Melatonin nanoparticles, methods of making and uses thereof

Abstract

The application discloses a kind of melatonin nanoparticles and preparation method and purposes thereof.The nanoparticles are composed of melatonin, bilirubin and protamine, and the mass ratio of melatonin, bilirubin and protamine is 3:(1-5):(1-10).The preparation method comprises first preparing a mixed solution of melatonin and bilirubin, then slowly dropping the mixed solution into a protamine solution, removing organic solvent after ultrasonic treatment, and freeze-drying to obtain melatonin nanoparticles.The prepared melatonin nanoparticles are simple to prepare, have high drug loading capacity and good stability, can significantly improve embryo implantation rate, pregnancy rate and birth rate by improving local microenvironment after co-transplantation with embryos, exhibit significant embryo protection effect, and have good clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanopharmaceutical carriers and medical technology, and particularly relates to a melatonin nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Human assisted reproductive technology has a history of more than 40 years, but the failure of assisted reproduction is still one of the biggest challenges faced by assisted reproductive technology. There are many factors affecting the clinical outcome of in vitro fertilization-embryo transfer, including age, ovarian reserve, maternal immune response to embryos, endometrial receptivity, embryo quality, ovulation induction scheme, embryo transfer technology, etc. During the embryo transfer process, different transfer fluids will inevitably have an impact on embryo implantation and clinical pregnancy rate. For example, the embryo transfer fluid (EmbryoGlue) developed by the Swedish Vitrolife company simulates the environment of the oviduct and uterus, and has a significant positive and promoting effect in the embryo transfer process.

[0003] Melatonin is one of the hormones secreted by the pineal gland, and also exists in other tissues and organs, such as skin, retina, hematopoietic cells, testes, etc. Studies have shown that melatonin has the functions of relieving insomnia, adjusting jet lag, and regulating circadian rhythm. Recent studies suggest that melatonin and its metabolites have a wide range of antioxidant and free radical scavenging effects, and can regulate cell activity under various physiological and pathological conditions, including apoptosis, inflammatory response, proliferation, etc. Bilirubin is usually considered to be the cause of jaundice and should be excreted as a harmful metabolite. However, in fact, bilirubin has a strong antioxidant effect and can directly interact with active oxygen. Protamine is a basic protein that mainly exists as a nucleoprotein combined with DNA in mature sperm cell nuclei of fish (such as salmon, trout, herring, etc.).

[0004] So far, there has been no related report on the preparation of an embryo transfer fluid for the process of in vitro fertilization and embryo transfer by combining melatonin, bilirubin and protamine preparations. SUMMARY

[0005] In order to solve the problems and deficiencies existing in the prior art, the present application provides a melatonin nanoparticle and a preparation method thereof, and the use of an embryo transfer fluid / preservation fluid in the process of in vitro fertilization and embryo transfer.

[0006] The technical scheme of the present application is as follows:

[0007] I. A melatonin nanoparticle:

[0008] The melatonin nanoparticle is mainly composed of melatonin, bilirubin and protamine.

[0009] The mass ratio of the melatonin, bilirubin and protamine is 3:(1-5):(1-10).

[0010] Preferably, the mass ratio of the melatonin, the bilirubin and the protamine is 3:2:5.

[0011] The particle size of the melatonin nanoparticles is 100-300 nm.

[0012] Through the above arrangement, the present application first proposes a melatonin nanoparticle composed of melatonin, bilirubin and protamine. Melatonin and bilirubin are both endogenous antioxidant anti-inflammatory substances, which synergistically regulate the local microenvironment to achieve embryo protection. Protamine can further improve the stability of the melatonin / bilirubin complex and improve its bioadhesion, increase the drug enrichment concentration around the embryo, and synergistically exert the protective effect with the active drug. This effective melatonin nanoparticle system has potential in embryo transfer fluid / preservation fluid. The present application also discloses a preparation method of the nanoparticle, which is simple, easy to operate and has high operability. At present, there is no report on the combination of melatonin, bilirubin and protamine to construct a nanoparticle delivery system and its protective effect in embryo transfer or preservation.

[0013] II. A preparation method of melatonin nanoparticles, the method comprising the following steps:

[0014] (1) Melatonin and bilirubin are dissolved in dimethyl sulfoxide to prepare a mixed solution I;

[0015] (2) Protamine is dissolved in water to obtain a protamine solution II;

[0016] (3) The mixed solution I is slowly dropped into the protamine solution II, and after continuous stirring for 8-24 hours, ultrasonic treatment is performed to obtain a solution III;

[0017] (4) The solution III is dialyzed to remove the organic solvent dimethyl sulfoxide, and the melatonin nanoparticles are obtained after freeze-drying after dialysis.

