Perfluorocarbon-based nanosystems with oxygen-carrying properties

Abstract

The invention relates to a nanosystem for the transport and release of vital gases, comprising a nanocapsule with a negative zeta potential and formed by an outer polymer selected from hyaluronic acid or modified derivatives thereof, a surfactant, an oil core, and a gas-carrying molecule belonging to the perfluorocarbon family.

Description

[0001] PERFLUOROCARBON-BASED NANOSYSTEMS WITH OXYGEN-TRANSPORTING PROPERTIES: DESCRIPTIVE MEMORANDUM

[0002] SCOPE

[0003] The present invention falls within the field of nanotechnology and its applications for the transport and release of vital gases. More specifically, the invention addresses artificial oxygen carriers (AOCs). This field encompasses everything from oxygen-based therapies to the prevention of associated diseases such as decompression sickness. However, this field should not be limited to clinical applications or a specific area of ​​human health. The invention also incorporates the oxygenation of non-biological systems, including the storage and transport of gases in confined spaces or using suitable devices, at different scales, with applications ranging from veterinary and agricultural fields to serving as a supplementary reservoir for blood banks.

[0004] BACKGROUND OF THE INVENTION

[0005] Oxygen (O2) is a vital gas for the cells in our body, and its absence in our system, known as hypoxia, causes stress and cell death. A lack of oxygen creates an imbalance in cellular metabolism, and if this condition persists, it eventually leads to cell damage and death. Hypoxia can be caused by any alteration in gas exchange (respiration) or blood flow (circulation). Given this, the need arises to prevent or reduce hypoxia in various tissues and conditions, with the aim of minimizing its effects and subsequent consequences. Throughout history, various solutions to this problem have been addressed, primarily the use of blood transfusions, which present challenges related to access, availability, storage, and potential associated infections.Other systems utilize blood derivatives, such as synthetic hemoglobin, which have led to a group of products called artificial oxygen carriers (AOCs). Current AOCs are based on the use of synthetic molecules called perfluorocarbons (PFCs). PFCs have a high O2 transport capacity, where the amount of O2 transported is directly proportional to the partial pressure of the gas and does not depend on the types of bonds between atoms or conformational changes, unlike synthetic hemoglobin. This characteristic makes PFC-based AOCs a better alternative than hemoglobin-based oxygen carriers.

[0006] Regarding the biological behavior of PFCs, it has been shown that only some of them have appropriate physicochemical properties and adequate oxygen transport capacity in vivo, such as 1-bromoperfluorooctane (1-BPFO). With respect to oxygen transport and release, the total amount of oxygen dissolved in a PFC emulsion depends linearly on the PFC concentration. Considering this, it can be hypothesized that the release of oxygen by PFCs involves a much simpler mechanism than that of hemoglobin. Furthermore, the release of oxygen by PFCs into tissues is understood as a mass transfer produced by an oxygen pressure gradient (pO2) between a tissue with a low oxygen concentration and arterial blood.Therefore, a current challenge remains to have a stable system with adequate PFC content to increase its O2 transport capacity, but without disturbing the properties of said PFCs.

[0007] Thus, at this point, the study, understanding, and use of nanomedicine proves ideal for solving the problems associated with the use of unencapsulated PFCs. Among its advantages, nanosystems protect the transported compound from premature degradation in the environment, improve bioavailability, and prolong its presence in fluids and its interaction with cells.

[0008] In the prior art, different approaches to oxygen transport and release have been addressed. In particular, in the patent literature, the inventors of international application WO2010077671 protected perfluorocarbon-based emulsions with high stability and good oxygen release. These emulsions comprise a dispersed phase of the perfluorocarbon and an emulsion stabilizer, as well as a continuous phase. The stabilization of the perfluorocarbon as an emulsion is achieved primarily by including the polyethylene oxide (POE) copolymer, also known as polyethylene glycol (PEG), in the dispersed phase. This document states in its eighth claim that the PFC suitable for the function can be selected from several alternatives: "... from the group consisting of perfluoro crown ethers, perfluoroalkanes, perfluoropentane, perfluorooctyl bromide, perfluorocycloalkanes, and mixtures thereof."''where one of them corresponds to “Perflubron” or 1-bromoperfluorooctane. Additionally, and with respect to the continuous aqueous phase of the emulsion, the document indicates in its eighteenth claim that it may include a hydrogel, where said hydrogel may be selected from: "... hyaluronic acid hydrogel, collagen, silk, acrylates, alginate, fibrin, fibronectin, chitosan, chondroitin sulfate, GAG proteins, proteoglycans, gelatin, protein, poly-NIPAam or combination thereof." Thus, a hyaluronic acid-based hydrogel is only one of several alternatives to form the aqueous phase of the emulsion.

