CN117860690A - Self-assembled nano anti-inflammatory drug, preparation method and application thereof - Google Patents
Self-assembled nano anti-inflammatory drug, preparation method and application thereof Download PDFInfo
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- CN117860690A CN117860690A CN202410011990.2A CN202410011990A CN117860690A CN 117860690 A CN117860690 A CN 117860690A CN 202410011990 A CN202410011990 A CN 202410011990A CN 117860690 A CN117860690 A CN 117860690A
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- 230000036266 weeks of gestation Effects 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
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Abstract
The invention discloses a self-assembled nano anti-inflammatory drug, a preparation method and application thereof, wherein the self-assembled nano anti-inflammatory drug consists of low-molecular heparin, an anti-inflammatory drug containing carboxyl and an active oxygen scavenger, the mass ratio of the low-molecular heparin to the anti-inflammatory drug containing carboxyl is between 0.3:1 and 40:1, the mass ratio of the low-molecular heparin to the active oxygen scavenger is between 0.5:1 and 8:1, and the particle size of the nano anti-inflammatory drug is between 60nm and 360 nm. The self-assembled nano anti-inflammatory drug is used for local delivery of drug delivery to vagina through intravenous injection or loading in hydrogel, and is targeted to the decidua layer of uterus, so that the purposes of anti-inflammation, anti-oxidative stress, and inhibiting uterine contraction are achieved, and further, premature labor is prevented and treated. The nanometer anti-inflammatory drug has the advantages of simple synthesis, low cost, simple and easy self-assembly method, easy removal of organic solvent, good stability and no immunogenicity, and the nanometer anti-inflammatory drug does not penetrate placenta barrier to enter fetus.
Description
Technical Field
The invention relates to the field of nano material and maternal and fetal medicine application, in particular to a self-assembled nano anti-inflammatory drug, a preparation method of the nano anti-inflammatory drug and application of the nano anti-inflammatory drug in preventing and treating premature delivery of pregnant women, and verifying safety of the nano anti-inflammatory drug to a mother and a fetus.
Background
Premature labor (PTB) refers to delivery before 37 weeks of gestation. PTB incidence is about 5% -18% of all gestations worldwide [1] And is associated with many bad pregnancy outcomes. PTB is the main cause of morbidity and mortality in newborns, accounting for 35% of the total neonatal mortality [2] . Extensive clinical studies have shown that intrauterine/systemic infections and inflammation are the major pathophysiological factors of PTB [3-5] . Neutrophils and macrophages are the primary inflammatory cells of the maternal-fetal interface and their pro-inflammatory activation is associated with many complications of pregnancy. In addition, overproducing Reactive Oxygen Species (ROS) are also closely related to the occurrence of PTB [6-9] . However, the exact cellular and molecular mechanisms of PTB are not known, thereby limiting the development of strategies to effectively control PTB. The current treatments commonly used in clinic include mechanical intervention and drug treatment.
Cervical cerclage and cervical pessary as mechanical interventions for premature delivery, which treatments may cause serious side effects to the mother, such as increased risk of bleeding and infection [10] . On the other hand, progestins, oxytocin antagonists, calcium channel blockers, betamethamine-mimetic and non-steroidal anti-inflammatory drugs are used to delay premature labor by reducing, preventing or slowing uterine contractions, but they provide only a short score Delayed delivery, no therapeutic effect on the etiologic mechanisms of premature delivery, or the benefit of providing substantially improved perinatal health outcomes [11] . Furthermore, the effectiveness and deleterious effects of anti-uterine agents remain elusive. These drugs may cause various adverse reactions to the mother and fetus, have safety problems, and the like.
