Preparation method and application of self-assembled nanodrug for improving fetal growth restriction

Abstract

The application discloses a preparation method of self-assembled nanomedicine for improving fetal growth restriction and application thereof. Aspirin raw medicine, polyethyleneimine and phenyl borate are dissolved in dimethyl sulfoxide, and after being mixed uniformly, the mixture is placed into an ultrasonic cleaner until the medicine powder is completely dissolved, then the mixed solution is placed in a dialysis bag, and the organic solvent is removed by rotating dialysis in deionized water, finally, the solution in the dialysis bag is freeze-dried, and the self-assembled nanomedicine is obtained. The medicine is injected through a vein to improve the pathological pregnancy environment of hypoxia, oxidative stress and inflammation related to fetal growth restriction in a gestation period, and promote long-term growth and development of the development-restricted fetus. The self-assembling method is simple and easy to operate, low in price, and the organic solvent is easy to remove, and the stability is good and the immunogenicity is free. More importantly, under a treatment dose much higher than the treatment dose, the self-assembled nanomedicine has no toxicity to the mother and fetus, and the safety development of the fetus in the gestation period is ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of self-assembled nanomedicine, in particular to the field of nanomedicine for improving fetal growth restriction. BACKGROUND

[0002] Fetal growth restriction (FGR) during pregnancy is a common complication of pregnancy, which threatens about 20 million infants worldwide each year, leading to adverse pregnancy outcomes. Its characteristics are impaired fetal development and lower birth weight than 90% of fetuses of the same gestational age. Fetal growth restriction, as a major cause of neonatal morbidity and mortality, leads to nearly 30% of stillbirth consequences, and is the second largest perinatal death cause after preterm birth, which brings serious challenges to public health and social economy. The etiology of fetal growth restriction is related to many factors, including placental dysfunction (such as abnormal invasion of trophoblasts, reduced uteroplacental blood flow), maternal vascular disease (such as gestational hypertension, chronic hypertension), intrauterine infection, genetic abnormalities and environmental factors (such as malnutrition, smoking). Although associated with a variety of risk factors, the pathomechanism of placenta is the same in many cases of fetal growth restriction. Chronic dysfunction of placenta is a common cause of fetal growth restriction, and insufficient blood flow to the placenta during pregnancy will lead to insufficient supply of nutrients and oxygen, which cannot maintain the normal growth of the fetus. It is known that during normal placental formation, placental trophoblast cells are responsible for coordinating the invasion and maturation of complex vascular networks within placental villi, while blood is responsible for transporting nutrients, oxygen, etc. from the mother to the fetus and transporting fetal metabolic waste, etc. to the mother for excretion. However, fetal growth restriction will hinder angiogenesis within placental villi, which in turn cannot transport enough oxygen and nutrients to the fetus, leading to poor fetal growth. In addition, studies have shown that placental hypoxia, oxidative stress and immune inflammation during pregnancy are usually accompanied by the occurrence and 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 prevention measures and optimal treatment options for effectively improving fetal growth restriction.

[0003] Prevention of FGR has mainly focused on interventions before and during pregnancy. Some studies have attempted to prevent FGR by enhancing maternal health status. However, current evidence shows that supplementation of multiple nutrients does not effectively prevent FGR or birth of SGA. In addition, some clinical recommendations have suggested prophylactic interventions with heparin for specific pregnancies at risk for FGR, but recent studies have shown that heparin administration does not effectively prevent prior severe or early onset FGR and patients with thrombophilia. As for the fetus, studies have attempted to supplement glucose to the fetus, but there is no good preventive benefit, and it may increase the risk of potential acidosis in the fetus. For all FGR fetuses delivered before 34 weeks of gestation, clinical guidelines recommend antenatal administration of corticosteroids; and for FGR fetuses born after 34 weeks of gestation, especially those delivered by selective cesarean section, the use of corticosteroids may be beneficial, but the number of FGR fetuses suffering from FGR syndrome does not show a statistically significant decrease. Currently, most guidelines recommend the use of low-dose aspirin before 16 weeks of gestation to prevent FGR.

[0004] The key to therapeutic intervention for FGR is 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 FGR is multifactorial, including chronic hypoxia, oxidative stress, impaired vascularization, inflammation imbalance, and placental hypoplasia. Therefore, a combination of multiple therapeutic strategies is needed to simultaneously target these interrelated pathological mechanisms in order to effectively treat FGR and provide breakthrough new insights for the prevention and management of related pregnancy complications.

[0005] Currently, traditional aspirin drug therapy for FGR is still limited by dose-limited non-selective biodistribution and bleeding risk. In recent years, some new nanodrugs for improving FGR have also been developed, including nanoparticles specifically targeting placental trophoblast cells and non-viral polymer nanoparticles. However, most of the above nanodrugs only target a single target to improve FGR, and the etiology of FGR is diverse and complex, and single-target therapy cannot effectively improve the pregnancy outcome of FGR. In addition, most studies rarely investigate the short-term and long-term development of FGR offspring after treatment. SUMMARY

[0006] The technical problem solved by the present application is how to prepare a self-assembled nanodrug for improving FGR, which is safe for both the pregnant mother and the fetus during pregnancy.

