Immunoregulation drug delivery system for specifically targeting focus MDSC cells and preparation method and application of immunoregulation drug delivery system

Abstract

The invention discloses an immunoregulation drug delivery system for specifically targeting focus MDSC cells as well as a preparation method and application of the immunoregulation drug delivery system. The delivery system is of a double-layer vesicle structure, an inner-layer vesicle is a polymer vesicle of an antibody targeting MDSC cell surface marker, the outer wall of the inner-layer vesicle is positively charged and loads an immunoregulation therapeutic gene, and a therapeutic drug is loaded in the inner-layer vesicle; the outer-layer vesicle is an enzyme substrate polypeptide-PEG (Polyethylene Glycol) modified liposomal vesicle which can be disintegrated in a targeted manner under the action of a placenta interstitial fluid high-expression enzyme. The delivery system provided by the invention can realize regulation and control of MDSC in the placenta and regulation and control of immunity in the placenta, avoids influence on functions of other cells in the placenta, can effectively reduce medicine leakage through maternal and fetal barriers, and reduces toxicity to fetuses and maternal bodies.

Description

Technical Field

[0001] The present invention relates to the fields of immunology, chemistry and biomedical engineering, and in particular to an immunomodulatory drug delivery system specifically targeting lesion MDSC cells, 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.

[0003] The latest research has found that MDSC plays an early, inducing, and dominant role in the changes of immune-related diseases, and inhibits immune responses through multiple mechanisms. Abnormal MDSC function often induces immune stress in the lesions, guides changes in downstream T cells, dNK cells, and other cells, and leads to immune killing; conversely, pathological activation of MDSC function leads to an immunosuppressive state. Therefore, MDSC can be used as an effective target for immunotherapy.

[0004] Existing drugs kill a variety of other immune cells when killing MDSC, making it difficult to achieve specific inhibition, with strong side effects and difficult application. Numerous studies have shown that COX-2 inhibition therapy can achieve intervention on MDSC through a pathway dominated by PG-E2 (prostaglandin E2). 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.]. Some non-steroidal anti-inflammatory drugs have specific COX-2 inhibitory effects and are theoretically suitable for targeted inhibition of MDSC-related inflammation. Non-steroidal anti-inflammatory immune drugs that specifically target COX-2 mainly include acetylsalicylic acid, celecoxib, oxaprozin, etodolac, etc. However, when existing immune drugs including COX-2 non-steroidal anti-inflammatory drugs are used in vivo, there is a common problem of producing immune regulation effects both inside and outside the lesion, limiting efficacy and causing toxicity. The distribution of immune drugs outside the lesions leads to widespread immunotoxicity, which limits the increase in drug dosage; it also significantly reduces the distribution ratio of drugs in lesions such as the placenta, thereby reducing the efficacy.

[0005] The placenta is the only channel for the exchange of substances between the mother and the fetus, and 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), diabetes, etc. 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, let alone distribution in the placenta to be effective. If the scope of immune cell function regulation can be limited to the local diseased placenta, it will effectively reduce the toxicity of immune drugs to pregnant women, making effective immunotherapy of placental diseases possible, and significantly improving the current situation where there is no treatment for placental dysfunction.

[0006] At present, many clinical trials related to preeclampsia at home and abroad have confirmed that non-steroidal anti-inflammatory immune drugs specifically targeting COX-2, represented by aspirin (acetylsalicylic acid), have a relatively clear therapeutic effect on preeclampsia. However, in clinical trials, when non-steroidal anti-inflammatory immune drugs targeting COX-2, such as aspirin, were used to treat pregnant women with PE, it was found that the use of conventional doses for non-pregnant women may lead to side effects including maternal bleeding, fetal bleeding, and immune disorders. Therefore, the current mainstream clinical trials are all exploring the lowest dose that can be effective in PE to avoid side effects [Lancet. 2020; 395(10220): 285-293]. However, this low-dose drug dosage restriction leads to low treatment efficiency, and many clinical trials have failed to observe improvements in the overall survival rate of pregnant women with PE [JAMA Pediatr. 2019; 173(7): 619-620.; JAMA. 2021; 326(12): 1153-1155]. Therefore, it is necessary to improve the distribution of non-steroidal anti-inflammatory drugs targeting COX-2 in the placenta of pregnant women with PE through drug dosage forms and administration methods to achieve better therapeutic effects. Summary of the invention

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide an immune regulatory drug delivery system that specifically targets MDSC cells in lesions, as well as a preparation method and application thereof, so as to use the delivery system targeting MDSCs in lesions to simultaneously deliver HIF-1α pathway and COX-2 pathway regulatory drugs, thereby achieving regulation of MDSCs in the placenta and regulation of immunity in the placenta, reducing toxicity to the fetus and mother, and ultimately achieving an in vivo therapeutic effect on PE.