In order to overcome the above problems, advanced drug delivery systems in recent years, such as liposomes, solid lipid Nanoparticles (NPs), nanostructured lipid carriers, nanoemulsions, polymer NPs, nanosuspensions, and the like, have a great clinical application value by increasing targeted absorption of uterus placenta and reducing fetal distribution, improving pharmacokinetics/pharmacology and safety, increasing therapeutic effects on PTB while reducing side effects, and have been paid attention to researchers. For example, progesterone nanosuspension coated with Pluronic F127 improves delivery of vaginal progesterone, thereby improving PTB prevention efficacy [12] . Likewise, vaginal delivery of mucous inert nanosuspensions of Histone Deacetylase (HDAC) inhibitors, with the addition of progesterone in some cases, can prevent premature birth and typical neurodevelopmental manifestations in living mice. In vitro human myometrium cells, progesterone/HDAC inhibitor combinations inhibit cell contraction by increasing the stability of progesterone receptor B [13] . And researches show that the oxytocin receptor targeted liposome inhibits or enhances human uterine contraction in vitro, is positioned in uterine tissue of pregnant mice in vivo, effectively delivers medicines for preventing premature labor caused by inflammation, and can effectively reduce the premature labor rate of the mice [14] 。
Although the above-described nanomedicine delivery systems have ideal uterine targeting and anti-uterine contractility, their benefits in preventing PTB are not significant due to the limited efficacy of existing drugs for preventing premature delivery per se. In particular, the related formulations do not fully regulate maternal-fetal interface inflammation, oxidative stress, and uterine myometrial contractions, thereby purposefully improving the etiologically related mechanisms that lead to PTB. In addition, the nanometer materials studied at present have the problems of poor storage stability, complex synthesis process, high preparation cost, obvious side effect and the like.
Disclosure of Invention
The invention aims to provide a composition and a preparation method of a self-assembled nano anti-inflammatory drug, and also to verify the effect of the nano anti-inflammatory drug in preventing and treating premature labor by adjusting the micro environment of a maternal-fetal interface through anti-inflammatory and anti-oxidative stress, and to evaluate the safety of the nano anti-inflammatory drug on a mother and a fetus.
The invention couples low molecular heparin, the anti-inflammatory drug containing carboxyl and the active oxygen scavenger through covalent bonds to form a novel material with anti-inflammatory and ROS scavenging functions, and the material has the characteristic of amphiphilicity and can construct a novel nano anti-inflammatory drug through self-assembly. The nanometer anti-inflammatory drug can target the uterus and placenta decidua of a PTB mouse, reduce the local active oxygen of the uterus and placenta and reduce the infiltration of neutrophils and macrophages. Further inhibit contraction of Uterine Smooth Muscle Cells (USMCs)/myometrium, protect USMCs from apoptosis induced by premature pathological microenvironment, and improve uterine arterial blood flow, thereby preventing and treating PTB and inhibiting PTB-related bad pregnancy outcome. Meanwhile, the low molecular heparin which is a component of the nano anti-inflammatory medicament has the effects of anti-inflammation, anticoagulation and thrombolysis, can improve uterine artery blood flow, and has positive effects on preventing and treating premature delivery. Compared with other means for preventing and treating premature delivery, the nano anti-inflammatory medicament prepared by self-assembly has the advantages of simple synthesis, anti-inflammatory and anti-oxidative stress functions, blood flow improvement, strong targeting, safety, low cost and the like, is expected to realize clinical transformation, provides technical reference for other gestational diseases, and has wider application prospect.
In view of this, the technical scheme adopted by the invention is as follows: a self-assembled nano anti-inflammatory drug is formed by self-assembling low-molecular heparin, an anti-inflammatory drug containing carboxyl and an active oxygen scavenger, wherein the mass ratio of the low-molecular heparin to the anti-inflammatory drug containing carboxyl is between 0.3:1 and 40:1, the mass ratio of the low-molecular heparin to the active oxygen scavenger is between 0.5:1 and 8:1, and the particle size of the nano drug is between 60nm and 360 nm. Wherein the low molecular heparin is selected from enoxaparin, nadroparin, dalteparin; the average molecular weight of the low molecular heparin is between 3000Da and 8000 Da; the anti-inflammatory drug containing carboxyl is selected from Ac-SDKP, AC2-26, aspirin, naproxen, ibuprofen, diclofenac, indomethacin, and aceclofenac; the active oxygen scavenger is selected from pinacol phenylborate (PBAP), tempol, resveratrol (RSV), salvianic Acid (SA), protocatechuic aldehyde, alpha-lipoic acid, and curcumin.
The invention also provides a preparation method of the self-assembled nano anti-inflammatory drug, which comprises the following steps: firstly, dissolving an active oxygen scavenger, low molecular heparin and N-hydroxysuccinimide (NHS), N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC) in preheated formamide, adding Triethanolamine (TEA), stirring the mixture at room temperature under the protection of nitrogen for 12 hours to obtain a polymer solution, placing the polymer solution in a dialysis bag, dialyzing in deionized water for 24 hours to remove an organic solvent, and freeze-drying and collecting; and then dissolving the collected material in dimethyl sulfoxide (DMSO), adding N-hydroxysuccinimide, N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide, triethanolamine and the anti-inflammatory drug containing carboxyl to react for 12 hours, dialyzing and freeze-drying to collect the self-assembled nano anti-inflammatory drug.