[0007] The application applies aspirin, polyethyleneimine (PEI) and reactive oxygen species (ROS) scavenger phenylboronic acid ester (PBE) to form a nano material with anti-inflammatory, anti-oxidative stress and other biological activities through covalent coupling, the material can be constructed into a new nano drug for improving fetal growth restriction through self-assembly. The nano drug can improve oxidative stress under placental hypoxic environment through the ROS scavenging capacity of PBE, relieve vascular injury and inflammation in placental trophoblast cells and vascular endothelial cells through the aspirin-mediated inflammatory protection effect, promote the targeted delivery of drugs to the placenta through PEI nanotherapy, reduce the risk of systemic bleeding caused by aspirin at the same time, restore the normal embryonic development environment, and thus play a role in treating fetal growth restriction.

[0008] The related experiments prove that the placenta of the fetal growth restriction patient is in a persistent hypoxic environment, and hypoxia can induce the production of a large amount of ROS through mitochondria, amplify oxidative damage; at the same time, the expression of certain immune inflammatory factors is up-regulated, and the pro-inflammatory placental microenvironment is triggered. The self-assembled nano drug can play an anti-oxidative stress role to inhibit the production of reactive oxygen species (ROS), achieve the purpose of improving placental hypoxia; at the same time, it reduces the hypoxia / active oxygen-induced apoptosis, restores the invasion ability of placental trophoblast cells and the angiogenesis ability of vascular endothelial cells, and inhibits the overexpression of related immune inflammatory factors, thereby playing an anti-inflammatory and anti-vascular injury role. Finally, it is verified that the nano drug cannot penetrate the placental barrier and has high safety to the mother and fetus. Therefore, the self-assembled nano drug can inhibit the occurrence and development of fetal growth restriction by improving hypoxia, reducing oxidative stress, anti-inflammation, anti-vascular injury and other effects, and promote the healthy development of the fetus. Compared with other nano drugs applied to improve fetal growth restriction, the nano drug prepared by self-assembly has multiple biological activities compared with the targeted drug with only a single function, not only can accurately and efficiently achieve targeted delivery to the placenta, but also can solve the effects of hypoxia, oxidative stress and inflammation, and verify the effectiveness of the "hypoxia-reactive oxygen-inflammation axis" as a therapeutic target; at the same time, the self-assembled nano drug also proves the safety to the mother and fetus. In addition, in addition to the model of this study, the adverse effects of hypoxia and inflammation are also related to the occurrence and development of other gestational diseases, so the self-assembled nano drug with anti-oxidative stress, anti-inflammatory and other multiple biological activities of the application has broad application prospects.

[0009] In view of this, the technical scheme adopted by the present application is as follows: a preparation method of a self-assembled nanodrug for improving fetal growth restriction, which is obtained by self-assembly of active oxygen reaction / clearance device phenylboronic acid ester (PBE) and aspirin (Aspirin) through polyethyleneimine (PEI), 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 nanodrug is between 100 nm and 200 nm.

[0010] Specifically, the above method comprises the following steps: dissolving aspirin raw material, polyethyleneimine and phenylboronic acid ester in an organic solvent, placing the mixture in an ultrasonic cleaner until the drug powder is completely dissolved, then placing the organic solvent containing completely dissolved aspirin raw material, polyethyleneimine and phenylboronic acid ester in a 3500 Da dialysis bag, rotating dialysis 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℃ for more than three hours, and then transferring it to a freeze dryer for freeze-drying to obtain the self-assembled nanodrug.

[0011] The organic solvent can be dimethyl sulfoxide. Other organic solvents commonly used in pharmaceutical methods, such as methanol and N,N-dimethylformamide, can also be used.

[0012] Specifically, the concentration of the phenylboronic acid ester solution is between 6 mg / mL and 20 mg / mL, the concentration of the 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 application, the average molecular weight of the polyethyleneimine is 1800 Da.

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

[0015] The present application also provides a self-assembled nanodrug for improving fetal growth restriction prepared by the above method.

[0016] The above self-assembled nanodrug is used in the preparation of drugs for preventing and treating fetal growth restriction and related pregnancy complications during pregnancy. The fetal growth restriction during pregnancy includes fetal growth restriction caused by hypoxia during pregnancy. The related pregnancy complications include preeclampsia and venous thrombosis during pregnancy.

[0017] The present application further provides the application of the self-assembled nanodrug, which comprises intravenously injecting the nanodrug into a fetal growth restriction model rat to improve fetal growth restriction caused by hypoxia during pregnancy.

[0018] The self-assembled nanomedicine for improving fetal growth restriction has multiple functions of improving placental hypoxia, reducing oxidative stress, anti-inflammation, anti-vascular injury and the like, can effectively prevent and treat the occurrence and development of fetal growth restriction in the gestation period, and is the first research of applying multiple bioactive nanomedicines to treat fetal growth restriction in the gestation period at home and abroad. Meanwhile, the self-assembled nanomedicine has wide application prospects in preventing and 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, has no toxicity to the mother and fetus at a higher treatment dose, ensures the safety of the mother and fetus during the gestation period, and thus can be used for preventing and treating fetal growth restriction in the gestation period and preventing and treating other related pregnancy diseases.

