A sustainable oxygen-producing composite carrier and its preparation method and application

Abstract

The present invention discloses a sustainable oxygen-producing composite carrier and a preparation method and application thereof. The sustainable oxygen-producing composite carrier includes an oxygen-producing source and a natural polysaccharide gel, and calcium peroxide is wrapped in a core-shell structure of a polylactic acid microsphere to slow down the reaction of calcium peroxide with water to achieve the effect of continuous oxygen release. The present invention disperses polylactic acid microspheres (CP) loaded with calcium peroxide in a solution of sodium alginate (SA), and prepares a sustainable oxygen-producing composite carrier (CP / SA) by chelating reaction with metal ions. The CP microspheres react with water inside the microcarrier to generate hydrogen peroxide, and oxygen is generated under the action of catalase, thereby increasing the oxygen content inside the composite carrier, and bone marrow mesenchymal stem cells can proliferate and differentiate inside the composite carrier, while reducing the expression of hypoxia-inducible factor (HIF‑1α). The sustainable oxygen-producing composite carrier has a good application prospect in simulating hypoxia-related diseases in vitro and building an oxygen production model.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical polymer materials, and in particular relates to a sustainable oxygen-producing composite carrier and a preparation method and application thereof. Background Art

[0002] Oxygen is the source of life for most organisms on Earth, and humans are no exception. Humans inhale oxygen through the respiratory system, and then transport oxygen to cells in various organs and tissues through blood circulation. Cells use oxygen to perform oxidative phosphorylation reactions to produce adenosine triphosphate (ATP), which provides energy for cell life activities. Therefore, oxygen is essential for human survival.

[0003] Cells are the basic units of the human body and life. When cells are cultured in vitro, the gas environment in the body needs to be simulated to ensure the normal growth and function of the cells. The gas environment mainly includes two gases, oxygen and carbon dioxide, which play an important role in the survival and metabolism of cells. Oxygen is an oxidant for cells, participates in the tricarboxylic acid cycle of cells, and provides energy for cells; carbon dioxide is a reductant for cells, a metabolic product of aerobic respiration of cells, and an essential substance for cell growth. At the same time, carbon dioxide can also react with water to form carbonic acid, which affects the pH value of the culture medium and maintains the acid-base balance of cells.

[0004] At present, the oxygen required for in vitro cell culture is mainly supplied by the following methods: first, oxygen is stored in gas cylinders, and then transported to the cell culture device through pipes and other tools; second, oxygen in the air is exchanged with cells in the culture medium by a membrane oxygenator to achieve the purpose of in vitro oxygen production; third, microorganisms in the bioreactor are photosynthesized or respired to produce oxygen to achieve in vitro oxygen production; fourth, inorganic peroxides are used to react with water to produce oxygen. However, these methods all have disadvantages. The gas cylinder oxygen production method requires frequent replacement of gas cylinders, which is expensive; the membrane oxygenator method requires professional equipment and technology; the bioreactor method requires special microorganisms and culture environments, and cannot be applied to all cells; the inorganic peroxide method produces oxygen too quickly and cannot continuously supply oxygen.

[0005] Therefore, developing a simple, low-cost cell growth matrix material that can continuously release oxygen is of great significance and value to improving the efficiency and quality of cell culture. Summary of the invention

[0006] In order to overcome the shortcomings and disadvantages of the prior art, the primary purpose of the present invention is to provide a method for preparing a composite carrier for sustainable oxygen production. The preparation method is simple and easy, the raw materials are readily available, the cost is low, and no complicated equipment and technology are required. It can be mass-produced and has good industrial application prospects.

[0007] The second object of the present invention is to provide a sustainable oxygen-producing composite carrier prepared by the above preparation method.

[0008] The third object of the present invention is to provide an application of the above-mentioned sustainable oxygen-producing composite carrier.

