Drug system for regulating and controlling oxidation reduction in immune cells through ion delivery as well as preparation method and application of drug system

Abstract

The invention discloses a drug system for regulating and controlling oxidation reduction in immune cells through ion delivery as well as a preparation method and application of the drug system. The drug system comprises polymer vesicles on the inner layer and liposome vesicles on the outer layer, the polymer vesicles are formed by self-assembly of diblock polymers PEI-b-PLA or PEI-b-PLA, and the surfaces of the polymer vesicles are modified by antibodies targeting MDSC cell surface markers. The outer wall of the polymer vesicle is positively charged and is compounded with a therapeutic gene with a cell redox regulation function through electrostatic interaction, and an iron ion therapeutic drug capable of realizing cell redox regulation is loaded in the polymer vesicle; the liposomal vesicles are modified with a targeted disintegrating enzyme substrate polypeptide-PEG. According to the medicine system, regulation and control of MDSC in the placenta and regulation and control of immunity in the placenta can be achieved, influences on functions of other cells in the placenta are avoided, the situation that medicine leaks through a maternal-fetal barrier can be effectively reduced, toxicity to a fetus and a maternal body is reduced, and a new thought is provided for effective immunotherapy of placenta dysfunction diseases.

Description

Technical Field

[0001] The present invention relates to the fields of immunology, chemistry and biomedical engineering, and in particular to a drug system for regulating redox in immune cells through ion delivery, and a preparation method and application thereof. Background Art

[0002] Myeloid-derived suppressor cells (MDSCs) are a group of immature, poorly differentiated cells with immunosuppressive functions such as T cell suppression. MDSCs are a group of heterogeneous cells, mainly composed of bone marrow hematopoietic stem cells and immature myeloid cells (IMCs). After activation, MDSCs transform the local immune microenvironment by expressing excessive oxidants (ROS and NO) and a large number of immunosuppressive factors (such as ARG1 and iNOS), inhibiting the activation of inflammatory responses by immune cells in lesions. The latest studies have shown that MDSCs are key regulatory cells for a variety of immunosuppressive / activation diseases and may become a powerful target for immunotherapy. However, existing drugs kill a variety of other immune cells when killing MDSCs, making it difficult to achieve specific inhibition, with strong side effects and difficult application.

[0003] It has been clarified that immunogenic cell death, which is closely related to cellular redox regulation, is a powerful regulatory target for immune cells and immune signaling pathways in vivo [Nature. 2019 May; 569 (7754): 73-78]. The signals generated by the redox changes in immune cells co-stimulate antigen-presenting cells to present antigens, thereby inducing immune cells including MDSC to produce adaptive immune responses [Nature. 2022 Dec; 612 (7939): 338-346]. Different types of immune cells undergo redox stimulation, leading to different changes in immune cells and secreted proteomes. The imbalance caused by MDSC redox has become a clear sign of infection, autoimmune diseases, cancer, and organismal development. This dynamic and sensitive balance is crucial for the physiological regulation of the immune system and is a good therapeutic target for immune diseases [Acta Biomater. 2023 Mar 1; 158: 708-724]. Ferroptosis is a form of redox-induced death driven by iron, reactive oxygen species (ROS), and cell membrane lipid peroxidation [Gut. 2023 Sep; 72(9): 1774-1782.]. The mitochondria of ferroptotic cells shrink, the cristae decrease, the cell volume decreases, the cell nucleus volume is normal, and membrane lipid peroxides accumulate [Trends Pharmacol Sci. 2023 Aug; 44(8): 542-552.]. Cell membrane lipid peroxidation is the final event of ferroptosis. The immune cell function related to reactive oxygen species (ROS) can be regulated by regulating the concentration of iron particles.

[0004] It is now clear that iron ion concentration regulating drugs can significantly affect the immune function of MDSCs through redox regulation [Redox Biol. 2022Jun; 52: 102317.]. There are many types of iron ion drugs currently available in the clinic, including ferrous sulfate, ferrous fumarate, ferrous gluconate, ferrous succinate, polysaccharide iron complex, sucrose iron, dextran iron, sorbitol iron, ferrous citrate, ferrous chloride, etc. [J Exp Clin Cancer Res. 2021Jun 23; 40(1): 206.]. Since these iron ion drugs cannot achieve cell-targeted delivery, the dosage used in vivo is limited and effective immune regulation cannot be achieved. Therefore, they are currently mostly used for iron supplementation therapy [JCI Insight. 2020May 7; 5(9): e132747.].

[0005] The placenta is the only channel for the exchange of substances between the mother and the fetus. 90% of prenatal and postpartum diseases are related to pathological changes such as immune inflammation in the placenta. The activation of immune inflammation in the placenta causes a variety of pregnancy diseases, including hypertension in pregnant women (preeclampsia) and diabetes. However, there is currently a lack of placental-targeted therapeutic drugs for pregnancy diseases. This is because the mother and fetus cannot tolerate the potential toxic side effects of placental therapeutic drugs. The vast majority of drugs cannot avoid potential toxicity caused by distribution in the mother and fetus, and it is even more impossible to achieve only distribution in the placenta to take effect. Currently used immunosuppressive drugs (such as glucocorticoids, tacrolimus, etc.) all target immune cells throughout the body. If used in pregnant women, in addition to suppressing immune cells in the placenta, it will also suppress immune cells throughout the body, resulting in systemic immunosuppression and toxicity. There are currently no immunomodulatory drugs on the market that can be used for diseases caused by abnormal placental function.