[0018] The ultrasonic treatment is performed at 60 W, ultrasonic treatment for 2 s, intermittent treatment for 2 s, and ultrasonic treatment for 5 min.

[0019] In the solution III obtained in step (3), the mass ratio of the melatonin, the bilirubin and the protamine is 3:(1-5):(1-10).

[0020] In the mixed solution I, the concentration of the melatonin is 5 mg / mL.

[0021] The volume ratio of the solution I to the solution II is 1:2.

[0022] The melatonin nanoparticles of the present application can be used in the preparation of embryo transfer fluid / preservation fluid for in vitro fertilization and embryo transfer.

[0023] The embryo transfer liquid / preservation liquid also comprises a pharmaceutically acceptable carrier, excipient or acceptable adjuvant for culture liquid.

[0024] The embryo transfer liquid / preservation liquid is used for fertilization operation, culture of fertilized eggs, culture operation before embryo transfer and embryo transfer operation.

[0025] The innovative mechanism of the present application: melatonin and bilirubin can improve the embryo transfer or culture microenvironment by regulating oxidative stress and inflammatory response in the local microenvironment, and improve the embryo implantation rate, pregnancy rate and birth rate. Protamine improves the stability of the melatonin / bilirubin complex and improves the bioadhesion effect, enriches the nanoparticles on the surface of the embryo, avoids loss after transfer, and realizes long-acting protection.

[0026] The melatonin nanoparticle composition provided by the present application is accurate, all of which are endogenous components, and has good biocompatibility.

[0027] The beneficial effects of the present application are as follows:

[0028] The nanoparticles of the present application can simultaneously exert the protective effects of melatonin, bilirubin and protamine, and solve the bottleneck problem of easy damage in embryo transfer and preservation.

[0029] The present application prepares nanoparticles by co-solvent induced self-assembly and cross-linking denaturation, which plays a role in solubilization, stabilization and local enrichment and release of melatonin and bilirubin. Moreover, the whole preparation process is simple in operation, stable in condition, good in reproducibility and high in drug loading. The present application finds through a large number of tests that the melatonin nanoparticles have a significant and good protective effect on embryo transfer and embryo preservation, have good biocompatibility, small toxic and side effects, low cost, and have a good clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The transmission electron microscope image of the melatonin nanoparticles prepared in Example 1 of the present application;

[0031] Figure 2 The result graph of the influence of adding different samples in the culture medium on the blastocyst development rate. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below with reference to examples.

[0033] The examples and comparisons of the present application are as follows:

[0034] 1. Preparation of melatonin nanoparticles

[0035] According to the following Table 1, 30 mg of melatonin and bilirubin are dissolved in dimethyl sulfoxide to form solution I; an appropriate amount of protamine is dissolved in 12 mL of deionized water to form solution II; solution I is slowly dripped into solution II, and after continuous stirring for 12 hours, probe ultrasonic treatment is performed with the following parameters: 60 W, ultrasonic treatment for 2 s, intermittent treatment for 2 s, and ultrasonic treatment for 5 min to obtain solution III; after dialysis of the obtained mixture III to remove organic reagents, freeze-drying is performed to obtain melatonin nanoparticles.

[0036] The morphology of the melatonin nanoparticles obtained above is examined by transmission electron microscopy. Specifically, the melatonin nanoparticles are diluted to an appropriate concentration, dropped on a carbon-coated copper grid to prepare a sample, and then the morphology of the sample is observed under a transmission electron microscope.

[0037] According to the same method, the proportion of the materials is changed to obtain melatonin nanoparticles, and a proportion control group and a replacement control group are set.

[0038] Table 1 Formulation of melatonin nanoparticles

[0039]

[0040]

[0041] The above 1-6 are examples of the present application, and 7-16 are comparative examples.

[0042] 2. Test process of examples and comparative examples:

[0043] 2.1) Characterization of the properties of melatonin nanoparticles

[0044] The sample prepared in Example 1 is resuspended with PBS, and then appearance observation is performed, and the particle size of the sample that is clear and has a Tyndall effect is measured. The results are shown in Table 2. The sample that meets the requirements is further tested for storage stability, i.e., the sample resuspended with PBS is placed at room temperature for 1 month, the average size of the nanoparticles is detected by dynamic light scattering, and the appearance change is observed.

