Preparation method and application of self-assembled nano-drug for improving fetal growth limitation

Abstract

The invention discloses a preparation method and application of a self-assembled nano-drug for improving fetal growth limitation. The preparation method comprises the following steps: dissolving an aspirin raw material medicine, polyethyleneimine and phenylboronic acid ester in dimethyl sulfoxide, reversing, uniformly mixing, putting into an ultrasonic cleaning machine until medicine powder is completely dissolved, putting a mixed solution into a dialysis bag, carrying out rotary dialysis in deionized water to remove an organic solvent, and finally, freeze-drying the solution in the dialysis bag to obtain the aspirin-polyethyleneimine-phenylboronic acid ester freeze-dried powder. The self-assembled nano-drug can be obtained. Through intravenous injection, the medicine can improve the pathological pregnancy environment of hypoxia, oxidative stress and inflammation related to growth restriction of a gestational fetus and promote long-term growth and development of the growth-restricted fetus. The self-assembly method is simple and easy to implement, low in price, good in stability and free of immunogenicity, and an organic solvent is easy to remove. More importantly, under the condition that the dosage is far higher than the therapeutic dosage, the self-assembled nano-drug has no toxicity to a maternal body and a fetus, and safe development of the fetus in the gestation period is guaranteed.

Description

Technical Field

[0001] The present invention relates to the field of self-assembling nanomedicines, in particular to the field of nanomedicines for improving fetal growth restriction. Background Art

[0002] Fetal growth restriction (FGR) during pregnancy is a common pregnancy complication that threatens approximately 20 million infants worldwide each year, leading to adverse pregnancy outcomes. It is characterized by impaired fetal development and a birth weight that is below the 90th percentile for gestational age. FGR is a major cause of neonatal morbidity and mortality, accounting for nearly 30% of stillbirths and the second leading cause of perinatal death after preterm birth, posing a significant public health and socioeconomic challenge. The etiology of FGR is multifactorial, including placental insufficiency (e.g., abnormal trophoblast invasion and decreased uteroplacental blood flow), maternal vascular disease (e.g., pregnancy-induced hypertension, chronic hypertension), intrauterine infection, genetic abnormalities, and environmental factors (e.g., malnutrition and smoking). Despite the association with multiple risk factors, the pathological mechanisms underlying FGR are hallmarks of the placenta. Chronic placental dysfunction is a common cause of FGR. Inadequate placental blood flow during pregnancy leads to inadequate nutrient and oxygen supply, thus preventing normal fetal growth. It is known that during normal placental formation, placental trophoblasts are responsible for coordinating the invasion and maturation of the complex vascular network within the placental villi, while the blood is responsible for transporting nutrients, oxygen, etc. from the mother to the fetus and transporting fetal metabolic waste to the mother for excretion. However, fetal growth restriction can hinder angiogenesis in the placental villi, which in turn makes it impossible to deliver sufficient oxygen and nutrients to the fetus, resulting in poor fetal growth. In addition, studies have shown that placental hypoxia, oxidative stress, and immune inflammation during pregnancy are often accompanied by the development of fetal growth restriction. However, the underlying mechanisms connecting hypoxia, oxidative stress, angiogenesis, inflammation, and placental dysfunction have not been fully elucidated, thus limiting the development of new preventive measures and optimal treatment options to effectively improve fetal growth restriction.

[0003] Prevention of fetal growth restriction primarily focuses on interventions before and during pregnancy. Some studies have attempted to prevent fetal growth restriction by improving maternal health. However, current evidence suggests that multinutrient supplementation is not effective in preventing fetal growth restriction or low-birth-weight-for-gestational-age infants. Furthermore, some clinicians recommend providing heparin as a preventive intervention for selected pregnancies at risk for fetal growth restriction, but recent studies have shown that heparin administration is not effective in patients with a history of severe or early-onset fetal growth restriction and thrombophilia. Regarding the fetus, some studies have attempted to administer glucose supplements, but this has not been shown to be effective and may exacerbate the potential risk of fetal acidosis. Clinical guidelines recommend antenatal steroids for all growth-restricted fetuses delivered before 34 weeks of gestation. While steroids may be beneficial for growth-restricted fetuses born after 34 weeks of gestation, particularly those delivered by elective cesarean section, these fetuses have not been shown to experience a statistically significant reduction in fetal respiratory distress syndrome. Currently, most guidelines recommend the use of low-dose aspirin as a first-line treatment before 16 weeks of gestation to prevent fetal growth restriction.

