Carrier system for achieving targeted drug delivery on basis of glutathione in response to microenvironmental reactive oxygen species, preparation method therefor, and use thereof

Abstract

A carrier system for achieving targeted drug delivery on the basis of glutathione in response to microenvironmental reactive oxygen species, a preparation method therefor, and use thereof in in vivo delivery of a therapeutic gene such as FSTL3. The inner core is a micelle formed by self-assembly of a diblock copolymer PLys-S-PPhe. Glutathione and the therapeutic gene are combined on the surface of the micelle by means of electrostatic adsorption. The present invention enables a lesion-targeting-like distribution in the placenta and provides a carrier for a drug for treating oxidative stress-related diseases in the placenta.

Description

Carrier system for targeted drug delivery based on glutathione-responsive microenvironment active oxygen and preparation method and application thereof

[0001] TECHNICAL FIELD

[0002] The present application belongs to the field of biological medicine and nanomedicine technology, and particularly relates to a carrier system for targeted drug delivery based on glutathione-responsive microenvironment active oxygen and a preparation method and application thereof.

[0003] BACKGROUND

[0004] Preeclampsia is a placental disease that seriously endangers the health of mother and infant, and lacks therapeutic targets in clinic. The etiology and pathogenesis of preeclampsia have been an important research topic in obstetrics. Over the years, it has been found that there are various pathological changes in the development of preeclampsia, such as impaired endothelial cell function, insufficient remodeling of spiral arteries, inflammation activation, inability to maintain immune tolerance, and even genetic factors. Although there is still no effective pathogenesis of preeclampsia that can comprehensively guide the diagnosis and treatment of PE to improve the efficacy of preeclampsia.

[0005] The first-line recommended antihypertensive drugs and antispasmodic drugs in the current clinical guidelines only have a symptomatic effect, and have not shown the effect of improving the prognosis of preeclampsia in clinical trials

Annu Rev Pathol. 2010; 5: 173-92.

Clin Sci. 2021 Sep 17; 135(17): 2049-2066.

[0006] The existing various targeted drug delivery systems such as PEI have obvious biological toxicity, or are difficult to excrete and degrade, and cannot be applied to placenta 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

Pharmacol Ther. 2022 Feb; 230: 107964.

Hum Reprod Update. 2015 Jan-Feb; 21(1): 97-118.

[0007] 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 and other classic high positive materials, the gene loading efficiency of polylysine is low. In order to improve the gene loading efficiency, high-generation dendritic polylysine nanoparticles inevitably increase the positive charge and enhance the cytotoxicity

Adv Drug Deliv Rev. 2020; 160: 199-211.

[0008] In order to overcome the deficiencies and shortcomings of the prior art, the purpose of the present application is to provide a carrier system for targeted drug delivery based on glutathione-responsive microenvironment active oxygen and its preparation method and application. In the present application, GSH is used as a shell to shield the strong positive charge of the amino acid gene carrier core. After reacting with ROS in the lesion, the shell falls off, the positive charge of the drug core is exposed, the gene transfection ability to diseased cells is enhanced, and the diseased cells obtain the treatment of the loaded FSTL3 gene. The therapeutic effect and the antioxidant treatment effect of the ROS loaded on the drug shell have mutual promotion beneficial effects.

[0009] The purpose of the present application is achieved by the following technical solutions:

[0010] A carrier system for targeted drug delivery based on glutathione-responsive microenvironment active oxygen, the inner core of the carrier system is a micelle formed by self-assembly of a two-block copolymer PLys-S-PPhe, and glutathione and a therapeutic gene are complexed on the surface of the micelle by electrostatic adsorption.

[0011] It is found that the ischemia and hypoxia caused by the placenta oxidative stress and ROS increase is the basic metabolic performance in the placenta. The ROS level in the preeclampsia placenta microenvironment is much higher than that in normal tissue. The excessive ROS can be regarded as a recognition target for targeted drug delivery. The ROS-responsive carrier material is designed and synthesized by introducing a ROS-responsive group, which is an effective method to achieve the targeted drug delivery in the preeclampsia placenta. Glutathione (r-glutamyl cysteingl + glycine, GSH) is a kind of tripeptide containing γ-amide bond and sulfydryl, which is composed of glutamic acid, cysteine and glycine, and exists in almost every cell in the body, and has antioxidant and detoxification effects. The present application realizes the specific release of drugs in the preeclampsia lesion by introducing the ROS-responsive glutathione (r-glutamyl cysteingl + glycine, GSH) in the carrier. The disintegrated GSH in the lesion reacts with ROS at the same time, and plays a microenvironment antioxidant therapy role. The antioxidant effect in the microenvironment can enhance the therapeutic effect of the gene loaded by the nanocarrier. At the same time, the reaction of the GSH shell leads to the release of the positively charged carrier loaded with the gene, which produces an effect similar to that of lesion-targeted delivery.

[0012] The two-block copolymer PLys-S-PPhe has the structural formula as follows:

[0013]

[0014] In the formula, x=40-60, and y=20-30.