[0009] While document WO2010077671 demonstrates the emulsion's biocompatibility through cytocompatibility testing in cell models, the difference between that international application and the present invention lies in the fact that the former still operates within the paradigm of dispersed-phase PFC emulsions, without incorporating a suitable nanosystem to ensure stability and controlled oxygen release over time. In this respect, WO2010077671 addresses the general applications of PFCs, including the technical challenge of their complex use as an artificial substitute, and therefore, the need to include additional components to stabilize emulsions containing them.

[0010] On the other hand, the scientific literature also shows that efforts are underway to provide formulations and devices for oxygen delivery, including nanoformulations. Along these lines, the publication by Skiba, M. et al. (2002) entitled “Design of Nanocapsules Based on Novel Fluorophile Cyclodextrin Derivatives and Their Potential Role in Oxygen Delivery” highlights the drawbacks of current PFC emulsions, including stability problems, large globules, and toxicity at very high surfactant concentrations. For these reasons, the maximum PFC loading in emulsions would be around 60% w / v, but this would result in highly unstable emulsions. In contrast, the authors designed nanocapsules that could achieve a maximum loading of 100% w / v by simply adding 5% surfactant stabilizers.The authors compared the oxygen solubility between aqueous emulsions with high PFC content and nanocapsules [PFC-NPs] designed with 25% perfluorodecalin by weight / volume, achieving a solubility of 20.72 ml O2 / 100 ml. Finally, the authors conclude that their results suggest that the new “PFC-NCs” would overcome the drawbacks of emulsions and could represent a novel alternative for fluorophilic carriers in various pharmaceutical dosage forms in the near future.

[0011] However, the teachings of Skiba, M. et al. (2002) differ from the present invention at least in the nature of the polymer used for the “PFC-NC” coating, using perfluoro-beta-cyclodextrin instead of hyaluronic acid. Of course, the inventors' choice of the present nanosystem is not obvious. This is because, in the nanocapsule synthesis process, some properties of these nanocapsules can have negative effects on the biocompatibility of the entire nanosystem. Conversely, in the present invention, the nanosystem with hyaluronic acid as a polymer coating achieves not only better biocompatibility but also improved performance in oxygen capture, transport, and release. In fact, while Skiba, M. et al.(2002) suggest that their PFC-NC system could constitute a new vehicle for in vivo oxygen delivery; unfortunately, they provide no data regarding its compatibility with biological systems, much less any cytotoxicity assays. This last point is key because, although p-cyclodextrin is a polymer widely used in the textile, food, and pharmaceutical industries, it has the disadvantage of low water solubility. Furthermore, this polymer can interact with compounds present in humans, such as cholesterol, forming low-solubility complexes that also exhibit nephrotoxic effects. Therefore, these potential negative effects of a p-cyclodextrin derivative alone make the nanosystem's compatibility more relevant in the context of a clinical application in humans or animals.

[0012] Given all this background, there remains interest in developing and biologically evaluating nanometric systems capable of encapsulating PFCs, which present, among other advantages, a greater capacity to dissolve O2 along with prolonged release over time; that is, a nanosystem that presents functional oxygenation characteristics comparable, for example, to blood.

[0013] Notwithstanding the foregoing, the nanosystem of the invention can be used as a gaseous oxygen reservoir in contexts where its storage and transport are safe for handling at different scales, from devices such as gas tanks in submarines, mining operations, and other applications where sensitive handling of this vital gas is required. Even veterinary and / or agronomic applications are compatible with the nanosystem of the invention, where the aim is to safely and continuously distribute oxygen through arrays, such as patches or other compatible devices for these applications in animals; or through its incorporation into controlled atmosphere devices to prevent the ripening of certain fruits or vegetables during handling and transport, where regulating or maintaining a certain level of surrounding oxygen is critical.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings are included to provide a further understanding of the invention and form part of this description and further illustrate a preferred embodiment of the invention, where it can be seen that:

[0016] Figure 1 shows a representative schematic of the structure of the nanosystem of the invention, illustrating each of the relevant components, such as a polymeric coating (PC) or external polymer, a surfactant (S), an oil phase (OP) or oil core, and a perfluorocarbon (PFC) compound. Figure 2A is a reproduction of Figure 2 showing the behavior of the nanosystem designed and reported by Skiba, M. et al. (2002) with respect to oxygen delivery over time. As a control, the release of O2 in water is illustrated.

[0017] Figure 2B graphically describes the behavior of the nanosystem of the invention with 10% PFCs with respect to oxygen release, where it can be seen that it exhibits a maximum initial O2 concentration of 46.04 mg / l (solid line, A). Meanwhile, a nanosystem based on (equivalent to the prior art) shows a maximum initial O2 concentration of 37.03 mg / l (dashed line, B). As a control, the release of O2 in water is also illustrated (dotted line, C).

