CN114891727A - A NO microvesicle preparation for promoting in vitro maturation of immature oocytes and a method for promoting in vitro maturation of oocytes - Google Patents

Abstract

The invention belongs to the field of reproductive biology, and particularly relates to an NO microbubble preparation for promoting in-vitro maturation of immature oocytes and a method for promoting in-vitro maturation of oocytes, wherein the NO microbubble preparation consists of a shell membrane layer and an inner layer cavity, and the inner layer cavity is filled with gas containing NO; the shell membrane layer comprises a lipid material, a foaming agent and a freeze-drying propping agent, and the lipid material, the foaming agent and the freeze-drying propping agent are mixed and then added with a solvent for freeze drying to prepare freeze-dried powder so as to obtain a shell membrane layer; the lipid material component is selected from one or more of natural phospholipid, hydrogenated phospholipid, synthetic phospholipid and polyethylene glycol modified derivatives thereof; the foaming agent is tween-80; the lyophilized proppant is Poloxamer 188. The NO microbubble preparation provided by the invention can realize long-acting release of NO when being added into a conventional oocyte in-vitro culture solution, has high biological safety, promotes the in-vitro maturation rate of the oocyte in the GV stage, and has good application prospect.

Description

A NO microvesicle preparation for promoting in vitro maturation of immature oocytes and method for promoting in vitro maturation of oocytes

technical field

The invention belongs to the field of reproductive biology, and in particular relates to a NO microbubble preparation for promoting the in vitro maturation of immature oocytes and a method for promoting the in vitro maturation of oocytes.

Background technique

Infertility has become a common disease that cannot be ignored in today's society. In vitro maturation of immature oocytes (in vitro maturation, IVM) refers to obtaining immature eggs from infertile patients, simulating the mature environment of in vivo oocytes, culturing the eggs in vitro to maturity, and then fertilizing the embryos in vitro. Transplant (IVF-ET) technique for the treatment of infertility.

As a cell signaling molecule, nitric oxide (NO) is widely present in organisms. Studies have shown that NO is involved in various stages of female reproduction, such as follicle development, oocyte maturation, ovulation, luteal degeneration, fertilization, embryo implantation Ingestion, pregnancy maintenance, labor regulation, and menstrual/estrous cycle regulation. The promoting effect of NO on oocyte maturation may be due to increased cGMP levels and decreased cAMP activation. However, excess NO also inhibits oocyte maturation. These indicate that NO participates in and regulates the oocyte maturation process.

Given the diversity of NO functions, it has great medicinal potential in cardiovascular therapy, anticancer therapy, and antibacterial therapy. However, since the half-life of NO in the body is very short, only a few seconds, it generally only plays a local role, and the route of inhaled NO administration is inconvenient, and the commonly used NO donor substances such as sodium nitroprusside (SNP) have certain cytotoxicity. It is not conducive to the in vitro culture of oocytes. Therefore, the development of a clinically useful NO administration method can be used as a form of NO transport in vivo and as a storage form, and has broad clinical application prospects. One of the attempts is a drug carrier based on lipid materials. system.

From a clinical point of view, an ideal NO microbubble preparation for promoting oocyte maturation in vitro should meet the following requirements: (1) high biosafety, non-toxic and non-irritating materials; (2) stable preparation properties, easy to transport and (3) After adding into the culture system, it can release NO gas stably and long-term, and provide exogenous NO during the in vitro maturation and culture of immature oocytes. Currently, drug delivery with high biosafety microbubbles and lipid materials is rarely used in the field of reproduction.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a NO microvesicle preparation for promoting the in vitro maturation of immature oocytes and a method for promoting the in vitro maturation of oocytes.

The technical solutions adopted in the present invention are as follows: A NO microbubble preparation for promoting the in vitro maturation of immature oocytes, which is composed of a shell membrane layer and an inner layer cavity, and the inner layer cavity is filled with gas containing NO; The shell film layer includes lipid material, foaming agent, and freeze-dried proppant. After mixing the lipid material, foaming agent, and freeze-dried support, tert-butanol is added for freeze-drying to prepare freeze-dried powder to obtain a shell. film layer;

The foaming agent is Tween-80;

The lyophilized proppant was Poloxamer 188.

The lipid material components are selected from one or more of natural phospholipids, hydrogenated phospholipids, synthetic phospholipids and their polyethylene glycol modified derivatives.

The preparation process of the shell membrane layer includes the following steps: a mixture of hydrogenated egg yolk phospholipid and natural phospholipid PC is used as a film-forming material for blank lipid microbubbles, Tween-80 is a foaming agent, and Poloxamer 188 is a freeze-dried proppant, according to 4: The mixture is mixed at a ratio of 10:100 to obtain a mixture, tert-butanol is added, and freeze-dried to obtain a freeze-dried powder.

2 mL of tert-butanol was added per 114 mg of the mixture.

NO gas was added per 20 mg of lyophilized powder.

In a method for promoting the in vitro maturation of oocytes, the above-mentioned NO microvesicle preparation for promoting the in vitro maturation of immature oocytes is used in the process of in vitro culturing of the oocytes.