[0020] The self-assembled nanomedicine for improving fetal growth restriction has the following advantages:

[0021] 1) The polyethyleneimine, aspirin and phenylborate used in the application have commercial products, are relatively low in price, and are simple in preparation, so that the industrialization of the corresponding preparation is easy to realize. Moreover, the aspirin raw material involved has high safety, and the clinical guidelines show that it can be used for pregnancy-related diseases.

[0022] 2) The self-assembly method adopted in the application is simple and easy to operate, and the organic solvent used is easy to remove, so that the feasibility and safety of the application of the nanomedicine are ensured.

[0023] 3) The self-assembled nanomedicine for improving fetal growth restriction prepared in the application is a solid, which is convenient for storage and carrying.

[0024] 4) The self-assembled nanomedicine for improving fetal growth restriction prepared in the application has good stability and no immunogenicity, so that the in-vivo safety is ensured.

[0025] 5) The self-assembled nanomedicine for improving fetal growth restriction prepared in the application has multiple functions of improving placental hypoxia, reducing oxidative stress, anti-inflammation, anti-vascular injury and the like, can effectively prevent and treat fetal growth restriction in the gestation period, and reduce perinatal complications. The application is the first research of applying multiple bioactive nanomedicines to treat fetal growth restriction in the gestation period at home and abroad. Meanwhile, the self-assembled nanomedicine is expected to prevent and treat other pregnancy complications related to hypoxia.

[0026] 6) The self-assembled nanomedicine for improving fetal growth restriction prepared in the application does not penetrate the placental barrier to enter the fetus, so that the safety of the mother in the gestation period and the normal development of the fetus are ensured, and thus the self-assembled nanomedicine can be applied to fetal growth restriction in the gestation period and the prevention and treatment of other related pregnancy diseases. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1Scanning electron microscope and transmission electron microscope images of the nano-drug prepared by self-assembly of aspirin (180.16 Da), polyethyleneimine (PEI, 1800 Da) and phenylboronic acid ester (PBE), in which the scale is 200 nm.

[0028] Figure 2 Particle size distribution and surface potential detection images of the self-assembled nano-drug.

[0029] Figure 3 Nuclear magnetic resonance hydrogen spectrum and Fourier transform infrared spectrum images of the self-assembled nano-drug.

[0030] Figure 4 Detection statistical chart of the scavenging ability of the self-assembled nano-drug to different active oxygen free radicals, in which, from left to right, the nano-drug scavenges superoxide anion, free radical DPPH·, H2O2, and hypochlorite.

[0031] Figure 5 Apoptosis chart of the blank control group (0.01M phosphate buffer), the hydrogen peroxide group (0.01M phosphate buffer), the treatment group (25 μg / mL, 50 μg / mL, 100 μg / mL of the self-assembled nano-drug group), and the aspirin group (100 μM).

[0032] Figure 6 Sodium superoxide detection probe (DHE) staining chart of the blank control group (0.01M phosphate buffer), the hydrogen peroxide group (0.01M phosphate buffer), the treatment group (25 μg / mL, 50 μg / mL, 100 μg / mL of the self-assembled nano-drug group), and the aspirin group (100 μM), in which the scale is 40 μm.

[0033] Figure 7 Cell migration chart of the blank control group (0.01M phosphate buffer), the hydrogen peroxide group (0.01M phosphate buffer), the treatment group (25 μg / mL, 50 μg / mL, 100 μg / mL of the self-assembled nano-drug group), and the aspirin group (100 μM), in which the scale is 100 μm.

[0034] Figure 8 Quantitative chart of interleukin 6 (IL-6) gene expression of the blank control group (0.01M phosphate buffer), the LPS group (100 ng / mL LPS), the treatment group (25 μg / mL, 50 μg / mL, 100 μg / mL of the self-assembled nano-drug group), and the aspirin group (100 μM) of placental trophoblast cells and vascular endothelial cells under lipopolysaccharide (LPS) stimulation.

[0035] Figure 9 The fluorescence distribution and quantification of Cy5-labeled self-assembled nanodrugs in the placenta and fetus of pregnant rats in the blank control group and the model group are shown in the figure, where the scale is 5 mm; in the figure, the two groups of columns from left to right are sham operation group + PBS, model group + PBS, sham operation group + Cy5-labeled nanodrug, and model group + Cy5-labeled nanodrug.

[0036] Figure 10 The statistical graphs of fetal weight, fetal length, and placental weight of the blank control group (0.01M phosphate buffer), the model group (0.01M phosphate buffer), the treatment group (5 mg / kg, 10 mg / kg, 20 mg / kg of self-assembled nanodrug group), and the aspirin group (4 mg / kg) after treating the fetal growth restriction rat model are shown in the figure. DETAILED DESCRIPTION

[0037] The invention content of the present application will be further described in detail in combination with specific embodiments. It should be understood that the embodiments of the present application are only used to illustrate the present application but not to limit the present application. Various replacements and changes made according to the ordinary technical knowledge and conventional means in the art without departing from the technical thought of the present application should be included in the scope of the present application.