[0018] Figure 3 graphically depicts cell viability using the Alamar blue method at different contact times for empty NCs and NCs with 1-BPFO in HEK293 cells. HEK293 cells were treated with NCs (A, B) without PFCs (empty) and (C, D) with 10% 1-BPFO for 1, 2, 4, and 24 h of contact time. For a positive control of cell death, cell lines were treated with 1% Triton X-100 for 10 min. The pre-treated cells were then subjected to the Alamar blue viability assay. (A, C) Native NCs (not isolated). (B, D) Isolated NCs (triplicate assays). The Mann-Whitney U test was performed comparing the control group (untreated cells) with the treated cells. Asterisks (*) indicate statistically significant differences (p<0.05).

[0019] Figure 4 shows cell viability by the Alamar blue method at different contact times of empty NCs and NCs with 1-BPFO in HUH7 cells. HUH7 cells were treated with NCs (A, B) without PFCs (empty) and (C, D) with 10% 1-BPFO for 1, 2, 4, and 24 h of contact. For the positive control of cell death, cell lines were treated with 1% Triton X-100 for 10 min. The pre-treated cells were then subjected to the Alamar blue viability assay. (A, C) Native NCs (not isolated). (B, D) Isolated NCs (triplicate assays). The Mann-Whitney U test was performed comparing the control group (untreated cells) with the treated cells. Asterisks (*) represent statistically significant differences (p<0.05).

[0020] Figure 5 graphically illustrates cell viability by the Alamar blue method after 24 h of hypoxia in HEK293 and HUH7 cells. Both cell lines were treated with empty NCs and with 1-BPFO at two different concentrations (1 / 2 and 1 / 5) with and without oxygen. The pre-treated cells were then subjected to the Alamar blue viability assay (triplicate assays). The Mann-Whitney U test was performed comparing the NC groups with and without oxygen. Asterisks (*) represent statistically significant differences (p<0.05).

[0021] DETAILED DESCRIPTION OF THE INVENTION

[0022] Taking into account the persistence of the aforementioned technical problems, the inventors of this application have developed a nanometric system, or nanosystem, capable of encapsulating PFCs, which features:

[0023] high capacity to dissolve gaseous oxygen

[0024] lower percentage use (weight by volume) of PFCs

[0025] Greater stability over time at room temperature and refrigerated; ability to recharge the system with oxygen once it is transported and the first used oxygen is released

[0026] Very low toxicity in cell models. In certain embodiments of the invention, the nanosystem includes at least a polymeric shell, also called an outer polymer; an oily core, also called an oily phase; and a surfactant. The surfactant comprises non-ionic surfactants to achieve stability and appropriate size for the system, as well as at least one cationic surfactant to achieve adhesion of the polymeric shell to the surface of the nanosystem. Preferably, the nanosystem is structurally configured as a nanocapsule.

[0027] In a preferred embodiment of the invention, the polymer coating is selected from suitable and biocompatible polymers, specifically from the group consisting of hyaluronic acid (HA) and modified HA derivatives. In further preferred embodiments, the concentration of said polymer for obtaining a nanocapsule is in the range of 3 to 9 mg / ml, and preferably between 5 and 7 mg / ml. In some embodiments of the invention, said HA is preferably selected from a low molecular weight hyaluronic acid, in the estimated range of 100 to 150 kDa, to achieve lower viscosity and a suitable particle size.

[0028] In some embodiments of the invention, the oily core incorporates compounds selected from the group consisting of: tocopherols, mineral oil, squalene oil, flavor oils, silicone oil, essential oils, water-insoluble vitamins, isopropyl stearate, butyl stearate, octyl palmitate, cetyl palmitate, decyl behenate, diisopropyl adipate, dioctyl sebacate, menthol anthranilate, cetyl octanoate, octyl salicylate, isopropyl myristate, neopentyl glycol ketoles, Cerafilos®, decyl oleate, C12-C15 alkyl lactates, cetyl lactate, lauryl lactate, isostearyl neopentanoate, myristyl lactate, isocetyl stearoyl stearate, octyldodecyl stearoyl stearate, hydrocarbon oils, isoparaffin, fluid paraffins, isododecane, petrolatum, argan oil, rapeseed oil, chili oil, coconut oil, corn oil, cottonseed oil, flaxseed oil, grapeseed oil, mustard oil, olive oil, palm oilfractionated palm oil, peanut oil, castor oil, pine kernel oil, poppy seed oil, pumpkin seed oil, rice bran oil, safflower, tea tree oil, truffle oil, vegetable oil, apricot oil, jojoba oil, macadamia oil, wheat germ oil, almond oil, soybean oil, sesame oil, hazelnut oil, sunflower oil, hemp oil, wood oil, kukui nut oil, avocado oil, walnut oil, fish oil, berry oil, allspice oil, juniper oil, seed oil, almond kernel oil, anise seed oil, celery seed oil, cumin seed oil, nutmeg seed oil, basil leaf oil, bay leaf oil, cinnamon leaf oil, sage leaf oil, eucalyptus leaf oil, lemon leaf oil, oil of melaleuca leaf, oregano oil,patchouli leaf oil, peppermint leaf oil, pine needle oil, rosemary leaf oil, spearmint oil, tea tree leaf oil, thyme leaf oil, Canadian tea leaf oil, flower oil, chamomile oil, clary sage oil, clove oil, geranium flower oil, hyssop flower oil, jasmine flower oil, lavender flower oil, manuka flower oil, marjoram flower oil, orange flower oil, rose flower oil, ylang-ylang flower oil, bark oil, cassia bark oil, cinnamon bark oil, sassafras bark oil, wood oil, camphor wood oil, cedarwood oil, rosewood oil, sandalwood oil, ginger wood oil, resin oil, castor oil, myrrh oil, skin oil, bergamot skin oil, grapefruit peel oil, skin oil of lemon, lime peel oil, orange peel oil,Mandarin peel oil, root oil, valerian oil, oleic acid, linoleic acid, oleyl alcohol, isostearyl alcohol, ethyl oleate, Miglyol®, Labrafil®, Labrafac®, Peceol® and Maisine®, synthetic or semi-synthetic derivatives thereof or combinations thereof. Whereas in preferred embodiments of the invention the concentration of these compounds for the oil phase is in the range between 1 and 50 mg / ml.