[0006] It has been confirmed that immune cells can produce iNOS to regulate the ferroptosis of other immune cells in the microenvironment [Nat Chem Biol. 2020 Mar; 16 (3): 278-290.]. This is considered to be a target for cellular immunotherapy. Some studies have used genetic or pharmacological means to reduce the expression of inducible nitric oxide synthase (iNOS), and found that it enhances the sensitivity of immune cells to ferroptosis and redox therapy [Redox Biol. 2022 Apr; 50: 102229.; Phytomedicine. 2022 Oct; 105: 154347]. In iNOS-deficient cells in the lesion microenvironment, resistance to redox is reduced. iNOS plays a role in the ferroptosis of immune cells in the lesion microenvironment [Redox Biol. 2021 Sep; 45: 102045]. In addition, iNOS is an important functional target of MDSC [Cancer Lett. 2023Aug 28; 570: 216330.]. MDSC can produce NO through iNOS to inhibit T cell proliferation and migration [Cancer Res. 2017Jun 1; 77(11): 2834-2843.]. At the same time, MDSC absorbs a large amount of arginine in the environment through iNOS, which reduces the expression of T cell CD3ζ and weakens the proliferation ability, thereby achieving immunosuppression [Hepatology. 2022Sep; 76(3): 759-774.]. Therefore, co-regulation of iNOS expression in MDSC can effectively promote iron-induced immune cell function regulation therapy [ACS Nano. 2024Mar 5; 18(9): 6863-6886.]

[0007] However, how to limit the scope of immune cell function regulation to the local area of ​​the diseased placenta, effectively reduce the toxicity of immune drugs to pregnant women, and significantly improve the treatment effect of placental dysfunction diseases is an urgent problem that needs to be solved. Summary of the invention

[0008] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide a drug system for regulating redox in immune cells through ion delivery. This drug system can limit the scope of immune cell function regulation to the local area of ​​the diseased placenta, effectively avoid affecting immune cells outside the lesion, effectively reduce the toxicity of immune drugs to pregnant women, and provide a new idea for the effective immunotherapy of placental dysfunction diseases.

[0009] The present invention is achieved through the following technical solutions:

[0010] On the one hand, the present invention provides a drug system for regulating redox in immune cells by ion delivery, the drug system comprising an inner layer of polymer vesicles and an outer layer of liposome vesicles, the polymer vesicles being self-assembled from diblock polymers PEI-b-PLA or PEI-b-PLA, the surface of the polymer vesicles being modified with antibodies targeting MDSC cell surface markers, the outer wall of the polymer vesicles being positively charged and compounding therapeutic genes having cell redox regulating functions through electrostatic action, the polymer vesicles being loaded with iron ion therapeutic drugs that can achieve cell redox regulation; the liposome vesicles being modified with a targeted disintegration enzyme substrate polypeptide-PEG.

[0011] Preferably, the iron ion therapeutic drug is ferrous sulfate, ferrous fumarate, ferrous gluconate, ferrous succinate, polysaccharide iron complex, iron sucrose, iron dextran, sorbitol iron, ferrous citrate or ferrous chloride. Iron ion concentration regulating drugs can significantly affect the immune function of MDSC through redox regulation.

[0012] Preferably, the therapeutic gene is siRNA of iNOS. The expression of inducible nitric oxide synthase (iNOS) is reduced by genetic or pharmacological means, and it is found that it can enhance the sensitivity of immune cells to ferroptosis and redox therapy. In iNOS-deficient cells in the lesion microenvironment, resistance to redox can be reduced. iNOS plays a role in ferroptosis of immune cells in the lesion microenvironment. In addition, iNOS is an important functional target of MDSC. MDSC can produce NO through iNOS, inhibit T cell proliferation and migration. At the same time, MDSC absorbs arginine in the environment in large quantities through iNOS, reduces the expression of T cell CD3ζ, and weakens the proliferation capacity to achieve immunosuppression. Therefore, MDSC is subjected to iNOS expression co-regulation, which can effectively promote the regulation and treatment of immune cell function induced by iron ions.

[0013] Preferably, the antibody targeting the MDSC cell surface marker is CD33.

[0014] Preferably, the enzyme substrate polypeptide is MCA-Lys-Pro-Leu-Gly-Leu-DNP-Dpa-Ala-Arg-NH2.

[0015] Further preferably, the drug system further comprises a molecular imaging contrast agent between the inner layer of polymer vesicles and the outer layer of liposome vesicles, which can realize imaging tracing of the distribution of MDSC-regulating drugs in vivo, and the molecular imaging contrast agent comprises an MRI T1 contrast agent, an MRI T2 contrast agent or a near-infrared fluorescent contrast agent.

