Chloroplast-sodium alginate microspheres and preparation method and use thereof

Abstract

The present invention discloses a chloroplast-sodium alginate microsphere and its preparation method and use. The preparation method of the chloroplast-sodium alginate microsphere comprises: mixing chloroplasts, sodium alginate and distilled water to obtain a mixed solution of chloroplasts and sodium alginate as a dispersed phase; dissolving a nonionic surfactant and CaI2 or CaCl2 in liquid paraffin to form an oil phase as a continuous phase; the dispersed phase and the continuous phase are cross-linked under the operation of a microfluidic device to form chloroplast-sodium alginate microspheres. The chloroplast-sodium alginate microspheres of the present invention have the ability to generate oxygen under illumination and are sustainable for up to two weeks. Chloroplasts are immunomodulated by reprogramming sugar metabolism to achieve anti-inflammatory and repair-promoting effects. The chloroplast-sodium alginate microspheres are suitable for wound repair, especially for chronic hypoxic wound repair similar to diabetic wounds.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a chloroplast-sodium alginate microsphere and a preparation method and application thereof. Background Art

[0002] Diabetic ulcers are the most common public health problem in the world, which causes delayed wound healing and even amputation due to long-term chronic hypoxia and excessive inflammation, and seriously affects the quality of life of patients. Current therapies to improve oxygen supply to diabetic wounds include: hyperbaric oxygen therapy, local oxygen therapy, and some biomaterial therapies that produce or carry oxygen such as hemoglobin and algae. However, hyperbaric oxygen therapy has the side effect of systemic toxicity, local oxygen therapy is difficult to achieve long-term continuous oxygen supply, and biomaterial therapy has potential safety issues. At present, the problem of curing diabetic ulcers remains thorny. Summary of the invention

[0003] In order to solve the deficiencies in the prior art, the present invention aims to provide a chloroplast-sodium alginate microsphere and a preparation method and use thereof.

[0004] The specific technical solutions of the present invention are as follows:

[0005] The present invention provides a method for preparing chloroplast-sodium alginate microspheres, comprising the following steps:

[0006] (1) mixing chloroplasts, sodium alginate and distilled water to obtain a mixed solution of chloroplasts and sodium alginate, wherein the mixed solution is used as a dispersed phase;

[0007] (2) dissolving a nonionic surfactant and CaI2 or CaCl2 in liquid paraffin to form an oil phase, and the obtained oil phase serves as a continuous phase;

[0008] (3) The dispersed phase and the continuous phase are cross-linked under the operation of a microfluidic device to form chloroplast-sodium alginate microspheres.

[0009] Furthermore, in step (2), the nonionic surfactant is selected from Span 80 or tween.

[0010] Furthermore, in step (2), a nonionic surfactant and CaI2 are dissolved in liquid paraffin to form an oil phase.

[0011] Furthermore, in step (3), the flow rate of the dispersed phase is 1-5 μL / min, and the flow rate of the continuous phase is 40-60 μL / min.

[0012] Furthermore, in step (1), the mass ratio of the chloroplast to sodium alginate is 1:1;

[0013] And / or, the nonionic surfactant is selected from Span 80, and the volume fraction of Span 80 in the oil phase is 2.5%-5%.

[0014] Further, step (1) is specifically: mixing 20 mg of chloroplasts, 20 mg of sodium alginate and 1 mL of distilled water to obtain a mixed solution of chloroplasts and sodium alginate;

[0015] Step (2) is specifically as follows: 20 mg of CaI2 and 500 μL of Span 80 are dissolved in 20 mL of liquid paraffin to form an oil phase.

[0016] Furthermore, the chloroplasts are extracted from fresh water hyacinth.

[0017] Furthermore, the formed chloroplast-sodium alginate microspheres are repeatedly washed with excess hexane, isopropanol and distilled water to separate the microspheres.

[0018] The present invention also provides chloroplast-sodium alginate microspheres prepared by the preparation method.

