Nano oxygen delivery preparation for enhancing physical ability in anoxic environment as well as preparation method and application of nano oxygen delivery preparation

Abstract

The invention discloses a nano oxygen delivery preparation for enhancing physical ability in an anoxic environment as well as a preparation method and application of the nano oxygen delivery preparation, and belongs to the technical field of biological medicines. The nano oxygen delivery preparation disclosed by the invention is oxygen nano bubbles; the oxygen nanobubbles are wrapped by a polymer or lipid; the polymer is at least one of polyoxyethylene polyoxypropylene block polyether, polyacrylic acid, fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, fatty acid polyoxyethylene ester, sorbitol ester polyoxyethylene ether and sorbitan fatty acid ester; the lipid is phospholipid. The P-ONBs prepared by the invention has strong stability in an aqueous solution, can continuously convey oxygen, and is beneficial to reversion of reduction of blood oxygen saturation in an anoxic environment, improvement of mitochondrial dysfunction of a cell model, enhancement of physical fitness condition of an animal model and improvement of biological safety of a preparation; the requirement on equipment is low, large-scale high-end equipment is not needed, the preparation process is simple, convenient and feasible, and large-scale production is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a nano-oxygen delivery agent for enhancing physical performance in hypoxic environments, its preparation method, and its application. Background Technology

[0002] Blood oxygen saturation is a key indicator reflecting the body's respiratory and circulatory functions, directly related to whether the body's tissues and organs receive sufficient oxygen to maintain normal physiological metabolism and function. In acute hypoxic scenarios such as high-altitude environments, blood oxygen saturation drops significantly, and simply relying on inhaling air or ordinary oxygen therapy cannot quickly reverse the hypoxic state. At this time, the body's tissues do not receive timely and sufficient oxygen supply, leading to mitochondrial metabolic disorders and functional impairment, resulting in increased levels of reactive oxygen species (ROS), decreased physical performance, and even more serious organ damage. For patients with severe respiratory diseases such as chronic obstructive pulmonary disease (COPD) and acute respiratory distress syndrome (ARDS), their lung gas exchange function is severely impaired. Conventional oxygen therapy often fails to deliver oxygen efficiently and in sufficient quantities to the systemic blood circulation. Even with prolonged high-flow oxygen therapy, due to diffusion impairment and ventilation / perfusion mismatch caused by lung lesions, they may still be in a state of hypoxemia, greatly affecting the recovery process and prognosis.

[0003] Existing materials for alleviating hypoxia mainly include: red blood cells or white blood cells as oxygen carriers; perfluorocarbons for loading oxygen; and strategies for generating oxygen through in-situ reactions have also been developed. However, the biosafety and degradability of existing oxygen carrier components remain issues, and many inorganic nanoparticles promote the release of pro-inflammatory cytokines. Therefore, developing a nano-delivery formulation that can overcome these shortcomings and improve blood oxygen saturation has become a pressing technical problem that needs to be solved. Summary of the Invention

[0004] The technical problem this invention aims to solve is to provide a P-ONBs with strong stability in aqueous solution, capable of continuously delivering oxygen and beneficial for reversing the decline in blood oxygen saturation under hypoxic conditions; to provide a nano-oxygen delivery agent that is biosafe, requires minimal equipment, does not require large-scale high-end equipment, and has a simple and easy preparation process. Furthermore, it proposes a nano-oxygen delivery agent for enhancing physical performance in hypoxic environments, its preparation method, and its application.

[0005] One objective of this invention is to provide a nano-oxygen delivery agent for enhancing physical performance in hypoxic environments. The nano-oxygen delivery agent is an oxygen nanobubble; the oxygen nanobubble is encapsulated by a polymer or lipid; the polymer is at least one of polyoxyethylene / polyoxypropylene block polyethers, polyacrylic acid polymers, fatty alcohol polyoxyethylene ethers, alkylphenol polyoxyethylene ethers, fatty acid polyoxyethylene esters, sorbitan ester polyoxyethylene ethers, and dehydrated sorbitan fatty acid esters; the lipid is a phospholipid.

