid release or hydrogels with more controlled oxygen release. Significance p<0.05 (unpaired t-test, One-way Anova). Three independent experiments in triplicate n=3. Control_Gel (control hydrogel). ViaCox_Gell (formulation with oxygen MNBs + surfactant). ViaCox Gel2 (formulation with oxygen MNBs + surfactant + oxygen MNDs + PFC [1% v / v]). FIG. 5 is a graph showing the porosity in hydrogels made with the composition of the present invention. Significance p<0.05 (One-way Anova). Three independent experiments in triplicate n=3. Ctr_Gel (Control hydrogel). ViaCox Gel2 (Hydrogel (10, 25, 35 % v / v) + MNBs + surfactant + MNDs).

[0018] FIG. 6 is a diagram showing the mobility of albumin conjugated to the anionic fluorophore FITC as an indirect diffusivity marker in hydrogels made with the composition of the present invention. Significance p<0.05 (unpaired t-test, one-way ANOVA). Three independent experiments in triplicate n=3. Ctrl (Control gel without MNBs or MNDs). ViaCox Gel na no (nanobubble-enriched hydrogel). ViaCox Gellmicro (microbubble-enriched hydrogel).

[0019] FIG. 7 is a diagram showing the effect of the composition of the present invention on the topography and nanoroughness of hydrogels. Ctr_Gel (hydrogel only). VC_Gel2 (hydrogel with MNBs + Surfactant + MNDs).

[0020] FIG. 8 is a diagram showing improvements in MSC cell survival and their reduction in apoptosis and necrosis after prolonged hypoxia of 3 days in mesenchymal cells cultured with the composition of the present invention DMEM ctri(Half solo). VC_L 2¡io%] (Medium with MNBs+MNDs at 10% v / v). VC_L 2po%] (Medium with MNBs+MNDs at 50%).VC_L 2[ioo%](Medium with MNBs+MNDs at 100%).

[0021] FIG. 9 is a diagram showing ATP levels when culturing MSCs with the composition of the present invention. Significance p<0.05 (unpaired t-test). Three independent experiments in triplicate, n=3. Untreated (medium alone). ViaCox_Liquid (medium + oxygen MNBs + surfactant). RLU (Relative Light Unit).

[0022] FIG. 10 is a diagram showing the antioxidant action (reducing mitochondrial ROS levels) of the composition of the present invention in MSCs cells. Significance p<0.05 (One-way ANOVA). Three independent experiments. N=3. DMEM. c tri(Medium alone). Menadione (positive mitochondrial stress control). VC_L[io%] (Medium with 10% v / v MNBs). VC_L[5o%] (Medium with 50% v / v MNBs). VC_L[wo%](Medium with 100% v / v MNBs).

[0023] FIG. 11 is a graph showing that the composition of the present invention promotes the growth of MSCs cells cultured in hypoxia (l% O2) ■ Significance p<0.05 (unpaired T-test) Three independent experiments in triplicate n=3. Untreated (medium alone). ViaCox_Liquid (medium + oxygen MNBs + Surfactant).

[0024] FIG 12 is a diagram showing the increase in viability and metabolic activity of cells grown on hydrogels exposed to PRL and hypoxia. Significance p<0.05 (One Way Anova) Three independent experiments in triplicate n=3. Dead_Gel (Death control hydrogel). Ctrl_Gel (untreated hydrogel). VC_Gel2 (hydrogel with oxygen MNBs + Surfactant + oxygen MNDs).

[0025] FIG. 13 is a diagram showing the increase in proliferation of cells cultured on hydrogels. The cultured cell density for each hydrogel was 0.25, 0.5, 1, 2 x 10 6 / ml), each hydrogel was subsequently crosslinked by UV light and incubated for 3 days at 37°C under normoxic and hypoxic conditions, respectively. To determine the cell number, a standard curve was previously determined at different cell densities. Significance p<0.05 (One Way Anova) Three independent experiments in triplicate n=3. Ctrl_Gel (untreated hydrogel). VC_Gel2 (hydrogel with oxygen MNBs + Surfactant + oxygen MNDs)

[0026] GENERAL DESCRIPTION OF THE INVENTION:

[0027] The present invention relates to a composition containing micro / nanobubbles (MNBs) and / or micro / nanodroplets of gases (MNDs), such as oxygen, and their use. More specifically, the present invention relates to a composition containing MNBs and MNDs in liquid solutions and in polymer solutions; capable of forming matrices or hydrogels after a polymerization reaction. Wherein, the composition of the invention is capable of supplying gases, such as oxygen, and thus increasing the availability or solubility of this gas in both liquid fluids and hydrogels.Additionally, the composition of the invention is capable of modulating porosity, diffusivity, and mechanical properties in hydrogels. Furthermore, the composition of the invention increases oxygen solubility and availability and mitigates cellular deficiencies related to hypoxia. It also activates mitochondria contained in the hydrogel and / or in the cells within the hydrogel, thereby reducing mitochondrial stress.

[0028] In one embodiment, the invention discloses a composition for promoting the survival and growth of cultured cells under hypoxic conditions by dissolving a gas in a liquid and in matrices using MNB and MND technology in hydrogels. The invention addresses the growing demand for the development and use of compositions, methods, and products requiring MNBs and MNDs. The present invention also addresses the demand for a more efficient method for promoting oxygen-releasing biomaterials (ORBs) under hypoxic and / or anoxic conditions.

[0029] On the other hand, mitochondria are energy-producing organs, and it is believed that increasing mitochondrial activity can increase energy production and consequently increase cell proliferation. However, hydrogels that promote mitochondrial activity have not yet been developed, and there is a demand for the development of liquid and polymeric solutions that activate mitochondria and reduce hypoxic stress. The present invention addresses this deficiency, as the composition of the invention allows mitochondria to be activated within the hydrogel by maintaining normoxic conditions within the hydrogel containing the mitochondria.

[0030] According to this specification, the following invention is provided.

