Nanocomposite as well as preparation method and application thereof

Abstract

The invention relates to the field of high polymer materials and medical engineering, in particular to a nano-composite as well as a preparation method and application thereof. The preparation method of the nano compound comprises the following steps: uniformly mixing the PEANT-Fer-1, the phospholipid-ketal thiol-polyethylene glycol, the phospholipid-polyethylene glycol and the dimethyl sulfoxide to obtain the nano compound. The preparation method comprises the following steps: uniformly mixing the PEANT-Fer-1, the phospholipid-ketal thiol-polyethylene glycol, the phospholipid-polyethylene glycol and the dimethyl sulfoxide to obtain the nano compound; the PEANT-Fer-1 has a structure as shown in a formula I which is described in the specification; in the formula I, p is any integer from 3 to 12, q is any integer from 3 to 10, and r is any integer from 1 to 5. The nano-composite prepared by the invention can realize treatment of an EAE model, has good biological safety and ROS responsiveness, so that the drug can realize higher drug loading efficiency, prolong the drug circulation time, avoid premature release, reduce side effects and enhance the treatment effect.

Description

Technical Field

[0001] The present invention relates to the fields of polymer materials and medical engineering, and in particular to a nanocomposite, a preparation method and application thereof. Background Art

[0002] The experimental autoimmune encephalomyelitis (EAE) model is a mouse alternative model for multiple sclerosis (MS). Multiple sclerosis is an immune-mediated chronic inflammatory disease of the central nervous system, characterized by demyelination and neurodegeneration of the nervous system. It mainly damages the spinal cord, brain, and optic nerves, and has a high disability rate. Previous studies have shown that a decrease in Treg cells (or dysfunction) and an increase in Th17 are the main pathogenic mechanisms of MS, but in recent years, some scholars have found through research that ferroptosis is also one of the pathogenesis of MS. In essence, MS is driven by the auto-amplification mechanism of inflammation and cell death. Current therapies mainly improve disease symptoms through immunosuppression, and there is no treatment for controlling cell death damage.

[0003] Ferroptosis is a newly discovered programmed cell death mode, which is mainly driven by iron imbalance and lipid peroxidation, leading to cellular oxidative stress, thereby affecting processes such as protein, nucleic acid, and lipid metabolism, and ultimately leading to cell death. In recent years, ferroptosis has been found to be involved in the occurrence and development of MS. The mechanism of ferroptosis involves iron homeostasis, lipid metabolism, and redox system; the auto-oxidation process of polyunsaturated fatty acids (PUFA) is the driving factor of ferroptosis; free radical trapping antioxidants (RTA) can terminate free radical chain reactions, form stable non-free radical products, and prevent cell membrane damage; ideally, better RTAs inhibit ferroptosis by directly terminating auto-oxidation reactions with high free radical trapping ability. Ferrostatin-1 (Fer-1) is one of the earliest reported synthetic RTAs that can inhibit ferroptosis and is widely used in ferroptosis research; ferroptosis inhibitors are expected to become new therapeutic drugs for MS, but small molecule ferroptosis inhibitors have a short half-life and cannot be targeted and released at the lesion. Therefore, it is very necessary to explore new ferroptosis inhibitor delivery systems, prolong the time that ferroptosis inhibitors circulate in the body, release drugs more accurately at the lesions, reduce side effects, and relieve EAE symptoms.

[0004] Reactive oxygen species (ROS) are the main molecules produced when the body is under oxidative stress, which can cause DNA damage and genetic instability. Changes in ROS often lead to various diseases, such as chronic gastrointestinal diseases, autoimmune diseases, fatty liver, tumors, etc. In recent years, the difference in ROS levels between normal tissues and EAE model lesions has been used to design ROS-responsive nanodelivery systems, which can release drugs in a high ROS environment at the lesion, thereby improving the targeting and efficacy of drugs, becoming a new direction. Similarly, the application of ROS-responsive nanoparticles in the delivery of ferroptosis inhibitors has also attracted great attention. For example, the use of ROS-responsive nanoparticles to deliver ferroptosis inhibitor prodrugs can effectively promote mesenchymal stem cell-mediated spinal cord injury repair, significantly inhibit ferroptosis and inflammatory responses after spinal cord injury, and promote neurological function recovery. In addition, nanomedicines can also improve the bioavailability of drugs and reduce the side effects of drugs. In recent years, research on targeting ferroptosis to prevent and treat major diseases has also been deepening. Studies have shown that a variety of diseases, including tumors and neurodegenerative diseases, can be treated by targeting ferroptosis. Ferroptosis inhibitors can also be used in combination with other treatments to improve the therapeutic effect.

[0005] In recent years, disease-modifying drugs for MS have been approved, but there is still no drug that can completely prevent or reverse progressive neurodegeneration, and MS patients are still in urgent need of more effective new therapies. Ferroptosis is a newly discovered form of programmed cell death, and focusing on ferroptosis inhibitors is expected to provide new diagnostic and treatment ideas for MS patients; based on the short half-life of small molecule ferroptosis inhibitors, frequent injections are required to maintain effective drug concentrations, and the key problem is that the efficiency of action at the lesion site is low. There is an urgent need to develop a nano-delivery system that can release ferroptosis inhibitors in a targeted manner. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a nanocomposite, a preparation method and application thereof. The nanocomposite prepared by the present invention can achieve the treatment of EAE model, and at the same time, has good biosafety and ROS responsiveness, so that the drug can achieve a higher drug loading efficiency, prolong the drug circulation time, avoid premature release, reduce side effects, and enhance the therapeutic effect.

[0007] The present invention provides a method for preparing a nanocomposite, comprising the following steps:

[0008] After PEANT-Fer-1, phospholipid-thioketal-polyethylene glycol, phospholipid-polyethylene glycol and dimethyl sulfoxide are mixed, a nanocomposite is obtained;

[0009] The PEANT-Fer-1 has the structure shown in Formula I;

[0010]

[0011] In formula I, p is any integer from 3 to 12, q is any integer from 3 to 10, and r is any integer from 1 to 5.

[0012] Preferably, the preparation method of PEANT-Fer-1 comprises the following steps:

[0013] Under protective gas conditions, a mixed solution including PEG-PAN-TK, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and tetrahydrofuran is mixed with a mixed solution including ethyl 3-amino-4-cyclohexylaminobenzoate and triethylamine, and then reacted to obtain PEANT-Fer-1;

[0014] The PEG-PAN-TK has a structure shown in Formula II;

[0015]

[0016] In formula II, m is any integer from 3 to 12, and n is any integer from 4 to 13.

