A carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species, and a preparation method and application thereof

Abstract

The present invention discloses a carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species, its preparation method, and its application in in vivo delivery of therapeutic genes such as FSTL3. The core is a micelle formed by self-assembly of a diblock copolymer PLys-S-PPhe, and glutathione and the therapeutic gene are complexed on the surface of the micelle through electrostatic adsorption. After the carrier system of the present invention enters the lesion, the adsorbed GSH on the outer shell reacts with the lesion ROS, producing a microenvironment antioxidant treatment effect, resulting in obvious dissociation of the outer shell and release of the strongly positively charged core "PLys-S-PPhe". After the release of the core and the increase in the overall positive charge on the surface of the drug, it is easy to promote the endocytosis of diseased cells in the placenta, producing the cell-targeted delivery effect of drugs such as FSTL3, and also producing the lesion-targeted retention effect of the positively charged core, achieving efficient distribution similar to lesion targeting in the placenta, reducing the toxicity to the mother or fetus, and providing a new carrier for drugs for treating placenta oxidative stress-related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and nanomedicine, and particularly relates to a carrier system for realizing targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species, a preparation method thereof, and an application thereof. Background Art

[0002] As a placental disease that seriously endangers the health of mothers and infants, preeclampsia lacks treatment targets clinically. The etiology and pathogenesis of preeclampsia have always been important research topics in obstetrics. Over the years, it has been found that there are various pathological changes during the occurrence and development of preeclampsia, such as impaired endothelial cell function, insufficient remodeling of spiral arteries, activation of inflammation, inability to maintain immune tolerance, and even genetic factors. Although there is still no effective theory on the pathogenesis of preeclampsia that can comprehensively guide the diagnosis and treatment of PE to improve the curative effect of preeclampsia.

[0003] Currently, the first-line recommended antihypertensive drugs and antispasmodic drugs in clinical guidelines only have the effect of relieving symptoms 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.

[0004] Existing various targeted delivery drugs such as PEI have obvious biological toxicity, or are difficult to excrete and degrade, and cannot be applied to the placenta and maternal and infant diseases. Biogenic carriers (amino acids, cholesterol, bile acids) 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.

[0005] Polylysine nanoparticles have a high positive charge, can be completely degraded in vivo, have a controllable structure and good monodispersity, so they are also used as gene delivery vectors widely studied in clinics. However, compared with classical highly positively charged materials such as PEI, their gene loading efficiency is low. To improve the gene loading efficiency, the positive charge of high-generation dendritic polylysine nanoparticles inevitably increases, and the cytotoxicity is enhanced [Adv Drug Deliv Rev. 2020; 160: 199-211.]. Appropriate structural design and modification methods are needed to reduce the charge and toxicity of polylysine. In addition, the lack of targeting in the in vivo distribution of lysine carriers may reduce the efficacy of drugs in the placenta and increase the potential toxicity of drugs outside the placenta. Therefore, it is necessary to improve their targeting to the lesion distribution. Summary of the Invention

[0006] In order to overcome the deficiencies and drawbacks of the prior art, the purpose of the present invention is to provide a carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species, its preparation method and application. The present invention uses GSH as the outer shell to shield the strong positive charge of the amino acid gene carrier core. After reacting with ROS in the lesion, the outer shell falls off, the positive charge of the drug core is revealed, and the gene transfection ability of the diseased cells is enhanced. The diseased cells obtain the treatment of genes such as FSTL3 loaded. This curative effect has a mutually promoting beneficial effect with the antioxidant treatment effect of ROS loaded on the drug outer shell.

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

[0008] A carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species, the core of the carrier system is a micelle formed by self-assembly of a diblock copolymer PLys-S-PPhe, and glutathione and therapeutic genes are complexed on the surface of the micelle through electrostatic adsorption.

[0009] At present, it has been found that the increased oxidative stress and ROS in the placenta caused by ischemia and hypoxia are the basic metabolic manifestations in the placenta. The level of ROS in the placental microenvironment of preeclampsia is much higher than that in normal tissues. The over-produced ROS can be regarded as the recognition target for targeted drug delivery. Designing and synthesizing ROS-responsive carrier materials by introducing ROS-responsive groups is an effective method to achieve targeted drug delivery to the placenta in preeclampsia. Glutathione (r-glutamyl cysteingl+glycine, GSH) is a tripeptide containing γ-amide bond and sulfhydryl group, composed of glutamic acid, cysteine and glycine, existing in almost every cell of the body, and having antioxidant and detoxification effects. In the present invention, by introducing glutathione (r-glutamyl cysteingl+glycine, GSH) responsive to ROS in the placental microenvironment into the carrier, specific release of the drug in the preeclampsia lesion is achieved. The disintegrated GSH in the lesion plays a role in antioxidant treatment of the microenvironment while reacting with ROS. This 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, producing an effect similar to targeted delivery to the lesion.

