ROS (reactive oxygen species) targeted modified antioxidant drug delivery system as well as preparation method and application thereof

Abstract

The invention discloses an antioxidant drug delivery system with ROS (reactive oxygen species) targeted modification. The antioxidant drug delivery system comprises nanoparticles with a core-shell structure and fat-soluble drugs loaded in the nanoparticles, wherein glutathione is used as a shell, polyethyleneimine-polycaprolactone is used as a core, and the fat-soluble drugs are loaded in the nanoparticles. Oxidized PEI-PCL is connected and modified by adopting GSH through a disulfide bond, a delivery system which is formed by self-assembly and has a core-shell structure with GSH as a functional anti-oxidation shell and (oxi) PEI-PCL as a drug loading core to load a fat-soluble drug can be used for realizing specific drug delivery of cells in a placenta in treatment of gestational diseases, and the delivery system has a wide application prospect. Non-specific drug absorption of a maternal body and a fetus is effectively avoided, a relatively exact drug distribution effect and an in-vivo treatment effect are achieved, toxic and side effects on the maternal body and the fetus are remarkably reduced, and drug treatment of gestational diseases is facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of chemistry, biomedical engineering technology, in particular to a ROS-targeted modified antioxidant drug delivery system and a preparation method and application thereof. BACKGROUND

[0002] Pregnancy diseases (such as preeclampsia, gestational diabetes, fetal growth restriction, etc.) caused by placental dysplasia or dysfunction have great damage to both pregnant women and fetuses, and even seriously endanger the lives of pregnant women and fetuses. Research has found that the root cause of such diseases is the impaired function of trophoblast cells (TB cells), and some drugs with TB cell function regulation have been developed for the treatment of such diseases. For example, the initial factor of gestational diabetes may be related to the inactivation of the Wnt / β-catenin pathway, insulin resistance related to the secretion of tumor necrosis factor, and the placental and systemic inflammatory response induced by interleukin 6 (IL-6). Using Wnt / β-catenin pathway activators such as WAY-262611 and IL-6 expression inhibiting siRNA to treat the above factors at the same time is expected to have a good therapeutic effect on gestational diabetes.

[0003] However, the use of drugs by pregnant women and the development of new drugs need to consider the distribution of drugs in both the mother and the fetus itself, as well as the toxicity problems of both. Most drugs can pass through the placenta and distribute into the fetus, affecting fetal development. Therefore, there are many contraindications for pregnant women to use drugs, including emergency drugs. Pregnant women are divided into five categories according to the teratogenicity of drugs. Except for a few drugs with minimal toxicity, which are classified as a and b, most of the remaining drugs are classified as c, d, and e, which have obvious damage to the fetus. Pregnant women have a heavy metabolic burden during pregnancy, and their immune changes are complex. Even if a drug has no obvious toxicity during non-pregnancy, it may have significant side effects on pregnant women. Currently, drugs that may have good effects on gestational diseases in vitro experiments have physiological and pathological obstacles in both the mother and the fetus when used in vivo, making it difficult to ensure the safety of the mother and the fetus.

[0004] Therefore, it is of great significance to provide a low-toxicity drug delivery system that can specifically deliver and functionally regulate drugs to cells in the placenta and effectively avoid non-specific drug absorption by the mother and the fetus for the treatment of gestational diseases. SUMMARY

[0005] To overcome the above deficiencies of the prior art, the ROS-targeted modified antioxidant drug delivery system of the present application can efficiently load liposoluble drugs, has a low surface potential, can avoid phagocytosis by the reticuloendothelial system, realize long circulation in the body, and can also be shed in response to an inflammatory microenvironment, realizing microenvironment-targeted drug release and microenvironment antioxidant therapy. The ROS-targeted modified antioxidant drug delivery system of the present application can be used to achieve specific drug delivery to cells in the placenta during the treatment of gestational diseases, effectively avoiding non-specific drug absorption by the mother and fetus, and has high safety. The ROS-targeted modified antioxidant drug delivery system of the present application can make drugs achieve passive targeting aggregation in lesions and microenvironment redox-sensitive distribution, while exhibiting antioxidant effects in the lesion microenvironment and intracellularly, realizing more accurate drug distribution and in vivo therapeutic effects, significantly reducing the toxic and side effects on the mother and fetus, and being conducive to the drug treatment of gestational diseases.

[0006] A further object of the present application is to provide a preparation method and application of the ROS-targeted modified antioxidant drug delivery system.

[0007] The ROS-targeted modified antioxidant drug delivery system of the present application is prepared by the following technical scheme:

[0008] The ROS-targeted modified antioxidant drug delivery system of the present application comprises nanoparticles having a core-shell structure with glutathione as the shell and polyethyleneimine-poly(caprolactone) as the core, and a liposoluble drug loaded in the nanoparticles.

[0009] The glutathione and the polyethyleneimine-poly(caprolactone) block copolymer are connected by a disulfide bond; and part of the units in the polyethyleneimine-poly(caprolactone) block copolymer are oxidized to units.

[0010] Preferably, the oxidation degree of the polyethyleneimine-poly(caprolactone) block copolymer is 30% to 70%, more preferably 40% to 65%. The oxidation degree of the polyethyleneimine-poly(caprolactone) block copolymer represents the percentage content of the number of oxidized units in the total number of units.

[0011] The application provides a method for testing the oxidation degree of polyethyleneimine-polycaprolactone block copolymer, comprising the following steps: using hydrogen nuclear magnetic spectrum to characterize the polyethyleneimine-polycaprolactone block copolymer before and after oxidation respectively, calculating the characteristic peak integral to obtain the PEI unit number and the non-oxidized PEI unit number in (oxi) PEI-PCL, wherein the characteristic peak of PCL near δ=4.05 ppm is integrated and normalized for calculating the unit number of PCL and further verifying the unit number of PEI through the integral ratio; for the PEI-PCL before and after oxidation, the characteristic peak of PEI near δ=2.6-2.8 ppm is taken out and the integral is calculated, so that the total PEI unit number in the PEI-PCL before oxidation and the non-oxidized PEI unit number in the (oxi) PEI-PCL after oxidation can be obtained. The oxidation degree is calculated according to the following formula.

[0012]

[0013] In the application, PEI-PCL represents non-oxidized polyethyleneimine-polycaprolactone block copolymer, and its chemical structure is as follows:

[0014] Wherein x=100-200, y=40-50.

[0015] In the application, (oxi) PEI-PCL represents polyethyleneimine-polycaprolactone block copolymer after oxidation.

[0016] In the application, the hydrogen nuclear magnetic spectrum 1 The H NMR is preferably analyzed by using a Varian Unity 400MHz nuclear magnetic resonance instrument, and the solvent is CDCl3.

[0017] Preferably, the particle size of the antioxidant drug delivery system is in the range of 80-120 nm.

[0018] Preferably, the surface potential of the antioxidant drug delivery system is 10-30 mV, and more preferably 12-25 mV.

[0019] The surface potential can be measured by using a Zeta potential analyzer, the temperature is set to 25°C, and the electric field strength is in the automatic mode (5-20 V / cm).