[0009] The primary purpose of the present invention is achieved through the following technical solutions:

[0010] A method for preparing a composite carrier for sustainable oxygen production comprises the following steps:

[0011] (1) Preparation of calcium peroxide-loaded polylactic acid microspheres (CP): polylactic acid particles are dissolved in chloroform, calcium peroxide particles are added, and the mixture is subjected to ultrasonic treatment to obtain a suspension; during the stirring process, the suspension is added to a high-concentration polyvinyl alcohol solution of calcium peroxide nanoparticles for emulsification, and then added to a low-concentration polyvinyl alcohol solution for stirring at room temperature, washed, and freeze-dried to obtain calcium peroxide-loaded polylactic acid microspheres (CP);

[0012] (2) Preparation of a sustainable oxygen-producing composite carrier: polylactic acid microspheres (CP) loaded with calcium peroxide and catalase are added to a sodium alginate (SA) solution, mixed and rapidly stirred to obtain a sodium alginate solution containing catalase and polylactic acid microspheres, which is then sprayed into a calcium chloride solution by voltage jetting to obtain a sustainable oxygen-producing composite carrier (CP / SA).

[0013] Preferably, in step (1), the concentration of the polylactic acid in chloroform is 50 mg / mL, the mass concentration of the high-concentration polyvinyl alcohol solution is 20 mg / mL, and the mass concentration of the low-concentration polyvinyl alcohol solution is 5 mg / mL; the volume ratio of the chloroform to the high-concentration polyvinyl alcohol is 1:2; and the volume ratio of the suspension to the low-concentration polyvinyl alcohol is 1:10.

[0014] Preferably, the mass concentration of the calcium peroxide nanoparticles in the high concentration polyvinyl alcohol solution in step (1) is 5-20 mg / mL. Low concentrations of calcium peroxide have poor oxygen production effects, and high concentrations of calcium peroxide cannot be evenly dispersed during the dispersion process, resulting in poor stability of the microspheres during the preparation of CP. In addition, the excessive oxygen produced by high concentrations of calcium peroxide will cause excessive production of reactive oxygen species inside cells, destroying the cells, so the preferred concentration of calcium peroxide is 10 mg / mL.

[0015] Furthermore, in the oxygen-generating system, calcium peroxide nanoparticles can be loaded into the core or shell of the polylactic acid microspheres.

[0016] Calcium peroxide is loaded into the shell, and after contact with water, the calcium peroxide in the shell reacts quickly with water. Calcium peroxide is loaded into the core of the polylactic acid microsphere, and the hydrophobic surface of the polylactic acid cannot make calcium peroxide react with water in a short time. Calcium peroxide is loaded in the core and shell, and the calcium peroxide on the surface of the microsphere reacts to produce oxygen in the early stage, and then the calcium peroxide in the core contacts water to produce oxygen, achieving long-term oxygen release.

[0017] Preferably, the mass concentration of the sodium alginate (SA) solution in step (2) is 10-40 mg / mL, preferably 20 mg / mL; the mass concentration of the catalase is 5 mg / mL; the mass concentration of the polylactic acid microspheres (CP) loaded with calcium peroxide is 2 mg / mL; and the mass concentration of the calcium chloride solution in step (2) is 40 mg / mL.

[0018] The gel prepared with low concentration of SA has higher porosity and lower strength, the gel prepared with higher concentration of SA has lower porosity and higher mechanical strength, and the gel prepared with medium concentration of SA can maintain appropriate porosity and mechanical strength.

[0019] Preferably, the stirring rate in step (2) may be 1000-1500 r / min, preferably 1500 r / min.

[0020] The second object of the present invention is achieved by the following technical solutions:

[0021] A composite carrier (CP / SA) capable of sustainably producing oxygen is prepared by the above preparation method.

[0022] Preferably, the particle size of the composite carrier (CP / SA) capable of sustainable oxygen production is 100-500 μm.

[0023] Preferably, the voltage is 20 kV-7 kV, and the size distribution of the composite carrier is kept within a reasonable particle size range within the controllable voltage range.

[0024] The composite carrier capable of sustainable oxygen production comprises an oxygen production source and a natural polysaccharide gel.

[0025] The third object of the present invention is achieved by the following technical solutions:

[0026] A composite carrier capable of sustainably producing oxygen is used in organoids. Specifically, a suspension of bone marrow mesenchymal stem cells is added to a composite carrier solution capable of sustainably producing oxygen, and then electrostatically sprayed into a calcium chloride solution to obtain a composite carrier capable of sustainably producing oxygen that carries cells. After culturing for 3 days, the cells inside the composite carrier are fluorescently stained to observe the division and differentiation of the cells inside. After culturing for 7 days, the living cells inside the carrier are fluorescently stained and the intracellular hypoxia fluorescently stained, and quantitative statistics are performed to prove the potential utilization value of the oxygen-producing composite carrier.