[0016] Preferably, the average total particle size of the drug system is 210-290 nm.

[0017] On the other hand, the present invention provides a method for preparing the above-mentioned drug system for regulating redox in immune cells by ion delivery, comprising the following steps:

[0018] S1. Preparation of PEI-b-PLA or PEI-b-PLA polymer;

[0019] S2, adding the therapeutic drug to the polymer, and preparing polymer vesicles loaded with the therapeutic drug by a double emulsification method;

[0020] S3, linking the antibody to the polymer vesicle to prepare the antibody-modified polymer vesicle;

[0021] S4, compounding the polymer vesicles prepared in S2 or the antibody-modified polymer vesicles prepared in S3 with the therapeutic gene through electrostatic interaction to obtain polymer vesicles loaded with therapeutic drugs and / or therapeutic genes;

[0022] S5, preparing enzyme substrate polypeptide-PEG;

[0023] S6, preparing enzyme substrate polypeptide-PEG, phospholipid and cholesterol to obtain enzyme substrate polypeptide-PEG-modified liposome vesicles;

[0024] S7. Assemble the polymer vesicles loaded with therapeutic drugs and / or therapeutic genes in S4 with the liposome vesicles modified with enzyme substrate polypeptide-PEG in S6 to prepare a drug system for regulating redox in immune cells through ion delivery.

[0025] In another aspect, the present invention provides the use of the above-mentioned drug system for regulating redox in immune cells by ion delivery in the preparation of drugs for regulating diseases related to immune dysfunction. Preferably, the disease related to immune dysfunction is preeclampsia.

[0026] Compared with the prior art, the present invention has the following technical effects:

[0027] The drug system of the present invention comprises an inner layer of polymer vesicles and an outer layer of liposome vesicles, wherein the outer layer of vesicles is sensitive to the microenvironment of the lesion, and the distribution of the MDSC function regulating drug is limited to the lesion by selectively releasing the outer layer of vesicles limited to the microenvironment of the lesion, thereby avoiding the distribution of the drug in the immune system outside the lesion and the influence on the immune system outside the lesion, and avoiding the potential immune side effects of the drug outside the lesion. After the inner layer of vesicles is released by the outer layer of vesicles that disintegrate into the lesion, the MDSC cells in the lesion are anchored by antibodies targeting MDSC cell membrane markers, effectively reducing the leakage of drugs through the maternal-fetal barrier, reducing the fetal toxicity caused by the therapeutic genes and drugs after reaching the embryo, and also reducing the reflux of the therapeutic genes and drugs to the maternal blood through the uterine vein, and causing maternal toxicity after reaching the normal tissue of the mother; utilizing the positive potential on the surface of the inner layer of vesicles, the genes of the cyst wall of the MDSC cells and the therapeutic drugs in the vesicles are promoted to be internalized into the MDSC cells, thereby realizing MDSC function regulation, and the distribution of the therapeutic genes and therapeutic drugs in other cells of the placenta is reduced, thereby avoiding the influence on the functions of other cells in the placenta. In addition, the inner layer of polymer vesicles can load and transport both hydrophobic and hydrophilic drugs, expanding the scope of application of drug carriers.

[0028] The drug system prepared by the specific design of the present invention can achieve the regulation of MDSCs in the placenta and the regulation of immunity in the placenta, avoid affecting the functions of other cells in the placenta, and can effectively reduce the leakage of drugs across the maternal-fetal barrier, reducing toxicity to the fetus and the mother, providing a new idea for the effective immunotherapy of placental dysfunction diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the structure of the drug system prepared by the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described below by means of specific implementation methods. The following examples are preferred implementation methods of the present invention, but the implementation methods of the present invention are not limited to the following examples.

[0031] The main raw material sources of the present invention are as follows:

[0032]

[0033]

[0034] Particle size test method:

[0035] The particle size of the sample was measured using a Zeta-Plus potentiometric particle size analyzer (Brooken Haven), with incident laser wavelength λ=532 nm, incident angle θ=90°, and temperature 25° C. The average of three measurements was taken.

[0036] Embodiment 1:

[0037] S1. Synthesis of poly(acetimide-co-polylactic acid) (PEI-b-PLA)

[0038] First, polylactic acid is synthesized, and then its terminal hydroxyl group is activated with CDI, and then reacted with polyacetimide to synthesize polyacetimide copolymer polylactic acid (PEI-b-PLA). The details are as follows:

[0039] In a 250 mL reaction bottle, add 135 μL of dried n-dodecanol (0.60 mmol), 11.52 g of newly recrystallized D, L-lactide (80 mmol) and a small amount of stannous octoate (0.5% of the mass of lactide), dry under vacuum at room temperature for 4 h, add 40 mL of freshly distilled anhydrous toluene to dissolve, and heat at 120 ○ C for 12 h. After cooling, a large amount of anhydrous ethanol was used for precipitation, and the liquid was placed in a refrigerator for low-temperature freezing, then centrifuged and dried, and then dissolved with dichloromethane, precipitated with anhydrous ethanol again, filtered, and dried to obtain the product PLA.