[0017] Preferably, the preparation method of PEG-PAN-TK comprises the following steps:

[0018] Under protective gas conditions, a mixed solution including 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and tetrahydrofuran is mixed with PEG-PANH and triethylamine, and then reacted to obtain PEG-PAN-TK;

[0019] The PEG-PANH has a structure shown in Formula III;

[0020]

[0021] In formula III, k is any integer from 7 to 25.

[0022] Preferably, the preparation method of PEG-PANH comprises the following steps:

[0023] Dissolve PEG-PAN-BOC in a hydrochloric acid-tetrahydrofuran solution, continue to introduce HCl gas and stir the reaction at room temperature to obtain PEG-PANH;

[0024] The PEG-PAN-BOC has a structure shown in Formula IV;

[0025]

[0026] In formula IV, k is any integer from 7 to 25.

[0027] Preferably, the preparation method of PEG-PAN-BOC comprises the following steps:

[0028] PEG-PAGE, 2-tert-butyloxycarbonylaminoethanethiol, benzoin dimethyl ether and dichloromethane were mixed, and stirred for reaction under protective gas conditions and ultraviolet light irradiation to obtain PEG-PAN-BOC;

[0029] The PEG-PAGE has a structure shown in Formula V;

[0030]

[0031] In formula V, k is any integer from 7 to 25.

[0032] Preferably, the preparation method of PEG-PAGE comprises the following steps:

[0033] Under carbon dioxide conditions, allyl glycidyl ether, (1S,2S)-(+)-1,2-cyclohexanediamine-N,N'-bis(3,5-di-T-butylsalicylaldehyde)cobalt(III)-trifluoroacetate, bis(triphenylphosphoryl)ammonium trifluoroacetate, a chain transfer agent, dichloromethane and toluene are mixed and then polymerized to obtain a polycarbonate polymer PEG-PAGE.

[0034] Preferably, the molar ratio of allyl glycidyl ether, salen Co-TFA, PPN-TFA and chain transfer agent is 400:18-22:0.5-1.5:0.5-1.5;

[0035] The concentration of the carbon dioxide is 2.5-3.5 MPa;

[0036] The polymerization reaction temperature is 20-30°C.

[0037] Preferably, the molar ratio of PEG-PAGE, 2-tert-butyloxycarbonylaminoethanethiol and benzoin dimethyl ether is 8-12:125-135:0.5-1.5;

[0038] The reaction temperature is 20-30°C;

[0039] After the reaction, the method further comprises: mixing the reaction solution with ether for precipitation, removing the supernatant by centrifugation, dissolving the solid with dichloromethane, precipitating with ether, and drying the obtained solid under reduced pressure to obtain PEG-PAN-BOC.

[0040] Preferably, the dosage ratio of the PEG-PAN-BOC to the hydrochloric acid-tetrahydrofuran solution is 380-420 mg: 3-7 mL;

[0041] After the stirring reaction, the method further comprises: mixing the reaction solution with ether for precipitation, removing the supernatant by centrifugation, dissolving the solid with dichloromethane, precipitating with ether, and drying the obtained solid under reduced pressure to obtain PEG-PANH.

[0042] Preferably, the molar ratio of the 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, PEG-PANH and triethylamine is 13-17: 0.5-1.5: 13-17: 13-17: 28-32;

[0043] After the reaction, the method further comprises: freeze-drying the reaction solution, dialyzing it in deionized water, and then freeze-drying the dialyzate to obtain PEG-PAN-TK.

[0044] Preferably, the molar ratio of PEG-PAN-TK, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, ethyl 3-amino-4-cyclohexylaminobenzoate and triethylamine is 2.5-3.5:8-12:31-35:31-35;

[0045] After the reaction, the method further comprises: freeze-drying the reaction solution, dialyzing it in deionized water, and then freeze-drying the dialyzate to obtain PEANT-Fer-1.

[0046] Preferably, the mass ratio of PEANT-Fer-1, phospholipid-thioketal-polyethylene glycol and phospholipid-polyethylene glycol is 0.5-1.5:0.5-1.5:0.5-1.5;

[0047] After the mixing, the method further comprises: adding double distilled water dropwise, performing ultrasound and performing dialysis to obtain a nanocomposite.

[0048] The present invention also provides a nanocomposite prepared by the preparation method described above.

[0049] The present invention also provides a use of the nanocomposite described above in at least one of the following aspects:

[0050] 1) preparing drug-carrying materials;

[0051] 2) Preparation of anti-inflammatory drugs;

[0052] 3) Prepare ferroptosis inhibitory drugs.

[0053] Preferably, the drug-carrying material is a nano-delivery material for directed release of ferroptosis inhibitors or a ROS-responsive nano-delivery material;

[0054] The anti-inflammatory drug is an anti-myelitis drug.

[0055] The invention provides a method for preparing a nanocomposite, comprising the following steps: mixing PEANT-Fer-1, phospholipid-thioketal-polyethylene glycol (DSPE-TK-PEG), phospholipid-polyethylene glycol (DSPE-PEG) and dimethyl sulfoxide (DMSO) to obtain a nanocomposite; the PEANT-Fer-1 has a structure shown in formula I; in formula I, p is any integer from 3 to 12, q is any integer from 3 to 10, and r is any integer from 1 to 5.

[0056] The present invention provides a method for preparing a ferroptosis-inhibiting nanocomposite with ROS responsiveness, wherein the carrier is a biodegradable polymer, PEG is used as an initiator, and the TK bond stimulates the release of drugs in response; utilizing the difference in ROS between lesions and normal tissues, the release of Fer-1 is stimulated under the high ROS level in the lesions, thereby achieving multiple effects such as ferroptosis inhibition, anti-inflammation, and ROS consumption, thereby achieving the treatment of the EAE model, and at the same time, the nanocomposite has good biosafety.

