Folic acid-polylactic acid double-layer vesicle delivery system for targeting MDSC cells in focus as well as preparation method and application of folic acid-polylactic acid double-layer vesicle delivery system

Abstract

The invention discloses a folic acid-polylactic acid double-layer vesicle delivery system for targeting MDSC cells in a focus and a preparation method and application of the folic acid-polylactic acid double-layer vesicle delivery system, the folic acid-polylactic acid double-layer vesicle delivery system comprises an inner-layer vesicle and an outer-layer vesicle, and a contrast agent is contained between the inner-layer vesicle and the outer-layer vesicle; the inner-layer vesicle is formed by self-assembling two block polymers, the outer wall of the inner-layer vesicle is positively charged, the inner-layer vesicle is compounded with an immunoregulation therapeutic gene through electrostatic interaction, the surface of the inner-layer vesicle is modified by an antibody targeting an MDSC cell surface marker, and a therapeutic drug is loaded in the inner-layer vesicle; the outer vesicle is formed by a liposome modified by targeted disintegration enzyme substrate polypeptide-PEG (Polyethylene Glycol). According to the double-layer vesicle delivery system, the delivery regulation range of the COX-2 inhibitor and folic acid targeted immunotherapy can be limited to local lesion placenta, the toxicity of immune drugs to pregnant women is effectively reduced, effective immunotherapy of placental diseases is made possible, the current situation that no treatment means are provided for placental dysfunction diseases is remarkably improved, and the application prospect is wide. 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 folic acid-polylactic acid double-layer vesicle delivery system targeting MDSC cells in lesions, and a preparation method and application thereof. Background Art

[0002] Fetal growth restriction (FGR) is one of the most common complications in obstetrics and is associated with placental hypoperfusion, adverse pregnancy outcomes, and long-term poor prognosis. 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. 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 in the mother and fetus, and it is even more impossible to achieve distribution only in the placenta to be effective.

[0003] 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, they 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.

[0004] Myeloid-derived suppressor cells (MDSC) are a group of immature, poorly differentiated cells with immunosuppressive functions such as T cell suppression. The latest studies have found that MDSC plays an early, inducing, and dominant role in the changes of immune-related diseases, and suppresses immune responses through multiple mechanisms. Abnormalities in MDSC function often induce immune stress in lesions, guide changes in downstream T cells, dNK and other cells, and lead 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. However, 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.

[0005] Drugs that inhibit COX2 can achieve immunotherapy by inhibiting PGE2 to inhibit MDSC function [Eur J Immunol. 2021 May; 51(5): 1110-1125.]. PGE2 is a pro-inflammatory molecule associated with G-protein coupled receptors (GPCRs), and is also an important bioactive lipid and active product of cyclooxygenase 2 (COX-2) [Immunol Invest. 2012; 41(6-7): 635-57.]. The COX-2 catalytic metabolic pathway involves the conversion of arachidonic acid into an unstable intermediate PGG2, and then into endoperoxide H2 (PGH2) [Cancer Immunol Res. 2016 Apr; 4(4): 303-11.]. Subsequently, it is converted into five primary prostaglandins (TXA2, PGD2, PGE2, PGF2a, and PGI2) by cell-specific synthases [Proc Natl Acad Sci US A. 2017 Jan 31; 114(5): 1117-1122.]. In MDSCs, PGE2 signals through the PGE2 receptor E-prostaglandin 4 (EP4) and upregulates the activity of arginase 1 in the cells, thereby enhancing its immunosuppressive effect [Cancers (Basel). 2021 Mar 16; 13(6): 1323.]. The production of COX-2 can increase the proliferation of MDSCs and is associated with the upregulation of arginase-1 and iNOS expression in leukocytes. PGE2 promotes the expression of COX2 in MDSCs [Blood. 2011 Nov 17; 118(20): 5498-505; Exp Hematol Oncol. 2022 Nov 8; 11(1): 88.]. COX-2 selective inhibitors and traditional non-steroidal anti-inflammatory drugs (NSAIDS) have been shown to inhibit immune escape. COX-2 inhibitors may stimulate type I immune responses by inhibiting MDSCs function. Regulating the metabolic pathway of arachidonic acid to PGE2 through COX-2 may be a therapeutic approach to control lipid accumulation and regulate the immunosuppressive function of MDSCs [Cancer Res. 2018 Oct 1; 78(19): 5586-5599.; BMC Cancer. 2010 Aug 30; 10: 464.].