[0004] The key to therapeutic intervention for fetal growth restriction lies in its feasibility during pregnancy. Currently, no therapeutic strategy has been identified that can effectively enhance placental function and promote fetal growth, and timely termination of pregnancy remains the best treatment. However, the etiology of fetal growth restriction is multifactorial, including chronic hypoxia, oxidative stress, impaired angiogenesis, inflammatory disorders, and placental hypoplasia. Therefore, it is necessary to combine multiple therapeutic strategies in the hope of simultaneously targeting these interrelated pathological mechanisms to achieve effective treatment of fetal growth restriction and provide breakthrough new insights into the prevention and management of related pregnancy complications.

[0005] Currently, traditional aspirin therapy for fetal growth restriction remains limited by the risk of bleeding due to its dose-limiting, nonselective biodistribution. In recent years, several novel nanoparticles have been developed to improve fetal growth restriction, including nanoparticles specifically targeting placental trophoblasts and non-viral polymer nanoparticles. However, most of these nanoparticles target only a single target to improve fetal growth restriction. Given the diverse and complex etiology of fetal growth restriction, single-target therapy is not sufficient to improve pregnancy outcomes in fetal growth restriction. Furthermore, most studies rarely examine the short-term and long-term development of offspring from treated fetuses with growth restriction. Summary of the Invention

[0006] The technical problem solved by the present invention is how to prepare a self-assembling nano drug for improving fetal growth restriction, so that the drug is safe for both the pregnant mother and the fetus.

[0007] The present invention utilizes aspirin, polyethyleneimine (PEI), and the reactive oxygen species (ROS) scavenger phenylboronic acid ester (PBE) via covalent bonding to form a nanomaterial with multiple biological activities, including anti-inflammatory and anti-oxidative stress. This material can be self-assembled to form a novel nanomedicine for improving fetal growth restriction. This nanomedicine can alleviate oxidative stress in the hypoxic environment of the placenta through the ROS scavenging ability of PBE. It can also alleviate vascular damage and inflammation in placental trophoblast cells and vascular endothelial cells through aspirin-mediated inflammation protection. PEI nanotherapy promotes targeted drug delivery to the placenta, while reducing the risk of systemic bleeding caused by aspirin and restoring a normal embryonic developmental environment, thereby effectively treating fetal growth restriction.

[0008] Related experiments have demonstrated that the placenta of patients with fetal growth restriction is subjected to a persistent hypoxic environment. Hypoxia induces the production of large amounts of reactive oxygen species (ROS) via mitochondria, amplifying oxidative damage. It also upregulates the expression of certain immune and inflammatory factors, triggering a pro-inflammatory placental microenvironment. This self-assembled nanodrug can exert its antioxidant effects, inhibiting the production of reactive oxygen species (ROS), thereby improving placental hypoxia. It also mitigates hypoxia- / ROS-induced apoptosis, restores the invasive capacity of placental trophoblast cells and the angiogenic capacity of vascular endothelial cells, and inhibits the overexpression of related immune and inflammatory factors, thereby exerting anti-inflammatory and anti-vascular damage effects. Finally, the nanodrug was demonstrated to be unable to cross the placental barrier, demonstrating its high safety for both mother and fetus. Therefore, this self-assembled nanodrug can inhibit the development and progression of fetal growth restriction by ameliorating hypoxia, reducing oxidative stress, and promoting healthy fetal development through its anti-inflammatory and anti-vascular damage effects. Compared to other nanodrugs used to improve fetal growth restriction, the self-assembled nanodrug prepared in this invention exhibits multiple biological activities, unlike targeted drugs with only a single function. Not only does it enable precise and efficient delivery to the placenta, it also addresses the effects of hypoxia, oxidative stress, and inflammation, validating the effectiveness of the "hypoxia-ROS-inflammation axis" as a therapeutic target. Furthermore, this self-assembled nanodrug has demonstrated safety for both mother and fetus. Furthermore, beyond this research model, the adverse effects of hypoxia and inflammation are associated with the development and progression of other pregnancy-related diseases. Therefore, this self-assembled nanodrug, with its multiple biological activities, including anti-oxidative stress and anti-inflammatory properties, holds broad application prospects.