[0029] On the other hand, regarding the surfactant, and in particular the non-ionic surfactants, these are selected from the group consisting of lecithin, lipocol, eryethanolamine oleate, tragacanth gum, polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene sorbitan monolaurate (Tween 20), sodium oleate, sodium cholate, PEG stearate, Solutol HS15®, or mixtures thereof. These surfactants provide stability and a suitable size (measured under IPD) to the nanosystem and are used in certain embodiments at concentrations between 5 and 45 mg / ml, and in preferred embodiments at concentrations between 10 and 40 mg / ml. Whereas, in relation to at least one additional surfactant, in particular in relation to a cationic surfactant, this allows the adhesion of the polymeric coating to the surface of the nanocapsule.In preferred embodiments of the invention, said at least one cationic surfactant is selected from the group consisting of oleylamine, stearylamine, benzalkonium chloride, benzethonium chloride, cetylpindinium chloride, cetyltindinium bromide, dodecyltrimethylammonium bromide, trimethyltetradecylammonium bromide, hexadecyltrimethylammonium bromide and poloxamines (e.g. Tetronic®), Varisoft or mixtures thereof, which is used in a range between 0.1 and 2.0 mg / ml, and preferably in a range between 0.2 and 1.8 mg / ml.

[0030] While several prior art procedures are acceptable for obtaining the nanosystem of the invention, the preferred embodiment involves the solvent displacement method. This method is widely used in the chemical field, and in the case of the present invention, it is suitable because PFC is insoluble in most solvents. In the solvent displacement method, using organic solvents such as acetone, ethanol, or other suitable solvents, the PFC is solubilized and incorporated into the core of a nanocapsule. Subsequently, these organic solvents are removed through rotary evaporation to reach the desired volume.

[0031] For illustrative and general purposes only, a solvent displacement method applied to obtaining the nanosystem of the invention comprises: pre-preparing the external polymer, preferably hyaluronic acid, at a high initial concentration, which is then diluted in ultrapure water and kept under stirring. In parallel, one or more compounds of the oil phase are combined and mixed for a sufficient time. A surfactant is added to the oil phase, and the mixture is homogenized. An aliquot of PFCs sufficient to load the core of the nanosystem is then added to the oil phase, followed by a volume of organic solvents, such as acetone. The oil phase is then collected using a suitable device, such as a sterile syringe, and poured into the previously stirred external polymer solution.The entire mixture is kept under agitation for a period of time, and then the organic solvents are evaporated, using a suitable device, until a sufficient volume is reduced. Finally, the pH of the formulation is adjusted by adding a strong base until it is buffered to a pH of approximately 7.2.

[0032] The nanosystem prepared with one of the methods available in the prior art, preferably by the solvent displacement method, provides highly stable nanocapsules (NCs) with a size ranging from 10 to 500 nanometers, and preferably with a size ranging from 150 to 350 nanometers; with a negative zeta potential ranging from -18 mV to -30 mV.

[0033] Furthermore, even though white or empty (PFC-free) nanocapsules (NCs) prepared using the solvent displacement method are already good candidates for capturing, transporting, and delivering oxygen, the incorporation of PFCs achieves greater long-term stability of the nanosystem, both for systems requiring oxygenation for extended periods and for devices designed for prolonged release of gaseous oxygen. In fact, in tests of the present invention on cellular hypoxia rescue models, surprisingly, empty but oxygen-filled NCs were the only ones to reverse hypoxia-induced cell death compared to hypoxic cells treated with the same empty NCs without oxygen. This occurred in less than 24 hours under static conditions, with a single oxygen load.