[0020] Preferably, the fat-soluble drug can be aspirin, nimesulide, vitamin E, vitamin C, etc.

[0021] Preferably, the ROS-targeted modified antioxidant drug delivery system of the present application further comprises a fluorescent marker loaded in the nanoparticles. The fluorescent marker can realize in vivo fluorescence imaging. The fluorescent marker is preferably a liposoluble fluorescent dye, such as long-chain carbon cyanine dye DIR, etc.

[0022] The present application also provides a preparation method of the ROS-targeted modified antioxidant drug delivery system, comprising the following steps:

[0023] S1, preparing an oxidized polyethyleneimine-poly-caprolactone block copolymer (oxi) PEI-PCL by oxidizing a polyethyleneimine-poly-caprolactone block copolymer with H2O2;

[0024] S2, modifying the (oxi) PEI-PCL with mercaptoacetic acid to obtain a thiolated oxidized polyethyleneimine-poly-caprolactone block copolymer SH-(oxi) PEI-PCL;

[0025] S3, forming a disulfide bond by oxidizing the thiol group of SH-(oxi) PEI-PCL with the thiol group of glutathione to prepare a glutathione-coupled modified polyethyleneimine-poly-caprolactone block copolymer GSH-(oxi) PEI-PCL;

[0026] S4, dissolving a liposoluble drug and the GSH-(oxi) PEI-PCL in an organic solvent to obtain an organic phase solution, dropping the organic phase solution into an aqueous surfactant solution drop by drop, and simultaneously performing ultrasonic dispersion stirring, obtaining an emulsion after the stirring is completed, removing the organic solvent from the emulsion by rotary evaporation, centrifuging, and washing to prepare the antioxidant drug delivery system.

[0027] Further, the step S1 comprises: under stirring conditions, adding a polyethyleneimine-poly-caprolactone block copolymer aqueous solution and an H2O2 aqueous solution into ultrapure water in a proportion to obtain a mixed solution, stirring and reacting, removing excess H2O2 by ultrafiltration after the reaction is completed, and freeze-drying to obtain the oxidized polyethyleneimine-poly-caprolactone block copolymer (oxi) PEI-PCL.

[0028] Preferably, the weight average molecular weight Mw of the polyethyleneimine-poly-caprolactone block copolymer is 10kDa-15kDa. W Preferably, the weight average molecular weight Mw of the polyethyleneimine-poly-caprolactone block copolymer is 10kDa-15kDa.

[0029] Preferably, the molar content of the polyethyleneimine-poly-caprolactone block copolymer in the mixed solution is 4-6mM, and the molar ratio of the polyethyleneimine-poly-caprolactone block copolymer to H2O2 is 1:5-10; the ultrafiltration step uses an ultrafiltration tube with a molecular weight cutoff of 3kDa.

[0030] Further, the step S2 comprises: adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into the aqueous mercaptoacetic acid solution, stirring, fully activating the carboxyl group of mercaptoacetic acid, then adding the (oxi) PEI-PCL prepared in step S1, stirring and reacting, after the reaction is completed, performing dialysis treatment, freeze-drying, to obtain the thiolated oxidized polyethyleneimine-poly-caprolactone block copolymer SH-(oxi) PEI-PCL.

[0031] Preferably, the aqueous mercaptoacetic acid solution has a molar content of mercaptoacetic acid of 10-15 mM; and the molar ratio of mercaptoacetic acid to (oxi) PEI-PCL is 1:1-1.2.

[0032] Preferably, in the step S2, the dialysis treatment step uses a dialysis bag with a molecular weight cut-off of 3.4 kDa.

[0033] Further, the step S3 comprises: adding the SH-(oxi) PEI-PCL prepared in step S2 and glutathione into pure water in a certain proportion, stirring and reacting, after the reaction is completed, removing the unreacted glutathione by dialysis treatment, freeze-drying, to obtain the polyethyleneimine-poly-caprolactone block copolymer GSH-(oxi) PEI-PCL modified by glutathione coupling.

[0034] Preferably, the glutathione is reduced glutathione.

[0035] Preferably, the ratio of the amount of SH-(oxi) PEI-PCL to pure water is 0.1 mmol: 10-20 mL; and the molar ratio of SH-(oxi) PEI-PCL to glutathione is 1:8-15.

[0036] Preferably, in the step S3, the dialysis treatment step uses a dialysis bag with a molecular weight cut-off of 0.5 kDa.

[0037] Preferably, in the step S4, the organic solvent is selected from ethanol; the molar concentration of GSH-(oxi) PEI-PCL in the organic phase solution is 0.2-0.5 M; and the molar ratio of GSH-(oxi) PEI-PCL to the fat-soluble drug is 10-20:1.

[0038] Preferably, in the step S4, the concentration of the surfactant aqueous solution is 0.1-0.5% w / v; and the surfactant can be Tween 80.

[0039] Preferably, in the step S4, the ultrasonic dispersion step has an ultrasonic power of 60-75 W and an ultrasonic time of 5-10 min.

[0040] Further, in the preparation of the antioxidant drug delivery system loaded with fluorescent markers in the nanoparticles, the fluorescent markers are dissolved in the organic solvent together with the GSH-(oxi)PEI-PCL and the fat-soluble drug to obtain an organic phase solution.

[0041] The application also provides the ROS-targeted modified antioxidant drug delivery system in the preparation of a pharmaceutical preparation for treating gestational diseases.

[0042] The application provides a preparation method of a pharmaceutical preparation for treating gestational diseases: the ROS-targeted modified antioxidant drug delivery system is added into PBS of a suitable volume to be resuspended to obtain a drug solution of a suitable concentration for treatment.

[0043] The application has the following beneficial effects:

[0044] The ROS-targeted modified antioxidant drug delivery system of the application uses the oxidized polyethyleneimine-poly-caprolactone block copolymer, which can effectively reduce the toxicity in the body and reduce the surface potential, and the glutathione is modified by a disulfide bond, which can further reduce the surface potential of the drug, avoid the phagocytosis of the reticuloendothelial system, realize the long circulation in the body, and can be shed in the inflammatory microenvironment with obvious oxidative stress, realize the microenvironment targeted drug release, realize the microenvironment antioxidant therapy, can be used for specific drug delivery to the placental cells in the treatment of gestational diseases, effectively avoid the non-specific drug absorption of the mother and the fetus, and has high safety.

