CN116459276A - Application of nano material in preparation of contraceptive preparation, contraceptive preparation and contraceptive method - Google Patents

Abstract

The invention provides an application of a nano material in preparing a contraceptive preparation, the contraceptive preparation and a contraceptive method, and particularly relates to the technical field of biological medicine. The nano material is a nano material with a killing effect on trophoblast cells of an embryo; the nanomaterial is used to kill trophoblast cells of the embryo, preventing normal implantation of the embryo. The invention transfers the contraceptive object from the human body to the blastula in the cell state, thereby avoiding systematic or local injury to the human body to the maximum extent; the nano material acts on the embryo trophoblast cells, so that the embryo cells are limited in development and naturally shed, and the contraceptive purpose is realized; the self-cleaning function of the physiological cycle of the uterine cavity can also avoid the risk of nano-material aggregation in the uterine cavity and even in the body.

Description

Application of nanomaterials in the preparation of contraceptive preparations, contraceptive preparations and contraceptive methods

technical field

The invention relates to the technical field of biomedicine, in particular to the application of a nanometer material in the preparation of contraceptive preparations, contraceptive preparations and contraceptive methods.

Background technique

Unintended pregnancy is an important public health problem, which will have adverse effects on women's bodies, families, and society. Miscarriage is generally the final outcome of an unwanted pregnancy, usually occurring in adolescents, adult couples outside the family planning. Miscarriage can have a very large health impact on a woman's body.

Contraception is one of the effective ways to prevent unwanted pregnancy. The female contraceptive devices currently used clinically mainly include oral hormonal contraceptives, epidermal patches, subcutaneous patches, vaginal rings, IUDs (hormone-loaded IUDs and copper IUDs), implants, and tubal ligation. However, hormonal contraceptives can lead to disruption of hormone homeostasis and endocrine homeostasis in women, thereby affecting women's normal menstrual cycle. Secondly, hormonal contraceptives achieve the purpose of contraception by inhibiting the function of the ovary and the production of eggs, which will affect the normal function of the ovary. In addition, hormonal contraceptives also achieve the purpose of contraception by promoting the secretion of uterine mucus and hindering the migration of sperm, which also interferes with the normal function of the uterus. Patches, vaginal rings, implants, IUDs, etc. are all loaded with different amounts of hormonal contraceptives to exert contraceptive effects, which not only have the side effects of hormonal contraceptives, but also cause immune stress reactions caused by foreign substances in the body. After the copper IUD is implanted in the uterus, it can play a long-term contraceptive effect, but there is a phenomenon of copper ion bursting in the early stage of use of the copper IUD, which leads to irregular uterine bleeding and even uterine perforation. Tubal ligation can cause pain, infection, permanent loss of fertility and other injuries.

Therefore, a new contraceptive method is urgently needed at present.

Contents of the invention

The invention provides an application of a nanometer material in the preparation of a contraceptive preparation, a contraceptive preparation and a contraceptive method, so as to solve the problem that the contraceptive measures currently used in clinics cause harm to the human body.

In the first aspect, the present invention proposes an application of a nanomaterial in the preparation of a contraceptive preparation, the nanomaterial is a nanomaterial that has a killing effect on the trophoblast cells of the embryo;

The nanometer material is used to kill the trophoblast cells of the embryo and prevent the normal implantation of the embryo.

Optionally, the nanomaterial damages trophoblast cells through oxidative stress;

The organelle and DNA damage of the trophoblast cells in turn mediates the death of the trophoblast cells.

Optionally, the nanomaterial is CuO nanoparticles. After the CuO nanoparticles are taken up by embryonic trophoblast cells, they react with hydrogen ions to generate copper ions under the action of lysosomes, and the copper ions induce a Fenton-like reaction in the cells, resulting in an increase in the production of reactive oxygen species, resulting in oxidative stress in the cells;

The oxidative stress response causes depolarization of the mitochondrial membrane in the embryonic trophoblast cells, increases the permeability of the mitochondrial membrane, and causes mitochondrial damage;

Under the action of the oxidative stress response, the DNA in the embryonic trophoblast cells is damaged and broken;

The DNA damage causes the division cycle of the embryonic trophoblast cells to arrest in the G2 phase.