[0038] The present application will be described in detail in combination with non-limiting examples. In the self-assembly process, the particle size of the self-assembled nanodrug can be controlled to be between 100 nm and 200 nm.

[0039] In example 1, 100 mg of PBE, 100 mg of aspirin raw material (average molecular weight 180.16 Da), and 100 mg of PEI were dissolved in DMSO (5 mL). After mixing uniformly by inverting up and down, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in DMSO. The mixed solution was transferred to a 3500 Da dialysis bag using a pipette gun. The dialysis bag was clamped with a sealing clamp and then placed in a 1L beaker filled with deionized water and rotated. The deionized water was replaced every 1h. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube, frozen into a solid at -80℃ (at least freeze-dried for more than three hours), and then transferred to a freeze dryer for drying. Finally, a white powder was obtained, which was the 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.

[0040] Example 2, 50 mg aspirin drug substance (average molecular weight 180.16 Da), 100 mg PBE and 100 mg PEI were dissolved in DMSO (5 mL), after mixing upside down, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred to a 3500 Da dialysis bag with a pipette, the dialysis bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water, and dialysis was performed by rotating, and the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred to a 50 mL centrifuge tube, and the liquid was frozen into a solid at -80°C (at least freeze-dried for more than three hours), then transferred to a freeze dryer for drying, finally a white powder was obtained, which was a self-assembled nanomedicine for improving fetal growth restriction, and 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] Example 3, 80 mg aspirin drug substance (average molecular weight 180.16 Da), 100 mg PBE and 100 mg PEI were dissolved in DMSO (5 mL), after mixing upside down, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred to a 3500 Da dialysis bag with a pipette, the dialysis bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water, and dialysis was performed by rotating, and the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred to a 50 mL centrifuge tube, and the liquid was frozen into a solid at -80°C (at least freeze-dried for more than three hours), then transferred to a freeze dryer for drying, finally a white powder was obtained, which was a self-assembled nanomedicine for improving fetal growth restriction, and 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] Example 4, 96 mg aspirin drug substance (average molecular weight 180.16 Da), 80 mg PBE and 80 mg PEI were dissolved in DMSO (5 mL), after mixing upside down, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred to a 3500 Da dialysis bag with a pipette, the dialysis bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water, and dialysis was performed by rotating, and the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred to a 50 mL centrifuge tube, and the liquid was frozen into a solid at -80°C (at least freeze-dried for more than three hours), then transferred to a freeze dryer for drying, finally a white powder was obtained, which was a self-assembled nanomedicine for improving fetal growth restriction, and 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] Example 5, 120 mg aspirin drug substance (average molecular weight 180.16 Da), 80 mg PBE and 80 mg PEI were dissolved in DMSO (5 mL), after mixing upside down, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred to a 3500 Da dialysis bag with a pipette, the dialysis bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water, and dialysis was performed by rotating, and the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred to a 50 mL centrifuge tube, and the liquid was frozen into a solid at -80°C (at least freeze-dried for more than three hours), then transferred to a freeze dryer for drying, finally a white powder was obtained, which was a self-assembled nanomedicine for improving fetal growth restriction, and 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] Example 6, 135 mg aspirin drug substance (average molecular weight 180.16 Da), 85 mg PBE and 85 mg PEI were dissolved in DMSO (5 mL), after mixing upside down, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred to a 3500 Da dialysis bag with a pipette, the dialysis bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water, and dialysis was performed by rotating, and the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred to a 50 mL centrifuge tube, and the liquid was frozen into a solid at -80°C (at least freeze-dried for more than three hours), then transferred to a freeze dryer for drying, finally a white powder was obtained, which was a self-assembled nanomedicine for improving fetal growth restriction, and 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] Example 7, 140 mg aspirin drug substance (average molecular weight 180.16 Da), 70 mg PBE and 70 mg PEI were dissolved in DMSO (5 mL), after mixing upside down, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred to a 3500 Da dialysis bag with a pipette, the dialysis bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water, and dialysis was performed by rotating, and the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred to a 50 mL centrifuge tube, and the liquid was frozen into a solid at -80°C (at least freeze-dried for more than three hours), then transferred to a freeze dryer for drying, finally a white powder was obtained, which was a self-assembled nanomedicine for improving fetal growth restriction, and 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] Example 8, 143 mg aspirin drug substance (average molecular weight 180.16 Da), 65 mg PBE and 65 mg PEI were dissolved in DMSO (5 mL), after mixing upside down, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred to a 3500 Da dialysis bag with a pipette, the dialysis bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water, and dialysis was performed by rotating, and the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred to a 50 mL centrifuge tube, the liquid was frozen into a solid at -80°C (at least freeze-dried for more than three hours), then transferred to a freeze dryer for drying, finally a white powder was obtained, which was a self-assembled nanomedicine for improving fetal growth restriction, and 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] Example 9, 150 mg aspirin drug substance (average molecular weight 180.16 Da), 60 mg PBE and 60 mg PEI were dissolved in DMSO (5 mL), after mixing upside down, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred to a 3500 Da dialysis bag with a pipette, the dialysis bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water, and dialysis was performed by rotating, and the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred to a 50 mL centrifuge tube, the liquid was frozen into a solid at -80°C (at least freeze-dried for more than three hours), then transferred to a freeze dryer for drying, finally a white powder was obtained, which was a self-assembled nanomedicine for improving fetal growth restriction, and 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] Example 10, 168 mg aspirin drug substance (average molecular weight 180.16 Da), 60 mg PBE and 60 mg PEI were dissolved in DMSO (5 mL), after mixing upside down, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred to a 3500 Da dialysis bag with a pipette, the dialysis bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water, and dialysis was performed by rotating, and the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred to a 50 mL centrifuge tube, the liquid was frozen into a solid at -80°C (at least freeze-dried for more than three hours), then transferred to a freeze dryer for drying, finally a white powder was obtained, which was a self-assembled nanomedicine for improving fetal growth restriction, and 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 11, 180 mg of aspirin raw material (average molecular weight 180.16 Da), 60 mg of PBE, and 60 mg of PEI were dissolved in DMSO (5 mL). After mixing by inverting the container, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The solution was transferred to a 3500 Da dialysis bag using a pipette. The dialysis bag was then clamped and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water changed every 1 hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen at -80°C (for at least three hours). The solution was then transferred to a freeze dryer for further drying, yielding a white powder, which was the 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 this self-assembled nanomedicine was between 100 nm and 200 nm.