[0006] Further, the polyoxyethylene polyoxypropylene block polyether is at least one of Pluronic F-127, Pluronic F-68, and Pluronic F-108; the polyacrylic acid is at least one of Carbomer 940, Carbomer 980, and sodium polyacrylate; the fatty alcohol polyoxyethylene ether is at least one of AEO-3, AEO-5, AEO-9, and AEO-15; the alkylphenol polyoxyethylene ether is at least one of NP-4, NP-10, NP-30, and OP-10; the fatty acid polyoxyethylene ester is at least one of SG-40, O-9, O-12, and O-15; the sorbitan ester polyoxyethylene ether is at least one of Tween20 and Tween80; and the dehydrated sorbitan fatty acid ester is at least one of Span80 and Span60.

[0007] Further, the phospholipid is at least one of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, sphingomyelin, and sphingomyelin-choline; preferably, the phospholipid is at least one of DSPC, DPPG, egg yolk lecithin, and DSPE-MPEG2000.

[0008] A second objective of this invention is to provide a method for preparing a nano-oxygen delivery agent for enhancing physical performance in hypoxic environments, comprising: S1. Oxygen and water are mixed to obtain a supersaturated gas solution; the supersaturated solution is depressurized under atmospheric pressure to generate bubble nucleation, yielding ONBs water; S2. Dissolve the polymer or phospholipid in an organic solvent, remove the organic solvent, generate a film, add water to the ONBs for hydration, seal, incubate in the dark, filter, granulate to make the particles uniform, take the filtrate to obtain P-ONBs.

[0009] Furthermore, the oxygen and water are mixed by a gas-liquid mixing pump; further, the pressure of the gas-liquid mixing pump is 0.3~0.4MPa; further, the supersaturated gas solution enters a water storage tank through a pipeline to reduce pressure.

[0010] Furthermore, the oxygen content in the ONBs water is not less than 15 mg / L at room temperature.

[0011] Furthermore, the polymer or phospholipid is dissolved in an organic solvent, and the organic solvent is removed by rotary evaporation.

[0012] Furthermore, the dark incubation time is 6-10 hours; the filtration is performed using a 0.45μm or 0.22μm filter membrane.

[0013] Further, the mass-to-volume ratio of the polymer to the ONBs water is (118~138mg):(20~30ml); the mass-to-volume ratio of the phospholipid to the ONBs water is (118~138mg):(20~30ml).

[0014] The third objective of this invention is to provide an application of a nano-oxygen delivery agent for enhancing physical performance in hypoxic environments, specifically for the application of nano-oxygen delivery agents in improving blood oxygen saturation and enhancing physical performance.

[0015] Compared with existing technologies, this invention proposes a nano-oxygen delivery agent for enhancing physical performance in hypoxic environments, its preparation method, and its application, which has the following beneficial effects: The P-ONBs prepared in this invention have strong stability in aqueous solution and can continuously deliver oxygen, which is beneficial for reversing the decrease in blood oxygen saturation under hypoxic conditions.

[0016] Furthermore, the P-ONBs prepared by this invention significantly improve blood oxygen saturation in animal models under hypoxic conditions; significantly improve mitochondrial dysfunction in cell models under hypoxic conditions; and significantly enhance the physical condition of animal models under hypoxic conditions.

[0017] Furthermore, the preparation process of P-ONBs in this invention avoids the use of toxic carriers under existing technology conditions, effectively improving the biosafety of the formulation; furthermore, the preparation method of P-ONBs proposed in this invention has low equipment requirements, does not require large-scale high-end equipment, and the preparation process is simple and easy to implement, and can be applied to large-scale production. Attached Figure Description

