Essential amino acid vector having functions of micro-environment redox response and Anti-oxidation treatment on diseased cells, preparation method therefor, and use thereof

Abstract

An essential amino acid vector which is capable of delivering therapeutic genes including MXRA5 and has functions of micro-environment redox response and anti-oxidation treatment on diseased cells, a preparation method therefor and the use thereof. The vector is a micelle formed by self-assembly of a multi-block polymer in water, the multi-block polymer being mPEG-GSH-PLys-PPhe; a hydrophilic shell of the vector is mPEG-GSH; a core is PLys-PPhe that encapsulates therapeutic drugs or therapeutic genes, such as MXRA5, for a placenta disease; and the shell and the core are linked by means of an amide bond. The vector can serve as a drug vector system used for treating intraplacental oxidative stress-related diseases, such as preeclampsia.

Description

Essential amino acid carrier for microenvironment redox response and anti-oxidative treatment function of diseased cells, and preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine and nanomedicine technology, and particularly relates to an essential amino acid carrier for microenvironment redox response and anti-oxidative treatment function of diseased cells, and a preparation method and application thereof. BACKGROUND

[0002] The increased incidence of diseases during pregnancy affects the health of the mother and fetus, causes the "pregnancy phobia" of women of childbearing age and their families, and is one of the important medical factors affecting the birth rate. The placenta is the only channel connecting the mother and fetus. Most (90% or more) of the diseases during pregnancy (pregnancy-induced hypertension, diabetes, fetal maldevelopment, miscarriage) are related to the basic pathological changes of the placenta. However, there is no specific drug targeting the diseases during pregnancy. This is mainly due to the lack of clinical treatment targets for placental diseases, including preeclampsia. Even if the treatment targets are extracted, the drugs cannot be retained in the expected treatment targets, i.e. the placenta, to produce efficacy using the previous technology. Drugs that cannot be targeted to the placenta have potential maternal / fetal toxicity and cannot be applied.

[0003] In the placenta, the "fetal barrier" between the maternal blood and the fetal blood mainly performs the function of material exchange, and nutrients, drugs and even pathogens entering the maternal blood can very easily pass through the placental barrier. Once the potentially toxic drugs enter the placenta and cannot be effectively retained in the placenta, they can easily enter the fetus, affecting its growth and development, and even causing malformation, stillbirth and miscarriage. The maternal uterine vein cooperates with the uterine artery blood supply characteristics, and the amount of venous blood flowing out is very large. Once the potentially toxic drugs enter the placenta and cannot be effectively retained in the placenta, they can also easily flow back to the maternal circulation through the uterine vein, causing toxicity to the mother. Therefore, achieving targeted delivery of drugs to the placenta and their retention in the placenta is a key technology for the development of drugs for the treatment of placental diseases. The lack of a carrier for retaining drugs in the placenta is a key problem why there is no targeted drug for diseases during pregnancy so far.

[0004] Various targeted delivery carriers, such as PEI, have obvious biological toxicity or are difficult to be excreted and degraded, and cannot be applied to placental and maternal and infant diseases. Biologically derived carriers (amino acids, cholesterol, cholic acid) have high biological safety, are easily degraded after being absorbed by cells, and have high bioavailability. However, they are easily degraded during distribution, have poor stability, and have low drug loading efficiency.

[0005] Polylysine nanocarriers are widely studied as gene delivery carriers in clinic due to their high positive charge, complete degradation in vivo, controllable structure and good monodispersity. However, compared with PEI, the gene loading efficiency of polylysine is low. In order to improve the gene loading efficiency, the positive charge and cytotoxicity of high-generation dendritic polylysine nanoparticles inevitably increase. Therefore, appropriate structural design and modification methods are needed to reduce the charge and toxicity of polylysine. In addition, the in vivo distribution of lysine carriers lacks targeting, which may reduce the placental efficacy of drugs and increase the potential toxicity of drugs outside the placenta, so it is necessary to improve the targeting of lesion distribution. SUMMARY

[0006] In order to overcome the deficiencies and shortcomings of the prior art, the purpose of the present application is to provide a microenvironment redox response and lesion cell antioxidant therapy function essential amino acid carrier and its preparation method and application. The nanocarrier prepared by appropriate structural design and modification method not only has high efficiency targeting for placental lesions, reduces the toxicity to the mother or fetus, but also realizes the response to the intracellular and extracellular ROS microenvironment of oxidative stress disease cells, especially the intracellular ROS microenvironment, plays an antioxidant role, and provides a new drug carrier system for treating preeclampsia and other oxidative stress diseases.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] A microenvironment redox response and lesion cell antioxidant therapy function essential amino acid carrier, the carrier is a micelle formed by self-assembly of a multi-block polymer in water, the multi-block polymer is mPEG-GSH-PLys-PPhe, the hydrophilic shell is mPEG-GSH, the core is PLys-PPhe wrapping drugs or therapeutic genes, and the shell and the core are connected by an amide bond.

[0009] The present application designs a microenvironment sensitive drug delivery carrier according to the characteristics of placental lesion microenvironment, which is an effective means to realize the treatment of placental diseases. The main function of the placenta is to supply blood and oxygen, and the main manifestation of placental lesions in diseases such as preeclampsia is ischemia and hypoxia, as well as oxidative stress after ischemia and hypoxia. Therefore, oxidative stress is an important feature of the lesion placental microenvironment and an important target for realizing placental microenvironment targeted delivery. Based on the unique redox microenvironment of the lesion placental site, the redox sensitive drug delivery system can cause the decomposition of the redox sensitive chemical bond on the shell, leading to the rapid release of the wrapped drug in the lesion microenvironment, showing good placental targeting and low toxicity.