[0009] In view of this, the technical solution adopted by the present invention is as follows: a method for preparing a self-assembled nanomedicine for improving fetal growth restriction, which is obtained by self-assembly of an active oxygen reaction / scavenging device phenylboronate (PBE) and aspirin via polyethyleneimine (PEI), wherein the mass ratio of aspirin:phenylboronate:polyethyleneimine is between 0.5:1:1 and 8:1:1, and the particle size of the self-assembled nanomedicine is between 100 nm and 200 nm.

[0010] Specifically, the above method includes the following steps: dissolving aspirin raw material, polyethyleneimine, and phenylboronic acid ester in an organic solvent, mixing them upside down, and then placing them in an ultrasonic cleaning machine until the drug powder is completely dissolved; then placing the organic solvent in which the aspirin raw material, polyethyleneimine, and phenylboronic acid ester are completely dissolved in a 3500 Da dialysis bag, rotating and dialyzing in deionized water to remove the organic solvent, replacing the deionized water every hour, and finally freeze-drying the solution in the dialysis bag at -80°C for more than three hours, and then transferring it to a freeze dryer for freeze drying to obtain the self-assembled nanomedicine.

[0011] The organic solvent may be dimethyl sulfoxide, or other organic solvents commonly used in pharmaceutical preparations, such as methanol and N,N-dimethylformamide.

[0012] Specifically, the concentration of the phenylboronic acid ester solution is between 6 mg / mL and 20 mg / mL, the concentration of aspirin is between 10 mg / mL and 48 mg / mL, and the concentration of the polyethyleneimine solution is between 6 mg / mL and 20 mg / mL.

[0013] In the present invention, the average molecular weight of the polyethyleneimine is 1800 Da.

[0014] Specifically, the average molecular weight of aspirin is 180.16 Da.

[0015] The present invention also provides a self-assembly nanomedicine prepared by the above method for improving fetal growth restriction.

[0016] The use of the self-assembled nanomedicine in the preparation of a drug for preventing and treating fetal growth restriction during pregnancy and related pregnancy complications. The fetal growth restriction during pregnancy includes fetal growth restriction caused by gestational hypoxia. The related pregnancy complications include preeclampsia and venous thrombosis during pregnancy.

[0017] The present invention further provides an application of the self-assembled nanomedicine, comprising intravenously injecting the nanomedicine into a fetal growth restriction model rat to improve fetal growth restriction caused by gestational hypoxia.

[0018] This self-assembled nanomedicine for improving fetal growth restriction has multiple functions, including alleviating hypoxia, reducing oxidative stress, and providing anti-inflammatory and anti-vascular damage benefits. It can effectively prevent and treat the development of fetal growth restriction during pregnancy. This is the first study in China and abroad to use a multi-bioactive nanomedicine to treat fetal growth restriction during pregnancy. It also has broad application prospects for treating other pregnancy complications related to hypoxia.

[0019] The self-assembled nanomedicine for improving fetal growth restriction cannot penetrate the placental barrier to enter the fetus and is non-toxic to the mother and fetus at higher therapeutic doses, thereby ensuring the safety of the mother and fetus when the nanomedicine is used for treatment during pregnancy. Therefore, it can be used for the prevention and treatment of fetal growth restriction during pregnancy, as well as the prevention and treatment of other related pregnancy diseases.

[0020] The present invention has the following advantages: 1) The polyethyleneimine, aspirin, and phenylboronic acid esters used in this invention are all commercially available, relatively inexpensive, and simple to synthesize, making them readily commercializable. Furthermore, the aspirin API is highly safe, and clinical guidelines indicate its use for pregnancy-related conditions.

[0021] 2) The self-assembly method used in the present invention is simple and easy, and the organic solvent used is easy to remove, ensuring the feasibility and safety of the final application of the nanomedicine.

[0022] 3) The self-assembled nanomedicine for improving fetal growth restriction prepared by the present invention is solid and easy to store and carry.

[0023] 4) The self-assembled nanomedicine for improving fetal growth restriction prepared by the present invention has good stability and no immunogenicity, ensuring in vivo safety.

[0024] 5) The self-assembled nanomedicine for improving fetal growth restriction prepared by this invention exhibits multiple functions, including improving placental hypoxia, reducing oxidative stress, and providing anti-inflammatory and anti-vascular damage benefits. It can effectively prevent and treat fetal growth restriction during pregnancy and reduce perinatal complications. This invention is the first domestic and international study to utilize a multi-bioactive nanomedicine to treat fetal growth restriction during pregnancy. It also has the potential to prevent and treat other pregnancy complications related to hypoxia.

