Preparation of polydopamine nano composite material and application of polydopamine nano composite material in treatment of endometriosis

Abstract

The invention is applied to the technical field of medicine of nano-drugs for treatment, and particularly discloses preparation of a polydopamine nanocomposite and application of the polydopamine nanocomposite in treatment of endometriosis, the polydopamine nanocomposite is polydopamine nanoparticles which are modified by polyethylene glycol and laminin and carry didrogesterone, and the polydopamine nanoparticles are polydopamine nanoparticles which are modified by polyethylene glycol and laminin and carry didrogesterone. The disease is endometriosis. Polydopamine nano-particles which are modified by polyethylene glycol and laminin and carry didrogesterone are constructed and applied to in-vitro and in-vivo experiments related to treatment of endometriosis, and the polydopamine nano-particles have the effects of improving oxidative stress, inhibiting inflammatory microenvironment, promoting ectopic endometrial atrophy, inhibiting inflammation and treating endometriosis. And a good treatment effect is achieved in the aspect of reducing the dosage of the didrogesterone.

Description

Technical Field

[0001] The present invention relates to the medical technical field of therapeutic nanomedicines, and in particular to the preparation of a polydopamine nanocomposite material and its application in treating endometriosis. Background Art

[0002] Endometriosis refers to the appearance, growth, and infiltration of endometrial tissue glands and stroma in the endometrium lining the uterine cavity and areas outside the uterus, with repeated bleeding, followed by pain, infertility, nodules, masses, etc. It is an estrogen-dependent inflammatory disease that affects 5-10% of women of childbearing age. The treatment goals of endometriosis are to reduce lesions, relieve pain, improve fertility and avoid recurrence. Clinically, endometriosis is mainly treated with drugs or surgery. The drug method is to use drugs to produce a low estrogen or high progesterone environment, and to achieve a stable steroid hormone environment to inhibit the proliferation of ectopic endometrial cells. The surgical method is to remove the lesions, make a clear diagnosis and restore the pelvic anatomical structure. It is generally considered to be the best treatment for ovarian and deep infiltrating endometriosis, and can reduce the postoperative recurrence rate.

[0003] However, drug treatment cannot cure endometriosis and may cause frequent or intolerable side effects. The resulting ovulation suppression effect will also affect the patient's natural reproductive process. It mainly creates a low estrogen or high progesterone environment to inhibit the proliferation of ectopic endometrial cells. However, this type of treatment cannot cure the disease and may cause frequent or intolerable side effects. For example, drug-induced ovulation suppression will directly affect the patient's natural reproductive process;

[0004] Furthermore, for the most common type of ovarian endometriosis, surgical resection may damage normal ovarian tissue and reduce ovarian reserve function. Furthermore, medication or surgical treatment may also lead to an increase in the patient's reproductive age, further reducing fertility. Although surgical treatment can remove lesions, confirm the diagnosis, and restore the pelvic anatomy, it is considered the preferred option for ovarian and deep infiltrating endometriosis and can reduce postoperative recurrence rates. However, for the most common type of ovarian endometriosis, surgical resection may damage normal ovarian tissue and lead to a decrease in ovarian reserve function. Furthermore, both medication and surgical treatment may further reduce fertility as the patient's reproductive age increases. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of a polydopamine nanocomposite material and its application in the treatment of endometriosis, so as to solve the problem in the above background technology that drug treatment cannot cure endometriosis and surgical resection may damage the normal function of ovarian tissue.

[0006] To achieve the above objectives, the present invention provides the following technical solutions: preparation of a polydopamine nanocomposite material and application thereof in the treatment of endometriosis, wherein the polydopamine nanocomposite material is polydopamine nanoparticles modified with polyethylene glycol and laminin and loaded with dydrogesterone, and the disease is endometriosis;

[0007] Preferably, the endometriosis is endometriosis caused by transplanting human ectopic endometrial tissue subcutaneously into female nude mice.

[0008] By using the above technical solution, the cause of endometriosis was clarified.

[0009] Preferably, the application is to use the polydopamine nanocomposite particles to reduce the inflammation level and oxidative stress level of endometriosis lesions and promote the atrophy of ectopic endometrial lesions.

[0010] By adopting the above technical solution, the application effect of polydopamine nanocomposite particles is clarified.

[0011] Preferably, the steps of preparing the polydopamine nanocomposite material include:

[0012] S1. Dissolving dopamine hydrochloride solid in ultrapure water to obtain a dopamine hydrochloride aqueous solution, then adding 5 mol / L NaOH solution to adjust the pH, heating and stirring in an oil bath, and then centrifuging. Washing with deionized water three times, the solid precipitate is freeze-dried to obtain polydopamine nanoparticles for later use;

[0013] S2. At room temperature, polydopamine nanoparticles and dydrogesterone are mixed and stirred in methanol, centrifuged, washed three times with deionized water, and freeze-dried to obtain polydopamine nanoparticles loaded with dydrogesterone, which are then set aside.

[0014] S3. At room temperature, polydopamine nanoparticles loaded with dydrogesterone and methoxypolyethylene glycolamine were mixed and stirred in an aqueous solution overnight, the precipitate was collected by centrifugation, redissolved in water, mixed and stirred with the laminin solution, centrifuged and freeze-dried to obtain a polydopamine nanocomposite material.

[0015] By adopting the above technical solution and following the above steps, a polydopamine nanocomposite material can be prepared.

[0016] Preferably, in the S1 process, the dopamine hydrochloride dissolution system is 200 ml of ultrapure water, NaOH solution is added dropwise to adjust the pH to 9, the oil bath heating temperature is 60° C., the stirring reaction time is 6 hours, the centrifugal speed is 18500 rpm, the centrifugal time is 30 minutes, and the dopamine hydrochloride solid mass is 200 mg.

[0017] By adopting the above technical solution, the specific parameters of step 1 in the preparation process of the polydopamine nanocomposite material are limited to ensure the reliability of the experimental results.

[0018] Preferably, in the S2 process, the methanol system is 2 ml, the mass of the polydopamine nanoparticles is 10 mg, the mass of the dydrogesterone is 0.25 mg, the centrifugal speed is 18500 rpm, and the centrifugation time is 30 minutes.

[0019] By adopting the above technical solution, the specific parameters of step 2 in the preparation process of the polydopamine nanocomposite material are limited to ensure the reliability of the experimental results.

[0020] Preferably, in the S3 process, the mass ratio of the polydopamine nanoparticles loaded with dydrogesterone to methoxypolyethylene glycolamine is 1:2, and the volume ratio of the nanoparticle solution to the laminin solution is 1:1. If nanoparticles not loaded with dydrogesterone need to be prepared in S3, the polydopamine nanoparticles obtained in step S1 are mixed with methoxypolyethylene glycolamine in a mass ratio of 1:2, and the volume ratio of the nanoparticle solution to the laminin solution is 1:1.

[0021] By adopting the above technical solution, the parameters of the surface modification process in step S3 are limited, and parameters such as mass ratio and volume ratio are specified, so as to accurately control the degree of modification of nanoparticles by polyethylene glycol and laminin.

[0022] Preferably, the concentration of the nanoparticle solution is 500 μg / ml, the concentration of the laminin solution is 50 μg / ml, the centrifugal speed is 18500 rpm, and the centrifugation time is 30 minutes.

[0023] The above technical solution was used to further refine the solution concentration and centrifugation parameters in step S3.

[0024] Preferably, the size of the dydrogesterone-loaded composite nanoparticles (DYD@PDA-PEG-LN NPs) is 185.5 nm, and the size of the un-drug-loaded modified nanoparticles (PDA-PEG-LN NPs) is 177.7 nm. Both the dydrogesterone-loaded composite nanoparticles and the un-drug-loaded modified nanoparticles are monodisperse spherical structures.

[0025] By adopting the above technical solution, the size and structural characteristics of nanoparticles loaded with dydrogesterone and those without the drug are clearly described.