[0008] The present invention is achieved through the following technical solutions:

[0009] The invention discloses an immunomodulatory drug delivery system specifically targeting lesion MDSC cells. The delivery system is a double-layer vesicle structure. The inner vesicle is a polymer vesicle with an antibody targeting an MDSC cell surface marker. The outer wall of the inner vesicle is positively charged and contains a composite immunomodulatory therapeutic gene. The inner vesicle is loaded with a therapeutic drug. The outer vesicle is an enzyme substrate polypeptide-PEG-modified liposome vesicle that can be targeted and disintegrated under the action of an enzyme highly expressed in the interstitial fluid of the placental tissue.

[0010] 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.

[0011] Preferably, the polymer is poly(ethyleneimine)-co-poly(lactic acid) (PEI-b-PLA) or poly(ethyleneimine)-co-poly(caprolactone) (PEI-b-PLA).

[0012] Preferably, the therapeutic drug is a non-steroidal anti-inflammatory immune drug that can target COX-2. Some non-steroidal anti-inflammatory drugs have a specific COX-2 inhibitory effect and are theoretically suitable for targeted inhibition of MDSC-related inflammation. The non-steroidal anti-inflammatory immune drug that can target COX-2 is preferably acetylsalicylic acid, celecoxib, oxaprozin or etodolac.

[0013] The HIF-1α pathway and the COX-2 pathway have a close interaction in the cell. Moreover, the HIF-1α pathway is closely related to the immune regulation related to MDSC in the body and is an effective therapeutic target for MDSC. By regulating the HIF-1α pathway, the COX-2 targeted MDSC treatment is enhanced. Therefore, preferably, the therapeutic gene is siRNA of HIF-1α.

[0014] Preferably, the antibody is a CD11b antibody.

[0015] Further preferably, the molecular imaging contrast agent is loaded between the inner layer vesicle and the outer layer vesicle, and the effect of the drug distribution confined to the lesion can be traced by the molecular imaging contrast agent loaded between the inner layer and the outer layer vesicle. The molecular imaging contrast agent includes MRI T1 contrast agent, MRI T2 contrast agent or near-infrared fluorescent contrast agent.

[0016] Preferably, the average total particle size of the delivery system is 200-300 nm.

[0017] The present invention also provides a method for preparing the above-mentioned immune regulation drug delivery system specifically targeting lesion MDSC cells, comprising the following steps:

[0018] S1, reacting polylactic acid or polycaprolactone with polyacetimide to synthesize a 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 polymer vesicle with the antibody targeting the MDSC cell surface marker;

[0021] S4, compounding the polymer vesicles prepared in S2 or the polymer vesicles with antibodies targeting MDSC cell surface markers prepared in S3 with therapeutic genes to prepare polymer vesicles loaded with therapeutic drugs and / or therapeutic genes;

[0022] S5, reacting the enzyme substrate polypeptide with mPEG-NHS to obtain 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 enzyme substrate polypeptide-PEG-modified liposome vesicles in S6 to prepare an immunomodulatory drug delivery system that specifically targets lesion MDSC cells.

[0025] The present invention also provides the use of the above-mentioned immune regulation drug delivery system specifically targeting lesion MDSC cells in the preparation of drugs for regulating diseases related to immune dysfunction.

[0026] Preferably, the immune dysfunction-related disease is preeclampsia.