[0057] The small molecule Fer-1 has a short half-life. The present invention forms nanoparticles (i.e., nanocomplexes) through self-assembly and packaging. At the same time, it has ROS responsiveness, so that the drug can achieve a higher drug loading efficiency, prolong the drug circulation time, avoid premature release, reduce side effects, and enhance the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 A complete set of synthesis steps of PEG-PAN-TK of the present invention;

[0059] Figure 2 The polycarbonate polymer PEG-PAGE synthesized in Example 1 of the present invention 1 H NMR spectrum;

[0060] Figure 3 PEG-PAN-BOC synthesized in Example 1 of the present invention 1 H NMR spectrum;

[0061] Figure 4 PEG-PANH synthesized in Example 1 of the present invention 1 H NMR spectrum;

[0062] Figure 5 PEG-PAN-TK synthesized in Example 1 of the present invention 1H NMR spectrum;

[0063] Figure 6 The figure is a synthetic step diagram of PEANT-Fer-1 of the present invention;

[0064] Figure 7 PEANT-Fer-1 synthesized in Example 1 of the present invention 1 H NMR spectrum;

[0065] Figure 8 The cytotoxicity test results of Fer-1 NPs;

[0066] Fig. 9 The content of ferrous ions and H2O2 in the lesion of the EAE model of the present invention;

[0067] Fig.10 The in vivo pharmacodynamic verification scheme and effect in Example 4 of the present invention;

[0068] Fig.11 The pathological section diagram in Example 4;

[0069] Fig.12 This is the in vivo efficacy and anti-inflammatory effect in Example 5 of the present invention. DETAILED DESCRIPTION

[0070] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0071] The present invention provides a method for preparing a nanocomposite, comprising the following steps:

[0072] The nanocomplex was obtained by mixing PEANT-Fer-1, phospholipid-thioketal-polyethylene glycol (DSPE-TK-PEG), phospholipid-polyethylene glycol (DSPE-PEG) and dimethyl sulfoxide (DMSO);

[0073] The PEANT-Fer-1 has the structure shown in Formula I;

[0074]

[0075] In formula I, p is any integer from 3 to 12, q is any integer from 3 to 10, and r is any integer from 1 to 5.

[0076] Specifically, p=5, q=5, r=2.

[0077] In some embodiments of the present invention, the method for preparing PEANT-Fer-1 comprises the following steps:

[0078] Under protective gas conditions, a mixed solution including PEG-PAN-TK, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS) and tetrahydrofuran is mixed with a mixed solution including 3-amino-4-cyclohexylaminobenzoic acid ethyl ester (Ferrostatin-1, Fer-1) and triethylamine, and then reacted to obtain PEANT-Fer-1;

[0079] The PEG-PAN-TK has a structure shown in Formula II;

[0080]

[0081] In formula II, m is any integer from 3 to 12, and n is any integer from 4 to 13.

[0082] Specifically, m=5, n=7.

[0083] In some embodiments of the present invention, the method for preparing PEG-PAN-TK comprises the following steps:

[0084] Under protective gas conditions, a mixed solution including 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid (TK), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS) and tetrahydrofuran is mixed with PEG-PANH and triethylamine, and then reacted to obtain PEG-PAN-TK;

[0085] The PEG-PANH has a structure shown in Formula III;

[0086]

[0087] In formula III, k is any integer from 7 to 25.

[0088] Specifically, k=12.

[0089] In some embodiments of the present invention, the method for preparing PEG-PANH comprises the following steps:

[0090] Dissolve PEG-PAN-BOC in a hydrochloric acid-tetrahydrofuran solution, continue to introduce HCl gas and stir the reaction at room temperature to obtain PEG-PANH;

[0091] The PEG-PAN-BOC has a structure shown in Formula IV;

[0092]

[0093] In formula IV, k is any integer from 7 to 25.

[0094] Specifically, k=12.

[0095] In some embodiments of the present invention, the preparation method of PEG-PAN-BOC comprises the following steps:

[0096] PEG-PAGE, 2-tert-butyloxycarbonylaminoethanethiol, benzoin dimethyl ether and dichloromethane were mixed, and stirred for reaction under protective gas conditions and ultraviolet light irradiation to obtain PEG-PAN-BOC;

[0097] The PEG-PAGE has a structure shown in Formula V;

[0098]

[0099] In formula V, k is any integer from 7 to 25.

[0100] Specifically, k=12.

[0101] In some embodiments of the present invention, the method for preparing PEG-PAGE comprises the following steps:

[0102] Under carbon dioxide conditions, allyl glycidyl ether (AGE monomer), (1S,2S)-(+)-1,2-cyclohexanediamine-N,N'-bis(3,5-di-T-butylsalicylaldehyde)cobalt(III)-trifluoroacetate (salen Co-TFA), bis(triphenylphosphoryl)ammonium trifluoroacetate (PPN-TFA), a chain transfer agent, dichloromethane and toluene are mixed and polymerized to obtain a polycarbonate polymer PEG-PAGE.

[0103] The present invention has no special restrictions on the source of (1S, 2S)-(+)-1,2-cyclohexanediamine-N, N'-bis(3,5-di-T-butyl salicylaldehyde) cobalt (III)-trifluoroacetate (salen Co-TFA). In an embodiment of the present invention, the salen Co-TFA is prepared by reacting (1S, 2S)-(+)-1,2-cyclohexanediamine-N, N'-bis(3,5-di-T-butyl salicylaldehyde) cobalt (II) (Salen-Co) and trifluoroacetic acid under oxygen conditions in dichloromethane. Specifically, the preparation method of the salen Co-TFA comprises the following steps:

[0104] 1g of (1S,2S)-(+)-1,2-cyclohexanediamine-N,N'-bis(3,5-di-T-butyl salicylaldehyde) cobalt(II) (Salen-Co) was dissolved in 10mL of dichloromethane, stirred and dissolved at room temperature, and then 0.3g of trifluoroacetic acid was added, and the reaction was stirred for 12h under oxygen conditions. The molar ratio of the Salen Co to the trifluoroacetic acid was 1:1.8. Then, the dichloromethane was removed by reduced pressure distillation at 25°C, and the obtained solid was washed with 3mL of n-hexane, and the liquid was removed by filtration to obtain 0.8g of (1S,2S)-(+)-1,2-cyclohexanediamine-N,N'-bis(3,5-di-T-butyl salicylaldehyde) cobalt(III)-trifluoroacetate (salen Co-TFA), with a yield of 69%.

