Immunomodulatory drug delivery system specifically targeting MDSC in lesion, and preparation method therefor and application thereof

Abstract

Disclosed in the present invention are an immunomodulatory drug delivery system specifically targeting an MDSC in a lesion, and a preparation method therefor and an application thereof. The delivery system has a bilayer vesicle structure. An inner vesicle is a polymeric vesicle containing an antibody that targets an MDSC cell surface marker, the outer wall of the inner vesicle is positively charged and is loaded with an immunomodulatory therapeutic gene, and a therapeutic drug is loaded within the inner vesicle. An outer vesicle is a liposome-like vesicle modified with enzyme-substrate peptide-PEG, which can undergo targeted disintegration under the action of an enzyme highly expressed in placenta tissue interstitial fluid. The delivery system of the present invention can enable modulation of MDSCs in a placenta and of the immune environment within the placenta, avoiding affecting the functions of other cells in the placenta; and in addition, the delivery system can effectively reduce the leakage of drugs across a placental barrier, reducing the toxicity to both the fetus and the mother.

Description

An immune regulation drug delivery system specifically targeting lesion MDSC cells and a preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of immunology, chemistry, biomedical engineering, and in particular to an immune regulation drug delivery system specifically targeting lesion MDSC cells and a preparation method and application thereof. BACKGROUND

[0002] Myeloid-derived suppressor cells (MDSC) are a group of immature, poorly differentiated cells with T cell inhibition and other immunosuppressive functions. MDSCs are a group of heterogeneous cells mainly composed of bone marrow hematopoietic stem cells and immature myeloid cells (IMCs). After activation, MDSCs modify the local immune microenvironment by expressing excessive oxidants (ROS and NO) and a large number of immunosuppressive factors (such as ARG1 and iNOS), and inhibit the activation of immune cells in the lesion to induce inflammatory response.

[0003] Recent studies have found that MDSC plays an early, inducing and leading role in immune-related disease changes, and inhibits immune response through various mechanisms. Abnormal function of MDSC often induces immune stress in the lesion, guides the changes of downstream T cells, dNK cells and other cells, and causes immune killing; on the contrary, pathological activation of MDSC function leads to immune suppression. Therefore, MDSC can be used as an effective target for immunotherapy.

[0004] Existing drugs kill a variety of other immune cells when killing MDSC, and it is difficult to achieve specific inhibition, has strong side effects, and is difficult to apply. Numerous studies have shown that COX-2 inhibition therapy can intervene in MDSC through the PG-E2 (prostaglandin E2) dominated pathway. These immunosuppressive effects have been confirmed by a series of preclinical trials

J Clin Invest. 2014; 124 (9): 3704-7.; Blood. 2011; 118 (20): 5498-505.; Cancer Res. 2011; 71 (7): 2664-74.

[0005] However, the existing immune drugs, including COX-2, have the problem of producing immune regulation in and out of the lesion when used in vivo, which limits the therapeutic effect and causes toxicity. The distribution of immune drugs outside the lesion leads to extensive immunotoxicity, which limits the increase of drug dosage; it also significantly reduces the distribution ratio of the drug in the placenta and other lesions, reducing the therapeutic effect.

[0006] The placenta is the only channel for maternal and fetal material exchange, and 90% of pregnancy diseases are related to pathological changes such as immune inflammation in the placenta. The activation of immune inflammation in the placenta causes various pregnancy diseases, including maternal hypertension (pre-eclampsia), diabetes, etc. However, there is currently a lack of therapeutic drugs targeting the placenta for gestational diseases. This is because the mother and fetus cannot tolerate the potential toxic side effects of placental therapeutic drugs. Most drugs cannot avoid distribution in the mother and fetus to produce potential toxicity, and it is even more impossible to achieve distribution only in the placenta to take effect. If the scope of immune cell function regulation can be limited to the local lesion of the placenta, it will effectively reduce the toxicity of immune drugs to pregnant women, making effective immune therapy of placental diseases possible, and significantly improving the current situation of no treatment for placental dysfunction diseases.

[0007] Many clinical trials related to pre-eclampsia at home and abroad have confirmed that non-steroidal anti-inflammatory immune drugs targeting COX-2, represented by aspirin (acetylsalicylic acid), have a relatively clear therapeutic effect on pre-eclampsia. However, when treating PE pregnant women with aspirin and other non-steroidal anti-inflammatory immune drugs targeting COX-2 in clinical trials, it was found that the use of non-pregnant conventional doses may cause side effects such as maternal bleeding, fetal bleeding, and immune disorders. Therefore, the current mainstream related clinical trials are all exploring the minimum dose that can take effect in PE to avoid side effects

Lancet. 2020; 395(10220) :285-293

JAMA Pediatr 2019; 173(7): 619-620.; JAMA. 2021; 326(12): 1153-1155

[0008] In order to overcome the defects and deficiencies of the prior art, the purpose of the present application is to provide an immune regulation drug delivery system specifically targeting lesion MDSC cells and a preparation method and application thereof, so as to realize the regulation of MDSC in the placenta and the regulation of placental immunity by means of the delivery system targeting MDSC in the lesion, reduce the toxicity to the fetus and the mother, and finally realize the in-vivo treatment effect on PE.