The final concentration of NO microvesicle preparation in the oocyte in vitro culture system is 0.05-0.5 mg/mL.

The beneficial effects of the present invention are as follows: the NO microbubble preparation provided by the present invention can realize long-term release of NO when added to the in vitro culture solution of conventional oocytes, and has high biological safety, which promotes the in vitro maturation rate of GV stage oocytes, Moreover, it reduces the level of oxygen free radicals and calcium ions in cells, protects mitochondrial function, improves the quality of oocytes, and has good application prospects.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention, and for those of ordinary skill in the art, obtaining other drawings according to these drawings still belongs to the scope of the present invention without any creative effort.

In Figure 1, A is the schematic diagram of the construction process of NO microbubble preparation, B is the comparison of the appearance of lipid microbubble (MB) without NO encapsulation and NO microbubble (NO-MB) preparation, and C is the uncoated NO-encapsulated lipid microbubble (MB) preparation. The comparison of lipid microbubble (MB) and NO microbubble (NO-MB) preparations on the microscopic morphology;

In Figure 2, A is the particle size distribution and particle number of NO microbubbles, and B is the stability test results of lipid microbubble (MB) and NO microbubble (NO-MB) preparations without NO encapsulation;

In Figure 3, A is the safety test result of the NO microbubble preparation, and B is the test result of the change of intracellular NO content with time after the cells were treated with NO microbubble;

In Figure 4, A is the cytoplasmic morphology of oocytes in the control group (Control), NO microbubble treatment group (NO-MB), and SNP administration group (SNP) after culturing for 16-18 h under the microscope, and B is the control group (Control), the comparison of the maturation rate of the NO microbubble treatment group (NO-MB) and the SNP administration group (SNP) after culturing for 16-18 h;

Figure 5 is the detection of (A) NO content (B) calcium ion content (C) mitochondrial membrane potential change (D) ROS content in oocytes treated with NO microvesicles using fluorescent probes.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

(1) Preparation of lipid microbubbles

Use different types and proportions of phospholipid materials or mixtures of phospholipid materials, foaming agents and lyophilized proppants to prepare lipid microbubbles, and determine the lyophilized powder form and resolubility, lipid microbubble concentration, lipid microbubble morphology, The lipid microvesicle particle size and particle size distribution were used as indicators to evaluate the quality of lipid microvesicles. The phospholipid components are selected from one or more of natural phospholipids, hydrogenated phospholipids, synthetic phospholipids and their polyethylene glycol modified derivatives.

Mix the phospholipid material or phospholipid material mixture, foaming agent and freeze-dried proppant according to the raw materials shown in Table 1 to obtain a mixture, add tert-butanol (the amount of tert-butanol is: 2 mL per 114 mg of the mixture), and heat the mixture to 65 ° C Completely dissolve and mix, and freeze-dried to obtain freeze-dried powder.

Preliminary method for evaluating the quality of microvesicles: draw a small amount of microvesicle freeze-dried powder sample suspension and spot it on a cell counting plate, observe the number of microvesicles in each cell of the cell counting plate through an optical microscope (400 times) and convert it into The concentration of microbubbles, the percentage A of microbubbles in the range of particle size of 2-8 μm, and the percentage of microbubbles in the range of particle size of 2-8 μm after 10 minutes were used to evaluate the quality of microbubbles.

Among them, the quality of the microbubbles prepared in Example 1 is the best, and the microbubbles prepared in Example 1 are selected for the following experiments.

(2) Preparation and stability experiments of NO microbubbles

Weigh 20mg of the prepared blank lipid microbubble freeze-dried powder, add it to a vial and seal it, and extract the gas in the bottle, add 1ml NO gas with a syringe, it can be kept for later use, and 1ml working solution can be added when using, and it can be prepared after mixing. to the required concentration. As shown in Figure 1, there was no significant difference in appearance between the lipid microbubble (MB) and NO microbubble (NO-MB) preparations without encapsulating NO. As shown in Figure 2, the particle size of NO microbubbles was 90% <3.681 μm. Compared with lipid microbubbles without NO encapsulation, NO microbubbles had higher stability at room temperature.

(3) NO microbubble cytotoxicity test and detection of intracellular NO concentration

Cytotoxicity experiment: collect human granulosa cells, digest them into single cells, and then carry out primary cell culture. When the cell density is appropriate, prepare a cell suspension for cell counting, and then plate 96-well plates according to the appropriate cell number (about 4×104). About 100ul of cell suspension in the well, cultured at 37°C, after the cells adhered to the wall, different concentrations of NO microbubbles could be added for treatment, and 4-6 replicate wells could be made for each concentration. Cell viability was determined.

Detection of intracellular NO concentration by fluorescent probe method: primary culture of granulated cells was performed as described above, and cell suspension was prepared for cell counting when the cell density was appropriate, and then a 48-well plate was plated according to the appropriate number of cells (about 1.2×105). Taking SNP as a control, different concentrations of preparations were treated for different time (such as 4h, 6h, 12h, 24h, 48h). DAF-FMDA was diluted 1:1000 to a final concentration of 5 μmol/L. Remove the cell culture medium and add an appropriate volume of diluted DAF-FM DA. The volume to be added should be sufficient to cover the cells. Incubate in a 37ºC cell incubator for 20 minutes. Cells were washed three times with PBS (pH 7.4) to sufficiently remove DAF-FM DA that did not enter the cells. Samples loaded with probes in situ can be directly observed with a laser confocal microscope, or cells can be collected and detected with a fluorescence microplate reader or flow cytometer.