[0015] Preferably, the therapeutic gene is FSTL3. Follistatin-like 3 (FSTL3) gene, also known as FLRG (Follistatin related-gene), is a binding protein of a member of the TGF-beta superfamily, which can specifically bind to myostatin, activin, bone morphogenetic protein (BMP), GDF-11 and other members of the TGF-beta superfamily, inhibit Smad-mediated intracellular signal transduction, and then inhibit the biological function of the TGF-beta superfamily member. Follistatin-like 3 is significantly highly expressed in placenta, mature testis, heart and pancreas tissue. The human FSTL3 gene is located in the human chromosome 19p13 region, and the FSTL3 gene is composed of 5 exons and 4 introns, encoding a signal peptide, an N-terminal domain, two FS regions and a C-terminal domain. FSTL3 overexpression may promote F-actin expression and BMP / SMAD signaling to activate epithelial-mesenchymal transition (EMT). FSTL3 binds to activin A in the placenta, reduces the ability of activin A to bind to its receptor, and regulates cell growth and differentiation, immune response through TGF-beta pathway activity. FSTL3 has been found to be a key factor in the pathogenesis of preeclampsia and can be used as a therapeutic target for preeclampsia. The present application is compounded on the inner core surface through electrostatic adsorption, which realizes the effective treatment of preeclampsia.

[0016] Preferably, the surface potential of the two-block copolymer PLys-S-PPhe is 20-42 mV.

[0017] Preferably, the surface potential of the carrier system is 3-15 mV.

[0018] The present application also provides a preparation method of the above-mentioned carrier system for targeted drug delivery based on glutathione response microenvironment active oxygen, comprising the following steps:

[0019] (1) using n-butylamine as an initiator, ring-opening polymerization of N6-benzyloxy carbonyl-L-lysine cyclic anhydride Lys-NCA to obtain poly(N-benzyloxy carbonyl lysine) PZLL;

[0020] (2) connecting PZLL and acrylic acid by amidation reaction to introduce double bond, and synthesizing double bond-terminated poly(N-benzyloxy carbonyl lysine)-vinyl PZLL-vinly;

[0021] (3) PZLL-vinly is synthesized by adding double bond of PZLL-vinly with sulfhydryl of cysteamine under the initiation of azobisisobutyronitrile, to obtain an amino-terminated polymer PZLL-S-NH2 connected by a thioether bond;

[0022] (4) PZLL-S-PPhe is obtained by ring-opening polymerization of L-phenylalanine-N-carboxylic anhydride (Phe-NCA) under the initiation of the terminal amino group of PZLL-S-NH2;

[0023] (5) After the side chain benzyl ester group of PZLL-S-PPhe is removed, the self-assembly of PZLL-S-PPhe is carried out in an aqueous solution to form polylysine-thioether bond-polyphenylalanine (PLys-S-PPhe) nanoparticles;

[0024] (6) The PLys-S-PPhe nanoparticles, glutathione and a therapeutic gene are combined to form a carrier system for targeted drug delivery based on glutathione-responsive microenvironment active oxygen.

[0025] Preferably, in step (2), the molar ratio of PZLL to acrylic acid is 1:1 to 1:2.

[0026] Preferably, in step (3), the molar ratio of PZLL-vinly to cysteamine is 1:1 to 1:2.

[0027] In another aspect, the application also provides a use of the above-mentioned carrier system for targeted drug delivery based on glutathione-responsive microenvironment active oxygen in the preparation of a medicine for treating placental oxidative stress-related diseases.

[0028] Preferably, the placental oxidative stress-related disease is preeclampsia.

[0029] Compared with the prior art, the application has the following advantages:

[0030] The synthesized two-block copolymer PLys-S-PPhe has amphiphilicity, self-assembles into nanomicelles in an aqueous solution, and due to the high positive nature of the self-chain polylysine, glutathione and therapeutic genes can be complexed on the surface of the micelles by electrostatic adsorption. After entering the lesion, the GSH adsorbed on the shell reacts with the lesion ROS, and plays an extracellular antioxidant therapy role in the placental microenvironment. After the antioxidant reaction occurs, the GSH shell dissociates, the strong positive of the drug-containing PLys-S-PPhe carrier core is exposed, and after the positive of the overall surface of the nanocarrier increases, it is easy to promote the endocytosis of the placental lesion cells, after endocytosis, the "sulfide bond" of the drug core in the cell is targeted to dissociate, further promoting the disintegration of the core and the release of the drug, producing the effect of cell-targeted delivery of drugs, and also producing the effect of lesion-targeted retention of the positive core, achieving similar lesion-targeted efficient distribution in the placenta, low toxicity to the liver and kidney, reducing the toxicity to the mother or fetus, providing a new carrier for treating placental oxidative stress-related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0031] Fig. 1 is a synthesis route diagram of the PLys-S-PPhe core;

[0032] Fig. 2 is a structure schematic diagram of the carrier system for targeted drug delivery based on glutathione response microenvironment active oxygen prepared in the embodiment of the present application;

[0033] Fig. 3 is an electron microscope diagram of the carrier system for targeted drug delivery based on glutathione response microenvironment active oxygen prepared in the embodiment of the present application. DETAILED DESCRIPTION

[0034] The present application will be further described by specific embodiments, and the following embodiments are specific embodiments of the present application, but the embodiments of the present application are not limited by the following embodiments.