[0031] (1) A composition containing MNBs with a size in (a) liquid (ViaCox_Liquid) of 200-400 nm and a composition of MNBs + MNDs + biopolymers + photoinitiator in (b) hydrogels (ViaCox_Gel) of 90 microns or less. The liquid phase MNBs used contain 10-12 ppm of oxygen, while the combination of MNBs and MNDs for hydrogels contains 18-22 ppm of oxygen. Here, the gas concentration is the concentration of both the dissolved gas and the content in the MNBs and MNDs.

[0032] (2) a composition in liquids (ViaCox_L¡quid) or hydrogels (ViaCox_Gel) defined in (la) or (Ib) capable of increasing cell survival.

[0033] (3) a composition in liquids (ViaCox_Liquid) or hydrogels (ViaCox_Gel) defined in (la) or (Ib) capable of reducing cell death.

[0034] (4) a composition in liquids or hydrogels defined in (la) or (Ib) capable of increasing ATP production.

[0035] (5) a composition in liquids or hydrogels defined in (la) or (Ib) capable of reducing mitochondrial stress due to hypoxia.

[0036] (6) a hydrogel composition defined in (Ib) capable of providing a higher concentration of oxygen in matrices.

[0037] (7) a hydrogel composition defined in (Ib) capable of mitigating anoxia by free radical polymerization (FRP) during hydrogel formation.

[0038] (8) a hydrogel composition defined in (Ib) capable of providing oxygen transfer coefficients to mitigate free radical polymerization (FRP) anoxia during hydrogel formation.

[0039] (9) a hydrogel composition defined in (Ib) capable of providing differential oxygen transfer coefficients (hydrogels with rapid release or hydrogels with more controlled oxygen release) to mitigate free radical polymerization (FRP) anoxia during hydrogel formation.

[0040] (10) a hydrogel composition defined in (Ib) capable of retaining oxygen during exposure to UV light, thus preventing anoxia by free radical polymerization (FRP) during hydrogel formation.

[0041] (11) a hydrogel composition defined in (Ib) capable of generating pores in constructs (hydrogels)

[0042] (12) a hydrogel composition defined in (Ib) capable of increasing the diffusion coefficient of gases and water-soluble molecules. (13) a hydrogel composition defined in (Ib) capable of generating differential topographies with respect to the control in terms of nano-roughness.

[0043] (14) a hydrogel composition defined in (Ib) capable of improving survival in mesenchymal cells encapsulated in hydrogels and cultured in hypoxia.

[0044] (15) a hydrogel composition defined in (Ib) capable of improving proliferation in mesenchymal cells encapsulated in hydrogels and cultured in hypoxia.

[0045] Depending on the composition of the present invention used, various effects can be obtained on the properties of liquids and hydrogels formed, the composition of the invention being capable of improving oxygenation, porosity, diffusion or mechanical properties of the liquid or hydrogel. Additionally, the composition of the invention has an effect on the cellular response of cells that are in contact with said composition, by promoting metabolic activation, increasing ATP production, promoting cell proliferation and decreasing cell damage or death in seeded cells: in 2D through composition (Ia) and in 3D through composition (Ib), during hypoxic conditions.

[0046] METHODOLOGY AND IMPLEMENTATION:

[0047] The invention relates to aqueous or polymeric solutions with micro / nanobubbles (MNBs) and micro / nanodroplets of gases (MNDs), such as oxygen, capable of acting as oxygen-releasing biomaterials (ORBs), useful for use in reactions for the formation of biopolymers, hydrogels, or other matrices that act as scaffolds for living cells.

[0048] While one of the preferred embodiments is hydrogels, the scope of the present invention is not limited to these, as the technology can also be extended to different types of biopolymers, natural, synthetic, and / or combinations thereof. It can also be extended to different functionalization methods and / or polymerization strategies, whether physical, chemical, reversible, or irreversible, among others. In a preferred embodiment, the composition of the invention is used in the formation of hydrogels, and in a particularly preferred embodiment, to methacrylated hydrogels.

[0049] Regarding the use of the present invention in polymer solutions, the use of the composition formulated according to the invention to generate hydrogels with oxygen MNBs+MNDs embedded in a biological and / or synthetic matrix with different types of functionalizations and / or with different polymerization strategies is emphasized.

[0050] The composition of the invention comprises a solution comprising a polymer, either natural or synthetic, with different types of functionalizations and / or different polymerization strategies. Although the hydrogels presented by the inventors are related to methacrylated salmon gelatin (sGelMA) polymers, the scope of the present invention is not limited to this single type of hydrogel, nor to this single type of functionalization or polymerization.

[0051] The polymers of the composition of the invention may be natural or synthetic and may or may not be functionalized. Wherein the functional groups include but are not limited to amine, carboxylic acid, biotin, azide, thiol, hydrazide, alkyne, succinic ester, vinyl, folate, methacrylate, styrene, acrylonitrile, vinyl ether, vinyl l-ester, maleimide, vinyl ketone, acrylamide, isocyanate, etc. groups. The compositions with these polymers conveniently also have polymerization initiator compounds, such as photoinitiators. These can be chosen from both types of photoinitiators, classified according to the type of cleavage before UV light (Norrish type 1 and Norrish type 2). The polymerization initiator is usually an oxidizing agent. Polymerization initiators also include those that are activated by exposure to electromagnetic radiation or heat.Polymerization initiators may also be used, which are described, for example, in US patent application publication no. Q US2010 / 0137241, or any other known in the art.

[0052] The composition and general steps of the manufacturing method in polymer solutions exemplified in the present invention and the development of the formulation are described below.

[0053] In one embodiment, to obtain a composition according to the invention it is required: i) to provide a liquid composition formed by a low melting point gelatin, less than 15°C, determined by having a proline and hydroxyproline content of 18% or less with respect to the total amino acid content; which is additionally functionalized with methacryloyl or methacrylamide groups; and a photoinitiator;

[0054] ¡i) adding a surfactant (T) and a perfluorocarbon compound (PFC) directly to the composition defined in i in a proportion or ratio of between 0.001 and 0.1% v / v for the surfactant and a proportion of between 0.5 and 10% v / v for the PFC compound. i¡¡) carrying the composition defined in ¡ia to a closed tube or container (capable of isolating from atmospheric pressure) and allowing high purity oxygen to be injected at controlled pressure. iv) forming MNBs+MNDs of oxygen by means of compression and extension cycles in a closed system (vacuum degassing cavitation) obtaining MNBs+MNDs directly in the composition defined in ¡i iv) initiating the polymerization of the liquid dispersant phase by applying light radiation; and v) obtaining a hydrogel with micro / nanobubbles + micro / nanodroplets of oxygen embedded in a matrix or hydrogel.