[0006] When existing immunomodulatory drugs are used in vivo, there is a common problem of producing immunomodulatory effects both inside and outside the lesion, limiting efficacy and causing toxicity. The distribution of immune drugs outside the lesion 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, reducing efficacy.

[0007] Folic acid is a vitamin that is essential for human cell growth. It can be used to treat anemia caused by folic acid deficiency and is also a nutrient supplement for pregnant women. Folic acid participates in the metabolism of amino acids and nucleic acids and plays an important role in cell proliferation, tissue differentiation and body growth and development [Int J Mol Sci. 2023Jan 22; 24(3): 2220.] Folic acid cannot be synthesized in the human body and can only be obtained from food. The physiological changes of the mother during pregnancy and the growth and development of the fetus lead to an increase in the need for folic acid [Mech Ageing Dev. 2010Apr; 131(4): 236-41.]. Folic acid is essential for cell function and cell division, and can also assist in DNA and protein synthesis, DNA repair, and methylation [Antioxid Redox Signal. 2012Jul 15; 17(2): 302-26.; Eur J Pharm Biopharm. 2017Nov; 120: 133-145.].

[0008] Folic acid is also widely involved in the proliferation of immune cells and the regulation of immune function in the body. Folic acid deficiency can lead to low serine levels, thereby interfering with antibody production and the normal function of effector T cells [Cell. 2022 Mar 31; 185 (7): 1189-1207.e25.]. Folic acid supplementation can improve immune function, but excessive supplementation can also cause immune disorders. Folic acid can inhibit the function of Hcy-guided NF-kB (a factor that can genetically regulate proinflammatory cytokines) in human monocytes [J Immunother Cancer. 2019 Aug 8; 7 (1): 215.]. Folic acid and B12 deficiency can double the expression of inflammatory mediators (IL-1, IL-6, monocyte chemoattractant protein 1 [MCP-1], TNFa) at the RNA and protein levels [Nat Commun. 2021 Feb 9; 12 (1): 877.]. Folic acid and B12 deficiency also reduce T cell proliferation, causing their cell cycle to stop at the S phase, cell apoptosis, increase the level of uracil in DNA and then reduce CD8+ T cell proliferation, leading to an imbalance in the CD4+ / CD8+ ratio [Immunity. 2022Jan 11; 55(1): 1-3]. CD8+ is a marker of cytotoxic T cells. This type of T cell can release cytokines such as TNF-a and IFN-γ, or release toxic cell granules (perforin and granzyme) or Fas / FasL pathway to cause cell apoptosis [J Nutr Biochem 2017Mar; 41: 65-72.; Oncoimmunology. 2022Aug 20; 11(1): 2113697.]. Too high or too low folic acid and B12 levels will have a negative effect on NK cells [Melanoma Res. 2016 Aug; 26 (4): 329-37.], which will cause abnormal hematopoietic function, resulting in cell cycle dysfunction, DNA damage, and NK cell function damage [Cancer Immunol Res. 2016 Apr; 4 (4): 323-336.; Nat Commun. 2021 Mar 29; 12 (1): 1940.]. Therefore, the immunotherapy effect based on MDSC can be achieved by effectively regulating folic acid levels. At the same time, it is also possible to achieve the combined targeted regulation effect of COX-2 through folic acid targeted therapy [J Hazard Mater. 2022 Feb 15; 424 (Pt B): 127354.; J Physiol Pharmacol. 2018 Jun; 69 (3).; Biomaterials. 2019 Jul; 207: 23-38.].