[0035] The raw materials used in the embodiment of the present application are as follows in Table 1:

[0036] Table 1

[0037] 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) Mcclin (China) n-Butylamine (Cat. 109-73-9) Mcclin (China) Monomethoxy-polyethylene glycol-amino (mPEG-NH2, Mw = 2000 Da) (Cat. 80506-64-5) Mcclin (China) β-Mercaptoethylamine (MEA) (Cat. 60-23-1) Mcclin (China) Azobisisobutyronitrile (AIBN) (Cat. 78-67-1) Mcclin (China) Acrylic acid (Cat. 79-10-7) Mcclin (China) Glutathione (reduced form) (Cat. 70-18-8) Sigma-Aldrich N-Hydroxysuccinimide (NHS) (Cat. 6066-82-6) Sigma-Aldrich 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Cat. 25952-53-8) 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 FSTL3 overexpression plasmid (Cat. No. 20995021 / 20995024) Applied Biological Materials Inc.

[0038] Example 1: Preparation of a carrier system for targeted drug delivery based on glutathione-responsive microenvironmental reactive oxygen species

[0039] (1) Synthesis of poly(N-benzyloxycarbonyl lysine) (PZLL)

[0040] PZLL was synthesized by ring-opening polymerization of N6-benzyloxycarbonyl-L-lysine cyclic anhydride (Lys-NCA) using n-butylamine as initiator. The specific operation for synthesizing PZLL is as follows: Lys-NCA (1.53 g, 5 mmol, 50 eq.) was weighed into a Schlenk flask dried completely by an oven, and was replaced with nitrogen for 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 n-butylamine (7.3 mg, 0.1 mmol, 1.0 eq.) prepared in DMF was added. The reaction was stirred in an oil bath at 35°C for 72 h in a nitrogen environment to seal and isolate air. After the reaction was completed, the reactants were precipitated by an excessive amount of diethyl ether to obtain transparent viscous PZLL. The viscous material was redissolved in chloroform and precipitated by an excessive amount of diethyl ether to obtain white solid. The pure PZLL powder with terminal amino group was obtained by drying at 40°C under vacuum for 24 h, and was stored at 4°C for standby use.

[0041] (2) Synthesis of double bond-terminated poly(N-benzyloxycarbonyl lysine) (PZLL-vinly)

[0042] PZLL-vinly was synthesized by connecting PZLL and acrylic acid through amidation reaction to introduce a double bond. The specific operation for synthesis is as follows: 1.3 g of PZLL (6500 g / mol, 0.2 mmol, 1.0 eq.), 46.6 mg of EDC (155.25 g / mol, 0.3 mmol, 1.5 eq.) and 37 mg of NHS (115.09 g / mol, 0.3 mmol, 1.5 eq.) were added to a 100 mL reaction bottle under nitrogen protection, and were dissolved in 50 mL of freshly distilled chloroform. After being stirred at room temperature for 1 h, 14.4 mg of acrylic acid (72 g / mol, 0.2 mmol, 1.0 eq.) was added. After being reacted at room temperature for 24 h, the insoluble DCU was removed by filtration, and the product PZLL-vinly was obtained by precipitating in a large amount of cold anhydrous ether, filtering, and sequentially washing with anhydrous ethanol, pH 5.0 ultrapure water, anhydrous ethanol and anhydrous ether, and drying;

[0043] (3) Synthesis of amino-terminated poly(N-benzyloxycarbonyl lysine) with a thioether bond (PZLL-S-NH2)

[0044] PZLL-S-NH2 was synthesized by addition of the thiol group of cysteamine to the double bond under the initiation of azobisisobutyronitrile (AIBN). The synthesis was performed as follows: 655 mg of PZLL-vinly (6550 g / mol, 0.10 mmol, 1.0 eq.) synthesized above, 154 mg of cysteamine (77.15 g / mol, 2 mmol, 20 eq.) and 2.5 mg of AIBN (164.21 g / mol, 0.015 mmol, 0.15 eq.) were dissolved in 20 mL of anhydrous DMSO in a 50 mL reaction vial, and the reaction was stirred at 70 °C for 48 h. The reaction solution was dialyzed in a dialysis bag with a molecular weight cut-off of 3.4 kDa for two days in an oxygen-free water, and then freeze-dried to obtain PZLL-S-NH2.

[0045] (4) Synthesis of poly(N-carbobenzyloxy lysine)-polyphenylalanine (PZLL-S-PPhe) linked by a thioether bond

[0046] PZLL-S-PPhe was synthesized by ring-opening polymerization of L-phenylalanine-N-carboxyanhydride (Phe-NCA) using the terminal amino group of PZLL-S-NH2 as an initiator. The synthesis of PZLL was performed as follows: PZLL-S-NH2 (6600 g / mol, 0.66 g, 0.1 mmol, 1.0 eq.) synthesized above and Phe-NCA (0.573 g, 3 mmol, 30 eq.) were added to a Schlenk flask dried completely in an oven, and the flask was purged with nitrogen three times. After sealing, 25 mL of DMF (2 wt%) was added using a syringe, and the reaction was stirred in an oil bath at 35 °C for 24 h. After the reaction was completed, the reaction mixture was precipitated with excess ether to obtain PZLL-S-PPhe as a white solid. Pure PZLL-S-PPhe powder with a terminal amino group was obtained by vacuum drying at 40 °C for 24 h, and was stored at 4 °C for later use.