[0009] The present application is realized by the following technical solutions:

[0010] The present application is realized by the following technical solutions:

[0011] Preferably, the enzyme highly expressed in the placental interstitial fluid is matrix metalloproteinase, and the enzyme substrate polypeptide is MCA-Lys-Pro-Leu-Gly-Leu-DNP-Dpa-Ala-Arg-NH2.

[0012] Preferably, the polymer is polyacetylimine copolymerized polylactic acid (PEI-b-PLA) or polyacetylimine copolymerized polycaprolactone (PEI-b-PLA).

[0013] Preferably, the therapeutic drug is a non-steroidal anti-inflammatory immune drug targeting COX-2, and part of the non-steroidal anti-inflammatory drugs has specific COX-2 inhibition effect, which is theoretically suitable for targeted inhibition of MDSC-related inflammation. The non-steroidal anti-inflammatory immune drug targeting COX-2 is preferably acetylsalicylic acid, celecoxib, oxaprozin or etodolac.

[0014] The HIF-1α pathway and the COX-2 pathway have a close interaction relationship in cells. Moreover, the HIF-1α pathway is closely related to the immune regulation of MDSC in the body and is an effective treatment target of MDSC. Through the regulation of the HIF-1α pathway, the COX-2 targeted MDSC treatment is enhanced. Therefore, preferably, the therapeutic gene is siRNA of HIF-1α.

[0015] Preferably, the antibody is a CD11b antibody.

[0016] Further preferably, the molecular imaging contrast agent loaded between the inner layer vesicle and the outer layer vesicle can be used to track the effect of the drug localized in the lesion. The molecular imaging contrast agent includes MRI T1 contrast agent, MRI T2 contrast agent or near-infrared fluorescence contrast agent.

[0017] Preferably, the average total particle size of the delivery system is 200-300 nm.

[0018] The application also provides a preparation method of the above-mentioned immune regulation drug delivery system specifically targeting MDSC cells in lesions, comprising the following steps:

[0019] S1, reacting polylactic acid or polycaprolactone with polyacetylimine to synthesize a polymer;

[0020] S2, adding a therapeutic drug to the polymer, and using a double emulsion method to prepare a polymer vesicle loaded with the therapeutic drug;

[0021] S3, linking an antibody to the polymer vesicle to prepare a polymer vesicle targeting a surface marker of MDSC cells;

[0022] S4, complexing the polymer vesicle prepared in S2 or the polymer vesicle targeting the surface marker of MDSC cells prepared in S3 with a therapeutic gene to prepare a polymer vesicle loaded with a therapeutic drug and / or a therapeutic gene;

[0023] S5, reacting an enzyme substrate polypeptide with mPEG-NHS to obtain an enzyme substrate polypeptide-PEG;

[0024] S6, preparing an enzyme substrate polypeptide-PEG modified liposome vesicle from the enzyme substrate polypeptide-PEG, phospholipid and cholesterol;

[0025] S7, assembling the polymer vesicle loaded with a therapeutic drug and / or a therapeutic gene in S4 and the enzyme substrate polypeptide-PEG modified liposome vesicle in S6 to prepare an immune regulation drug delivery system specifically targeting MDSC cells in lesions.

[0026] The application also provides application of the above-mentioned immune regulation drug delivery system specifically targeting MDSC cells in lesions in preparation of a drug for regulating an immune function disorder related disease.

[0027] Preferably, the immune function disorder related disease is preeclampsia.

[0028] Compared with the prior art, the application has the following technical effects:

[0029] The present application uses the polypeptide-PEG modified lipid-like vesicle as the outer vesicle, and uses the polymer vesicle targeting the MDSC cell surface marker as the inner vesicle to synthesize a double-layer vesicle delivery system. The outer vesicle is sensitive to the lesion microenvironment, and the selective release of the outer vesicle limited to the lesion microenvironment limits the distribution of the MDSC function regulating drug to the lesion, avoids the distribution of the drug to the immune system outside the lesion and the influence on the immune system outside the lesion, and avoids the potential immune side effects of the drug outside the lesion. After the inner vesicle is released by the disintegrated outer vesicle entering the lesion, the antibody targeting the MDSC cell membrane marker is used to anchor the MDSC cells in the lesion, effectively reduces the leakage of the drug through the maternal-fetal barrier, reduces the fetal toxicity caused by the treatment gene and the drug reaching the fetus, and also reduces the maternal toxicity caused by the treatment gene and the drug flowing back to the maternal blood through the uterine vein and reaching the maternal normal tissue; the positive potential on the surface of the inner vesicle promotes the endocytosis of the genes in the vesicle wall and the treatment drug in the vesicle into the MDSC cells, and realizes the regulation of the MDSC function. The distribution of the treatment gene and the treatment drug in other cells in the placenta is reduced, and the influence on the functions of other cells in the placenta is avoided.