[0009] However, there is currently a lack of nanocarrier systems that can limit the delivery and regulation of COX-2 inhibitors and folic acid targeted immunotherapy to the local area of ​​the diseased placenta, avoid affecting immune cells outside the lesion, and then specifically deliver drugs and regulate the function of immune cells such as MDSC in the immune microenvironment within the lesion. Summary of the invention

[0010] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide a folic acid-polylactic acid double-layer vesicle delivery system targeting MDSC cells in the lesion. The double-layer vesicle delivery system can limit the scope of COX-2 inhibitor and folic acid targeted immunotherapy delivery regulation to the local area of ​​the diseased placenta, effectively reduce the toxicity of immune drugs to pregnant women, make effective immunotherapy of placental diseases possible, significantly improve the current situation that there is no treatment for placental dysfunction diseases, and provide new ideas for effective immunotherapy of placental dysfunction diseases.

[0011] The present invention is achieved through the following technical solutions:

[0012] On the one hand, the present invention provides a folic acid-polylactic acid bilayer vesicle delivery system targeting MDSC cells in lesions, the delivery system comprising an inner vesicle and an outer vesicle, wherein a molecular imaging contrast agent is contained between the inner vesicle and the outer vesicle; the inner vesicle is self-assembled from a diblock polymer PEI-b-PLA or PEI-b-PLA, the outer wall of the inner vesicle is positively charged and complexes the immunoregulatory COX-2 inhibitory therapeutic gene through electrostatic action, the surface of the inner vesicle is modified with an antibody targeting an MDSC cell surface marker, and the inner vesicle is loaded with a therapeutic drug; the outer vesicle is formed by a liposome modified with a protein kinase C substrate polypeptide-PEG targeted for disintegration.

[0013] Preferably, the therapeutic drug is folic acid, and the therapeutic gene is siRNA of COX-2. The present invention achieves the immunotherapy effect based on MDSC by effectively regulating the level of folic acid. At the same time, the combined targeted regulation effect of COX-2 is achieved through folic acid targeted therapy.

[0014] Preferably, the antibody targeting the MDSC cell surface marker is CD14.

[0015] Preferably, the protein kinase C substrate polypeptide is Glu-Arg-Met-Arg-Pro-Arg-Lys-Arg-Gln-Gly-Ser-Val-Arg-Arg-Arg-Val.

[0016] Preferably, the molecular imaging contrast agent comprises an MRI T1 contrast agent, an MRI T2 contrast agent or a near-infrared fluorescent contrast agent, preferably gadobenate dimeglumine, gadoxetate disodium or gadopentetate dimeglumine.

[0017] Preferably, the average total particle size of the delivery system is 220-290 nm.

[0018] In a second aspect, the present invention provides a method for preparing the above-mentioned folic acid-polylactic acid bilayer vesicle delivery system targeting MDSC cells in lesions, comprising the following steps:

[0019] S1. synthesizing PEI-b-PLA or PEI-b-PLA polymer;

[0020] S2, preparing inner layer vesicles loaded with therapeutic drugs by double emulsification method;

[0021] S3, linking antibodies targeting MDSC cell surface markers to the inner layer vesicles to prepare antibody-modified inner layer vesicles;

[0022] S4, compounding the inner layer vesicles prepared in S2 or the antibody-modified inner layer vesicles prepared in S3 with the therapeutic gene through electrostatic interaction to obtain inner layer vesicles loaded with therapeutic drugs and / or therapeutic genes;

[0023] S5, preparing enzyme substrate polypeptide-PEG;

[0024] S6, preparing enzyme substrate polypeptide-PEG-modified liposomes by using protein kinase C substrate polypeptide-PEG, phospholipid and cholesterol;

[0025] S7. Assemble the inner layer vesicles loaded with therapeutic drugs and / or therapeutic genes in S4 with the enzyme substrate polypeptide-PEG-modified liposomes in S6 to prepare a folic acid-polylactic acid bilayer vesicle delivery system targeting MDSC cells in the lesion.

[0026] In a third aspect, the present invention also provides the use of the above-mentioned folic acid-polylactic acid bilayer vesicle delivery system targeting MDSC cells in lesions in the preparation of drugs for regulating placental MDSC cell dysfunction diseases.

[0027] Preferably, the placental MDSC cell dysfunction disease is fetal growth restriction.