Further, the concentration of the low molecular heparin solution is between 5mg/mL and 90mg/mL, the concentration of the organic solution of the anti-inflammatory drug containing carboxyl is between 1mg/mL and 10mg/mL, and the concentration of the active oxygen scavenger solution is between 3mg/mL and 50 mg/mL.
The invention further provides application of the self-assembled nano anti-inflammatory drug in preparation of drugs for preventing and treating premature delivery.
The nanometer anti-inflammatory medicine is used for intravenous injection. Or the nano anti-inflammatory drug is loaded on Poloxamer 407 (PX) to form composite hydrogel, and the composite hydrogel is locally delivered to the vagina for drug delivery, so as to prevent and treat premature delivery and pregnancy related diseases.
The nano anti-inflammatory medicine is concentrated in the uterus and placenta. Has multiple functions of anti-inflammatory, antioxidation, uterine contraction inhibition and the like, and achieves the purpose of preventing and treating premature labor. Meanwhile, can also prevent and treat gestational related diseases such as preeclampsia, fetal growth restriction and the like. The self-assembled nano anti-inflammatory drug cannot penetrate placenta barrier, has no obvious side effect on a mother and a fetus, can realize clinical transformation, and is further applied to related diseases in gestation period.
The invention has the following advantages:
1) The heparin, anti-inflammatory drugs and active oxygen scavengers used in the invention are simple to synthesize and relatively low in price, so that industrialization of corresponding preparations is easy to realize. In addition, the heparin is low molecular heparin, has higher safety, and clinical guidelines indicate that the heparin can be used for gestation-related diseases.
2) The self-assembly method adopted by the invention is simple and easy to implement, and the used organic solvent is easy to remove, so that the feasibility and the safety of the application of the final nano material are ensured.
3) The nano anti-inflammatory drug prepared by the invention is passively targeted at the womb and placental decidua of pregnancy through the response of ROS, and has high targeting property. And the nano anti-inflammatory drug cannot cross the placenta barrier, thereby reducing possible side effects on the fetus. Has good stability and no immunogenicity, and ensures the safety of the mother fetus.
4) The nano-medicament prepared by the invention has multiple effects of anti-inflammatory, antioxidant stress and uterine contraction inhibition, and can effectively prevent premature delivery, improve survival rate of offspring and improve bad pregnancy outcome.
5) The nano medicine prepared by the invention is solid, is convenient to store and is flexible and convenient to use.
6) The nano medicine prepared by the invention can be used by intravenous injection, can also be prepared into hydrogel for local slow release administration of vagina, realizes non-invasive safe treatment, and can be further used for related gestational diseases such as preeclampsia, fetal growth restriction and the like.
Drawings
FIG. 1 is a transmission electron microscope image and a scanning electron microscope image of an LMWH/PBAP/Ac-SDKP nano-drug prepared by self-assembly of low molecular heparin (LMWH), phenylboronic acid pinacol ester (PBAP) and anti-inflammatory polypeptide Ac-SDKP, wherein the scale is 200nm;
FIG. 2 is a quantitative statistical chart of the ability of LMWH/PBAP/Ac-SDKP nano-drug to scavenge different reactive oxygen free radicals;
FIG. 3 shows a blank (0.01M phosphate buffer) and model(0.01M H 2 O 2 ) And treatment group (0.01M H) 2 O 2 In vitro inhibition of hydrogen peroxide (H) by +different doses of LMWH/PBAP/Ac-SDKP nanodrug group, atosiban group 2 O 2 ) Confocal microscopy images of the induction of reactive oxygen species produced by different cells (neutrophils, macrophages, human amniotic epithelial cells), with a scale of 10 μm;
FIG. 4 is a graph of in vitro inhibition of neutrophil and macrophage migration in a blank (fresh medium), model lipopolysaccharide LPS (1. Mu.g/mL) and treatment (1. Mu.g/mL LPS+different doses of LMWH/PBAP/Ac-SDKP nano-drug group, atosiban group), with scale of 100 μm;
FIG. 5 is a statistical graph of in vitro inhibition of neutrophil and macrophage inflammatory cytokines in a blank (fresh medium), model (1. Mu.g/mL) and treatment (1. Mu.g/mL LPS+different doses of LMWH/PBAP/Ac-SDKP nano-drug group, atosiban group);
FIG. 6 shows the fluorescence intensity of Cy5/LMWH/PBAP/Ac-SDKP nanomedicine over time in the uterus and placenta of a model of premature mice;
FIG. 7 is a statistical plot of gestation time, offspring survival rate, fetal weight and placenta thickness for a model of premature delivery of mice treated with different drugs;
FIG. 8 is a photograph of fetal HE staining after treatment of a model of premature mice with different drugs, with a scale of 5mm;
FIG. 9 is a photograph of fetal alizarin red and A Li Xinlan bone staining after treatment of a model of premature mice with different drugs, with a scale of 1cm;
FIG. 10 is a photograph of HE staining of placenta after treatment of a model of premature mice with different drugs, with scales of 1mm (upper) and 200 μm (lower), respectively;
figure 11 is a statistical plot of the inhibition of uterine and fetal membrane inflammation levels following treatment of a model of premature mice with different drugs.