[0026] Preferably, the nanocomposite material is used in the treatment of oxidative stress, chronic inflammatory environment and estrogen dependence of endometriosis.

[0027] The adoption of the above technical solution expands the application scope of the polydopamine nanocomposite material.

[0028] Compared with the prior art, the present invention has the following beneficial effects: the novel polydopamine nanocomposite material and its application in treating endometriosis:

[0029] 1. The present invention constructs polydopamine nanoparticles modified with polyethylene glycol and laminin and loaded with dydrogesterone, and applies them to in vitro and in vivo experiments related to the treatment of endometriosis. They have significant therapeutic effects in improving oxidative stress, inhibiting the inflammatory microenvironment, promoting atrophy of the ectopic endometrium, and reducing the dosage of dydrogesterone. PDA-PEG-LN NPs have good biosafety and blood compatibility; they can effectively scavenge extracellular superoxide anions (O2·-), hydroxyl radicals (·OH), and intracellular ROS that increase in response to exogenous stimulation; they can also reduce the expression of TNF-α and IL-6 levels of HESCs after IL-1β stimulation; PDA-PEG-LN NPs can also inhibit the expression of M1 macrophages and related genes and promote the expression of M2 macrophages and related genes;

[0030] Furthermore, DYD@PDA-PEG-LN NPs loaded with dydrogesterone can better inhibit the proliferation and migration of human endometrial stromal cells (HESCs), promote their apoptosis, and reduce the dosage of DYD compared to dydrogesterone alone. After injection into endometriosis model mice created by subcutaneously transplanting human ectopic endometrial lesions into nude mice, the nanomaterial not only aggregated and retained in the lesion endometrium, improving the local inflammatory microenvironment and oxidative stress levels, but also induced atrophy of the ectopic endometrium and inhibited its proliferation activity, without affecting the ovarian function and fertility of the model mice, and without obvious side effects.

[0031] 2. The polydopamine nanocomposite material constructed in the present invention can treat endometriosis without affecting ovarian function and fertility, does not affect the natural reproductive process, and will not lead to a further decline in fertility due to increasing the patient's childbearing age. It not only solves the contradiction between disease treatment and fertility needs, but also reduces the dosage of dydrogesterone, thereby reducing the incidence of adverse reactions, improving patient compliance, and making up for the shortcomings of existing treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the characterization results of nanoparticles at different stages in an embodiment of the present invention;

[0033] Figure 2Schematic diagram of the results of the cytotoxicity experiment of PDA-PEG-LN NPs and the biosafety experiment of DYD@PDA-PEG-LN NPs in the examples of the present invention;

[0034] Figure 3 Schematic diagram of the experimental results of the antioxidant and anti-inflammatory abilities of PDA-PEG-LN NPs in the examples of the present invention;

[0035] Figure 4 Schematic diagram of the effects of PDA-PEG-LN NPs on macrophage polarization and related gene expression in an embodiment of the present invention;

[0036] Figure 5 Schematic diagram of the effects of DYD@PDA-PEG-LN NPs on the proliferation, migration and apoptosis of endometrial stromal cells in an embodiment of the present invention;

[0037] Figure 6 Schematic diagram of the aggregation and distribution results of DYD@PDA-PEG-LN NPs and unmodified DYD@PDA NPs after subcutaneous or intravenous injection into endometriosis model mice in an embodiment of the present invention;

[0038] Figure 7 This is a schematic diagram of the effect of DYD@PDA-PEG-LN NPs on the area and weight of ectopic endometrial tissue in endometriosis model mice in an embodiment of the present invention;

[0039] Figure 8 This is a schematic diagram of the effect of DYD@PDA-PEG-LN NPs on the area and weight of ectopic endometrial tissue in endometriosis model mice in an embodiment of the present invention;

[0040] Figure 9 Schematic diagram of the improvement effect of DYD@PDA-PEG-LN NPs on the oxidative stress level and inflammatory microenvironment of ectopic endometrial tissue in endometriosis model mice in an embodiment of the present invention;

[0041] Figure 10 This is a schematic diagram of the results in the examples of the present invention showing that DYD@PDA-PEG-LN NPs have no damage to the ovarian function and fertility of mice and no teratogenic effect on mouse embryos. DETAILED DESCRIPTION

[0042] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0043] See also Figures 1-10 The present invention provides a technical solution: preparation of polydopamine nanocomposite materials and application in the treatment of endometriosis.

[0044] Example 1 - Preparation of Dydrogesterone-loaded Polydopamine Nanocomposite DYD@PDA-PEG-LN NPs

[0045] (1) 200 mg of dopamine hydrochloride solid was dissolved in 200 ml of ultrapure water to obtain a dopamine hydrochloride aqueous solution, followed by adding 5 mol / L NaOH solution to adjust the pH to 9, heating and stirring in an oil bath at 60°C for 6 hours, centrifuging at 18500 rpm for 30 minutes, washing three times with deionized water, and freeze-drying the solid precipitate to obtain polydopamine nanoparticles for later use;

[0046] (2) 10 mg of the polydopamine nanoparticles obtained in (1) above and 0.25 mg of dydrogesterone were mixed and stirred in 2 ml of methanol at room temperature for 24 hours, centrifuged at 18500 rpm for 30 minutes, washed, and freeze-dried to obtain polydopamine nanoparticles loaded with dydrogesterone, which were set aside;

[0047] (3) At room temperature, the polydopamine nanoparticles loaded with dydrogesterone obtained in (2) were mixed with methoxypolyethylene glycolamine in an aqueous solution at a mass ratio of 1:2 and stirred overnight. The precipitate was collected by centrifugation, redissolved in water (0.5 mg / ml), and mixed with an equal volume of laminin solution (50 μg / ml) and stirred for 3 hours. The mixture was centrifuged at 18500 rpm for 30 minutes and then freeze-dried to obtain polydopamine nanocomposites loaded with dydrogesterone (DYD@PDA-PEG-LN NPs).

[0048] Example 2 - Preparation of non-drug-loaded polydopamine nanocomposite PDA-PEG-LN NPs

[0049] (1) 200 mg of dopamine hydrochloride solid was dissolved in 200 ml of ultrapure water to obtain a dopamine hydrochloride aqueous solution, followed by adding 5 mol / L NaOH solution to adjust the pH to 9, heating and stirring in an oil bath at 60°C for 6 hours, centrifuging at 18500 rpm for 30 minutes, washing three times with deionized water, and freeze-drying the solid precipitate to obtain polydopamine nanoparticles for later use;

[0050] (2) at room temperature, the polydopamine nanoparticles obtained in (1) and methoxypolyethylene glycol amine were mixed in an aqueous solution at a mass ratio of 1:2 and stirred overnight, and the mixture was centrifuged at 18500 rpm for 30 min and the precipitate was collected to obtain polydopamine nanoparticles modified with PEG;

[0051] (3) At room temperature, the nanoparticles obtained in (2) were re-dissolved in water (0.5 mg / ml) and mixed with an equal volume of laminin solution (50 μg / ml) for 3 hours. The mixture was centrifuged at 18500 rpm for 30 minutes and then freeze-dried to obtain drug-free polydopamine nanocomposites (PDA-PEG-LN NPs).

[0052] Example 3 - Structural Characterization of Polydopamine Nanocomposites

[0053] In this example, the structure of the polydopamine nanocomposite DYD@PDA-PEG-LN NPs was characterized and compared with unmodified and preliminarily modified nanoparticles (PDA NPs, PDA-PEG NPs, and PDA-PEG-LN NPs).

[0054] The steps of the above structural characterization are briefly described as follows:

[0055] Scanning electron microscopy (SEM, ZEISS Sigma 300) and transmission electron microscopy (TEM, JEOL JEM-F200) were used to characterize the morphology of NPs at different stages. The hydrodynamic size distribution and zeta potential of NPs at different stages resuspended in distilled water were measured using a Malvern Panalytical particle size analyzer. The infrared and UV-Vis spectra of these materials were recorded using a Fourier transform near-infrared spectrometer (FTIR, Thermo Fisher Scientific Nicolet iS20) and a UV-visible spectrophotometer.