Optionally, the contraceptive preparation is administered before the embryo invades the uterus;

The administration method of the contraceptive preparation includes a back minimally invasive administration method and an abdominal minimally invasive administration method;

The administration mode of the contraceptive preparation also includes intrauterine perfusion administration mode.

Optionally, the concentration range of the contraceptive preparation is greater than 0 μg/mL and less than 1000 μg/mL.

Optionally, based on the application of the first aspect above, it also includes: controlling the release rate of the nanomaterials through pH response, or controlling the release rate of the nanomaterials in the contraceptive preparation by controlling the degradation rate of the contraceptive preparation itself.

In the second aspect, the present invention proposes a contraceptive preparation, which includes nanomaterials and carriers;

Wherein, the nanomaterial is a nanomaterial that has a killing effect on the trophoblast cells of the embryo, and the nanomaterial is used to kill the trophoblast cells of the embryo and prevent the normal implantation of the embryo;

The carrier is an organic polymer;

The concentration range of the contraceptive preparation is greater than 0 μg/mL and less than 1000 μg/mL.

Optionally, the nanomaterial damages trophoblast cells through oxidative stress;

organelle and DNA damage of said trophoblast cells which in turn mediates said trophoblast cell death;

The organic macromolecular polymer is used to maintain the slow release of nanomaterials.

Optionally, the nanomaterial is an inorganic nanomaterial;

The inorganic nanomaterials include single-component or multiple-component metal nanomaterials, single-component or multiple-component metal compound nanomaterials, single-component or multiple-component non-metallic materials;

Wherein, the elements constituting the nanomaterials are non-toxic and harmless elements for human body.

In a third aspect, the present invention proposes a contraceptive method for non-therapeutic purposes, said method comprising:

The contraceptive preparation is placed in the uterine cavity based on the intrauterine administration method, so that the contraceptive preparation described in any one of the above second aspects is placed in the uterine cavity based on the intrauterine administration method, so that the nanomaterial can kill the embryonic trophoblast cells based on the slow release effect, and prevent the normal implantation of embryonic cells.

Embodiments of the present invention include the following advantages:

1. The embodiments of the present invention provide an application of nanomaterials in the preparation of contraceptive preparations, contraceptive preparations and contraceptive methods, which transfer the contraceptive object from the human body (that is, the mother) to the blastocyst in the cell state, and avoid systemic or local damage to the human body to the greatest extent;

2. The contraceptive preparation proposed in the embodiment of the present invention can be administered through the uterine cavity, avoiding blood and organ metabolic circulation, and the natural self-clearing function of the physiological cycle can also avoid the risk of nanomaterials gathering in the uterine cavity or even in the body;

3. In the embodiment of the present invention, the use of nanomaterials to prepare contraceptive preparations can avoid excessive active oxygen damage to the uterine cavity to a certain extent, and at the same time avoid interfering with hormone homeostasis and endocrine balance in the body;

4. In the embodiment of the present invention, the elements of the nanomaterials used in the contraceptive preparation are non-toxic and harmless elements for the human body, which can play a vital role in normal life functions, thereby reducing the toxicity of the nanomaterials.

Description of drawings

Fig. 1 is a schematic diagram of a contraceptive method in which nanomaterials act on embryonic trophoblast cells provided in an embodiment of the present invention;

Fig. 2 is the characterization result and toxicity diagram of CuO nanomaterial in the embodiment of the present invention;

3 is a schematic diagram of phagocytosis and induction of Fenton-like reactions by embryonic trophoblast cells in an embodiment of the present invention;

4 is a schematic diagram of CuO nanomaterials mediating trophoblast cell death through oxidative stress in an embodiment of the present invention;

Fig. 5 is a schematic diagram of the death mechanism of trophoblast cells mediated by CuO nanomaterials revealed by transcriptomics in the embodiment of the present invention;

6 is a schematic diagram of the contraceptive effect of CuO nanomaterials in animals in the embodiment of the present invention;

Figure 7 is a schematic diagram of the characterization and contraceptive effect of thermosensitive hydrogel-loaded CuO nanomaterials in the embodiment of the present invention;

Fig. 8 is a schematic diagram of the safety of heat-sensitive hydrogel-loaded CuO nanomaterials in animals in an embodiment of the present invention.

Detailed ways

The following examples are provided in order to further understand the present invention better, and are not limited to the best implementation mode, and do not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the present invention with other features of the prior art, all falls within the protection scope of the present invention.