[0047] (5) Deprotection of PZLL-S-PPhe

[0048] PZLL-S-PPhe (2.0 g) prepared above was dissolved in trifluoroacetic acid (0.04 g / mL), and hydrogen bromide gas was introduced and bubbled for 30 min. The reaction was then stirred in an ice bath for 12 h, and then precipitated with excess ether to obtain the deprotected product. Deprotected polylysine-thioether bond-polyphenylalanine (PLys-S-PPhe) was obtained by vacuum drying at 40 °C for 24 h.

[0049] (6) Preparation of PLys-S-PPhe nanoparticles

[0050] The PLys-S-PPhe prepared above can be self-assembled into nanoparticles in an aqueous solution. The specific operation is as follows: 20 mg of PLys-S-PPhe is completely dissolved in 5 mL of an aqueous PBS solution, and then the nanoparticles are dispersed under ultrasonic treatment. After the ultrasonic treatment is completed, the solution is filtered with a water phase filter (pore size: 0.45 μm) to remove large aggregates, and then dialyzed in PBS (pH 7.4) for 2 d to remove the polymers that do not form particles, to obtain a PLys-S-PPhe nanoparticle solution. FIG. 1 shows the synthesis route of the PLys-S-PPhe nanoparticles.

[0051] (7) Preparation of FSTL3 plasmid composite PLys-S-PPhe nanoparticles

[0052] The FSTL3 plasmid composite nanoparticle PLys-S-PPhe carrier is prepared by electrostatic adsorption. The specific operation is as follows: 500 μg of FSTL3 plasmid is diluted with PBS to a final volume of 1.5 mL, and then the diluted FSTL3 plasmid solution is mixed with the nanoparticle PLys-S-PPhe solution. After being mixed and uniformly stirred by blowing, the solution is placed in a dialysis bag (molecular cut-off: 14 kDa) and dialyzed for 24 h to remove the unreacted plasmid, to obtain uniform FSTL3 plasmid composite PLys-S-PPhe nanoparticles.

[0053] (8) Preparation of a carrier system for targeted drug delivery based on glutathione-responsive microenvironment active oxygen

[0054] The positively charged FSTL3 plasmid composite PLys-S-PPhe nanoparticles and the negatively charged glutathione GSH can be combined into a nanocomposite by electrostatic interaction. The specific operation is as follows: The prepared GSH aqueous solution (0.2 g / mL, 10 mL) is added dropwise into the FSTL3 plasmid composite PLys-S-PPhe nanoparticle solution (200 mg / mL, 5 mL) at a rate of 0.5 mL / min, and stirred for 1 h. Finally, the solution is placed in a dialysis bag (molecular cut-off: 14 kDa) and dialyzed for 24 h to remove the unreacted GSH, and then concentrated with an ultrafiltration tube (molecular cut-off: 100 kDa). Finally, after washing with PBS (pH 7.4), the solution is filtered with a water phase filter (pore size: 0.22 μm) to obtain the carrier system for targeted drug delivery based on glutathione-responsive microenvironment active oxygen (GSH@PLys-S-PPhe).

[0055] Figure 2 shows a structural schematic diagram of the prepared carrier system, from which it can be seen that the core of the carrier system is a micelle formed by self-assembly of the two-block copolymer PLys-S-PPhe, and glutathione and the therapeutic gene are complexed on the surface of the micelle by electrostatic adsorption.

[0056] Figure 3 shows an electron microscope image of the prepared carrier system.

[0057] Comparative Example 1: Synthesis of PEG-PLys-S-PPhe and preparation of a nano-carrier loaded with a therapeutic gene plasmid

[0058] (1) Synthesis of polyethylene glycol-poly (N-benzyloxycarbonyl lysine) (mPEG-PZLL)

[0059] mPEG-PZLL was synthesized by ring-opening polymerization. mPEG-PZLL was obtained by ring-opening polymerization of N6-benzyloxycarbonyl lysine-L-carboxylic anhydride (Lys-NCA) using mPEG-NH2 as an initiator. The specific operation for synthesizing mPEG-PZLL is as follows: 1.53 g of Lys-NCA (5 mmol) was weighed into a Schlenk flask that was completely dried in an oven, and was replaced with nitrogen three times, then 20 mL of DMF was added by syringe after stirring and dissolving at 35°C, then 5 mL of the prepared mPEG-NH2 (0.23 g, 0.1 mmol) DMF solution was added, and the reaction was stirred in a 35°C oil bath under a nitrogen environment to seal out air 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 mPEG-PZLL powder with terminal amino groups was obtained by vacuum drying at 40°C for 24 h;

[0060] (2) Synthesis of double bond terminated polyethylene glycol-poly (N-benzyloxycarbonyl lysine) (mPEG-PZLL-vinly)

[0061] The double bond was introduced by connecting mPEG-PZLL with acrylic acid through amidation reaction to synthesize double bond-terminated polyethylene glycol-poly(N-benzyloxy carbonyl lysine)-vinyl (mPEG-PZLL-vinly). The specific operation of synthesis is as follows: under nitrogen protection, 1.7 g of mPEG-PZLL (8500 g / mol, 0.2 mmol, 1.0 eq.) was added to a 100 mL reaction bottle, 46.6 mg of EDC (155.25 g / mol, 0.3 mmol, 1.5 eq.) and 37 mg of NHS (115.09 g / mol, 0.3 mmol, 1.5 eq.) were added, and 50 mL of freshly distilled chloroform was dissolved. After stirring at room temperature for 1 h, 14.4 mg of acrylic acid (72 g / mol, 0.2 mmol, 1.0 eq.) was added, and after reaction at room temperature for 24 h, the insoluble DCU was removed by filtration, and the product mPEG-PZLL-vinly was obtained by precipitation in a large amount of cold anhydrous ether, filtration, and washing with anhydrous ethanol, pH 5.0 ultrapure water, anhydrous ethanol and anhydrous ether, and drying;