[0027] Compared with the prior art, the present invention has the following technical effects:

[0028] The present invention uses enzyme substrate polypeptide-PEG modified liposome vesicles as outer vesicles, and uses antibody-targeted MDSC cell surface marker polymer vesicles as inner vesicles to synthesize a double-layer vesicle delivery system. The outer vesicle is sensitive to the lesion microenvironment, and the selective release of the outer vesicles confined to the lesion microenvironment is used to confine the distribution of the MDSC function regulation drug to the lesion, 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 vesicle is released by the outer vesicle that enters the lesion and disintegrates, the antibody targeting the MDSC cell membrane marker is used to anchor the MDSC cells in the lesion, effectively reducing the drug from leaking through the maternal-fetal barrier, reducing the fetal toxicity caused by the therapeutic gene and drug after reaching the embryo, and also reducing the therapeutic gene and drug from flowing back to the maternal blood through the uterine vein, and causing maternal toxicity after reaching the normal tissue of the mother; using the positive potential on the surface of the inner vesicle, the genes of the cyst wall of the MDSC cell and the therapeutic drugs in the vesicle are promoted to be endocytosed into the MDSC cell, and the MDSC function regulation is realized. The distribution of therapeutic genes and therapeutic drugs in other cells of the placenta is reduced to avoid affecting the functions of other cells in the placenta.

[0029] The present invention uses a delivery system that targets MDSCs in lesions and simultaneously delivers HIF-1α pathway and COX-2 pathway regulating drugs, thereby achieving regulation of MDSCs in the placenta and regulation of immunity in the placenta, reducing toxicity to the fetus and mother, and ultimately achieving an in vivo therapeutic effect on PE. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the structure of the delivery system prepared by the present invention. DETAILED DESCRIPTION

[0031] 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.

[0032] The main raw material sources of the present invention are as follows:

[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 layer poly(acetimide-co-lactic acid) vesicles loaded with therapeutic drugs

[0043] The 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 in an ice bath and under ultrasonic action. The first emulsion was added dropwise to 20 mL of phosphate buffer (0.02 M, pH 7.4) under ultrasonic action to obtain a secondary emulsion, the chloroform was evaporated off, and large particles were removed by filtering through a syringe filter (0.45 μm), and the PEI-b-PLA polymer vesicles loaded with acetylsalicylic acid were obtained by ultrafiltration and concentration, and the volume was fixed to 20 mL for use.

[0044] S3. Preparation of antibody-targeted inner-layer poly(acetimide-co-lactic acid) vesicles

[0045] First, the CD11b antibody is cleaved by the method in the existing literature to obtain the Fab segment of CD11b and purify it. Then, CD11b-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 CD11b-Fab antibody-targeted poly(acetimide-co-polylactic acid) vesicles (Fab-PEI-PLA vesicle nanoparticles);

[0046] The specific operation is as follows: weigh 10 mg of CD11b antibody and enzymatically hydrolyze it for 4 hours under the conditions of 0.5 mg·ml-1 papain, 10 mmol·L-1 cysteine, 2 mmol·L-1 EDTA, pH 7.6. The enzymatic hydrolyzate is separated by Protein A affinity chromatography, and the penetration peak is further purified by DEAE anion exchange chromatography. After dialysis, desalting and freeze-drying, a CD11b Fab fragment with high purity is obtained;

[0047] Weigh 1 mg of CD11b Fab fragment (Mn = 45 kDa) and pretreat with EDTA solution (500 μL 0.5 M) at 4 ° C for 15 min. Add 5 ml of PBS solution to dissolve, add 1 mg of dithiothreitol, and react at 25 ° C for 30 min. After centrifugation with a centrifugal ultrafiltration tube with a molecular weight cutoff of 1k to remove dithiothreitol, add 5 ml of PBS solution to dissolve, then add mal-PEG-COOH (2 mg, Mn = 4k) and mix evenly, and place at 4 ° C overnight. Centrifuge with a centrifugal ultrafiltration tube with a molecular weight cutoff of 5k to remove excess mal-PEG-COOH. Use 500 μg of EDC and NHS respectively to activate the carboxyl groups in Fab-PEG-COOH for 15 minutes, then add 18 mL of PEI-b-PLA polymer vesicles prepared in step S2, 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 CD11b 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-targeted inner layer poly(acetimide) copolymer poly(lactic acid) vesicles (Fab-PEI-PLA).