[0055] Preferably, the invention proposes the use of fluorinated compounds such as the fluorosurfactant + liquid PFC as a liquid mixture of colloidal stability and suspended in sGelMA, both in solution and in hydrogel. In this way, we seek the integration of immiscible micro / nanoblots + micro / nanobubbles into 3D scaffolds, promoting not only the availability of oxygen in tissue engineering, but also gaining properties in porosity, a key parameter for promoting improved nutrient diffusion and gas coefficients.

[0056] The composition of the invention requires an agent capable of offering high efficiency in reducing surface tension, which is an important aspect in the field of bubble stability and bioprinting, and a relevant requirement for creating three-dimensional (3D) structures of living cells and biomaterials. Therefore, the preferred surfactant for this invention is an anionic, fluorinated, and branched surfactant, which translates to a fluorosurfactant.

[0057] In a preferred embodiment, the commercial fluoro-surfactant Tivida® FL 2300 is used, the first commercially available C2-Fluoro surfactant of the Tivida® FL technological platform, which has favorable properties not only due to its ionic and structural (branched) nature, but also due to its exceptional reduction in surface tension for a wide spectrum of liquid phases, offering several additional advantages such as the absence of oral toxicity, zero aquatic toxicity, lack of bioaccumulation.

[0058] On the other hand, the second fluorinated compound in the composition is preferably perfluorocarbon (PFC). Although PFCs have previously been recognized in biomedicine for their ability to function as potential artificial blood substitutes due to their chemical and biological inertness, ease of sterilization, and high oxygen solubility, PFCs are a type of oxygen carrier with a high oxygen mobilization capacity (20 times greater than that of water). This remarkable oxygen transport property allows PFCs to be used in artificial blood. PFCs have also been applied during tissue culture to increase the survival of mesenchymal stem cells (MSCs) under hypoxic conditions. However, PFCs alone have a short in vivo retention time due to their low molecular weight, which limits their applications in ischemic tissue regeneration, so it was not obvious to use them in the present invention.

[0059] There is very little literature available on the use of biomaterials combined or embedded with micro / nanodroplets and micro / nanobubbles, and the need for this resource is what motivated the inventors to develop the present invention.

[0060] In one embodiment, the invention consists of the development of a formulation capable of integrating MNBs+MNDs that, suspended in polymeric matrices + fluorosurfactant + liquid PFC, allow increasing the availability of oxygen in matrices to mitigate deficiencies related to hypoxia, thus improving short / moderate-term cell survival under hypoxic and / or anoxic conditions.

[0061] Specifically, both surfactants and PFC compounds are used under the conditions provided by the manufacturer, which are used in the proportions already indicated, that is, the liquid fluorosurfactant (T) is used in a proportion between 0.001 and 0.1% v / v, preferably between 0.005 and 0.05. And the liquid perfluorocarbon compound (PFC) is used in a proportion between 0.5 and 10% v / v, preferably between 1 and 5% v / v. For example, fluorinated perfluorodecalin can be used as a PFC, which the manufacturer provides as a 100% liquid solution with a density of 1.93 g / cm3. As a surfactant, Tivida FL 2300® can be used, where the manufacturer provides a stock solution at 33% w / v of the active component, anionic fluorosurfactant, and with a density of 1.05 g / cm3. This fluorosurfactate stock is prepared on the solvent l-methoxy-2-propanol.

[0062] The composition of the present invention is produced by converting dissolved oxygen gas containing micro / nanobubbles or micro / nanodroplets into liquid solutions (e.g., ultrapure water, culture medium, polymer solutions). Although the purity of the gas is not specifically limited, when used for drinking water, it is preferable to use high-purity gas.

[0063] The production of the present invention consists of a preparation method for non-polymeric and polymeric liquid solutions, containing MNBs, MNDs, or MNBs-MNDs either in colloidal suspension of liquid phases or confined within semi-solid phases such as polymeric matrices or hydrogels, capable of oxygenating, generating pores, increasing diffusivity, modulating crosslinking density, nanotopography and mechanical properties of the matrix.

[0064] In the present invention, the nanobubble generator can be used without particular limitation as long as it is a conventional nanobubble generator. For example, the present invention is obtained from a preparation method that requires a liquid fluid for use in a Venturi type hydrodynamic cavitation system (2a) or vacuum degassing cavitation (2b), the two-phase working fluid has a liquid phase and a gas phase, comprising:

[0065] • a fluid inlet;

[0066] • a fluid outlet;

[0067] • at least one gas inlet;

[0068] • and fine bubble generators or a combination of bubble generators 2a and 2b described below. 2a and 2b.

[0069] 2a. Venturi Method

[0070] • Fine bubbles can be generated in a pulsatile manner using a mechanical fluid pulsation controller to manipulate the inlet fluid or the outlet fluid of the fine bubble generating system.

[0071] • Gas and liquid are mixed in a generator component called a "Venturi tube" or valve, which includes a gas inlet, then the mixed gas-liquid flows through several loops in a Venturi valve and promotes the collapse of bubbles to form micro and nanobubbles (MNBs).

[0072] • The subsequent cavitation overflow is released into a container and recycled or recirculated back into the entire system.

[0073] • Microbubbles and nanobubbles (MNB) can be present in the fluid for different periods of time.

[0074] • This method allows working with large quantities of liquids (IL and 25L).

[0075] • Inlet water pressure can be as low as 0.08 MPa, with no additional gas required, and ambient air intake is sufficient. The number of fine bubbles can be as high as 100,000,000 per mL.

[0076] • The pressure source for the liquid can be a pump, which provides the energy needed to circulate the fluid. The operating pressure is a minimum of approximately 0.08 MPa, with a preferred range of approximately 0.2 MPa to approximately 0.5 MPa.