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

[0029] The delivery system of the present invention is a double-layer vesicle delivery system composed of an inner layer vesicle and an outer layer vesicle, the outer layer vesicle is sensitive to the lesion microenvironment, and the distribution of the MDSC function regulating drug is limited to the placenta by selectively releasing the outer layer vesicle confined to the lesion microenvironment, 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. The effect of the drug being confined to the distribution within the lesion can be traced by using the contrast agent loaded between the inner layer and the outer layer vesicle. After the inner vesicles are released by the outer vesicles that disintegrate into the lesion, antibodies targeting MDSC cell membrane markers are used to anchor the MDSC cells in the lesion, effectively reducing the leakage of drugs across the maternal-fetal barrier, reducing the fetal toxicity caused by therapeutic genes and drugs after reaching the embryo, and reducing the return of therapeutic genes and drugs to the maternal blood through the uterine veins, causing maternal toxicity after reaching the normal maternal tissues; utilizing the positive potential on the surface of the inner vesicles, the genes in the cyst wall and the therapeutic drugs in the vesicles are promoted to be internalized into the MDSC cells, thereby achieving MDSC function regulation, and reducing the distribution of therapeutic genes and therapeutic drugs in other cells of the placenta, avoiding affecting the functions of other cells in the placenta.

[0030] The double-layer vesicle delivery system prepared by the specific design of the present invention can limit the scope of delivery and regulation of COX-2 inhibitors and folic acid targeted immunotherapy to the local area of ​​the diseased placenta, effectively reducing the toxicity of immune drugs to pregnant women, making effective immunotherapy of placental diseases possible, significantly improving the current situation where there is no treatment for placental dysfunction diseases, and providing new ideas for effective immunotherapy of placental dysfunction diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a transmission electron microscopy image of the delivery system prepared in the present invention. DETAILED DESCRIPTION

[0032] 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.

[0033] The main raw material sources of the present invention are as follows:

[0034]

[0035] Particle size test method:

[0036] 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.

[0037] Embodiment 1:

[0038] S1. Synthesis of poly(acetimide-co-polylactic acid) (PEI-b-PLA)

[0039] 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:

[0040] Add 135 μL of dried n-dodecanol (0.60 mmol) to a 250 mL reaction bottle, add 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), vacuum dry at room temperature for 4 hours, add 40 mL of freshly distilled anhydrous toluene to dissolve, and reflux at 120°C for 12 hours. After cooling, precipitate with a large amount of anhydrous ethanol, freeze the liquid in a refrigerator at low temperature, then centrifuge and dry, then dissolve with dichloromethane, precipitate with anhydrous ethanol again, filter, and dry to obtain the product PLA.

[0041] Activate the terminal hydroxyl groups of PLA with CDI. Add 6.0g PLA to a 100mL reaction bottle, dry under vacuum at 70℃ for 4h to remove water, add 40mL freshly distilled tetrahydrofuran to dissolve, slowly add the polymer solution dropwise to the tetrahydrofuran solution of CDI (0.50g) under argon protection, react at room temperature for 24h, precipitate with a large amount of anhydrous ethanol, freeze the liquid in a refrigerator, centrifuge and dry, dissolve with dichloromethane, precipitate with anhydrous ethanol again, filter and dry to obtain PLA-CDI.

[0042] 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.

[0043] S2. Preparation of inner layer vesicles

[0044] The inner layer vesicles loaded with folic 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 DMSO / water (volume ratio 1:1) solution containing 0.5 mg of folic acid 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 filtration through a syringe filter (0.45 μm), and the PEI-b-PLA polymer vesicles loaded with folic acid were obtained by ultrafiltration and concentration, and the volume was fixed to 20 mL for use.

[0045] S3. Preparation of antibody-modified inner vesicles

[0046] First, the CD14 antibody is cleaved by the method in the existing literature to obtain the Fab segment of CD14 and purify it. Then, the CD14-Fab is linked to mal-PEG-COOH, and then the PEG linked to the antibody is reacted with the amino group on the poly(lactic acid) vesicle nanoparticles of poly(acetimide) copolymer by amidation reaction to prepare the inner layer vesicles (Fab-PEI-PLA vesicle nanoparticles) targeted by the CD14-Fab antibody.