[0027] Preferably, the cell concentration in the cell suspension is 1×10 6 The mass concentration of the calcium chloride solution is 40 mg / mL. The present invention has the following advantages and effects compared with the prior art:

[0028] (1) The oxygen release system provided by the present invention is polylactic acid microspheres (CP) loaded with calcium peroxide. Calcium peroxide has high-energy peroxide covalent bonds and can be decomposed in an aqueous medium to produce oxygen. Since it is wrapped by the polylactic acid microspheres, it can produce oxygen continuously and stably, and is suitable for in vitro cell culture. It can provide cells with sufficient oxygen and improve the efficiency and quality of cell culture.

[0029] (2) The method for preparing sustained oxygen-releasing materials provided by the present invention is simple and easy, the raw materials are readily available, the cost is low, and no complicated equipment and technology are required. It can be produced on a large scale and has good industrial application prospects.

[0030] (3) The continuous oxygen-producing composite microcarrier prepared by the present invention can be used as a matrix material for cell growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Scanning electron microscope observation images of polylactic acid (PLLA) and polylactic acid microspheres (CP) loaded with calcium peroxide.

[0032] Figure 2 This is the electron microscope energy spectrum mapping diagram of polylactic acid microspheres (CP) loaded with calcium peroxide.

[0033] Figure 3 Optical microscope image of the composite carrier (CP / SA) with sustainable oxygen production.

[0034] Figure 4 This is the real-time oxygen release diagram of sodium alginate (SA) and a composite carrier with sustainable oxygen production (CP / SA) within 14 days.

[0035] Figure 5 Confocal microscopy images of bone marrow mesenchymal stem cells cultured in a composite carrier with sustainable oxygen production (CP / SA) for 3 days.

[0036] Figure 6 This is a fluorescence quantitative image of live cells of bone marrow mesenchymal stem cells cultured in a composite carrier with sustainable oxygen production (CP / SA) for 7 days.

[0037] Figure 7 This is a fluorescence quantitative graph of hypoxia-inducible factor (HIF-1α) in bone marrow mesenchymal stem cells cultured in a composite carrier with sustainable oxygen production (CP / SA) for 7 days. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. The materials involved in the following examples can be obtained from commercial channels unless otherwise specified. The methods described are conventional methods unless otherwise specified.

[0039] Example 1 Preparation of calcium peroxide loaded polylactic acid microspheres (CP)

[0040] The steps for preparing polylactic acid microspheres (CP) loaded with calcium peroxide in this embodiment are as follows:

[0041] Polylactic acid particles are dissolved in chloroform (50mg / mL), then calcium peroxide nanoparticles (10mg / mL) are added in polylactic acid solution. Mixture was subjected to ultrasonic treatment for 10 minutes to obtain uniform suspension (20mL, w1). Under agitation (650rpm / min) this suspension was joined to the PVA aqueous solution containing 1% calcium peroxide (20mg / mL, 40mL, w1) after emulsification for 3 minutes, emulsion was joined to the polyvinyl alcohol aqueous solution of 5mg / mL (600mL, w2), and stirring speed was constant at 250rpm / min. After 4 hours, microspheres were washed 8 times with deionized water, and then freeze-dried to obtain the polylactic acid microspheres (CP) loaded with calcium peroxide.

[0042] Taking polylactic acid (PLLA) microspheres as a control, polylactic acid particles were dissolved in chloroform (50 mg / mL), and the suspension was added to a PVA aqueous solution (20 mg / mL, 40 mL, w1) under stirring (650 rpm / min) for emulsification for 3 minutes, and then the emulsion was added to a 5 mg / mL polyvinyl alcohol aqueous solution (600 mL, w2) with a constant stirring speed of 250 rpm / min. After 4 hours, the microspheres were washed 8 times with deionized water and then freeze-dried to obtain PLLA microspheres.