[0077] 2b Vacuum method or vacuum degassing

[0078] • For small volumes (less than 7 ml), the production of the compositions of the invention called ViaCox_Liquid or ViaCox Gel were preferably generated by the vacuum degassing method, from standardized parameters such as compression and extension cycles (60 cycles), and fluoro-surfactant (Tivida® 0.02%).

[0079] • A syringe is filled with pure water or other liquid and, after expelling any trapped air inside, the syringe tip is sealed with a Luer lock cap.

[0080] • Water or other liquid is subjected to a vacuum inside a syringe at a constant temperature of 20°C, creating a gas desaturation which, when followed by the release of the vacuum, leads to the formation of micro / nanobubbles (MNB) and micro / nanodroplets (MND).

[0081] • The water or other liquid is then depressurized by a rapid pull of the syringe plunger and then repressurized by the instantaneous release of the plunger, which travels at a relatively high velocity under the action of vacuum pressure (typically ~7 cm s-1). This is relevant for biomedical and in vitro research applications. For example, the generation of MNBs+MNDs for engineering oxygen-releasing matrices, especially for injectable and porous bio-inks. This bimodal platform of promoting oxygenated and porous hydrogels is primarily feasible through this invention, promoting enhanced diffusivity and modulation of surface nanotopography and mechanical properties of hydrogels.

[0082] • Regarding the advantages of this method are the fact that it shortens its production times, the versatility to work with different biopolymers and surfactants at small, low and moderate volumes, and it is also a clean, scalable, automatable and efficient method in terms of quantity, quality and stability of the MNBs+MNDs.

[0083] Thus, the invention relates to a method for stably incorporating nanobubbles and / or nanodroplets of gases into aqueous solutions for biological and regenerative medicine applications, which comprises the following steps: i) Providing an aqueous liquid composition of functionalized or non-functionalized natural or synthetic polymers, biologically compatible, and a polymerization initiator compound;

[0084] (i) adding directly to the composition defined in (a) or (b): a liquid fluorosurfactant (T) in a proportion between 0.001 and 0.1% v / v; and a liquid perfluorocarbon compound (PFC) in a proportion between 0.5 and 10% v / v; iii) carrying the composition obtained in (i) to a closed tube or container (capable of isolating from atmospheric pressure) and allowing oxygen to be injected at controlled pressure; iv) forming micro / nanobubbles (MNBs) and / or micro / nanodroplets (MNDs) of gas by means of compression and extension cycles in a closed system, by vacuum degassing cavitation obtaining MNBs and MNDs directly in the composition defined in (i); (v) obtaining an aqueous polymer solution with stable micro / nanobubbles and / or micro / nanodroplets of oxygen; vi) initiate polymerization of the solution either by reversible (physical) polymerization and / or by irreversible (chemical or photochemical) polymerization;and obtain a hydrogel with stable micro / nanobubbles and / or micro / nanodroplets of oxygen embedded in a matrix or hydrogel, providing porosity and oxygen availability within the hydrogel.

[0085] Where the anionic surfactant is a branched fluorosurfactant; and the oxygen is high-purity oxygen.

[0086] The liquid composition additionally comprises active compounds such as drugs, growth and / or migration factors, antibodies, peptides, vesicles, cell derivatives, and / or oligonucleotides; and / or additionally comprises living components such as cells or organelles, where the organelle is an active mitochondria.

[0087] In one embodiment, the liquid polymeric composition is a low-melting point gelatin, less than 15°C, determined by having a proline and hydroxyproline content of 18% or less with respect to the total amino acids, which is functionalized with methacryloyl or methacrylamide groups, in a proportion of 30% to 100% of lysine residues of the amino acid chain of said gelatin. Specifically, the low-melting point gelatin is derived from organisms of the genus Salmo or Oncorhynchus.

[0088] Where the polymerization of step vi) is initiated by UV radiation in the presence of a photoinitiator at a concentration between 0.01% and 5% (w / v). In a second aspect, the invention relates to the hydrogel formed according to the method described above, which is formed of gelatin polymerized with oxygen nanobubbles at a concentration of 10 6 to 10 9 micro / nanobubbles (MNBs) and / or micro / nanodroplets (MNDs) per cm 3, which gives it porosity and oxygen availability. Conveniently, the oxygen is high-purity oxygen.

[0089] This hydrogel of the invention additionally comprises active compounds such as drugs, growth and / or migration factors, antibodies, peptides, vesicles, cell derivatives, and / or oligonucleotides; and / or additionally comprises living components such as cells or organelles. Where the organelle is an active mitochondria.

[0090] In a third aspect, the invention relates to the use of the hydrogel as a biological support for tissue regeneration / generation. Where tissue regeneration is in a joint injury, cartilage injury, bone injury, ulcer injury, ligament injury, organ injury, spinal cord injury, or soft tissue injury. Or conveniently, tissue regeneration is in the reconstruction of skin tissue, muscle tissue, soft tissue, or post-surgical replacement tissue.

[0091] In a further aspect, the invention aims at the use of the hydrogel as a biological matrix as a support for cells, for cell invasion.

[0092] The invention is also useful in cases of blood perfusion blockage or trauma involving inflammation and / or ischemia of skin tissue, musculoskeletal tissue, vascular tissue, nervous tissue, brain tissue, spinal cord tissue, liver tissue, pancreatic tissue, intestinal tissue, lung tissue, or cardiac tissue, to deliver oxygen and active components. For example, the hydrogel of the invention can be placed, for example during a surgical procedure, over tissue damaged by blood perfusion blockage, so as to rapidly deliver oxygen and active components for tissue preservation.

[0093] Finally, the invention also relates to the use of the hydrogel as an acellular biological matrix, a biological matrix as a mechanical support, or a biological matrix for active components. The present invention will now be described with reference to examples, but the scope of the present invention is not limited by these examples.

[0094] EXAMPLES

[0095] Example 0.

[0096] Obtaining composition for hydrogel.

[0097] First, purified and filtered salmon gelatin was obtained, which was then dried and ground.