As shown in Figure 3, the MTT results showed that NO microbubbles were significantly less toxic than SNPs at a concentration of 5 mg/mL. In the intracellular NO detection test, the intracellular NO content was detected at different time points. It can be seen that compared with the blank control group, both the NO microbubble preparation and SNP can significantly increase the intracellular NO content, but the NO microbubble and SNP can significantly increase the intracellular NO content. There were no significant differences between SNPs.

(4) In vitro maturation culture of mouse immature oocytes

Female mice were intraperitoneally injected with PMSG 10IU and killed by cervical dislocation at 44-46 hours. Take out both ovaries, puncture the follicles with a 1ml needle under a stereo microscope, collect germinal vesicle GV oocytes, and place them in 37°C, CO2 incubators for 2-6 hours to equilibrate with different concentrations of NO microbubble preparations, SNPs Or cultured in general IVM medium without other substances.

(5) Evaluate egg quality and evaluate the effect of NO microbubble preparation and SNP on egg quality

Observation of in vitro maturation of immature oocytes: Observe the maturation rate of oocytes and the morphology of mature eggs after adding NO microbubbles or SNPs into the IVM culture system. As shown in Figure 4, observed under the microscope, the NO microvesicle treatment group had a higher maturation rate, and the cytoplasmic morphology of mature oocytes was better than that of the control group and the SNP administration group.

Determination of mitochondrial membrane potential: The mitochondrial membrane potential was measured with JC-1 lipophilic cationic fluorescent dye, observed and analyzed by fluorescence microscope. High membrane potential mitochondria are in red and low membrane potential in green. Detection of reactive oxygen species (ROS) levels: ROS detection was performed using the fluorescent probe DCFH-DA. Intracellular calcium ion level detection: The intracellular calcium ion fluorescent probe Fluo-4 AM is used to detect the calcium ion level. The above three fluorescent probes were diluted at a ratio of 1:1000 to make the final concentration 5 μmol/L. Remove the cell culture medium and incubate in a 37ºC cell incubator for 20 minutes. Cells were washed three times with PBS (pH 7.4) to sufficiently remove fluorescent probes that did not enter the cells. Samples can be directly observed with a laser confocal microscope, or cells can be collected and detected with a fluorescence microplate reader or flow cytometer. As shown in Figure 5, the NO microbubble formulation reduced the level of intracellular oxygen free radicals, increased the concentration of calcium ions, and protected mitochondrial function.

The above disclosures are only preferred embodiments of the present invention, and of course, the scope of the rights of the present invention cannot be limited by this. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims (7)

1. An NO microvesicle preparation for promoting in vitro maturation of immature oocytes, comprising: the gas-filled composite membrane consists of a shell membrane layer and an inner layer cavity, wherein the inner layer cavity is filled with gas containing NO; the shell membrane layer comprises a lipid material, a foaming agent and a freeze-drying propping agent, wherein the lipid material, the foaming agent and the freeze-drying propping agent are mixed and then added with tert-butyl alcohol for freeze drying to prepare freeze-dried powder so as to obtain the shell membrane layer;

the foaming agent is tween-80;

the lyophilized proppant is Poloxamer 188.

2. The preparation of NO microvesicles for promoting the in vitro maturation of immature oocytes according to claim 1, wherein: the lipid material component is selected from one or more of natural phospholipid, hydrogenated phospholipid, synthetic phospholipid and polyethylene glycol modified derivatives thereof.

3. The preparation of NO microvesicles for promoting the in vitro maturation of immature oocytes according to claim 1, wherein: the preparation process of the shell membrane layer comprises the following steps: the mixture of hydrogenated yolk phospholipid and natural phospholipid PC is used as a film forming material of blank lipid microbubbles, Tween-80 is used as a foaming agent, Poloxamer 188 is used as a freeze-dried proppant, and the weight ratio of the mixture is as follows 4: 10: mixing at a ratio of 100 to obtain a mixture, adding tert-butanol, and freeze-drying to obtain lyophilized powder.

4. The preparation of NO microvesicles for promoting the in vitro maturation of immature oocytes according to claim 3, wherein: 2mL of t-butanol was added per 114mg of the mixture.

5. The preparation of NO microvesicles for promoting the in vitro maturation of immature oocytes according to claim 1, wherein: NO gas was added per 20mg of lyophilized powder.

6. A method of promoting oocyte maturation in vitro, comprising: use of a NO microvesicle preparation according to any one of claims 1 to 5 for promoting the in vitro maturation of immature oocytes during the in vitro culture of oocytes.

7. The method of claim 6, wherein: the final concentration of the NO microvesicle preparation in the oocyte in-vitro culture system is 0.05-0.5 mg/mL.

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