[0010] Glutathione (GSH) is a key tripeptide consisting of glutamate-cysteine-glycine produced in the cytoplasm, and its molecular formula is C 10 H 17 O6SN3, wherein the main group that plays a role is the reducing group sulfhydryl (-SH). GSH is the main reducing agent in the body and is considered the main thiol-dithiol redox buffer system of cells. This makes GSH a redox reaction trigger in drug delivery. Redox-sensitive targeted nanodrug delivery systems rely on redox-sensitive chemical bonds to function. These drug delivery systems are modified by GSH, and after reaching the redox lesion site, the GSH chemical bond is broken due to the difference in redox potential, which can achieve precise drug release and delivery to the lesion. Therefore, the present application uses GSH to modify the shell of the positive gene vector to achieve the effect of delivering the placental microenvironment under ischemia, hypoxia and oxidative stress.

[0011] The placental drug design must consider the low toxicity of the drug delivery core. The low toxicity of the core can ensure the low toxicity and low teratogenicity of the drug to the fetus. Therefore, the present application selects "lysine" in "essential amino acids" which cannot be synthesized by the fetus and needs to be absorbed from the outside as the basic component of the drug core, to ensure that it is absorbed and degraded by the fetus without obvious toxicity, or even participates in the normal amino acid metabolism of the fetus. Polylysine nanocarriers have high positive charge, can be completely degraded in vivo, have controllable structure and good monodispersity, but the lysine carrier lacks targeting in vivo distribution and cannot ensure the release and efficacy of the drug in the placenta. The present application uses a GSH-modified placental targeted delivery shell to enhance the placental targeting distribution performance of the polylysine drug core.

[0012] The drug or therapeutic gene loaded in the carrier core is the key to the therapeutic effect of the nanodrug after achieving placental distribution. The present application selects the previously screened gene Matrix Remodelling Associated Protein 5 (MXRA5) which is lowly expressed in the placenta of preeclampsia as a therapeutic gene. MXRA5 is a secreted glycoprotein that contains 7 leucine-rich repeat sequences and 12 perlecan-related immunoglobulin-like C2-type domains. It has been predicted as a possible diagnostic and therapeutic target for preeclampsia in multiple center preeclampsia placental sequencing and proteomics data. The overexpression vector of the present application is complexed in the drug core, and after achieving placental targeting distribution, microenvironment effective release and cell effective transfection, the therapeutic effect of preeclampsia is achieved.

[0013] The structure of the multi-block polymer according to the present application is as follows:

[0014]

[0015] wherein, x=45-80, y=40-60, z=20-30.

[0016] The average particle size of the carrier is 80-120 nm.

[0017] The carrier can wrap the drug or therapeutic gene in the polylysine PLYS-PPhe with high positive property, the therapeutic gene is the gene with antioxidant effect in cells, preferably, the therapeutic gene is MXRA5.

[0018] The application further provides a preparation method of the microenvironment redox response and essential amino acid carrier for treating the function of pathological cells.

[0019] (1) synthesis of mPEG-s-s-GSH:

[0020] The monomethoxy-polyethylene glycol-mercapto mPEG-SH is subjected to oxidation reaction with glutathione in water at 20-25℃ for 12-24 h, and then dialysis is performed to obtain mPEG-s-s-GSH.

[0021] (2) synthesis of mPEG-GSH-PZLL:

[0022] The N6-benzyloxy carbonyl lysine-L-carbonyl anhydride Lys-NCA is dissolved with mPEG-s-s-GSH obtained in step (1) in DMF, and then stirring reaction is performed in an oil bath at 30-35℃ for 48-72 h; after the reaction is completed, the reaction product is precipitated with excessive diethyl ether to obtain mPEG-GSH-PZLL.

[0023] (3) synthesis of mPEG-GSH-PZLL-PPhe:

[0024] The mPEG-GSH-PZLL synthesized in step (2) and phenylalanine N-carbonyl carboxylic anhydride Phe-NCA are dissolved in DMF, and then stirring reaction is performed in an oil bath at 30-35℃ for 24-48 h; after the reaction is completed, the reaction product is precipitated with excessive diethyl ether to obtain mPEG-GSH-PZLL-PPhe.

[0025] (4) deprotection of mPEG-GSH-PZLL-PPhe

[0026] The mPEG-GSH-PZLL-PPhe obtained in step (3) is dissolved in trifluoroacetic acid, and hydrogen bromide gas is introduced to bubble for 20-40 min, and the reaction is continuously stirred in ice bath for 10-15 h, and the reactants are settled by excessive diethyl ether to obtain the deprotected mPEG-GSH-PZLL-PPhe;

[0027] (5) Preparation of the nano-carrier:

[0028] The deprotected mPEG-GSH-PLys-PPhe is dissolved in an aqueous solution, and then the nanoparticles are dispersed under ultrasonic action to obtain an mPEG-GSH-PLys-PPhe nanoparticle solution; a dilute solution of a drug or a therapeutic gene is mixed with the mPEG-GSH-PLys-PPhe nanoparticle solution, and after being mixed uniformly by blowing and standing, dialysis is performed to obtain the essential amino acid carrier with microenvironment redox response and anti-oxidation treatment function of diseased cells.

[0029] Preferably, in step (1), the molar ratio of mPEG-SH to glutathione is 1:1; and the molecular weight cut-off of the dialysis bag used for dialysis is 0.5-1 kDa.

[0030] Preferably, in step (2), the molar ratio of N6-benzyloxy carbonyl lysine-L-carbonyl anhydride Lys-NCA to mPEG-s-s-GSH is 40:1-60:1.

[0031] Preferably, in step (3), the molar ratio of phenylalanine N-carbonyl carboxylic anhydride Phe-NCA to mPEG-GSH-PZLL is 20:1-30:1.

[0032] Preferably, in step (5), after removing chloroform, filtration is performed by using a water phase filter head with a pore size of 0.4-0.6 μm to remove large aggregates, then dialysis is performed in pure water by using a dialysis bag with a molecular weight cut-off of 5-14 kDa to remove polymers that do not form particles, finally, the mPEG-GSH-PLys-PPhe nanoparticle solution is concentrated by using an ultrafiltration tube with a molecular weight cut-off of 100-200 kDa, and after washing with pure water, the mPEG-GSH-PLys-PPhe nanoparticle solution is obtained by filtration through a water phase filter head with a pore size of 0.1-0.22 μm.