[0025] 6) The self-assembled nanomedicine for improving fetal growth restriction prepared by the present invention does not pass through the placental barrier to enter the fetus, ensuring the safety of the mother during pregnancy and the normal development of the fetus. Therefore, it can be used to treat fetal growth restriction during pregnancy and prevent and treat other related pregnancy diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1These are scanning electron microscopy and transmission electron microscopy images of nanomedicines prepared by self-assembly of aspirin with a molecular weight of 180.16 Da, polyethyleneimine (PEI) with a molecular weight of 1800 Da, and phenylboronic acid ester (PBE). The scale bar is 200 nm.

[0027] Figure 2 This is the particle size distribution and surface potential detection diagram of the self-assembled nanomedicine.

[0028] Figure 3 These are the H NMR spectrum and Fourier transform infrared spectrum of the self-assembled nanomedicine.

[0029] Figure 4 This is a statistical chart showing the ability of the self-assembled nanomedicine to scavenge different reactive oxygen free radicals. In the figure, from left to right, the nanomedicine scavenges superoxide anion, free radical DPPH·, H2O2, and hypochlorite.

[0030] Figure 5 Cell apoptosis graphs of the blank control group (0.01M phosphate buffer), hydrogen peroxide group (0.01M phosphate buffer), treatment groups (25 μg / mL, 50 μg / mL, 100 μg / mL self-assembled nanodrug groups) and aspirin group (100 μM).

[0031] Figure 6 The superoxide anion detection probe (DHE) staining images of the blank control group (0.01M phosphate buffer), hydrogen peroxide group (0.01M phosphate buffer), treatment groups (25 μg / mL, 50 μg / mL, 100 μg / mL self-assembled nanodrug groups) and aspirin group (100 μM), where the scale bar is 40 μm.

[0032] Figure 7 These are the cell migration images of the blank control group (0.01M phosphate buffer), hydrogen peroxide group (0.01M phosphate buffer), treatment groups (25 μg / mL, 50 μg / mL, 100 μg / mL self-assembled nanodrug groups), and aspirin group (100 μM). The scale bar is 100 μm.

[0033] Figure 8 Quantitative graphs of interleukin-6 (IL-6) gene expression in placental trophoblast cells and vascular endothelial cells under lipopolysaccharide (LPS) stimulation in the blank control group (0.01M phosphate buffer), LPS group (100 μg / mL LPS), treatment groups (25 μg / mL, 50 μg / mL, and 100 μg / mL self-assembled nanodrug groups), and aspirin group (100 μM).

[0034] Figure 9 Figure 2 shows the fluorescence distribution and quantitative analysis of the Cy5-linked self-assembled nanodrug in the placenta and fetus of pregnant rats in the blank control group and the model group. The scale is 5 mm. In the figure, the two groups of bars from left to right are the sham operation group + PBS, the model group + PBS, the sham operation group + Cy5-linked nanodrug, and the model group + Cy5-linked nanodrug.

[0035] Figure 10 This is a statistical chart of fetal weight, fetal length, and placental weight after treatment of fetal growth restriction rat model in the blank control group (0.01M phosphate buffer), model group (0.01M phosphate buffer), treatment group (5 mg / kg, 10 mg / kg, 20 mg / kg self-assembled nanodrug group), and aspirin group (4 mg / kg). DETAILED DESCRIPTION

[0036] The following is a further detailed description of the invention content of the present invention in conjunction with specific embodiments. It should be understood that the embodiments of the present invention are only used to illustrate the present invention and are not intended to limit the present invention. Without departing from the technical concept of the present invention, various substitutions and modifications made according to common technical knowledge and customary means in the art should be included within the scope of the present invention.

[0037] The present invention is described in detail below with reference to non-limiting examples. During the self-assembly process, the particle size of the self-assembled nanomedicine can be controlled to be between 100 nm and 200 nm.