If no specific experimental steps or conditions are indicated in the examples, it can be carried out according to the operation or conditions of the conventional experimental steps described in the literature in this field. The reagents used and other instruments whose manufacturers are not indicated are all conventional reagent products that can be purchased from the market.

At present, the idea of contraceptive measures is to hinder the production of egg cells or hinder the combination of egg cells and sperm. Most of the clinically used contraceptive measures proposed by this idea (for example: oral hormonal contraceptives, epidermal patches, subcutaneous patches, vaginal rings, IUDs, implants, and tubal ligation) will cause harm to the human body. Therefore, the embodiments of the present invention change the thinking and carry out contraceptive treatment from the embryonic level.

The normal implantation process of the embryo is: after the fertilized egg is formed, the trophoblast cells of the embryo are formed, the embryo completes the invasion of the uterus, and finally grows and develops smoothly.

In recent years, with the rapid development of nanotechnology, nanomaterials have shown great application potential in the fields of biology and medicine. Therefore, the embodiment of the present invention only proposes the idea: after the formation of the trophoblast of the embryo and before the invasion, use nanomaterials to mediate the death of the embryo trophoblast cells, prevent the embryo from invading, and make the growth of the embryo cells be limited and naturally fall off, so as to achieve the purpose of contraception. Since unimplanted embryos belong to the state of cells, the killing effect on cells can also avoid moral and ethical concerns.

Based on this, in the first aspect, an embodiment of the present invention provides an application of a nanomaterial in a contraceptive preparation. The nanomaterial is a nanomaterial that has a killing effect on the trophoblast cells of the embryo; the nanomaterial is used to kill the trophoblast cells of the embryo and prevent the normal implantation of the embryo. Wherein, the nanomaterial damages the trophoblast cells through oxidative stress; the organelles and DNA damage of the trophoblast cells further mediate the death of the trophoblast cells.

Specifically, in this implementation case, CuO nanomaterials were used as verification materials, trophoblast HTR8/SVneo cells were used as model cells, and related experiments were carried out in a rat pregnancy model (see Example 4 below).

Specifically, the nanomaterial is CuO nanoparticles. After the CuO nanoparticles are taken up by embryonic trophoblast cells, they react with hydrogen ions to generate copper ions under the action of lysosomes, and the copper ions induce a Fenton-like reaction in the cells, resulting in an increase in the production of reactive oxygen species, resulting in oxidative stress in the cells; The DNA in the cell is damaged and broken; the DNA damage causes the division cycle of the embryonic trophoblast cells to stagnate in the G2 phase; wherein, the above-mentioned reactive oxygen species is H₂o₂, O^2-, -HO, etc.

Based on the first aspect, the contraceptive preparation is administered before the embryo invades the uterus; the administration method of the contraceptive preparation includes a minimally invasive administration method on the back and a minimally invasive administration method on the abdomen; the administration method of the contraceptive preparation also includes an intrauterine infusion administration method; wherein, the administration method of the contraceptive preparation is preferably an intrauterine infusion administration method.

After the fertilized egg is formed, the embryonic trophoblast cells are formed, and the embryonic trophoblast cells invade the uterus. At this time, the embryo has not yet begun to invade the uterus. Therefore, administration after the formation of embryonic trophoblast cells or before embryo invasion can act on the embryonic trophoblast cells to limit the growth of embryonic trophoblast cells and achieve the purpose of contraception.

The above-mentioned contraceptive preparation can be administered through the back minimally invasive administration method, the abdominal minimally invasive administration method, and the intrauterine infusion administration method. Due to the special environment of the uterine cavity, in the embodiments of the present invention, the intrauterine infusion administration method is preferred to directly administer the above-mentioned contraceptive preparation into the uterine cavity.

Based on the first aspect, the concentration range of the contraceptive preparation is greater than 0 μg/mL and less than 1000 μg/mL.

Exemplarily, the concentration used in the contraceptive preparation in the embodiment of the present invention can be 100 μg/mL, 200 μg/mL, 300 μg/mL, 400 μg/mL, 500 μg/mL, 600 μg/mL, 700 μg/mL, 800 μg/mL, 900 μg/mL, 1000 μg/mL, etc. Those skilled in the art can make settings according to requirements, and the embodiments of the present invention will not be described in detail.

