Phosphazene-based amphiphilic conjugate, anti-inflammatory nano-micelle as well as preparation method and application of phosphazene-based amphiphilic conjugate and anti-inflammatory nano-micelle

Abstract

The invention belongs to the technical field of biomedical nano materials, and particularly relates to a phosphazene-based amphiphilic conjugate, an anti-inflammatory nano-micelle as well as a preparation method and application of the phosphazene-based amphiphilic conjugate and the anti-inflammatory nano-micelle. The structural formula of the phosphazene-based amphiphilic conjugate is shown in the specification. The anti-inflammatory nano-micelle based on the phosphazene-based amphiphilic conjugate can spontaneously form a micelle-shaped nano-structure with uniform particle size and high stability in an aqueous solution, and the solubility and the dispersity of insoluble active ingredients such as luminol and the like are remarkably improved; and the anti-inflammatory nano-micelle has the synergistic active oxygen scavenging ability of luminol and Tempol, can effectively regulate the oxidative stress microenvironment of an inflammatory site and relieve inflammatory response, and can be used as an inflammatory response carrier to realize targeted delivery of anti-inflammatory drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine nanomaterials, and particularly relates to a phosphazene-based amphiphilic conjugate, an anti-inflammatory nanomicelle and a preparation method and application thereof. BACKGROUND

[0002] The prevention and treatment of inflammation-related diseases are the research focus in the field of biological medicine. The drug-free macromolecule therapy has become the core research direction in this field due to its unique advantages of being able to regulate inflammatory response and restore the homeostasis of pro-inflammatory microenvironment. Natural polysaccharides and their derivatives have been confirmed to have anti-inflammatory activity in various animal models, but they generally have the problem of low efficacy. The polysaccharide derivatives chemically modified by biological active sites can exert strong anti-inflammatory effect through multiple mechanisms such as scavenging reactive oxygen species (ROS), inhibiting nuclear factor kappa B (NF-κB) nuclear translocation, reducing inflammatory cell recruitment and activation, and blocking NETosis. In addition, synthetic glycoproteins and sugar polymers can inhibit the secretion of pro-inflammatory cytokines by neutrophils and macrophages; dendrimers conjugated with sulfate or phosphorus groups can block leukocyte infiltration as multivalent inhibitors; and rationally designed complement protein C3dg display peptide assemblies can effectively protect mice from lipopolysaccharide (LPS) or imiquimod-induced inflammatory injury. Such functional macromolecules provide a new technical path for the treatment of inflammatory diseases.

[0003] There are still many key bottlenecks in the prior art. On the one hand, ROS scavenging-related macromolecule therapies (such as polymers containing 2,2,6,6-tetramethylpiperidine-1-oxyl radical (Tempol), phenylboronic acid pinacol ester, and bilirubin-polymer conjugates) have achieved good efficacy in animal models of acute kidney / liver injury, colitis, atherosclerosis, etc., but the molecular structure is complex, the structure control and customization are difficult, and the large-scale production with low cost and high repeatability has become a core obstacle to clinical translation. On the other hand, luminol and its derivatives are widely used in forensic detection and inflammation imaging due to their chemiluminescence characteristics, and have potential value in treating alopecia areata and promoting wound healing, but their solubility in physiological pH aqueous solution is extremely poor and their biological barrier penetration ability is weak, resulting in low in vivo absorption and bioavailability. Existing luminol conjugated materials (such as cyclodextrin nanoparticles) have the defects of difficult structure regulation and can only be dissolved in specific high-polarity solvents, which cannot meet the needs of the development of conversion-type preparations.