[0062] (3) Synthesis of amino-terminated polyethylene glycol-poly(N-benzyloxy carbonyl lysine) with thioether bond (mPEG-PZLL-S-NH2) Under the initiation of azobisisobutyronitrile (AIBN), the thioether bond- connected amino-terminated polymer mPEG-PZLL-S-NH2 was synthesized by adding the thiol group of cysteamine to the double bond. The specific operation of synthesis is as follows: 855 mg of mPEG-PZLL-vinly (8550 g / mol, 0.10 mmol, 1.0 eq.) synthesized above, 154 mg of cysteamine (77.15 g / mol, 2 mmol, 20 eq.) and 2.5 mg of AIBN (164.21 g / mol, 0.015 mmol, 0.15 eq.) were dissolved in 20 mL of anhydrous DMSO in a 50 mL reaction bottle, and the reaction was stirred at 70°C for 48 h. The reaction solution was dialyzed in a dialysis bag with a molecular weight cutoff of 3.4 kDa in an oxygen-free water for two days, and then freeze-dried to obtain mPEG-PZLL-S-NH2;

[0063] (4) Synthesis of polyethylene glycol-poly(N-benzyloxy carbonyl lysine)-polyphenylalanine (mPEG-PZLL-S-PPhe) connected by thioether bond

[0064] The end amino group of mPEG-PZLL-S-NH2 was used as an initiator to initiate ring-opening polymerization of L-phenylalanine-N-carboxylic anhydride (Phe-NCA) to obtain mPEG-PZLL-S-PPhe. The specific operation of synthesizing mPEG-PZLL-S-PPhe is as follows: The mPEG-PZLL-S-NH2 (8600 g / mol, 0.86 g, 0.1 mmol, 1.0 eq.) product and Phe-NCA (0.573 g, 3 mmol, 30 eq.) synthesized above were added to a Schlenk flask completely dried in an oven, and the flask was replaced with nitrogen three times. After sealing, 25 mL of DMF (2 wt%) was added by a syringe, and 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 a white solid. The pure mPEG-PZLL-S-PPhe powder was obtained by vacuum drying at 40°C for 24 h and stored at 4°C for standby;

[0065] (5) Deprotection of mPEG-PZLL-S-PPhe

[0066] The mPEG-PZLL-S-PPhe (1.2 g) prepared above was dissolved in trifluoroacetic acid (0.04 g / mL), and hydrogen bromide gas was introduced and bubbled for 30 min. The reaction was continuously stirred in an ice bath for 12 h, and then precipitated with excess ether to obtain the deprotected product. The deprotected polyethylene glycol-polylysine-sulfur bond-polyphenylalanine (mPEG-PLys-S-PPhe) was obtained by vacuum drying at 40°C for 24 h;

[0067] (6) Preparation of mPEG-PLys-S-PPhe gene complex nanocarrier

[0068] The positively charged mPEG-PLys-S-PPhe nanoparticles and the negatively charged FSTL3 plasmid can be complexed by electrostatic interaction to form a nanocomposite. The specific operation is as follows: 500 μg of FSTL3 plasmid was diluted with PBS to a final volume of 1.5 mL and shaken uniformly. 25 mg of mPEG-PLys-S-PPhe was dissolved in 5 mL of PBS solution, and then the diluted solution of FSTL3 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 treatment, the solution was left to stand for 30 min after blowing, and large aggregates were removed by filtration with an aqueous filter head (pore size: 0.45 μm). Subsequently, the solution was placed in a dialysis bag (molecular weight cut-off: 14 kDa), and the unreacted plasmid was removed by dialysis for 24 h. After washing with PBS (pH 7.4), the uniform FSTL3 plasmid complexed mPEG-PLys-S-PPhe nanocarrier was obtained by filtration with an aqueous filter head (pore size: 0.22 μm).

[0069] Comparative Example 2: Synthesis of polylysine-polyphenylalanine (PLys-PPhe) and preparation of nanocarrier loaded with therapeutic gene plasmid

[0070] (1) The amino-terminated PZLL synthesized in S1 was used as an initiator to initiate ring-opening polymerization of phenylalanine N-carbonyl carboxylic anhydride (Phe-NCA) to obtain poly(N-carbobenzyloxy lysine)-polyphenylalanine (PZLL-PPhe). The specific operation is as follows: 1.5 g of the PZLL product synthesized above was weighed, the amino-terminated PZLL and Phe-NCA (0.573 g, 3 mmol) were added to a Schlenk flask completely dried in an oven, and the Schlenk flask was replaced with nitrogen three times. After sealing, 25 mL of DMF (2 wt%) was added by a syringe, and 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 a transparent viscous polymer product. The viscous material was redissolved in chloroform and precipitated with excess ether to obtain white solid PZLL-PPhe.

[0071] (2) After obtaining PZLL-PPhe, deprotection was performed: PZLL-PPhe (1.0 g) prepared above was dissolved in trifluoroacetic acid (0.04 g / mL), and hydrogen bromide gas was bubbled for 30 min. The reaction was continuously stirred in an ice bath for 12 h, and then precipitated with excess ether to obtain the deprotected product. Vacuum drying at 40°C for 24 h obtained deprotected polylysine-polyphenylalanine (PLys-PPhe).