[0040] Activate the terminal hydroxyl groups of PLA with CDI. Add 6.0 g PLA and 70 ○ C for 4 h to remove water, add 40 mL of freshly distilled tetrahydrofuran to dissolve, and under argon protection, slowly add the polymer solution dropwise to a tetrahydrofuran solution of CDI (0.50 g), react at room temperature for 24 h, precipitate with a large amount of anhydrous ethanol, freeze the liquid in a refrigerator, then centrifuge and dry, then dissolve with dichloromethane, precipitate with anhydrous ethanol again, filter and dry to obtain PLA-CDI.

[0041] 1.05 g PEI was added to a 100 mL reaction bottle and dissolved in 20 mL freshly distilled chloroform. 4 g PLA-CDI was dissolved in 30 mL freshly distilled chloroform, and then added dropwise to the PEI solution under argon protection, and stirred for reaction at room temperature for 24 h. The reaction solution was dialyzed in chloroform with a dialysis bag (MW = 3.5 kDa) to remove excess PEI, and precipitated with a large amount of anhydrous ethanol. The liquid was placed in a refrigerator for low-temperature freezing, and then centrifuged and dried to obtain the polymer PEI-b-PLA.

[0042] S2. Preparation of inner polymer vesicles

[0043] The polymer vesicles loaded with ferrous sulfate were prepared by double emulsification method, specifically: 30 mg of PEI-b-PLA polymer was weighed and dissolved in 3 mL of chloroform, and 0.3 mL of an aqueous solution containing 4 mg of ferrous sulfate (heptahydrate) was slowly added under ice bath and ultrasound. The first emulsion was added dropwise to 20 mL of phosphate buffer (0.02 M, pH 7.4) under ultrasound to obtain a secondary emulsion, the chloroform was evaporated, and the large particles were removed by filtering through a syringe filter (0.45 μm), and the PEI-b-PLA polymer vesicles loaded with ferrous sulfate were obtained by ultrafiltration and concentration, and the volume was fixed to 20 mL for use.

[0044] S3. Preparation of antibody-modified polymersomes

[0045] First, the CD33 antibody is cleaved by the method in the existing literature to obtain the Fab segment of CD33 and purify it. Then, CD33-Fab is linked to mal-PEG-COOH, and then the PEG linked to the antibody is reacted with the amino group on the poly(acetimide-co-polylactic acid) vesicle nanoparticles by amidation reaction to prepare the CD33-Fab antibody-targeted poly(acetimide-co-polylactic acid) vesicles (Fab-PEI-PLA vesicle nanoparticles);

[0046] The specific operation is as follows: 10 mg of CD33 antibody was weighed and enzymatically hydrolyzed for 4 h in 0.5 mg ml-1 papain, 10 mmol L-1 cysteine, 2 mmol L-1 EDTA, pH 7.6. The enzymatic hydrolysis product was separated by Protein A affinity chromatography, and the penetration peak was further purified by DEAE anion exchange chromatography. After dialysis, desalting and freeze-drying, a CD33 Fab fragment with high purity was obtained;

[0047] Weigh 1 mg of CD33 Fab fragment (Mn = 45 kDa) and pretreat with EDTA solution (500 μL 0.5 M) at 4°C for 15 min. Dissolve in 5 ml of PBS solution, add 1 mg of dithiothreitol, and react at 25°C for 30 min. Remove dithiothreitol by centrifugation with a 1k molecular weight cutoff ultrafiltration tube, add 5 ml of PBS solution to dissolve, add mal-PEG-COOH (2 mg, Mn = 4k) and mix evenly, and place at 4°C overnight. Remove excess mal-PEG-COOH by centrifugation with a 5k molecular weight cutoff ultrafiltration tube. Use 500 μg of EDC and NHS respectively to activate the carboxyl groups in Fab-PEG-COOH for 15 min, then add 18 mL of PEI-PLA nanovesicles prepared in step 2, react overnight at 4°C, and finally use an ultrafiltration centrifuge tube with a molecular weight cutoff of 100k to remove excess EDC, NHS and unconnected CD33 Fab fragments, collect the purified solution in the centrifuge tube, ultrasonically disperse it in distilled water, and dilute it to 18 mL for use, thereby obtaining antibody-modified polymer vesicles (Fab-PEI-PLA).

[0048] S4. Preparation of gene-complexed polymer vesicles

[0049] The positively charged PEI-b-PLA vesicle nanoparticles prepared by S2 or the Fab-PEI-PLA vesicle nanoparticles prepared by S3 were compounded with the negatively charged iNOS gene through electrostatic action to prepare a nanocomposite. The specific operation is as follows: 400 μg of iNOS gene was diluted with PBS to a final volume of 4 mL and oscillated evenly. 4 mL of the PEI-b-PLA vesicle nanoparticle solution prepared by S2 (or the Fab-PEI-PLA nanoparticle solution prepared by S3) was ultrasonically dispersed evenly, the iNOS gene diluted solution was mixed with the PEI-b-PLA (or Fab-PEI-PLA) nanoparticle solution, the complex system was fixed to 4 mL, blown to mix evenly and allowed to stand for 30 minutes to obtain a polymer vesicle of the composite gene.