[0043] The morphology and size of PLLA microspheres and CP microspheres were observed by scanning electron microscopy. Figure 1 As shown in Figure 2, there is no significant change in the size of CP microspheres compared to PLLA microspheres. On the surface of the microspheres, the surface of the microspheres becomes rough after loading calcium peroxide.

[0044] By qualitatively characterizing the elements of CP microspheres, Figure 2 The mapping diagram of the energy spectrum of CP microspheres is shown. The results show that the surface of the microspheres contains calcium elements, and the broken CP microspheres show more calcium elements distributed, indicating that calcium peroxide has been successfully loaded into the core and shell of the microspheres. The loading of polylactic acid microspheres can slow down the reaction of calcium peroxide with water and achieve slow oxygen release.

[0045] Example 2 Preparation of composite carrier (CP / SA) for sustainable oxygen production

[0046] The preparation of the composite carrier (CP / SA) for sustainable oxygen production described in this example is as follows:

[0047] Since calcium peroxide reacts with water to generate hydrogen peroxide, if hydrogen peroxide cannot be decomposed in time, it will cause damage to cells. Therefore, catalase is introduced into the oxygen-generating system to accelerate its decomposition. Prepare a SA solution with a mass concentration of 20 mg / mL, in which the concentration of catalase is 5 mg / mL and the concentration of CP microspheres is 2 mg / mL. Mix the three at a speed of 1500 rpm / min, spray it into a calcium chloride solution (concentration 40 mg / mL) through voltage, rinse with deionized water, and prepare a composite carrier (CP / SA). Since the carrier particle size is required to be between 100-500 μm, the voltage needs to be adjusted to a certain value. The high voltage is 20 kv and the low voltage is 7 kv to spray out the carrier with the particle size that meets the requirements.

[0048] Using SA as a control, a SA solution with a mass concentration of 20 mg / mL was prepared, sprayed into a calcium chloride solution (concentration 40 mg / mL) through voltage, and rinsed with deionized water to obtain the carrier SA. The voltage needs to be adjusted to a certain value, with the high voltage at 20 kV and the low voltage at 7 kV.

[0049] Observation of the composite carrier (CP / SA) for sustainable oxygen production by optical microscopy. Figure 3 It was shown that the particle size of the composite carrier (CP / SA) with sustainable oxygen production was about 300 μm and had good sphering performance. The dispersion of CP in the microgel was good, which could achieve the purpose of local oxygen supply and facilitate cell proliferation.

[0050] In the present invention, the release of oxygen is very critical. Oxygen is generated inside the composite carrier, and the long-term release maintains the stability of oxygen inside the composite carrier. The dissolved oxygen in the composite carrier medium was measured for 14 days ( Figure 4 ). The CP / SA sample could maintain the dissolved oxygen content in the composite carrier higher than that of the control group alginate (SA) until the 14th day.

[0051] Example 3 Preparation of a composite carrier (CP / SA) loaded with cells for sustainable oxygen production

[0052] Based on Example 2, the number of configured is 1×10 6 The suspension of bone marrow mesenchymal stem cells (BMSCs) was added to the SA solution containing CP and catalase, and mixed at a speed of 1500 r / min. It was sprayed into a calcium chloride solution (concentration 40 mg / mL) under the action of voltage, and rinsed with deionized water to obtain a composite carrier loaded with bone marrow mesenchymal stem cells that continuously produces oxygen.

[0053] SA loaded with bone marrow mesenchymal stem cells was used as a control, and the number of cells was 1×10 6 The suspension of bone marrow mesenchymal stem cells (BMSCs) of 100 mg / mL was added to the SA solution and mixed at a speed of 1500 r / min. The suspension was sprayed into a calcium chloride solution (concentration 40 mg / mL) under voltage and rinsed with deionized water to obtain SA loaded with bone marrow mesenchymal stem cells.

[0054] After culturing bone marrow mesenchymal stem cells in microgels for 3 days, the cell division in the composite carrier (CP / SA) with sustainable oxygen production was observed by confocal microscopy after staining the cell nucleus and cytoskeleton. Figure 5 It shows that cells are dividing in different observed areas, which indicates that cells can stably divide, differentiate and proliferate inside the composite carrier (CP / SA) with sustainable oxygen production.