Detailed Description
The summary of the invention is described in further detail below in conjunction with the detailed description. It should be understood that the embodiments of the present invention are only for illustrating the present invention and not for limiting the present invention, and various substitutions and modifications made according to the general knowledge and conventional means of the person skilled in the art should be included in the scope of the present invention without departing from the technical spirit of the present invention.
The invention is described in detail below with reference to non-limiting examples in which the selection of active oxygen scavengers, anti-inflammatory agents and low molecular heparin can be controlled during self-assembly to control the particle size of the nano anti-inflammatory agent to between 60nm and 360 nm. All raw materials used in the invention have commercial products.
Example 1
Enoxaparin (90 mg), PBAP (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all materials were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of AC-SDKP was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 2
Enoxaparin (42 mg), tempol (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added and all materials were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 14mL of DMSO in the presence of EDC, NHS and TEA, 140mg of AC-SDKP was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeated dialysis and lyophilization. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 3
Enoxaparin (400 mg), resveratrol (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 10mg of AC-SDKP was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 4
Enoxaparin (720 mg), salvianic acid (90 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all materials were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 18mL of DMSO in the presence of EDC, NHS and TEA, 18mg of AC-SDKP was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeated dialysis and lyophilization. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 5
Enoxaparin (40 mg), protocatechuic aldehyde (80 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 2mL of DMSO in the presence of EDC, NHS and TEA, 12mg of AC-SDKP was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 6
Enoxaparin (90 mg), alpha-lipoic acid (24 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added and all materials were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of AC-SDKP was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 7
Enoxaparin (200 mg), curcumin (400 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 5mg of AC-SDKP was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 8
Enoxaparin (40 mg), PBAP (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all materials were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 1mg of AC2-26 was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 9
Enoxaparin (42 mg), PBAP (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all materials were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 20mL of DMSO in the presence of EDC, NHS and TEA, 140mg of aspirin was added and the reaction was stirred at room temperature for 12h, and the sample was collected by repeated dialysis and lyophilization. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 10
Enoxaparin (400 mg), PBAP (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all materials were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of the sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 10mg of naproxen was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 11
Enoxaparin (400 mg), PBAP (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all materials were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 10mL of DMSO in the presence of EDC, NHS and TEA, 10mg of ibuprofen was added and the reaction was stirred at room temperature for 12h, and the sample was collected by repeated dialysis and lyophilization. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 12
Enoxaparin (90 mg), PBAP (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all materials were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of the sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of diclofenac was added thereto, and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 13
Enoxaparin (90 mg), PBAP (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all materials were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of indomethacin was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 14
Enoxaparin (90 mg), PBAP (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all materials were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of the sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of aceclofenac was added thereto and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 15
Natriin (90 mg), PBAP (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of AC-SDKP was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 16
Natrii droparin (90 mg), tempol (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of AC-SDKP was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 17
Natrii droparin (90 mg), resveratrol (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of AC-SDKP was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 18
Natriin (90 mg), salvianic acid (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of AC-SDKP was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 19
Natrii droparin (90 mg), protocatechuic aldehyde (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of AC-SDKP was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 20
Natriin (90 mg), alpha-lipoic acid (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of AC-SDKP was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 21