[0038] In Example 1, 100 mg of PBE, 100 mg of aspirin API (average molecular weight 180.16 Da), and 100 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every 1 hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0039] In Example 2, 50 mg of aspirin API (average molecular weight 180.16 Da), 100 mg of PBE, and 100 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0040] In Example 3, 80 mg of aspirin API (average molecular weight 180.16 Da), 100 mg of PBE, and 100 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0041] In Example 4, 96 mg of aspirin API (average molecular weight 180.16 Da), 80 mg of PBE, and 80 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0042] In Example 5, 120 mg of aspirin API (average molecular weight 180.16 Da), 80 mg of PBE, and 80 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0043] In Example 6, 135 mg of aspirin API (average molecular weight 180.16 Da), 85 mg of PBE, and 85 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0044] In Example 7, 140 mg of aspirin API (average molecular weight 180.16 Da), 70 mg of PBE, and 70 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every 1 hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0045] In Example 8, 143 mg of aspirin API (average molecular weight 180.16 Da), 65 mg of PBE, and 65 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0046] In Example 9, 150 mg of aspirin API (average molecular weight 180.16 Da), 60 mg of PBE, and 60 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every 1 hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0047] In Example 10, 168 mg of aspirin API (average molecular weight 180.16 Da), 60 mg of PBE, and 60 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every 1 hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0048] In Example 11, 180 mg of aspirin API (average molecular weight 180.16 Da), 60 mg of PBE, and 60 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis. The deionized water was changed every 1 hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0049] In Example 12, 176 mg of aspirin API (average molecular weight 180.16 Da), 55 mg of PBE, and 55 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0050] In Example 13, 175 mg of aspirin API (average molecular weight 180.16 Da), 50 mg of PBE, and 50 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0051] In Example 14, 190 mg of aspirin API (average molecular weight 180.16 Da), 50 mg of PBE, and 50 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanodrug for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanodrug was between 100 nm and 200 nm.

[0052] In Example 15, 200 mg of aspirin API (average molecular weight 180.16 Da), 50 mg of PBE, and 50 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0053] In Example 16, 207 mg of aspirin API (average molecular weight 180.16 Da), 46 mg of PBE, and 46 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0054] In Example 17, 215 mg of aspirin API (average molecular weight 180.16 Da), 43 mg of PBE, and 43 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0055] In Example 18, 220 mg of aspirin API (average molecular weight 180.16 Da), 40 mg of PBE, and 40 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanomedicine for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanomedicine was between 100 nm and 200 nm.

[0056] In Example 19, 210 mg of aspirin API (average molecular weight 180.16 Da), 35 mg of PBE, and 35 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanodrug for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanodrug was between 100 nm and 200 nm.

[0057] In Example 20, 240 mg of aspirin API (average molecular weight 180.16 Da), 30 mg of PBE, and 30 mg of PEI were dissolved in DMSO (5 mL). The mixture was mixed by inversion and then placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette. The bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water replaced every 1 hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen solid at -80°C (freeze-dried for at least three hours). The solution was then transferred to a freeze dryer for drying to obtain a white powder, i.e., a self-assembled nanodrug for improving fetal growth restriction. The shape and weight of the powder in the centrifuge tube were recorded. The particle size of the self-assembled nanodrug was between 100 nm and 200 nm.

[0058] The scanning electron microscope and transmission electron microscope images of the nanomedicine prepared according to the method of Example 1 are as follows: Figure 1 shown.

[0059] Figure 2 Figure 2 shows the particle size distribution and surface potential of the self-assembled nanodrug. Approximately 5 mg of dry nanodrug was added to 1 mL of deionized water, mixed thoroughly, and sonicated to fully dissolve the nanomicelles. The nanomicelles were then added to a sample cell, and the particle size distribution and surface potential of the nanodrug were measured using a Malvern laser particle size analyzer.

[0060] Figure 3 The following are the H NMR spectra (left) and FT-IR spectra (right) of the self-assembled nanodrug. Dried nanodrug (10 mg), PBE (10 mg), aspirin (10 mg), and PEI (10 mg) were each dissolved in 600 μL of deuterated methanol and scanned using a 600 MHz NMR spectrometer. Separately, appropriate amounts of dried nanodrug, PBE, aspirin, and PEI were placed on the Fourier transform infrared spectrometer's inspection platform for scanning.