[0030] The present application uses the delivery system targeting the MDSC in the lesion to simultaneously deliver the HIF-1α pathway and COX-2 pathway regulating drugs, realizes the regulation of the MDSC in the placenta and the regulation of the immunity in the placenta, reduces the toxicity to the fetus and the mother, and finally realizes the in vivo treatment effect on PE. BRIEF DESCRIPTION OF DRAWINGS

[0031] Fig. 1 is a structural schematic diagram of the delivery system prepared by the present application. DETAILED DESCRIPTION

[0032] The present application will be further described by specific embodiments, and the following embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the following embodiments.

[0033] The main raw materials of the present application are as follows:

[0034] The present application uses the delivery system targeting the MDSC in the lesion to simultaneously deliver the HIF-1α pathway and COX-2 pathway regulating drugs, realizes the regulation of the MDSC in the placenta and the regulation of the immunity in the placenta, reduces the toxicity to the fetus and the mother, and finally realizes the in vivo treatment effect on PE.

[0035] Monomethyl ether polyethylene glycol (mPEG, Mn=2000 Da) Sigma;

[0036] Linear polyethyleneimine (PEI Cas No.: 9002-98-6, Mn=2100 Da) BASF;

[0037] Lactide 99% Shenzhen Guanghua Weiyeh Industry Co., Ltd.

[0038] N,N-carbonyldiimidazole (CDI) AR Sigma-Aldrich

[0039] mal-PEG-COOH Beijing Kairong Pharmaceutical

[0040] Gadopentetic acid meglumine Bayer Healthcare

[0041] siRNA of HIF-1a (sc-35561) SANTA CRUZ

[0042] Acetylsalicylic acid (Cas No.: 50-78-2) MedChemExpress

[0043] mPEG-NHS Sigma-Aldrich

[0044] Anti-CD11b (ab133357) Abcam

[0045] Matrix metalloproteinase substrate polypeptide (SCP0193) MedChemExpress

[0046] Phospholipid Sigma-Aldrich

[0047] Cholesterol (Cas No.: 57-88-5) Sigma-Aldrich

[0048] Particle size test method:

[0049] The 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℃; the average value of three measurement values was taken. Example 1:

[0050] S1. Synthesis of polyacetylene copolymer polylactic acid (PEI-b-PLA)

[0051] First, polylactic acid was synthesized, and then the terminal hydroxyl group was activated with CDI, and then reacted with polyacetylene to synthesize polyacetylene copolymer polylactic acid (PEI-b-PLA). The specific process is as follows:

[0052] In a 250 mL reaction bottle, 135 μL of dried n-dodecanol (0.60 mmol) was added, 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) were added, vacuum dried at room temperature for 4 h, dissolved in 40 mL of freshly distilled anhydrous toluene, and then refluxed at 120°C for 12 h. After cooling, precipitate with a large amount of anhydrous ethanol, freeze in the refrigerator, then centrifugal dry, then dissolve in dichloromethane, precipitate with anhydrous ethanol again, filter and dry to obtain the product PLA.

[0053] The terminal hydroxyl group of PLA was activated with CDI. In a 100 mL reaction bottle, 6.0 g of PLA was added, vacuum dried at 70°C for 4 h to remove water, then dissolved in 40 mL of freshly distilled tetrahydrofuran, and then slowly added to the CDI (0.50 g) tetrahydrofuran solution under argon protection, and then reacted at room temperature for 24 h. Precipitate with a large amount of anhydrous ethanol, freeze in the refrigerator, then centrifugal dry, then dissolve in dichloromethane, precipitate with anhydrous ethanol again, filter and dry to obtain PLA-CDI.

[0054] In a 100 mL reaction bottle, 1.05 g of PEI was added, and then dissolved in 20 mL of freshly distilled chloroform. 4 g of PLA-CDI was dissolved in 30 mL of freshly distilled chloroform, and then added dropwise to the PEI solution under argon protection, and then stirred at room temperature for 24 h. The reaction solution was dialyzed in a dialysis bag (MW = 3.5 kDa) in chloroform to remove excess PEI, precipitated with a large amount of anhydrous ethanol, frozen in the refrigerator, then centrifugal dried to obtain the polymer PEI-b-PLA.

[0055] S2. Preparation of inner-layer polyacetylene copolymer polylactic acid vesicles loaded with therapeutic drugs

[0056] The PEI-b-PLA polymer vesicles loaded with acetylsalicylic acid 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 50% ethanol aqueous solution containing 5 mg of acetylsalicylic acid was slowly added dropwise under ice bath and ultrasonic action. The first emulsion was added dropwise into 20 mL of phosphate buffer (0.02 M, pH 7.4) under ultrasonic action to obtain a second emulsion. The chloroform was removed by rotary evaporation, and the large particles were removed by filtration through a needle cylinder filter (0.45 μm). The PEI-b-PLA polymer vesicles loaded with acetylsalicylic acid were obtained by ultrafiltration and concentration, and the volume was adjusted to 20 mL for standby.