[0098] Ground salmon gelatin was dissolved to a desired final concentration of 10–35% (w / v) in PBS IX (pH 7.4) at 60 °C. Once completely dissolved, methacrylic anhydride (276685, Sigma, USA) was slowly added while still stirring to a final concentration of 8% (v / v). Different levels of methacryloyl and / or methacrylamide functionalization require different concentrations of methacrylic anhydride. After 3 h of reaction, a 5X dilution in PBS IX was made and the reacted gelatin was dialyzed against deionized water at 40 °C for 1 week. Daily replacements of fresh deionized water were performed to remove any unreacted methacrylic anhydride during dialysis. Finally, the dialyzed mixture was filtered with 20 pm porous filter paper, lyophilized and stored at -20 °C for later use.

[0099] This process produces the base biomaterial, methacrylated salmon gelatin (sGelMA), at different concentrations. This is then combined with a photoinitiator, such as Irgacure® 2959 [BASF Resins], preferably at a concentration of 0.01% to 5% (w / v), which is activated with UV light to form a hydrogel. The photoinitiator concentration used was 0.5% w / v.

[0100] For the validation of the composition of the invention ViaCox Liquid, or ViaCox_L, different conditions were evaluated in the following examples, which have the following components:

[0101] Control Liquid: ultrapure water, PBS or culture medium

[0102] ViaCox_L¡quid_0: ultrapure water, PBS or culture medium + uncoated oxygen MNBs.

[0103] ViaCox_Liquid_l: ultrapure water, PBS or culture medium + surfactant-coated oxygen MNBs

[0104] ViaCox_Liquid_2: Ultrapure water, PBS or culture medium + surfactant-coated oxygen MNBs + oxygen and PFC MNDs [1-5%].

[0105] (PBS: Phosphate Buffered Saline) For the validation of the composition of the invention ViaCox Gel, ViaCox G, different conditions were evaluated in the following examples, which have the following components:

[0106] Control: sGelMA (10% v / v) +photoinitiator 0.1%,

[0107] ViaCox_GelO: sGelMA (10% v / v), 0.1% photoinitiator, uncoated oxygen MNBs.

[0108] ViaCox_Gell: sGelMA (10% v / v), 0.1% photoinitiator, 0.02% v / v surfactant-coated oxygen MNBs.

[0109] ViaCox Gel2: sGelMA (10% v / v), 0.1% photoinitiator, 0.02% v / v surfactant-coated oxygen MNBs, PFC-oxygen MNDs [1-5% v / v]

[0110] Example 1:

[0111] The method preferably used in the present invention is based on the principle of Henry's law and the use of vacuum degassing, and is evaluated in this example.

[0112] In liquid culture media, micro / nanobubbles coded as ViaCox_L0 (or VC_L0) and ViaCox_Li (or VC_Li) were generated as defined in 2b, with the generation of MNBs being the final production process. ViaCox_L2 is described further in Example 8.

[0113] Specifically, as indicated in 2b, 5 mL of pre-oxygenated DMEM medium (for 2 minutes) was taken either oxygenated medium alone (ViaCox_L0) and / or with Tivida 0.02% v / v (ViaCox_Li ) into a syringe, once any trapped air was expelled inside, the syringe tip was sealed with a Luer lock cap. The syringe was connected to a vacuum degassing cavitation system, comprising a fluid inlet; a fluid outlet; and a gas inlet. The system has a constant temperature controlled at 20°C. Each test, ViaCox_Lo and ViaCox_Li, was subjected to 60 compression and extension cycles. In the case of ViaCox_Li, oxygen was combined with the fluoro-surfactant Tivida® at a concentration of 0.02% (v / v)

[0114] Both compositions showed the presence of billions of nanoentities per NTA and with colloidal stability favorable to remain in suspension by zeta potential.

[0115] It is observed that ViaCox_Ll is the composition with the highest production (2.0xl0 A 9 bubbles mL-1) and zeta potential (-51.9 mV) compared to the l,4xlO A 9 mL-1 bubbles and -TI , 9 mV. The results are graphed in Figure 1, for Normoxia. It is observed that the inventive method condition VC_Li forms more nanobubbles with a lower z potential.

[0116] Additionally, the effect of VC_L was tested, by favoring a higher level of oxygenation in liquids subjected to hypoxia or oxygen-deficient environments [1% O2] for 4 hours at 37°C Figure 1, for Hypoxia. This demonstrates that the nanobubbles formed are stable in a liquid solution, where the condition of the invention has a significantly greater stability in an aqueous solution, such as a culture medium.

[0117] Example 2:

[0118] Oxygen dynamics were measured simultaneously with UV polymerization of a hydrogel.

[0119] As we have indicated, it is known that UV hydrogel polymerization under normal conditions generates a highly anoxic environment. Therefore, the ability of the nanobubbles in the inventive method to remain intact during the polymerization process will be evaluated.

[0120] For this, 5 mL compositions were prepared as indicated in example 0, for ViaCox Gel: Control, VC_Gell, VC_Gel2 and oxygen saturated control. 5 mL of the liquid of interest of each of the compositions were added to a 50 mL falcon tube, said tube was connected by a natural #30 thin wall tubing to an adapter that connects to a 99.9% oxygen cylinder. This connection allowed oxygen to be carried from the cylinder to the medium or liquid with an oxygen flow of 0.5 L / min, at an oxygen pressure of 0.05 Mpa, and for 2 min). As indicated, VC_Gell comprises 0.02% Tivida surfactant, and VC_Gel2 comprises 0.02% Tivida surfactant v / v and PFC [l% v / v]

[0121] As indicated, polymerization was initiated with UV light, and the dissolved oxygen concentration in the various compositions was measured up to 400 minutes after polymerization. The results are shown in Figure 2.

[0122] Both inventive compositions evaluated, VC_Gel 1 and 2, maintained dissolved oxygen in the biopolymer matrix after polymerization. VC_Gel 1 contributed to a reduction in the anoxic period, and ViaCoxGel2 showed the best anoxia protection after UV-mediated free radical polymerization. Interestingly, oxygen-saturated sGelMA did not differ from the control sGelMA without prior oxygenation; in both cases, no dissolved oxygen was detected after polymerization. This reflects that oxygen saturation alone is not sufficient to improve oxygen levels after UV polymerization. Consequently, it is suggested that the presence of ViaCox_Gel contributes to a shorter anoxia window, while ViaCoxGel2 showed the best anoxia protection, thus mitigating the anoxic period entirely in sGelMA hydrogels.