[0018] Figure 1 A schematic diagram illustrating the preparation and application of P-ONBs according to an embodiment of the present invention is shown; Figure 2 An oxygen concentration diagram of different materials according to an embodiment of the present invention is shown; Figure 3 A particle size variation diagram of P-ONBs according to an embodiment of the present invention is shown; Figure 4 The diagram shows the particle size distribution variation of P-ONBs according to an embodiment of the present invention. Figure 5The diagram illustrates the effect of P-ONBs on the blood oxygen saturation of experimental animals according to an embodiment of the present invention. Figure 6 This invention illustrates the effect of P-ONBs on the mitochondrial membrane potential of hypoxic cardiomyocytes according to an embodiment of the present invention. Figure 7 The figure shows the effect of a type of P-ONBs from an embodiment of the present invention on the weight-bearing swimming time of hypoxic Balb / C mice. Detailed Implementation

[0019] Exemplary embodiments of the present application will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present application are shown in the drawings, it should be understood that the present application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this application will be thorough and complete, and will fully convey the scope of the present application to those skilled in the art.

[0020] Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the reagents and materials in this invention are obtained from the market or other public channels.

[0021] The present invention discloses a nano-oxygen delivery agent for enhancing physical performance in hypoxic environments, its preparation method, and its application. The main technical principles of this invention include: Nanobubble water is a solution formed by dispersing gas in water as free bubbles with nanometer diameters using specific equipment. The resulting system contains a large number of bulk nanobubbles (BNBs), which can remain stable in the aqueous phase for days, weeks, or even months without any surfactants. BNBs also possess high specific surface area, high internal pressure, negative surface charge, good biocompatibility, and properties related to force, heat, light, and sound. Most importantly, BNBs exhibit good biocompatibility and can be applied in the biomedical field. The resulting nanobubble solution increases gas solubility in the aqueous phase, expands the gas diffusion interface area, and reduces the possibility of gas embolism in the bloodstream. This invention applies this to oxygen transport, using aqueous BNBs as a dispersion medium for drug delivery. The BNBs encapsulate and carry oxygen for long-term storage, providing more space for drug loading. This invention also studies the effects of polymer and phospholipid encapsulation on the stability of ONBs in water, preparing a lipid-encapsulated nano-oxygen delivery formulation, P-ONBs, which can improve blood oxygen saturation and enhance physical performance at high altitudes through injection.

[0022] Based on the above principles, this invention proposes a method for preparing a nano-oxygen delivery agent for enhancing physical performance in hypoxic environments, comprising the following steps: ①Preparation of ONBs water: Oxygen and water are mixed by passing them into a gas-liquid mixing pump to form a supersaturated gas solution, which is then piped into a water storage tank. The supersaturated gas solution is depressurized under atmospheric pressure, generating bubbles that nucleate and produce ONBs water. ②Preparation of P-ONBs: The polymer or phospholipid is dissolved in an organic solvent, and then the organic solvent is removed by rotary evaporation to form a film at the bottom of the bottle. ONBs water is added for hydration, the bottle is sealed and incubated in the dark for 8 hours, filtered and granulated to make the particles uniform in size, and the filtrate is collected to obtain P-ONBs.

[0023] Preferably, in step ①, the pressure of the gas-liquid mixing pump is 0.3-0.4 MPa.

[0024] Preferably, in ①, the oxygen content in the ONBs water at room temperature is not less than 15 mg / L.

[0025] Preferably, in step ②, the filtration operation specifically involves filtration using a 0.45μm or 0.22μm filter membrane.

[0026] Preferably, in step ②, the polymer includes at least one of Pluronic F-127, Pluronic F-68, Carbomer 940, Tween 20, and Span 80.

[0027] Preferably, in step ②, the phospholipids include DPPG, egg yolk lecithin, and DSPE-MPEG 2000, wherein the weight ratio of DPPG, egg yolk lecithin, and DSPE-MPEG2000 is 2:20:1.

[0028] Preferably, in step ②, the ratio of the polymer or phospholipid mixture to ONBs water is 128 mg: 25 ml.