[0057] S3. Preparation of inner-layer polyacetylene co-poly-lactic acid vesicles targeted by antibodies

[0058] First, the CD11b antibody was cleaved by the method in the existing literature to obtain the Fab fragment of CD11b, and then the CD11b-Fab was linked to mal-PEG-COOH, and the PEG with the antibody was reacted with the amino group on the polyacetylene co-poly-lactic acid vesicle nanoparticles by amidation reaction to prepare the polyacetylene co-poly-lactic acid vesicles (Fab-PEI-PLA vesicle nanoparticles) targeted by CD11b-Fab antibodies.

[0059] Specifically, 10 mg of CD11b antibody was weighed and subjected to enzymolysis under the conditions of 0.5 mg·ml-1 of papain, 10 mmol·L-1 of cysteine, 2 mmol·L-1 of EDTA, and pH 7.6 for 4 h. The enzymolysis product was separated by Protein A affinity chromatography, and the breakthrough peak was further purified by DEAE anion exchange chromatography. After dialysis desalting and freeze-drying, the Fab fragment of CD11b with high purity was obtained.

[0060] Weigh 1 mg of the CD11b Fab fragment (Mn = 45 kDa) and pretreat it 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. After removing the dithiothreitol by centrifugation using an ultrafiltration tube with a molecular weight cutoff of 1 kDa, dissolve in 5 mL of PBS solution, add 2 mg of mal-PEG-COOH (Mn = 4 kDa), mix well, and incubate overnight at 4 °C. Finally, remove excess mal-PEG-COOH by centrifugation using an ultrafiltration tube with a molecular weight cutoff of 5 kDa. The carboxyl groups in Fab-PEG-COOH were activated with 500 µg each of EDC and NHS for 15 min. Then, 18 mL of the PEI-b-PLA polymer vesicles prepared in step S2 were added, and the reaction was carried out overnight at 4 °C. Finally, an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kJ was used to remove excess EDC, NHS, and unlinked CD11b Fab fragments. The purified solution in the centrifuge tube was collected, ultrasonically dispersed in distilled water, and brought to a final volume of 18 mL for later use, yielding antibody-targeted inner-layer polyacetylimide copolymer polylactic acid vesicles (Fab-PEI-PLA).

[0061] S4. Preparation of polyacetylimide copolymer polylactic acid vesicles with the inner layer of the composite gene

[0062] A nanocomposite was prepared by electrostatically combining positively charged PEI-b-PLA vesicle nanoparticles prepared in S2 or Fab-PEI-PLA vesicle nanoparticles prepared in S3 with a negatively charged HIF-1α gene. The specific procedure was as follows: 400 μg of the HIF-1α gene was diluted with PBS to a final volume of 4 mL and shaken thoroughly. 4 mL of the PEI-b-PLA vesicle nanoparticle solution prepared in S2 (or the Fab-PEI-PLA nanoparticle solution prepared in S3) was ultrasonically dispersed. The diluted HIF-1α gene solution was mixed with the PEI-b-PLA (or Fab-PEI-PLA) nanoparticle solution. The composite system was then brought to a final volume of 4 mL, mixed thoroughly by pipetting, and allowed to stand for 30 minutes to obtain the inner layer of the composite gene, which is a polyacetylimide copolymer polylactic acid vesicle.

[0063] S5, Synthesis of enzyme substrate polypeptide-PEG

[0064] 0.05 mmol of matrix metalloproteinase substrate peptide can be selected

[0065] MCA-Lys-Pro-Leu-Gly-Leu-DNP-Dpa-Ala-Arg-NH 2，Molecular weight: 1221.32 Da, 5 mmol of EDC and 5 mmol of DMAP were dissolved in 10 mL of acetonitrile aqueous solution (acetonitrile: water = 1:1), activated the peptide under N2 protection on ice water bath, magnetic stirring at 500 rpm for 2 h. After 2 h, 0.5 mmol of mPEG-NHS (molecular weight about 3000 Da) was added, and the reaction was continued for 72 h. After the reaction was completed, the reaction solution was placed in a dialysis bag (MWCO = 3.5 kDa), dialyzed for 72 h, freeze-dried to obtain the product, enzyme substrate polypeptide-PEG.

[0066] S6, enzyme substrate polypeptide-PEG modified liposome-like vesicle@polyacetylene imine copolymerized polylactic acid vesicle

[0067] Enzyme substrate polypeptide-PEG 10 mg, phospholipid 3 mg and cholesterol 20 mg were dissolved in 5 mL of dichloromethane, and the dichloromethane was spun dry by a vacuum rotary evaporator to form a liposome film of enzyme substrate polypeptide-PEG, phospholipid and cholesterol on the wall of a round-bottom flask. Gadopentetic acid meglumine 0.2 mL 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 mixture was added to the liposome film of enzyme substrate polypeptide-PEG, phospholipid and cholesterol at a rate of 0.5 mL / min. After the addition was completed, the stirring was continued for 30 min to allow the liposome and Fab-PEI-PLA vesicle nanoparticles to fully assemble, and then the high-speed centrifugation method was used to separate the liposome loaded with the complex nanoparticles of the therapeutic gene and the empty liposome. Finally, 2 mL of normal saline (0.9% NaCl) solution was added to dissolve the enzyme substrate polypeptide-PEG modified liposome-like vesicle@polyacetylene imine copolymerized polylactic acid vesicle particles, filtered by a 220 nm pore size needle filter, and the volume was adjusted to 2 mL. The particles were stored at 4°C for standby, and an immune regulation drug delivery system specifically targeting MDSC cells in lesions was obtained.