Natriin (90 mg), curcumin (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of AC-SDKP was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 22
Natriin (90 mg), PBAP (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of the sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of AC2-26 was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 23
Natriin (90 mg), PBAP (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of aspirin was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeated dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 24
Natriin (90 mg), PBAP (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of the sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of naproxen was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 25
Natriin (90 mg), PBAP (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of the sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 3mg of ibuprofen was added and the reaction was stirred at room temperature for 12h, and the sample was collected by repeated dialysis and lyophilization. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 26
Natrii droparin (200 mg), PBAP (400 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of the sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 10mg of diclofenac was added thereto, and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 27
Natrii droparin (40 mg), PBAP (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of the sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 1mg of indomethacin was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 28
Natriin (42 mg), PBAP (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of the sample was dissolved in 14mL of DMSO in the presence of EDC, NHS and TEA, 140mg of aceclofenac was added thereto and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 29
Daheparin (400 mg), protocatechuic aldehyde (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all substances were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of the sample was dissolved in 1mL of DMSO in the presence of EDC, NHS and TEA, 10mg of ibuprofen was added and the reaction was stirred at room temperature for 12h, and the sample was collected by repeated dialysis and lyophilization. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 30
Daheparin (400 mg), alpha-lipoic acid (50 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all materials were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of the sample was dissolved in 10mL of DMSO in the presence of EDC, NHS and TEA, 10mg of diclofenac was added thereto, and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
Example 31
Daheparin (40 mg), curcumin (80 mg), NHS (48 mg) and EDC (100 mg) were dissolved in 8mL formamide, 30mg TEA was added, and all materials were dissolved by heating at 50℃for 0.5 h. The resulting mixture was magnetically stirred at room temperature under nitrogen for 12h. The final solution was dialyzed against deionized water at 25 ℃. Water was changed every 30 minutes. After 24h of dialysis, samples were collected by lyophilization. Subsequently, the above 50mg of sample was dissolved in 2mL of DMSO in the presence of EDC, NHS and TEA, 2mg of AC-SDKP was added and the reaction was stirred at room temperature for 12 hours, and the sample was collected by repeating dialysis and freeze-drying. The sample is self-assembled nano anti-inflammatory drug, and the particle size of the self-assembled nano anti-inflammatory drug is between 60nm and 360 nm.
A transmission electron microscope and a scanning electron microscope of the nano anti-inflammatory drug prepared according to the method of the above example 1 are shown in FIG. 1.
FIG. 2 is a statistical graph of the detection of the ability of LMWH/PBAP/Ac-SDKP nano-drug to scavenge different reactive oxygen free radicals. LMWH/PBAP/Ac-SDKP nano-drug scavenging superoxide anion assay prepared according to the method of example 1 above: preparing nano medicines with different concentrations of 0.05,0.1,0.15,0.25, 0.5-1 mg/mL and xanthine oxidationExcess superoxide anions generated by the enzyme anion system are co-incubated. A constant temperature water bath at 37 ℃ for 40min, and the remaining superoxide anions are measured by using a superoxide anion radical detection kit; and (3) detecting the DPPH-capability of the LMWH/PBAP/Ac-SDKP nano drug for scavenging free radicals: according to the DPPH detection kit instruction, 1.5mL of DPPH (100 mug/mL) reagent and 3mL of nano-drugs (0.05, 0.1,0.15,0.25,0.5,1 mug/mL) with different concentrations are incubated for 30min under the dark condition, the absorbance at 517nm is measured by an ultraviolet spectrophotometer, and the DPPH clearance capacity is calculated; LMWH/PBAP/Ac-SDKP nano-drug scavenging H 2 O 2 And (3) detection: according to H 2 O 2 Detection kit, LMWH/PBAP/Ac-SDKP nano-drug (0, 1,2,4,6 mg/mL) with different concentrations and 2mL containing 50nM H 2 O 2 Is incubated for 24H with PBS (0.01M), and residual H is measured by measuring absorbance at 405nm 2 O 2 And calculates the eliminated H 2 O 2 The method comprises the steps of carrying out a first treatment on the surface of the And (3) detecting the hypochlorite removal capability of the LMWH/PBAP/Ac-SDKP nano-drug: homemade nanoprobes (Lu-bCD NP) were synthesized according to literature for detection of hypochlorite (ClO) - ) Clearance ability 25. Mu.L of the various concentrations of the nanopharmaceuticals (1, 2,3,4,5 mg/mL) were mixed with 475. Mu.L of 100mM NaClO solution for 15min, 50. Mu.L of the mixed reaction supernatant was aspirated and reacted with 50. Mu.L of Lu-bCD NP solution (10 mg/mL), and ClO-was calculated according to the standard curve. The result shows that the nano medicine effectively eliminates superoxide anion, free radical and H 2 O 2 And hypochlorite, show the broad-spectrum ROS-eliminating ability of nano-drugs in a dose-dependent manner.