[0045] The ROS-targeted modified antioxidant drug delivery system of the application has a core-shell structure with glutathione as a functional antioxidant shell and oxidized polyethyleneimine-poly-caprolactone as a drug loading inner core, can efficiently load fat-soluble drugs, has a particle size range of 80-120 nm, is suitable for passive targeting retention and distribution by using the pores of placental endothelial cells. After endocytosis by cells, the residual GSH in the shell can play an antioxidant role in the cells, and the drug delivery system structure of the (oxi)PEI-PCL inner core combined with the antioxidant GSH shell can make the drug realize passive targeted aggregation in the lesion and microenvironment redox sensitive distribution, and at the same time, the antioxidant effect in the lesion microenvironment and the cells is realized, the drug distribution effect and the in-vivo treatment effect are more accurate, the toxic and side effects on the mother and the fetus are significantly reduced, and the drug treatment of gestational diseases is facilitated. DETAILED DESCRIPTION

[0046] To describe the technical content of the present application, the purposes and effects achieved, the technical solutions of the present application are described clearly and completely in combination with embodiments below, but the described embodiments are only some embodiments of the present application, and the implementation and protection of the present application are not limited thereto. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor belong to the scope of protection of the present application. It should be noted that if the processes are not specifically described below, they can be implemented or understood by those skilled in the art according to the prior art. If the reagents or instruments used are not marked with the manufacturer, they are considered to be conventional products that can be purchased on the market.

[0047] The raw material sources used in the embodiments of the present application are as follows:

[0048] The raw material sources of the present application are as follows

[0049]

[0050] Example 1

[0051] A ROS-targeted modified antioxidant drug delivery system and a preparation method thereof are as follows:

[0052] S1, synthesis of (oxi)PEI-PCL

[0053] Under the condition of vigorous stirring, 1 mL of PEI-PCL aqueous solution (10 kDa, 10 mM, pH = 9), 40 μL of freshly prepared H2O2 aqueous solution (2 M) were added to 960 μL of ultrapure water to obtain a mixed solution, and the reaction was stirred at 35°C and 1300 rpm for 60 min. After the reaction was completed, the excess H2O2 in the solution was removed by using an ultrafiltration tube with a molecular weight cut-off of 3 kDa, and freeze-drying was performed to obtain a light yellow solid (oxi)PEI-PCL;

[0054] S2, synthesis of SH-(oxi)PEI-PCL

[0055] 9.2 mg of mercaptoacetic acid was dissolved in 10 mL of ultrapure water to obtain a mercaptoacetic acid aqueous solution (10 mM), and an excess of EDC (44.5 mg, 15 mM) and NHS (17 mg, 15 mM) were added. After stirring for 2 h to fully activate the carboxyl group of mercaptoacetic acid, 1 g of (oxi)PEI-PCL (10 mM) was added, and the reaction was stirred at 35°C for 24 h. After the reaction was completed, the dialysis treatment was performed for two days by using a dialysis bag with a molecular weight cut-off of 3.4 kDa, and freeze-drying was performed to obtain SH-(oxi)PEI-PCL, which was stored at 4°C for standby;

[0056] S3, synthesis of GSH-(oxi)PEI-PCL

[0057] GSH-(oxi)PEI-PCL (0.15 g, 1 mmol), DIR (50 mg, 50 μmol) and aspirin (9 mg, 50 μmol) were dissolved in 5 mL ethanol to get organic phase solution, the organic phase solution was added dropwise into 0.1% w / v Tween 80 aqueous solution, and was dispersed and stirred under ultrasonic wave with 60 W power for 5 min. After stirring, an emulsion was obtained, and the emulsion was placed in a rotary evaporator, and the organic solvent was evaporated under reduced pressure until the organic solvent was completely removed to obtain a drug-loaded nanoparticle suspension. The drug-loaded nanoparticles were collected by centrifugation at 10,000 rpm for 15 min, and the nanoparticles were washed with ultrapure water to remove unencapsulated drugs and surfactants, to prepare a ROS-targeted antioxidant drug delivery system. A suitable volume of PBS was added to resuspend to obtain a drug solution with a suitable concentration for treatment.

[0058] S4, Preparation of ROS-targeted antioxidant drug delivery system

[0059] GSH-(oxi)PEI-PCL (0.15 g, 1 mmol), DIR (50 mg, 50 μmol) and aspirin (9 mg, 50 μmol) were dissolved in 5 mL ethanol to get organic phase solution, the organic phase solution was added dropwise into 0.1% w / v Tween 80 aqueous solution, and was dispersed and stirred under ultrasonic wave with 60 W power for 5 min. After stirring, an emulsion was obtained, and the emulsion was placed in a rotary evaporator, and the organic solvent was evaporated under reduced pressure until the organic solvent was completely removed to obtain a drug-loaded nanoparticle suspension. The drug-loaded nanoparticles were collected by centrifugation at 10,000 rpm for 15 min, and the nanoparticles were washed with ultrapure water to remove unencapsulated drugs and surfactants, to prepare a ROS-targeted antioxidant drug delivery system. A suitable volume of PBS was added to resuspend to obtain a drug solution with a suitable concentration for treatment.

[0060] Example 2

[0061] A ROS-targeted antioxidant drug delivery system was prepared by the following method:

[0062] S1, Synthesis of (oxi)PEI-PCL

[0063] Under vigorous stirring, 1 mL of PEI-PCL aqueous solution (10 kDa, 10 mM, pH = 9), 40 μL of freshly prepared H2O2 aqueous solution (2 M) were added to 960 μL of ultrapure water to obtain a mixed solution, which was stirred at 35°C and 1300 rpm for 30 min. After the reaction was completed, the excess H2O2 in the solution was removed by ultrafiltration tube with a molecular weight cut-off of 3 kDa, and freeze-drying was performed to obtain a light yellow solid (oxi)PEI-PCL.

[0064] S2 and S3 steps were the same as in Example 1 to prepare GSH-(oxi)PEI-PCL.

[0065] S4, Preparation of ROS-targeted antioxidant drug delivery system

[0066] The GSH-(oxi)PEI-PCL (0.15 g, 1 mmol), DIR (50 mg, 50 μmol) and aspirin (9 mg, 50 μmol) were dissolved in 5 mL of ethanol to obtain an organic phase solution, and the organic phase solution was added dropwise into a 0.1% w / v Tween 80 aqueous solution while being stirred and ultrasonically dispersed at a power of 75 W for 5 min. After stirring, an emulsion was obtained, and the emulsion was placed in a rotary evaporator, and the organic solvent was evaporated under reduced pressure until the organic solvent was completely removed, to obtain a drug-loaded nanoparticle suspension. The drug-loaded nanoparticles were then collected by centrifugation at 10,000 rpm for 15 min, and the nanoparticles were washed with ultrapure water to remove unencapsulated drugs and surfactants, to prepare a ROS-targeted antioxidant drug delivery system. A suitable volume of PBS was added to resuspend the nanoparticles to obtain a drug solution of a suitable concentration for treatment.

[0067] Example 3

[0068] A ROS-targeted antioxidant drug delivery system was prepared as follows:

[0069] S1, Synthesis of (oxi)PEI-PCL

[0070] Under vigorous stirring, 1 mL of a PEI-PCL aqueous solution (10 kDa, 10 mM, pH = 9), 40 μL of freshly prepared H2O2 aqueous solution (2 M) were added to 960 μL of ultrapure water to obtain a mixed solution, which was stirred at 35°C and 1300 rpm for 75 min. After the reaction, the excess H2O2 in the solution was removed using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, and the (oxi)PEI-PCL was obtained as a light yellow solid after freeze-drying;

[0071] S2 and S3 were the same as in Example 1, and GSH-(oxi)PEI-PCL was prepared.