[0033] The application further provides application of the essential amino acid carrier with microenvironment redox response and anti-oxidation treatment function of diseased cells in the preparation of a drug for treating placental oxidative stress related diseases such as preeclampsia.

[0034] Compared with the prior art, the application has the following excellent effects:

[0035] The application provides a microenvironment redox response and necessary amino acid carrier for treating functions of lesion cells against oxidation, taking strong positive PLys-PPhe as an inner core to ensure the gene loading efficiency; and adopting negative polyethylene glycol-glutathione mPEG-GSH as an outer shell for surface modification, which can shield the strong positive PLys-PPhe and reduce the toxicity. In terms of improving lesion targeting, the negative outer shell shields the positive inner core, in the blood circulation process before entering the lesion, avoids the positive stimulation of the reticuloendothelial system dominated by the liver, is not easily recognized, intercepted and phagocytosed by the reticuloendothelial system dominated by the liver, so that the drug reaches the uterus with high efficiency, and is easily entered into the placenta after reaching the uterus. In terms of ROS response, the glutathione GSH of the outer shell can realize the response to the ROS microenvironment inside and outside the oxidative stress disease cells, especially the intracellular ROS microenvironment, after the nano-carrier enters the lesion, and plays an antioxidant role. Part of the GSH that occurs antioxidant reaction reduces the stability of the outer shell, promotes the dissociation of the outer shell and the positive exposure of the drug-containing carrier inner core, and even releases part of the inner core, produces a similar lesion-targeted high-efficiency distribution effect, reduces the toxicity to the mother or fetus, and provides a new drug carrier system for treating preeclampsia and other oxidative stress diseases.

[0036] BRIEF DESCRIPTION OF DRAWINGS

[0037] Fig. 1 is a synthesis route diagram of the multi-block polymer mPEG-GSH-PLys-PPhe;

[0038] Fig. 2 is a transmission electron microscope image of the mPEG-GSH-PLys-PPhe nano-carrier prepared in the example;

[0039] Fig. 3 is a structure schematic diagram of the mPEG-GSH-PLys-PPhe nano-carrier prepared in the example. DETAILED DESCRIPTION

[0040] The application will be further described below through specific embodiments, and the following examples are specific embodiments of the application, but the embodiments of the application are not limited by the following examples.

[0041] The raw materials used in the embodiments of the application are shown in the following table 1:

[0042] Table 1

[0043] N6-benzyloxycarbonyl-L-lysine cyclic anhydride (Lys-NCA) (Cat. 1676-86-4) Mcclin (China) L-phenylalanine-N-carboxy cyclic anhydride (Phe-NCA) (Cat. 14825-82-2) Aladdin (China) Cyclohexylamine (Cat. 108-91-8) Sigma-Aldrich Monomethoxy-polyethylene glycol-thiol (mPEG-SH, Mw = 2000 Da) (Cat. 134874-49-0) Sigma-Aldrich Monomethoxy-polyethylene glycol-amino (mPEG-NH2, Mw = 2000 Da) (Cat. 80506-64-5) Sigma-Aldrich Glutathione (GSH, reduced form) (Cat. 70-18-8) Sigma-Aldrich Trichloromethane (CH3Cl) (Cat. 67-66-3) Sigma-Aldrich N,N-dimethylformamide (DMF) (Cat. 68-12-2) Sigma-Aldrich Hydrobromic acid (HBr) (Cat. 10035-10-6) Sigma-Aldrich Trifluoroacetic acid (TFA) (Cat. 76-05-1) Sigma-Aldrich MXRA5 plasmid (Cat. No. 31189021) Applied Biological Materials Inc.

[0044] Example 1: Preparation of microenvironment redox-responsive and essential amino acid carrier for lesion cell antioxidant therapy function

[0045] The synthesis route map of the multi-block polymer mPEG-GSH-PLys-PPhe is shown in Figure 1, and the specific operation steps are as follows:

[0046] (1) Synthesis of polyethylene glycol-disulfide-glutathione (mPEG-s-s-GSH)

[0047] The mPEG-s-s-GSH polymer is obtained by oxidation reaction of the active thiol group of mPEG-SH with the thiol group on glutathione GSH. The specific operation is as follows: weigh mPEG-SH (2 g, 1 mmol), and glutathione (0.307 g, 1 mmol) in a 100 mL round-bottom flask with 20 mL aqueous solution, stir at 23°C for 18 h. After the reaction is completed, remove the unreacted GSH by dialysis in a dialysis bag (molecular weight cut-off: 0.5 kDa) for 1 d, freeze-dry the product to obtain a white powder, and the product is mPEG-s-s-GSH (2.3 g).

[0048] (2) Synthesis of polyethylene glycol-glutathione-poly(N-carbobenzyloxy lysine) (mPEG-GSH-PZLL)

[0049] mPEG-GSH-PZLL was synthesized by ring-opening polymerization. mPEG-GSH-NH2 was used as an initiator to initiate ring-opening polymerization of N6-carbobenzyloxy lysine-L-carbonyl anhydride (Lys-NCA) to obtain mPEG-GSH-PZLL. The specific operation for synthesizing mPEG-GSH-PZLL is as follows: 1.53 g of Lys-NCA (5 mmol) was weighed into a Schlenk flask completely dried by an oven, and was replaced with nitrogen three times. After sealing, 20 mL of DMF was added by a syringe, and was dissolved by stirring at 35°C. Then, 5 mL of the above-prepared DMF solution of mPEG-s-s-GSH (0.23 g, 0.1 mmol) was added. The reaction was carried out in a nitrogen environment by sealing to isolate air, and was stirred in an oil bath at 35°C for 72 h. After the reaction was completed, the reaction product was precipitated by an excessive amount of diethyl ether to obtain transparent viscous mPEG-GSH-PZLL. The viscous substance was redissolved in chloroform, and was precipitated by an excessive amount of diethyl ether to obtain a white solid. The pure mPEG-GSH-PZLL powder with an amino group at the end (1.75 g) was obtained by vacuum drying at 40°C for 24 h, and was stored at 4°C for standby use.