[0056] The above characterization results are as follows Figure 1 As shown, Figure 1 As shown in A, SEM and TEM results show that the nanoparticles at different modification stages have uniform spherical morphology;

[0057] like Figure 1 B, 1C, DLS measurement results show that PDA NPs, PDA-PEG NPs, PDA-PEG-LN NPs, and DYD@PDA-PEG-LN NPs are stable in water, with particle sizes of 125.3 nm, 134.6 nm, 177.7 nm, and 185.5 nm, respectively. There is no obvious aggregation in water, and their zeta potentials are -26.8 mV, -7.4 mV, -10.7 mV, and -8.7 mV, respectively;

[0058] like Figure 1 As shown in D, the FTIR results provide additional evidence for the formation of PDA NPs. In the dopamine monomer, the absorption peak at 3343 cm-1 corresponds to the stretching vibration of NH and OH, 3041 cm-1 corresponds to the stretching vibration of CH in the benzene ring, 2926 cm-1 and 2855 cm-1 correspond to the stretching vibration of methyl and methylene, 1617 cm-1 and 1501 cm-1 correspond to the stretching vibration of C=C in the benzene ring, the absorption peak at 1501 cm-1 is also mixed with NH bending vibration, and the absorption peak at 1286 cm-1 corresponds to the stretching vibration of CO. In NPs, 3403 cm-1 corresponds to NH and OH stretching vibrations, 1608 cm-1 and 1514 cm-1 correspond to C=C stretching vibrations in the benzene ring, the absorption peak at 1514 cm-1 also corresponds to NH bending vibration, and the absorption peak at 1288 cm-1 corresponds to CO stretching vibration. The main chemical bonds in dopamine are benzene rings, phenolic hydroxyl groups, and amino groups, among which the benzene ring has obvious peaks at 1617 cm-1 and 1501 cm-1; however, after polymerization to form dopamine, the structure of the benzene ring will change, and new C=C double bonds may be formed. The peaks of these C=C are all in the 1650-1500 cm-1 band. Since the absorption peaks of C=C from different sources overlap with each other, the absorption peak in this band becomes wider. Therefore, the successful synthesis of polydopamine is judged by the increase in the width of the absorption peak at this location.

[0059] exist Figure 1 In Figure E, the infrared spectrum of PDA-PEG NPs retained the characteristic absorption peaks of PDA at 3392 cm-1, 1612 cm-1, and 1505 cm-1, while the stretching vibration of CH of the methylene group in polyethylene glycol was increased at 3000-2800 cm-1, and the CO stretching vibration peak on polyethylene glycol was increased at 1094 cm-1. These phenomena proved that the preparation of PDA-PEG NPs was successful. In the infrared spectrum of PDA-PEG-LN NPs, not only the characteristic absorption peaks of PDA-PEG were retained, but also the stretching vibration peak of CN of amide at 1402 cm-1, the stretching vibration peak of Ar-O at 1202 cm-1, and the stretching vibration peak of CN of amine at 1132 cm-1 of laminin (LN) were increased. The above can prove that the loading of laminin was successful.

[0060] In the infrared spectrum of dydrogesterone, 2942 and 2858 cm-1 are the C-H stretching vibration peaks of the alkyl group on dydrogesterone, 1701, 1660, and 1615 cm-1 are the C=O stretching vibration peaks of the carbonyl group on dydrogesterone and the C=C stretching vibration peaks of the double bond, and 1448 and 1378 cm-1 are the C-H bending vibration peaks of the alkyl group on dydrogesterone. In the infrared spectrum of DYD@PDA-PEG-LN NPs, the characteristic absorption peaks of PDA-PEG-LN NPs are still retained, and the characteristic absorption peak of dydrogesterone can be seen around 1660 cm-1. From this, we can judge that DYD@PDA-PEG-LN NPs were successfully prepared.

[0061] Figure 1 F recorded the changes in the UV-Vis spectra of the particles at different stages. DYD@PDA-PEG-LN NPs had a stronger absorbance at 260nm-300nm compared with PDA-PEG-LN NPs, which further proved the successful loading of dydrogesterone.

[0062] Example 4 - Cytotoxicity Verification of Unloaded Polydopamine Nanocomposites (PDA-PEG-LN NPs) and In Vivo Biosafety Characterization of Drug-Loaded Polydopamine Nanocomposites (DYD@PDA-PEG-LN NPs)

[0063] In order to verify that PDA-PEG-LN NPs have good cytotoxicity and DYD@PDA-PEG-LN NPs have good biosafety in vivo, the experimental steps used in this example are as follows:

[0064] (1) Verification of PDA-PEG-LN NPs cytotoxicity

[0065] The steps of the above cytotoxicity validation are briefly described as follows:

[0066] Human endometrial stromal cells (HESCs) were cultured in DMEM medium (Servicebio) containing 10% FBS and 1% penicillin-streptomycin in a constant temperature and humidity environment (37°C, 5% CO2).

[0067] MTT assay: HESCs were cultured in 96-well plates at a cell density of 104 cells per well for 6 h. PDA-PEG-LN NPs at different concentrations were added and incubated for 24 or 48 h. The cells were then treated with an MTT kit (Beyotime Biotechnology) and the absorbance change at 570 nm was measured on a microplate reader. To observe cell viability after NPs incubation, HESCs were cultured in 6-well plates for 12 h. NPs at different concentrations were added and incubated for 24 h. After washing with PBS, the cells were stained with Calcein AM and PI (Beyotime Biotechnology) for live and dead cells, and fluorescence images were collected using a fluorescence microscope (Leica).

[0068] LDH release assay: To verify the proportion of LDH released from cells after co-incubation with NPs, cells without NPs were lysed, the precipitate was removed by centrifugation, and the supernatant was collected after 24 or 48 hours of incubation with NPs. The total LDH level in the cells and the LDH concentration released in the supernatant after incubation with NPs were measured using an LDH detection kit after centrifugation to remove the precipitate.

[0069] ATP level determination (Beyotime Biotechnology): HESCs were incubated with different concentrations of NPs for 24 or 48 hours. The cells were washed and lysed, and ATP detection working solution was added. The chemiluminescence value of each group was measured using a multifunctional microplate reader to reflect the ATP level of each group.

[0070] Hemolysis assay: Red blood cells were separated from serum and diluted to four times the volume with normal saline. 0.2 ml of the diluted RBCs were mixed with 0.8 ml of 0.9% NaCl solution as the negative control group, and 0.2 ml of the diluted RBCs were mixed with 0.8 ml of water as the positive control group. RBCs were then incubated with 0.9% NaCl solution containing different concentrations of NPs. After 3 hours, the absorbance at 541 nm was measured using a multifunctional microplate reader.

[0071] The results of the above tests are as follows Figure 2 As shown, in Figure 2 In A, the relative viability of endometrial stromal cells (HESCs) at different concentrations of PDA-PEG-LN NPs was evaluated, and no significant cytotoxicity was observed;

[0072] like Figure 2 As shown in Figure B, under fluorescence excitation, dead cells will be stained with PI and emit red light, and living cells will be stained with CalceinAM and emit green light. Almost all cells in all groups appear green, which further proves that PDA-PEG-LN NPs have no obvious cytotoxicity.