[0010] The structural formula of the diblock copolymer PLys-S-PPhe described in the present invention is:

[0011]

[0012] Wherein, x = 40-60, y = 20-30.

[0013] Preferably, the therapeutic gene is FSTL3. Follistatin-like 3 (FSTL3) gene, also known as FLRG (Follistatin related-gene), is a binding protein of the TGF-β superfamily members. It can specifically bind to multiple members of the TGF-β superfamily such as myostatin, activin, bone morphogenetic protein (BMPs), and GDF-11, inhibit Smad-mediated intracellular signal transduction, and further inhibit the biological functions of TGF-β superfamily members. Follistatin-like3 is significantly highly expressed in placenta, mature testis, heart, and pancreatic tissues. The human FSTL3 gene is located in the human chromosome 19p13 region. The FSTL3 gene consists of 5 exons and 4 introns, encoding a signal peptide, an N-terminal domain, two FS regions, and a C-terminal domain. Overexpression of FSTL3 may activate epithelial-mesenchymal transition (EMT) by promoting F-actin expression and BMP / SMAD signal transduction. FSTL3 binds to activin A in the placenta, reducing the ability of activin A to bind to its receptor, thereby regulating cell growth and differentiation and immune response through the regulation of TGF-β 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. In the present invention, it is compounded on the surface of the inner core through electrostatic adsorption, achieving an effective therapeutic effect on preeclampsia.

[0014] Preferably, the surface potential of the diblock copolymer PLys-S-PPhe is 20 - 42 mV.

[0015] Preferably, the surface potential of the carrier system is 3 - 15 mV.

[0016] The present invention also provides a preparation method of the above-mentioned carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species, including the following steps:

[0017] (1) Using n-butylamine as an initiator, initiating the ring-opening polymerization of N6-carbobenzoxy-L-lysine cyclic anhydride Lys-NCA to obtain poly(N-carbobenzoxylysine) PZLL;

[0018] (2) Connecting PZLL and acrylic acid through an amidation reaction to introduce a double bond, and synthesizing a double bond-terminated poly(N-carbobenzoxylysine)-vinyl PZLL-vinly;

[0019] (3) Under the initiation of azobisisobutyronitrile, the double bond of PZLL-vinly was added with the mercapto group of cysteamine to synthesize an amino-terminated polymer PZLL-S-NH2 connected by a thioether bond;

[0020] (4) Using the terminal amino group of PZLL-S-NH2 as an initiator, the ring-opening polymerization of L-phenylalanine-N-carboxy anhydride Phe-NCA was initiated to obtain PZLL-S-PPhe;

[0021] (5) After the deprotection of the side-chain benzyl ester group of PZLL-S-PPhe, self-assembly was carried out in an aqueous solution to form polylysine-thioether bond-polyphenylalanine PLys-S-PPhe nanoparticles;

[0022] (6) PLys-S-PPhe nanoparticles, glutathione and a therapeutic gene were prepared by electrostatic adsorption to form a carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species.

[0023] Preferably, in step (2), the molar ratio of PZLL to acrylic acid is 1:1 to 1:2.

[0024] Preferably, in step (3), the molar ratio of PZLL-vinly to cysteamine is 1:1 to 1:2.

[0025] On the other hand, the present invention also provides the application of the above-mentioned carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species in the preparation of drugs for treating placenta-related oxidative stress diseases.

[0026] Preferably, the placenta-related oxidative stress disease is preeclampsia.

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

[0028] The diblock copolymer PLys-S-PPhe synthesized by the present invention has amphiphilicity and self-assembles into nano-micelles in aqueous solution. Due to the high positive charge property of its chain-like polylysine, glutathione and therapeutic genes can be complexed on the surface of the micelles through electrostatic adsorption. After entering the lesion, the adsorbed GSH on the outer shell reacts with the lesion ROS, playing an extracellular antioxidant therapy role in the placental microenvironment. After the antioxidant reaction occurs, the GSH outer shell dissociates, and the strong positive charge of the PLys-S-PPhe carrier inner core containing the drug is exposed. After the overall surface positive charge of the nano-carrier increases, it is easy to promote the endocytosis of diseased cells in the placenta. After endocytosis, the "sulfide bond" of the drug inner core dissociates specifically in the cell, further promoting the disintegration of the inner core and the release of the drug, producing the cell-targeted delivery effect of the drug and also producing the lesion-targeted retention effect of the positive charge inner core, achieving an efficient distribution similar to lesion targeting in the placenta, with low liver and kidney toxicity, reducing the toxicity to the mother or fetus, and providing a new carrier for drugs treating oxidative stress-related diseases in the placenta. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a synthetic route diagram of the PLys-S-PPhe inner core;

[0030] Figure 2 It is a schematic structural diagram of the carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species prepared in the examples of the present invention;

[0031] Figure 3 It is an electron micrograph of the carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species prepared in the examples of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention will be further described below through specific embodiments. The following examples are specific embodiments of the present invention, but the embodiments of the present invention are not limited by the following examples.