Based on the first aspect, the application of nanomaterials in contraceptive preparations also includes: controlling the release rate of nanomaterials through pH response, or controlling the release speed of nanomaterials in contraceptive preparations by controlling the degradation rate of the contraceptive preparation itself.

Among them, the mechanism of pH-responsive controlled release is as follows: the environment of the uterus is acidic, and the carrier of the contraceptive preparation can decompose and release the carrier-coated nanomaterials under acidic conditions; according to the pH value, the decomposition rate of the contraceptive preparation carrier can be adjusted, thereby realizing the controlled release of the carrier-coated nanomaterials.

The mechanism of the self-degradation of the contraceptive preparation is as follows: Uterine mucus is a hydrogel-like liquid containing various ions, which can chemically corrode the carrier of the contraceptive preparation and then decompose and release the carrier-coated nanomaterials. The chemical corrosion rate of the contraceptive preparation can be controlled by changing the physicochemical properties of the contraceptive preparation, and then the release of the carrier-coated nanomaterials can be controlled.

Based on the same inventive concept as above, in a second aspect, the present invention provides a contraceptive preparation, the contraceptive preparation comprising a nanomaterial and a carrier; wherein the nanomaterial is a nanomaterial having a killing effect on the trophoblast cells of the embryo, and the nanomaterial is used to kill the trophoblast cells of the embryo and prevent the normal implantation of the embryo; the carrier is an organic polymer; the concentration range of the contraceptive preparation is greater than 0 μg/mL and less than 1000 μg/mL.

Since the embodiment of the present invention can be administered after the formation of embryonic trophoblast cells, or before embryo invasion, it can act on embryonic trophoblast cells to limit the growth of embryo trophoblast cells and achieve the purpose of contraception. Therefore, the nanomaterials in the embodiments of the present invention are nanomaterials that have a killing effect on embryonic trophoblast cells; based on the above-mentioned pH-responsive controlled release or controlling the degradation rate of the contraceptive preparation itself to control the sustained release mechanism of the nanomaterials in the contraceptive preparation.

In the embodiment of the present invention, the carrier of the coated nanomaterial in the contraceptive preparation is selected as an organic high molecular polymer. Exemplarily, the above organic high molecular polymer can be thermosensitive hydrogel, sodium alginate, chitosan, polylactic acid, agarose, liposome, polyethylene glycol, gelatin, acrylic acid or other organic high molecular materials that can be gelled. In the embodiment of the present invention, it is preferable that the thermosensitive hydrogel is used as the carrier of the nanomaterial in the contraceptive preparation.

Wherein, the nanomaterial damages the trophoblast cells through oxidative stress; the organelles and DNA damage of the trophoblast cells further mediate the death of the trophoblast cells; the organic high molecular polymer is used to maintain the slow release of the nanomaterials.

Based on the second aspect, the nanomaterials are inorganic nanomaterials; the inorganic nanomaterials include single-component or multiple-component metal nanomaterials, single-component or multiple-component metal compound nanomaterials, single-component or multiple-component non-metallic materials; wherein, the elements constituting the nanomaterials are non-toxic and harmless elements for the human body.

In the embodiment of the present invention, illustratively, the above-mentioned inorganic nanomaterials are preferably copper oxide nanomaterials.

Exemplarily, the above-mentioned inorganic nanomaterials may be iron oxide nanomaterials, zinc oxide nanomaterials, magnesium oxide nanomaterials, gold nanomaterials, silver nanomaterials, copper nanomaterials, zinc nanomaterials, and the like.

In a third aspect, the present invention provides a contraceptive method for non-therapeutic purposes, the method comprising: placing the contraceptive preparation described in any one of the above-mentioned second aspects in the uterine cavity based on intrauterine administration, so that the nanomaterial can kill embryonic trophoblast cells based on a sustained release effect, and prevent normal implantation of embryonic cells.