[0123] Example 3:

[0124] The oxygen transfer coefficients in hydrogels developed with the composition of the present invention, specifically ViaCox_Gell, were studied.

[0125] Hydrogels with branched fluorosurfactant-stabilized nanobubbles (ViaCox_Gell) were shown to be 5 times more efficient in reducing the anoxic period than the sGelMA control (Fig. 3). The experimental determination of the oxygen transfer coefficient was performed using the gassing-out method or gas elimination as a consequence of exposing methacryl-functionalized hydrogels to UV light (365 nm). For the purpose of generic observations, in this first example we will dispense with cell encapsulation (acellular evaluation), so the gas elimination technique will be applied. We also consider the methacrylated hydrogel model and the degassing shown in the previous example (anoxia by exposure of functionalized gelatin to ultraviolet light or blue light).

[0126] Due to the restriction posed for the development of the measurement, equation (1), which refers to the oxygen transfer rate (OTR).

[0127] Equation 1: OTR= kLa ■ (C * — C) = kLa A C. C* - liquid oxygen saturation concentration, C=liquid oxygen concentration.

[0128] The terms kL are already difficult to measure directly; it is usually evaluated as kLa, which is called the volumetric transfer coefficient. Since equation (1) only shows the variation of C over time, integration between the limits at = o and C, = o gives the following equation:

[0129] Equation 2: liquid time zero. Therefore, Eq. (2) is a natural logarithm line Ln(l -C / C*ql vs t with slope equal to - kLa. The experimental measurements necessary to evaluate kLa must consist of variations of C, (t). We propose to use this method in a hydrogel in which, prior to any test, the dissolved oxygen in the hydrogel is eliminated by exposure to UV light (20 seconds) by bubbling an inert gas, nitrogen, until c, -O.

[0130] These data confirm that the presence of MNBs allows for a significantly higher K¿a value, which correlates with a higher capacity to facilitate oxygen transfer (OTR) from the nanobubble gas core to the liquid phase (Fig. 3).

[0131] Example 4:

[0132] The oxygen transfer coefficients in hydrogels developed with the composition of the present invention, ViaCox_Gell and ViaCox_Gel2, were studied.

[0133] As indicated, we developed a second formulation of the invention by incorporating perfluorocarbon (PFC) with the aim of improving the affinity of the polymer for oxygen. These compositions were called ViaCox_Gel2.

[0134] As shown in Example 2, the development of ViaCox_Gel2 hydrogels significantly reduced the anoxic period compared to ViaCox_Gell (Fig. 2). Furthermore, ViaCox_Gel2 showed 2 ppm of DO inside the hydrogel and remained constant throughout the oxygen release experiment (60 minutes). These results demonstrate that the ViaCoxGel2 composition protects against anoxia, generating a greater equilibrium in oxygen levels, showing a more controlled and continuous mass transfer coefficient, which is related to a more paused or slower oxygen release compared to the ViaCoxGell composition. Figure 4 shows the oxygen transfer coefficients (OTR) for Control, ViaCox_Gell and ViaCox_Gel2.

[0135] This remarkable difference between K La (mass transfer in a liquid phase) and OTR suggests that ViaCox_Gel2 have a slower oxygen transfer than ViaCox_Gell, which implies that when using the hydrogels with the formulations of the invention, they will be oxygenated for longer periods and have slow and fast oxygen release coefficients. Example 5:

[0136] The design of the present invention combines the use of micro / nanobubbles and micro / nanodroplets in natural polymers such as Salmon Methacrylated Gelatin (sGelMA). The objective of this strategy is to mitigate hypoxic stress in cells encapsulated in sGelMA matrices, an aspect that has not yet been evaluated or addressed and that currently represents a technical limitation in tissue engineering, especially in hydrogels formed by free radical polymerization. Simultaneously, this invention also contributes to the development of polymers with a hybrid composition (micro / nanobubbles and micro / nanodroplets) not only for a greater and efficient delivery of oxygen and soluble factors, but also as a strategy for the generation of pores within the structures (hydrogels).

[0137] Porosity is defined as the volumetric percentage of empty spaces or pores within a material. In the context of tissue regeneration, especially in hard tissues (bone and cartilage), porosity and diffusion play a crucial role in facilitating the infiltration of cells, nutrients, and blood vessels, which are essential for tissue formation. The range of porosity and diffusion can vary depending on several factors, including the specific defect being treated. In general, for hard tissues, ~40% is an adequate porosity range for proper regeneration.

[0138] The composition of the present invention increased the percentage of porosity in hydrogels with different gelatin concentrations, or hardnesses as analyzed by SEM microscopy and ImageJ software. ViaCox Gel2 compositions with micro / nanobubbles and micro / nanodroplets with compositions with different concentrations of hydrogel or sGelMA, coded here as VC_Gel2 [10%], VC_Gel2 [25%], VC_Gel2 [35%], were obtained with the method of the invention, showed a wide range of up to 40% porosity as shown in Figure 5, with a pore size distribution between 210 nm and 90 pm.

[0139] Example 6:

[0140] The composition of the present invention provides improvements in the diffusivity of hydrogels for soluble factors such as albumin conjugated to anionic fluorophores such as FITC. ViaCox_Gel shows higher fluorescence intensity and suggests higher diffusivity. The composition of this invention also showed a greater distance traveled through the hydrogel, showing mobility 2.6-9.1 times greater than control hydrogels (Figure 6). Example 7

[0141] Hydrogels fabricated with the composition of the present invention were scanned with the Oxford Jupiter XR AFM at room temperature using the resonance mode and a soft tip (AC240TS-R3). Micro / nano-entities compatible with MNBs or MNDs (ViaCox_Gel2) are observed, which provide the hydrogels with different nano-roughnesses compared to the control and suggest their existence after gel polymerization (Figure 7). Therefore, the hybrid combination of micro / nanobubbles and micro / nanodroplets generates topographical differences compared to the control hydrogels. These results are in agreement with SEM (Fig. 5) and the diffusion test (Fig. 6). AFM allows to confirm the presence of micro / nanobubbles and micro / nanodroplets in situ, since it does not require any additional processing compared to other microscopy techniques (e.g., SEM).