[0033] The sources of the raw materials used in the examples of the present invention are as shown in Table 1 below:

[0034] Table 1

[0035]

[0036]

[0037] Example 1: Preparation of a Carrier System for Targeted Drug Delivery Based on Glutathione-Responsive Microenvironment Reactive Oxygen Species

[0038] (1) Synthesis of poly(N-carbobenzoxylysine) (PZLL)

[0039] Using n-butylamine as an initiator, the ring-opening polymerization of N6-carbobenzoxy-L-lysine cyclic anhydride (Lys-NCA) was initiated to obtain PZLL. The specific operation for synthesizing PZLL is as follows: Weigh Lys-NCA (1.53 g, 5 mmol, 50 eq.), put it into a Schlenk flask completely dried in an oven, purge with nitrogen three times, seal it, and then add 20 mL of DMF with a syringe. After stirring and dissolving at 35 °C, add the prepared 5 mL of a DMF solution of n-butylamine (7.3 mg, 0.1 mmol, 1.0 eq.). Seal and isolate the air under a nitrogen environment, and stir and react in an oil bath at 35 °C for 72 h. After the reaction is completed, the reaction product is precipitated with an excess of diethyl ether to obtain a transparent viscous PZLL. This viscous substance is redissolved in chloroform and precipitated with an excess of diethyl ether to obtain a white solid. Vacuum dry at 40 °C for 24 hours to obtain a pure PZLL powder with a terminal amino group, and store it at 4 °C for later use.

[0040] (2) Synthesis of double bond-terminated poly(N-carbobenzoxylysine) (PZLL-vinly)

[0041] PZLL was connected with acrylic acid through an amidation reaction to introduce a double bond, and double bond-terminated poly(N-carbobenzoxylysine)-vinyl (PZLL-vinly) was synthesized. The specific operation for synthesis is as follows: Under nitrogen protection, add 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.) into a 100 mL reaction flask. After dissolving in 50 mL of freshly distilled chloroform, stir and react at room temperature for 1 h. Add 14.4 mg of acrylic acid (72 g / mol, 0.2 mmol, 1.0 eq.), react at room temperature for 24 h, filter to remove the insoluble substance DCU, precipitate in a large amount of cold anhydrous diethyl ether, filter, wash successively with absolute ethanol, ultrapure water with pH 5.0, absolute ethanol and anhydrous diethyl ether, and then dry to obtain the product PZLL-vinly;

[0042] (3) Synthesis of amino-terminated poly(N-carbobenzoxylysine) with a thioether bond (PZLL-S-NH2)

[0043] Under the initiation of azobisisobutyronitrile (AIBN), the thiol group of cysteamine was added to the double bond to synthesize the amino-terminated polymer PZLL-S-NH2 connected by a thioether bond. The specific synthesis operation is as follows: In a 50 mL reaction flask, dissolve 655 mg of the above-synthesized PZLL-vinly (6550 g / mol, 0.10 mmol, 1.0 eq.), 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.) in 20 mL of anhydrous DMSO, heat and stir the reaction at 70 °C for 48 h. Dialyze the reaction solution in anaerobic water for two days using a dialysis bag with a molecular cut-off of 3.4 kDa, and freeze-dry to obtain PZLL-S-NH2;

[0044] (4) Synthesis of poly(N-carbobenzoxylysine)-polyphenylalanine (PZLL-S-PPhe) connected by a thioether bond

[0045] Using the terminal amino group of PZLL-S-NH2 as an initiator, ring-opening polymerization of L-phenylalanine-N-carboxyanhydride (Phe-NCA) was initiated to obtain PZLL-S-PPhe. The specific operation for synthesizing PZLL is as follows: Add the above-synthesized PZLL-S-NH2 (6600 g / mol, 0.66 g, 0.1 mmol, 1.0 eq.) product and Phe-NCA (0.573 g, 3 mmol, 30 eq.) into a Schlenk flask completely dried in an oven, replace the air with nitrogen three times, seal it, and add 25 mL of DMF (2 wt%) with a syringe. Stir the reaction in an oil bath at 35 °C for 24 h. After the reaction, precipitate the reactant with an excess of ether to obtain a white solid of PZLL-S-PPhe. Vacuum dry it at 40 °C for 24 h to obtain a pure powder of PZLL-S-PPhe with a terminal amino group, and store it at 4 °C for later use.

[0046] (5) Deprotection of PZLL-S-PPhe

[0047] Dissolve the above-prepared PZLL-S-PPhe (2.0 g) in trifluoroacetic acid (0.04 g / mL), then introduce hydrogen bromide gas, bubble and react for 30 min, continue to stir the reaction in an ice bath for 12 h, and then precipitate with an excess of ether to obtain the deprotected product. Vacuum dry it at 40 °C for 24 h to obtain deprotected polylysine-thioether bond-polyphenylalanine (PLys-S-PPhe).