[0048] S4. Preparation of poly(lactic acid)-co-acetimidylene vesicles containing genes

[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 HIF-1α gene through electrostatic interaction to form a nanocomposite. The specific operation is as follows: 400 μg of HIF-1α 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 HIF-1α 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 the inner layer of poly(acetimide-co-polylactic acid) vesicles 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 @ poly(acetimide) copolymerized poly(lactic acid) 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 by vacuum rotary true hair dryer to form a liposome film on the wall of round-bottom flask with enzyme substrate polypeptide-PEG, phospholipid and cholesterol. 0.2 mL of gadopentetate dimeglumine 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 @ poly(acetimide) copolymer poly(lactic acid) vesicle particles, filtered through a syringe filter with a pore size of 220 nm, fixed to 2 mL, and stored at 4°C for later use to obtain an immune regulatory drug delivery system that specifically targets lesion MDSC cells.

[0054] Examples 2-3 and Comparative Examples 1-5:

[0055] 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:

[0056] Table 1: Examples and Comparative Examples

[0057]

[0058] Functional evaluation experiment

[0059] 1. Magnetic resonance imaging (MRI) experiments to evaluate the placenta-specific delivery function of drugs

[0060] Model building:

[0061] 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.

[0062] MRI imaging to detect the placental distribution of drugs:

[0063] 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.

[0064] 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 T1map 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. 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.

[0065] Table 2 Results of placenta-specific delivery function evaluation

[0066]

[0067] 2. Establishing an animal model of preeclampsia to evaluate the therapeutic effect

[0068] 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:

[0069] 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;

[0070] 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℃.

[0071] Table 3 Evaluation of therapeutic effects in animal models of preeclampsia

[0072]

[0073]

[0074] From the results of Tables 2 and 3 above, it can be seen that there is no CD11b targeting antibody fragment linked to the inner vesicles loaded with HIF-1α siRNA and acetylsalicylic acid in Comparative Example 1. HIF-1α siRNA and acetylsalicylic acid can enter the lesion as a whole. After entering the lesion, the matrix metalloproteinase substrate polypeptide group of the outer vesicle is degraded, the outer vesicle disintegrates, and the contrast agent between the inner and outer vesicles, as well as 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 vesicles targeted by the matrix metalloproteinase substrate polypeptide, and obtained 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. The MDSC cells in the animal model were not treated with targeted therapy, so the efficacy was poor. The blood pressure of the animals was detected and it was 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 non-targeted HIF-1α siRNA and acetylsalicylic acid in the lesions failed to enter the MDSC cells, but were engulfed by other functional cells, causing further damage. In addition, since the inner vesicles loaded with HIF-1α siRNA and acetylsalicylic acid failed to anchor to MDSC, some of them passed through the placental barrier and flowed into the embryo, causing embryonic tissue toxicity and reducing the size and number of embryos.

[0075] 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 HIF-1α siRNA and acetylsalicylic acid cannot be targeted and distributed in the lesions. MRI imaging detected the distribution of contrast agents and found that HIF-1α siRNA and acetylsalicylic acid were not wrapped by the outer vesicles and failed to load the contrast agent. Compared with Example 1, no imaging signal was detected in the lesions, no imaging signal was detected in the embryos, and no imaging signal was detected in the liver. The lesions in the animal model were not targeted for treatment, so the efficacy was poor. Animal blood pressure was detected and found to be higher than the PBS treatment control group of the disease model. This may be because the HIF-1α siRNA and acetylsalicylic acid that failed to be targeted and distributed in the lesions were engulfed by functional cells in other organs throughout the body, causing further systemic side effects. Furthermore, since there were no pathological changes in the lesions and systemic side effects occurred, the size and number of embryos were significantly reduced.

[0076] In comparative example 3, the outer vesicles do not have enzyme-sensitive matrix metalloproteinase substrate polypeptide-PEG, and the inner vesicles do not have CD11b targeting antibody fragments. The outer vesicles are difficult to disintegrate in the lesion, and the contrast agent between the inner and outer vesicles and the HIF-1α siRNA and acetylsalicylic acid loaded by the inner vesicles are released slowly, and the lesion-targeted distribution cannot be achieved. The drug-loaded inner vesicles outside the lesion cannot achieve MDSC targeted distribution. MRI imaging detected the distribution of the contrast agent 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 was extremely low, the imaging signal detected by the embryo was extremely low, and the imaging signal caused by the non-targeted distribution contrast agent detected in the liver was extremely high. The lesions in the animal model were not targeted for treatment, so the efficacy was poor. The blood pressure of the animals was detected and found to be higher than that of the PBS treatment control group of the disease model. This may be due to the failure of HIF-1α siRNA and acetylsalicylic acid to be targeted and distributed in the lesion to be engulfed by functional cells in other organs throughout the body, causing further systemic side effects. Furthermore, since there were no pathological changes in the lesions and systemic side effects occurred, the size and number of embryos were significantly reduced.