[0142] Example 8:

[0143] In this experiment, umbilical cord blood mesenchymal stem cells were cultured in hypoxia [1%O2] for 3 days and in the presence of medium + MNBs-MNDs (v / v) at different concentrations VC_L_2[10% v / v], VC_L_2[50%v / v], VC_L_2[100%v / v]. The results of the analysis for viability, apoptosis and necrosis are shown in Fig. 8, where all the VC_L_2 formulations evaluated improved the survival rate, decreased the apoptosis and necrosis rate. Prolonged hypoxia time shows a decrease in the viability of control cells, reflecting that even in hypoxic-resistant cells such as UC-MSCs, viability can start to be affected after 3 days of hypoxia. However, cells exposed to increased oxygenation via the ViaCox_L_2 composition showed significant improvements in promoting viability and decreasing apoptosis and necrosis.

[0144] Example 9:

[0145] Serum deprivation and hypoxia are two common experimental conditions used in cell culture to simulate specific physiological or pathological conditions for the study of cellular responses. Therefore, this patent focuses on conditions that mimic the reduced availability of oxygen and nutrients, as well as the limitations of growth factors, that cells may experience during certain physiological or pathological situations (e.g., tissue injury, ischemia, or specific stages of cell differentiation). Based on this, cells are treated with different concentrations of MNBs under low serum (FBS 1%) and hypoxic (1% O2) conditions.

[0146] It was observed that under hypoxic conditions (1% O2), VC_L1 can transport and supply oxygen to cultures. Mesenchymal cells in the presence of ViaCox_L_l significantly increased ATP production (Fig. 9) regardless of the percentage of serum availability used in the medium. We found a 1.7 and 2.5 fold increase in ATP generation in hypoxia-FBS1% and hypoxia-FBS10%, respectively (Figure 9). All of the above suggests an important energetic effect on cellular metabolism mediated in part by increased oxygenation that would favor greater mitochondrial function and consequently a metabolism more oriented to the participation of mitochondria through the electron transport chain or OXPHOS metabolism.

[0147] Example 10:

[0148] Experiment in which mitochondrial stress levels were measured in 2D UC-MSCs cultured in hypoxia for 24 hours (Fig. 10) by flow cytometry. Hypoxia induction at 1% O2 increased mitochondrial ROS levels (880 FU) compared to untreated or normoxic cells (466 FU). Furthermore, all UC-MSCs exposed to ViaCox_Ll decreased their ROS levels, showing a dose-dependent profile, making VC_L1 [100%] the most efficient composition in mitigating mitochondrial ROS (653 FU) and reaching values ​​similar to untreated cells under normoxic conditions (data not shown). These results are the first evidence of the positive effect of ViaCox_Ll in reducing mitochondrial stress under 1% hypoxia.

[0149] Example 11:

[0150] We measured the cell proliferation kinetics of UC-MSCs cultured in 2D using colorimetric metabolic indicators. The proliferation curve is mainly exponential during the first 3 days, with a maximum plate at 5 days. (Data not shown). We highlight that only the hypoxic condition reveals a notable effect of the ViaCox_Ll composition. Cells exposed to ViaCox_Ll (0.23 abs) during the expansion phase (days 1-3) reached a higher metabolic activity than untreated hypoxic cells (0.19 abs), but similar to untreated cells under normoxic conditions (0.24 abs). Subsequently, manual cell counting was performed after 24 hours of incubation (Fig. 11), showing that the VC_L1 composition increases the total number of cells as well as their growth rate. This result suggests that ViaCox_Liquid promotes 1.8 times more cell proliferation of UC-MSCs in hypoxia.

[0151] Example 12:

[0152] For biological and cellular evaluation in hydrogels, cell density, volume of medium used and FBS concentration were previously standardized for 3D culture (data not shown). Samples with different amounts of ViaCox_Gel2_with three different concentrations of PFC [1, 2, 5%] were tested. We selected a DMEM volume of 400 |_d per well as the best option for future experiments. Hydrogels with VC_Gel2 significantly increased mitochondrial activity, but in a dose-dependent manner. According to the PFC concentrations evaluated [1%, 2.5%, 5%], levels of 0.27, 0.31, 0.35 abs respectively were observed. The data in Fig 12 only show the formulation with the highest metabolic activity compared to the control (0.19 abs), maintaining cell viability above 80% while cells encapsulated in control sGelMA showed 65% viability.Although these results are limited to short-term hypoxia (24 h, n=3), it is suggested that hydrogels fabricated with the composition of the present invention protect UC-MSCs against hypoxic stress, increasing their survival rate and promoting higher metabolic activity, the latter an indirect indicator of viability.

[0153] Example 13:

[0154] To determine cell proliferation in 3D cultures, hydrogels were manufactured with the ViaCoxGel 2 composition of the present invention. After 3 days of follow-up under normoxic and hypoxic conditions, a significant effect p<0.05 (paired T-test) was observed in cell proliferation for each of the cell densities evaluated (0.25, 0.5, 1, 2 xl0 6 / ml). Being, ViaCox _Gel2 the most efficient condition to increase the cell growth rate. The above suggests that ViaCoxGel2 optimizes proliferation even in normoxia, demonstrating an evident dual benefit, not only in its protective role by mitigating the deficiencies related to hydrogels incubated in hypoxia [1% O2], but also as an efficient hydrogel in increasing metabolic activity and cell number, even under normoxic conditions. The above could favor future cell regeneration analyses. Significance p < 0.05 (paired T- test) Three independent experiments in triplicate n = 3. VC_Gel2 [10% m / v] PERSPECTIVES / APPLICATIONS

[0155] The present invention creates fine bubbles and / or Itrafines and alternatively nanodroplets in biopolymer-based constructs, highlighting the production method and potential use thereof. Furthermore, the composition of the micro / nanobubbles with a gas core or micro / nanodroplets with a liquid PFC core that shows enhanced affinity for oxygen and is capable of reducing the anoxic period after free radical polymerization. This integration of micro / nanobubbles and nanodroplets with the various biomaterials provides the polymers with a very useful composition for oxygen delivery and porogenic purposes in constructs (hydrogels). Furthermore, the biopolymer-based constructs with fine bubbles and pores obtained by the present invention can be produced on a micro / nano scale, furthermore, it presents a structural configuration that would be adapted to satisfy mechanical and biofunctional requirements.Furthermore, these biopolymers would promote cell survival by reducing cell death induced by hypoxia or hypoxic stress after free radical polymerization, due to the increased availability of oxygen trapped in the fine bubble / nanoblot mixture.