[0048] (6) Preparation of PLys-S-PPhe nanoparticles

[0049] The prepared PLys-S-PPhe above can form nanoparticles by self-assembly in an aqueous solution. The specific operation is as follows: Dissolve 20 mg of PLys-S-PPhe completely in 5 mL of PBS aqueous solution, then disperse the nanoparticles under ultrasonic treatment. After the ultrasonic treatment ends, filter with an aqueous phase filter head (pore size: 0.45 μm) to remove large aggregates, and then dialyze with a dialysis bag (molecular cut-off: 14 kDa) in PBS (pH 7.4) for 2 days to remove the polymers that have not formed particles, obtaining a PLys-S-PPhe nanoparticle solution. Figure 1 The synthetic route of PLys-S-PPhe nanoparticles is shown.

[0050] (7) Preparation of FSTL3 plasmid complexed with PLys-S-PPhe nanoparticles

[0051] Prepare an FSTL3 plasmid complexed nanoparticle PLys-S-PPhe carrier through electrostatic adsorption. The specific operation is as follows: Dilute 500 μg of FSTL3 plasmid with PBS to a final volume of 1.5 mL. Subsequently, mix the diluted solution of FSTL3 plasmid with the nanopolylysine particle solution, pipette and mix well, let it stand for 30 min, then place the solution in a dialysis bag (molecular cut-off: 14 kDa) and dialyze for 24 h to remove the unreacted plasmid, obtaining uniform FSTL3 plasmid complexed PLys-S-PPhe nanoparticles.

[0052] (8) Preparation of a carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species

[0053] The positively charged FSTL3 plasmid complexed PLys-S-PPhe nanoparticles and the negatively charged glutathione GSH can be complexed through electrostatic interaction to form a nanocomplex. The specific operation is as follows: Dropwise add the prepared GSH aqueous solution (0.2 g / mL, 10 mL) into the FSTL3 plasmid complexed PLys-S-PPhe nanoparticle solution (200 mg / mL, 5 mL) at a rate of 0.5 mL / min and stir for 1 h. Finally, place the solution in a dialysis bag (molecular cut-off: 14 kDa) and dialyze for 24 h to remove the unreacted GSH, then concentrate with an ultrafiltration tube (molecular cut-off: 100 kDa), and finally wash with PBS (pH 7.4) and filter through an aqueous phase filter head (pore size: 0.22 μm) to obtain a carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species (GSH@PLys-S-PPhe).

[0054] Figure 2The structural schematic diagram of the prepared carrier system is shown. It can be seen from the figure that the core of the carrier system is a micelle formed by the self-assembly of the diblock copolymer PLys-S-PPhe, and glutathione and the therapeutic gene are complexed on the surface of the micelle through electrostatic adsorption.

[0055] Figure 3 The electron micrograph of the prepared carrier system is shown.

[0056] Comparative Example 1: Synthesis of PEG-PLys-S-PPhe and Preparation of Nanocarriers Loaded with Therapeutic Gene Plasmid

[0057] (1) Synthesis of Methoxypolyethylene glycol-poly(N-carbobenzoxy-L-lysine) (mPEG-PZLL)

[0058] mPEG-PZLL was synthesized by ring-opening polymerization. Using mPEG-NH2 as an initiator, N6-carbobenzoxy-L-lysine-L-carboxylic anhydride (Lys-NCA) was initiated to carry out ring-opening polymerization to obtain mPEG-PZLL. The specific operation for synthesizing mPEG-PZLL is as follows: Weigh 1.53 g of Lys-NCA (5 mmol) and put it into a Schlenk flask completely dried in an oven. Replace the air with nitrogen three times, seal it, and then add 20 mL of DMF with a syringe. After stirring and dissolving at 35 °C, add 5 mL of the DMF solution of the above-prepared mPEG-NH2 (0.23 g, 0.1 mmol). Seal it in a nitrogen environment to isolate air, and stir and react in an oil bath at 35 °C for 72 h. After the reaction is completed, the reactant is precipitated with an excessive amount of ether to obtain a transparent viscous mPEG-PZLL. This viscous substance is redissolved in chloroform and precipitated with an excessive amount of ether to obtain a white solid. Vacuum dry at 40 °C for 24 h to obtain pure mPEG-PZLL powder with a terminal amino group;

[0059] (2) Synthesis of Double Bond-Terminated Methoxypolyethylene glycol-poly(N-carbobenzoxy-L-lysine) (mPEG-PZLL-vinly)