[0050] (3) Synthesis of polyethylene glycol-glutathione-poly(N-carbobenzyloxy lysine)- polyphenylalanine (mPEG-GSH-PZLL-PPhe)

[0051] mPEG-GSH-PZLL-PPhe was obtained by using the mPEG-GSH-PZLL powder with an amino group at the end as an initiator to initiate ring-opening polymerization of phenylalanine N-carbonyl carboxylic anhydride (Phe-NCA). The specific operation is as follows: 1.75 g of the above-synthesized mPEG-GSH-PZLL product and Phe-NCA (0.573 g, 3 mmol) were weighed into a Schlenk flask completely dried by an oven, and were replaced with nitrogen three times. After sealing, 25 mL of DMF (2 wt%) was added by a syringe, and the reaction was carried out by stirring in an oil bath at 35°C for 24 h. After the reaction was completed, the reaction product was precipitated by an excessive amount of diethyl ether to obtain transparent viscous mPEG-GSH-PZLL-PPhe white solid. The pure mPEG-GSH-PZLL-PPhe powder with an amino group at the end (2.3 g) was obtained by vacuum drying at 40°C for 24 h, and was stored at 4°C for standby use.

[0052] (4) Deprotection of mPEG-GSH-PZLL-PPhe

[0053] The mPEG-GSH-PZLL-PPhe was dissolved in trifluoroacetic acid (0.04 g / mL), and then hydrogen bromide gas was introduced to bubble for 30 min, and the reaction was continuously stirred in ice bath for 12 h, and then the deprotected product was obtained by precipitation with excess ether. The deprotected polyethylene glycol-glutathione-polylysine-polyphenylalanine (mPEG-GSH-PLys-PPhe) was obtained by vacuum drying at 40°C for 24 h.

[0054] (5) Preparation of MXRA5 plasmid composite nanocarrier

[0055] The positively charged mPEG-GSH-PLys-PPhe nanoparticles and the negatively charged MXRA5 plasmid can be combined to form a nanocomposite through electrostatic interaction. The specific operation is as follows: 500 μg of MXRA5 plasmid was diluted with PBS to a final volume of 1.5 mL and uniformly oscillated. 20 mg of mPEG-GSH-PLys-PPhe was dissolved in 5 mL of PBS solution, and then the diluted solution of MXRA5 plasmid was added to the PBS solution (pH 7.4) of the nanocarrier at a rate of 0.5 mL / min under ultrasonic action. After ultrasonic, stand for 30 min, filter with water phase filter head (pore size: 0.45 μm) to remove large aggregates. Then, the solution was placed in a dialysis bag (molecular weight cut-off: 14 kDa), and dialyzed for 24 h to remove unreacted plasmid, and then filtered through a water phase filter head (pore size: 0.22 μm) after washing with PBS (pH 7.4) to obtain a uniform MXRA5 plasmid composite microenvironment redox response and essential amino acid carrier for oxidative therapy of diseased cells (mPEG-GSH-PLys-PPhe nanocarrier) with an average particle size of 102.3 nm.

[0056] The morphology of the obtained nanocarrier was observed under a transmission electron microscope (TEM). The obtained nanocarrier solution (10 μL, 1 mg / mL) was dropped onto a 200-mesh pure carbon film copper mesh, and then dried at room temperature. Then 10 μL of 1% uranyl acetate solution was added to the copper mesh, and after 1 min, the copper mesh was dried in a desiccator overnight (room temperature). The morphology was observed under a TEM. FIG. 2 is a transmission electron micrograph of the prepared mPEG-GSH-PLys-PPhe nanocarrier. As can be clearly seen from the circular structure with a diameter of about 100 nm in the figure, the nanocarrier prepared in the present application is a micellar structure, which is self-assembled from the amphiphilic polymer mPEG-GSH-PLys-PPhe.

[0057] Figure 3 is a schematic diagram of the structure of the prepared mPEG-GSH-PLys-PPhe nanocarrier, the hydrophilic shell is mPEG-GSH, and the core is PLys-PPhe encapsulating drugs or therapeutic genes.

[0058] Example 2:

[0059] In step (2), the feeding amount of Lys-NCA was 1.865 g, and in step (3), the feeding amount of Phe-NCA was 0.657 g, and the rest was the same as in Example 1. The average particle size of the prepared nanocarrier was 118.9 nm.

[0060] Example 3:

[0061] In step (2), the feeding amount of Lys-NCA was 1.227 g, and in step (3), the feeding amount of Phe-NCA was 0.488 g, and the rest was the same as in Example 1. The average particle size of the prepared nanocarrier was 81.7 nm.

[0062] Preparation of mPEG-PLys-PPhe nanocarrier

[0063] With amino-terminated PZLL as initiator, phenylalanine N-carbonyl carboxylic anhydride (Phe-NCA) ring-opening polymerization was carried out to obtain polyethylene glycol-glutathione-poly (N-carbobenzyloxy lysine) -polyphenylalanine (mPEG-PZLL-PPhe), and then a deprotection step was carried out to obtain mPEG-PLys-PPhe.

[0064] The specific operation is as follows:

[0065] mPEG-PZLL was synthesized by ring-opening polymerization. With mPEG-NH2 as initiator, N6-carbobenzyloxy lysine-L-carbonyl anhydride (Lys-NCA) ring-opening polymerization was carried out to obtain mPEG-PZLL. The specific operation for synthesizing mPEG-PZLL is as follows: 1.53 g of Lys-NCA (5 mmol) was weighed into a Schlenk flask completely dried in an oven, and was replaced with nitrogen three times, then 20 mL of DMF was added with a syringe after stirring and dissolving at 35°C, then 5 mL of the above-prepared mPEG-NH2 (0.23 g, 0.1 mmol) DMF solution was added, and the reaction was carried out in a nitrogen environment under 35°C oil bath stirring for 72 h. After the reaction was completed, the reaction was precipitated with excess ether to obtain transparent viscous mPEG-PZLL, which was redissolved in chloroform and precipitated with excess ether to obtain white solid. The pure amino-terminated mPEG-PZLL powder (1.75 g) was obtained by vacuum drying at 40°C for 24 h.