[0068] Example 2-3 and Comparative Example 1-5:

[0069] Compared with Example 1, changing the amount of polymer or drug in step S2 or omitting a certain step in steps S3, S4, S5 or S6 can prepare Example 2-3 or Comparative Example 1-5. See Table 1 below for details:

[0070] Table 1: Examples and Comparative Examples

[0071] S2 polymer feed amount (mg) S2 acetylsalicylic acid feed amount (mg) S3 linked antibody S4 gene S6 contrast agent S6 liposome thin film coating S5 enzyme substrate polypeptide product final particle size (nm) Example 1 15 CD11b-Fab HIF-1a gene gadopentetate meglumine enzyme substrate polypeptide-PEG 10 mg yes 223.5 Example 2 15 CD11b-Fab HIF-1a gene gadopentetate meglumine enzyme substrate polypeptide-PEG 20 mg yes 286.3 Example 3 15 CD11b-Fab HIF-1a gene no enzyme substrate polypeptide-PEG 15 mg yes 256.1 Comparative Example 1 15 no HIF-1a gene gadopentetate meglumine enzyme substrate polypeptide-PEG 10 mg yes 218.8 Comparative Example 2 15 CD11b-Fab HIF-1a gene no no S6 step no 138.2 Comparative Example 3 15 no HIF-1a gene gadopentetate meglumine enzyme substrate polypeptide-PEG 0 mg no 210.4 Comparative Example 4 15 no HIF-1a gene no no S6 step no 135.6 Comparative Example 5 15 CD11b-Fab HIF-1a gene gadopentetate meglumine enzyme substrate polypeptide-PEG 40 mg yes 350.4

[0072] Functional evaluation experiment

[0073] 1. MRI experiment to evaluate placenta-specific delivery function of the drug

[0074] Model establishment:

[0075] SPF level C57BL / 6 mice 8 weeks old (purchased from Guangdong Medical Laboratory Animal Center), female mice and male mice 2:1 in estrus period, the next day the vaginal smear of female mice was stained with Papnikolaou, and the specimen was observed under an optical microscope. The positive specimen of vaginal sperm was diagnosed as pregnancy, and was marked as the 0th day of pregnancy (D0). Pregnant mice were fed with 2 mg / mL nitroso-L-arginine methyl ester in sterile water to establish the pre-eclampsia / pregnancy-induced hypertension model, and the same amount of pregnant mice fed with double distilled water were used as normal control group.

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

[0077] The pre-eclampsia model animals were anesthetized with chloral hydrate on the 11th day, and MRI T1 scanning was performed at the time points of before drug injection (0 h) and 2 hours after injection (2 h) to observe the in vivo distribution of the nano-drug. The dose of nano-drug injected through the tail vein was 0.1 mL of the example drug, or the comparative example drug, or the same volume of PBS.

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

[0079] Table 2 Evaluation results of placenta-specific delivery function

[0080] Placenta RSI (signal relative multiple) Embryo RSI (signal relative multiple) Liver RSI (signal relative multiple) Example 1 1.0 1.0 1.0 Example 2 0.9 1.0 1.2 Example 3 0 0 0 Example 4 0.2 0.2 1.6 Example 5 0 0 0 Example 6 0.4 0.5 1.7

[0081] 2. Establishing a pre-eclampsia animal model to evaluate the treatment effect

[0082] On D3, D6, D9, D12, D15, inject the nanodrug. The dose of the nanodrug injected into the tail vein is 0.1 mL of the example drug, or the comparative drug, or the same volume of PBS. On D17, perform a series of tests, and the test results are shown in Table 3:

[0083] Blood pressure detection: using BP-2000 blood pressure analysis system, using tail cuff method to non-invasively measure the systolic blood pressure (Systolic blood pressure, SBP) of pregnant mice,

[0084] Keep the room temperature at 26 degrees Celsius, set channel 1 to 1V (1V corresponds to 300mmHg), and channel 2 to SmV. Fix the mouse in the mouse cage, place the mouse tail in the 17mm tail cuff, and the bottom of the mouse tail is in the middle of the sensor. Measure 10 times continuously under the quiet state of the pregnant mouse, with an interval of 1s each time, take the average value and make a record;

[0085] Placenta and fetal examination: placenta tissue: anesthetize the pregnant mouse, open the abdominal cavity, dissect the uterus, and then take out the fetus and placenta, and record the number of live fetuses. Remove the fetal membranes and umbilical cord on the placenta, cut off the umbilical cord at the end of the fetus, and place the placenta and fetus on sterile gauze to absorb the surface amniotic fluid, and analyze the balance to weigh the placenta and fetus. Cut the placental tissue and place it in liquid nitrogen, and store it at -80℃.