[0156] The range of applications based on MNBs and nanodroplets is highly relevant for the development of hypoxia-sensitive natural or synthetic biopolymers capable of simultaneously mitigating hypoxic stress and integrating the design of porous matrices. Furthermore, this invention could have an impact on other fields of biomedicine, such as tissue engineering, the development of bioinks based on free radical polymerization, materials science, biofabrication, bioprinting, along with the development of pharmaceutical products, organs-on-chips, organoids, cell therapy, regenerative therapy, among others.

Claims

CLAIMS 1. Method for stably incorporating oxygen nanobubbles and / or nanodroplets into hydrogels for biological and regenerative medicine applications CHARACTERIZED in that it comprises the following steps: i) Providing an aqueous liquid composition of functionalized or non-functionalized natural or synthetic polymers, biologically compatible, and a polymerization initiator compound; (i) adding directly to the composition defined in (a) or (b): a liquid fluorosurfactant (T) in a proportion between 0.001 and 0.1% v / v; and a liquid perfluorocarbon compound (PFC) in a proportion between 0.5 and 10% v / v; iii) carrying the composition obtained in (i) to a closed tube or container (capable of isolating from atmospheric pressure) and allowing oxygen to be injected at controlled pressure; iv) forming micro / nanobubbles (MNBs) and / or micro / nanodroplets (MNDs) of gas by means of compression and extension cycles in a closed system, by vacuum degassing cavitation obtaining MNBs and MNDs directly in the composition defined in (i); (v) obtaining an aqueous polymer solution with stable micro / nanobubbles and / or micro / nanodroplets of oxygen; vi) initiate polymerization of the solution either by reversible polymerization (physical) and / or by irreversible polymerization (chemical or photochemical);and obtain a hydrogel with stable micro / nanobubbles and / or micro / nanodroplets of oxygen embedded in a matrix or hydrogel, providing porosity and oxygen availability within the hydrogel.

2. Method according to claim 1 CHARACTERIZED in that the anionic surfactant is a branched fluorosurfactant.

3. Method according to claim 1 CHARACTERIZED in that the oxygen is high purity oxygen.

4. Method according to claim 3 CHARACTERIZED in that the liquid composition additionally comprises active compounds such as medicines, growth and / or migration factors, antibodies, peptides, vesicles, cellular derivatives and / or oligonucleotides.

5. Method according to claim 3 CHARACTERIZED in that the liquid composition additionally comprises living components such as cells or organelles.

6. Method according to claim 5 CHARACTERIZED in that the organelle is an active mitochondria.

7. Method according to claim 1, CHARACTERIZED in that the liquid polymeric composition is a low melting point gelatin, less than 15°C, determined because it has a proline and hydroxyproline content of 18% or less with respect to the total amino acids, which is functionalized with methacryloyl or methacrylamide groups, in a proportion of 30% to 100% of lysine residues of the amino acid chain of said gelatin.

8. Method according to claim 7 CHARACTERIZED in that the low melting point gelatin is derived from organisms of the genus Salmo or Oncorhynchus.

9. Method according to claim 1 CHARACTERIZED in that the polymerization of step vi) is initiated by UV radiation in the presence of a photoinitiator in a concentration between 0.01% to 5% (w / v).

10. Hydrogel formed according to the method described in any of claims 1 to 9, CHARACTERIZED in that it is formed of polymerized gelatin with oxygen nanobubbles at a concentration of 10 6 to 10 9 micro / nanobubbles (MNBs) and / or micro / nanodroplets (MNDs) per cm 3 , which gives it porosity and oxygen availability.

11. Hydrogel of claim 10 CHARACTERIZED in that the oxygen is high purity oxygen.

12. Hydrogel of claim 10 CHARACTERIZED in that it additionally comprises active compounds such as medicines, growth and / or migration factors, antibodies, peptides, vesicles, cellular derivatives and / or oligonucleotides.

13. Hydrogel of claim 10 CHARACTERIZED in that it additionally comprises living components such as cells or organelles.

14. Hydrogel claim 13 CHARACTERIZED in that the organelle is an active mitochondria.

15. Use of the hydrogel of claim 10 CHARACTERIZED in that it serves as a biological support for tissue regeneration / generation.

16. Use of claim 15 CHARACTERIZED in that the tissue regeneration is in a joint injury, cartilage injury, bone injury, ulcerous injury, ligament injury, organ injury, spinal cord injury, or soft tissue injury.

17. Use of claim 15 CHARACTERIZED in that the tissue regeneration is in a reconstitution of skin tissue, muscle tissue, soft tissue or post-surgical replacement tissue.

18. Use of claim 15 CHARACTERIZED in that it serves as a biological matrix as a support for cells, for cell invasion.

19. Use of claim 15 CHARACTERIZED in that it serves as an acellular biological matrix, biological matrix as a mechanical support, biological matrix for active components.

20. Use of the hydrogel of claim 10 CHARACTERIZED in that it serves in events of blood perfusion blockage or trauma that involve inflammation and / or ischemia of skin tissue, musculoskeletal tissue, vascular tissue, nervous tissue, brain tissue, spinal cord, liver tissue, pancreatic tissue, intestinal tissue, lung tissue, cardiac tissue, to supply oxygen and active components.

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