[0060] Connect mPEG-PZLL with acrylic acid through amidation reaction to introduce double bonds and synthesize vinyl-capped polyethylene glycol-poly(N-benzyloxycarbonyl lysine)-vinyl (mPEG-PZLL-vinly). The specific synthesis operations are as follows: Under nitrogen protection, add 1.7 g of mPEG-PZLL (8500 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.) into a 100 mL reaction flask. After dissolving in 50 mL of freshly distilled chloroform, stir the reaction at room temperature for 1 h. Add 14.4 mg of acrylic acid (72 g / mol, 0.2 mmol, 1.0 eq.), and after reacting at room temperature for 24 h, filter to remove the insoluble DCU, then precipitate in a large amount of cold anhydrous ether, filter, wash successively with absolute ethanol, ultrapure water with pH 5.0, absolute ethanol and anhydrous ether, and then dry to obtain the product mPEG-PZLL-vinly;

[0061] (3) Synthesis of amino-capped polyethylene glycol-poly(N-benzyloxycarbonyl lysine) with thioether bond (mPEG-PZLL-S-NH2)

[0062] Under the initiation of azobisisobutyronitrile (AIBN), use the thiol group of cysteamine to add to the double bond to synthesize the thioether bond-linked amino-capped polymer mPEG-PZLL-S-NH2. The specific synthesis operations are as follows: In a 50 mL reaction flask, dissolve 855 mg of the above-synthesized mPEG-PZLL-vinly (8550 g / mol, 0.10 mmol, 1.0 eq.), 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.) in 20 mL of anhydrous DMSO, heat and stir the reaction at 70 °C for 48 h. Dialyze the reaction solution in anaerobic water for two days using a dialysis bag with a molecular cut-off of 3.4 kDa, and then freeze-dry to obtain mPEG-PZLL-S-NH2;

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

[0064] Using the terminal amino group of mPEG-PZLL-S-NH2 as an initiator, ring-opening polymerization of L-phenylalanine-N-carboxyanhydride (Phe-NCA) was initiated to obtain mPEG-PZLL-S-PPhe. The specific operation for synthesizing mPEG-PZLL-S-PPhe is as follows: The product of mPEG-PZLL-S-NH2 (8600 g / mol, 0.86 g, 0.1 mmol, 1.0 eq.) synthesized above and Phe-NCA (0.573 g, 3 mmol, 30 eq.) were added into a Schlenk flask completely dried in an oven, purged with nitrogen three times, sealed, and 25 mL of DMF (2 wt%) was added with a syringe. The reaction was stirred in an oil bath at 35 °C for 24 h. After the reaction, the reactants were precipitated with excess ether to obtain a white solid. Vacuum drying at 40 °C for 24 h gave pure mPEG-PZLL-S-PPhe powder, which was stored at 4 °C for later use;

[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 then hydrogen bromide gas was introduced. The bubbling reaction was carried out for 30 min, and the reaction was continued with stirring in an ice bath for 12 h. Subsequently, it was precipitated with excess ether to obtain the deprotected product. Vacuum drying at 40 °C for 24 h gave deprotected polyethylene glycol-polylysine-thioether bond-polyphenylalanine (mPEG-PLys-S-PPhe);

[0067] (6) Preparation of mPEG-PLys-S-PPhe gene composite nanoparticles

[0068] Positively charged mPEG-PLys-S-PPhe nanoparticles and negatively charged FSTL3 plasmid can form nanocomposites 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 shaken evenly. 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 dropwise to the PBS solution (pH 7.4) of the nanocarrier at a rate of 0.5 mL / min under ultrasonic action. After the ultrasonic treatment, it was pipetted and allowed to stand for 30 min, and then filtered through a water phase filter head (pore size: 0.45 μm) to remove large aggregates. Subsequently, the solution was placed in a dialysis bag (molecular cut-off: 14 kDa) and dialyzed for 24 h to remove unreacted plasmid, washed with PBS (pH 7.4), and then filtered through a water phase filter head (pore size: 0.22 μm) to obtain a uniform FSTL3 plasmid-complexed mPEG-PLys-S-PPhe nanocarrier.

[0069] Comparative Example 2: Synthesis of Polylysine-Phenylalanine (PLys-PPhe) and Preparation of Nanocarriers Loaded with Therapeutic Gene Plasmids

[0070] (1) Using the amino-terminated PZLL synthesized by S1 as an initiator, ring-opening polymerization of phenylalanine N-carboxyanhydride (Phe-NCA) was initiated to obtain poly(N-carbobenzoxylysine)-phenylalanine (PZLL-PPhe). The specific operation is as follows: Weigh 1.5 g of the above-synthesized PZLL product, add the amino-terminated PZLL and Phe-NCA (0.573 g, 3 mmol) into a Schlenk flask completely dried in an oven, replace the air with nitrogen three times, seal it, and then add 25 mL of DMF (2 wt%) with a syringe. Stir and react in an oil bath at 35 °C for 24 h. After the reaction, the reactant was precipitated with an excess of diethyl ether to obtain a transparent viscous polymer product. This viscous substance was redissolved in chloroform and precipitated with an excess of diethyl ether to obtain a white solid PZLL-PPhe.