[0105] The present invention has no particular restrictions on the source of bis(triphenylphosphorane)ammonium trifluoroacetate (PPN-TFA). In an embodiment of the present invention, the PPN-TFA is prepared by reacting bis(triphenylphosphorane)ammonium chloride (PPN-Cl) and trifluoroacetic acid in water. Specifically, the preparation method of the PPN-TFA comprises the following steps:

[0106] Take 1g of bis(triphenylphosphorane)ammonium chloride (PPN-Cl) and add it to 10mL of water, heat it to 60°C to make it completely dissolved, then add 0.3g of trifluoroacetic acid and stir to react for 5min, the molar ratio of PPN-Cl to trifluoroacetic acid is 1:1.8, filter while hot to obtain 0.8g of bis(triphenylphosphorane)ammonium trifluoroacetate solid, with a yield of 70%.

[0107] The chain transfer agent may be polyethylene glycol; specifically, it may be PEG-5000.

[0108] The molar ratio of the AGE monomer, salenCo-TFA, PPN-TFA and chain transfer agent is 400:18-22:0.5-1.5:0.5-1.5, such as 400:20:1:1.

[0109] The dichloromethane and toluene are solvents. The present invention has no special limitation on the usage of the dichloromethane and toluene. In some embodiments, the volume ratio of the dichloromethane and toluene is 0.5-1.5:0.5-1.5, such as 1:1.

[0110] The concentration of the carbon dioxide is 2.5-3.5 MPa, for example 3.0 MPa.

[0111] The polymerization reaction temperature is 20-30° C., such as 25° C., and the polymerization reaction time is 45-55 h, such as 48 h.

[0112] After obtaining PEG-PAGE, PEG-PAGE, 2-tert-butyloxycarbonylaminoethanethiol, benzoin dimethyl ether and dichloromethane are mixed, and stirred for reaction under protective gas conditions and ultraviolet light irradiation to obtain PEG-PAN-BOC.

[0113] The protective gas is nitrogen.

[0114] The molar ratio of PEG-PAGE, 2-tert-butyloxycarbonylaminoethanethiol and benzoin dimethyl ether is 8-12:125-135:0.5-1.5, such as 10:120:1.

[0115] The dichloromethane is a solvent, and the present invention has no particular limitation on the amount of the solvent.

[0116] The reaction temperature is 20-30°C, such as 25°C; the reaction time is 2-6h, such as 4h.

[0117] After the reaction, the method further comprises: mixing the reaction solution with ether for precipitation, removing the supernatant by centrifugation, dissolving the solid with dichloromethane, precipitating with ether, and drying the obtained solid under reduced pressure to obtain PEG-PAN-BOC.

[0118] In the first precipitation after the reaction, the volume of ether is 9 to 11 times, for example, 10 times, the volume of the reaction solution.

[0119] In the precipitate obtained after the solid is dissolved in dichloromethane, the volume of ether is 9 to 11 times, for example, 10 times, the volume of the dissolving solution.

[0120] After obtaining PEG-PAN-BOC, PEG-PAN-BOC is dissolved in a hydrochloric acid-tetrahydrofuran solution, HCl gas is continuously introduced and the reaction is stirred at room temperature to obtain PEG-PANH.

[0121] The concentration of the hydrochloric acid-tetrahydrofuran solution is 0.08-0.12 g / mL, such as 0.1 g / mL.

[0122] The dosage ratio of the PEG-PAN-BOC to the hydrochloric acid-tetrahydrofuran solution is 380-420 mg: 3-7 mL, such as 400 mg: 5 mL.

[0123] The stirring reaction time is 4 to 8 hours, such as 6 hours.

[0124] After the stirring reaction, the method further comprises: mixing the reaction solution with ether for precipitation, removing the supernatant by centrifugation, dissolving the solid with dichloromethane, precipitating with ether, and drying the obtained solid under reduced pressure to obtain PEG-PANH.

[0125] In the first precipitation after the reaction, the volume of ether is 9 to 11 times, for example, 10 times, the volume of the reaction solution.

[0126] In the precipitate obtained after the solid is dissolved in dichloromethane, the volume of ether is 9 to 11 times, for example, 10 times, the volume of the dissolving solution.

[0127] After obtaining PEG-PANH, under protective gas conditions, a mixed solution including 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid (TK), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS) and tetrahydrofuran is mixed with PEG-PANH and triethylamine, and then reacted to obtain PEG-PAN-TK.

[0128] Specifically, they include:

[0129] Under protective gas conditions, 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid (TK), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS) and tetrahydrofuran were stirred to react, and then PEG-PANH and triethylamine were added and stirred to react to obtain PEG-PAN-TK.

[0130] The protective gas is nitrogen.

[0131] 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid (TK), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS) and tetrahydrofuran are stirred for reaction at a temperature of 20 to 30°C, such as 25°C, and for a reaction time of 0.5 to 1.5 hours, such as 1 hour.

[0132] The temperature for stirring the reaction with PEG-PANH and triethylamine is 20-30° C., such as 25° C., and the time is 2-4 hours, such as 3 hours.

[0133] The molar ratio of 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid (TK), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS), PEG-PANH and triethylamine is 13-17:0.5-1.5:13-17:13-17:28-32, such as 15:1:15:15:30.

[0134] The tetrahydrofuran is a solvent, and the present invention has no particular limitation on the amount of the solvent.

[0135] After the reaction, the method further comprises: freeze-drying the reaction solution, dialyzing it in deionized water, and then freeze-drying the dialyzate to obtain PEG-PAN-TK.

[0136] Figure 1 1 is a complete set of synthetic steps for PEG-PAN-TK of the present invention.

[0137] After obtaining PEG-PAN-TK, under protective gas conditions, a mixed solution including PEG-PAN-TK, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS) and tetrahydrofuran is mixed with a mixed solution including 3-amino-4-cyclohexylaminobenzoic acid ethyl ester (Ferrostatin-1, Fer-1) and triethylamine, and then reacted to obtain PEANT-Fer-1.

[0138] Specifically, they include:

[0139] Under protective gas conditions, PEG-PAN-TK, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS) and tetrahydrofuran are stirred to react, and then 3-amino-4-cyclohexylaminobenzoic acid ethyl ester (Ferrostatin-1, Fer-1) and triethylamine are added and stirred to react to obtain PEANT-Fer-1.

[0140] The protective gas is nitrogen.

[0141] The temperature of stirring the reaction of PEG-PAN-TK, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS) and tetrahydrofuran is 20-30° C., such as 25° C., and the reaction time is 0.5-1.5 h, such as 1 h.