[0072] S4, Preparation of a ROS-targeted antioxidant drug delivery system

[0073] GSH-(oxi)PEI-PCL (0.15 g, 1 mmol), DIR (50 mg, 50 μmol) and nimesulide (18 mg, 50 μmol) were dissolved in 5 mL ethanol to form an organic phase solution, which was added dropwise into 0.1% w / v Tween 80 aqueous solution under ultrasonic dispersion stirring at a power of 60 W for 5 min. After stirring, an emulsion was obtained, which was placed in a rotary evaporator to evaporate the organic solvent under reduced pressure until the organic solvent was completely removed, to obtain a drug-loaded nanoparticle suspension, which was then collected by centrifugation at 10,000 rpm for 15 min, and the nanoparticles were washed with ultrapure water to remove unencapsulated drugs and surfactants, to prepare drug-loaded nanoparticle drug particles. A suitable volume of PBS was added to resuspend the nanoparticles to obtain a drug solution of a suitable concentration for treatment.

[0074] Example 4

[0075] A ROS-targeted modified antioxidant drug delivery system was prepared as follows:

[0076] S1, synthesis of (oxi)PEI-PCL

[0077] Under vigorous stirring, 1 mL of PEI-PCL aqueous solution (15 kDa, 10 mM, pH = 9), 40 μL of freshly prepared H2O2 aqueous solution (2 M) were added to 960 μL of ultrapure water to form a mixed solution, which was stirred at 35°C and 1300 rpm for 60 min. After the reaction, excess H2O2 was removed by ultrafiltration with an ultrafiltration tube with a molecular weight cut-off of 3 kDa, and the product was freeze-dried to obtain a light yellow solid (oxi)PEI-PCL;

[0078] S2, synthesis of SH-(oxi)PEI-PCL

[0079] 9.2 mg of mercaptoacetic acid was weighed into 10 mL of ultrapure water to form a mercaptoacetic acid aqueous solution (10 mM), and an excess of EDC (44.5 mg, 15 mM) and NHS (17 mg, 15 mM) were added, and stirred for 2 h to activate the carboxyl group of mercaptoacetic acid. Then, 1.5 g of (oxi)PEI-PCL (10 mM) was added, and the mixture was stirred at 35°C for 24 h. After the reaction, the product was dialyzed for two days using a dialysis bag with a molecular weight cut-off of 3.4 kDa, and freeze-dried to obtain SH-(oxi)PEI-PCL, which was stored at 4°C for later use;

[0080] S3, synthesis of GSH-(oxi)PEI-PCL

[0081] GSH-(oxi)PEI-PCL (0.2 g, 1 mmol), DIR (50 mg, 50 μmol) and aspirin (9 mg, 50 μmol) were dissolved in 5 mL ethanol to get organic phase solution, the organic phase solution was added dropwise into 0.1% w / v Tween 80 aqueous solution, and ultrasonic dispersion stirring was carried out at 60 W for 3 min. After stirring, an emulsion was obtained, and the emulsion was placed in a rotary evaporator, and the organic solvent was evaporated under reduced pressure until the organic solvent was completely removed, to obtain a drug-loaded nanoparticle suspension, which was then centrifuged at 10,000 rpm for 15 min to collect the drug-loaded nanoparticles. The nanoparticles were washed with ultrapure water to remove unencapsulated drugs and surfactants, to obtain drug-loaded nanoparticle drug particles. A suitable volume of PBS was added to resuspend the nanoparticles to obtain a drug solution of a suitable concentration for treatment.

[0082] S4, Preparation of ROS-targeted modified antioxidant drug delivery system

[0083] GSH-(oxi)PEI-PCL (0.2 g, 1 mmol), DIR (50 mg, 50 μmol) and aspirin (9 mg, 50 μmol) were dissolved in 5 mL ethanol to get organic phase solution, the organic phase solution was added dropwise into 0.1% w / v Tween 80 aqueous solution, and ultrasonic dispersion stirring was carried out at 60 W for 3 min. After stirring, an emulsion was obtained, and the emulsion was placed in a rotary evaporator, and the organic solvent was evaporated under reduced pressure until the organic solvent was completely removed, to obtain a drug-loaded nanoparticle suspension, which was then centrifuged at 10,000 rpm for 15 min to collect the drug-loaded nanoparticles. The nanoparticles were washed with ultrapure water to remove unencapsulated drugs and surfactants, to obtain drug-loaded nanoparticle drug particles. A suitable volume of PBS was added to resuspend the nanoparticles to obtain a drug solution of a suitable concentration for treatment.

[0084] Comparative Example 1

[0085] A drug delivery system was prepared as follows:

[0086] S1, under vigorous stirring, 1 mL of PEI-PCL aqueous solution (10 kDa, 10 mM, pH = 9), 40 μL of freshly prepared H2O2 aqueous solution (2 M) were added to 960 μL of ultrapure water to obtain a mixed solution, which was stirred at 35°C and 1300 rpm for 60 min. After the reaction, the excess H2O2 in the solution was removed by using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, and freeze-drying was performed to obtain a light yellow solid (oxi)PEI-PCL;

[0087] S2, dissolve (oxi)PEI-PCL (0.1 g, 1 mmol), DIR (50 mg, 50 μmol) and aspirin (9 mg, 50 μmol) in 5 mL ethanol to obtain an organic phase solution, drop the organic phase solution into 0.1% w / v Tween 80 aqueous solution, and stir and disperse ultrasonically at a power of 60 W for 5 min. After stirring, an emulsion is obtained, which is placed in a rotary evaporator, and the organic solvent is evaporated under reduced pressure until the organic solvent is completely removed, to obtain a drug-loaded nanoparticle suspension, which is then collected by centrifugation at 10,000 rpm for 15 min, and the nanoparticles are washed with ultrapure water to remove unencapsulated drugs and surfactants, to prepare a drug delivery system. Add a suitable volume of PBS to resuspend to obtain a drug solution of a suitable concentration for treatment.

[0088] Comparative Example 2

[0089] A drug delivery system is prepared by the following method:

[0090] S1, weigh 9.2 mg of mercaptoacetic acid into 10 mL ultrapure water to obtain a mercaptoacetic acid aqueous solution (10 mM), add excess EDC (44.5 mg, 15 mM) and NHS (17 mg, 15 mM), stir for 2 h to fully activate the carboxyl group of mercaptoacetic acid, then add 1 g of PEI-PCL (10 mM), stir at 35°C for 24 h, after the reaction is completed, dialyze with a dialysis bag with a molecular weight cut-off of 3.4 kDa for two days, freeze-dry to obtain SH-PEI-PCL, store at 4°C, and use as needed;

[0091] S2, weigh SH-PEI-PCL (1 g, 0.1 mmol) and glutathione (0.307 g, 1 mmol) into 20 mL pure water, and react at 23°C for 18 h. After the reaction is completed, dialyze with a dialysis bag with a molecular weight cut-off of 0.5 kDa in pure water for one day to remove unreacted GSH, and freeze-dry to obtain GSH-PEI-PCL.