[0050] In Example 12, 176 mg of aspirin raw material (average molecular weight 180.16 Da), 55 mg of PBE, and 55 mg of PEI were dissolved in DMSO (5 mL). After mixing by inverting the container, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The solution was transferred to a 3500 Da dialysis bag using a pipette. The dialysis bag was then clamped and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water changed every 1 hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen into a solid at -80°C (for at least three hours). The solid was then transferred to a freeze dryer for further drying, yielding a white powder, which was the 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 this self-assembled nanomedicine was between 100 nm and 200 nm.

[0051] In Example 13, 175 mg of aspirin raw material (average molecular weight 180.16 Da), 50 mg of PBE, and 50 mg of PEI were dissolved in DMSO (5 mL). After mixing by inverting the container, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in the DMSO. The solution was transferred to a 3500 Da dialysis bag using a pipette. The dialysis bag was then clamped and placed in a 1 L beaker filled with deionized water for rotary dialysis, with the deionized water changed every 1 hour. The solution in the dialysis bag was transferred to a 50 mL centrifuge tube and frozen into a solid at -80°C (for at least three hours). The solid was then transferred to a freeze dryer for further drying, yielding a white powder, which was the 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 this self-assembled nanomedicine was between 100 nm and 200 nm.

[0052] Example 14, 190 mg aspirin drug substance (average molecular weight 180.16 Da), 50 mg PBE and 50 mg PEI were dissolved in DMSO (5 mL), after mixing upside down, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred to a 3500 Da dialysis bag with a pipette, the dialysis bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water, and dialysis was performed by rotating, and the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred to a 50 mL centrifuge tube, the liquid was frozen into a solid at -80 ℃ (at least freeze-dried for more than three hours), then transferred to a freeze dryer for drying, finally a white powder was obtained, which was a self-assembled nanomedicine for improving fetal growth restriction, and 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] Example 15, 200 mg aspirin drug substance (average molecular weight 180.16 Da), 50 mg PBE and 50 mg PEI were dissolved in DMSO (5 mL), after mixing upside down, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred to a 3500 Da dialysis bag with a pipette, the dialysis bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water, and dialysis was performed by rotating, and the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred to a 50 mL centrifuge tube, the liquid was frozen into a solid at -80 ℃ (at least freeze-dried for more than three hours), then transferred to a freeze dryer for drying, finally a white powder was obtained, which was a self-assembled nanomedicine for improving fetal growth restriction, and 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] Example 16, 207 mg aspirin drug substance (average molecular weight 180.16 Da), 46 mg PBE and 46 mg PEI were dissolved in DMSO (5 mL), after mixing upside down, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred to a 3500 Da dialysis bag with a pipette, the dialysis bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water, and dialysis was performed by rotating, and the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred to a 50 mL centrifuge tube, the liquid was frozen into a solid at -80 ℃ (at least freeze-dried for more than three hours), then transferred to a freeze dryer for drying, finally a white powder was obtained, which was a self-assembled nanomedicine for improving fetal growth restriction, and 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] Example 17, 215 mg aspirin drug substance (average molecular weight 180.16 Da), 43 mg PBE and 43 mg PEI were dissolved in DMSO (5 mL), after mixing upside down, the mixture was put into an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred into a dialysis bag of 3500 Da with a pipette, after clamping the dialysis bag with a sealing clamp, it was put into a beaker filled with 1 L of deionized water and rotated for dialysis, the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred into a 50 mL centrifuge tube, the liquid was frozen into a solid at -80 ℃ (at least freeze-dried for more than three hours), then transferred into a freeze dryer for drying, finally a white powder was obtained, which was 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 - 200 nm.