[0047] The specific operation is as follows: 10 mg of CD14 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 CD14 Fab fragment with high purity was obtained;

[0048] Weigh 1 mg of CD14 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 centrifugal ultrafiltration tube with a molecular weight cutoff of 1k, 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 centrifugal ultrafiltration tube with a molecular weight cutoff of 5k. 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 nanovesicles prepared in step S2, react overnight at 4°C, and finally remove excess EDC, NHS and unconnected CD14 Fab fragments using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100k. 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).

[0049] S4. Preparation of inner vesicles containing complex genes

[0050] 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 COX-2 gene through electrostatic action to prepare a nanocomposite. The specific operation is as follows: 400 μg of COX-2 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 the inner layer vesicle of the composite gene.

[0051] S5. Synthesis of enzyme substrate peptide-PEG

[0052] 0.05mmol protein kinase C substrate peptide can be selected Glu-Arg-Met-Arg-Pro-Arg-Lys-Arg-Gln-Gly-Ser-Val-Arg-Arg-Arg-Val, molecular weight: 2067.43Da. 5mmol EDC and 5mmol DMAP are dissolved in 10mL 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 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.

[0053] S6, Enzyme substrate peptide-PEG modified outer vesicles@inner vesicles

[0054] 10 mg of enzyme substrate polypeptide-PEG, 3 mg of phospholipid and 20 mg of cholesterol are dissolved in 5 mL of dichloromethane, and the dichloromethane is 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 gadobenate dimeglumine with a concentration of 469.01 mg / ml is added to 2 mL of Fab-PEI-PLA vesicle nanoparticles prepared in step S4 under slow stirring, and then the mixed solution is 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 is completed, continue to stir for 30 minutes to fully assemble the liposomes and Fab-PEI-PLA vesicle nanoparticles, and then separate the liposomes carrying the therapeutic gene composite nanoparticles from the empty liposomes 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 to obtain a folic acid-polylactic acid bilayer vesicle delivery system targeting MDSC cells in the lesion.

[0055] like Figure 1 A transmission electron micrograph of the prepared delivery system is shown. As can be seen from the figure, the delivery system includes a double-layer vesicle structure consisting of an inner vesicle and an outer vesicle.

[0056] Embodiment 2:

[0057] The difference from Example 1 is that the enzyme substrate polypeptide-PEG in S6 is 20 mg, and the rest is the same as Example 1.

[0058] Embodiment 3:

[0059] The difference from Example 1 is that no contrast agent is added in S6, and the rest is the same as Example 1.

[0060] Comparative Example 1:

[0061] The difference from Example 1 is that the S3 step is missing (the inner layer vesicles are not linked to CD14-Fab), and the rest is the same as Example 1.

[0062] Comparative Example 2:

[0063] The difference from Example 1 is that steps S5 and S6 are missing (the delivery system has no outer layer of liposome vesicles and no contrast agent), and the rest is the same as Example 1.

[0064] Comparative Example 3:

[0065] The difference from Example 1 is that the S3 step is missing (the inner layer vesicle is not linked to CD14-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.

[0066] Comparative Example 4:

[0067] The difference from Example 1 is that the S3 step is missing (the polymer vesicle is not linked to CD14-Fab), the S5 and S6 steps are missing (the delivery system has no outer vesicle and no contrast agent), and the rest is the same as Example 1.

[0068] Comparative Example 5:

[0069] The difference from Example 1 is that the enzyme substrate polypeptide-PEG in S6 is 40 mg, and the rest is the same as Example 1.

[0070] 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:

[0071] Table 1: Examples and Comparative Examples

[0072]

[0073]

[0074] Functional evaluation experiment

[0075] 1. Magnetic resonance imaging (MRI) experiments to evaluate the placenta-specific delivery function of drugs

[0076] Model building:

[0077] 8-week-old SPF-grade C57BL / 6 mice (purchased from Guangdong Medical Experimental Animal Center) were mated in the estrus period at a ratio of 2:1 between female mice and male mice. On the second day, vaginal secretion smears of female mice were stained with Papanicolaou staining. Those with positive vaginal sperm under an optical microscope were diagnosed as pregnant and marked as day 0 of pregnancy (D0).