[0086] Table 3 Evaluation of therapeutic effect of preeclampsia animal model

[0087] Blood pressure (mmHg) Fetal weight (g) Number of offspring per litter Disease PBS 165.1 0.6 2.5 Example 1 109.2 1.6 8.5 Example 2 115.6 1.4 8.2 Example 3 113.3 1.5 8.4 Comparative Example 1 168.1 0.5 2.4 Comparative Example 2 179.3 0.5 2.0 Comparative Example 3 169.5 0.6 2.6 Comparative Example 4 165.3 0.5 2.5 Comparative Example 5 141.3 1.1 5.3

[0088] From the above results of Table 2 and Table 3, it can be seen that in Comparative Example 1, the inner vesicles loaded with HIF-1α siRNA and acetylsalicylic acid did not have the CD11b targeting antibody fragment linked. The HIF-1α siRNA and acetylsalicylic acid as a whole can enter the lesion, and after entering the lesion, the matrix metalloproteinase substrate polypeptide group of the outer vesicle is degraded, the outer vesicle is disintegrated, the contrast agent between the inner and outer vesicles, and the inner vesicle are released in the lesion. The distribution of the contrast agent detected by MRI imaging shows that the contrast agent is targeted to the distribution in the placenta after the disintegration of the outer vesicle with the matrix metalloproteinase substrate polypeptide. Compared with Example 1, the imaging signal is not significantly reduced, the distribution in the embryo and the imaging signal are not significantly increased, and the distribution in the liver and the imaging signal are not significantly increased. The MDSC cells in the animal model are not targeted for treatment, so the therapeutic effect is poor. The detection of the blood pressure of the animal shows that compared with the PBS treatment control group of the disease model, there is no significant improvement, and even a slight increase. This can be due to the non-targeted distribution of HIF-1α siRNA and acetylsalicylic acid in the lesion, which fails to target the MDSC cells, but is phagocytosed by other functional cells, triggering further damage. Further, since the inner vesicles loaded with HIF-1α siRNA and acetylsalicylic acid fail to anchor to the MDSC, part of them pass through the placental barrier and flow into the embryo, resulting in embryo tissue toxicity and reducing the size and number of embryos.

[0089] In Comparative Example 2, there is no outer vesicle, and no contrast agent between the inner and outer vesicles. The inner vesicles loaded with HIF-1a siRNA and acetylsalicylic acid cannot be targeted to the lesion. MRI imaging to detect the distribution of the contrast agent shows that HIF-1a siRNA and acetylsalicylic acid fail to be encapsulated by the outer vesicle and fail to be loaded with the contrast agent. Compared with Example 1, no imaging signal is detected in the lesion, no imaging signal is detected in the embryo, and no imaging signal is detected in the liver. The lesion in the animal model fails to be treated by targeting, so the curative effect is poor. Detection of the blood pressure of the animal shows that the blood pressure is higher than that of the PBS treatment control group in the disease model. This can be due to the fact that HIF-1a siRNA and acetylsalicylic acid, which fail to be targeted to the lesion, are phagocytosed by functional cells in other organs of the whole body, causing further systemic side effects. Further, because the lesion has no pathological changes, and systemic side effects occur, the size and number of embryos are significantly reduced.

[0090] In Comparative Example 3, the outer vesicle has no enzyme-sensitive matrix metalloproteinase substrate polypeptide-PEG, and the inner vesicle has no CD11b-targeting antibody fragment. The outer vesicle is more difficult to disintegrate in the lesion, and the contrast agent between the inner and outer vesicles and the HIF-1a siRNA and acetylsalicylic acid loaded in the inner vesicle are released more slowly, and cannot be targeted to the lesion. The drug-loaded inner vesicles outside the lesion also fail to be targeted to MDSC. MRI imaging to detect the distribution of the contrast agent shows that the contrast agent is encapsulated by the non-lesion-targeting outer vesicle. Compared with Example 1, the imaging signal detected in the lesion is extremely low, the imaging signal detected in the embryo is extremely low, and the imaging signal detected in the liver due to the non-targeted distribution of the contrast agent is extremely high. The lesion in the animal model fails to be treated by targeting, so the curative effect is poor. Detection of the blood pressure of the animal shows that the blood pressure is higher than that of the PBS treatment control group in the disease model. This can be due to the fact that HIF-1a siRNA and acetylsalicylic acid, which fail to be targeted to the lesion, are phagocytosed by functional cells in other organs of the whole body, causing further systemic side effects. Further, because the lesion has no pathological changes, and systemic side effects occur, the size and number of embryos are significantly reduced.