[0029] The P-ONBs prepared by this invention have strong stability in aqueous solution and can continuously deliver oxygen, which is beneficial to reversing the decrease in blood oxygen saturation under hypoxic conditions. They can significantly improve blood oxygen saturation in hypoxic animal models, improve mitochondrial dysfunction in cell models, enhance the physical condition of animal models, and improve the biosafety of the formulation. The equipment requirements are low, no large-scale high-end equipment is required, the preparation process is simple and easy to implement, and it is convenient for large-scale production.

[0030] Example 1 This invention proposes a method for preparing oxygen nanobubbles (ONBs).

[0031] Mainly includes: Oxygen is used to form a supersaturated gas solution by passing it through a gas-liquid mixing pump (20QY-1DS, China Southern Pump Industry, 0.3-0.4MPa). The solution is then fed into a water storage tank through a nozzle at the pipeline outlet. The supersaturated gas solution is depressurized under atmospheric pressure, generating bubbles that nucleate to produce ONBs water.

[0032] result: ONBs mix nano-sized oxygen into water. Due to their tiny volume, they float very slowly and can remain stably suspended in water for a long time, forming "supersaturated" oxygen-rich bubble water, which has application value in many fields.

[0033] Example 2 This invention proposes a method for preparing nano-oxygen delivery agents P-ONBs.

[0034] Mainly includes: The thin-film hydration method was adopted, specifically as follows: a polymer or phospholipid mixture (DPPG, egg yolk lecithin and DSPE-MPEG2000 in a weight ratio of 2:20:1) was dissolved in chloroform, placed in a distillation flask, and then the chloroform was removed by rotary evaporation, producing a thin film at the bottom of the flask. ONBs water was added for hydration, the flask was sealed and incubated in the dark for 8 hours, and then the particles were filtered through a 0.45 μm filter membrane to make the particles uniform in size.

[0035] Among them, Pluronic F-127, Pluronic F-68, Carbomer 940, Tween20 and Span80 were purchased from Beijing Innocare Technology Co., Ltd.; DPPG and DSPE-MPEG2000 were purchased from Jiangsu Southeast Nanomaterials Co., Ltd.; and egg yolk lecithin (ePC, PC-98T) was sourced from AVT (Shanghai) Pharmaceutical Technology Co., Ltd.

[0036] result: Please see Figure 1 The polymer material forms a stable membrane structure, which further enhances the stability of oxygen bubbles in P-ONBs.

[0037] Example 3 This invention proposes an experiment to characterize oxygen-carrying capacity.

[0038] Mainly includes: The oxygen-carrying capacity of Examples 1 and 2 was tested using a dissolved oxygen microelectrode. 4 mL of the stock solution was added to a penicillin bottle, and the oxygen concentration at a depth of 0.5 cm from the bottom of the bottle was measured using a dissolved oxygen microelectrode. To prevent oxygen leakage, a paraffin oil seal was applied. Oxygen release was monitored over time at a predetermined temperature (20°C).

[0039] result: Please see Figure 2 It can be seen that the oxygen content of ONBs is 28 mg / L and that of P-ONBs is 25 mg / L, thus retaining the oxygen-carrying capacity of ONBs. Both ONBs and P-ONBs can continuously release oxygen within 7 days.

[0040] Example 4 This invention presents a particle size characterization experiment for P-ONBs, a nano-oxygen delivery agent.

[0041] Mainly includes: The particle size and particle size distribution (PDI) of the P-ONBs prepared in Example 2 were determined by dynamic light scattering (DLS). 2 mL of the diluent was analyzed using a Nanobrook 90 Plus Zata nanoparticle analyzer.

[0042] result: Please see Figure 3 and Figure 4 The results showed that P-ONBs remained stable for at least a week, with the average hydrodynamic particle size decreasing from 628.0 nm to 502.0 nm and the PDI increasing from 0.289 to 0.318.

[0043] Example 5 This invention presents an experimental study on the effect of a nano-oxygen delivery agent, P-ONBs, on blood oxygen saturation in model animals.