[0092] S3, GSH-PEI-PCL (0.15 g, 1 mmol), DIR (50 mg, 50 μmol) and aspirin (9 mg, 50 μmol) were dissolved in 5 mL ethanol to get organic phase solution, the organic phase solution was added dropwise into 0.1% w / v Tween 80 aqueous solution, and dispersed by ultrasonic stirring at a power of 60 W for 5 min. After stirring, an emulsion was obtained, and the emulsion was placed in a rotary evaporator, and the organic solvent was evaporated under reduced pressure until the organic solvent was completely removed, to obtain a drug-loaded nanoparticle suspension, which was then collected by centrifugation at 10,000 rpm for 15 min, and the nanoparticles were washed with ultrapure water to remove unencapsulated drugs and surfactants, to prepare a drug delivery system. The appropriate volume of PBS was added to resuspend to obtain a drug solution of a suitable concentration for treatment.

[0093] Comparative Example 3

[0094] A drug delivery system was prepared by the following method:

[0095] GSH-(oxi)PEI-PCL was prepared according to Example 1.

[0096] GSH-PEI-PCL (0.15 g, 1 mmol), DIR (50 mg, 50 μmol) and aspirin (9 mg, 50 μmol) were dissolved in 5 mL ethanol to get organic phase solution, the organic phase solution was added dropwise into 0.1% w / v Tween 80 aqueous solution, and dispersed by ultrasonic stirring at a power of 75 W for 10 min. After stirring, an emulsion was obtained, and the emulsion was placed in a rotary evaporator, and the organic solvent was evaporated under reduced pressure until the organic solvent was completely removed, to obtain a drug-loaded nanoparticle suspension, which was then collected by centrifugation at 10,000 rpm for 15 min, and the nanoparticles were washed with ultrapure water to remove unencapsulated drugs and surfactants, to prepare a drug delivery system. The appropriate volume of PBS was added to resuspend to obtain a drug solution of a suitable concentration for treatment.

[0097] Comparative Example 4

[0098] A drug delivery system was prepared by the following method:

[0099] GSH-(oxi)PEI-PCL was prepared according to Example 4.

[0100] GSH-(oxi)PEI-PCL (0.2 g, 1 mmol), DIR (50 mg, 50 μmol) and aspirin (9 mg, 50 μmol) were dissolved in 5 mL ethanol to get organic phase solution, the organic phase solution was added dropwise into 0.1% w / v Tween 80 aqueous solution, and dispersed by ultrasonic stirring at a power of 45 W for 3 min. After stirring, an emulsion was obtained, and the emulsion was placed in a rotary evaporator to evaporate the organic solvent under reduced pressure until the organic solvent was completely removed, to obtain a drug-loaded nanoparticle suspension, which was then collected by centrifugation at 10,000 rpm for 15 min, and the nanoparticles were washed with ultrapure water to remove unencapsulated drugs and surfactants, to prepare a drug delivery system. The appropriate volume of PBS was added to resuspend to obtain a suitable concentration of drug solution for treatment.

[0101] Comparative Example 5

[0102] A drug delivery system was prepared by the following method:

[0103] S1, synthesis of (oxi)PEI-PCL

[0104] Under vigorous stirring, 1 mL of PEI-PCL aqueous solution (10 kDa, 10 mM, pH = 9), 40 μL of freshly prepared H2O2 aqueous solution (2 M) were added to 960 μL of ultrapure water to obtain a mixed solution, which was stirred at 35°C and 1300 rpm for 15 min. After the reaction, the excess H2O2 in the solution was removed by ultrafiltration tube with a molecular weight cut-off of 3 kDa, and freeze-dried to obtain a light yellow solid (oxi)PEI-PCL;

[0105] S2 and S3 steps were the same as in Example 1, to obtain GSH-(oxi)PEI-PCL;

[0106] S4, GSH-(oxi)PEI-PCL (0.15 g, 1 mmol), DIR (50 mg, 50 μmol) and aspirin (9 mg, 50 μmol) were dissolved in 5 mL ethanol to obtain an organic phase solution, which was added dropwise into 0.1% w / v Tween 80 aqueous solution, and dispersed by ultrasonic stirring at a power of 60 W for 5 min. After stirring, an emulsion was obtained, and the emulsion was placed in a rotary evaporator to evaporate the organic solvent under reduced pressure until the organic solvent was completely removed, to obtain a drug-loaded nanoparticle suspension, which was then collected by centrifugation at 10,000 rpm for 15 min, and the nanoparticles were washed with ultrapure water to remove unencapsulated drugs and surfactants, to prepare a drug delivery system. The appropriate volume of PBS was added to resuspend to obtain a suitable concentration of drug solution for treatment.

[0107] Comparative Example 6

[0108] A drug delivery system is prepared by the following method:

[0109] S1, synthesis of (oxi)PEI-PCL

[0110] Under vigorous stirring, 1 mL of PEI-PCL aqueous solution (10 kDa, 10 mM, pH = 9), 40 μL of freshly prepared H2O2 aqueous solution (2 M) were added into 960 μL of ultrapure water to obtain a mixed solution, which was stirred at 35 °C and 1300 rpm for 90 min. After the reaction, the excess H2O2 in the solution was removed by ultrafiltration tube with a molecular weight cut-off of 3 kDa, and then freeze-dried to obtain a light yellow solid (oxi)PEI-PCL;

[0111] S2 and S3 steps are the same as in Example 1 to obtain GSH-(oxi)PEI-PCL;

[0112] S4, GSH-(oxi)PEI-PCL (0.15 g, 1 mmol), DIR (50 mg, 50 μmol) and aspirin (9 mg, 50 μmol) were dissolved in 5 mL of ethanol to obtain an organic phase solution, which was added dropwise into an aqueous solution of 0.1% w / v Tween 80, and simultaneously ultrasonically dispersed and stirred for 5 min at a power of 60 W. After stirring, an emulsion was obtained, which was placed in a rotary evaporator and evaporated under reduced pressure until the organic solvent was completely removed to obtain a drug-loaded nanoparticle suspension. The drug-loaded nanoparticles were then collected by centrifugation at 10000 rpm for 15 min, and the nanoparticles were washed with ultrapure water to remove unencapsulated drugs and surfactants, thereby preparing a drug delivery system. The appropriate volume of PBS was added to resuspend the nanoparticles to obtain a drug solution of appropriate concentration for treatment.

[0113] Related tests of the examples and comparative examples:

[0114] I. Characterization of ROS-targeted antioxidant drug delivery system

[0115] Potential test method: Zeta potential analyzer (Brookhaven ZetaPALS) was used, the temperature was set to 25 °C, the electric field intensity was in automatic mode (5-20 V / cm) and at least 3 repeated values were averaged. The results are shown in Table 1.

[0116] Particle size test method: the average particle size of the sample was tested by Zeta-Plus potential particle size instrument (Brooken Haven), the incident laser wavelength λ = 532 nm, the incident angle θ = 90°, and the temperature was 25 °C; the average value of three measurement values was taken.