[0073] The concentration of lactate dehydrogenase (LDH) in the supernatant after co-incubation of cells with PDA-PEG-LN NPs was detected to reflect the release of cell LDH caused by PDA-PEG-LN NPs. Figure 2 C shows that the LDH release level of the experimental group cells was not significantly different from that of the control group, which proves that NPs do not cause potential cell damage;

[0074] like Figure 2 As shown in D, there was no significant difference in the ATP levels of cells in each group, and PDA-PEG-LN NPs did not affect the mitochondrial function of cells;

[0075] Hemolysis experiments were used to verify the interaction between PDA-PEG-LN NPs and blood components. After incubation of NPs with red blood cells for 3 h, no obvious hemolysis was observed at all concentrations, e.g. Figure 2 As shown in E, the above experiments demonstrated that PDA-PEG-LNNPs have good biocompatibility in vitro;

[0076] (2) Verification of the in vivo biosafety of DYD@PDA-PEG-LN NPs

[0077] The steps of the above-mentioned in vivo biosafety verification are briefly described as follows:

[0078] DYD@PDA-PEG-LN NPs were injected into mice at a dose of 10 mg / kg body weight via subcutaneous injection and tail vein injection, once every two days. After 21 days, the subcutaneous injection group, tail vein injection group and control group were compared to demonstrate that DYD@PDA-PEG-LN NPs also have good biosafety in vivo.

[0079] The above test results, such as Figure 2 As shown in F, there was no significant difference in the weight changes of mice in each group during the injection process. Figure 2 As shown in G, there was no significant difference in the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) among the mice groups;

[0080] The main organs of mice in each group were removed and stained with hematoxylin and eosin (H&E). Figure 2 As shown in Figure 3, the results showed that no obvious morphological changes or signs of inflammation were found in the heart, liver, spleen, lungs and kidneys of mice after either subcutaneous or intravenous injection. The above experiments can prove that DYD@PDA-PEG-LN NPs also have good biosafety in vivo.

[0081] Example 5 - Characterization of the Antioxidant and Anti-inflammatory Capabilities of Polydopamine Nanocomposites

[0082] In order to verify the antioxidant capacity and ability to reduce the expression of inflammatory factors of the modified polydopamine nanocomposite (PDA-PEG-LN NPs), the experimental steps used in this example are as follows:

[0083] (1) Verification of the scavenging ability of PDA-PEG-LN NPs for superoxide anions and hydroxyl radicals

[0084] The steps for verifying the superoxide anion and hydroxyl radical scavenging ability are briefly described as follows:

[0085] Riboflavin (20 μM), methionine (12.5 mM) and NBT (75 μM) were added to 25 mM PBS (pH 7.4) and irradiated under ultraviolet (UV) for 15 minutes. After NBT was reduced, a strong absorption signal could be detected at 600 nm, indicating that a high concentration of superoxide anions (O2·-) were generated. Adding different concentrations of PDA-PEG-LN NPs to the above solution and then irradiating with UV could reduce the absorbance at 600 nm. The photoreduction inhibition rate of NBT was measured to reflect the scavenging ability of PDA-PEG-LN NPs for superoxide anions (O2·-). The fluorescent 2-hydroxyterephthalic acid generated by terephthalic acid (TA) in the presence of ·OH could be detected at 425 nm. Terephthalic acid (0.5 mM, NaOH solution), H2O2 (10 mM) and NPs of different concentrations were added to PBS (pH 7.4). After incubation in the dark for 18 hours, the fluorescence signal value of 2-hydroxyterephthalic acid (Ex: 320 nm, Em: 425 nm) was detected to reflect the change in ·OH concentration, thereby obtaining the scavenging efficiency of PDA-PEG-LN NPs of different concentrations on ·OH.

[0086] like Figure 3 As shown in A, the absorbance at 600 nm decreases with the increase of NPs concentration. When the concentration of PDA-PEG-LN NPs is 0.05 mg / ml, it can almost remove 60% of superoxide anions (O2·-). When the concentration of PDA-PEG-LN NPs is greater than 0.5 mg / ml, the decrease in absorbance is no longer obvious, which may be that O2·- in the solution is completely removed, which proves that PDA-PEG-LN NPs have good scavenging ability for O2·-.

[0087] like Figure 3 As shown in Figure 3B, adding different concentrations of PDA-PEG-LN NPs to the solution can significantly reduce the fluorescence value at 425 nm. The fluorescence intensity also decreases with the increase of PDA-PEG-LN NPs concentration, which proves that PDA-PEG-LN NPs also has a good scavenging effect on ·OH.

[0088] (2) Uptake of PDA-PEG-LN NPs by HESCs

[0089] The steps for verifying the uptake capacity of HESCs for PDA-PEG-LN NPs are briefly described as follows:

[0090] After culturing HESCs in a 6-well plate for 12 h, 0.1 mg / ml ICG fluorescently labeled PDA-PEG-LN NPs were added and incubated with the cells. The cells were washed three times after incubation for 1, 2, 4, 6, 24, and 48 h. The cells were digested with trypsin to prepare a cell suspension, and the fluorescence intensity of the cells was detected by flow cytometry to reflect the uptake of PDA-PEG-LN NPs by HESCs.

[0091] like Figure 3 As shown in Figure C, the fluorescence signal in the cells can be detected after 1 hour of incubation, and the intensity of the fluorescence signal increases with the increase of incubation time, which means that HESCs can not only take up PDA-PEG-LN NPs, but also the longer the incubation time, the more the amount taken up.

[0092] (3) PDA-PEG-LN NPs clear ROS in HESCs and inhibit the expression of inflammatory factors

[0093] The steps for verifying the ability of PDA-PEG-LN NPs to scavenge ROS in HESCs and inhibit the expression of inflammatory factors in HESCs are briefly described as follows:

[0094] First, PDA-PEG-LN NPs were co-incubated with HESCs to allow the cells to fully absorb them, and then the DCFH-DA probe was added. Flow cytometry was used to determine whether PDA-PEG-LN NPs would affect the ROS level in normal cells. Secondly, DCFH-DA probe was added to the control group cells and cells that had absorbed PDA-PEG-LN NPs, and then Rosup reagent was added to the cell culture medium to promote the generation of intracellular ROS. The changes in ROS levels in the two groups of cells were detected by flow cytometry. Similarly, 5 ng / ml IL-1β was added to the culture medium of the control group cells and the cell culture medium of cells that had absorbed PDA-PEG-LN NPs. After co-culture for 24 hours, the changes in intracellular ROS were determined by DCFH-DA probe and flow cytometry. The cell supernatants of each group were collected, and the levels of IL-6 and TNF-α in the supernatants were determined by enzyme-linked immunosorbent assay.

[0095] like Figure 3As shown in Figure D, the above experimental results show that the addition of PDA-PEG-LN NPs does not affect the ROS level in normal cells. The addition of exogenous Rosup reagent can significantly increase the ROS content in both groups of cells. After the cells that have taken up PDA-PEG-LN NPs are stimulated, the increase in their intracellular ROS level is significantly less than that in the control group cells. Incubation with IL-1β for 24 hours will indeed cause an increase in intracellular ROS in HESCs, and PDA-PEG-LN NPs can also reduce the ROS level of cells after being stimulated by IL-1β. Figure 3 E display;

[0096] IL-1β can also increase the secretion of IL-6 and TNF-α in HESCs cells, and PDA-PEG-LN NPs can also reduce the expression of IL-6 and TNF-α. Figure 3 F can show the expression of IL-6, Figure 3 G can show the expression of TNF-α.