For example, refer to FIG. 1 . FIG. 1 is a schematic diagram of a new female contraceptive method in which nanomaterials act on embryonic trophoblast cells provided in an embodiment of the present invention. Specifically, using copper-based nanomaterials as an illustration, it can be seen from part B1 in Figure 1 that the normal implantation process of the embryo is: after the fertilized egg is formed, the trophoblast cells of the embryo are formed, the trophoblast cells of the embryo complete the invasion of the uterus, and the embryo grows and develops smoothly. When the embryonic trophoblast cells die, the embryonic cells will fall off naturally to achieve the purpose of contraception. Wherein, after the contraceptive preparation is placed in the uterine cavity, since the contraceptive preparation can realize the slow release of copper-based nanomaterials through pH regulation or control the degradation rate of the contraceptive preparation itself, when the embryo trophoblast cells are formed, the released copper-based nanomaterials will act on the embryo trophoblast cells, and the embryo trophoblast cells absorb the released copper-based nanomaterials through phagocytosis. The increase in material production leads to oxidative stress in the cells, resulting in apoptosis and ferroptosis of the embryonic trophoblast cells. Due to the copper homeostasis, the copper death of the embryonic trophoblast cells is caused. Part B2 in Figure 1 is the fallopian tube without the action of copper-based nanomaterials. It can be seen that the embryo develops normally without the action of copper-based nanomaterials. Part B3 in Figure 1 is the fallopian tube with the action of copper-based nanomaterials. It can be seen that embryonic cells fall off; and the process of embryo shedding and normal embryonic development in the fallopian tube is reversible.

In order for those skilled in the art to better understand the present invention, the following specific examples illustrate the application of the nanomaterials provided by the present invention in the preparation of contraceptive preparations, contraceptive preparations and contraceptive methods.

Embodiment 1: Nanomaterial Characterization:

In the embodiments of the present invention, nanomaterials, including inorganic nanomaterials and their loaded drugs, are purchased or synthesized, and their physical and chemical properties such as morphology, size, elements, and optics are characterized.

Among them, the purchase sources of nanomaterials include Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai Macklin Biochemical Technology Co., Ltd., Merck Co., Ltd., or other companies with the qualification to produce nanomaterials.

Among them, the synthesis methods of nanomaterials include physical methods and chemical methods; physical methods include vacuum condensation method, physical pulverization method, mechanical ball milling method; chemical methods include gas phase synthesis method, liquid phase synthesis method, solid phase synthesis method, ultrasonic assisted method, high temperature assisted method, surfactant method, microwave oven assisted method, etc.

Wherein, the nanomaterial is an inorganic nanomaterial; the inorganic nanomaterial includes a single or multiple metal nanomaterial, a single component or a multicomponent metal compound nanomaterial, a single component or a multicomponent non-metallic material; the constituent elements of the nanomaterial are all non-toxic and harmless elements for human body.

In the embodiments of the present invention, optical microscopy, scanning electron microscopy, transmission electron microscopy, particle size instruments, X-ray diffraction spectroscopy, differential scanning calorimetry, thermogravimetric analysis, or infrared spectroscopy are used to characterize the physical and chemical properties of nanomaterials such as morphology, size, elements, and optics.

As can be seen from Fig. 2, the CuO nanomaterial shown in Fig. 2 (A-D parts) was purchased from Aladdin Company; A part in Fig. 2 is to adopt the transmission electron microscope to characterize the morphology of the CuO nanomaterial, and it can be seen that the CuO nanomaterial is granular; B part among Fig. ) to analyze the elements of the CuO nanomaterials, the CuO nanomaterials have 11 characteristic peaks; part D in Figure 2 is to use a UV spectrophotometer to characterize the CuO nanomaterials, showing that the CuO nanomaterials have absorption peaks around 400nm. Referring to part E in Fig. 2, the present invention uses the CCK8 method to explore the difference in the toxicity of CuO nanoparticles to trophoblast cells (HTR8/SVneo cells) and normal endometrial stromal cells (HESC cells). It can be seen that the toxicity of CuO nanoparticles to trophoblast cells is significantly higher than that of normal endometrial stromal cells at the same dosage concentration.

Example 2: Nanomaterials mediate toxicity to embryonic trophoblast cells:

Mix the CuO nanomaterials with the cell culture medium, pour them into a T75 cell culture bottle that is 90% full of cells, and culture for 4 hours. The cells are scraped off to make ultra-thin slices of the cells. The CuO nanomaterials are phagocytized and taken up by embryonic trophoblast cells under a transmission electron microscope. For the treated cells, the mRNA of the cells was extracted by the spin column method, and the cell protein was extracted by the broken centrifugation method, and the expression of genes and proteins were detected by qPCR (fluorescent quantitative PCR) technology and Western Blot (Western blotting) technology, respectively. Wherein, in the embodiment of the present application, the above-mentioned CuO nanomaterials are CuO nanoparticles.