[0061] Figure 4The following is a statistical chart showing the ability of the self-assembled nanomedicine to scavenge different reactive oxygen species free radicals. Nanomedicine superoxide anion scavenging assay: The nanomedicine methanol solutions were prepared at concentrations of 0.05, 0.1, 0.15, 0.25, 0.5, and 1 mg / mL. According to the instructions of the superoxide anion detection kit, the prepared reagent was mixed with the nanodrugs of various concentrations, incubated in a constant temperature water bath at 37 ºC for 40 min, and then the colorimetric reagent was added. The OD value at 550 nm was measured, and the superoxide anion scavenging ability of the nanodrug was calculated according to the instructions of the kit. The DPPH· free radical scavenging ability of the nanodrug was tested: According to the instructions of the DPPH· detection kit, 1.5 mL of DPPH· (100 μg / mL) reagent was incubated with 3 mL of nanodrugs of different concentrations (0.05, 0.1, 0.25, 0.5, 1, and 2 mg / mL) in the dark for 30 min. The absorbance at 517 nm was measured using a UV spectrophotometer to calculate the DPPH· scavenging ability. The H2O2 scavenging ability of the nanodrug was tested: According to the H2O2 detection kit, different concentrations of nanodrugs (0, 1, 2, 4, and 6 mg / mL) were incubated with 2 mL of PBS (0.01 M) containing 50 nM H2O2 for 24 h. The OD value at 405 nm was measured. The residual H2O2 was measured by the absorbance at nm, and the eliminated H2O2 was calculated; the hypochlorite scavenging ability of nanomedicines was tested: according to the literature, a homemade nanoprobe (Lu-bCD NP) was synthesized to detect the hypochlorite scavenging ability. 25 μL of nanomedicines with different concentrations (1, 2, 3, 4, 5 mg / mL) was mixed with 475 μL of 100 mM NaClO solution for 15 min, 50 μL of the supernatant of the mixed reaction was aspirated and reacted with 50 μL of Lu-bCD NP solution (10 mg / mL), and ClO was calculated according to the standard curve. - Removal efficiency. Figure 4 It can be seen that the self-assembled nanomedicine can scavenge different reactive oxygen free radicals.

[0062] Figure 5 The following are cell apoptosis diagrams for the blank control group (0.01M phosphate buffer), hydrogen peroxide group (0.01M phosphate buffer), treatment group (25μg / mL, 50μg / mL, 100μg / mL self-assembled nanodrug group) and aspirin group (100μM). Human vascular endothelial cells (HUVECs) were seeded in 6-well plates and incubated with PBS, self-assembled nanodrugs (25μg / mL, 50μg / mL, 100μg / mL), and aspirin (100μM) for 24 hours. Except for the blank group, the cells were treated with 100μM H2O2 for 12 hours. The cells were then digested with 0.25% trypsin, collected by centrifugation, and analyzed by flow cytometry after Annexin V and PI staining. Figure 5 It can be concluded that the nanomedicine group inhibited H2O2-induced cell apoptosis in a concentration-dependent manner, and the number of inhibited cell apoptosis was more significant than that of the aspirin group.

[0063] Figure 6 Figure 3 shows superoxide anion detection probe (DHE) staining of the blank control group (0.01M phosphate buffer), hydrogen peroxide group (0.01M phosphate buffer), treatment group (25μg / mL, 50μg / mL, 100μg / mL self-assembled nanodrug group), and aspirin group (100μM). The scale bar is 40μm. HTR8 (placental trophoblast cells) were seeded in 12-well plates (1×10 cells per well). 5 Cells) were cultured overnight, and treated with fresh culture medium without fetal bovine serum, fresh culture medium containing nanodrugs (25μg / mL, 50μg / mL, 100μg / mL of self-assembled nanodrug group), and aspirin (100μM) for 24 hours. Except for the blank group, the culture medium was replaced with fresh culture medium containing 100μM H2O2 and incubated for 12 hours to induce ROS production. The cells were then washed with PBS, and 1 mL of culture medium containing 5μM DHE was added. The cells were incubated in a cell culture incubator for 30 min, washed with sterile PBS, fixed with 4% paraformaldehyde, and the cell nuclei were stained with DAPI for 5 min. After sealing, the cell fluorescence intensity was observed using laser confocal microscopy. Cells were only added with PBS without hydrogen peroxide induction as a blank control group. From Figure 6 It can be concluded that compared with the aspirin group, the nanomedicine group more significantly reduced the production of cellular reactive oxygen species induced by hydrogen peroxide.