[0117] NMR test method: 1HNMR was analyzed by Varian Unity 400MHz nuclear magnetic resonance instrument, and the solvent was CDCl3.

[0118] Particular note: The polyethyleneimine-poly-caprolactone block copolymer before and after oxidation was characterized by hydrogen nuclear magnetic spectrum, and the number of PEI units and the number of unoxidized PEI units in (oxi) PEI-PCL were calculated by integrating the characteristic peaks. The characteristic peak of PCL at δ = 4.05 ppm was integrated and normalized for calculating the number of PCL units and further verifying the number of PEI units by integral ratio. For PEI-PCL before and after oxidation, the characteristic peak of PEI was taken from the nuclear magnetic hydrogen spectrum near δ = 2.6-2.8 ppm, and the integral was calculated to obtain the total number of PEI units in PEI-PCL before oxidation and the number of unoxidized PEI units in (oxi) PEI-PCL after oxidation. The degree of oxidation of (oxi) PEI-PCL was calculated according to the following formula.

[0119]

[0120] The above results are shown in Table 1.

[0121] Table 1:

[0122]

[0123] II. Near-infrared in vivo fluorescence (NIRF) molecular imaging experiment to evaluate the placenta-specific delivery function of the drug

[0124] Model establishment:

[0125] 8-week-old SPF C57BL / 6 mice (purchased from Guangdong Medical Laboratory Animal Center) were mated in a cage at a ratio of 2:1 between female and male mice during the estrus period. The next day, the vaginal secretion smear of the female mouse was stained with Papnikolaou, and the specimen was observed under an optical microscope. The specimen with positive vaginal sperm was diagnosed as pregnant, and was marked as the 0th day of pregnancy (D0). Pregnant mice fed with 2 mg / mL nitroso-L-arginine methyl ester in sterile water were used to establish a preeclampsia / pregnancy-induced hypertension (PE) model, and pregnant mice fed with an equal amount of double-distilled water were used as normal control group.

[0126] 1. Evaluation of placenta-specific delivery function

[0127] To evaluate the efficiency of the injected drug in distributing in solid organs such as placenta, embryo, and liver, the preeclampsia model animals were injected with fluorescently labeled drugs (0.5 mg PCL equivalent / drug per injection) 2 hours after anesthesia with chloral hydrate. The organs were then separated, and the targeting distribution efficiency in each organ was evaluated using an in vivo fluorescence imaging instrument. The results are shown in Table 2.

[0128] Table 2 Results of placenta-specific delivery function evaluation

[0129]

[0130] From the above results, it can be seen that:

[0131] The drug delivery system of Comparative Example 1 lacks a GSH shell, has a high surface potential, has high cytotoxicity, and after destroying the placental barrier cells in the lesion, it is distributed in large quantities to the fetus; and during systemic blood distribution, it stimulates the non-specific phagocytosis of liver macrophages and other systemic reticuloendothelial systems. The total placental fluorescence intensity is significantly reduced, the total embryonic fluorescence intensity is significantly increased, and the total liver fluorescence intensity is significantly increased compared with the embodiment.

[0132] The drug delivery system of Comparative Example 2 has a PEI-PCL core in the carrier that is not oxidized, has a high surface potential, has high cytotoxicity, and after destroying the placental barrier cells in the lesion, it is distributed in large quantities to the fetus; and during systemic blood distribution, it stimulates the non-specific phagocytosis of liver macrophages and other systemic reticuloendothelial systems. The total placental fluorescence intensity is significantly reduced, the total embryonic fluorescence intensity is significantly increased, and the total liver fluorescence intensity is significantly increased compared with the embodiment.

[0133] The particle size of the drug delivery system of Comparative Example 3 is too small, and it can almost freely enter and exit the pores of the placental lesion endothelial cells; it is difficult to produce non-specific aggregation due to the EPR effect after entering the lesion, and there is less aggregation in the lesion, and thus there is less distribution to the fetus, and the drug is aggregated in organs rich in reticuloendothelial systems such as the liver in the body. The total placental fluorescence intensity is significantly reduced, the total embryonic fluorescence intensity is significantly reduced, and the total liver fluorescence intensity is significantly increased compared with the embodiment.

[0134] The particle size of the drug delivery system of Comparative Example 4 is too large, and the efficiency of entering the placental lesion is low, it is difficult to produce non-specific aggregation due to the EPR effect, there is less aggregation in the lesion, and thus there is less distribution to the fetus, and the drug is aggregated in organs rich in reticuloendothelial systems such as the liver in the body, and its large particle size easily induces endocytosis by liver macrophages. The total placental fluorescence intensity is reduced, the total embryonic fluorescence intensity is reduced, and the total liver fluorescence intensity is significantly increased compared with the embodiment.

[0135] The drug delivery system of Comparative Example 5 has a PEI-PCL core in the carrier that is not oxidized, has a high surface potential, has high cytotoxicity, and after destroying the placental barrier cells in the lesion, it is distributed in large quantities to the fetus; and during systemic blood distribution, it stimulates the non-specific phagocytosis of liver macrophages and other systemic reticuloendothelial systems. The total placental fluorescence intensity is significantly reduced, the total embryonic fluorescence intensity is significantly increased, and the total liver fluorescence intensity is significantly increased compared with the embodiment.

[0136] The drug delivery system of Comparative Example 6 has a higher degree of oxidation treatment of the carrier core PEI-PCL, a low surface potential, a low cytotoxicity, a smaller damage to placental barrier cells, a smaller distribution to fetuses, and a smaller stimulation to liver macrophages and other reticuloendothelial systems in the whole body blood distribution process, and a lower non-specific phagocytosis. Compared with the examples, the total fluorescence intensity of the placenta is obviously increased, the total fluorescence intensity of the embryo is not obviously increased, and the total fluorescence intensity of the liver is obviously decreased.

[0137] The ROS-targeted modified antioxidant drug delivery system of Examples 1-4 uses (oxi)PEI-PCL as the carrier core, which can reduce the surface positive potential and is beneficial to the implementation of low-toxicity drug delivery and treatment while reducing the in-vivo toxicity. Further, the GSH modification on the surface of the carrier can further reduce the surface potential of the drug, avoid phagocytosis by the reticuloendothelial system, and achieve long circulation in the body. The drug delivery system with a particle size range of 80-120 nm is suitable for passive targeting retention and distribution by using the pores of placental endothelial cells. After endocytosis by cells, the residual GSH of the outer shell can play an antioxidant role in the cells. The drug delivery system structure of the combination of the (oxi)PEI-PCL core and the antioxidant GSH outer shell can enable the drug to achieve passive targeting aggregation and microenvironment redox-sensitive distribution in the lesion, and achieve a more accurate drug targeting distribution effect.