[0019] The present invention also provides use of the chloroplast-sodium alginate microspheres in preparing wound repair biological materials.

[0020] Furthermore, the wound repair is diabetic wound repair.

[0021] The beneficial effects of the present invention are:

[0022] The chloroplast-sodium alginate microspheres provided by the present invention have the ability to generate oxygen under light and are sustainable for up to two weeks. Chloroplasts perform immune regulation through sugar metabolism reprogramming to achieve anti-inflammatory and repair-promoting effects. The chloroplast-sodium alginate microspheres are suitable for wound repair, especially for the repair of chronic hypoxic wounds similar to diabetic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The graphs of different chloroplast contents and oxygen release in chloroplast-sodium alginate microspheres;

[0024] Figure 2 It is a fluorescence image of the immunomodulatory effect of chloroplast-alginate microspheres (20 mg / mL CP in MRM-EX) on macrophages;

[0025] Figure 3 for the recyclable release of oxygen and long-term stability in chloroplast-alginate microspheres;

[0026] Figure 4 Hemolytic analysis of chloroplast-alginate microspheres (20 mg / mL CP in MRM-EX);

[0027] Figure 5 The effect of chloroplast-alginate microspheres (20 mg / mL CP in MRM-EX) on the cell viability of RS-1 and RAW 264.7;

[0028] Figure 6 The wound repair effect of chloroplast-alginate microspheres (20 mg / mL CP in MRM-EX) on diabetic pigs;

[0029] Among them, MRM-EX stands for chloroplast-sodium alginate microspheres; SA stands for sodium alginate microspheres, MRM-END stands for metformin microspheres, and MRM-M stands for chloroplast-metformin microspheres. DETAILED DESCRIPTION

[0030] In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and accompanying drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. In the examples, each raw material reagent is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0031] Example 1

[0032] This embodiment provides a chloroplast-sodium alginate microsphere, which mainly contains two components: chloroplast and sodium alginate (SA). Sodium alginate can react with calcium ions to form a three-dimensional complex, which is cross-linked under the operation of a microfluidic device to form a chloroplast-sodium alginate microsphere. The specific preparation process is as follows:

[0033] (1) Extraction of chloroplasts: Take 5 g of fresh water hyacinth, tear off the veins, cut as much as possible, and put it into a mortar. Add pre-cooled chloroplast extraction reagent, continue grinding, filter with a mesh, centrifuge at 300 g for 2 minutes, collect the supernatant, centrifuge at 1000 g for 2 minutes, collect the precipitate as chloroplasts, and suspend in PBS and store at 4°C.

[0034] (2) SA (20 mg) and extracted chloroplasts (20 mg) were dissolved in 1 mL of distilled water, and the resulting mixed solution was used as the dispersed phase.

[0035] (3) 20 mg of CaI2 and 500 μL of Span 80 were dissolved in 20 mL of liquid paraffin to form an oil phase, which was used as the continuous phase.

[0036] (4) The dispersed phase and the continuous phase are cross-linked to form chloroplast-sodium alginate microspheres under the operation of a microfluidic device, wherein the flow rate of the dispersed phase is 1 μL / min, and the flow rate of the continuous phase is 50 μL / min. The microspheres are repeatedly washed and separated with excess hexane, isopropanol and distilled water.

[0037] Example 2

[0038] The chloroplast-sodium alginate microspheres prepared by the present invention are metabolic regulation hydrogel microspheres containing natural chloroplasts, and have good oxygen supply capacity and immunoregulatory effect. The chloroplast-sodium alginate microspheres (MRM-EX) with different chloroplast contents were prepared by microfluidics using the preparation method of Example 1, and the oxygen content (MRM-EX) generated by the chloroplast-sodium alginate microspheres with different chloroplast contents under 650nm light irradiation within one hour was detected by an oxygen content detector. Figure 1 ). Macrophages were first cultured in anoxic culture for 2 days, and then stimulated with LPS and IFN-γ for 24 hours to induce a pro-inflammatory phenotype of macrophages. Then PBS, sodium alginate microspheres and chloroplast-sodium alginate microspheres were placed in tranwells and co-cultured with cells for 24 hours, and irradiated with 650nm light for 30 minutes every day. Subsequently, the cells were fixed, permeabilized, incubated with primary antibodies, incubated with secondary antibodies, and stained with DAPI, and the expression of CD206 / CD86 in each group was photographed by confocal microscopy ( Figure 2 ).