[0072] (3) The positively charged PLys-PPhe nanoparticles and the negatively charged FSTL3 plasmid can form a nanocomposite through electrostatic interaction. The specific operation is as follows: 500 μg of FSTL3 plasmid was diluted with PBS to a final volume of 1.5 mL and uniformly oscillated. 20 mg of PLys-PPhe was dissolved in 5 mL of PBS solution, and then the diluted solution of FSTL3 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 action, the solution was blown and left for 30 min, and then filtered with an aqueous filter head (pore size: 0.45 μm) to remove large aggregates. Subsequently, the solution was placed in a dialysis bag (molecular weight cut-off: 14 kDa), and dialysis was performed for 24 h to remove unreacted plasmid. After washing with PBS (pH 7.4), the solution was filtered with an aqueous filter head (pore size: 0.22 μm) to obtain uniformly FSTL3 plasmid-complexed PLys-PPhe nanocarrier.

[0073] (4) The positively charged FSTL3 plasmid complexed with PLys-PPhe nanoparticles and the negatively charged GSH can form a nanocomposite by electrostatic interaction. The specific operation is as follows: The prepared GSH aqueous solution (0.2 g / mL, 10 mL) is added into the PLys-PPhe nanoparticle solution (200 mg / mL, 5 mL) at a rate of 0.5 mL / min, and stirred for 1 h. Finally, the solution is placed in a dialysis bag (molecular weight cut-off: 14 kDa), and dialyzed for 24 h to remove unreacted GSH, and then the GSH@PLys-PPhe nanoparticle solution is concentrated by an ultrafiltration tube (molecular weight cut-off: 100 kDa), and finally washed with PBS (pH 7.4) and filtered through a water phase filter head (pore size: 0.22 μm) to obtain the GSH@PLys-PPhe nanocarrier.

[0074] Example 2-3 and Comparative Examples 3-5:

[0075] As shown in Table 2, the Lys-NCA feeding amount in step (1), the Phe-NCA feeding amount in step (4), and the GSH feeding amount are changed, and the rest is the same as Example 1.

[0076] Table 2: Examples and Comparative Examples

[0077] GSH feeding amount (g) Lys-NCA feeding amount in step (1) (g) Phe-NCA feeding amount in step (4) (g) Plasmid FSTL3 feeding amount (μg) Surface potential of block copolymer (mV) Surface potential of carrier system (mV) Example1 2.00 1.53 0.573 500 30.6 7.8 Example2 2.00 1.22 5 0.486 500 21.1 3.0 Example3 2.00 1.86 5 0.573 500 42.2 14.9 Comparative Example1-1 5.30 0.573 500 5.4 5.3 Comparative Example2 2.00 1.53 0.573 500 39.5 10.9 Comparative Example3 2.00 0.80 9 0.486 500 15.3 2.4 Comparative Example4 2.00 2.04 0 0.573 500 52.0 22.0 Comparative Example5 0.50 1.53 0 0.573 500 30.5 19.6

[0078] The carrier systems prepared in the examples and comparative examples are subjected to functional evaluation experiments as follows:

[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 during the estrus period. The next day, the vaginal secretion of female mice was smeared and Papainikolaou staining was performed. The specimen was observed under an optical microscope. The positive sample of vaginal sperm was diagnosed as pregnancy, and was marked as the 0th day of pregnancy (D0). Pregnant mice were fed with 2 mg / mL nitroso-L-arginine methyl ester in sterile water to establish a preeclampsia / hypertensive pregnancy model. Pregnant mice fed with an equal amount of double-distilled water served as a normal control group.

[0082] NIRF imaging to detect the placental distribution of drugs:

[0083] Near-infrared dye CY7 was used for carrier staining and tracking. The preeclampsia model animals were scanned at 0 h (before drug injection) and 2 h (2 h after drug injection) time points after chloral hydrate anesthesia on the 11th day to observe the in vivo distribution of drugs containing near-infrared fluorescent dyes. The dose of nm drug injected through the tail vein was: (treatment dose 12.5 μg nucleic acid equivalent drug, or an equal volume of PBS);

[0084] In vivo FX live fluorescence scanner was used for live imaging of C57BL / 6j mice. 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, and the results are shown in Table 3.

[0085] Table 3 Evaluation results of placental specific delivery function

[0086] Group Placenta RSI (relative signal multiple) Liver RSI (relative signal multiple) Example 1 1.0 1.0 Example 2 0.9 1.3 Example 3 1.1 0.9 Comparative Example 1 0.8 1.0 Comparative Example 2 1.0 1.0 Comparative Example 3 1.1 0.9 Comparative Example 4 0.7 1.5 Comparative Example 5 0.6 1.5

[0087] 2. Establishing a preeclampsia animal model to evaluate the therapeutic effect

[0088] On D3, D6, D9, D12, and D15, 12.5 μg nucleic acid equivalent drug or an equal volume of normal saline was injected, and serial tests were performed on D17. The test results are shown in Table 4:

[0089] Blood pressure detection: BP-2000 blood pressure analysis system was used,

[0090] The tail cuff method was used to non-invasively measure the systolic blood pressure (SBP) of pregnant mice. The room temperature was maintained at 26°C. Channel 1 was set to 1V (1V corresponds to 300 mmHg), and channel 2 was set to SmV. The mice were fixed in the mouse cage, and the tail was placed in a 17 mm tail cuff. The bottom of the mouse tail was in the middle of the sensor. The pregnant mice were continuously pressurized for 10 times at an interval of 1 s in a calm state, and the average value was taken and recorded;

[0091] Placenta and fetal examination: The pregnant mice were sacrificed, the abdominal cavity was opened, the uterus was dissected, and the fetuses and placenta were sequentially removed. The number of surviving fetuses was recorded. The fetal membranes and umbilical cords on the placenta were removed, the fetal end of the umbilical cord 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. The fetus was weighed on an analytical balance.