[0056] Example 18, 220 mg aspirin drug substance (average molecular weight 180.16 Da), 40 mg PBE and 40 mg PEI were dissolved in DMSO (5 mL), after mixing upside down, the mixture was put into an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred into a dialysis bag of 3500 Da with a pipette, after clamping the dialysis bag with a sealing clamp, it was put into a beaker filled with 1 L of deionized water and rotated for dialysis, the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred into a 50 mL centrifuge tube, the liquid was frozen into a solid at -80 ℃ (at least freeze-dried for more than three hours), then transferred into a freeze dryer for drying, finally a white powder was obtained, which was 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 - 200 nm.

[0057] Example 19, 210 mg aspirin drug substance (average molecular weight 180.16 Da), 35 mg PBE and 35 mg PEI were dissolved in DMSO (5 mL), after mixing upside down, the mixture was put into an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred into a dialysis bag of 3500 Da with a pipette, after clamping the dialysis bag with a sealing clamp, it was put into a beaker filled with 1 L of deionized water and rotated for dialysis, the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred into a 50 mL centrifuge tube, the liquid was frozen into a solid at -80 ℃ (at least freeze-dried for more than three hours), then transferred into a freeze dryer for drying, finally a white powder was obtained, which was 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 - 200 nm.

[0058] Example 20, 240 mg of aspirin raw material (average molecular weight 180.16 Da), 30 mg of PBE and 30 mg of PEI were dissolved in DMSO (5 mL), after mixing well by inverting up and down, the mixture was placed in an ultrasonic cleaner until the powder was completely dissolved in DMSO; the mixed solution was transferred to a 3500 Da dialysis bag with a pipette, the dialysis bag was clamped with a sealing clamp and placed in a 1 L beaker filled with deionized water, and dialysis was performed by rotating, the deionized water was replaced every 1 h; the solution in the dialysis bag was transferred into a 50 mL centrifuge tube, the liquid was frozen into a solid at -80 ℃ (at least freeze-dried for more than three hours), then transferred to a freeze dryer for drying, finally a white powder was obtained, which was 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.

[0059] The scanning electron microscope and transmission electron microscope images of the nanodrug prepared according to the method of Example 1 above are shown in Figure 1

[0060] Figure 2 The particle size distribution and surface potential of the self-assembled nanodrug were detected. About 5 mg of dried nanodrug was added to 1 mL of deionized water and mixed well, and then ultrasonically dissolved to form nanomicelles. The nanomicelles were added to the sample cell, and a Malvern laser particle size analyzer was used to detect the particle size distribution and surface potential of the nanodrug.

[0061] Figure 3 The nuclear magnetic resonance spectrum of hydrogen (left) and the Fourier transform infrared spectrum (right) of the self-assembled nanodrug are shown. Dry nanodrug (10 mg), PBE (10 mg), aspirin (10 mg), and PEI (10 mg) were dissolved in 600 μL of deuterated methanol, respectively, and scanned and detected using a nuclear magnetic resonance instrument at 600 MHz. In addition, an appropriate amount of dry nanodrug, PBE, aspirin and PEI were placed on the detection table of a Fourier transform infrared spectrometer for scanning and detection.

[0062] Figure 4 ​The detection statistical chart of the self-assembled nanodrug scavenging different active oxygen free radicals. Nanodrug scavenging superoxide anion detection: prepare methanol solution of nanodrug with concentration of 0.05, 0.1, 0.15, 0.25, 0.5, 1 mg / mL. According to the superoxide anion detection kit instructions, mix the prepared reagent with each concentration of nanodrug, incubate in 37 °C constant temperature water bath for 40 min, then add chromogenic agent, measure the OD value at 550 nm, calculate the scavenging ability of superoxide anion according to the kit instructions; Nanodrug scavenging free radical DPPH· ability detection: according to the DPPH· detection kit instructions, 1.5 mL DPPH· (100 μg / mL) reagent and 3 mL different concentration of nanodrug (0.05, 0.1, 0.25, 0.5, 1, 2 mg / mL) are incubated in the dark for 30 min, and the absorbance at 517 nm is measured by ultraviolet spectrophotometer, and the DPPH· scavenging ability is calculated; Nanodrug scavenging H2O2 detection: according to the H2O2 detection kit, different concentrations of nanodrug (0, 1, 2, 4, 6 mg / mL) are incubated with 2 mL PBS (0.01 M) containing 50 nM H2O2 for 24 h, the residual H2O2 is measured by measuring the absorbance at 405 nm, and the eliminated H2O2 is calculated; Nanodrug scavenging hypochlorite detection: according to the literature, self-made nanoprobes (Lu-bCD NP) are used to detect the scavenging ability of hypochlorite, 25 μL of different concentrations of nanodrug (1, 2, 3, 4, 5 mg / mL) are mixed with 475 μL of 100 mM NaClO solution for 15 min, 50 μL of mixed reaction supernatant is taken and reacted with 50 μL of Lu-bCD NP solution (10 mg / mL), and the ClO - scavenging efficiency. From Figure 4 It can be seen that the self-assembled nanodrug can scavenge different active oxygen free radicals.