[0034] Therefore, in embodiments of the present invention, and without prejudice to the fact that the nanosystem is already stable and capable of capturing, transporting, and releasing oxygen, the incorporation of small amounts of PFCs is key to stabilizing the transport of gaseous oxygen. In certain embodiments of the invention, these small amounts of PFCs range from 1% to 14% (weight / volume) in the nanosystem, preferably from 3% to 12% (weight / volume), with a more preferred embodiment involving the incorporation of 10% (weight / volume) of PFCs in the nanosystem. At higher amounts, above 15% (weight / volume), flocculation of the nanosystem occurs.

[0035] Thus, the present invention preferentially addresses in its design the incorporation of PFCs, where said PFCs can be selected from the group consisting of 1-bromoperfluorooctane (1-BPFO), perfluorotributylamine, perfluorohexane, perfluorobutyltetrahydrofuran, perfluorotripropylamine, perfluoro-n-octane, perfluorodecane, fluoro-dimethyladamantane (F-dimethyladamantane), fluoro-methyladamantane (F-methyladamantane), perfluoro- / V-(4-methylcyclohexyl)-pipedidine, or mixtures thereof. In highly preferred embodiments of the invention, the PFC selected for incorporation into the NCs is 1-BPFO.

[0036] Based on the previously described findings, a novel and innovative nanometric system (nanocapsules, NCs) was formulated that exhibits functional oxygenation characteristics comparable to blood. It was remarkable that the use of all components allowed for an almost 14% increase in the percentage of PFCs in the nanocapsule, granting it stability over time and across different pH levels, without agglomeration and maintaining its nanometric structure.

[0037] Regarding the applications of the nanosystem of the invention, this nanosystem can be used in a wide range of industries, especially those where the storage and transport of gases, particularly gaseous oxygen, must be handled safely and stably at different scales, including devices of significant volumes such as gas tanks in submarines, mining operations, naval operations, among others.

[0038] In this regard, the present nanosystem is not only compatible with applications in human health, but also in veterinary and / or agricultural fields. In these latter cases, the nanosystem represents an ideal alternative for the safe and prolonged distribution of oxygen through matrices, such as patches or other products compatible with these applications in animals; or through its incorporation into controlled atmosphere devices to prevent the ripening of certain fruits or vegetables during handling and transport. Thus, the nanosystem can be combined with, or incorporated into, solvents and matrices suitable for use in veterinary, human health, agricultural, environmental, and marine applications, and even in the oxygenation of closed systems.

[0039] Therefore, the present invention, including more complex formulations that incorporate the nanosystem, addresses its use in the preparation of medicines or products useful for the treatment and / or prevention of events related to hypoxia, including but not limited to immersion hypoxia, among other pathologies and / or conditions, such as decompression syndrome.

[0040] Similarly, the nanosystem or formulations containing it can be useful for addressing almost any clinical condition involving hypoxia as a central problem, i.e., wound healing, intervention in ischemic territories (myocardial or cerebral infarctions), preservation of organs for transplantation, as a cytotoxic agent in solid tumors, among others.

[0041] EXAMPLES

[0042] Example 1 - Nanosystem design and synthesis

[0043] Initially, 1-BPFO-based nanocapsules with an oil core coated with protamine (nuclear proteins used in biomedicine and approved by the FDA) were generated. These particles exhibited nanometric size (<400 nm) and a suitable positive zeta potential (>+20), in addition to being homogeneous (Ipd < 0.3). However, they showed in vitro cytotoxicity against HEK-293 and HUH7 cell lines, which was mainly associated with the positively charged polymer, and therefore this developed nanosystem was discarded. To reduce cytotoxicity, the aforementioned nanocapsules were modified by replacing the external polymer with hyaluronic acid (HA, a component used in biomedicine and also approved by the FDA). by optimizing the core of the nanocapsules and evaluating different lipid components (oily core containing DL-a-tocopherol, Lecithin, Lipocol, Varisoft) and encapsulating 1-bromoperfluorooctane (1-BPFO).On the other hand, the methods for preparation, O2 transport, and in vitro evaluation of the designed nanosystems were optimized. The preparation method consisted of:

[0044] 1. The day before the preparation of the formulation, a 30 mg / ml solution in water Mili Q of the AH polymer was left in a total volume of 4 ml under magnetic stirring overnight.

[0045] 2. The excipients were weighed together in a 10 ml glass beaker to form the oily phase: 180 mg of Vitamin E, 150 mg of Lipocol and 54 mg of lecithin.

[0046] 3. A 1 ml solution of 10 mg / ml of Varisoft BT85 in ethanol was prepared in an Eppendorf tube and homogenized by vortexing.

[0047] 4. Add 450 ml of Varisoft BT85 solution to the oil phase and homogenize using a vortex mixer.

[0048] 5. To the above solution, 500 pl of PFCs were added to prepare the charged nanocapsules; otherwise (white nanocapsules, as a control), the above solution was left as it was in point 4 above.