[0044] Mainly includes: Female Balb / C mice weighing 20-22g were selected and placed in a hypobaric chamber at an altitude of 5000m. A pulse oximeter was used to monitor the blood oxygen saturation of the mice in real time while they were awake. At the start of the experiment, the pulse oximeter was turned on, all parameters were set, the mice's tails were clamped, and the blood oxygen saturation of each group of mice was monitored. After 24 hours, P-ONBs (40mL / kg) were injected, and changes in blood oxygen saturation in each group of mice were monitored.

[0045] result: Please see Figure 5 P-ONBs effectively improved blood oxygen saturation in mice. Compared with before injection, P-ONBs significantly increased blood oxygen saturation in mice from 71.9% to 80.5%.

[0046] Example 6 This invention presents an experimental study on the effect of a nano-oxygen delivery agent, P-ONBs, on the mitochondrial membrane potential of model cells.

[0047] Mainly includes: Cardiomyocytes were seeded at appropriate densities, and blank control, hypoxia control, and P-ONBs groups were set up. The hypoxia control and P-ONBs groups were cultured in 1% oxygen for 24 h, while the blank control group was cultured in a normal incubator. After 24 h, the culture medium was discarded, and the cells were gently washed twice with pre-warmed PBS. JC-1 staining working solution was prepared, added to well plates, and incubated at 37°C in the dark for 20-30 minutes. After staining, the staining solution was discarded, and the cells were washed twice with PBS to remove unbound dye. Adherent cells were trypsinized and collected by centrifugation, while suspension cells were directly centrifuged and resuspended in PBS. Cell slides were immediately analyzed, with excitation light at 488 nm and 585 nm. Normal cells showed predominantly red light, while green light increased as membrane potential decreased. The membrane potential level was assessed by changes in the red-green fluorescence ratio.

[0048] result: Please see Figure 6 Compared with the hypoxia group, P-ONBs significantly increased the mitochondrial membrane potential from 61.2% to 89.6%, indicating that P-ONBs can effectively alleviate mitochondrial dysfunction caused by hypoxia and reduce the impact of hypoxia on cellular energy metabolism.

[0049] Example 7 This invention presents an experimental study on the effects of a nano-oxygen delivery agent, P-ONBs, on the physical performance of model animals.

[0050] Mainly includes: Balb / C mice were used in the experiment. A blank control, a hypoxia control, and a P-ONBs group were set up. First, the blank control mice were placed in a plain environment, while the hypoxia control and P-ONBs group mice were placed in an environment equivalent to 5000m altitude for at least 3 days to acclimatize, maintaining room temperature (22±2℃) and a 12-hour light cycle. Mice were fasted for 4 hours before the formal experiment but had free access to water. The P-ONBs group received a tail vein injection of an adjuvant (40mL / kg) 10 minutes before the experiment. The other two groups received an equal volume of physiological saline as controls before the weighted swimming test. A lead weight equal to 5% of the mouse's body weight was suspended from the mouse's tail, and the mice were placed in a pool with a depth ≥30cm (water temperature 25±0.5℃), avoiding excessive tightness that could affect blood circulation. A timer was started, and the mice's movement was observed. Exhaustion was defined as when the head was completely submerged in water for more than 10 seconds, and the total swimming time was recorded. If a mouse floated motionless during the test, it could be gently touched with a stick to encourage it to continue swimming, but human interference should be avoided. After the experiment, the mice were provided with ample food and water, and their condition was observed.

[0051] result: Please see Figure 7 Compared with the hypoxia group, P-ONBs significantly increased swimming time from 202.9s to 287.1s, indicating that P-ONBs can effectively alleviate the decline in physical fitness caused by hypoxia and enhance athletic performance.

[0052] In summary, this invention presents a nano-oxygen delivery agent for enhancing physical performance in hypoxic environments, along with its preparation method and application. The P-ONBs prepared by this invention can efficiently deliver oxygen, increase blood oxygen saturation, improve hypoxia at high altitudes, alleviate mitochondrial dysfunction, and enhance physical performance at high altitudes.