[0097] Example 6 - Characterization of the effects of polydopamine nanocomposites on macrophage polarization and related gene expression

[0098] In order to verify that PDA-PEG-LN NPs can inhibit the expression of M1 macrophages and their related genes and promote the expression of M2 macrophages and their related genes, the experimental steps used in this example are as follows:

[0099] THP-1 cells were cultured in RPMI-1640 medium (Servicebio) containing 10% FBS, 1% penicillin-streptomycin, and 0.05 mM β-mercaptoethanol (Solarbio) in a constant temperature and humidity environment (37°C, 5% CO2). THP-1 cells were stimulated with 100 ng / ml PMA (MCE) and induced into M0 macrophages 48 h later. After replacing the medium with fresh medium, 100 ng / ml PMA, 100 ng / ml LPS (Sigma), and 20 ng / ml IFN-γ (MCE) were added. After 72 h, M1 macrophages were obtained. Alternatively, after replacing the medium with fresh medium, 100 ng / ml PMA, 20 ng / ml IL-4 (MCE), and 20 ng / ml IL-13 (MCE) were added. After 72 h, M2 macrophages were obtained. When inducing M1 and M2 macrophages, PDA-PEG-LN was added to the culture medium, respectively. NPs, and after 72 hours, whether the addition of PDA-PEG-LNNPs affects the polarization of M1 or M2 macrophages was evaluated. After each group of cells was fixed and permeabilized (Elabscience), they were stained with FITC anti-human CD68 (Biolegend) antibody, and the fluorescence intensity of the cells in the FITC channel was detected by flow cytometry to reflect the expression of CD68 in each group of cells. After the successful induction of M0 macrophages, APC anti-human CD86 antibody was used to stain the surface of each group of cells, and the fluorescence intensity of the cells in the APC channel was detected by flow cytometry to reflect the expression of M1 macrophage marker CD86 in each group of cells. In order to detect the expression of M2 macrophage marker CD206 in each group of cells, PE was added to the cell suspension after fixation and permeabilization. Anti-human CD206 antibodies were used, and the fluorescence of the cells in the PE channel was detected by flow cytometry. RNA was extracted from each cell group using a centrifugal column RNA extraction method. After removing genomic DNA, reverse transcription reaction (Takara) was performed. The resulting cDNA solution was used using the TBGreen chimeric fluorescence method (Takara) and Real-Time PCR experiments using an Applied Biosystems 7500 to detect changes in macrophage-related genes in each group.

[0100] The above experimental results, such as Figure 4 As shown in A, the expressions of CD68 and CD14 in the induced cells were higher than those in THP-1, proving that these groups of cells were all macrophages;

[0101] like Figure 4B. Flow cytometry results showed that the positive rate of surface marker CD86 of M1 macrophages induced by PMA, LPS and IFN-γ was significantly higher than that of M0 and M2 groups, while the addition of PDA-PEG-LN NPs during M1 induction could significantly reduce the positive rate of CD86.

[0102] The expression of M1 macrophage-related genes was further evaluated by qRT-PCR. The results showed that the gene expression of TNF-α, IL-6, CXCL10, CD80, and CD86 in M1 macrophages was significantly higher than that in the M0 and M2 groups, while the addition of PDA-PEG-LN NPs could reduce the expression of TNF-α, IL-6, CXCL10, CD80, and CD86 genes in M1 macrophages.

[0103] like Figure 4 As shown in C, when inducing M2 macrophages, flow cytometry results showed that the CD206 positivity rate of M2 macrophages was significantly higher than that of M0 and M1 macrophages, and the addition of PDA-PEG-LN NPs significantly increased the CD206 positivity rate of M2 macrophages;

[0104] As shown by the qRT-PCR results, the expression of CCL22, CD206 and TGF-β genes in M2 macrophages was higher than that in M0 and M1 macrophages. The M2 macrophages with the addition of PDA-PEG-LN NPs showed a significant increase in CD206 expression at the flow cytometry and qRT-PCR levels. The qRT-PCR results also showed that PDA-PEG-LN NPs could upregulate the expression of CCL22 gene in M2 macrophages, but the expression of TGF-β was not significantly different from that of normally induced M2 macrophages. Therefore, PDA-PEG-LN NPs can effectively downregulate the expression of inflammatory genes and CD86 in M1 macrophages, inhibit the polarization of M0 to M1, and at the same time upregulate the expression of M2 macrophage-related genes and CD206, promoting the polarization of M0 to M2 macrophages.

[0105] Example 7 - Characterization of the Effects of Polydopamine Nanocomposites on Endometrial Stromal Cells

[0106] In order to verify that DYD@PDA-PEG-LN NPs can better inhibit the proliferation and migration of HESCs cells and promote their apoptosis than dydrogesterone alone, the experimental steps used in this example are as follows:

[0107] MTT assay: HESCs were co-incubated with dydrogesterone and DYD@PDA-PEG-LN NPs carrying the same dose of dydrogesterone for 48 h, and the relative viability of HESCs cells at different drug concentrations was evaluated using the MTT assay.

[0108] EdU proliferation assay: Using the EdU Cell Proliferation Kit with Alexa Fluor 555, the thymidine analog EdU (5-ethynyl-2'-deoxyuridine) is incorporated during DNA synthesis. EdU is then labeled with the fluorescent dye Alexa Fluor 555 via a click reaction. After digestion and resuspending, the EdU-555 ratio is measured by flow cytometry, thereby measuring the rate of cell proliferation.

[0109] Apoptosis cell detection: The proportion of apoptotic cells after co-incubation was detected using the FITC-Annexin V / PI Apoptosis Detection Kit and flow cytometry. In the early stages of apoptosis, phosphatidylserine (PS) is externalized to the cell surface and labeled with FITC-Annexin V, emitting green light. In necrotic cells or cells that have lost cell membrane integrity in the late stages of apoptosis, the nuclei are bound by propidium iodide (PI), resulting in red fluorescence.

[0110] Mitochondrial membrane potential detection: A mitochondrial membrane potential assay kit with JC-1 and fluorescence microscopy were used to detect changes in mitochondrial membrane potential in each group of cells. A decrease in mitochondrial membrane potential is also one of the early hallmark events of cell apoptosis. When the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix and exists as JC-1 monomers, which can produce green fluorescence. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form polymers J-aggregates, which can produce red fluorescence.

[0111] Cell migration assay: HESCs were incubated with PDA-PEG-LN NPs, dydrogesterone, and DYD@PDA-PEG-LN NPs for 48 hours before being replated. A 200-μl pipette tip was then used to scratch the cells. Changes in scratch width were observed after 24 and 48 hours to reflect cell migration ability.

[0112] The above experimental results are as follows Figure 5 As shown in A, when the dosage of dydrogesterone was 0.1 μg / ml and 1 μg / ml, the relative viability of cells was significantly reduced, and the effect of DYD@PDA-PEG-LN NPs on HESCs was more significant than that of the same dose of dydrogesterone alone;

[0113] like Figure 5 As shown in Figure B, the use of PDA-PEG-LN NPs did not affect cell proliferation, while dydrogesterone and DYD@PDA-PEG-LN NPs reduced the proportion of proliferating cells. However, the effect of DYD@PDA-PEG-LN NPs was significantly better than that of using the same dose of dydrogesterone alone.

[0114] like Figure 5 As shown in Figure C, the use of non-drug-loaded NPs did not affect cell apoptosis, but after co-incubation with dydrogesterone and DYD@PDA-PEG-LN NPs, the proportion of cells in the early stage of apoptosis, late stage of apoptosis or necrosis increased;

[0115] like Figure 5 As shown in D, co-incubation with dydrogesterone and DYD@PDA-PEG-LN NPs can reduce the mitochondrial membrane potential of cells and increase the proportion of cells in the early stage of apoptosis. Figure 5 In C, we can also find that the proportion of cells in the early stage of apoptosis increased significantly, which is consistent with Figure 5 The experimental results shown in D correspond to;

[0116] like Figure 5 As shown in E, the scratch assay evaluated the effect of drugs on cell migration. The results showed that the use of DYD@PDA-PEG-LN NPs could better inhibit the apoptosis of HESCs compared with the use of dydrogesterone alone.