[0049] 6. A 6 mg / ml polymer solution was prepared from the solution described in point 1 in 10 ml of Milli Q water and left under magnetic stirring.

[0050] 7. 4.75 ml of acetone was added to the oil phase (point 5).

[0051] 8. The oil phase was taken with a sterile syringe and subsequently poured onto the polymer solution (point 6) that was under magnetic stirring.

[0052] 9. The formulation (point 8) was left under constant agitation for approximately 10 min.

[0053] 10. The organic solvents were evaporated using a rotary evaporator to a final volume of 5 ml. 11. The pH of the formulation was changed by using 0.1 N NaOH until a value of 7.2 was reached (dropwise).

[0054] 12. The capsules were characterized by their size, polydispersity index and zeta potential using the DLS-zetasizer instrument.

[0055] As can be seen in the following table, a nanometric-sized capsule was obtained with a high percentage of encapsulation and a negative zeta potential.

[0056]

[0057] Additionally, some of the HA nanocapsules were isolated by ultracentrifugation filtration for 30 min at 3000 g using 15 ml Amicon® tubes, thus obtaining the isolated hyaluronic acid nanocapsules (NCs). Nanocapsules not subjected to this process are then referred to as native (i.e., non-isolated) NCs.

[0058] Example 2 - Comparison of an Equivalent Nanosystem to the Prior Art The inventors of this application conducted further tests to compare the oxygen dissolution results between the nanosystem of the invention and a nanocapsule with p-cyclodextrin (BCD) as the external polymer. These tests determined that, for this nanocapsule, a higher concentration of p-cyclodextrin (PCT) reduces the maximum oxygen concentration it can dissolve. In contrast, compared to nanosystems with hydrochloric acid (HA), the BCD nanocapsules release oxygen more quickly, resulting in uncontrolled oxygen release.

[0059] In fact, as can be seen in Figures 2A and 2B, a prior art nanocapsule, such as that described by Skiba, M. et al. (2002), while derived from BCD, exhibits similar behavior to a BCD nanocapsule. Compared to a nanocapsule based on AH as the external polymer, the former shows a maximum initial O2 concentration of 37.03 mg / L (dashed line, B); whereas an AH-based nanocapsule, as in the present invention, achieves a surprisingly high maximum initial oxygen concentration of 46.04 mg / L (solid line, A). Furthermore, the curves in both graphs, and especially the curve compared in Figure 2B, show that the present invention achieves a nanosystem with a much more controlled and prolonged release. This is ideal and advantageous for the oxygenation of biological systems.

[0060] Example 3 - Evaluations of the invention's nanosystem

[0061] To evaluate the cytotoxicity of the developed hyaluronic acid nanocapsules, the human cell lines HEK-293 and HUH7 (ATCC, USA) were used. Empty nanocapsules and nanocapsules containing 1-BPFO, in both their native and isolated forms, were then subjected to different nanocapsule dilutions and contact times to assess the effects of concentration and contact time, as well as the effect of the native or isolated form. Finally, the cells exposed to the nanosystems were subjected to the Alamar blue method to assess their viability.

[0062] Cells were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Biological Industries) and a 100X antibiotic-antifungal agent (Biological Industries). For the assay, 5000 cells were seeded per well in a 96-well plate. After 24 h incubation, the medium was changed, leaving the necessary volume in each well for the addition of the different nanocapsule concentrations, to a final volume of 100 pL (e.g., for a 1 / 2 dilution, 50 pL of the complete medium and 50 pL of the nanosystem were added). Subsequently, the nanosystems were added at the study dilutions of 1 / 2 and 1 / 5 for contact times of 4, 8, 12, and 24 h, depending on the experiment. Then, 10 min before the end of the incubation time, Triton X-100 was added at a final concentration of 1% to the wells that were used as a positive control for cell death.After the incubation period, the medium containing the nanocapsules or Triton was removed, and Alamar Blue was added at a concentration of 0.15 mg / mL from a stock solution to 100 pL of complete medium. The final concentration of Alamar Blue was 0.03 mg / mL per well, and the mixture was incubated for 2 h. Cell viability was measured by fluorescence (560 nm excitation and 590 nm emission) using a Cytation 5 instrument (Biotek, USA). Experiments were performed in triplicate for each condition.