[0078] When inducing FGR formation, mice were given tobacco and alcohol treatment groups starting from 7 days before mating until delivery to establish a fetal intrauterine growth restriction model. The tobacco and alcohol treatment method was to place mice in a 5L sealed container for passive smoking for 2 hours every day (9 cigarettes were lit every hour; the cigarette brand was Liqun (soft blue) purchased from Hangzhou Cigarette Factory, tar content: 11mg; nicotine content: 1.0mg; carbon monoxide content: 11mg), and each mouse was given 45% ethanol 2ml (ethanol purchased from Sigma) by oral gavage every day. The control group mice were placed in a smoke-free sealed container for 2 hours every day, and an equal volume of distilled water was gavaged. The pregnant mice placed in a 5L sealed container every day without passive smoking treatment served as the normal control group.

[0079] MRI imaging to detect the placental distribution of drugs:

[0080] On the 11th day, the fetal growth restriction 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.

[0081] 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 / 6 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 gadobenate 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.

[0082] Table 2 Results of placenta-specific delivery function evaluation

[0083]

[0084]

[0085] 2. Establishing an animal model of fetal growth restriction to evaluate the therapeutic effect

[0086] 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:

[0087] 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℃.

[0088] The weights of all fetuses in the normal control group were counted, and the fetuses with weights below the tenth percentile of all fetuses were defined as FGR. The incidence of FGR in each treatment group was then calculated as (number of FGR / total number of animals in this group) × 100%.

[0089] Table 3 Evaluation of therapeutic effects in animal models of fetal growth restriction

[0090]

[0091]

[0092] Embodiment 1-3 prepares the delivery system including inner layer vesicle and outer layer vesicle, and the particle size range of the delivery system is 200-300nm. Its particle size of about 200nm and the negatively charged liposome vesicle of the outer layer are convenient to avoid being engulfed in large quantities by the reticuloendothelial system, so as to achieve the extension of the circulation time in vivo and the effective circulation in vivo. Its enzyme substrate polypeptide-PEG-modified liposome vesicle is stable in the circulation of other tissues and organs in the body. When it reaches the placental microenvironment with high specific expression of protein kinase C, the protein kinase C substrate polypeptide is degraded and the outer layer vesicle disintegrates. 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, so as to avoid the induction of systemic immunotoxicity. After the inner layer vesicles encapsulating therapeutic drugs or genes are released, the therapeutic drugs or genes containing antibody fragments are exposed. Antibody fragments can be anchored to the membrane markers of MDSC cells in the microenvironment. Furthermore, the positive electrical properties of the inner vesicles are utilized to stimulate the specific endocytosis of the inner vesicles by MDSC cells, so that therapeutic drugs or genes can specifically enter MDSC. 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 CD14 targeted antibody fragment is modified so that the inner capsule containing therapeutic drugs or genes is anchored to the 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 veins, and after reaching the normal tissues of the mother, maternal toxicity is caused. In Example 1-3, after the lesions are treated, the incidence of FGR in 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.

[0093] In comparative example 1, the outer vesicles are modified with targeted disintegration enzyme substrate polypeptide-PEG, and the drug system as a whole can enter the lesion. After entering the lesion, the protein kinase C substrate polypeptide group of the liposome vesicles is degraded, the outer liposome vesicles disintegrate, 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 vesicles targeted by the protein kinase C 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. However, since the CD14 targeting antibody fragment was not linked to the inner polymer vesicle wall of comparative example 1, the MDSC cells in the animal model were not targeted for treatment, so the efficacy was poor. The incidence of animal FGR 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 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.

[0094] 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 distributed in a targeted manner to 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 incidence of FGR in animals was detected and it was found that the size and number of embryos were significantly reduced compared with the PBS treatment control group of the disease model.

[0095] In comparative example 3, the outer vesicles do not have the enzyme-sensitive 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 targeted distribution of the lesion cannot be achieved. The distribution of the contrast agent detected by MRI imaging 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 CD14 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, so the efficacy is poor. The detection of the incidence of FGR in animals found that the size and number of embryos were significantly reduced compared to the PBS treatment control group of the disease model.

[0096] 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. There is no CD14 targeting antibody fragment in the inner vesicles. 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 incidence of FGR in animals was detected and it was found that the size and number of embryos were significantly reduced compared with the PBS treatment control group of the disease model.