[0142] Then, the mixture is stirred with 3-amino-4-cyclohexylaminobenzoic acid ethyl ester (Ferrostatin-1, Fer-1) and triethylamine at a temperature of 20 to 30° C., such as 25° C., and a reaction time of 2 to 4 hours, such as 3 hours.

[0143] The molar ratio of PEG-PAN-TK, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS), 3-amino-4-cyclohexylaminobenzoic acid ethyl ester (Ferrostatin-1, Fer-1) and triethylamine is 2.5-3.5:8-12:31-35:31-35:48-52, for example, 3:10:33:33:50.

[0144] The tetrahydrofuran is a solvent, and the present invention has no particular limitation on the amount of the solvent.

[0145] After the reaction, the method further comprises: freeze-drying the reaction solution, dialyzing it in deionized water, and then freeze-drying the dialyzate to obtain PEANT-Fer-1.

[0146] After PEANT-Fer-1 is obtained, PEANT-Fer-1, phospholipid-thioketal-polyethylene glycol (DSPE-TK-PEG), phospholipid-polyethylene glycol (DSPE-PEG) and dimethyl sulfoxide (DMSO) are mixed to obtain nanocomplexes (Fer-1 NPs).

[0147] The phospholipid-thioketal-polyethylene glycol (DSPE-TK-PEG) is DSPE-TK-PEG 2000. The phospholipid-polyethylene glycol (DSPE-PEG) is DSPE-PEG 2000.

[0148] The mass ratio of PEANT-Fer-1, phospholipid-thioketal-polyethylene glycol (DSPE-TK-PEG) and phospholipid-polyethylene glycol (DSPE-PEG) is 0.5-1.5:0.5-1.5:0.5-1.5, such as 1:1:1.

[0149] The dimethyl sulfoxide is a solvent, and the present invention has no particular limitation on the amount of the solvent.

[0150] The mixing is performed at room temperature.

[0151] After the mixing, the method further includes: dripping double distilled water, ultrasonicating, and dialysis to obtain a nanocomposite. The dripping of double distilled water is to prepare nanoparticles by nanoprecipitation method. The ultrasonication time is 3 to 7 minutes, such as 5 minutes. The dialysis uses a 5000Da dialysis bag. The dialysis time is 22 to 26 hours, such as 24 hours. The concentration of the nanocomposite (Fer-1 NPs) is 1.5 to 2.5 mg / mL, such as 2 mg / mL.

[0152] The present invention also provides a nanocomposite prepared by the preparation method described above.

[0153] The present invention also provides a use of the nanocomposite described above in at least one of the following aspects:

[0154] 1) Preparation of drug-loaded materials; such as nano-delivery materials for targeted release of ferroptosis inhibitors or ROS-responsive nano-delivery materials;

[0155] 2) Preparation of anti-inflammatory drugs; such as anti-myelitis drugs, specifically anti-experimental autoimmune encephalomyelitis drugs;

[0156] 3) Prepare ferroptosis inhibitory drugs.

[0157] The present invention modifies a small molecule ferroptosis inhibitor (Fer-1) drug onto the side chain of a high molecular weight polymer, and connects it through a thioketal to give the drug ROS responsiveness, thereby achieving targeted release of the drug at the lesion, effectively enriching the lesion site, increasing the bioavailability of the drug, consuming ROS while inhibiting ferroptosis, synergistically acting on the lesion, and improving the therapeutic effect.

[0158] The present invention provides a new idea for the treatment of EAE models.

[0159] The nanocomplex exhibits a 100% disease inhibition rate on mice, and can reduce inflammation in the spinal cord of mice, induce an increase in M2 microglia, and reduce inflammation.

[0160] The present invention has no particular limitation on the sources of the raw materials used above, and they can be generally commercially available.

[0161] In order to further illustrate the present invention, a nanocomposite provided by the present invention, its preparation method and application are described in detail below in conjunction with examples, but it should not be understood as limiting the scope of protection of the present invention.

[0162] Example 1

[0163] Preparation of ROS-responsive ferroptosis-inhibiting nanocomplexes:

[0164] 1) Synthesis of PEG-PAGE:

[0165] 962 mg of AGE monomer, 15.1 mg of salenCo-TFA, 13.7 mg of PPN-TFA, 2.11 g of chain transfer agent PEG-5000, 0.5 mL of dichloromethane and 0.5 mL of toluene were placed in a 10 mL autoclave, wherein the molar ratio of the AGE monomer, salenCo-TFA, PPN-TFA and chain transfer agent PEG-5000 was 400:20:1:1, the CO2 concentration was adjusted to 3.0 MPa, and the polymerization was carried out at 25° C. for 48 h to obtain a polycarbonate polymer PEG-PAGE having a structure shown in formula V (wherein k=12). The synthesis steps are shown in Figure 1 , 1 H NMR spectrum is shown in Figure 2 .

[0166] Figure 2 The polycarbonate polymer PEG-PAGE synthesized in Example 1 of the present invention 1 H NMR spectrum.

[0167] 2) Synthesis of PEG-PAN-BOC:

[0168] 1 g of PEG-PAGE, 308 mg of 2-tert-butyloxycarbonylaminoethanethiol, 3.7 mg of benzoin dimethyl ether and 10 mL of dichloromethane were placed in a round-bottom flask, wherein the molar ratio of the PEG-PAGE, 2-tert-butyloxycarbonylaminoethanethiol and benzoin dimethyl ether was 10:120:1. The mixture was stirred at 25°C for 4 h under nitrogen atmosphere and UV irradiation. The reaction solution was mixed with ether having a volume of 10 times that of the solution for precipitation. The supernatant was removed by centrifugation. The solid was dissolved in dichloromethane and precipitated with ether having a volume of 10 times that of the solution for 3 times. The obtained solid was dried under reduced pressure to obtain PEG-PAN-BOC having a structure shown in Formula IV (where k=12). The synthesis steps are shown in Figure 1 , 1 H NMR spectrum is shown in Figure 3 .

[0169] Figure 3 PEG-PAN-BOC synthesized in Example 1 of the present invention 1 H NMR spectrum.