[0053] It should be noted that the term "comprising," or any other variation thereof, is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0054] The above are merely embodiments of this application and are not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A nano-oxigen delivery formulation for physical fitness enhancement in hypoxic environment, characterized in that, The nano-oxygen delivery preparation is oxygen nano-bubbles; The oxygen nano-bubbles are polymer or lipid coated; The polymer is at least one of polyoxyethylene polyoxypropylene block polyether, polyacrylic acid, fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, fatty acid polyoxyethylene ester, sorbitol ester polyoxyethylene ether, and sorbitan fatty acid ester; The lipid is phospholipid.

2. The nano-oxigen delivery formulation for hypoxic environment physical fitness enhancement as claimed in claim 1, wherein, The polyoxyethylene polyoxypropylene block polyether is at least one of Pluronic F-127, Pluronic F-68, and Pluronic F-108; The polyacrylic acid is at least one of Carbomer 940, Carbomer 980, and sodium polyacrylate; The fatty alcohol polyoxyethylene ether is at least one of AEO-3, AEO-5, AEO-9, and AEO-15; The alkylphenol polyoxyethylene ether is at least one of NP-4, NP-10, NP-30, and OP-10; The fatty acid polyoxyethylene ester is at least one of SG-40, O-9, O-12, and O-15; The sorbitol ester polyoxyethylene ether is at least one of Tween 20 and Tween 80; The sorbitan fatty acid ester is at least one of Span 80 and Span 60.

3. The nano-oxigen delivery formulation for hypoxic environment physical fitness enhancement as claimed in claim 1, wherein, The phospholipid is at least one of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, sphingomyelin, and sphingomyelin choline. Preferably, the phospholipid is at least one of DSPC, DPPG, egg yolk lecithin, and DSPE-MPEG2000.

4. A method for preparing a nano-oxygen delivery agent for enhancing physical performance in hypoxic environments, as described in any one of claims 1 to 3, characterized in that, The method comprises: S1, mixing oxygen and water to obtain a supersaturated gas solution; reducing the pressure of the supersaturated solution under atmospheric pressure to generate bubble nucleation, and obtaining ONBs water; S2, dissolving a polymer or phospholipid in an organic solvent, removing the organic solvent to generate a film, adding the ONBs water for hydration, sealing and dark incubation, filtering, granulating to make the particles uniform, taking the filtrate, and obtaining P-ONBs.

5. The process for the preparation of nano-oxigen delivery formulation for hypoxic environment physical fitness enhancement as claimed in claim 4 wherein, The oxygen and water are mixed by a gas-liquid mixing pump; The pressure of the gas-liquid mixing pump is 0.3-0.4 MPa; The supersaturated gas solution enters a water storage tank through a pipeline for pressure reduction.

6. The process for the preparation of nano-oxigen delivery formulation for hypoxic environment physical fitness enhancement as claimed in claim 4 wherein, In the ONBs water, the oxygen content at room temperature is not less than 15 mg / L.

7. The method for preparing the nano-oxygen delivery agent for enhancing physical performance in hypoxic environments according to claim 4, characterized in that, The polymer or phospholipid is dissolved in an organic solvent, and the organic solvent is removed by rotary evaporation.

8. The method for preparing the nano-oxygen delivery agent for enhancing physical performance in hypoxic environments according to claim 4, characterized in that, The dark incubation time is 6-10 h; The filtration is 0.45 μm or 0.22 μm membrane filtration.

9. The process for the preparation of nano-oxigen delivery formulation for hypoxic environment physical fitness enhancement as claimed in claim 4 wherein, The mass-volume ratio of the polymer to the ONBs water is (118-138 mg):(20-30 ml); The mass-volume ratio of the phospholipid to the ONBs water is (118-138 mg):(20-30 ml).

10. Use of a nano-oxigen delivery formulation for physical fitness enhancement in hypoxic environments, characterized by, The application of the nano-oxygen delivery preparation prepared by the method for preparing a nano-oxygen delivery preparation for hypoxic environment physical fitness enhancement according to any one of claims 1-3 or 4-9 in improving blood oxygen saturation and enhancing physical fitness.

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