[0045] Table 2 Characterization of the properties of melatonin nanoparticles

[0046]

[0047]

[0048] The melatonin nanoparticles prepared by the present application have a size range of about 100-300 nm, and under appropriate formulation proportions and preparation conditions, the melatonin nanoparticles can be successfully prepared, so the preparation method is reasonable and simple. The transmission electron micrograph of the sample 1 melatonin nanoparticles prepared according to the present application is as follows: Figure 1As shown, the appearance thereof is spherical, the particle size distribution is uniform, and the basic characteristics of nanoparticles are exhibited.

[0049] When the ratio of melatonin, bilirubin and protamine is not appropriate, the nanoparticles cannot be formed or cannot be stably present. When the volume of the selected organic reagent is not appropriate, the nanoparticles can also not be formed. Therefore, referring to the provided preparation method (referring to sample groups 1-6), melatonin nanoparticles with suitable particle size can be successfully prepared.

[0050] The melatonin nanoparticles prepared by the present application have good storage stability. After being stored at room temperature for 1 month, the nanoparticles still maintain good nanostructure.

[0051] 2.2) Protective effect of melatonin nanoparticles on oxidative stress damage of mouse embryos

[0052] The melatonin nanoparticle sample prepared in Example 1 or the free drug was dispersed in KSOM culture medium. ICR mouse zygotes were randomly divided into groups, and the zygotes were moved to a four-well plate containing the corresponding culture medium for culture (37°C, 5% CO2), and then statistical analysis was performed after the zygotes developed to the blastocyst stage.

[0053] The grouping conditions are as follows:

[0054] (1) Normal group: normal KSOM culture medium

[0055] (2) H2O2 group: normal KSOM culture medium + 25 μM H2O2

[0056] (3) Control group 1: normal KSOM culture medium + 25 μM H2O2 + N-acetyl-L-cysteine (3 μg / mL)

[0057] (4) Control group 2: normal KSOM culture medium + 25 μM H2O2 + melatonin (3 μg / mL)

[0058] (5) Control group 3: normal KSOM culture medium + 25 μM H2O2 + melatonin (3 μg / mL) + bilirubin (2 μg / mL) + protamine (5 μg / mL)

[0059] (6) Experimental group 1: normal KSOM culture medium + 25 μM H2O2 + sample group 1

[0060] (7) Experimental group 2: normal KSOM culture medium + 25 μM H2O2 + sample group 2

[0061] (8) Experimental group 3: normal KSOM culture medium + 25 μM H2O2 + sample group 3

[0062] (9) Experiment group 4: normal KSOM medium + 25 μM H2O2 + sample group 4

[0063] (10) Experiment group 5: normal KSOM medium + 25 μM H2O2 + sample group 5

[0064] (11) Experiment group 6: normal KSOM medium + 25 μM H2O2 + sample group 6

[0065] (12) Experiment group 7: normal KSOM medium + 25 μM H2O2 + sample group 9

[0066] (13) Experiment group 8: normal KSOM medium + 25 μM H2O2 + sample group 16

[0067] It was found through in vitro culture that the blastocyst morphology of the normal group was full, and the blastocyst rate and development rate were more than 85%. Compared with the normal group, the blastocyst development rate of the H2O2 group was significantly lower than that of the normal group, and the blastocyst development rate was reduced to 58%, which had a significant difference. Compared with the normal group, the blastocyst morphology of the H2O2 group was smaller than that of the normal group, and the morphology difference was larger.

[0068] The results of the blastocyst development rate of each group are shown in Table 1. Figure 2 As shown in Table 1, compared with H2O2, the blastocyst development rate was improved after adding the control sample of the present application, but the difference was not significant. Only the experiment groups adding sample groups 1-6 and sample 9 of the present application had significance.

[0069] 2.3) In vivo application of the melatonin nanoparticle embryo transfer fluid

[0070] After the embryos were pre-treated by incubating in the prepared embryo transfer fluid for 3 hours, they were ready for transplantation. 10-week-old female ICR mice were selected and caged with ligated male mice, and the next day the positive was recorded as pseudopregnant. After 3 days, the pseudopregnant mice were anesthetized, a 1.5 cm incision was made in the middle of the back, and the skin and subcutaneous tissue were bluntly separated with forceps: a 1 cm small opening was made in the muscle layer corresponding to the uterine horn, the fat pad was pulled out and fixed; a 1 mL injection needle was used to make a small opening in the uterine horn at the avascular site towards the uterine body, the needle was pulled out while being inserted into the mouth of the suction tube, and the blastocysts (8 in each uterine horn) were gently blown into the uterine cavity; then the mouth of the suction tube was pulled out, the uterus and fat pad were returned, the blood stasis was cleaned up, and the incision was sutured.