[0063] The results of these trials showed that the formulation without PFCs (empty NCs) has a viability greater than 71% in HEK-293 cells up to 4 h of contact (native NCs concentration 1 / 2 p: 0.23; concentration 1 / 5 p: 0.32. Isolated NCs concentration 1 / 2 p: 0.4; concentration 1 / 5 p: 0.53) and only at 24 h of contact can a considerable drop be observed with a viability greater than 32% (native NCs concentration 1 / 2 p: 0.023; concentration 1 / 5 p: 0.1. Isolated NCs concentration 1 / 2 p: 0.0011; concentration 1 / 5 p: 0.0023) (Figures 3A, 3B). Furthermore, no significant differences were observed between native empty NCs and isolated empty NCs (24 h contact time, concentration 1 / 2 p: 0.41; concentration 1 / 5 p: 0.36). Additionally, NCs formulated with 1-BPFO showed HEK293 cell viability greater than 76% up to 4 h contact time (native NCs concentration 1 / 2 p: 0.027; concentration 1 / 5 p: 0.19).Isolated NCs (1 / 2 p concentration: 0.031; 1 / 5 p concentration: 0.28) and a viability greater than 19% at 24 h of contact (native NCs 1 / 2 p concentration: 0.0018; 1 / 5 p concentration: 0.017. Isolated NCs 1 / 2 p concentration: 0.029; 1 / 5 p concentration: 0.091) (Figures 3C, 3D). Furthermore, it can be observed that there were no significant differences between native PFC NCs and isolated PFC NCs (24 h of contact, 1 / 2 p concentration: 0.17; 1 / 5 p concentration: 0.081).

[0064] Subsequently, in HUH7 liver cells, it was observed that the empty NC formulation has a viability greater than 89% up to 4 h of contact (native NCs concentration 1 / 2 p: 0.42; concentration 1 / 5 p: 0.51. Isolated NCs concentration 1 / 2 p: 0.38; concentration 1 / 5 p: 0.43), and only at 24 h of contact can a considerable drop be observed with a viability greater than 41% (native NCs concentration 1 / 2 p: 0.019; concentration 1 / 5 p: 0.072. Isolated NCs concentration 1 / 2 p: 0.025; concentration 1 / 5 p: 0.032) (Figures 4A, 4B). Furthermore, no significant differences were observed between native empty NCs and isolated empty NCs (24 h contact time, concentration 1 / 2 p: 0.26; concentration 1 / 5 p: 0.17). Additionally, NCs formulated with 1-BPFO showed HUH7 cell viability greater than 91% up to 4 h contact time (native NCs concentration 1 / 2 p: 0.024; concentration 1 / 5 p: 0.089).Isolated NCs (concentration 1 / 2 p: 0.028; concentration 1 / 5 p: 0.51) and a viability greater than 45% at 24 h of contact (native NCs concentration 1 / 2 p: 0.0052; concentration 1 / 5 p: 0.0063. Isolated NCs concentration 1 / 2 p: 0.0092; concentration 1 / 5 p: 0.087) (Figures 4C, 4D). In addition, it can be observed that there were no significant differences between native PFC NCs and isolated PFC NCs (24 h of contact, concentration 1 / 2 p: 0.38; concentration 1 / 5 p: 0.41).

[0065] Observing similar cell viability behaviors between native and isolated nanocapsules (NCs), it was decided to use only native NCs for the remaining biological assays. Additionally, the reduction of hypoxia with oxygen-charged nanocapsules was determined by assessing cell viability using the Alamar Blue assay. For this, the HEK293 and HUH7 cell lines (ATCC, USA) were seeded at a density of 5000 per well in a 96-well plate. Subsequently, 24 h later, the complete DMEM medium supplemented with 10% SBF was replaced, leaving the necessary volume in each well for the addition of the different nanocapsule concentrations, to a final volume of 100 pL. The plates with cells were then placed in a hypoxia chamber, which, after being sealed, was connected to a gaseous nitrogen tank at a flow rate of 40 L / min for 8 min.After this time, the plates were removed from the hypoxia chamber, treated with the oxygen-containing and oxygen-free formulations, and then returned to the hypoxia chamber. Oxygen was purged using a pulse of nitrogen gas at a flow rate of 40 L / min for 3 min, and the sealed hypoxia chamber was placed in the incubator at 37 °C for the study period (4, 8, 12, 24 h). Then, 10 min before the end of the incubation period, Triton X-100 was added at a final concentration of 1%. After the incubation period, the medium containing the nanocapsules or Triton was removed, and Alamar Blue was added (20 lp of a stock solution at a concentration of 0.15 mg / ml in 100 lp of complete medium). The mixture was then incubated for 2 h. Cell viability was measured by fluorescence (560 nm excitation and 590 nm emission) using the Cytation 5 instrument (Biotek, USA). Experiments were performed in triplicate for each condition.To determine the biological effects of the nanocapsules, and whether oxygen-charged nanocapsules could reduce hypoxia-induced cell death, the following approach was used: HEK293 and HUH7 cell lines were subjected to complete hypoxia for 24 h. During the first hour of hypoxia, the nanosystems, previously charged with oxygen (at a flow rate of 100 ml / min for 5 min), were added in two dilutions (1 / 2 and 1 / 5). Surprisingly, the empty oxygen-charged nanocapsules diluted to 1 / 5 were the only ones to reverse hypoxia-induced cell death compared to hypoxic cells treated with the same empty, uncharged nanocapsules (HEK293 cells p: 0.021; HUH7 cells p: 0.036). The other conditions showed no significant differences when comparing oxygen-charged and uncharged nanocapsules (Figure 5).