[0064] Figure 7 Figure 1 shows cell migration patterns for the blank control group (0.01M phosphate buffer), hydrogen peroxide group (0.01M phosphate buffer), treatment groups (25μg / mL, 50μg / mL, and 100μg / mL self-assembled nanodrug groups), and aspirin group (100μM). The scale bar represents 100μm. Fresh 1640 medium was added to the bottom compartment. HTR8 (placental trophoblast cells) were cultured at 4 × 10 4 The cells were seeded at a density of 100 cells / well in the upper chamber of the cell chamber, and the following culture media were added: 100 mL of fresh culture medium without fetal bovine serum, fresh culture medium containing nanodrugs (25μg / mL, 50μg / mL, 100μg / mL of self-assembled nanodrugs), and culture medium containing aspirin (100μM). After 24 hours of treatment, the culture medium was replaced with fresh culture medium containing 100μMH2O2 and incubated for 12 hours, except for the blank group. The invaded cells were fixed with 4% paraformaldehyde, stained with crystal violet, and images were taken by optical microscopy. Figure 7It can be concluded that compared with the aspirin group, the nanodrug group significantly promoted the cell invasion ability under hydrogen peroxide inhibition in a concentration-dependent manner, and the ability of the 100 μg / mL nanodrug treatment group to promote cell invasion was comparable to that of the control group.

[0065] Figure 8 Figure 1 shows the quantitative expression of interleukin-6 (IL-6) in placental trophoblast cells (HTR8) and vascular endothelial cells (HUVECs) under lipopolysaccharide (LPS) stimulation in the blank control group (0.01M phosphate buffer), LPS group (100ng / mL LPS), treatment group (25μg / mL, 50μg / mL, 100mg / mL self-assembled nanodrug group), and aspirin group (100μM). Placental trophoblast cells (HTR8) and vascular endothelial cells (HUVECs) were seeded into six-well cell culture plates (2 × 10 cells per well). 5 Cells were incubated overnight with fresh culture medium without fetal bovine serum, fresh culture medium containing nanodrugs (25μg / mL, 50μg / mL, 100μg / mL of nanodrugs), and aspirin (100μM) for 24 hours. Except for the blank group, the culture medium was replaced with fresh culture medium containing 100 ng / mL of LPS and incubated for 12 hours. The cells were then washed with PBS, and the cell RNA was extracted using a rapid RNA extraction kit according to the manufacturer's instructions. At the same time, the extracted cell RNA was reverse transcribed to synthesize cDNA and qPCR reaction was performed according to the manufacturer's instructions to analyze gene expression. Figure 8 It can be concluded that the nanomedicine group more significantly reduced the LPS-induced IL-6 gene expression level than the aspirin group.

[0066] Figure 9 The fluorescence distribution and quantitative graph of Cy5-linked self-assembled nanodrugs in the placenta and fetus of pregnant rats in the sham operation group and the model group, where the scale is 5 mm. After anesthesia, SD female rats on the 14.5th day of pregnancy were made a 1.5 cm midline incision in the lower abdomen. The model group used 3-0 silk thread to ligate the uterine blood vessels to induce a rat model of fetal growth restriction; the pregnant female rats in the sham operation group only had an abdominal opening without ligating the uterine blood vessels. One day after the surgical model was established, 10 mg / kg of Cy5-linked self-assembled nanodrugs or an equivalent dose of PBS were injected into the tail vein of pregnant rats in the fetal growth restriction model group and the sham operation group. The rats were killed 4 hours later, and the uterus was removed after irrigating with normal saline. The fetus and placenta were separated, and the residual blood on the surface of the fetus and placenta was removed. The distribution of Cy5 fluorescence in the fetus and placenta was observed by small animal live imaging, and the fluorescence intensity was counted. Figure 9It can be concluded that the self-assembled nanodrug is only enriched in the basal decidua of the placenta (i.e., the maternal side of the placenta), and no fluorescent signal can be detected in the fetus, indicating that the drug cannot pass through the placental barrier to affect the fetus.

[0067] Figure 10 The statistical chart shows the fetal weight, fetal length and placenta weight after nanomedicine treatment of the fetal growth restriction rat model. After anesthesia, SD female rats on the 14.5th day of pregnancy were made a 1.5 cm midline incision in the lower abdomen. The model group used 3-0 silk thread to ligate the uterine blood vessels to induce a fetal growth restriction rat model; the pregnant female rats in the sham operation group only underwent abdominal opening without ligating the uterine blood vessels. One day after the surgical model was established, the self-assembled nanodrug (5 mg / kg, 10 mg / kg, 20 mg / kg) was injected into the model rats through the tail vein or aspirin (4 mg / kg) was orally administered. The model group and the sham operation group were injected with the same dose of normal saline through the tail vein. Each group was injected or orally once a day. After 5 days of treatment, the pregnant rats were killed and the fetal length, fetal weight and placenta weight were measured. From Figure 10 It can be concluded that the fetal growth restriction rat model reduces the weight of the fetus and placenta. After treatment with nanomedicine, the nanomedicine group can increase the weight of the fetus and placenta to a certain extent and restore the fetal length compared with the aspirin group, indicating that nanomedicine can improve fetal growth restriction.