[0050] S5. Synthesis of enzyme substrate peptide-PEG

[0051] 0.05mmol of matrix metalloproteinase substrate peptide can be selected MCA-Lys-Pro-Leu-Gly-Leu-DNP-Dpa-Ala-Arg-NH2, molecular weight: 1221.32Da, 5mmol of EDC and 5mmol of DMAP are dissolved in 10mL of acetonitrile aqueous solution (acetonitrile: water = 1:1), and magnetically stirred at 500rpm for 2h in an ice water bath under N2 protection to activate the peptide. After 2h, 0.5mmol of mPEG-NHS (molecular weight of about 3000Da) is added and the reaction is continued for 72h. After the reaction is completed, the reaction solution is placed in a dialysis bag (MWCO = 3.5kDa), dialyzed for 72h, and freeze-dried to obtain the product enzyme substrate peptide-PEG.

[0052] S6, Enzyme substrate peptide-PEG modified liposome vesicles@polymer vesicles

[0053] 10 mg of enzyme substrate polypeptide-PEG, 3 mg of phospholipid and 20 mg of cholesterol were dissolved in 5 mL of dichloromethane, and the dichloromethane was dried with a vacuum rotary true hair dryer to form a liposome film on the wall of the round-bottom flask with the enzyme substrate polypeptide-PEG, phospholipid and cholesterol. 0.2 mL of disodium gadoxetate with a concentration of 469.01 mg / ml was added to 2 mL of Fab-PEI-PLA vesicle nanoparticles prepared in step S4 under slow stirring, and then the mixed solution was added dropwise to the liposome film formed by the above enzyme substrate polypeptide-PEG, phospholipid and cholesterol at a speed of 0.5 mL / min. After the addition was completed, stirring was continued for 30 minutes to fully assemble the liposomes and Fab-PEI-PLA vesicle nanoparticles, and then the liposomes carrying the therapeutic gene composite nanoparticles and the empty liposomes were separated by high-speed centrifugation. Finally, 2 mL of normal saline (0.9% NaCl) solution was added to dissolve the enzyme substrate polypeptide-PEG-modified liposome vesicles@polymer vesicle particles, filtered through a syringe filter with a pore size of 220 nm, the volume was fixed to 2 mL, and stored at 4°C for later use, thereby obtaining a drug system for regulating redox in immune cells by ion delivery.

[0054] like Figure 1 A schematic structural diagram of the prepared drug system is shown. As can be seen from the figure, the drug system includes an inner layer of polymer vesicles and an outer layer of liposome vesicles. The surface of the inner layer vesicles is modified with antibodies targeting MDSC cell surface markers, the outer wall of the inner layer vesicles is composited with therapeutic genes, and the inner layer vesicles are loaded with iron ion therapeutic drugs; the outer layer vesicles are modified with a targeted disintegration enzyme substrate polypeptide-PEG.

[0055] Embodiment 2:

[0056] The drug in S2 is ferrous gluconate, the enzyme substrate polypeptide-PEG in S6 is 20 mg, and the rest is the same as in Example 1.

[0057] Embodiment 3:

[0058] No contrast agent is added in S6, and the rest is the same as in Example 1.

[0059] Comparative Example 1:

[0060] The S3 step was missing (the polymersome was not linked to CD33-Fab), and the rest was the same as Example 1.

[0061] Comparative Example 2:

[0062] The steps S5 and S6 are missing (the drug system has no outer layer of liposome vesicles and no contrast agent), and the rest is the same as Example 1.

[0063] Comparative Example 3:

[0064] The S3 step is missing (the polymer vesicle is not linked to CD33-Fab), the S5 step is missing (the liposome vesicle is not modified with enzyme substrate polypeptide-PEG), and the rest is the same as Example 1.

[0065] Comparative Example 4:

[0066] The S3 step is missing (the polymer vesicle is not linked to CD33-Fab), the S5 and S6 steps are missing (the drug system has no outer layer of liposome vesicles and no contrast agent), and the rest is the same as Example 1.

[0067] Comparative Example 5:

[0068] The enzyme substrate polypeptide-PEG in S6 is 40 mg, and the rest is the same as in Example 1.