In the embodiment of the present invention, cell viability, scratch, and cell counting methods were used to detect cell proliferation and invasion ability, fluorescent probe method (fluorescence microscope + flow cytometry) was used to detect the basic level of reactive oxygen species (reactive oxygen species, ROS) in two normal cells, flow cytometry was used to detect cell cycle and organelle damage, Coomassie brilliant blue method was used to detect protein content, hydroxylamine method was used to detect superoxide dismutase, and visible light colorimetry was used to detect catalase and superoxide anion (O^2-), hydrogen peroxide (H₂o₂), hydroxyl radical (OH-), reduced glutathione, oxidized glutathione, mitochondrial respiratory chain complex to verify cellular DNA damage.

It can be seen from part A in Fig. 3 that CuO nanomaterials can be phagocytized by cells. Part B of Figure 3 shows that copper ion transport-related genes (MT1F, MT1X, MT1E, etc.) and copper ion-binding protein-related genes (ATP7A, ATP7B, etc.) have undergone significant changes over time: when CuO nanomaterials are taken up by embryonic trophoblast cells, under the action of lysosomes, CuO nanomaterials react with hydrogen ions to generate copper ions, and then copper ions induce a Fenton-like reaction (involving redox reactions) in the cells, resulting in reactive oxygen species (H ₂ O ₂ , ·O ^2- , -HO) production increases, which in turn leads to oxidative stress in cells. As shown in part C of Figure 3, after the cells were stained, the pH _changes of _the lysosomes were observed using a fluorescence microscope. The increase in pH means that the _H ⁺ in the lysosomes was consumed and copper ions were generated at the same time; this process required a large amount of ^energy consumption, resulting in the continuous decomposition of O ₂ into O ^2- in the cells. At the same time, ^{toxic -HO is generated; finally, oxidized glutathione oxidizes Cu + to Cu 2+ ;} _the above reaction repeats a cycle to produce a large amount of -HO ^{and Cu + ,} which leads to aggravated cell damage. The above analysis is the result of detection and analysis of the relevant components involved in the Fenton-like reaction by using the trace method.

Example 3: revealing the mechanism of toxicity of nanomaterials to embryonic trophoblast cells:

In the embodiment of the present invention, gene sequencing-RNA transcriptome analysis, gas phase mass spectrometry (GCMS), liquid phase mass spectrometry (LCMS), gas phase-liquid chromatography (GCLC), electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), secondary ion mass spectrometry (SIMS)-cell metabolome analysis, qPCR analysis of the relative expression of related genes, and Western Blot (WB, western blotting) analysis of related protein expression.

It can be seen from Figure 4 that the embryonic trophoblast cells underwent oxidative stress under the action of CuO nanomaterials. The cells were collected and stained and then observed under a fluorescent inverted microscope. Figure 4 (Part A-C) showed an increase in reactive oxygen species; oxidative stress caused the depolarization of the mitochondrial membrane in the embryonic trophoblast cells, and increased the permeability of the mitochondrial membrane, resulting in mitochondrial damage. Part F in Figure 4 shows that the red fluorescence in the mitochondria gradually converted to green fluorescence; Part D in Figure 4 and Part E in Figure 4 It shows that under the action of oxidative stress, the DNA in the cells is damaged, and the electrophoretic comet experiment results show that the smears are small fragments of DNA fragments; the DNA damage causes the cell division cycle to arrest in the G2 phase, and the flow cytometry method is used to determine the arrest period of the cell division phase. In summary, CuO nanomaterials mediate trophoblast cell death by damaging organelles and DNA through oxidative stress.

It can be seen from Figure 5 that KEGG (Kyoto Encyclopedia of Genes and Genomes, Kyoto Encyclopedia of Genes and Genomes) enrichment analysis was performed on the results of transcriptomics, and the results showed that under the action of CuO nanomaterials, the death modes of embryonic trophoblast cells included apoptosis and ferroptosis.