[0170] 3) Synthesis of PEG-PANH:

[0171] 400 mg of PEG-PAN-BOC was dissolved in 5 mL of 0.1 g / mL hydrochloric acid-tetrahydrofuran solution, HCl gas was continuously introduced and stirred at room temperature for 6 h, the reaction solution was mixed with 10 times the volume of ether for precipitation, the supernatant was removed by centrifugation, the solid was dissolved in dichloromethane and precipitated with 10 times the volume of ether for 3 times, the obtained solid was dried under reduced pressure to obtain PEG-PANH having a structure shown in Formula III (wherein k=12), the synthesis steps are shown in Figure 1 , 1 H NMR spectrum is shown in Figure 4 .

[0172] Figure 4 PEG-PANH synthesized in Example 1 of the present invention 1 H NMR spectrum.

[0173] 4) Synthesis of PEG-PAN-TK:

[0174] 48.3 mg of 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid (TK), 36.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 22 mg of N-hydroxysuccinimide (NHS) and 3 ml of tetrahydrofuran were placed in a high vacuum reaction tube and stirred at 25°C for 1 h under a nitrogen environment. Then, 100 mg of PEG-PANH and 38.8 mg of triethylamine were added. The molar ratio of TK, EDCI, NHS, PEG-PANH and triethylamine was 15:1:15:15:30. The reaction was continued under nitrogen environment at 25°C with stirring for 3 h. The reaction solution was freeze-dried and dialyzed in deionized water for 48 h. The dialyzate was freeze-dried to obtain PEG-PAN-TK having a structure shown in Formula II (wherein m=5 and n=7). The synthesis steps are shown in FIG. Figure 1 , 1 H NMR spectrum is shown in Figure 5 .

[0175] Figure 5 PEG-PAN-TK synthesized in Example 1 of the present invention 1 H NMR spectrum.

[0176] 5) Synthesis of PEANT-Fer-1:

[0177] 100 mg PEG-PAN-TK, 19.5 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 14.5 mg N-hydroxysuccinimide (NHS) and 3 ml tetrahydrofuran were placed in a high vacuum reaction tube and stirred at 25° C. for 1 h under a nitrogen environment. Then, 100 mg 3-amino-4-cyclohexylaminobenzoic acid ethyl ester (Ferrostatin-1, Fer-1) and 19.3 mg triethylamine were added. The molar ratio of TK, EDCI, NHS, PEG-PAN-TK and triethylamine was 3:10:33:33:50. Stirring was continued at 25° C. for 3 h under a nitrogen environment. After the reaction solution was freeze-dried, it was dialyzed in deionized water for 48 h, and the dialyzate was freeze-dried to obtain PEANT-Fer-1 having a structure shown in Formula I (wherein p=5, q=5, r=2). The synthesis steps are shown in Figure 6 , 1 H NMR spectrum is shown in Figure 7 .

[0178] Figure 6 Schematic diagram of the synthesis steps of PEANT-Fer-1 of the present invention.

[0179] Figure 7 PEANT-Fer-1 synthesized in Example 1 of the present invention 1 H NMR spectrum.

[0180] 6) Preparation of Nanocomposite Fer-1 NPs:

[0181] 30 mg PEANT-Fer-1, 30 mg DSPE-TK-PEG2000 and 30 mg DSPE-PEG2000 were dissolved in 300 μL DMSO, mixed at room temperature, and then 14.1 mL double distilled water was added dropwise. After ultrasonication for 5 min, the mixture was transferred to a 5000 Da dialysis bag and dialyzed for 24 h to obtain nanocomplex Fer-1 NPs. The concentration of the nanocomplex (Fer-1 NPs) was 2 mg / mL.

[0182] Example 2

[0183] Investigation of ROS responsiveness, stability and cytotoxicity of Fer-1 NPs:

[0184] 1) In the EAE lesions, there is a high level of H2O2 locally due to oxidative stress. Therefore, different H2O2 concentrations can be used to simulate the state of the lesions in vitro. By detecting the dynamic light scattering (DLS) particle size under different H2O2 concentrations, it is confirmed that Fer-1NPs can release Fer-1 in response to high ROS conditions. Therefore, it can be concluded that Fer-1 NPs stably carry drugs in the blood circulation and quickly release drugs after reaching the lesions, thereby extending the half-life of the drugs and better exerting their therapeutic effects.

[0185] 2) Fer-1 NPs were placed in phosphate buffer (PBS, pH 7.2-7.4, 0.01 mol / L), saline, fetal bovine serum (FBS), and water, incubated at 37°C, and the DLS particle size at different time points was dynamically detected. It was found that the particle size of Fer-1 NPs did not change significantly within 48 hours, indicating that Fer-1 NPs have good stability.

[0186] 3) BV2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were incubated with different concentrations of Fer-1 and Fer-1 NPs for 24 h, and then CCK8 was added. The absorbance at 450 nm was detected by an ELISA reader. The results are as follows: Figure 8 shown. Figure 8 The cytotoxicity test results of Fer-1NPs. Figure 8 It can be seen that at the concentrations of 0.1μmol / L, 0.5μmol / L, 1μmol / L, 2μmol / L, 5μmol / L, 10μmol / L, and 20μmol / L, there was no significant difference in the survival rate of BV2 cells after Fer-1 NPs compared with Fer-1. The results show that Fer-1 NPs have no obvious cytotoxicity.

[0187] Example 3

[0188] Investigate the content of ferrous ions and H2O2 in the lesions of EAE model:

[0189] The EAE model was established in C57BL / 6J mice (18-20 g, female, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) by using myelin oligodendrocyte glycoprotein 35-55 (MOG35-55, Gill Biochemical), inactivated Mycobacterium tuberculosis (H37Ra) (BD Difco, 231141), complete Freund's adjuvant, and pertussis toxin (List Biological Labs, 179A). The spinal cord and brain of the mice were removed at the peak of the disease, and the ferrous ion and H2O2 contents were detected by the kit. The results are as follows: Fig. 9 shown. Fig. 9 The content of ferrous ions and H2O2 in the EAE model lesion of the present invention. Fig. 9 It can be seen that compared with the WT group, the content of ferrous ions and H2O2 in the lesions of the EAE model was significantly increased.

[0190] Example 4

[0191] Investigating the in vivo efficacy of Fer-1 NPs in the EAE model:

[0192] In order to verify the in vivo efficacy of Fer-1 NPs in the early and peak stages of EAE model establishment, Fer-1 NPs were injected through the tail vein 3 days after establishment and after onset (14 days after establishment). The specific dosing regimen is shown in Fig.10 A, 3 C57BL / J mice (18-20 g, female, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) in each group. Fig.10 This is the in vivo pharmacodynamic verification scheme and effect in Example 4 of the present invention.