[0066] Take 1.75 g (0.1 mmol) of the mPEG-PZLL product synthesized above and Phe-NCA (0.573 g, 3 mmol) into a Schlenk flask dried completely in the oven, replace the air with nitrogen three times, and then seal the flask. Add 25 mL of DMF (2 wt%) into the flask using a syringe, and stir the reaction at 35°C for 24 h in an oil bath. After the reaction is completed, precipitate the reaction product with excess ether to obtain the white product mPEG-PZLL-PPhe.

[0067] Dissolve the mPEG-PZLL-PPhe in trifluoroacetic acid (0.04 g / mL), and then pass hydrogen bromide gas through the solution. Bubble the solution for 30 min, and then continue stirring the reaction in an ice bath for 12 h. Subsequently, precipitate the product with excess ether to obtain the deprotected product. Dry the deprotected product in a vacuum at 40°C for 24 h to obtain the deprotected polyethylene glycol-polylysine-polyphenylalanine (mPEG-PLys-PPhe).

[0068] The positively charged mPEG-PZLL-PPhe nanoparticles can be combined with the negatively charged MXRA5 plasmid to form a nanocomplex through electrostatic interaction. The specific operation is as follows: dilute 500 μg of MXRA5 plasmid with PBS to a final volume of 1.5 mL, and then shake the solution until it is uniform. Dissolve 20 mg of mPEG-PZLL-PPhe in 5 mL of PBS solution, and then add the diluted MXRA5 plasmid solution to the PBS solution (pH 7.4) of the nanocarrier at a rate of 0.5 mL / min under ultrasonic action. After the ultrasonic action is completed, blow and stand for 30 min, and then filter the solution using a water phase filter head (pore size: 0.45 μm) to remove large aggregates. Subsequently, place the solution in a dialysis bag (molecular weight cut-off: 14 kDa), and dialyze the solution for 24 h to remove the unreacted plasmid. Wash the solution with PBS (pH 7.4), and then filter the solution using a water phase filter head (pore size: 0.22 μm) to obtain the uniform MXRA5 plasmid-combined mPEG-PZLL-PPhe nanocarrier, which has an average particle size of 107.6 nm.

[0069] Comparative Example 2: Preparation of PLys-PPhe nanocarrier

[0070] Polylysine-polyphenylalanine (PLys-PPhe) was synthesized by two-step ring-opening polymerization. First, poly(N-carbobenzyloxy lysine) (PZLL) was synthesized by ring-opening polymerization of N6-carbobenzyloxy lysine-L-carboxyanhydride (Lys-NCA) using enamine as initiator. Then, after removing the carbobenzyloxy protection group of lysine, polylysine-polyphenylalanine (PLys-PPhe) was obtained. The specific operation is as follows: 2.040 g of Lys-NCA (10 mmol) was weighed into a Schlenk flask completely dried by an oven, and was replaced with nitrogen three times. After being sealed, 20 mL of DMF was added by a syringe. After being dissolved by stirring at 35°C, 5 mL of enamine (10 mg, 0.1 mmol) DMF solution was added. The reaction was stirred in a 35°C oil bath under nitrogen environment to seal off air for 72 h. After the reaction was completed, the reaction was precipitated with excess ether to obtain transparent viscous PZLL. The viscous material was redissolved in chloroform and precipitated with excess ether to obtain white solid. The pure PZLL powder with terminal amino group was obtained by vacuum drying at 40°C for 24 h and was stored at 4°C for standby.

[0071] Poly(N-carbobenzyloxy lysine)-polyphenylalanine (PZLL-PPhe) was synthesized by ring-opening polymerization of phenylalanine N-carboxyanhydride (Phe-NCA) using PZLL with terminal amino group as initiator. The specific operation is as follows: 1.5 g of the above-synthesized PZLL product and Phe-NCA (0.783 g, 5 mmol) were weighed into a Schlenk flask completely dried by an oven, and were replaced with nitrogen three times. After being sealed, 25 mL of DMF (2wt%) was added by a syringe. The reaction was stirred in a 35°C oil bath for 24 h. After the reaction was completed, the reaction was precipitated with excess ether to obtain white product PZLL-PPhe.

[0072] PZLL-PPhe was dissolved in trifluoroacetic acid (0.04 g / mL), and hydrogen bromide gas was introduced to bubble for 30 min. The reaction was continuously stirred in ice bath for 12 h. Then, the deprotected product was obtained by precipitating with excess ether. The deprotected PLys-PPhe was obtained by vacuum drying at 40°C for 24 h.

[0073] The positively charged PLys-PPhe nanoparticles and the negatively charged MXRA5 plasmid can be combined to form a nanocomplex through electrostatic interaction. The specific operation is as follows: 500 μg of MXRA5 plasmid is diluted with PBS to a final volume of 1.5 mL and shaken uniformly. 18 mg of PLys-PPhe is dissolved in 5 mL of PBS solution, and then the diluted solution of MXRA5 plasmid is added to the PBS solution (pH 7.4) of the nanocarrier at a rate of 0.5 mL / min under ultrasonic action. After ultrasonic action, blow and stand for 30 min, filter with an aqueous filter head (pore size: 0.45 μm) to remove large aggregates. Subsequently, the solution is placed in a dialysis bag (molecular weight cut-off: 14 kDa) and dialyzed for 24 h to remove unreacted plasmid, and then filtered through an aqueous filter head (pore size: 0.22 μm) with PBS (pH 7.4) to obtain a uniform MXRA5 plasmid-complexed PLys-PPhe nanocarrier with an average particle size of 160.7 nm. Comparative Example 3:

[0074] In step (1), the amount of mPEG is 0.400 g, in step (2), the amount of Lys-NCA is 2.040 g, and in step (3), the amount of Phe-NCA is 1.146 g,

[0075] The rest is the same as Example 1, and the average particle size of the prepared nanocarrier is 200.2 nm. Comparative Example 4:

[0076] In step (1), the amount of mPEG is 0.100 g, in step (2), the amount of Lys-NCA is 1.020 g, and in step (3), the amount of Phe-NCA is 0.382 g,

[0077] The rest is the same as Example 1, and the average particle size of the prepared nanocarrier is 51.3 nm.