[0092] Table 4 Evaluation of therapeutic effect of preeclampsia animal model

[0093] Group Blood pressure (mmHg) Fetal weight (g) Number of offspring per litter No disease, pbs injection control 103.5 1.7 8.0 Example 1 109.2 1.5 7.5 Example 2 112.3 1.4 7.0 Example 3 113.6 1.3 7.1 Comparative Example 0, disease group PBS 161.3 0.9 3.5 Comparative Example 1 131.0 1.2 6.2 Comparative Example 2 138.5 1.2 6.0 Comparative Example 3 139.3 1.3 6.2 Comparative Example 4 145.2 1.1 5.7 Comparative Example 5 152.3 1.0 5.5

[0094] As can be seen from the results of Table 3 and Table 4, the strong positive potential (+20~42mV) of the inner core of the PLys-S-PPhe carrier in Examples 1-3 is shielded by the adsorbed GSH shell to a weaker positive potential. During the blood circulation process before entering the lesion, the strong positive electric stimulation of the reticuloendothelial system dominated by the liver is avoided. The overall potential of these drugs is (+3~15mV), which is not easy to be intercepted and phagocytosed by the reticuloendothelial system dominated by the liver, and the retention of organs outside the lesion represented by the liver is less. The molecular contrast agent complexed with the drug distributes with the drug, and the imaging signal of the aggregation in the liver is weak. After the drug easily enters the placental blood vessels, it passes through the endothelial gap of the newborn capillary, reaches and targets the placental blood sinus (intercapillary space), and the drug concentration in the placenta is higher. The molecular contrast agent complexed with the drug distributes in the placenta, and obvious aggregation imaging signal is generated.

[0095] In addition, after the drug of Example 1-3 enters the lesion, the GSH shell reacts with the ROS in the lesion to produce a microenvironment antioxidant treatment effect, and obvious shell dissociation and release of the strong positive electric inner core "PLys-S-PPhe" occur. After the inner core is released and the overall surface positive electricity of the drug increases, the endocytosis of the placental lesion cells is easily promoted, the cell-targeted delivery effect of the drug is produced, and the lesion-targeted retention effect of the positive electric inner core is produced. After endocytosis, the "sulfide bond" of the inner core of the drug is targeted to dissociate in the cell, promoting the disintegration of the inner core, the release of the drug, and the exertion of the treatment effect. Therefore, the blood pressure of the animals in this group is obviously restored, and the weight of the fetuses and the number of offspring per pregnancy are obviously restored.

[0096] The shell of Comparative Example 1 is a PEG shell connected by a chemical bond, and the electric potential is low, which is not easy to be phagocytosed by the reticuloendothelial system represented by the liver. Moreover, the drug shell does not have GSH that can react with ROS in the lesion, and the drug shell cannot react with ROS in the lesion after the drug enters the lesion, and no microenvironment antioxidant treatment effect is produced, and no obvious shell dissociation and release of the strong positive electric inner core occur. No release effect of the lesion-specific PLys-S-PPhe carrier inner core is produced, and less aggregation in the placenta is produced compared with the example. The surface electric potential of the drug in the lesion is low, and the endocytosis efficiency is low compared with the example, and the treatment effect is weak. Therefore, compared with the example, the blood pressure of the animals in this group is less restored, and the weight of the fetuses and the number of offspring per pregnancy are less restored.

[0097] The shell of Comparative Example 2 is an adsorbed GSH shell, which can shield the strong positive electric inner core, and the overall drug has a weak positive electric potential, which is not easy to be phagocytosed by the reticuloendothelial system represented by the liver. After entering the lesion, the GSH shell reacts with the ROS in the lesion to produce a microenvironment antioxidant treatment effect, and obvious shell dissociation and release of the strong positive electric inner core "PLys-PPhe" occur. After the inner core is released and the overall surface positive electricity of the drug increases, the endocytosis of the placental lesion cells is easily promoted, the cell-targeted delivery effect of the drug is produced, and the lesion-targeted retention effect of the positive electric inner core is produced. However, compared with the example, the inner core of the drug does not have a "sulfide bond" that is targeted to dissociate in the cell after endocytosis, and the disintegration of the inner core and the release of the drug are difficult, and the treatment effect is weaker compared with the example. Therefore, compared with the example, the blood pressure of the animals in this group is not obviously restored, and the weight of the fetuses and the number of offspring per pregnancy are less restored.

[0098] In Comparative Example 3, the electric potential of the inner core is low, and the overall combination treatment plasmid is less, and the treatment effect is weaker compared with the example. Therefore, compared with the example, the blood pressure of the animals in this group is less restored, and the weight of the fetuses and the number of offspring per pregnancy are less restored.