[0193] Fig.10 A is a schematic diagram of the specific animal experiment scheme. The mice were euthanized on the 28th day after modeling. Fig.10 B represents the clinical scores of mice. The results showed that compared with the EAE group, the clinical scores of the Fer-1 NPs prevention group and the Fer-1 NPs treatment group were significantly lower; Fig.10 C represents the incidence rate of mice. The results showed that the mice in the WT group became ill on the 15th day after modeling, while the mice in the Fer-1 NPs prevention group began to become ill on the 18th day after modeling, and the mice in the Fer-1 NPs treatment group did not become ill until the 28th day after modeling, indicating that the Fer-1 NPs treatment group showed a 100% disease inhibition rate in mice. Fig.10D represents the change in mouse weight. The results showed that there was no significant difference in mouse weight among the groups, indicating that Fer-1 NPs had no significant effect on mouse weight. This suggests that treatment can delay the peak of disease onset and alleviate clinical symptoms, and the effect of the Fer-1 NPs treatment group is more significant.

[0194] To further verify the therapeutic effect, the spinal cord and brain of mice were dissected and tested for ferrous ion, malondialdehyde (MDA), superoxide dismutase (SOD) and other test kits. Fig.10 (EJ), Fig.10 E is mouse spinal cord Fe 2+ content, Fig.10 H is Fe in mouse brain 2 + The results showed that compared with the EAE group, the Fe content in the spinal cord and brain of the mice in the Fer-1NPs prevention and treatment groups was 2+ The contents of both groups were decreased with significant differences, especially in the Fer-1NPs treatment group. Fig.10 F is the MDA content in the mouse spinal cord, Fig.10 I is the MDA content in the mouse brain. The results showed that compared with the WT group, the MDA content in the EAE group was significantly increased. After treatment with Fer-1NPs, the MDA content was significantly decreased, and there were significant differences. Fig.10 G is the SOD content in the mouse spinal cord, Fig.10 J is the SOD content in the mouse brain. The results showed that compared with the WT group, the SOD content in the EAE group was significantly decreased. After treatment with Fer-1NPs, the SOD content was significantly increased, and there were significant differences. The above results indicate that Fer-1 NPs can significantly improve ferroptosis in the mouse brain and spinal cord.

[0195] Pathological sections were also performed, including H&E staining, fast blue staining, and Nissl staining. Fig.11 It is the pathological section diagram in Example 4. Fig.11 It can be seen that compared with the WT group, the EAE group had increased spinal cord inflammatory infiltration, demyelination, and neuronal damage; the Fer-1NPs treatment group was more effective in maintaining myelin integrity and reducing inflammatory cell infiltration and neuronal damage.

[0196] Combined with the above results, it can be concluded that Fer-1 NPs have significant therapeutic effect.

[0197] Example 5

[0198] Investigation of the anti-inflammatory effect of Fer-1 NPs:

[0199] In order to further compare the efficacy of Fer-1 NPs and Fer-1, and to explore the anti-inflammatory effect of Fer-1 NPs, the EAE model was re-established, and Fer-1 and Fer-1 NPs were injected through the tail vein 3 days after modeling and 14 days after onset of the disease, respectively. The specific dosing regimen is shown in Fig.12 A, C57BL / 6J mice (18-20 g, female, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were euthanized until the peak of the disease (20 days after modeling). Fig.12 This is the in vivo efficacy and anti-inflammatory effect in Example 5 of the present invention. Fig.12 A is a schematic diagram of the specific animal experiment plan. Fig.12 B is the clinical score of mice. The results showed that compared with the Fer-1 prevention group, the clinical score of the Fer-1 NPs prevention group was significantly reduced, and compared with the Fer-1 treatment group, the clinical score of the Fer-1 NPs treatment group was significantly reduced, both with significant differences; Fig.12 C is the incidence rate of mice. The results showed that compared with the Fer-1 prevention group and treatment group, the onset time of the Fer-1 NPs prevention group and treatment group was delayed, and no mice in the Fer-1 NPs treatment group became ill after administration; Fig.12 D is the change in mouse weight. The results show that there is no significant difference in the weight of mice among the groups, indicating that Fer-1 NPs has no significant effect on the weight of mice. The results show that the efficacy of the Fer-1 NPs group is better than that of the Fer-1 group, whether for preventive or therapeutic administration.

[0200] To verify the effect of drugs on microglia, mouse spinal cords were dissected for flow cytometry, following the following steps: tissues were collected and single-cell suspensions were prepared, cell suspensions were filtered to remove clumps and debris, cells were centrifuged and resuspended in staining solution, cell counting and activity analysis were performed, anti-CD11b-PE / Cy7 (purchased from Biolegend), anti-F4 / 80-APC (purchased from Biolegend), and anti-CD86-PE antibodies (purchased from Biolegend) were incubated, membranes were broken and fixed, anti-CD206-FITC antibodies were incubated, and the results were detected on the machine; the results are shown in Figure 2 . Fig.12 (EF). Fig.12 E is CD86 + The results showed that compared with the EAE group, the Fer-1 NPs prevention group and the treatment group significantly reduced the proportion of CD86 + Compared with the Fer-1 prevention group and treatment group, the Fer-1 NPs prevention group and treatment group significantly reduced the CD86 + There were significant differences in the proportion of microglia. Fig.12 F is CD206 +The results showed that compared with the EAE group, the Fer-1 NPs prevention group and the treatment group significantly increased the CD206 + Compared with the Fer-1 prevention group and treatment group, the Fer-1 NPs prevention group and treatment group significantly increased CD206 + The proportion of microglia showed significant differences. The results showed that Fer-1 NPs can promote the polarization of microglia to M2, enhance anti-inflammatory effects and tissue repair ability.

[0201] Example 6

[0202] Investigation of the biosafety of Fer-1 NPs:

[0203] At the end of the in vivo experiment, orbital venous blood was collected from C57BL / 6J mice (18-20g, female, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) to detect blood routine and blood biochemical indicators of mice. It was found that there was no significant difference in each treatment group compared with the WT group; the heart, liver, spleen, lung, and kidney of C57BL / 6J mice (18-20g, female, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were taken for H&E staining, indicating that there was no obvious pathological damage in each group, indicating that Fer-1 NPs have good biosafety.