[0117] Example 8 - Characterization of the Targeting Effect of Polydopamine Nanocomposites on Ectopic Endometrial Lesions in Endometriosis Model Mice

[0118] To verify that the modified DYD@PDA-PEG-LN NPs can better target and retain in ectopic endometrial tissue lesions than unmodified DYD@PDA NPs, whether injected subcutaneously or intravenously, the experimental steps used in this example are as follows:

[0119] (1) Establishment of endometriosis mouse model and sham operation mouse model

[0120] The steps for establishing the above-mentioned endometriosis mouse model and sham-operated mouse model are briefly described as follows:

[0121] Female BALB / c nude mice, 6 weeks old and weighing approximately 14 g, were used. All nude mice were housed in an SPF barrier environment with constant temperature and humidity, 12 hours of light and 12 hours of darkness. Feed and water were sterilized by high pressure. Typical ectopic endometrial tissue from patients with ovarian endometriosis undergoing surgical treatment was selected and transplanted subcutaneously into the interscapular region of female Balb / c nude mice (one graft per mouse). Patient inclusion criteria were: women of childbearing age aged 20-45 years, with regular menstrual cycles, who underwent laparoscopic or laparotomy for dysmenorrhea or infertility, who were diagnosed with endometriosis by postoperative pathology, and who were diagnosed with For stage III-IV endometriosis, the ex vivo tissue was transferred to a clean bench, washed three times with PBS, and cut into approximately 6mm*6mm tissue blocks. The nude mice were anesthetized with 2% isoflurane inhalation, and the back skin was disinfected with iodine. Sterile ophthalmic scissors were used to make an incision of approximately 0.5cm between the shoulder blades. The subcutaneous space was bluntly freed to 1cm, and the endometrial tissue was buried at the deepest point. The incision was sutured with 4-0 silk sutures. After the incision was disinfected, the mice were returned to the cage and continued to be raised. Starting on the day of surgery and every 48 hours, estradiol benzoate was injected into the leg muscles of the nude mice to maintain lesion growth. Sham-operated mice were only incised and sutured at the same site, and no endometrial tissue was implanted.

[0122] (2) Evaluation of the distribution and aggregation of modified DYD@PDA-PEG-LN NPs and unmodified DYD@PDA NPs in endometriosis model mice after subcutaneous injection

[0123] The steps for evaluating the distribution and aggregation ability of the above-mentioned DYD@PDA-PEG-LN NPs and DYD@PDA NPs in endometriosis model mice after subcutaneous injection are as follows:

[0124] On the 7th day after surgical transplantation, ICG fluorescent-labeled DYD@PDA-PEG-LN NPs or DYD@PDA NPs were injected into mice at a dose of 10 mg / kg body weight. Local administration of NPs was achieved by subcutaneous injection into the endothelial graft. Fluorescent images of the mice were recorded using an in vivo imaging system 1, 2, 4, 6, 24, and 48 hours after injection.

[0125] The above experimental results, such as Figure 6 As shown in A, strong fluorescence signals can be observed at the graft site 1, 2, and 4 hours after injection. Starting from the 6th hour, it can be observed that the fluorescence signal of the DYD@PDA NPs group is slightly lower than that of the DYD@PDA-PEG-LN NPs group. At the 24th and 48th hours, it can be observed that the fluorescence signal of the DYD@PDA NPs group is significantly lower than that of the DYD@PDA-PEG-LN NPs group. The DYD@PDA-PEG-LN NPs modified with PEG and LN can be better taken up by the ectopic endometrium and retained in the lesion tissue.

[0126] (3) Evaluation of the distribution and aggregation of modified DYD@PDA-PEG-LN NPs and unmodified DYD@PDA NPs in endometriosis model mice after intravenous injection

[0127] The steps for evaluating the distribution and aggregation ability of the above-mentioned DYD@PDA-PEG-LN NPs and DYD@PDA NPs in endometriosis model mice after tail vein injection are as follows:

[0128] On the 7th day after surgical transplantation, ICG fluorescently labeled DYD@PDA-PEG-LN NPs or DYD@PDA NPs were injected into mice at a dose of 10 mg / kg body weight. Systemic administration of NPs was achieved by tail vein injection. Fluorescent images of the mice were recorded using an in vivo imaging system 1, 2, 4, 6, 24, and 48 hours after injection.

[0129] The above experimental results, such as Figure 6 As shown in Figure 2, 4 hours after injection, fluorescence signals generated by the aggregation of NPs were observed in the endometriosis lesions of both groups of mice, but the fluorescence signal of the DYD@PDA NPs group was lower. At 6, 24, and 48 hours, the fluorescence signals at the lesions of the DYD@PDA-PEG-LN NPs group decreased compared with those at 2 and 4 hours, but a strong fluorescence signal generated by the aggregation of nanoparticles could still be seen. The fluorescence signals of the DYD@PDA NPs group at 6, 24, and 48 hours were significantly lower than those of the DYD@PDA-PEG-LN NPs group. Even at 48 hours, almost no fluorescence signals were detected in the lesions of some mice. Compared with unmodified DYD@PDA NPs, DYD@PDA-PEG-LN NPs modified with PEG and LN can better target, aggregate, and retain in ectopic endometrial tissue.

[0130] (4) Evaluation of the distribution and aggregation of modified DYD@PDA-PEG-LN NPs after subcutaneous or intravenous injection into sham-operated mice

[0131] The steps for evaluating the distribution and aggregation ability of the above-mentioned DYD@PDA-PEG-LN NPs in endometriosis model mice after subcutaneous injection or tail vein injection are as follows:

[0132] On the seventh day after sham surgery, DYD@PDA-PEG-LN NPs were injected subcutaneously or intravenously into the wound, and the in vivo fluorescence images of the mice were recorded at 1, 2, 4, 6, 24, and 48 hours using an in vivo imaging system.

[0133] The above experimental results, such as Figure 6As shown in Figure C, after local injection of DYD@PDA-PEG-LN NPs into the wounds of sham-operated mice, it was found that the local fluorescence signal of the wound gradually weakened with the extension of time, especially 6 hours after injection, the fluorescence intensity was significantly lower than that of endometriosis model mice. When DYD@PDA-PEG-LN NPs were injected into the sham-operated mice through the tail vein, no obvious aggregation was found at the wound site, which means that the aggregation of DYD@PDA-PEG-LN NPs in endometriosis model mice was mainly caused by the transplanted ectopic endometrium, rather than surgical factors.

[0134] Example 9 - Characterization of the Effect of Polydopamine Nanocomposite on Lesion Tissue in Endometriosis Model Mice

[0135] To verify that DYD@PDA-PEG-LN NPs can better promote ectopic endometrial atrophy and inhibit its hormone responsiveness and proliferation activity compared with PBS, PDA-PEG-LN NPs or dydrogesterone alone, whether injected subcutaneously or intravenously, the experimental steps used in this example are as follows:

[0136] Endometriosis model mice were randomly divided into a subcutaneous injection group and a tail vein injection group. Each group was further divided into four groups, namely the control group (PBS injection), the PDA-PEG-LN group (10 mg / kg body weight), the dydrogesterone group (102.6 μg / kg body weight), and the DYD@PDA-PEG-LN group (10 mg / kg body weight). Injection treatment began on the 7th day after transplantation and ended 21 days after injection (28 days after transplantation). The area of ​​the subcutaneous graft of each group of mice was recorded every 4 days after endometrial transplantation. After the end of treatment, the grafts were removed and weighed. The removed grafts were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. Immunohistochemical staining (IHC) of ESR1, PGR, LI-67, and VEGF was then performed. After dewaxing and antigen retrieval, blocking, incubation with primary and secondary antibodies, section washing, and DAB development, the immunohistochemical staining of ESR1, PGR, LI-67, and VEGF was observed under a microscope.

[0137] The above experimental results are as follows Figure 7 、 8 As shown, Figure 7 A is a comparison of the changes in graft area and graft weight after treatment in four groups of mice after 21 days of local injection of grafts using subcutaneous injection. Figure 7B is a comparison of the graft area change and graft weight after 21 days of treatment by tail vein injection. Regardless of whether it is local injection or tail vein injection, the area and weight of the graft in the dydrogesterone group and the DYD@PDA-PEG-LN NPs group decreased significantly. Although the two groups carried the same amount of dydrogesterone, the degree of decrease in the DYD@PDA-PEG-LN NPs group was significantly greater than that in the dydrogesterone group. The clinical dosage of dydrogesterone for the treatment of endometriosis is about 0.5 mg / kg / d, while the dydrogesterone injected into the mice of the DYD@PDA-PEG-LN NPs group is about 0.05 mg / kg / d. This means that the use of DYD@PDA-PEG-LN NPs to treat endometriosis may only require a lower dose of dydrogesterone to achieve better therapeutic effects, which may also mean a lower incidence of side effects.