[0055] After culturing bone marrow mesenchymal stem cells in microgels for 7 days, the nuclei were stained, and the living cells were stained and observed and quantitatively analyzed using confocal microscopy. Figure 6 As shown in the figure, through quantitative statistics, the fluorescence intensity of living cells in the composite carrier (CP / SA) with sustainable oxygen production is higher than that in SA. HIF-1α in cells was stained and observed by confocal microscopy and quantitatively analyzed. The HIF-1α factor will show strong fluorescence in a relatively hypoxic environment. When the oxygen content is relatively high, the fluorescence intensity of HIF-1α is low. Figure 7 As shown, the fluorescence intensity of CP / SA is lower than that of SA, which indicates that the intracellular oxygen content of the composite carrier (CP / SA) with sustainable oxygen production is relatively high.

[0056] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a composite carrier capable of sustainable oxygen production, characterized in that: The following steps are included: (1) Preparation of calcium peroxide-loaded polylactic acid microspheres: polylactic acid particles are dissolved in chloroform, calcium peroxide particles are added, the mixture is mixed and ultrasonically treated to obtain a suspension; During the stirring process, the suspension is added to a high-concentration polyvinyl alcohol solution of calcium peroxide nanoparticles for emulsification, and then added to a low-concentration polyvinyl alcohol solution for stirring at room temperature, washed, and freeze-dried to obtain polylactic acid microspheres loaded with calcium peroxide; (2) Preparation of a composite carrier for sustainable oxygen production: adding polylactic acid microspheres loaded with calcium peroxide and catalase to a sodium alginate solution, mixing and rapidly stirring to obtain a sodium alginate solution containing catalase and polylactic acid microspheres, and spraying the solution into a calcium chloride solution by voltage jetting to obtain a composite carrier for sustainable oxygen production; In step (1), the concentration of polylactic acid in chloroform is 50 mg / mL, the mass concentration of the high-concentration polyvinyl alcohol solution is 20 mg / mL, and the mass concentration of the low-concentration polyvinyl alcohol solution is 5 mg / mL; the volume ratio of chloroform to high-concentration polyvinyl alcohol is 1:2; the volume ratio of the suspension to low-concentration polyvinyl alcohol is 1:10; The mass concentration of the calcium peroxide nanoparticles in the high concentration polyvinyl alcohol solution in step (1) is 5-20 mg / mL; In step (2), the mass concentration of the sodium alginate solution is 10-40 mg / mL, the mass concentration of the catalase is 5 mg / mL, the mass concentration of the polylactic acid microspheres loaded with calcium peroxide is 2 mg / mL, and the mass concentration of the calcium chloride solution is 40 mg / mL.

2. The method for preparing a sustainable oxygen-producing composite carrier according to claim 1, characterized in that: The stirring rate in step (2) may be 1000-1500 r / min.

3. A composite carrier capable of sustainable oxygen production, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 2.

4. The composite carrier for sustainable oxygen production according to claim 3, characterized in that: The particle size of the composite carrier capable of sustainably producing oxygen is 100-500 μm.

5. The composite carrier for sustainable oxygen production according to claim 4, characterized in that: The voltage is 20 kv-7 kv.

6. Use of the composite carrier for sustainable oxygen production according to any one of claims 3 to 5 in organoids, characterized in that: The application specifically includes adding a bone marrow mesenchymal stem cell suspension into a composite carrier solution that can sustainably produce oxygen, and then electrostatically spraying it into a calcium chloride solution to obtain a composite carrier that can sustainably produce oxygen and load cells. After culturing for 3 days, the cells inside the composite carrier are fluorescently stained to observe the division and differentiation status of the cells inside. After culturing for 7 days, the living cells inside the carrier are fluorescently stained as well as the intracellular hypoxia fluorescent staining, and quantitative statistics are performed to prove the potential utilization value of the oxygen-producing composite carrier.

7. Use of the composite carrier for sustainable oxygen production in organoids according to claim 6, characterized in that: The cell concentration in the cell suspension is 1×10 6 / mL, and the mass concentration of the calcium chloride solution is 40 mg / mL.

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