[0138] 2. Evaluation of treatment effect on the preeclampsia animal model

[0139] On D3, D6, D9, D12, and D15, the drug was injected (the treatment dose was 0.5 mg PCL equivalent / time of drug), and on D17, a series of tests were performed, and the test results are shown in Table 3:

[0140] Blood pressure detection: The BP-2000 blood pressure analysis system was used to non-invasively measure the systolic blood pressure (SBP) of the pregnant mice by tail cuff method, the room temperature was kept at 26 degrees Celsius, channel 1 was set to 1V (1V was equivalent to 300 mmHg), and channel 2 was set to SmV. The mice were fixed in the mouse cage, the tail was placed in the 17 mm tail cuff, and the bottom of the tail was in the middle of the sensor. After the pregnant mice were in a calm state, 10 continuous pressure measurements were performed, each with an interval of 1 s, and the average value was recorded.

[0141] Fetal examination: The pregnant mice were anesthetized, the abdominal cavity was opened, the uterus was cut open, the fetuses were taken out in turn, the fetuses were cut off at the umbilical cord root, the fetuses were placed on sterile gauze to absorb the surface amniotic fluid, and the fetuses were weighed on an analytical balance.

[0142] Table 3 Evaluation of treatment effect on the preeclampsia animal model

[0143] Blood pressure (mmHg) Fetal weight (g) Normal rat untreated group 102.3 1.4 PE rat untreated group 163.5 0.6 Example 1 108.3 1.3 Example 2 115.2 1.2 Example 3 109.7 1.3 Example 4 109.1 1.2 Comparative Example 1 163.1 0.7 Comparative Example 2 178.4 0.5 Comparative Example 3 142.7 0.9 Comparative Example 4 135.2 1.0 Comparative Example 5 152.3 0.8 Comparative Example 6 143.9 0.9

[0144] From the above results, it can be seen that:

[0145] The drug delivery system of Comparative Example 1 lacks a GSH shell, has a high surface potential, has high cytotoxicity, and is distributed in large amounts to the fetus after destroying the placental barrier cells in the lesion; and in the process of systemic blood distribution, stimulates the non-specific phagocytosis of liver macrophages and other systemic reticuloendothelial systems; and the lack of a GSH shell leads to a lack of correspondence with the oxidative stress inflammatory lesion microenvironment, insufficient lesion targeting distribution efficiency, and insufficient therapeutic effect in the lesion; and the lack of a shell GSH can produce an antioxidant effect in the cell after the drug is phagocytosed into the cell, and the therapeutic effect is correspondingly reduced. Compared with the example, the blood pressure is significantly increased, the weight of the fetal pups is significantly decreased, the overall growth trend is far from that of normal mice, and tends to the untreated PE mouse group.

[0146] The drug delivery system of Comparative Example 2 has a non-oxidized PEI-PCL in the core of the carrier, has a high surface potential, has high cytotoxicity, and is distributed in large amounts to the fetus after destroying the placental barrier cells in the lesion; and in the process of systemic blood distribution, stimulates the non-specific phagocytosis of liver macrophages and other systemic reticuloendothelial systems; and the non-oxidized PEI-PCL produces high toxicity in the cell after the drug is phagocytosed into the cell, antagonizes the therapeutic effect of the therapeutic group, the therapeutic effect is correspondingly reduced, and even further killing is produced due to the toxicity. Compared with the example, the blood pressure is significantly increased, the weight of the fetal pups is significantly decreased, the overall growth trend is far from that of normal mice, and tends to the untreated PE mouse group, and is even more deteriorated.

[0147] The particle size of the drug delivery system of Comparative Example 3 is too small, and can almost freely enter and exit the placental lesion endothelial cell pores; it is difficult to produce non-specific aggregation due to the EPR effect after entering the lesion, and is less aggregated in the lesion, and further less distributed to the fetus, and the drug is aggregated in the liver and other organs rich in reticuloendothelial systems in the body; at the same time, the small size leads to a sharp decrease in drug loading efficiency, and the therapeutic effect is poor. Compared with the example, the blood pressure is significantly increased, the weight of the fetal pups is significantly decreased, the overall growth trend is far from that of normal mice, and tends to the untreated PE mouse group.

[0148] The particle size of the drug delivery system of Comparative Example 4 is too large, has low efficiency in entering the placental lesion, is difficult to produce non-specific aggregation due to the EPR effect, is less aggregated in the lesion, and further less distributed to the fetus, and the drug is aggregated in the liver and other organs rich in reticuloendothelial systems in the body, and the large particle size easily induces endocytosis of liver macrophages; at the same time, the large size leads to low drug loading stability in the body, and the therapeutic effect is poor. Compared with the example, the blood pressure is significantly increased, the weight of the fetal pups is significantly decreased, the overall growth trend is far from that of normal mice, and tends to the untreated PE mouse group.

[0149] The drug delivery system of Comparative Example 5 has a lower degree of oxidation treatment of the inner core of the carrier PEI-PCL, a higher surface potential, a large cytotoxicity, a large distribution to the fetus after destroying the placental barrier cells in the lesion, and a high potential to stimulate the liver macrophages and other reticuloendothelial system in the whole body during the whole blood distribution. Compared with the embodiment, the placental drug distribution is obviously reduced, and the embryo drug distribution is obviously increased. Compared with the embodiment, the blood pressure is obviously increased, the weight of the fetus is obviously decreased, the overall growth trend is far away from the normal mouse, and tends to the untreated PE mouse.

[0150] The drug delivery system of Comparative Example 6 has a higher degree of oxidation treatment of the inner core of the carrier PEI-PCL, a low surface potential, a low cytotoxicity, a small destruction to the placental barrier cells, and a small distribution to the fetus. However, the low potential leads to a significant reduction in drug loading, and a significant reduction in drug concentration at the target delivery site. During the whole blood distribution, the stimulation to the liver macrophages and other reticuloendothelial system is small, and the non-specific phagocytosis is low. Compared with the embodiment, the placental drug distribution efficiency is slightly higher, and the embryo drug distribution is not obviously increased. Compared with the embodiment, the blood pressure is obviously increased, the weight of the fetus is obviously decreased, the overall growth trend is far away from the normal mouse, and tends to the untreated PE mouse.

[0151] The ROS-targeted modified antioxidant drug delivery system of Examples 1-4 uses (oxi)PEI-PCL as the inner core of the carrier, which can reduce the toxicity in the body, reduce the high surface positive potential, and be beneficial to the implementation of low-toxicity drug delivery and treatment. Further, the GSH modification on the surface of the carrier can further reduce the surface potential of the drug, avoid phagocytosis by the reticuloendothelial system, and achieve long circulation in the body. In addition, the oxidation stress can be obviously reacted in the inflammatory microenvironment, the positive inner core can be exposed during the anti-inflammatory process, the microenvironment-targeted drug release can be achieved, and the microenvironment antioxidant therapy can be achieved. The drug delivery system with a particle size range of 80-120 nm is suitable for utilizing the pores of placental endothelial cells to achieve passive targeting retention and distribution. After endocytosis by cells, the residual GSH on the shell can play an antioxidant role in the cells. The drug delivery system structure of the (oxi)PEI-PCL inner core combined with the antioxidant GSH shell can make the drug achieve passive targeting aggregation in the lesion and microenvironment oxidation-reduction sensitive distribution, exhibit antioxidant effect in the lesion microenvironment and cells, and achieve accurate drug targeting distribution effect and in vivo treatment effect. Compared with the untreated PE mouse, the blood pressure is obviously decreased, the weight of the fetus is obviously increased, the overall growth trend tends to the normal mouse, and the obvious good treatment effect is embodied.