[0138] Figure 8 A is to inject the graft locally subcutaneously, Figure 8 B is the result of immunohistochemical staining of ESR1, PGR, KI-67 and VEGF on the endometrial graft removed after tail vein injection treatment. It can be found that regardless of the injection method, the expression of ESR1 in the endometrial tissue after DYD@PDA-PEG-LN NPs treatment is decreased. The reduction of steroid receptors means that the hormone responsiveness of the graft is decreased, and the ability to grow and proliferate under the action of estrogen is lost. At the same time, the decrease in KI-67 and VEGF expression also proves the reduction in the proliferation ability and angiogenesis activity of the endometrial tissue after treatment. Whether it is subcutaneous injection or intravenous injection, DYD@PDA-PEG-LN NPs can promote the atrophy of the ectopic endometrium and inhibit its proliferation activity, thereby achieving the treatment of endometriosis lesions.

[0139] Example 10 - Characterization of the effect of polydopamine nanocomposites on oxidative stress and inflammatory microenvironment of the ectopic endometrium in endometriosis model mice

[0140] In order to verify that DYD@PDA-PEG-LN NPs can better improve the oxidative stress and inflammation levels of ectopic endometrial tissue, reduce the content of M1 macrophages and increase the proportion of M2 macrophages in ectopic endometrium, whether injected subcutaneously or intravenously, the experimental steps used in this example are as follows:

[0141] (1) Evaluation of hydrogen peroxide content in endometriosis lesions of mice in each group after treatment with different drugs

[0142] The steps for evaluating the hydrogen peroxide content in the ectopic endometrial tissue of endometriosis mice after treatment in the above groups are as follows:

[0143] Grafts injected with PBS alone and injected with PDA-PEG-LN, dydrogesterone, and DYD@PDA-PEG-LN 21 days later were collected and prepared into tissue homogenates. A portion of the homogenate was lysed with RIPA and the protein concentration in the grafts was determined using the Bradford method. The protein concentration in the grafts was determined using ferric-orange xylenol The FOX system (FOX) was used to detect hydrogen peroxide levels in each group, reflecting the changes in ROS in the lesion tissue of each group. FOX working solution: ferrous ammonium sulfate was added to 250 mM sulfuric acid solution to make working solution 1 at a concentration of 25 mM. 62.5 μM xylenol orange and 150 mM sorbitol were used to make working solution 2. The ratio of working solutions 1 and 2 was 1:100. The working solutions should be prepared freshly for use. The appropriately diluted test solution was mixed with the working solution in a 1:2 ratio and incubated in the dark for 30 minutes. After centrifugation at 3500 rpm for 10 minutes, the absorbance of the supernatant was measured at 580 nm using a microplate reader. The amount of hydrogen peroxide in the test solution was calculated using a standard curve. This standard curve was obtained by diluting 30% hydrogen peroxide solution to different concentrations ranging from 0.0136 μg / ml to 0.17 μg / ml. The R2 correlation coefficient was 0.99996. The final hydrogen peroxide content of the graft was expressed in μg / mg protein.

[0144] The above experimental results, such as Figure 9 As shown in A, after treatment with PDA-PEG-LN NPs and DYD@PDA-PEG-LN NPs, the content of hydrogen peroxide in the grafts decreased significantly compared with that before treatment, regardless of whether it was subcutaneous injection or intravenous injection, while there was no significant difference in the other groups. This proves that the oxidative stress level of the ectopic endometrium treated with PDA-PEG-LN NPs and DYD@PDA-PEG-LN NPs decreased after treatment;

[0145] (2) Evaluation of changes in serum inflammatory factor levels in each group of mice after treatment with different drugs

[0146] The steps for evaluating the changes in serum inflammatory factors in endometriosis mice after treatment in each of the above groups are as follows:

[0147] The serum of normal mice, mice injected with PBS alone, and mice injected with PDA-PEG-LN, dydrogesterone, and DYD@PDA-PEG-LN 21 days later was collected, and the levels of THN-α, IL-1β, and IL-6 in the mouse serum were determined by enzyme-linked immunosorbent assay.

[0148] The above experimental results are as follows Figure 9Figures B, 9C, and 9D show the changes in the levels of TNF-α, IL-1β, and IL-6 in mouse serum, respectively. Non-operated mice are normal mice without modeling, PBS mice are mice injected with PBS for 21 days starting from the seventh day after modeling, and the other groups are mice injected with PDA-PEG-LN NPs, dydrogesterone, and DYD@PDA-PEG-LN NPs subcutaneously and intravenously for 21 days. The inflammatory factor levels of mice in the PBS group were significantly higher than those of mice without modeling. The inflammatory factor levels of mice can be reduced by injection of PDA-PEG-LN NPs, DYD, and DYD@PDA-PEG-LN NPs for 21 days, but the effect of DYD@PDA-PEG-LN treatment is the best.

[0149] (3) Evaluation of the changes in the proportion of M1 and M2 macrophages in the ectopic endometrial tissue of each group of mice after treatment with different drugs

[0150] The steps for evaluating the changes in the ratio of M1 and M2 macrophages in the ectopic endometrial tissue of endometriosis mice after treatment in the above groups are as follows:

[0151] The endothelial grafts removed 21 days after treatment were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. All cell nuclei were stained with DAPI, all macrophages were stained with F4 / 80 antibody, M1 macrophages were stained with iNOS antibody, and M2 macrophages were labeled with CD206 antibody. The staining results were observed using a fluorescence microscope.

[0152] The above experimental results are as follows Figure 9 E and 9F show the ectopic endometrial grafts of model mice treated with PDA-PEG-LN NPs, DYD, and DYD@PDA-PEG-LN NPs for 21 days, as well as the grafts of the control group. Regardless of subcutaneous or intravenous injection, the expression of iNOS in the endometrium treated with PDA-PEG-LN NPs and DYD@PDA-PEG-LN NPs decreased, while the expression of CD206 increased. In the endometrium of these two groups after treatment, the expression of pro-inflammatory macrophages M1 decreased, while the expression of anti-inflammatory macrophages M2 increased. However, there was no significant change in the expression of M1 and M2 in the endometrium treated with only PBS and DYD.

[0153] Example 11 - Effects of polydopamine nanocomposites on mouse ovarian function and fertility, and characterization of teratogenic effects on embryonic mice

[0154] To verify that DYD@PDA-PEG-LN NPs do not affect the ovarian function and fertility of mice, and do not cause teratogenic effects on embryonic mice, whether injected subcutaneously or intravenously, the experimental steps used in this example are as follows:

[0155] (1) Effect of injection of DYD@PDA-PEG-LN NPs on ovarian function in mice

[0156] The steps for evaluating the effect of DYD@PDA-PEG-LN NPs on mouse ovarian function are as follows:

[0157] The ovaries and serum of mice that were not injected with drugs and those that were injected with DYD@PDA-PEG-LN NPs subcutaneously and intravenously at a dose of 10 mg / kg body weight every two days for 21 days were collected. The structural changes of the ovaries were observed by H&E staining, and the AMH levels in the serum were measured by enzyme-linked immunosorbent assay to reflect the ovarian reserve function of mice in each group.

[0158] The above experimental results are as follows Figure 10 As shown in A, 10B, and 10C, Figure 10 A shows the ovaries of mice in each group after injection. No thickening or structural disorder was observed in the ovarian cortex. Figure 10 As shown in B, there was no significant difference in the number of follicles between the ovaries of mice in the control group, and no obvious ovarian atrophy was found; Figure 10 As shown in C, no matter which type of injection, it will not cause a significant decrease in the AMH level of mice. DYD@PDA-PEG-LN NPs injection treatment for 21 days will not have an adverse effect on ovarian reserve function.