[0069] Compared with Example 1, by changing the feeding amount of the polymer or drug in step S2 or omitting one of steps S3, S4, S5 or S6, Examples 2-3 or Comparative Examples 1-5 can be prepared, as shown in Table 1 below:

[0070] Table 1: Examples and Comparative Examples

[0071]

[0072] Functional evaluation experiment

[0073] 1. Magnetic resonance imaging (MRI) experiments to evaluate the placenta-specific delivery of drugs

[0074] Model building:

[0075] 8-week-old SPF-grade C57BL / 6 mice (purchased from Guangdong Medical Experimental Animal Center) were mated with male mice in a 2:1 ratio during estrus. The vaginal secretion smears of female mice were stained with Papanicolaou on the second day. The specimens with positive vaginal sperm were observed under an optical microscope and diagnosed as pregnancy, which was marked as pregnancy day 0 (D0). Pregnant mice were fed with 2 mg / mL nitroso-L-arginine methyl ester dissolved in sterile water to establish the preeclampsia / gestational hypertension model, and pregnant mice fed with an equal amount of double distilled water served as the normal control group.

[0076] MRI imaging to detect the placental distribution of drugs:

[0077] On the 11th day, the preeclampsia model animals were anesthetized with chloral hydrate and MRI T1 scans were performed before drug injection (0h) and 2 hours after injection (2h) to observe the distribution of the nanodrug in vivo. The dose of the nanodrug injected into the tail vein was 0.1 mL of the example drug, or the comparative example drug, or an equal volume of PBS.

[0078] Philips Intera 1.5T MRI scanner and its animal-specific coil were used to perform MRI imaging of the uterus of C57BL / 6j mice. [The signal intensity evolution of the uterus and embryonic region of C57BL / 6j mice was observed on the MRI FFE sequence, and the T1 map imaging technique was used to measure the changes in T1 relaxation time caused by the distribution of gadopentetate dimeglumine in the uterus, placenta, embryo and other organs in the body, and the relaxation rate R1 at 0h and 2h was calculated respectively]. The relative increase ratio of R1 at 2h after drug injection (RSI (Relative Signal Intensity) % = R12h / R10h) was calculated, and the results are shown in Table 2.

[0079] Table 2 Results of placenta-specific delivery function evaluation

[0080]

[0081]

[0082] 2. Establishing an animal model of preeclampsia to evaluate the therapeutic effect

[0083] Nano drugs were injected on D3, D6, D9, D12, and D15. The dosage of nano drugs injected into the tail vein was 0.1 mL of the example drug, or the comparative example drug, or an equal volume of PBS. A series of tests were performed on D17, and the test results are shown in Table 3:

[0084] Blood pressure test: Use the BP-2000 blood pressure analysis system to non-invasively measure the systolic blood pressure (SBP) of pregnant mice using the tail cuff method. Maintain the room temperature at 26 degrees Celsius, set channel 1 to 1V (1V is equivalent to 300mmHg), and channel 2 to SmV. Fix the mouse in a cage, place the mouse's tail in a 17mm tail cuff, and make sure the bottom of the tail contacts the sensor. When the pregnant mouse is in a quiet state, continuously apply pressure and measure 10 times, with an interval of 1s each time, take the average value and keep a record;

[0085] Placenta and fetus examination: Placenta tissue: Anesthetize the pregnant mouse, open the abdominal cavity, dissect the uterus, take out the fetus and placenta in turn, and record the number of surviving fetuses. Remove the fetal membrane and umbilical cord on the placenta, cut off the umbilical cord at the fetal end along the root of the umbilical cord, place the placenta and fetus on sterile gauze to absorb the surface amniotic fluid, and weigh the placenta and fetus on an analytical balance. Cut the placenta tissue and put it in liquid nitrogen and store it at -80℃.

[0086] Table 3 Evaluation of therapeutic effects in animal models of preeclampsia

[0087]

[0088]

[0089] The drug system prepared in Example 1-3 includes an inner layer of polymer vesicles and an outer layer of liposome vesicles, and the particle size range of the delivery system is 200-300nm. Its particle size of about 200nm and the negatively charged liposome vesicles in the outer layer are convenient to avoid being engulfed in large quantities by the reticuloendothelial system, and the circulation time in the body is prolonged, and the effective circulation in the body is achieved. The liposome vesicles modified by its substrate matrix metalloproteinase substrate polypeptide-PEG are stable in the circulation of other tissues and organs in the body. When reaching the placental microenvironment with high specific expression of matrix metalloproteinase, the matrix metalloproteinase substrate polypeptide is degraded and the outer layer vesicles disintegrate. The contrast agent between the inner and outer layer vesicles and the inner layer vesicles encapsulating therapeutic drugs or genes are released to achieve the specific distribution of contrast agents and therapeutic drugs or genes in placental tissue. The distribution of contrast agents and therapeutic drugs or genes outside the lesion is avoided, and systemic immunotoxicity is avoided. After the inner layer vesicles encapsulating therapeutic drugs or genes are released, the therapeutic drugs or genes containing antibody fragments are exposed. The antibody fragment can be anchored to the membrane marker of MDSC cells in the microenvironment. Furthermore, the positive electrical properties of the inner vesicles are used to stimulate the specific endocytosis of the inner vesicles by MDSC cells, so that therapeutic drugs or genes can enter MDSC specifically. After the therapeutic drugs or genes are specifically endocytosed by MDSC cells, MDSC functional regulation is achieved. The distribution of therapeutic drugs or genes in other cells of the placenta is reduced to avoid affecting the functions of other cells in the placenta. In addition, the CD33 targeting antibody fragment is modified so that the inner capsule containing therapeutic drugs or genes is anchored to MDSC cells, effectively reducing the leakage of therapeutic drugs or genes through the maternal-fetal barrier, and reducing the fetal toxicity caused by the therapeutic drugs or genes after reaching the embryo. In addition, the therapeutic drugs or genes are also reduced from flowing back to the maternal blood through the uterine vein, and after reaching the normal tissues of the mother, maternal toxicity is caused. In Example 1-3, after the lesions are treated, the blood pressure of the animals is significantly reduced, and the fetal condition is significantly improved, and the weight and number of fetuses are significantly recovered. The main difference between Example 3 and Examples 1-2 is that there is no contrast agent between the outer layer vesicles and the inner layer vesicles, and no enhanced imaging signal can be formed in the specific accumulation lesions and non-specific accumulation liver during the entire medication process.