In addition, through clustering and classification of related death genes, it was found that the death mode of embryonic trophoblast cells also includes copper death; further, as shown in part C of Figure 5, the interaction analysis of DNA damage, mitochondrial membrane damage, apoptosis, ferroptosis and copper death related marker genes was conducted to explore the degree of close correlation between them; finally, the gene expression and protein expression of several death modes were verified by qPCR technology and WB technology. Parts D, F, and H in Figure 5 are changes in expression of marker genes related to apoptosis, ferroptosis, and copper death, respectively, while parts E, G, and I in Figure 5 are changes in expression of marker proteins related to apoptosis, ferroptosis, and copper death, respectively.

Embodiment 4: Rat pregnancy model experiment:

Experimental animals were used to establish animal pregnancy models to verify the contraceptive effect of nanomaterials; the selected experimental animals were SD female mice, and the animal pregnancy model was SD female mice in a fertilized state after successful mating; the administration method was minimally invasive surgery on the back of rats, as shown in part A of Figure 6.

Among them, on the 10th day after fertilization of rats, the contraceptive effect of CuO nanomaterials was verified by touching the abdomen to see if there was a grainy feeling of the embryo and performing abdominal minimally invasive surgery.

The above-mentioned animal pregnancy model is administered through a minimally invasive administration method in the abdomen. The administration concentration is 200 μg/mL, 500 μg/mL, and 1000 μg/mL of CuO nanomaterials. Pregnancy, but it stimulates the uterus and causes the uterus to produce decidualization.

Example 5: Preparation of contraceptive preparations and research on sustained release of nanomaterials:

In the embodiment of the present invention, the organic polymer is used as the drug delivery carrier of the nanomaterial, and the contraceptive time of the nanomaterial is prolonged and the toxicity of the nanomaterial is reduced through the effect of controlled release and slow release.

Among them, the loading materials of nanomaterials include thermosensitive hydrogel, sodium alginate, chitosan, polylactic acid, agarose, liposome, polyethylene glycol, gelatin, acrylic acid, and other organic polymer materials that can form gels; the loading methods of nanomaterial loading materials include direct loading, in-situ synthesis, etc.

In this embodiment, thermosensitive hydrogel is used to load CuO nanomaterials to achieve the purpose of controlled and sustained release of nanomaterials, because thermosensitive hydrogel is a gel with good biocompatibility and does not affect cell proliferation.

The thermosensitive hydrogel loaded with CuO nanomaterials realizes the sustained release of nanomaterials through the following methods: pH-responsive controlled release: the uterus is in an acidic environment, and thermosensitive hydrogel materials can decompose and release drugs under acidic conditions. The decomposition rate of the materials can be adjusted according to the pH value, thereby controlling the release of nanomaterials; carrier self-degradation: uterine mucus is a hydrogel-like liquid containing various ions, which can chemically corrode thermosensitive hydrogel materials and then decompose and release nanomaterials. Controlled release of nanomaterials.

From part A in Figure 7, it can be seen that the thermosensitive hydrogel is in the injectable solution state at room temperature of 25°C, and is too porous in a solid state at 37°C. In addition, the infrared characterization of the thermosensitive hydrogel was carried out. At the same time, the experimental results of CCK8 (Cell Counting Kit-8, cell proliferation detection kit) showed that the thermosensitive hydrogel has good biocompatibility and does not affect the growth and proliferation of cells. From part B in Figure 7, it can be seen that after the thermosensitive hydrogel is loaded with CuO nanomaterials, the properties of the thermosensitive hydrogel do not change, and at the same time, it can degrade and release CuO nanomaterials in simulated intrauterine fluid (SUF) with different pH. As shown in part C of Figure 7, the thermosensitive hydrogel can play a contraceptive role by releasing the loaded CuO nanomaterials, and the results of HE slices also show that the stimulation to the uterus is greatly reduced.

Embodiment 6: the safety of contraceptive preparation in rat body:

In the embodiment of the present invention, by administering drugs to a rat pregnancy model, the safety in vivo of the rat pregnancy model is investigated, including the aggregation and degradation of nanomaterials in rats, inflammatory factors in the blood, blood concentration of nanomaterials in blood, cell morphology and structural changes in various metabolic organs, etc.