[0091] In Comparative Example 4, there were no outer vesicles and no contrast agent between the inner and outer vesicles. The inner vesicles had no CD11b-targeted antibody fragment. The inner vesicles loaded with HIF-1a siRNA and acetylsalicylic acid failed to achieve focal targeting distribution. The drug-loaded inner vesicles failed to achieve MDSC targeting distribution in and outside the lesion. MRI imaging detected the distribution of the contrast agent, and found that the drug failed to be encapsulated by the outer vesicles and failed to be loaded with the contrast agent. Compared with Example 1, the imaging signal was not detected in the lesion, the imaging signal was not detected in the embryo, and the imaging signal was not detected in the liver. The lesion in the animal model failed to be targeted for treatment, so the therapeutic effect was poor. Detection of animal blood pressure found that the blood pressure was higher than that of the PBS treatment control group in the disease model. This can be due to the HIF-1a siRNA and acetylsalicylic acid that failed to be targeted for distribution in the lesion being phagocytosed by functional cells in other organs of the body, triggering further systemic side effects. In turn, because the lesion had no pathological changes and systemic side effects occurred, the size and number of embryos were significantly reduced.

[0092] In Comparative Example 5, the particle size of the delivery system was > 300 nm, which was not suitable for in vivo distribution, and the efficiency of entering the lesion was low, and the phagocytosis by the reticuloendothelial system was high. A small amount of drug entering the lesion still completed the action of the outer membrane enzyme-sensitive disintegration to release the contrast agent and the inner vesicles loaded with HIF-1a siRNA and acetylsalicylic acid. 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 imaging signal of the lesion was significantly reduced, the distribution and imaging signal in the embryo were significantly reduced, the phagocytosis of the contrast agent by the reticuloendothelial system in the liver was increased, and the distribution and imaging signal in the liver were significantly increased. The lesion in the animal model distributed less inner vesicles and HIF-1a siRNA and acetylsalicylic acid, and the targeted therapy had a poor therapeutic effect. Detection of animal blood pressure found that the blood pressure was significantly lower than that of the PBS treatment control group in the disease model. The embryo improved compared with the disease group, and the size and number were significantly increased.

[0093] Example 1-3 uses matrix metalloproteinase substrate polypeptide-PEG modified niosomes as the outer vesicle, and CD11b-targeted antibody fragment modified polymersomes as the inner vesicle, to synthesize a double-layer vesicle delivery system. The particle size of the delivery system is in the range of 200-300 nm. Its particle size of about 200 nm, and the negative charge of the outer niosome, facilitate avoiding being phagocytosed by the reticuloendothelial system, achieving longer circulation time in vivo, and achieving effective circulation in vivo. Its matrix metalloproteinase substrate polypeptide-PEG modified niosome is stable in the circulation of other tissues and organs in vivo. When it reaches the specific high expression of matrix metalloproteinase in the placental microenvironment, the matrix metalloproteinase substrate polypeptide degrades, and the outer vesicle disintegrates. The contrast agent between the inner and outer vesicles, as well as the inner vesicle loaded with HIF-1α siRNA and acetylsalicylic acid, is released, achieving specific distribution of the contrast agent and HIF-1α siRNA and acetylsalicylic acid in the placental tissue. Avoiding the distribution of the contrast agent and HIF-1α siRNA and acetylsalicylic acid outside the lesion, avoiding its induction of systemic immunotoxicity. After the release of the inner vesicle loaded with HIF-1α siRNA and acetylsalicylic acid, the antibody fragment-containing HIF-1α siRNA and acetylsalicylic acid is exposed. The antibody fragment can be anchored to the membrane marker of MDSC cells in the microenvironment. Further, by taking advantage of the positive charge of the inner vesicle, the MDSC cells specifically endocytose the inner vesicle, and the HIF-1α siRNA and acetylsalicylic acid is specifically introduced into the MDSC. After the HIF-1α siRNA and acetylsalicylic acid is specifically endocytosed by the MDSC cells, the MDSC function regulation is achieved. The distribution of HIF-1α siRNA and acetylsalicylic acid in other cells of the placenta is reduced, avoiding affecting the function of other cells in the placenta. In addition, the CD11b-targeted antibody fragment modification allows the inner vesicle loaded with HIF-1α siRNA and acetylsalicylic acid to be anchored to the MDSC cells, effectively reducing the leakage of HIF-1α siRNA and acetylsalicylic acid through the maternal-fetal barrier, reducing the fetal toxicity caused by HIF-1α siRNA and acetylsalicylic acid reaching the fetus. In addition, it also reduces the HIF-1α siRNA and acetylsalicylic acid backflowing to the maternal blood through the uterine vein, reaching the maternal normal tissues, and causing maternal toxicity. In Example 1-3, after the lesion is treated, the animal's blood pressure is significantly reduced, and the fetal condition is significantly improved, with a significant increase in fetal weight and number. The main difference between Example 3 and Examples 1-2 is that there is no contrast agent between the outer vesicle and the inner vesicle, and no enhanced imaging signal is formed in the specific aggregation lesion and the liver during the entire medication process.