[0063] Figure 5 Apoptosis chart for blank control group (0.01M phosphate buffer), hydrogen peroxide group (0.01M phosphate buffer), treatment group (25 μg / mL, 50 μg / mL, 100 μg / mL of self-assembled nanodrug group) and aspirin group (100 μM). Vascular endothelial cells (HUVECs) were seeded in a 6-well plate and incubated with PBS, self-assembled nanodrug (25 μg / mL, 50 μg / mL, 100 μg / mL), aspirin (100 μM) respectively for 24 hours, then treated with 100 μM H2O2 for 12 hours except the blank group. Then the cells were digested with 0.25 % trypsin, centrifuged to collect the cells, and then analyzed by flow cytometry after Annexin V and PI staining.Figure 5 It can be concluded that the nano-drug group inhibited H2O2-induced apoptosis in a concentration-dependent manner, and the number of inhibited apoptosis was more significant compared with the aspirin group.

[0064] Figure 6 The superoxide anion detection probe (DHE) staining chart for the blank control group (0.01M phosphate buffer), the hydrogen peroxide group (0.01M phosphate buffer), the treatment group (25 μg / mL, 50 μg / mL, 100 μg / mL of the self-assembled nano-drug group), and the aspirin group (100 μM), wherein the scale is 40 μm. HTR8 (placental trophoblast cells) were inoculated in a 12-well plate (1 × 10 5 After being treated with fresh medium without fetal bovine serum, fresh medium containing nano-drugs (25 μg / mL, 50 μg / mL, 100 μg / mL of the self-assembled nano-drug group), and aspirin (100 μM) for 24 hours, the medium of the blank group was replaced with fresh medium containing 100 μM H2O2 for 12 hours to induce ROS production. Then the cells were washed with PBS, and 1 mL of medium containing 5 μM DHE was added, and the cells were incubated in a cell incubator for 30 min, washed with sterile PBS, fixed with 4% paraformaldehyde, and the cell nucleus was stained with DAPI for 5 min. After mounting, the cell fluorescence intensity was observed using laser confocal. The blank control group was obtained by adding only PBS without adding hydrogen peroxide induction in the cells. From Figure 6 It can be concluded that the nano-drug group more significantly reduced the production of cellular reactive oxygen species under hydrogen peroxide induction compared with the aspirin group.

[0065] Figure 7 The cell migration chart for the blank control group (0.01M phosphate buffer), the hydrogen peroxide group (0.01M phosphate buffer), the treatment group (25 μg / mL, 50 μg / mL, 100 μg / mL of the self-assembled nano-drug group), and the aspirin group (100 μM), wherein the scale is 100 μm. Fresh 1640 medium was added to the bottom compartment. HTR8 (placental trophoblast cells) were inoculated in the upper chamber of the cell chamber at a density of 4 × 10 4 4 × 10 Figure 7It can be concluded that the nano-drug group significantly promoted the cell invasion ability under the inhibition of hydrogen peroxide in a concentration-dependent manner compared with the aspirin group, and the cell invasion ability promoted by the 100 μg / mL nano-drug treatment group was comparable to that of the control group.

[0066] Figure 8 The figure of interleukin 6 (IL-6) gene expression quantification of placental trophoblast cells (HTR8) and vascular endothelial cells (HUVECs) under the blank control group (0.01M phosphate buffer), LPS group (100 ng / mL of LPS), treatment group (25 μg / mL, 50 μg / mL, 100 μg / mL of self-assembled nano-drug group) and aspirin group (100 μM) under the stimulation of lipopolysaccharide (LPS). Placental trophoblast cells (HTR8) and vascular endothelial cells (HUVECs) were inoculated into a six-well cell culture plate (2 ×10 5 After being treated with fresh culture medium without fetal bovine serum, fresh culture medium containing nano-drugs (25 μg / mL, 50 μg / mL, 100 μg / mL of nano-drugs), aspirin (100 μM) for 24 hours, the culture medium of the blank group was replaced with fresh culture medium containing 100 ng / mL of LPS for 12 hours. Then the cells were washed with PBS, and the cell RNA was extracted with a rapid RNA extraction kit according to the manufacturer's instructions, and the extracted cell RNA was reversely transcribed into cDNA and subjected to qPCR reaction and gene expression analysis according to the manufacturer's instructions. From Figure 8 It can be concluded that the nano-drug group more significantly reduced the LPS-induced IL-6 gene expression level compared with the aspirin group.