[0078] Application Example: Functional Evaluation Experiment

[0079] 1. Near-infrared in vivo fluorescence (NIRF) molecular imaging experiment to evaluate the placenta-specific delivery function of the drug

[0080] Model establishment:

[0081] 8-week-old SPF C57BL / 6 mice (purchased from Guangdong Medical Laboratory Animal Center) were mated with male mice at a ratio of 2:1 at the estrus stage. The next day, the vaginal secretion of female mice was smeared and Papnikolaou staining was performed. The specimen was observed under an optical microscope. The specimen was diagnosed as pregnant if the vaginal sperm was positive, and 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 a preeclampsia / hypertension during pregnancy model. Pregnant mice fed with an equal amount of double-distilled water served as a normal control group.

[0082] NIRF imaging detection of drug placental distribution:

[0083] Molecular contrast agent (near-infrared dye CY7) was used for carrier staining tracing. The preeclampsia model animals were scanned at the time points of before drug injection (0 h) and 2 h after injection on the 11th day after anesthesia with chloral hydrate. The in vivo distribution of nanomedicine containing near-infrared fluorescent dye was observed. The dose of nanomedicine (Examples 1-3 and Comparative Examples 1-4) injected through the tail vein was: (treatment dose 12.5 μg nucleic acid equivalent drug, or an equal volume of PBS); C57BL / 6 mice were imaged in vivo using an In vivo FX live fluorescence scanner. The relative signal intensity (RSI (Relative Signal Intensity) % = R2h / R0h) of the placental region (mouse lower abdominal uterine region) or liver region was calculated 2 h after drug injection, respectively. The results are shown in Table 2.

[0084] Table 2 Evaluation results of placenta-specific delivery function

[0085] Group Placenta RSI (relative signal multiple) Liver RSI (relative signal multiple) Example 1 1.0 1.0 Example 2 1.3 0.9 Example 3 1.1 0.9 Comparative Example 1 0.5 1.4 Comparative Example 2 0.1 1.9 Comparative Example 3 0.3 1.3 Comparative Example 4 0.4 0.7

[0086] From the above results, it can be seen that the positive electricity of the inner core of the PLys-PPhe carrier in Examples 1-3 is shielded by the mPEG-GSH shell. During the blood circulation before entering the lesion, the positive electricity stimulates the reticuloendothelial system mainly in the liver to be avoided. The particle size of these drug carriers is 80-120 nm, which is not easily recognized, intercepted and phagocytosed by the reticuloendothelial system mainly in the liver, and the liver retention is less; the particle size is smaller than the endothelial gap of the uterine spiral artery capillary supplying blood to the placenta, and it is easy to enter the placenta after reaching the uterus, and the drug concentration in the placenta is higher. The molecular contrast agent complexed with the drug carrier is distributed with the drug, and an obvious imaging signal is generated in the placenta.

[0087] The shell of Comparative Example 1 has no GSH that can react with ROS in the lesions in the placenta. After the nanocarriers enter the lesions, no obvious shell dissociation and release of the drug-containing core of the nanocarriers can occur. The drug cores that cannot be released flow out of the placenta with the venous blood, and the retention of the drug cores in the lesions of the placenta is very small. The drug-complexed molecular contrast agent is distributed with the drug, and the aggregation of the drug-complexed molecular contrast agent in the placenta is less, resulting in a significantly reduced imaging signal compared with the embodiment, and the aggregation of the drug-complexed molecular contrast agent in the liver is more, resulting in a significantly higher RSI imaging signal of the liver.

[0088] The shell of Comparative Example 2 lacks the shell that can mask the strong positive charge of the core. During the blood circulation before entering the lesions, the obvious positive charge on the surface of the drug PLys-PPhe carrier core stimulates the reticuloendothelial system mainly in the liver. Further, the drug is obviously intercepted and phagocytosed by the reticuloendothelial system, the blood drug concentration is reduced, and the drug entering the placenta is very small. The drug-complexed molecular contrast agent is distributed with the drug, and the aggregation of the drug-complexed molecular contrast agent in the placenta is very small, resulting in a significantly reduced imaging signal compared with the embodiment, and the aggregation of the drug-complexed molecular contrast agent in the liver is less, resulting in a significantly higher RSI imaging signal of the liver.

[0089] The nanocarriers of Comparative Example 3 have a particle size that is too large, are easily recognized, intercepted and phagocytosed by the reticuloendothelial system mainly in the liver, have a high drug concentration in the liver, and the blood drug concentration is obviously reduced during in vivo distribution. In addition, the particle size is larger than the endothelial gap of the newly formed capillaries of the uterine spiral arteries supplying blood to the placenta, so that even if the nanocarriers reach the uterus, the ratio of the nanocarriers entering the placenta is very low, and the drug concentration in the placenta is low. The drug-complexed molecular contrast agent is distributed with the drug, and the aggregation of the drug-complexed molecular contrast agent in the placenta is less, resulting in a significantly reduced imaging signal compared with the embodiment, and the aggregation of the drug-complexed molecular contrast agent in the liver is more, resulting in a significantly higher RSI imaging signal of the liver.