[0071] 14 days after transplantation, whether the transplanted female mice were pregnant could be observed, and the pregnancy rate = the number of pregnant female mice / the total number of transplanted female mice x 100%. 19-21 days after transplantation, young ICR mice were born, and the number of litters and the litter rate were counted, and the litter rate = the number of litters / the number of transplanted embryos x 100%.

[0072] The grouping is as follows:

[0073] (1) Common group: CZB transplantation culture solution

[0074] (2) Control group 1: CZB transplantation culture solution + melatonin (3 μg / mL)

[0075] (3) Control group 2: CZB transplantation culture solution + melatonin (3 μg / mL) + bilirubin (2 μg / mL) + protamine (5 μg / mL)

[0076] (4) Experimental group 1: CZB transplantation culture solution + sample group 1

[0077] (5) Experimental group 2: CZB transplantation culture solution + sample group 3

[0078] (6) Experimental group 3: CZB transplantation culture solution + sample group 6

[0079] (7) Experimental group 4: CZB transplantation culture solution + sample group 16

[0080] The research findings (as shown in Table 4) are as follows:

[0081] Table 4 Effects of different embryo transplantation solutions on post-transplantation pregnancy rate and litter production rate

[0082] Average number of pregnancies Pregnancy rate (%) Average number of litters per sow Litter size (%) Common group 11.25 70.31 4.25 26.56 Control group 1 11.75 73.44 5.25 32.81 Control group 2 12.25 76.56 5.50 34.38 Experimental group 1 13.50 84.38 8.00 50.00 Experimental group 2 13.25 82.81 8.50 53.13 Experimental group 3 13.00 81.25 9.00 56.25 Experimental group 4 11.75 73.44 4.75 29.69

[0083] After artificial embryo transplantation, the 4 mice in the common group produced a total of 17 young mice, and the litter production rate was 26.56. After the addition of the samples in the experimental groups 1-3, the pregnancy rate and the litter production rate were significantly improved.

[0084] After the addition of the control sample 16 in Example 1 which is not a component of the present application in the experimental group 4, the pregnancy rate and the litter production rate are similar to the effect of free drugs, and do not play a significant role in improving the transplantation effect.

[0085] Through the comparison of the common group, the control group and the experimental group, it is found that the sample prepared by the preferred component, the preferred ratio and the specific process of the present application can significantly improve the implantation rate of embryos, and can provide sustained protection for the transplanted embryos.

[0086] The above disclosure is only the preferred embodiments of the present application, and of course cannot limit the scope of the rights of the present application, so the equivalent changes made according to the claims of the present application still fall within the scope covered by the present application.

Claims

1. A melatonin nanoparticle, characterized in that: The melatonin nanoparticles are composed of melatonin, bilirubin and protamine; the mass ratio of the melatonin, bilirubin and protamine is 3: (1-5): (1-10).

2. The melatonin nanoparticle of claim 1, wherein: The mass ratio of the melatonin, bilirubin and protamine is 3:2:

5.

3. The melatonin nanoparticle of claim 1, wherein: The particle size of the melatonin nanoparticles is 100-300 nm.

4. A method of preparing melatonin nanoparticles as claimed in any one of claims 1 to 3, characterized by: The method comprises the following steps: (1) Melatonin and bilirubin are dissolved in dimethyl sulfoxide to prepare a mixed solution I; (2) Protamine is dissolved in water to obtain a protamine solution II; (3) The mixed solution I is dropped into the protamine solution II, and the solution is stirred for a period of time and then ultrasonically treated to obtain a solution III; (4) The solution III is dialyzed, and the dialyzed solution is freeze-dried to obtain the melatonin nanoparticles.

5. The preparation method of the melatonin nanoparticles according to claim 4, characterized in that: In the solution III obtained in the step (3), the mass ratio of the melatonin, bilirubin and protamine is 3: (1-5): (1-10).

6. The preparation method of the melatonin nanoparticles according to claim 4, characterized in that: In the mixed solution I, the concentration of the melatonin is 5 mg / mL.

7. Use of melatonin nanoparticles as claimed in any one of claims 1 to 3, characterized in that: In the application of preparing embryo transfer fluid / preservation fluid.

8. The application of the melatonin nanoparticles according to claim 7, characterized in that: The embryo transfer fluid / preservation fluid further comprises a pharmaceutically acceptable carrier, excipient or adjuvant.

9. The application of the melatonin nanoparticles according to claim 7, characterized in that: The embryo transfer fluid / preservation fluid is used for fertilization operation, culture of fertilized eggs, culture operation before embryo transfer and embryo transfer operation.

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