[0094] 3. Toxicity evaluation of nanomedicine for animal models

[0095] The normal control group of mice was injected with nano-drugs for 72 hours, and blood was taken from the tail vein to detect liver function indicators alanine transaminase (ALT), total bilirubin (TBil) and kidney function indicators blood urea nitrogen (BUN) and serum creatinine (sCr). The detection instrument is Hitachi 7600 automatic biochemical analyzer, and the detection results are shown in Table 4.

[0096] Table 4 Toxicity evaluation results

[0097] Liver toxicity (liver function) ALT (U / L) Liver toxicity (liver function) TBIL (μmol / L) Kidney toxicity (kidney function) BUN (mg / dL) Kidney toxicity (kidney function) sCr (mg / dL) Example 1 35.8 0.32 3.7 0.1 Example 2 37.5 0.32 4.1 0.1 Example 3 36.5 0.32 3.9 0.1

[0098] From the above results, in Examples 1-3, the inner vesicles loaded with HIF-1α siRNA and acetylsalicylic acid are first targeted to the lesions due to the matrix metalloproteinase substrate polypeptide modification of the outer vesicles, and then targeted to MDSC in the lesions due to the CD11b targeting antibody fragment on the inner vesicles, avoiding leakage into the fetus or returning to the maternal circulation. Therefore, the liver and kidney in Examples 1-3 are not affected by the side effects of HIF-1α siRNA and acetylsalicylic acid, and the liver function (ALT, TBIL) and kidney function (BUN, sCr) are normal. No obvious toxic side effects on the mother and fetus were observed.

Claims

An immunomodulatory drug delivery system specifically targeting lesion MDSC cells, characterized in that, The delivery system is a double-layer vesicular structure, the inner vesicle is a polymer vesicle targeting MDSC cell surface markers with antibodies, the outer wall of the inner vesicle is positively charged and complexed with an immunomodulatory therapeutic gene, and the inner vesicle is loaded with a therapeutic drug; the outer vesicle is a lipid-like vesicle modified with an enzyme substrate polypeptide-PEG, which can be targeted and disintegrated by a placental tissue interstitial fluid highly expressed enzyme. The immunomodulatory drug delivery system for specifically targeting lesion MDSC cells according to claim 1, wherein, The placental tissue interstitial fluid highly expressed enzyme is a matrix metalloproteinase, and the enzyme substrate polypeptide is MCA-Lys-Pro-Leu-Gly-Leu-DNP-Dpa-Ala-Arg-NH2. The immunomodulatory drug delivery system specifically targeting lesion MDSC cells according to claim 1, characterized in that, The polymer is polyacetylimine copolymerized with polylactic acid or polycaprolactone. The immunomodulatory drug delivery system specifically targeting lesion MDSC cells according to claim 1, characterized in that, The therapeutic drug is a non-steroidal anti-inflammatory immune drug that can target COX-2, preferably acetylsalicylic acid, celecoxib, oxaprozin or etodolac; and the therapeutic gene is an siRNA of HIF-1α. The immunomodulatory drug delivery system for specifically targeting lesion MDSC cells according to claim 1, wherein, The antibody is a CD11b antibody. The immunomodulatory drug delivery system specifically targeting lesion MDSC cells according to claim 1, characterized in that, The inner vesicle and the outer vesicle are loaded with a molecular imaging contrast agent, which includes an MRI T1 contrast agent, an MRI T2 contrast agent or a near-infrared fluorescent contrast agent. The immunomodulatory drug delivery system for specifically targeting lesion MDSC cells according to claim 1, wherein, The average total particle size of the delivery system is 200-300 nm. Process for the preparation of an immunomodulatory drug delivery system specifically targeting lesion MDSC cells according to any one of claims 1 to 7, characterized in that, The method comprises the following steps: S1. Synthesizing a polymer by reacting polylactic acid or polycaprolactone with polyacetylimine; S2. Adding a therapeutic drug to the polymer to prepare a polymer vesicle loaded with the therapeutic drug by double emulsification; S3. Linking an antibody to the polymer vesicle to prepare a polymer vesicle targeting MDSC cell surface markers with antibodies; S4. Complexing the polymer vesicle prepared in S2 or the polymer vesicle targeting MDSC cell surface markers with antibodies prepared in S3 with a therapeutic gene to prepare a polymer vesicle loaded with a therapeutic drug and / or a therapeutic gene; S5. Reacting an enzyme substrate polypeptide with mPEG-NHS to obtain an enzyme substrate polypeptide-PEG; S6. Preparing an enzyme substrate polypeptide-PEG modified lipid-like vesicle from the enzyme substrate polypeptide-PEG, phospholipids and cholesterol; S7. Assembling the polymer vesicle loaded with a therapeutic drug and / or a therapeutic gene in S4 with the enzyme substrate polypeptide-PEG modified lipid-like vesicle in S6 to prepare an immunomodulatory drug delivery system specifically targeting lesion MDSC cells. The use of the immunomodulatory drug delivery system specifically targeting lesion MDSC cells according to any one of claims 1-7 in the preparation of a drug for regulating immune dysfunction related diseases. The use according to claim 9, characterized in that The immune dysfunction related disease is preeclampsia. The immune dysfunction related disease is preeclampsia.

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