[0090] The nanocarriers of Comparative Example 4 have a particle size that is too small, are not easily recognized, intercepted and phagocytosed by the reticuloendothelial system mainly in the liver, have less retention in the liver, have a low drug concentration, and the reduction of the blood drug concentration is not obvious during distribution. However, the particle size is much smaller than the endothelial gap of the capillaries of the placenta and the uterine vein, so that even if the nanocarriers enter the placenta, they enter the uterine vein with the blood flow, are discharged from the placenta, and have a low drug concentration in the placenta. The drug-complexed molecular contrast agent is distributed with the drug, and the aggregation of the drug-complexed molecular contrast agent in the placenta is less, resulting in a significantly reduced imaging signal compared with the embodiment, and the aggregation of the drug-complexed molecular contrast agent in the liver is less, resulting in a significantly lower RSI imaging signal of the liver.

[0091] 2. Establishing a pre-eclampsia animal model to evaluate the therapeutic effect

[0092] On D3, D6, D9, D12 and D15, 12.5 μg of nucleic acid equivalent drug or an equal volume of PBS was injected, and a series of tests were performed on D17. The test results are shown in Table 3:

[0093] Blood pressure detection: BP-2000 blood pressure analysis system was used to noninvasively measure the systolic blood pressure (SBP) of pregnant rats by tail cuff method. The room temperature was kept at 26 degrees Celsius, channel 1 was set at 1V (1V equivalent to 300 mmHg), and channel 2 was set at SmV. The mice were fixed in the mouse cage, and the tail was placed in the 17 mm tail cuff. The bottom of the mouse tail was in the middle of the sensor. The pregnant rats were continuously pressurized for 10 times under the condition of quiet state, with an interval of 1 s each time. The average value was taken and recorded;

[0094] Placenta and fetal examination: placental tissue: the pregnant rats were sacrificed, the abdominal cavity was opened, the uterus was dissected, the fetuses and placenta were taken out in turn, and the number of surviving fetuses was recorded. The fetal membranes and umbilical cords on the placenta were removed, the umbilical cord at the end of the fetus was cut off along the root of the umbilical cord, and the placenta and fetus were placed on sterile gauze to absorb the surface amniotic fluid, and the fetus was weighed on an analytical balance. The placental tissue was cut and placed in liquid nitrogen, and stored at -80℃.

[0095] Table 3 Evaluation of therapeutic effect of preeclampsia animal model

[0096] Group Blood pressure (mmHg) Fetal weight (g) Number of offspring per litter Normal group, pbs injection control 101.6 1.6 8.2 Example 1 114.0 1.4 7.8 Example 2 115.3 1.5 7.6 Example 3 113.7 1.5 7.9 Untreated disease model, PBS 156.0 0.8 3.2 Comparative example 1 143.5 0.9 4.6 Comparative example 2 151.3 0.9 3.5 Comparative example 3 138.5 1.2 5.8 Comparative example 4 132.3 1.3 6.1

[0097] From the above results, it can be seen that the drug carrier in examples 1-3 reacts with the ROS in the lesion after entering the lesion, and plays a partial extracellular antioxidant role. The part of GSH that occurs antioxidant reaction reduces the stability of the shell, promotes the dissociation of the shell and the exposure of the positive electricity of the drug carrier core, and even part of the core is released, producing an effect similar to lesion targeting. The overall positive surface of the drug carrier is easily endocytosed by the placental lesion cells. The residual GSH of the shell in the cells can also play an antioxidant function. The antioxidant therapy MXRA5 plasmid and the residual GSH of the shell are distributed into the lesion cells with the drug, and play an antioxidant role in the cells. Compared with the untreated disease model group, the blood pressure decreased significantly to approach the normal control group; the fetal weight increased significantly to approach the normal control group; the number of offspring per litter increased significantly to approach the normal control group.

[0098] The shell in Comparative Example 1 lacks GSH that can react with ROS in the lesions in the placenta. The shell cannot play an extracellular antioxidant role after entering the lesions, and no obvious shell dissociation and release of the drug-containing core occur. The drug core that cannot be released flows out of the placenta with the venous blood, and stays in the lesions in the placenta very little. The small amount of drug that stays in the placenta is endocytosed by the diseased cells, and the shell also cannot play an antioxidant function in the cells. The antioxidant therapy MXRA5 plasmid and the residual GSH in the shell have a low efficiency of distributing into the diseased cells with the drug, and the blood pressure is obviously higher close to the untreated disease model; the fetal weight is obviously lower close to the untreated disease model; and the number of offspring per pregnancy is obviously higher close to the untreated disease model.

[0099] The shell in Comparative Example 2 lacks GSH that can shield the strong positive charge of the core. During the blood circulation before entering the lesions, the obvious positive charge on the surface of the drug PLys-PPhe carrier core stimulates the reticuloendothelial system mainly in the liver. Further, the drug is obviously intercepted and phagocytosed by the reticuloendothelial system, the blood drug concentration is reduced, and the drug entering the placenta is very little. The antioxidant therapy plasmid and the residual GSH in the shell have a very low efficiency of distributing into the placenta with the drug, resulting in a very low efficiency of entering the diseased cells, and the blood pressure is obviously higher close to the untreated disease model; the fetal weight is obviously lower close to the untreated disease model; and the number of offspring per pregnancy is obviously higher close to the untreated disease model.

[0100] The particle size of the delivery system in Comparative Example 3 is too large, the antioxidant therapy plasmid and the residual GSH in the shell have a low efficiency of distributing into the placenta with the drug, the drug concentration in the placenta is reduced, the efficiency of the drug entering the diseased cells is very low, and the blood pressure is obviously higher close to the untreated disease model; the fetal weight is obviously lower close to the untreated disease model; and the number of offspring per pregnancy is obviously higher close to the untreated disease model.

[0101] The particle size in Comparative Example 4 is too small, the antioxidant therapy plasmid and the residual GSH in the shell distribute with the drug, a large amount of the drug entering the placenta flows out of the placenta, cannot achieve effective retention in the placenta, and the drug concentration in the placenta is very low. Therefore, the efficiency of the drug finally entering the diseased cells is low, and the blood pressure is obviously higher close to the untreated disease model; the fetal weight is obviously lower close to the untreated disease model; and the number of offspring per pregnancy is obviously higher close to the untreated disease model.