[0071] (2) After obtaining PZLL-PPhe, the deprotection operation was carried out: Dissolve the above-prepared PZLL-PPhe (1.0 g) in trifluoroacetic acid (0.04 g / mL), then introduce hydrogen bromide gas, and bubble and react for 30 min. Continue to stir and react in an ice bath for 12 h, and then precipitate with an excess of diethyl ether to obtain the deprotected product. Vacuum dry at 40 °C for 24 h to obtain deprotected polylysine-phenylalanine (PLys-PPhe).

[0072] (3) Positively charged PLys-PPhe nanoparticles and negatively charged FSTL3 plasmid can form a nanocomplex through electrostatic interaction. The specific operation is as follows: Dilute 500 μg of FSTL3 plasmid with PBS to a final volume of 1.5 mL and shake well. Dissolve 20 mg of PLys-PPhe in 5 mL of PBS solution, and then drop the diluted solution of FSTL3 plasmid into the PBS solution (pH 7.4) of the nanocarrier at a rate of 0.5 mL / min under ultrasonic action. After the ultrasonic treatment, blow and let it stand for 30 min, and filter with an aqueous filter head (pore size: 0.45 μm) to remove large aggregates. Subsequently, place the solution in a dialysis bag (molecular cut-off: 14 kDa) and dialyze for 24 h to remove unreacted plasmids. Wash with PBS (pH 7.4) and then filter through an aqueous filter head (pore size: 0.22 μm) to obtain a uniform PLys-PPhe nanocarrier complexed with FSTL3 plasmid.

[0073] (4) Positively charged FSTL3 plasmid complexed with PLys-PPhe nanoparticles and negatively charged GSH can form nanocomposites through electrostatic interaction. The specific operation is as follows: The prepared GSH aqueous solution (0.2 g / mL, 10 mL) was added dropwise 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 was placed in a dialysis bag (molecular cut-off: 14 kDa) and dialyzed for 24 h to remove unreacted GSH. Subsequently, the GSH@PLys-PPhe nanoparticle solution was concentrated using an ultrafiltration tube (molecular cut-off: 100 kDa), and finally washed with PBS (pH 7.4) and filtered through an aqueous filter head (pore size: 0.22 μm) to obtain the GSH@PLys-PPhe nanocarrier.

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

[0075] As shown in Table 2, the feed amounts of Lys-NCA in step (1), Phe-NCA in step (4), and GSH were changed, and the rest was the same as in Example 1.

[0076] Table 2: Examples and Comparative Examples

[0077]

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

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

[0080] Model establishment:

[0081] 8-week-old SPF-grade C57BL / 6 mice (purchased from Guangdong Provincial Medical Experimental Animal Center), female and male mice were caged and mated at a ratio of 2:1 during estrus. The next day, the vaginal secretions of the female mice were smeared and stained with Papanicolaou stain. Those with positive vaginal sperm were diagnosed as pregnant under an optical microscope and marked as day 0 of pregnancy (D0). The pregnant mice were fed with 2 mg / mL methyl L-arginine nitroso in sterile water to establish a preeclampsia / gestational hypertension model, and the pregnant mice fed with an equal amount of double-distilled water were used as the normal control group.

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

[0083] Near-infrared dye CY7 was used for carrier staining and tracing. On the 11th day, preeclampsia model animals were anesthetized with chloral hydrate and scanned at the time points before drug injection (0 h) and 2 hours after injection (2 h) to observe the in vivo distribution of the drug containing the near-infrared fluorescent dye. The dose of the nm drug injected via the tail vein was: (therapeutic dose of 12.5 μg nucleic acid equivalent drug, or an equal volume of PBS); an In vivo FX in vivo fluorescence scanner was used for in vivo imaging of C57BL / 6j mice. The relative signal increase ratio (RSI (Relative Signal Intensity) % = R2h / R0h) in the placenta region (lower abdominal uterine region of the mouse) or liver region was calculated 2 hours after drug injection, and the results are shown in Table 3.

[0084] Table 3 Results of the evaluation of placental-specific delivery function

[0085] Group Placenta RSI (Relative Signal Multiples) Liver RSI (Relative Signal Multiples) 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

[0086] 2. Establishment of a preeclampsia animal model to evaluate the treatment effect

[0087] On D3, D6, D9, D12, and D15, 12.5 μg nucleic acid equivalent drug, or an equal volume of normal saline, was injected, and a series of tests were performed on D17. The test results are shown in Table 4:

[0088] Blood pressure detection: Using a BP-2000 blood pressure analysis system, the systolic blood pressure (SBP) of pregnant mice was measured non-invasively using the tail cuff method. The room temperature was maintained at 26 °C, channel 1 was set to 1V (1V is equivalent to 300 mmHg), and channel 2 was set to SmV. The mouse was fixed in a mouse cage, and the mouse tail was placed in a 17 mm tail cuff, with the center of the bottom of the mouse tail touching the sensor. The pregnant mouse was continuously pressurized and measured 10 times in a quiet state, with an interval of 1 s each time, and the average value was taken and recorded.