[0090] In comparative example 1, the outer layer of the liposome vesicles is modified with a targeted disintegration enzyme substrate polypeptide-PEG, and the drug system as a whole can enter the lesion. After entering the lesion, the matrix metalloproteinase substrate polypeptide group of the liposome vesicle is degraded, the outer layer of the liposome vesicle disintegrates, and the contrast agent between the inner and outer vesicles and the inner vesicles are released together in the lesion. MRI imaging detected the distribution of the contrast agent and found that the contrast agent disintegrated with the outer layer of the vesicle targeted by the matrix metalloproteinase substrate polypeptide, and obtained the targeted distribution in the placenta. Compared with Example 1, the imaging signal did not decrease significantly, the distribution and imaging signal in the embryo did not increase significantly, and the distribution and imaging signal in the liver did not increase significantly. However, since the CD33 targeting antibody fragment was not linked to the inner layer of the polymer vesicle wall of comparative example 1, the MDSC cells in the animal model were not targeted for treatment, so the efficacy was poor. Animal blood pressure was detected and found that there was no significant improvement compared with the PBS treatment control group of the disease model, and even a slight increase. This may be due to the fact that the therapeutic genes or drugs distributed non-targeted in the lesion failed to target the MDSC cells, but were engulfed by other functional cells, causing further damage. Furthermore, since the loaded therapeutic genes or drugs in the inner layer vesicles fail to anchor to MDSCs, some of them pass through the placental barrier and flow into the embryo, causing embryonic tissue toxicity and reducing the size and number of embryos.

[0091] There is no outer vesicle in Comparative Example 2, and there is no contrast agent between the inner and outer vesicles. The inner vesicles loaded with therapeutic genes or drugs cannot be targeted and distributed in the lesions. MRI imaging detected the distribution of contrast agents and found that the therapeutic genes or drugs were not wrapped by the outer vesicles and could not load the contrast agent. Compared with Example 1, no imaging signals were detected in the lesions, no imaging signals were detected in the embryos, and no imaging signals were detected in the liver. Due to the absence of the outer layer of liposome vesicles, the lesions in the animal model could not be targeted for treatment, so the efficacy was poor. The blood pressure of the animals was detected and it was found that the size and number of the embryos were significantly reduced compared with the PBS treatment control group of the disease model.

[0092] In comparative example 3, the outer vesicles do not have enzyme-sensitive matrix metalloproteinase substrate polypeptide-PEG, and the outer vesicles are difficult to disintegrate in the lesion. The contrast agent between the inner and outer vesicles and the therapeutic gene or drug loaded by the inner vesicles are released slowly, and the lesion-targeted distribution cannot be achieved. MRI imaging detected the distribution of contrast agents and found that the contrast agent was wrapped by the outer vesicles without lesion targeting. Compared with Example 1, the imaging signal detected by the lesion is extremely low, the imaging signal detected by the embryo is extremely low, and the imaging signal caused by the non-targeted distribution contrast agent detected in the liver is extremely high. The inner vesicles do not have CD11b targeting antibody fragments, and the drug-loaded inner vesicles outside the lesion cannot achieve MDSC targeted distribution. The lesions in the animal model have not been targeted for treatment, so the efficacy is poor. The blood pressure of animals was detected and found to be increased compared with the PBS treatment control group of the disease model, and the size and number of embryos were significantly reduced.

[0093] There are no outer vesicles in Comparative Example 4, and there is no contrast agent between the inner and outer vesicles. MRI imaging detected the distribution of the contrast agent and found that the drug was not wrapped by the outer vesicles and failed to load the contrast agent. Compared with Example 1, no imaging signal was detected in the lesion, no imaging signal was detected in the embryo, and no imaging signal was detected in the liver. The inner vesicles do not have CD11b targeting antibody fragments. The inner vesicles loaded with therapeutic genes or drugs cannot achieve targeted distribution of lesions. The drug-loaded inner vesicles inside and outside the lesion cannot achieve MDSC targeted distribution. The lesions in the animal model were not treated with targeted therapy, so the efficacy was poor. The blood pressure of the animals was detected and found to be higher than that of the PBS-treated control group in the disease model, and the size and number of the embryos were significantly reduced.