FIG. 3 shows a blank (0.01M phosphate buffer PBS), model (0.01M H) 2 O 2 ) And treatment group (0.01M H) 2 O 2 +LMWH/PBAP/Ac-SDKP nano-drug group, atosiban group) in vitro to inhibit hydrogen peroxide from inducing different cells to produce active oxygen, wherein the scale is 10 μm. Cells (macrophages, human amniotic epithelial cells) at 2X 10 5 The individual wells were inoculated into slide 24-well plates, and after overnight incubation, blank medium, fresh medium (dose from 100, 200 to 400. Mu.g/mL) with LMWH/PBAP/Ac-SDKP nanomedicine prepared as described in example 1 above, or atosiban was added, respectivelyCells were treated with ban (0.27. Mu.g/mL) for 12 hours, and after removal of the medium, 200. Mu.M H was added 2 O 2 Solution treatment for 6h induced ROS production, removal of cell culture broth, addition of 1mL of medium containing 2.5 μm DHE, incubation in cell incubator for 30min, washing with sterile PBS, fixation of 4% paraformaldehyde, staining of nuclei with DAPI for 5min, laser confocal observation of ROS levels in samples after sealing, addition of PBS alone without hydrogen peroxide induction as a blank control. Similarly, neutrophils were grown at 2X 10 5 Each cell/ml was seeded in 24-well plates and incubated for 30min. After 2 hours of treatment of cells with fresh medium containing nanomedicine (doses 100, 200 and 400. Mu.g/mL) or atosiban (0.27. Mu.g/mL), cells were treated with medium containing 200. Mu. M H 2 O 2 Replaced the original medium and incubated for 1h. Subsequent experiments were performed as described above. The results show that the nano-drug effectively inhibits H in a dose dependent manner 2 O 2 Induced ROS production by neutrophils, macrophages and human amniotic epithelial cells.
FIG. 4 is a graph of in vitro inhibition of neutrophil and macrophage migration in a blank (fresh medium), model lipopolysaccharide LPS (1. Mu.g/mL) and treatment (1. Mu.g/mL LPS+LMWH/PBAP/Ac-SDKP nano-drug group, atosiban group), with a scale of 100. Mu.m. Primary neutrophils and macrophages at 4X 10 4 100. Mu.L of medium containing LMWH/PBAP/Ac-SDKP nanomedicine (100, 200 and 400. Mu.g/mL) or atosiban (0.27. Mu.g/mL) prepared as described in example 1 above was added to the upper chamber of the Transwell chamber, respectively. Fresh medium is added to the bottom compartment. After 2 hours of neutrophil incubation, macrophages were incubated for 12 hours and LPS (1. Mu.g/mL) was added to the upper chamber separately from the control group, which was treated with PBS. After 24h of macrophage treatment, neutrophils were treated for 4h, the cells were fixed after invading the membrane, stained with crystal violet, and observed with an optical microscope. It was found that nano-drugs can inhibit lipopolysaccharide mediated migration of neutrophils and macrophages in a dose dependent manner.
FIG. 5 is a blank (fresh medium), model LPS (1. Mu.g/mL) and treatment (1. Mu.g/mL LPS+LMWH/PBAP/Ac-SDKP nanopharmaceutical group, atosiban) Group) in vitro, inhibiting neutrophil and macrophage inflammatory cytokines. Neutrophils were grown at 2X 10 5 Each cell/ml was seeded in 24-well plates and incubated for 30min. After 2 hours of treatment of cells with fresh medium containing LMWH/PBAP/Ac-SDKP nanomaterials prepared as in example 1 above (doses of 100, 200 and 400 μg/mL) or atosiban (0.27 μg/mL), the medium was replaced with fresh medium containing LPS (1 μg/mL) and incubated for 1 hour. The control group was treated with PBS alone. For macrophages and human amniotic epithelial cells hAECs, the cells were grown at 2X 10 5 Individual cells/ml were seeded in 24-well plates and incubated overnight. Cells were treated with fresh medium containing LMWH/PBAP/Ac-SDKP nanomaterials (doses 100, 200 and 400 μg/mL) or atosiban (0.27 μg/mL) for 12 hours, then the medium was replaced with fresh medium containing LPS (1 μg/mL) and incubated for 6 hours. Both treatments were assayed for IL-1β, TNF- α and IL-8 content in the supernatants by ELISA. The results demonstrate that in lipopolysaccharide stimulated neutrophils, macrophages and hAECs, the nanomedicine significantly reduced the secretion of inflammatory factors IL-1β, TNF- α and IL-8 in a dose dependent manner.