[0089] Placenta and fetal examination: Placental tissue: The pregnant mouse was sacrificed, the abdominal cavity was opened, the uterus was dissected, and the fetus and placenta were taken out in sequence, and the number of surviving fetuses was recorded. The fetal membranes and umbilical cord on the placenta were removed, the umbilical cord at the fetal end was cut off at the root of the umbilical cord, and the placenta and fetus were placed on sterile gauze to absorb the surface amniotic fluid, and then weighed on an analytical balance. Placental tissue was cut and placed in liquid nitrogen and stored at -80 °C.

[0090] Table 4 Evaluation of the treatment effect of the preeclampsia animal model

[0091] Group Blood Pressure (mmHg) Fetal Weight (g) Litter Size per Birth Disease-free, 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

[0092] As can be seen from the results of Table 3 and Table 4, the strong positive potential (+20 - 42 mV) of the PLys-S-PPhe carrier core in Examples 1 - 3 is shielded by the adsorbed GSH shell into a weaker positive potential, which can avoid being phagocytosed after strongly positively stimulating the reticuloendothelial system mainly composed of the liver during the blood circulation before entering the lesion. The overall potential of these drugs is within (+3 - 15 mV), and they are not easily intercepted and phagocytosed by the reticuloendothelial system mainly composed of the liver, and there is less retention in the organs outside the lesion represented by the liver. The molecular contrast agent combined with the drug is distributed along with the drug, and the imaging signal of aggregation in the liver is weak. After the drug easily enters the placental blood vessels, it passes through the gaps between the neonatal capillary endothelial cells, reaches and targets and retains in the placental blood sinus (intervillous space), and the drug concentration in the placenta is relatively high. The molecular contrast agent combined with the drug is distributed along with the drug, and obvious aggregation imaging signals are generated in the placenta.

[0093] In addition, after the drugs in Examples 1 - 3 enter the lesion, the GSH shell reacts with the ROS in the lesion to produce a microenvironment antioxidant treatment effect, accompanied by obvious shell dissociation and release of the strong positive charge core "PLys-S-PPhe". After the release of the core and the increase of the overall surface positive charge of the drug, it is easy to promote the endocytosis of the diseased cells in the placenta, produce the cell-targeted delivery effect of the drug, and generate the lesion-targeted retention effect of the positive charge core. After endocytosis, the "thioether bond" of the drug core dissociates intracellularly in a targeted manner, promoting the disintegration of the core, drug release, and the exertion of the treatment effect. Therefore, the blood pressure of this group of animals is significantly restored, and the fetal weight and the number of offspring per litter are significantly restored.

[0094] The shell of Comparative Example 1 is a PEG shell connected by chemical bonds, with a relatively low potential and is not easily phagocytosed by the reticuloendothelial system represented by the liver. Moreover, this drug shell does not have GSH that can react with ROS in the lesion. After the drug enters the lesion, the shell cannot react with the ROS in the lesion, does not produce a microenvironment antioxidant treatment effect, and does not show obvious shell dissociation and release of the strong positive charge core. There is no release effect of the lesion-specific PLys-S-PPhe carrier core, and there is less aggregation in the placenta compared with the examples. The surface potential of the drug in the lesion is relatively low, and the endocytosis efficiency is lower and the treatment effect is weaker compared with the examples. Therefore, compared with the examples, the blood pressure recovery of this group of animals is worse, and the fetal weight and the number of offspring per litter are worse restored.

[0095] The outer shell of Comparative Example 2 is an adsorbed GSH outer shell, which can shield the strongly positively charged inner core. The overall drug has a relatively weak positive potential and is not easily phagocytosed by the reticuloendothelial system represented by the liver. After entering the lesion, the GSH outer shell reacts with the lesion ROS, producing a microenvironment antioxidant therapy effect, accompanied by obvious outer shell dissociation and release of the strongly positively charged inner core "PLys-PPhe". After the release of the inner core and the increase in the positive charge on the overall surface of the drug, it is easy to promote the endocytosis of diseased cells in the placenta, resulting in cell-targeted delivery of the drug and also the effect of targeted retention of the positively charged inner core in the lesion. However, compared with the Example, after endocytosis, the drug inner core does not have the "thioether bond" for targeted dissociation in the cell, and the disintegration of the inner core and the release of the drug are relatively difficult. Compared with the Example, the therapeutic effect is weaker. Therefore, compared with the Example, the blood pressure recovery of this group of animals is not obvious, and the recovery of fetal weight and litter size per pregnancy is poor.