[0068] Figure 9-10 The method for establishing the fetal growth restriction model in gestational rats is as follows: Female Sprague-Dawley rats (11-12 weeks, 240-250 g) and male Sprague-Dawley rats (12-13 weeks, 250-260 g) were purchased from the Experimental Animal Center of Chongqing Medical University. Rats were housed in a standard shed, maintained at a constant circadian rhythm and temperature. After one week of housing, they were mated at a female:male ratio of 2:1. Female rats underwent vaginal smear examination, and those with sperm were designated as gestational day 0 (G0) and used for subsequent experiments. On gestational day 14.5, rats were anesthetized intraperitoneally with 0.3% pentobarbital at a dose of 1 mL / 100 g. The abdomen was shaved, and the skin was disinfected with iodine before being draped. In the model group, a midline incision was made in the lower abdomen, dissecting the skin, subcutaneous tissue, rectus abdominis muscle, and peritoneum. The uterus was gently removed and placed on saline-soaked gauze. The uterine vessels on both sides were exposed. 2-0 silk thread was used as a cushion, and the uterine vessels were ligated with 3-0 silk thread. The 2-0 silk thread was then removed, and the uterus was returned to the abdominal cavity. After the abdominal cavity was flushed with saline, 4-0 silk thread was used to suture the area layer by layer to close the abdominal cavity. In the sham group, only the abdominal tissue layers were incised, and the uterine artery vessels were not ligated. After the abdominal cavity was opened, the abdomen was flushed with saline, and sutured layer by layer to close the abdominal cavity.

Claims

1. A method for preparing a self-assembled nanomedicine for improving fetal growth restriction, characterized by: The self-assembled nanomedicine is obtained by connecting phenylboronic acid ester and aspirin through polyethyleneimine, wherein the mass ratio of aspirin:phenylboronic acid ester:polyethyleneimine is between 0.5:1:1 and 8:1:1, and the particle size of the self-assembled nanomedicine is between 100 nm and 200 nm.

2. The method for preparing a self-assembled nanomedicine for improving fetal growth restriction according to claim 1, characterized in that: The following steps are involved: Aspirin raw material, polyethyleneimine, and phenylboronic acid ester are dissolved in an organic solvent, mixed upside down, and then placed in an ultrasonic cleaner until the drug powder is completely dissolved. The organic solvent in which the aspirin raw material, polyethyleneimine, and phenylboronic acid ester are completely dissolved is then placed in a 3500 Da dialysis bag and rotary dialyzed in deionized water to remove the organic solvent. The deionized water is replaced every hour. Finally, the solution in the dialysis bag is freeze-dried at -80°C for more than three hours and then transferred to a freeze dryer for freeze drying to obtain the self-assembled nanomedicine.

3. The method for preparing a self-assembled nanomedicine for improving fetal growth restriction according to claim 2, characterized in that: The organic solvent is dimethyl sulfoxide.

4. The method for preparing a self-assembled nanomedicine for improving fetal growth restriction according to claim 2 or 3, characterized in that: The concentration of the phenylboronic acid ester solution is between 6 mg / mL and 20 mg / mL, the concentration of aspirin is between 10 mg / mL and 48 mg / mL, and the concentration of the polyethyleneimine solution is between 6 mg / mL and 20 mg / mL.

5. The method for preparing a self-assembled nanomedicine for improving fetal growth restriction according to any one of claims 1 to 3, characterized in that: The average molecular weight of the polyethyleneimine is 1800 Da.

6. The method for preparing a self-assembled nanomedicine for improving fetal growth restriction according to any one of claims 1 to 3, characterized in that: The average molecular weight of aspirin is 180.16 Da.

7. A self-assembled nanomedicine for improving fetal growth restriction, characterized in that: The method is prepared by any one of claims 1 to 6.

8. Use of the self-assembled nanomedicine according to claim 7 in the preparation of drugs for preventing and treating fetal growth restriction and related pregnancy complications during pregnancy.

9. The application according to claim 8, characterized in that: The fetal growth restriction during pregnancy includes fetal growth restriction caused by hypoxia during pregnancy.

10. The application according to claim 8, characterized in that: The related pregnancy complications include preeclampsia and venous thrombosis during pregnancy.

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