FIG. 6 shows the fluorescence intensity of Cy5/LMWH/PBAP/Ac-SDKP nanomedicine over time in uterus and placenta of a model of premature mice.
Figure 7 is a statistical plot of gestation time, offspring survival, fetal weight and placenta thickness for the pre-term models of mice treated with different drugs.
Fig. 8 is a photograph of fetal HE staining after treatment of a model of premature mice with different drugs, with a scale of 5mm.
FIG. 9 is a photograph of fetal alizarin red and A Li Xinlan bone staining after treatment of a premature mouse model with different drugs, with a scale of 1cm.
Fig. 10 is a photograph of HE staining of placenta after treatment of a premature mouse model with different drugs, with scales of 1mm (upper) and 200 μm (lower), respectively.
Figure 11 is a statistical plot of the inhibition of uterine and fetal membrane inflammation levels following treatment of a model of premature mice with different drugs.
The establishment and efficacy evaluation of the mice pre-term model described in FIGS. 6-11 is as follows:
(1) Model of premature mouse: ICR pregnant mice were modeled for LPS-induced PTB at 16.5 days of gestation (E16.5). Pregnant mice were anesthetized by isoflurane inhalation, sterilized with abdominal iodophor, and subjected to laparotomy with a 1.5cm midline incision made in the lower abdomen. Mice were randomly assigned to intrauterine injections of LPS (model group) or normal saline (Sham, control group). Pregnant mice PTB were induced by injecting 100. Mu.L of LPS saline between the right uterine horn 1 and 2 embryos. For the normal control group, pregnant mice were injected with 100 μl of physiological saline in a similar manner, and subjected to abdominal closure.
(2) Pregnant mice were randomly divided into control group (intrauterine injection of physiological saline), model group (intrauterine injection of 100. Mu.L of LPS), nano-drug group (intrauterine injection of 100. Mu.L of LPS and intravenous injection of LMWH/PBAP/Ac-SDKP nano-drug 2,5 or 10mg/kg per mouse) and atosiban group (intrauterine injection of 100. Mu.L of LPS, intravenous injection of 0.17mg/kg of atosiban). For the nano-drug group and atosiban group, nano-drugs were prepared as in example 1 above, and intravenous injection of different doses of nano-drug or atosiban was performed after 30min of LPS induction, respectively.
(3) LMWH/PBAP/Ac-SDKP nano drug profile in pregnant mice: after LPS induction of the E16.5 pregnant mice PTB model, mice were randomly assigned for intravenous injection of Cy5-LMWH/PBAP/Ac-SDKP nanopharmaceuticals, wherein nanopharmaceuticals were prepared as described in example 1 above, using normal saline as a control. At various time points (0, 1, 2, 4, 8, 12 and 24 h), mice were euthanized, ex vivo uterus and placenta were imaged in vitro using an OI600 MF palpable multifunctional imager and fluorescence intensity of uterus and placenta was observed. The results indicate that the nanomedicine was passively targeted to the uterus and placental decidua layer of model mice, with increasing fluorescence intensity of uterus and placenta isolated from PTB mice over time.