[0096] In Comparative Example 3, due to the relatively low potential of its inner core, the overall amount of therapeutic plasmid bound is less. Compared with the Example, the therapeutic effect is weaker. Therefore, compared with the Example, the blood pressure recovery of this group of animals is poor, and the recovery of fetal weight and litter size per pregnancy is poor.

[0097] In Comparative Example 4, since the potentials of both the overall nano-carrier and the inner core are significantly higher than those of the Example, after microenvironment retention and endocytosis, too much drug is retained by the reticuloendothelial system, resulting in less drug reaching the placenta. Compared with the Example, the imaging signal in the placenta is reduced. And it has relatively high cytotoxicity, affecting the exertion of the therapeutic effect, and the therapeutic effect is poor. Therefore, compared with the Example, the blood pressure recovery of this group of animals is poor, and the recovery of fetal weight and litter size per pregnancy is poor.

[0098] In Comparative Example 5, the overall surface is complexed with less GSH, and the potential of the overall nano-carrier is relatively high. After microenvironment retention and endocytosis, too much drug is retained by the reticuloendothelial system, resulting in less drug reaching the placenta. Compared with the Example, the imaging signal in the placenta is reduced. And it has relatively high cytotoxicity, affecting the exertion of the therapeutic effect, and the therapeutic effect is poor. Therefore, compared with the Example, the blood pressure recovery of this group of animals is poor, and the recovery of fetal weight and litter size per pregnancy is poor.

[0099] 3. Toxicity evaluation of the drug for the animal model

[0100] At 72 hours after injecting the drug into the mice in the normal control group, blood was collected from the tail vein to detect the liver function indexes alanine transaminase (ALT), total bilirubin (TBil) and the kidney function indexes blood urea nitrogen (BUN) and serum creatinine (sCr). The detection instrument was a Hitachi 7600 type automatic biochemical analyzer, and the detection results are shown in Table 5.

[0101] Table 5 Toxicity evaluation results

[0102]

[0103]

[0104] As can be seen from the results in Table 5, the carrier system prepared by the present invention is not easily intercepted and phagocytosed by the reticuloendothelial system mainly composed of the liver, with less retention in extrahepatic organs represented by the liver and lower liver toxicity. These drugs achieve efficient distribution similar to lesion targeting in the placenta, with reduced blood drug concentration, no obvious toxic reaction in the kidneys, and no obvious change in renal function.

Claims

1. A carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species, characterized in that, The core of the carrier system is a micelle formed by the self-assembly of the diblock copolymer PLys-S-PPhe. Glutathione and the therapeutic gene are complexed on the surface of the micelle through electrostatic adsorption. The surface potential of the diblock copolymer PLys-S-PPhe is 20 - 42 mV, and the surface potential of the carrier system is 3 - 15 mV. The structural formula of the diblock copolymer PLys-S-PPhe is: ； where x = 40 - 60 and y = 20 - 30.

2. The carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species according to claim 1, characterized in that The therapeutic gene is FSTL3.

3. The preparation method of the carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species according to any one of claims 1-2, characterized in that, It includes the following steps: (1) Using n-butylamine as an initiator, initiating the ring-opening polymerization of N6-carbobenzoxy-L-lysine cyclic anhydride Lys-NCA to obtain poly(N-carbobenzoxylysine) PZLL; (2) Connecting PZLL and acrylic acid through an amidation reaction to introduce a double bond, and synthesizing a double-bond-capped poly(N-carbobenzoxylysine)-vinyl PZLL-vinly; (3) Under the initiation of azobisisobutyronitrile, adding the thiol group of cysteamine to the double bond of PZLL-vinly to synthesize an amino-capped polymer PZLL-S-NH2 connected by a thioether bond; (4) Using the terminal amino group of PZLL-S-NH2 as an initiator, initiating the ring-opening polymerization of L-phenylalanine-N-carboxyl cyclic anhydride Phe-NCA to obtain PZLL-S-PPhe; (5) After deprotecting and removing the side-chain benzyl ester group of PZLL-S-PPhe, self-assembling in an aqueous solution to form polylysine thioether bond-polyphenylalanine PLys-S-PPhe nanoparticles; (6) Preparing a carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species by electrostatic adsorption of PLys-S-PPhe nanoparticles with glutathione and the therapeutic gene.

4. The preparation method of the carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species according to claim 3, wherein, In step (2), the molar ratio of PZLL to acrylic acid is 1:1 - 1:

2.

5. The preparation method of the carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species according to claim 3, characterized in that, In step (3), the molar ratio of PZLL-vinly to cysteamine is 1:1 - 1:

2.

6. Use of the carrier system for targeted drug delivery based on glutathione-responsive microenvironment reactive oxygen species according to any one of claims 1 - 2 in the preparation of a drug for treating placenta intra-oxidative stress-related diseases, wherein the placenta intra-oxidative stress-related disease is preeclampsia.

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