[0004] Therefore, it is necessary to develop a phosphazene-based amphiphilic conjugate and nanomicelle with anti-inflammatory activity and high bioavailability. SUMMARY

[0005] Based on this, the present application aims to provide a phosphazene-based amphiphilic conjugate, an anti-inflammatory nanomicelle and a preparation method and application thereof. By covalently connecting luminol, 2, 2, 6, 6-tetramethylpiperidine-1-oxyl radical (Tempol) and polyethylene glycol (PEG) to a hydrolysable cyclic phosphazene skeleton, an amphiphilic conjugate with high bioactivity is formed; the amphiphilic conjugate spontaneously forms a nanomicelle with stable structure and uniform particle size distribution through intermolecular forces. The nanomicelle has an accurate chemical structure and is easy to functionally regulate, and at the same time has a high active oxygen scavenging capacity of luminol and Tempol, can promote the normalization of the inflammatory microenvironment, and realize the prevention and treatment of inflammation-related diseases.

[0006] In order to achieve the above-mentioned purpose, the present application can adopt the following technical solutions: The present application provides a phosphazene-based amphiphilic conjugate, and the structural formula is as follows: Among them, R is , n is an integer of 10-50; R1 is ; R2 is .

[0007] The present application provides an intermediate, and the structure is as follows: Intermediate 1: ; or Intermediate 2: ; or Intermediate 3: ; Among them, n is an integer of 10-50.

[0008] The present application provides an anti-inflammatory nanomicelle, which is obtained by dispersing the above-mentioned phosphazene-based amphiphilic conjugate in water.

[0009] Preferably, the preparation method of the above-mentioned anti-inflammatory nanomicelle comprises: placing the phosphazene-based amphiphilic conjugate in water, and obtaining the anti-inflammatory nanomicelle after ultrasonic treatment and standing; wherein the ultrasonic power is 40W-100W, the ultrasonic treatment time is 5min-30min; and / or the standing time is 5min-60min, Preferably, in the above-mentioned anti-inflammatory nanomicelle, the concentration of the phosphazene-based amphiphilic conjugate in water is ≥12.3μg / mL.

[0010] In still another aspect, the present application provides a preparation method of phosphazene-based amphiphilic conjugate, which comprises: (1) reacting hexachlorophosphazene and Tpl to obtain intermediate 1; (2) reacting intermediate 1 and methoxypolyethylene glycol amino to obtain intermediate 2; (3) reacting intermediate 2 and propargylamine to obtain intermediate 3; and (4) reacting intermediate 3 and azidoluninol to obtain the phosphazene-based amphiphilic conjugate; wherein the intermediate 1, the intermediate 2 and the intermediate 3 have the following structural formulas, respectively: ; the intermediate 2: ; and the intermediate 3: ; wherein n is an integer of 10-50.

[0011] Preferably, the preparation method comprises: (a) dissolving hexachlorophosphazene in anhydrous dichloromethane, adding triethylamine, and dropwise adding an anhydrous dichloromethane solution of Tpl to react to obtain the intermediate 1; (b) dissolving the intermediate 1 in anhydrous tetrahydrofuran, adding triethylamine, and dropwise adding an anhydrous tetrahydrofuran solution of methoxypolyethylene glycol amino to react to obtain the intermediate 2; (c) dissolving the intermediate 2, triethylamine and propargylamine in anhydrous tetrahydrofuran to react to obtain the intermediate 3; and (d) dissolving the intermediate 3 and azidoluninol in a dimethyl sulfoxide / water mixed solvent, using copper sulfate and sodium ascorbate as a click reaction catalyst to react to obtain the phosphazene-based amphiphilic conjugate.

[0012] More preferably, the preparation method satisfies one or more of conditions (i) to (iv): (i) in step (a): the molar ratio of hexachlorophosphazene, Tpl and triethylamine is 1: (0.5-1.2): (1-2.4); and / or the total amount of anhydrous dichloromethane is 10 mL / g-20 mL / g based on the total mass of hexachlorophosphazene and Tpl; (ii) in step (b): the molar ratio of the intermediate 1, methoxypolyethylene glycol amino and triethylamine is 1: (0.8-1): (1.6-2); and / or the total amount of anhydrous tetrahydrofuran is 10 mL / g-20 mL / g based on the total mass of the intermediate 1 and methoxypolyethylene glycol amino; (iii) in step (c): the molar ratio of the intermediate 2, propargylamine and triethylamine is 1: (4-6): (8-10); and / or the amount of anhydrous tetrahydrofuran is 10 mL / g-20 mL / g based on the total mass of the intermediate 2 and propargylamine; (iv) in step (d): the molar ratio of the intermediate 3, azidoluninol, copper sulfate and sodium ascorbate is 1: (4-5): (1-2): (2-4); the amount of dimethyl sulfoxide / water mixed solvent is 10 mL / g-20 mL / g based on the total mass of the intermediate 3 and azidoluninol; and / or the volume ratio of dimethyl sulfoxide to water in the mixed solvent is 3: (1-3).