[0102] 3. Toxicity evaluation of the drug for animal models

[0103] The normal control group mice were injected with drugs for 72 hours, and blood was taken from the tail vein to detect liver function indicators alanine transaminase (ALT), 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.

[0104] Table 4 Toxicity evaluation results

[0105] Group Hepatotoxicity (liver function) ALT (U / L) Hepatotoxicity (liver function) TBIL (μmol / L) Renal toxicity (kidney function) BUN (mg / dL) Renal toxicity (kidney function) Cr (mg / dL) Example 1 42.2 0.2 24.1 0.2 Example 2 42.1 0.2 24.3 0.2 Example 3 42.1 0.2 24.2 0.2

[0106] From the above results, it can be seen that the nano-carrier prepared in the application has less drug retention in the liver and lower hepatotoxicity. At the same time, these drugs achieve high-efficiency distribution similar to lesion targeting in the placenta, the blood drug concentration is reduced, the kidneys do not produce obvious toxic reactions, and the kidney function does not change significantly.

Claims

1. A microenvironment redox response and essential amino acid carrier for treating the function of diseased cells against oxidation, characterized in that, The carrier is micelles formed by self-assembly of a multi-block polymer in water, the multi-block polymer is mPEG-GSH-PLys-PPhe, the hydrophilic shell is mPEG-GSH, the core is PLys-PPhe wrapping drugs or therapeutic genes, and the shell and the core are connected by an amide bond.

2. The microenvironment redox response and essential amino acid carrier for diseased cell antioxidant therapy function of claim 1, wherein, The structural formula of the multi-block polymer is: ； wherein x = 45-80, y = 40-60, and z = 20-30.

3. The microenvironment redox response and essential amino acid carrier for diseased cell antioxidant therapy function of claim 1, wherein, The average particle size of the carrier is 80-120 nm.

4. The microenvironment redox response and essential amino acid carrier of claim 1, wherein, The therapeutic gene is a gene that plays an antioxidant role in cells, preferably MXRA5.

5. The method of claim 1-4, wherein the method is characterized in that, The method comprises the following steps: (1) synthesis of mPEG-s-s-GSH: Carry out an oxidation reaction of monomethoxy-polyethylene glycol-thiol mPEG-SH and glutathione in water at 20-25℃ for 12-24 h, dialyze, and obtain mPEG-s-s-GSH; (2) synthesis of mPEG-GSH-PZLL: Dissolve N6-benzyloxy carbonyl lysine-L-carbonyl anhydride Lys-NCA and mPEG-s-s-GSH obtained in step (1) in DMF, stir and react in an oil bath at 30-35℃ for 48-72 h, after the reaction is completed, precipitate the reaction product with excessive diethyl ether, and obtain mPEG-GSH-PZLL; (3) synthesis of mPEG-GSH-PZLL-PPhe: Dissolve mPEG-GSH-PZLL synthesized in step (2) and phenylalanine N-carbonyl carboxylic anhydride Phe-NCA in DMF, stir and react in an oil bath at 30-35℃ for 24-48 h, after the reaction is completed, precipitate the reaction product with excessive diethyl ether, and obtain mPEG-GSH-PZLL-PPhe; (4) deprotection of mPEG-GSH-PZLL-PPhe Dissolve mPEG-GSH-PZLL-PPhe obtained in step (3) in trifluoroacetic acid, then pass hydrogen bromide gas through it, bubble for 20-40 min, continue to stir and react in an ice bath for 10-15 h, precipitate the reaction product with excessive diethyl ether, and obtain deprotected mPEG-GSH-PZLL-PPhe; (5) preparation of a nano-carrier: Dissolve deprotected mPEG-GSH-PLys-PPhe in chloroform, then drop the mixed solution into PBS in an ice bath under ultrasonic action, remove the chloroform, filter, and obtain a mPEG-GSH-PLys-PPhe nanoparticle solution; mix a dilute solution of drugs or therapeutic genes with the mPEG-GSH-PLys-PPhe nanoparticle solution, mix uniformly by blowing, stand still, dialyze, and obtain an amino acid carrier necessary for the microenvironment redox response and the antioxidant treatment function of diseased cells.

6. The method for preparing the essential amino acid carrier for microenvironment redox response and antioxidant therapeutic function of diseased cells according to claim 5, characterized in that, In step (1), the molar ratio of mPEG-SH to glutathione is 1:1; and the molecular weight cut-off of the dialysis bag used for dialysis is 0.5-1 kDa.

7. The method for preparing the essential amino acid carrier for microenvironment redox response and antioxidant therapeutic function of diseased cells according to claim 5, characterized in that, In step (2), the molar ratio of N6-benzyloxy carbonyl lysine-L-carbonyl anhydride Lys-NCA to mPEG-s-s-GSH is 40:1-60:

1.

8. The method for preparing the essential amino acid carrier for microenvironment redox response and antioxidant therapeutic function of diseased cells according to claim 5, characterized in that, In step (3), the molar ratio of the phenylalanine N-carbonyl carboxylic anhydride Phe-NCA and mPEG-GSH-PZLL is 20:1-30:

1.

9. The method of claim 5, wherein the microenvironment redox response and diseased cell antioxidant therapy functional essential amino acid carrier is prepared by, In step (5), after removing chloroform, filtration is performed using a water phase filter head with a pore size of 0.4-0.6 μm to remove large aggregates, then dialysis is performed in pure water using a dialysis bag with a molecular cut-off of 5-14 kDa to remove polymers that do not form particles, finally, the mPEG-GSH-PLys-PPhe nanoparticle solution is concentrated using an ultrafiltration tube with a molecular cut-off of 100-200 kDa, washed with pure water, and filtered through a water phase filter head with a pore size of 0.1-0.22 μm to obtain the mPEG-GSH-PLys-PPhe nanoparticle solution.

10. Use of the microenvironment redox response and lesion cell antioxidant therapy function essential amino acid carrier according to any one of claims 1-4 in the preparation of a drug for treating placental oxidative stress related diseases such as preeclampsia.

Images