[0152] 3. Toxicity evaluation of the drug for animal models

[0153] The normal control group mice were injected with drugs for 72 hours, and blood was taken from the tail vein to detect liver function indicators alanine transaminase (ALT) and kidney function indicators blood urea nitrogen (BUN). The detection instrument was Hitachi 7600 automatic biochemical analyzer, and the detection results are shown in Table 4.

[0154] Table 4: Results of liver and kidney function detection

[0155] Liver function ALT (U / L) Kidney function BUN (mg / dL) Saline group 39.3 22.1 Example 1 39.5 22.5 Example 2 39.7 22.3 Example 3 39.2 22.7 Example 4 39.5 22.4

[0156] From the results of the above examples 1-4, it can be seen that the ROS-targeted modified antioxidant drug delivery system of the present application does not have obvious toxic side effects on the mother and fetus when treating gestational diseases. The oxidized polyethyleneimine-poly-caprolactone block copolymer is modified by glutathione through disulfide bond connection, and a core-shell structure drug delivery system with glutathione as the functional antioxidant shell, oxidized polyethyleneimine-poly-caprolactone as the drug-loaded inner core is formed by self-assembly, which shows a significant protective effect of reducing toxicity.

[0157] For those skilled in the art, the present application is not limited to the details of the above exemplary embodiments, and the present application can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application; therefore, in any aspect, the embodiments should be regarded as exemplary and non-limiting, and the scope of the present application is defined by the appended claims rather than the above description, and therefore all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present application. Any mark in the claims should not be regarded as limiting the involved claims.

[0158] All the above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, which are all included in the protection scope of the present application.

Claims

1. A ROS-targeted modified antioxidant drug delivery system, characterized in that, The antioxidant drug delivery system includes nanoparticles with a core-shell structure having glutathione as the outer shell and polyethyleneimine-polycaprolactone as the core, and a lipophilic drug loaded within the nanoparticles. The glutathione and the polyethyleneimine-polycaprolactone block copolymer are linked by disulfide bonds; a portion of the polyethyleneimine-polycaprolactone block copolymer... The unit is oxidized to unit.

2. The ROS-targeted modified antioxidant drug delivery system according to claim 1, characterized in that, The degree of oxidation of the polyethyleneimine-polycaprolactone block copolymer is 30%-70%, preferably 40%-65%.

3. The ROS-targeted modified antioxidant drug delivery system according to claim 1, characterized in that, The particle size range of the antioxidant drug delivery system is 80-120 nm.

4. The ROS-targeted modified antioxidant drug delivery system according to claim 1, characterized in that, The surface potential of the antioxidant drug delivery system is 10-30 mV, preferably 12-25 mV.

5. The ROS-targeted modified antioxidant drug delivery system according to claim 1, characterized in that, The antioxidant drug delivery system also includes a fluorescent label loaded within the nanoparticles; the fluorescent label is selected from lipid-soluble fluorescent dyes.

6. A method for preparing a ROS-targeted modified antioxidant drug delivery system as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Oxidized polyethyleneimine-polycaprolactone block copolymer (oxi)PEI-PCL was prepared by oxidizing the polyethyleneimine-polycaprolactone block copolymer with H2O2. S2. (oxi)PEI-PCL was modified with mercaptoacetic acid to obtain mercapto-oxidized polyethyleneimine-polycaprolactone block copolymer SH-(oxi)PEI-PCL; S3. By oxidizing the thiol groups of SH-(oxi)PEI-PCL with the thiol groups of glutathione to form disulfide bonds, a glutathione-coupled modified polyethyleneimine-polycaprolactone block copolymer GSH-(oxi)PEI-PCL was prepared. S4. Dissolve the lipid-soluble drug and GSH-(oxi)PEI-PCL in an organic solvent to obtain an organic phase solution. Add the organic phase solution dropwise into an aqueous surfactant solution while simultaneously performing ultrasonic dispersion and stirring. After stirring, an emulsion is obtained. Remove the organic solvent from the emulsion by rotary evaporation, centrifuge, and wash to prepare the antioxidant drug delivery system.

7. The preparation method according to claim 6, characterized in that, Step S1 includes: under stirring conditions, adding an aqueous solution of polyethyleneimine-polycaprolactone block copolymer and an aqueous solution of H2O2 to ultrapure water in a certain proportion to obtain a mixed solution, stirring and reacting, removing excess H2O2 by ultrafiltration after the reaction is completed, and freeze-drying to obtain oxidized polyethyleneimine-polycaprolactone block copolymer (oxi)PEI-PCL; the molar content of polyethyleneimine-polycaprolactone block copolymer in the mixed solution is 4-6 mM, and the molar ratio of polyethyleneimine-polycaprolactone block copolymer to H2O2 is 1:5-10; Step S2 includes: adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to an aqueous solution of mercaptoacetic acid, stirring, and after fully activating the carboxyl groups of mercaptoacetic acid, adding (oxi)PEI-PCL obtained in step S1, stirring and reacting, and after the reaction is completed, dialysis and freeze-drying to obtain thiolated oxidized polyethyleneimine-polycaprolactone block copolymer SH-(oxi)PEI-PCL; the molar content of mercaptoacetic acid in the aqueous solution of mercaptoacetic acid is 10-15 mM; the molar ratio of mercaptoacetic acid to (oxi)PEI-PCL is 1:1-1.2; Step S3 includes: adding SH-(oxi)PEI-PCL and glutathione obtained in step S2 to pure water in a certain proportion and stirring to react. After the reaction is completed, dialysis is performed to remove unreacted glutathione, and the mixture is freeze-dried to prepare glutathione-coupled polyethyleneimine-polycaprolactone block copolymer GSH-(oxi)PEI-PCL. The ratio of SH-(oxi)PEI-PCL to pure water is 0.1 mmol: 10-20 mL. The molar ratio of SH-(oxi)PEI-PCL to glutathione is 1:8-15.

8. The preparation method according to claim 6, characterized in that, In step S4, the organic solvent is selected from ethanol; the molar concentration of GSH-(oxi)PEI-PCL in the organic phase solution is 0.2-0.5 M, and the molar ratio of GSH-(oxi)PEI-PCL to the lipid-soluble drug is 10-20:1; the concentration of the surfactant aqueous solution is 0.1-0.5% w / v, and the surfactant is selected from Tween 80; the ultrasonic power of the ultrasonic dispersion step is 60-75W, and the ultrasonic time is 3-10 min.

9. The use of the ROS-targeted modified antioxidant drug delivery system according to any one of claims 1-5 in the preparation of pharmaceutical formulations for treating pregnancy-related diseases.

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