[0094] The particle size of the drug system in Comparative Example 5 is greater than 300nm, which is not suitable for distribution in the body, has a low efficiency in entering the lesion, and a high proportion of being engulfed by the reticuloendothelial system. MRI imaging detected the distribution of the contrast agent and found that the lesion distribution of the contrast agent was reduced. Compared with Example 1, the lesion imaging signal was significantly reduced, the distribution and imaging signal in the embryo were significantly reduced, the contrast agent was engulfed by the reticuloendothelial system in the liver, and the distribution and imaging signal in the liver were significantly increased. The targeted therapy efficacy in the animal model was poor, and the blood pressure of the animals was significantly reduced compared with the PBS treatment control group of the disease model. The embryos improved compared with the disease group, and the size and number were significantly recovered.

[0095] 3. Toxicity evaluation of nanodrug systems in animal models

[0096] 72 hours after the mice in the normal control group were injected with nanomedicine, blood was collected from the tail vein to detect liver function indicators alanine transaminase (ALT), total bilirubin (TBil) and renal function indicators blood urea nitrogen (BUN) and serum creatinine (sCr). The detection instrument was Hitachi 7600 automatic biochemical analyzer, and the test results are shown in Table 4.

[0097] Table 4 Toxicity evaluation results

[0098]

[0099] From the above results, it can be seen that the liver function (ALT, TBIL) and kidney function (BUN, sCr) in Examples 1-3 were normal, and no obvious toxic side effects on the mother and fetus were observed.

Claims

1. A drug system for regulating redox in immune cells by ion delivery, characterized in that: The drug system comprises an inner layer of polymer vesicles and an outer layer of liposome vesicles, wherein the polymer vesicles are self-assembled from diblock polymers PEI-b-PLA or PEI-b-PLA, the surface of the polymer vesicles is modified with antibodies targeting MDSC cell surface markers, the outer wall of the polymer vesicles is positively charged and is compounded with therapeutic genes having cell redox regulation function through electrostatic action, and the polymer vesicles are loaded with iron ion therapeutic drugs that can achieve cell redox regulation; the liposome vesicles are modified with a targeted disintegration enzyme substrate polypeptide-PEG.

2. The pharmaceutical system for regulating redox in immune cells by ion delivery according to claim 1, characterized in that: The iron ion therapeutic drug is ferrous sulfate, ferrous fumarate, ferrous gluconate, ferrous succinate, polysaccharide iron complex, iron sucrose, iron dextran, sorbitol iron, ferrous citrate or ferrous chloride, and the therapeutic gene is siRNA of iNOS.

3. The pharmaceutical system for regulating redox in immune cells by ion delivery according to claim 1, characterized in that: The antibody targeting the MDSC cell surface marker is CD33.

4. The pharmaceutical system for regulating redox in immune cells by ion delivery according to claim 1, characterized in that: The enzyme substrate polypeptide is MCA-Lys-Pro-Leu-Gly-Leu-DNP-Dpa-Ala-Arg-NH2.

5. The pharmaceutical system for regulating redox in immune cells by ion delivery according to claim 1, characterized in that: The drug system further comprises a molecular imaging contrast agent between the inner polymer vesicle and the outer liposome vesicle, wherein the molecular imaging contrast agent comprises an MRI T1 contrast agent, an MRI T2 contrast agent or a near-infrared fluorescent contrast agent.

6. The pharmaceutical system for regulating redox in immune cells by ion delivery according to claim 1, characterized in that: The average total particle size of the drug system is 210-290 nm.

7. The method for preparing the pharmaceutical system for regulating redox in immune cells by ion delivery according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1. Preparation of PEI-b-PLA or PEI-b-PLA polymer; S2, adding the therapeutic drug to the polymer, and preparing polymer vesicles loaded with the therapeutic drug by a double emulsification method; S3, linking the antibody to the polymer vesicle to prepare the antibody-modified polymer vesicle; S4, compounding the polymer vesicles prepared in S2 or the antibody-modified polymer vesicles prepared in S3 with the therapeutic gene through electrostatic interaction to obtain polymer vesicles loaded with therapeutic drugs and / or therapeutic genes; S5, preparing enzyme substrate polypeptide-PEG; S6, preparing enzyme substrate polypeptide-PEG, phospholipid and cholesterol to obtain enzyme substrate polypeptide-PEG-modified liposome vesicles; S7. Assemble the polymer vesicles loaded with therapeutic drugs and / or therapeutic genes in S4 with the liposome vesicles modified with enzyme substrate polypeptide-PEG in S6 to prepare a drug system for regulating redox in immune cells through ion delivery.

8. Use of the drug system for regulating redox in immune cells by ion delivery according to any one of claims 1 to 6 in the preparation of drugs for regulating diseases related to immune dysfunction.

9. The use according to claim 8, characterized in that: The immune dysfunction-related disease is preeclampsia.

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