[0067] Figure 9 The figure of fluorescence distribution and quantification of Cy5-labeled self-assembled nano-drugs in the placenta and fetus of pregnant rats in the sham operation group and the model group, in which the scale is 5 mm. After the 14.5-day pregnant SD female mice were anesthetized, a 1.5 cm midline incision was made on the lower abdomen, and the uterine vessels of the model group were ligated with 3-0 silk to induce a fetal growth restriction rat model; the pregnant female mice in the sham operation group were only opened and not ligated with uterine vessels. One day after the operation model was established, 10 mg / kg of Cy5-labeled self-assembled nano-drugs or the same dose of PBS were injected into the tail vein of the fetal growth restriction model group and the sham operation group pregnant mice, and the rats were sacrificed 4 hours later. After being washed with normal saline, the uterus was removed, the fetus and placenta were separated, 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. From Figure 9It can be concluded that the self-assembled nanodrug only enriches in the basal decidua of the placenta (i.e. the maternal surface of the placenta), and no fluorescence signal is detected in the fetus, indicating that the drug cannot penetrate the placental barrier to affect the fetus.

[0068] Figure 10 The bar charts of the weight of the fetal rats, the length of the fetal rats and the weight of the placenta after the nanodrug was used to treat the fetal growth restriction rat model are shown in FIG. 6. The SD female rats on the 14.5th day of pregnancy were anesthetized, and a 1.5 cm midline incision was made on the lower abdomen. The model group was ligated with 3-0 silk thread to induce the fetal growth restriction rat model. The sham operation group was only opened and not ligated. One day after the operation model was established, the self-assembled nanodrug (5 mg / kg, 10 mg / kg, 20 mg / kg) was injected intravenously or aspirin (4 mg / kg) was orally administered to the model rats. The model group and the sham operation group were injected with the same dose of normal saline. Each group was injected or orally administered once a day, and the pregnant rats were sacrificed after 5 days of continuous treatment. The length of the fetal rats, the weight of the fetal rats and the weight of the placenta were measured. Figure 10 It can be concluded that the fetal growth restriction rat model reduces the weight of the fetus and the placenta, and after the nanodrug treatment, the nanodrug group can increase the weight of the fetus and the placenta to a certain extent, and restore the length of the fetus, indicating that the nanodrug can improve fetal growth restriction.

[0069] Figures 9-10 The method for establishing the fetal growth restriction model of the pregnant rats is as follows:

[0070] 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. The rats were raised in standard cages, maintaining the circadian rhythm and constant temperature. After being raised for one week, the rats were mated according to the ratio of 2:1 (female:male). The female rats were examined by vaginal smear, and the presence of sperm was recorded as the 0th day of pregnancy (G0) for subsequent experiments. On the 14.5th day of pregnancy, 0.3% pentobarbital was used for intraperitoneal anesthesia at a dose of 1 mL / 100 g. The abdomen was shaved, and the skin was disinfected with iodophor before laying a towel. The model group was incised in the middle of the lower abdomen to expose the skin, subcutaneous tissue, rectus abdominis, and peritoneum. The uterus was gently removed and placed on gauze soaked with normal saline. The uterine blood vessels were exposed, and 2-0 silk thread was used as a pad. The uterine blood vessels were ligated with 3-0 silk thread, and then the 2-0 silk thread was pulled out. The uterus was returned to the abdominal cavity, and the abdominal cavity was washed with normal saline. Then, the abdominal cavity was closed layer by layer with 4-0 silk thread. The sham operation group was only incised in the abdominal tissues, and the uterine blood vessels were not ligated. After the abdomen was opened, the abdominal cavity was washed with normal saline, and the abdominal cavity was closed layer by layer.

Claims

1. A method for preparing a self-assembled nanomedicine to improve fetal growth restriction, characterized in that: Includes the following steps: Aspirin raw material, polyethyleneimine, and phenylboronic acid ester were dissolved in an organic solvent. After mixing by inverting the container, the solution was placed in an ultrasonic cleaner until the drug powder was completely dissolved. Then, the organic solvent containing the completely dissolved aspirin raw material, polyethyleneimine, and phenylboronic acid ester was placed in a 3500 Da dialysis bag and subjected to rotary dialysis in deionized water to remove the organic solvent. The deionized water was replaced every hour. Finally, the solution in the dialysis bag was 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. The mass ratio of aspirin raw material: phenylboronic acid ester: polyethyleneimine was between 0.5:1:1 and 8:1:1, and the particle size of the self-assembled nanomedicine was 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 organic solvent used is dimethyl sulfoxide.

3. The method for preparing a self-assembled nanomedicine for improving fetal growth restriction according to claim 1 or 2, characterized in that: The concentrations of phenylboronic acid ester solutions range from 6 mg / mL to 20 mg / mL, aspirin from 10 mg / mL to 48 mg / mL, and polyethyleneimine solutions from 6 mg / mL to 20 mg / mL.

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

5. The method for preparing a self-assembled nanomedicine for improving fetal growth restriction according to claim 1, characterized in that: The aspirin has an average molecular weight of 180.16 Da.

6. A self-assembled nanomedicine for improving fetal growth restriction, characterized in that, It is prepared by the method described in any one of claims 1-5.

7. The use of the self-assembled nanomedicine according to claim 6 in the preparation of drugs for preventing and treating fetal growth restriction during pregnancy.

8. The application according to claim 7, characterized in that: Fetal growth restriction during pregnancy includes fetal growth restriction caused by hypoxia during pregnancy.

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