[0066] In summary, regarding cytotoxicity, the formulations showed adequate cell viability after 4 hours of contact, with cell viability decreasing markedly after 24 hours of contact. However, in a model of complete cellular hypoxia, empty NCs were observed to be able to reverse hypoxia-induced cell death.

[0067] On the other hand, regarding stability, the tests showed that the nanosystems of the invention had adequate stability under storage conditions at 4 °C for at least 6 weeks. Furthermore, they exhibited adequate stability under cellular conditions using Dulbecco's Modified Eagle Medium (DMEM) with and without fetal bovine serum (Biological Industries) at 37 °C for at least 24 h. As for the evaluation of oxygen uptake and release by the nanosystems, both the empty nanosystems and those containing 1-BPFO were able to capture and release oxygen, with the nanosystems containing 1-BPFO capturing a greater quantity and releasing oxygen more slowly or over a longer period.

[0068] Furthermore, the reusability of the nanosystem was evaluated, meaning its ability to be recharged with oxygen for use and subsequent release up to 10 times without compromising its physicochemical properties. This characteristic was tested using nanosystems containing 3%, 10%, and 12% (weight / volume) of PFCs (1-BPFO), respectively, followed by charging with gaseous oxygen and complete release of the contained oxygen. This methodology was repeated 10 times for each nanosystem, and the stability of the nanosystem was verified before and after each use. The physicochemical properties (surface charge, size, polydispersity index) remained constant compared to the initially generated nanosystem, demonstrating excellent performance and the potential for reuse at least 10 times.

[0069] The examples described above are intended to illustrate the invention and some of its preferred embodiments, but in no circumstances should they be considered to restrict the scope of the invention, which will be defined by the terms of the claims presented below.

Claims

CLAIMS 1.- A nanosystem for the transport and release of gases, CHARACTERIZED in that it comprises a nanocapsule with negative zeta potential including: a polymeric coating or external polymer selected from hyaluronic acid or modified derivatives thereof, a surfactant, an oily phase or core and a gas carrier molecule belonging to the perfluorocarbon (PFC) family.

2. The nanosystem for the transport and release of gases according to claim 1, CHARACTERIZED in that its zeta potential varies between -18 mV and -30 mV.

3. The nanosystem for the transport and release of gases according to claim 1, CHARACTERIZED in that its size varies between 10 and 500 nanometers.

4. The nanosystem for the transport and release of gases according to claim 3, CHARACTERIZED in that its size varies between 150 and 350 nanometers.

5. The nanosystem for the transport and release of gases according to claim 1, CHARACTERIZED in that the perfluorocarbon (PFC) molecule is selected from the group consisting of 1-bromoperfluorooctane (1-BPFO), perfluorotributylamine, perfluorohexane, perfluorobutyltetrahydrofuran, perfluorotripropylamine, perfluoro-n-octane, perfluorodecane, fluoro-dimethyladamantane (F-dimethyladamantane), fluoro-methyladamantane (F-methyladamantane), perfluoro- / / -(4-methylcyclohexyl)-piperidine, or mixtures thereof. 6.- The nanosystem for the transport and release of gases according to claim 5, CHARACTERIZED in that the selected perfluorocarbon (PFC) molecule is 1-bromoperfluorooctane (1-BPFO). 7.- The nanosystem for the transport and release of gases according to claim 1, CHARACTERIZED in that the perfluorocarbon (PFC) molecule is incorporated in a range between 1% and 14% (weight / volume). 8.- The nanosystem for the transport and release of gases according to claim 7, CHARACTERIZED in that the perfluorocarbon (PFC) molecule is incorporated in a range between 3% and 12% (weight / volume).

9. A formulation for providing adequate levels of oxygen in the form of prolonged release, CHARACTERIZED in that it incorporates the nanosystem according to any of claims 1 to 8, in addition to solvents and matrices suitable for its application in the veterinary, human health, agricultural, environmental, naval fields, and for the oxygenation of closed systems. 10.- Use of the nanosystem for the transport and release of gases according to claim 1, CHARACTERIZED in that it serves to prepare a drug useful for the treatment and / or prevention of events related to hypoxia. 11.- The use of the nanosystem for the transport and release of gases according to claim 10, CHARACTERIZED in that the treatment and / or prevention of hypoxia-related events are such as immersion hypoxia, among other pathologies and / or conditions, including decompression syndrome. 12.- Use of the nanosystem for the transport and release of gases according to claim 1, CHARACTERIZED in that it serves to intervene in a clinical condition involving hypoxia as a central problem including wound healing, intervention of ischemic territories, preservation of organs for transplantation, cytotoxic in solid tumors, among others.

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