[0039] Example 3

[0040] The chloroplast-sodium alginate microspheres prepared by the present invention have good biosafety, can delay the activity of chloroplasts, and can be used in vitro circulation for up to 14 days. The chloroplast-sodium alginate microspheres (MRM-EX) with different chloroplast contents were prepared by microfluidics using the preparation method of Example 1, and the oxygen content produced by the chloroplast-sodium alginate microspheres with different chloroplast contents under 650nm light irradiation within 0.5 hours, as well as the oxygen content without light within 0.5 hours, were detected using an oxygen content detector. Three cycles were tested. The microspheres were stored in a 4-degree refrigerator for 7 days and 14 days, and the oxygen production after illumination within one hour was tested ( Figure 3 ). Through hemolysis detection, equal amounts of microspheres and red blood cells were incubated at 37 degrees for 4 hours, and then centrifuged to photograph the hemolysis of red blood cells ( Figure 4 ). The biocompatibility was investigated by staining macrophages incubated with microspheres for 24 hours using a live-dead cell staining kit ( Figure 5 ).

[0041] Example 4

[0042] The chloroplast-sodium alginate microspheres prepared by the present invention have excellent biocompatibility and significantly promote the repair of diabetic wounds. The effect is better than the 3M patch used clinically. One week after STZ was injected into the pig, random blood sugar was tested to determine the construction of a diabetic model. Subsequently, a 1cm*1cm wound was cut on the skin, and different materials were given, and photographed at various time points ( Figure 6 ).

[0043] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing chloroplast-sodium alginate microspheres, characterized in that: The steps include: (1) mixing chloroplasts, sodium alginate and distilled water to obtain a mixed solution of chloroplasts and sodium alginate, wherein the mixed solution is used as a dispersed phase; (2) dissolving a nonionic surfactant and CaI2 or CaCl2 in liquid paraffin to form an oil phase, and the obtained oil phase serves as a continuous phase; (3) The dispersed phase and the continuous phase are cross-linked under the operation of a microfluidic device to form chloroplast-sodium alginate microspheres.

2. The preparation method according to claim 1, characterized in that: In step (2), the nonionic surfactant is selected from Span 80 or tween.

3. The preparation method according to claim 1, characterized in that: In step (2), a nonionic surfactant and CaI2 are dissolved in liquid paraffin to form an oil phase.

4. The preparation method according to claim 1, characterized in that: In step (3), the flow rate of the dispersed phase is 1-5 μL / min, and the flow rate of the continuous phase is 40-60 μL / min.

5. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of the chloroplast to sodium alginate is 1:1; And / or, the nonionic surfactant is selected from Span 80, and the volume fraction of Span 80 in the oil phase is 2.5%-5%.

6. The preparation method according to claim 1, characterized in that: Step (1) is specifically: mixing 20 mg of chloroplasts, 20 mg of sodium alginate and 1 mL of distilled water to obtain a mixed solution of chloroplasts and sodium alginate; Step (2) is specifically as follows: 20 mg of CaI2 and 500 μL of Span 80 are dissolved in 20 mL of liquid paraffin to form an oil phase.

7. The preparation method according to claim 1, characterized in that: The chloroplast is extracted from fresh water hyacinth.

8. Chloroplast-sodium alginate microspheres prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the chloroplast-sodium alginate microspheres according to claim 8 in preparing wound repair biomaterials.

10. The use according to claim 9, characterized in that The wound repair is diabetic wound repair.

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