[0099] In the comparative example 4, the potential of the whole nanocarrier and the core is significantly higher than that of the examples, and after microenvironment retention and endocytosis, the drug retained by the reticuloendothelial system is excessive, resulting in less drug reaching the placenta, and the imaging signal in the placenta is lower than that of the examples. And the cytotoxicity is large, which affects the treatment effect, and the treatment effect is poor. Therefore, compared with the examples, the blood pressure recovery of the animals in this group is poor, and the weight of the fetal and the number of offspring per pregnancy are poor.

[0100] In the comparative example 5, the whole surface of the nanocarrier is complexed with less GSH, and the potential of the whole nanocarrier is higher. After microenvironment retention and endocytosis, the drug retained by the reticuloendothelial system is excessive, resulting in less drug reaching the placenta, and the imaging signal in the placenta is lower than that of the examples. And the cytotoxicity is large, which affects the treatment effect, and the treatment effect is poor. Therefore, compared with the examples, the blood pressure recovery of the animals in this group is poor, and the weight of the fetal and the number of offspring per pregnancy are poor.

[0101] 3. Toxicity evaluation of drugs for animal models

[0102] The normal control group of mice was 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 type automatic biochemical analyzer, and the detection results are shown in Table 5.

[0103] Table 5 Toxicity evaluation results

[0104] Group Hepatotoxicity (liver function) ALT (U / L) Hepatotoxicity (liver function) TBIL (μmol / L) Renal toxicity (renal function) BUN (mg / dL) Renal toxicity (renal function) Cr (mg / dL) Example 1 40.3 0.5 27.1 0.6 Example 2 43.5 0.7 27.5 0.7 Example 3 40.1 0.5 26.9 0.6

[0105] As can be seen from the results in Table 5, the carrier system prepared by the present application is not easily intercepted and phagocytosed by the reticuloendothelial system dominated by the liver, and the retention of the liver as a representative lesion in the extrale-sional organs is less, and the hepatotoxicity is lower. These drugs achieve efficient distribution similar to lesion targeting in the placenta, the blood drug concentration is reduced, the kidney does not produce obvious toxic reaction, and the kidney function does not change significantly.

Claims

1. A carrier system for targeted drug delivery based on glutathione-responsive microenvironment active oxygen, characterized by, The core of the carrier system is a micelle self-assembled by the diblock copolymer PLys-S-PPhe, and the glutathione and the therapeutic gene are combined on the surface of the micelle by electrostatic adsorption.

2. The carrier system for targeted drug delivery based on glutathione-responsive microenvironmental active oxygen according to claim 1, wherein, The structural formula of the diblock copolymer PLys-S-PPhe is as follows: wherein x = 40-60, and y = 20-30.

3. The carrier system for targeted drug delivery based on glutathione-responsive microenvironmental active oxygen according to claim 1, wherein, The therapeutic gene is FSTL3.

4. The carrier system for targeted drug delivery based on glutathione-responsive microenvironmental active oxygen according to claim 1, wherein, The surface potential of the diblock copolymer PLys-S-PPhe is 20-42 mV.

5. The carrier system for targeted drug delivery based on glutathione-responsive microenvironment active oxygen according to claim 1, wherein, The surface potential of the carrier system is 3-15 mV.

6. The method of claim 1-5, wherein the preparation of the glutathione- responsive microenvironment active oxygen-based carrier system for targeted drug delivery is characterized in that, The method comprises the following steps: (1) using n-butylamine as an initiator, open ring polymerization of N6-benzyloxy carbonyl-L-lysine cyclic anhydride Lys-NCA to obtain poly(N-benzyloxy carbonyl lysine) PZLL; (2) introducing a double bond by connecting PZLL and acrylic acid through amide reaction to synthesize double bond-terminated poly(N-benzyloxy carbonyl lysine)-vinyl PZLL-vinly; (3) using the thiol group of cysteamine to add the double bond of PZLL-vinly under the initiation of azobisisobutyronitrile to synthesize thioether bond connected amino-terminated polymer PZLL-S-NH2; (4) using the terminal amino group of PZLL-S-NH2 as an initiator, open ring polymerization of L-phenylalanine-N-carboxy cyclic anhydride Phe-NCA to obtain PZLL-S-PPhe; (5) after removing the side chain benzyl ester group of PZLL-S-PPhe, self-assemble in aqueous solution to form polylysine-thioether bond-polyphenylalanine nanoparticles PLys-S-PPhe; (6) preparing the carrier system based on glutathione-responsive microenvironment active oxygen to realize targeted drug delivery by electrostatic adsorption of PLys-S-PPhe nanoparticles, glutathione and therapeutic gene.

7. The method of claim 6, wherein the method is characterized by, In step (2), the molar ratio of PZLL to acrylic acid is 1:1-1:

2.

8. The method of claim 6, wherein the method is characterized by, In step (3), the molar ratio of PZLL-vinly to cysteamine is 1:1-1:

2.

9. Use of the carrier system based on glutathione-responsive microenvironment active oxygen to realize targeted drug delivery according to any one of claims 1-5 in the preparation of a drug for treating placental oxidative stress-related diseases.

10. Use according to claim 9, characterized in that, The placental oxidative stress-related disease is preeclampsia.

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