From part A in Figure 8, it can be known that the CuO nanomaterials supported by the thermosensitive hydrogel did not affect the normal growth and development of rats in the rat uterine cavity, specifically reflected in the body weight. Secondly, it can be seen from parts B-F in Figure 8 that copper ions, estrogen, and inflammatory factors in plasma did not change significantly, indicating that the CuO nanomaterials loaded with thermosensitive hydrogel did not cause systemic damage to the rat body; at the same time, it can be seen from part G in Figure 8 that the cell morphology and structure of major organs including the brain, heart, liver, spleen, kidney, and ovary did not change significantly. These results verified the safety of controlled release of CuO nanomaterials through thermosensitive hydrogel for contraceptive effect.

As for the system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and for the related parts, please refer to the part of the description of the method embodiment.

Each embodiment in this specification is described in a progressive manner, each embodiment focuses on the difference from other embodiments, and the same and similar parts of each embodiment can be referred to each other.

The application of a nanomaterial provided by the present invention in the preparation of contraceptive preparations, contraceptive preparations and contraceptive methods has been introduced in detail above. In this paper, specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the above examples are only used to help understand the methods and core ideas of the present invention; meanwhile, for those of ordinary skill in the art, according to the ideas of the present invention, there will be changes in the specific implementation methods and application ranges. In summary, the content of this specification should not be understood as limiting the present invention.

Claims (10)

1. the application of a kind of nano material in contraceptive preparation, it is characterized in that, described nano material is the nano material that the trophoblast cell of embryo is had killing effect; The nanometer material is used to kill the trophoblast cells of the embryo and prevent the normal implantation of the embryo. 2. The application according to claim 1, wherein the nanomaterial damages trophoblast cells by oxidative stress; The organelle and DNA damage of the trophoblast cells in turn mediates the death of the trophoblast cells. 3. The application according to claim 1, wherein the nanomaterial is CuO nanoparticles, and after the CuO nanoparticles are ingested by embryonic trophoblast cells, under the action of lysosomes, they react with hydrogen ions to generate copper ions, and the copper ions induce a Fenton-like reaction in the cells, resulting in an increase in the production of reactive oxygen species, leading to oxidative stress in the cells; The oxidative stress response causes depolarization of the mitochondrial membrane in the embryonic trophoblast cells, increases the permeability of the mitochondrial membrane, and causes mitochondrial damage; Under the action of the oxidative stress response, the DNA in the embryonic trophoblast cells is damaged and broken; The DNA damage causes the division cycle of the embryonic trophoblast cells to arrest in the G2 phase. 4. The application according to claim 1, wherein the contraceptive preparation is administered before the embryo invades the uterus; The method of administration of the contraceptive preparation includes a minimally invasive intrauterine administration method on the back and a minimally invasive intrauterine administration method on the abdomen; The administration mode of the contraceptive preparation also includes intrauterine perfusion administration mode. 5. The application according to claim 1, characterized in that the concentration range of the contraceptive preparation is greater than 0 μg/mL and less than 1000 μg/mL. 6. The application according to claim 1, further comprising: controlling the release rate of the nanomaterials through pH response, or controlling the release rate of the nanomaterials in the contraceptive preparation by controlling the degradation rate of the contraceptive preparation itself. 7. A contraceptive preparation, characterized in that, the contraceptive preparation comprises nanomaterials and carriers; Wherein, the nanomaterial is a nanomaterial that has a killing effect on the trophoblast cells of the embryo, and the nanomaterial is used to kill the trophoblast cells of the embryo and prevent the normal implantation of the embryo; The carrier is an organic polymer; The concentration range of the contraceptive preparation is greater than 0 μg/mL and less than 1000 μg/mL. 8. The contraceptive preparation according to claim 7, wherein the nanomaterial damages trophoblast cells by oxidative stress; organelle and DNA damage of said trophoblast cells which in turn mediates said trophoblast cell death; The organic macromolecular polymer is used to maintain the slow release of nanomaterials. 9. The contraceptive preparation according to claim 7, wherein the nanomaterial is an inorganic nanomaterial; The inorganic nanomaterials include single-component or multiple-component metal nanomaterials, single-component or multiple-component metal compound nanomaterials, single-component or multiple-component non-metallic materials; Wherein, the elements constituting the nanomaterials are non-toxic and harmless elements for human body. 10. A contraceptive method for non-therapeutic purposes, characterized in that the method comprises: The contraceptive preparation according to any one of the above claims 7-9 is placed in the uterine cavity based on intrauterine administration, so that the nanomaterial can kill embryonic trophoblast cells based on sustained release and prevent normal implantation of embryonic cells.