[0077] There are no outer vesicles in Comparative Example 4, and there is no contrast agent between the inner and outer vesicles. The inner vesicles do not have CD11b targeting antibody fragments. The inner vesicles loaded with HIF-1α siRNA and acetylsalicylic acid cannot achieve targeted distribution of lesions. The drug-loaded inner vesicles inside and outside the lesion cannot achieve MDSC targeted distribution. MRI imaging detected the distribution of contrast agents and found that the drug failed to be 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 lesions in the animal model were not treated with targeted therapy, so the efficacy was poor. Animal blood pressure was detected and found to be higher than the PBS treatment control group of the disease model. This may be because the HIF-1α siRNA and acetylsalicylic acid that failed to be targeted and distributed in the lesions were engulfed by functional cells in other organs throughout the body, causing further systemic side effects. Furthermore, since there were no pathological changes in the lesions and systemic side effects occurred, the size and number of embryos were significantly reduced.

[0078] The particle size of the delivery system in Comparative Example 5 is greater than 300nm, which is not suitable for in vivo distribution, has a low efficiency in entering the lesion, and a high proportion of being engulfed by the reticuloendothelial system. A small amount of drugs entering the lesion can still complete the sensitive disintegration of the outer membrane enzyme, release the contrast agent and the inner vesicles containing HIF-1α siRNA and acetylsalicylic acid. MRI imaging detected the distribution of the contrast agent and found that the distribution of the contrast agent in the lesion 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 lesions in the animal model were distributed with fewer inner capsules, HIF-1α siRNA and acetylsalicylic acid, and the targeted treatment effect was poor. Animal blood pressure was detected and found to be significantly lower than the PBS treatment control group of the disease model. The embryo improved compared with the disease group, and the size and number were significantly recovered.

[0079] Example 1-3 uses matrix metalloproteinase substrate polypeptide-PEG-modified liposome vesicles as outer vesicles, and uses CD11b targeting antibody fragment-modified polymer vesicles as inner vesicles to synthesize a double-layer vesicle delivery system. 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, thereby extending the in vivo circulation time and achieving effective circulation in the body. Its substrate matrix metalloproteinase substrate polypeptide-PEG-modified liposome vesicles are stable in the circulation of other tissues and organs in the body. When reaching the placental microenvironment with high expression of specific matrix metalloproteinases, the matrix metalloproteinase substrate polypeptide is degraded and the outer vesicles disintegrate. The contrast agent between the inner and outer vesicles and the inner vesicles encapsulating HIF-1α siRNA and acetylsalicylic acid are released to achieve the specific distribution of contrast agent, HIF-1α siRNA and acetylsalicylic acid in placental tissue. The contrast agent, HIF-1α siRNA and acetylsalicylic acid are avoided from being distributed outside the lesion, thus avoiding their induction of systemic immunotoxicity. After the inner vesicles encapsulating HIF-1α siRNA and acetylsalicylic acid are released, HIF-1α siRNA and acetylsalicylic acid containing antibody fragments are exposed. In the microenvironment, the antibody fragments can be anchored to the membrane markers of MDSC cells. Furthermore, the positive charge characteristics of the inner vesicles are utilized to stimulate the MDSC cells to specifically internalize the inner vesicles, so that HIF-1α siRNA and acetylsalicylic acid can specifically enter the MDSC. After HIF-1α siRNA and acetylsalicylic acid are specifically internalized by MDSC cells, the function of MDSC is regulated. The distribution of HIF-1α siRNA and acetylsalicylic acid in other cells of the placenta is reduced, avoiding the impact on the functions of other cells in the placenta. In addition, the CD11b targeting antibody fragment is modified to anchor the inner capsule containing HIF-1α siRNA and acetylsalicylic acid to the MDSC cells, effectively reducing the leakage of HIF-1α siRNA and acetylsalicylic acid through the maternal-fetal barrier, and reducing the fetal toxicity caused by HIF-1α siRNA and acetylsalicylic acid after reaching the embryo. In addition, it also reduces the return of HIF-1α siRNA and acetylsalicylic acid to the maternal blood through the uterine vein, and causes maternal toxicity after reaching the normal tissue of the mother. In Examples 1-3, after the lesions were treated, the blood pressure of the animals was significantly reduced, and the fetal condition was significantly improved, with a significant increase in fetal weight and number. The main difference between Example 3 and Example 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 specifically aggregated lesions and non-specifically aggregated livers during the entire medication process.