[0159] (2) Effect of injection of DYD@PDA-PEG-LN NPs on mouse fertility

[0160] The steps for evaluating the effect of DYD@PDA-PEG-LN NPs on mouse fertility are as follows:

[0161] One or two female mice were caged with a male mouse of the same age. To ensure successful conception, the male mice in each cage were swapped every four days. The morning when a vaginal plug appeared in the female mouse's vagina was considered day 0.5 of pregnancy. The time from the time mice were caged together to successful conception was recorded for each group. The female mice were euthanized on day 14.5 of pregnancy to accurately count the number of embryos in each group of pregnant mice.

[0162] The above experimental results are as follows Figure 10As shown in Figures 10D, 10E, and 10F, there was no significant difference in the time it took for the mice in each group to be confirmed pregnant. The mice in each group successfully became pregnant without miscarriage, and the number of embryos was not significantly different from that in the control group. Therefore, DYD@PDA-PEG-LN NPs did not affect the natural conception process and the number of embryos in the mice.

[0163] (3) Effects on the placenta of pregnant mice and evaluation of teratogenic effects

[0164] The steps for evaluating the effects of DYD@PDA-PEG-LN NPs injection on the placenta of pregnant mice and teratogenicity 21 days after injection are as follows:

[0165] The female mice were euthanized on gestational day 14.5, and the embryos and placentas of mice in each group were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned for H&E staining;

[0166] The above experimental results are as follows Figure 10 As shown in G and 10H, the embryos of pregnant mice injected with DYD@PDA-PEG-LN NPs showed no significant morphological abnormalities compared with the control group, and no abnormalities were observed in the placenta of the placenta decidua, trophoblast, and spiral arteries. This proves that DYD@PDA-PEG-LN NPs not only have good biocompatibility in mice, but also do not damage ovarian function and fertility, and have no obvious reproductive toxicity.

[0167] It can be seen from the above examples that the polydopamine nanocomposite material of the present invention has good antioxidant and anti-inflammatory capabilities, can also inhibit the polarization of M1 macrophages, and promote the expression of anti-inflammatory macrophages M2. In endometriosis model mice, DYD@PDA-PEG-LN NPs can target ectopic endometrial tissue and promote the atrophy of ectopic endometrial tissue while reducing the amount of DYD. DYD@PDA-PEG-LN NPs have high biocompatibility and low systemic toxicity. Using it to treat endometriosis does not damage ovarian reserve function and fertility, has no obvious toxic side effects, and has excellent biological application value.

[0168] Working principle: Dopamine hydrochloride solid is dissolved in ultrapure water to obtain a dopamine hydrochloride aqueous solution, and then 5 mol / L NaOH solution is added to adjust the pH. The mixture is heated in an oil bath and stirred, and then centrifuged. The mixture is washed three times with deionized water, and the solid precipitate is freeze-dried to obtain polydopamine nanoparticles. The polydopamine nanoparticles and dydrogesterone are mixed and stirred in methanol at room temperature, the mixture is centrifuged and washed three times with deionized water, and freeze-dried to obtain polydopamine nanoparticles carrying dydrogesterone. The polydopamine nanoparticles carrying dydrogesterone are mixed and stirred with methoxypolyethylene glycolamine in an aqueous solution overnight, the precipitate is collected by centrifugation, and the mixture is redissolved in water and mixed with laminin solution, centrifuged and freeze-dried to obtain a polydopamine nanocomposite material. The prepared polydopamine nanocomposite material can be used for endometriosis, can reduce the inflammation level and oxidative stress level of endometriosis lesions, promote the atrophy of ectopic endometrial lesions, and ensure the patient's ovarian function and fertility in the process of treating endometriosis.

[0169] While the embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that various changes, modifications, substitutions, and alterations can be made to the embodiments without departing from the principles and spirit of the invention.

Claims

1. Preparation of polydopamine nanocomposite materials and their application in the treatment of endometriosis, characterized by: The polydopamine nanocomposite material is polydopamine nanoparticles modified with polyethylene glycol and laminin and loaded with dydrogesterone, and the disease is endometriosis.

2. The use according to claim 1, characterized in that: The endometriosis is caused by transplanting human ectopic endometrial tissue into the subcutaneous tissue of female nude mice.

3. The use according to claim 1, characterized in that: The application is to use polydopamine nanocomposite particles to reduce the inflammation level and oxidative stress level of endometriosis lesions and promote the atrophy of ectopic endometrial lesions.

4. The use according to claim 1, characterized in that: The preparation steps of the polydopamine nanocomposite material include: S1. Dissolving dopamine hydrochloride solid in ultrapure water to obtain a dopamine hydrochloride aqueous solution, then adding 5 mol / L NaOH solution to adjust the pH, heating and stirring in an oil bath, and then centrifuging. Washing with deionized water three times, the solid precipitate is freeze-dried to obtain polydopamine nanoparticles for later use; S2. At room temperature, polydopamine nanoparticles and dydrogesterone are mixed and stirred in methanol, centrifuged, washed three times with deionized water, and freeze-dried to obtain polydopamine nanoparticles loaded with dydrogesterone, which are then set aside. S3. At room temperature, polydopamine nanoparticles loaded with dydrogesterone and methoxypolyethylene glycolamine were mixed and stirred in an aqueous solution overnight, the precipitate was collected by centrifugation, redissolved in water, mixed and stirred with the laminin solution, centrifuged and freeze-dried to obtain a polydopamine nanocomposite material.

5. The method for preparing the polydopamine nanocomposite material according to claim 4, wherein: In the S1 process, the dopamine hydrochloride dissolution system is 200 ml of ultrapure water, NaOH solution is added dropwise to adjust the pH to 9, the oil bath heating temperature is 60° C., the stirring reaction time is 6 hours, the centrifugal speed is 18500 rpm, the centrifugation time is 30 minutes, and the dopamine hydrochloride solid mass is 200 mg.

6. The method for preparing the polydopamine nanocomposite material according to claim 4, wherein: In the S2 process, the methanol system is 2 ml, the mass of the polydopamine nanoparticles is 10 mg, the mass of the dydrogesterone is 0.25 mg, the centrifugal speed is 18500 rpm, and the centrifugation time is 30 minutes.

7. The method for preparing the polydopamine nanocomposite material according to claim 4, wherein: In the S3 process, the mass ratio of the polydopamine nanoparticles loaded with dydrogesterone to methoxypolyethylene glycolamine is 1:2, and the volume ratio of the nanoparticle solution to the laminin solution is 1:

1. If nanoparticles not loaded with dydrogesterone need to be prepared in S3, the polydopamine nanoparticles obtained in step S1 are mixed with methoxypolyethylene glycolamine in a mass ratio of 1:2, and the volume ratio of the nanoparticle solution to the laminin solution is 1:

1.

8. The method for preparing the polydopamine nanocomposite material according to claim 7, wherein: The concentration of the nanoparticle solution is 500 μg / ml, the concentration of the laminin solution is 50 μg / ml, the centrifugal speed is 18500 rpm, and the centrifugation time is 30 minutes.

9. A polydopamine nanocomposite material prepared by the method of claim 7, characterized in that: The size of the dydrogesterone-loaded composite nanoparticles (DYD@PDA-PEG-LN NPs) is 185.5 nm, and the size of the un-drug-loaded modified nanoparticles (PDA-PEG-LN NPs) is 177.7 nm. Both the dydrogesterone-loaded composite nanoparticles and the un-drug-loaded modified nanoparticles are monodisperse spherical structures.

10. The use according to claim 9, characterized in that: It also includes that the nanocomposite material is used in the treatment of oxidative stress, chronic inflammatory environment and estrogen dependence of endometriosis.

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