[0097] 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 incidence of FGR in the animal was detected and found to be 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 both rebounded significantly.

[0098] 3. Toxicity evaluation of nanomedicine in animal models

[0099] 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.

[0100] Table 4 Toxicity evaluation results

[0101]

[0102] From the above results, it can be seen that the liver and kidneys in Examples 1-3 were not affected by the side effects of COX-2 siRNA and folic acid, and the liver function (ALT, TBIL) and kidney function (BUN, sCr) were normal, and no obvious toxic side effects were observed on the mother and fetus.

Claims

1. A folic acid-polylactic acid bilayer vesicle delivery system targeting MDSC cells in lesions, characterized in that: The delivery system includes an inner vesicle and an outer vesicle, wherein a molecular imaging contrast agent is contained between the inner vesicle and the outer vesicle; the inner vesicle is self-assembled from a diblock polymer PEI-b-PLA or PEI-b-PLA, the outer wall of the inner vesicle is positively charged and the immunoregulatory COX-2 inhibitory therapeutic gene is compounded through electrostatic action, the surface of the inner vesicle is modified with an antibody targeting an MDSC cell surface marker, and the inner vesicle is loaded with a therapeutic drug; the outer vesicle is formed by a liposome modified with a protein kinase C substrate polypeptide-PEG targeted for disintegration.

2. The folic acid-polylactic acid bilayer vesicle delivery system targeting MDSC cells in lesions according to claim 1, characterized in that: The therapeutic drug is folic acid, and the therapeutic gene is siRNA of COX-2.

3. The folic acid-polylactic acid bilayer vesicle delivery system targeting MDSC cells in lesions according to claim 1, characterized in that: The antibody targeting the MDSC cell surface marker is CD14.

4. The folic acid-polylactic acid bilayer vesicle delivery system targeting MDSC cells in lesions according to claim 1, characterized in that: The protein kinase C substrate polypeptide is Glu-Arg-Met-Arg-Pro-Arg-Lys-Arg-Gln-Gly-Ser-Val-Arg-Arg-Arg-Val.

5. The folic acid-polylactic acid bilayer vesicle delivery system targeting MDSC cells in lesions according to claim 1, characterized in that: The molecular imaging contrast agent includes MRI T1 contrast agent, MRI T2 contrast agent or near-infrared fluorescent contrast agent, preferably gadobenate dimeglumine, gadoxetate disodium or gadopentetate dimeglumine.

6. The folic acid-polylactic acid bilayer vesicle delivery system for targeting MDSC cells in lesions according to claim 1, characterized in that: The average overall particle size of the delivery system is 220-290 nm.

7. The method for preparing the folic acid-polylactic acid bilayer vesicle delivery system targeting MDSC cells in lesions according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1. synthesizing PEI-b-PLA or PEI-b-PLA polymer; S2, preparing inner layer vesicles loaded with therapeutic drugs by double emulsification method; S3, linking antibodies targeting MDSC cell surface markers to the inner layer vesicles to prepare antibody-modified inner layer vesicles; S4, compounding the inner layer vesicles prepared in S2 or the antibody-modified inner layer vesicles prepared in S3 with the therapeutic gene through electrostatic interaction to obtain inner layer vesicles loaded with therapeutic drugs and / or therapeutic genes; S5, preparing enzyme substrate polypeptide-PEG; S6, preparing enzyme substrate polypeptide-PEG-modified liposomes by using protein kinase C substrate polypeptide-PEG, phospholipid and cholesterol; S7. Assemble the inner layer vesicles loaded with therapeutic drugs and / or therapeutic genes in S4 with the enzyme substrate polypeptide-PEG-modified liposomes in S6 to prepare a folic acid-polylactic acid bilayer vesicle delivery system targeting MDSC cells in the lesion.

8. Use of the folic acid-polylactic acid bilayer vesicle delivery system targeting MDSC cells in lesions as described in any one of claims 1 to 6 in the preparation of drugs for regulating placental MDSC cell dysfunction diseases.

9. The use according to claim 8, characterized in that: The disease in which the placental MDSC cells are dysfunctional is fetal growth restriction.

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