(4) Evaluation of premature labor treatment effect: the time of delivery after the mice were modelled was recorded, the number of litters from term and premature births was calculated, and the percentage of live births was calculated from the number of litters counted at the time of surgery. After delivery, the fetus and placenta are collected, photographed and weighed. Placenta and half of fetuses of each fetus were fixed with 4% paraformaldehyde, histologically analyzed with H & E staining, structural changes of placenta and fetuses were observed, and placenta thickness was measured. The other half of the fetuses were preserved with 95% ethanol solution, eviscerated, bone stained with aliskirin blue and alizarin red, and skeletal development of the fetuses was observed. The results show that the treatment with the high-dose (10 mg/kg) nano-drug significantly inhibits premature delivery, and the treatment with the medium-dose (5 mg/kg) drug also prolongs the gestation time of the mice to a certain extent. However, the low dose (2 mg/kg) of the nano-drug was not significantly effective on premature delivery, and atosiban also inhibited premature delivery to some extent, but was not as effective as the high dose nano-drug. The nano medicine has the advantages of improving the survival rate of offspring in a dose-dependent mode, effectively increasing the weight of a fetus, restoring the integrity and thickness of a placenta structure and improving the abnormal bone development of the fetus, and shows better premature delivery treatment effect.
(5) Evaluation of premature delivery treatment mechanism: e16.5 pregnant mice 8h after LPS injection, pregnant mice were sacrificed, uterine and fetal membrane tissues were collected, mRNA levels of TNF-alpha, IL-1 beta and IL-8 were quantified by RT-PCR analysis, and an inflammation index was detected. The results prove that the expression of inflammatory factors (IL-1 beta, TNF-alpha and IL-8) in uterus and fetal membrane tissues of PTB mice treated by nano-drug treatment is obviously reduced, which proves that the nano-drug has better anti-inflammatory treatment effect.
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Claims (10)
1. A self-assembled nano anti-inflammatory drug is characterized in that: the nano anti-inflammatory drug is formed by self-assembly of low molecular heparin, an anti-inflammatory drug containing carboxyl and an active oxygen scavenger, wherein the mass ratio of the low molecular heparin to the anti-inflammatory drug containing carboxyl is between 0.3:1 and 40:1, the mass ratio of the low molecular heparin to the active oxygen scavenger is between 0.5:1 and 8:1, and the particle size of the nano drug is between 60nm and 360 nm.
2. The self-assembled nano anti-inflammatory drug according to claim 1, wherein: the low molecular heparin is selected from enoxaparin, nadroparin or dalteparin, and the average molecular weight of the low molecular heparin is between 3000Da and 8000 Da.
3. The self-assembled nano anti-inflammatory drug according to claim 1, wherein: the anti-inflammatory drug containing carboxyl is selected from Ac-SDKP, AC2-26, aspirin, naproxen, ibuprofen, diclofenac, indomethacin and aceclofenac.
4. The self-assembled nano anti-inflammatory drug according to claim 1, wherein: the active oxygen scavenger is selected from pinacol phenylborate, tempol, resveratrol, salvianic acid, protocatechuic aldehyde, alpha-lipoic acid or curcumin.
5. A method for preparing the self-assembled nano anti-inflammatory drug according to any one of claims 1 to 4, comprising the steps of: firstly, dissolving an active oxygen scavenger, low molecular heparin, N-hydroxysuccinimide and N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide in preheated formamide, adding triethanolamine, stirring the mixture at room temperature under the protection of nitrogen for 12 hours to obtain a polymer solution, placing the polymer solution in a dialysis bag, dialyzing in deionized water for 24 hours to remove an organic solvent, and freeze-drying and collecting; and then dissolving the collected material in dimethyl sulfoxide, adding N-hydroxysuccinimide, N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide, triethanolamine and the anti-inflammatory drug containing carboxyl to react for 12 hours, dialyzing and freeze-drying to collect the self-assembled nano anti-inflammatory drug.
6. The method for preparing the self-assembled nano anti-inflammatory drug according to claim 5, which is characterized in that: the concentration of the low molecular heparin solution is between 5mg/mL and 90mg/mL, the concentration of the organic solution of the anti-inflammatory drug containing carboxyl is between 1mg/mL and 10mg/mL, and the concentration of the active oxygen scavenger solution is between 3mg/mL and 50 mg/mL.
7. Use of the self-assembled nano anti-inflammatory drug according to any one of claims 1 to 4 for the manufacture of a medicament for the prevention and treatment of premature labor.
8. The use according to claim 7, characterized in that: the nanometer anti-inflammatory medicine is used for intravenous injection.
9. The use according to claim 7, characterized in that: the nano anti-inflammatory drug is loaded on Poloxamer 407 to form composite hydrogel, and is locally delivered to the vagina for administration.
10. The use according to claim 7, characterized in that: the nano anti-inflammatory medicine is concentrated in the uterus and placenta.
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