[0080] 3. Toxicity evaluation of nanomedicine in animal models

[0081] 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.

[0082] Table 4 Toxicity evaluation results

[0083]

[0084] From the above results, it can be seen that in Examples 1-3, the inner vesicles loaded with HIF-1α siRNA and acetylsalicylic acid are first targeted and distributed in the lesions due to the modification of the matrix metalloproteinase substrate polypeptide of the outer vesicles, and then due to the CD11b targeting antibody fragment on the inner vesicles, the MDSC in the lesions are targeted and distributed to avoid leakage into the fetus or return to the maternal circulation. Therefore, the liver and kidneys 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

1. An immune-regulating drug delivery system specifically targeting lesion MDSC cells, characterized in that: The delivery system is a double-layer vesicle structure, wherein the inner vesicle is a polymer vesicle that targets MDSC cell surface markers with antibodies, the outer wall of the inner vesicle is positively charged and contains a composite immune regulation therapeutic gene, and the inner vesicle is loaded with therapeutic drugs; and the outer vesicle is an enzyme substrate polypeptide-PEG-modified liposome vesicle that can be targeted and disintegrated under the action of an enzyme highly expressed in the interstitial fluid of the placental tissue.

2. The immune-regulating drug delivery system specifically targeting lesion MDSC cells according to claim 1, characterized in that: 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.

3. The immune-regulating drug delivery system specifically targeting lesion MDSC cells according to claim 1, characterized in that: The polymer is polyacetimide copolymerized with polylactic acid or polyacetimide copolymerized with polycaprolactone.

4. The immune-regulating 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; the therapeutic gene is siRNA of HIF-1α.

5. The immune-regulating drug delivery system specifically targeting lesion MDSC cells according to claim 1, characterized in that: The antibody is a CD11b antibody.

6. The immune-regulating drug delivery system specifically targeting lesion MDSC cells according to claim 1, characterized in that: Molecular imaging contrast agents are loaded between the inner layer vesicles and the outer layer vesicles, and the molecular imaging contrast agents include MRIT1 contrast agents, MRI T2 contrast agents or near-infrared fluorescent contrast agents.

7. The immune-regulating drug delivery system specifically targeting lesion MDSC cells according to claim 1, characterized in that: The average overall particle size of the delivery system is 200-300 nm.

8. The method for preparing the immunomodulatory drug delivery system specifically targeting lesion MDSC cells according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. synthesizing a polymer by reacting polylactic acid or polycaprolactone with polyacetimide; S2. adding the therapeutic drug to the polymer and preparing polymer vesicles loaded with the therapeutic drug by double emulsification method; S3. linking the antibody to the polymer vesicle to prepare the polymer vesicle with antibody targeting MDSC cell surface markers; S4. Compounding the polymer vesicles prepared in S2 or the polymer vesicles containing antibodies targeting MDSC cell surface markers prepared in S3 with therapeutic genes to prepare polymer vesicles loaded with therapeutic drugs and / or therapeutic genes; S5. reacting the enzyme substrate polypeptide with mPEG-NHS to obtain enzyme substrate polypeptide-PEG; S6. preparing enzyme substrate polypeptide-PEG, phospholipid and cholesterol to obtain enzyme substrate polypeptide-PEG-modified liposome vesicles; S7. The polymer vesicles loaded with therapeutic drugs and / or therapeutic genes in S4 are assembled with the liposome vesicles modified with enzyme substrate polypeptide-PEG in S6 to prepare an immunomodulatory drug delivery system that specifically targets lesion MDSC cells.

9. Use of the immune-regulating drug delivery system specifically targeting lesion MDSC cells according to any one of claims 1 to 7 in the preparation of drugs for regulating diseases related to immune dysfunction.

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

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