[0204] Example 7

[0205] Investigating the in vitro efficacy of Fer-1 NPs:

[0206] After clarifying the efficacy of Fer-1 NPs, we further verified the efficacy and explored its mechanism of action in vitro, using the ferroptosis inducer RSL3 to induce ferroptosis in BV2 cells. The cells were divided into DMSO group, RSL3 group, RSL3+Fer-1 group, and RSL3+Fer-1 NPs group, and CCK8, qPCR and other methods were used to detect the efficacy.

[0207] The description of the above embodiments is only used to help understand the method of the present invention and its core idea. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a nanocomposite, comprising the following steps: PEANT-Fer-1, phospholipid-thioketal-polyethylene glycol, phospholipid-polyethylene glycol and dimethyl sulfoxide were mixed to obtain a nanocomposite; The PEANT-Fer-1 has the structure shown in Formula I; In formula I, p is any integer from 3 to 12, q is any integer from 3 to 10, and r is any integer from 1 to 5.

2. The preparation method according to claim 1, characterized in that: The preparation method of PEANT-Fer-1 comprises the following steps: Under protective gas conditions, a mixed solution including PEG-PAN-TK, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and tetrahydrofuran is mixed with a mixed solution including ethyl 3-amino-4-cyclohexylaminobenzoate and triethylamine, and then reacted to obtain PEANT-Fer-1; The PEG-PAN-TK has a structure shown in Formula II; In formula II, m is any integer from 3 to 12, and n is any integer from 4 to 13.

3. The preparation method according to claim 2, characterized in that: The preparation method of the PEG-PAN-TK comprises the following steps: Under protective gas conditions, a mixed solution including 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and tetrahydrofuran is mixed with PEG-PANH and triethylamine, and then reacted to obtain PEG-PAN-TK; The PEG-PANH has a structure shown in Formula III; In formula III, k is any integer from 7 to 25.

4. The preparation method according to claim 3, characterized in that: The preparation method of the PEG-PANH comprises the following steps: Dissolve PEG-PAN-BOC in a hydrochloric acid-tetrahydrofuran solution, continue to introduce HCl gas and stir the reaction at room temperature to obtain PEG-PANH; The PEG-PAN-BOC has a structure shown in Formula IV; In formula IV, k is any integer from 7 to 25.

5. The preparation method according to claim 4, characterized in that: The preparation method of the PEG-PAN-BOC comprises the following steps: PEG-PAGE, 2-tert-butyloxycarbonylaminoethanethiol, benzoin dimethyl ether and dichloromethane were mixed, and stirred to react under protective gas conditions and ultraviolet light irradiation to obtain PEG-PAN-BOC; The PEG-PAGE has a structure shown in Formula V; In formula V, k is any integer from 7 to 25.

6. The preparation method according to claim 5, characterized in that: The preparation method of the PEG-PAGE comprises the following steps: Under carbon dioxide conditions, allyl glycidyl ether, (1S,2S)-(+)-1,2-cyclohexanediamine-N,N'-bis(3,5-di-T-butylsalicylaldehyde)cobalt(III)-trifluoroacetate, bis(triphenylphosphoryl)ammonium trifluoroacetate, a chain transfer agent, dichloromethane and toluene are mixed and then polymerized to obtain a polycarbonate polymer PEG-PAGE.

7. The preparation method according to claim 6, characterized in that: The molar ratio of allyl glycidyl ether, salen Co-TFA, PPN-TFA and chain transfer agent is 400: 18-22: 0.5-1.5: 0.5-1.5; The concentration of the carbon dioxide is 2.5-3.5 MPa; The polymerization reaction temperature is 20-30°C.

8. The preparation method according to claim 5, characterized in that: The molar ratio of PEG-PAGE, 2-tert-butyloxycarbonylaminoethanethiol and benzoin dimethyl ether is 8-12:125-135:0.5-1.5; The reaction temperature is 20-30°C; After the reaction, the method further comprises: mixing the reaction solution with ether for precipitation, removing the supernatant by centrifugation, dissolving the solid with dichloromethane, precipitating with ether, and drying the obtained solid under reduced pressure to obtain PEG-PAN-BOC.

9. The preparation method according to claim 4, characterized in that: The dosage ratio of the PEG-PAN-BOC to the hydrochloric acid-tetrahydrofuran solution is 380-420 mg: 3-7 mL; After the stirring reaction, the method further comprises: mixing the reaction solution with ether for precipitation, removing the supernatant by centrifugation, dissolving the solid with dichloromethane, precipitating with ether, and drying the obtained solid under reduced pressure to obtain PEG-PANH.

10. The preparation method according to claim 3, characterized in that: The molar ratio of 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, PEG-PANH and triethylamine is 13-17: 0.5-1.5: 13-17: 13-17: 28-32; After the reaction, the method further comprises: freeze-drying the reaction solution, dialyzing it in deionized water, and then freeze-drying the dialyzate to obtain PEG-PAN-TK.

11. The preparation method according to claim 2, characterized in that: The molar ratio of PEG-PAN-TK, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, ethyl 3-amino-4-cyclohexylaminobenzoate and triethylamine is 2.5-3.5:8-12:31-35:31-35; After the reaction, the method further comprises: freeze-drying the reaction solution, dialyzing it in deionized water, and then freeze-drying the dialyzate to obtain PEANT-Fer-1.

12. The preparation method according to claim 1, characterized in that: The mass ratio of PEANT-Fer-1, phospholipid-thioketal-polyethylene glycol and phospholipid-polyethylene glycol is 0.5-1.5:0.5-1.5:0.5-1.5; After the mixing, the method further comprises: adding double distilled water dropwise, performing ultrasound and performing dialysis to obtain a nanocomposite.

13. A nanocomposite prepared by the preparation method according to any one of claims 1 to 12.

14. A use of the nanocomposite according to claim 13 in at least one of the following aspects; 1) preparing drug-carrying materials; 2) Preparation of anti-inflammatory drugs; 3) Prepare ferroptosis inhibitory drugs.

15. The use according to claim 14, characterized in that: The drug-carrying material is a nano-delivery material for directed release of ferroptosis inhibitors or a ROS-responsive nano-delivery material; The anti-inflammatory drug is an anti-myelitis drug.

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