[0013] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-mentioned anti-inflammatory nanomicelles and an effective amount of the active pharmaceutical ingredient.

[0014] In another aspect, the present invention provides the application of the above-mentioned anti-inflammatory nanomicelles in regulating the inflammatory microenvironment and / or scavenging reactive oxygen species.

[0015] The beneficial effects of this invention include: the anti-inflammatory nanomicelles based on the phosphazene-based amphiphilic conjugates provided by this invention can spontaneously form a uniform and stable micelle-like nanostructure in aqueous solution, significantly improving the solubility and dispersibility of poorly soluble active ingredients such as luminol; and the anti-inflammatory nanomicelles also have the synergistic reactive oxygen species scavenging ability of luminol and tempol, which can effectively regulate the oxidative stress microenvironment at the site of inflammation, alleviate the inflammatory response, and at the same time, can serve as an inflammatory responsive carrier to achieve targeted delivery of anti-inflammatory drugs. Attached Figure Description

[0016] Figure 1 Synthetic route of phosphazene amphiphilic conjugate (PL4T1) in anti-inflammatory nanomicelles; Figure 2 The 1H NMR spectrum of the phosphazene amphiphilic conjugate (PL4T1) in anti-inflammatory nanomicelles; Figure 3 Fourier infrared spectra of the phosphazene amphiphilic conjugate (PL4T1) and intermediate products in anti-inflammatory nanomicelles; Figure 4 Transmission electron microscopy image and particle size distribution of anti-inflammatory nanomicelles (PL4T1NM); Figure 5 Critical micelle concentration curves for anti-inflammatory nanomicelles (PL4T1 NM) determined by pyrene fluorescence method; Figure 6 The total antioxidant capacity evaluation curve of the anti-inflammatory nanomicelles (PL4T1 NM) is shown. Figure 7 The DPPH and hydroxyl radical scavenging curves of anti-inflammatory nanomicelles (PL4T1 NM); Figure 8 Hydrogen peroxide scavenging curves of anti-inflammatory nanomicelles (PL4T1 NM); Figure 9 The superoxide anion scavenging curve of anti-inflammatory nanomicelles (PL4T1 NM) is shown. Detailed Implementation

[0017] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0018] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0019] In a first aspect, embodiments of the present invention provide a phosphazene-based amphiphilic conjugate, the structural formula of which is shown below: Where: R is n is an integer from 10 to 50; R1 is R2 is .

[0020] It should be noted that R in this invention represents methoxylated polyethylene glycol amino ( R1 is a 4-amino-2,2,6,6-tetramethylpiperidine-1-oxo radical ( R2 is connected to the phosphazene ring via a 1,2,3-triazole ring.

[0021] Secondly, embodiments of the present invention provide an intermediate, the structure of which is shown below: Intermediate 1: ;or Intermediate 2: ;or Intermediate 3: ; Where n is an integer from 10 to 50.

[0022] It should be noted that the intermediates 1 to 3 mentioned above are intermediates for preparing phosphazene-based amphiphilic conjugates. Phosphazene-based amphiphilic conjugates are prepared by using intermediates 1, 2 or 3 as raw materials.

[0023] Thirdly, embodiments of the present invention provide an anti-inflammatory nanomicelle, which is obtained by dispersing the above-mentioned phosphazene-based amphiphilic conjugate in water.

[0024] It should be noted that the phosphazene-based amphiphilic conjugates in this invention can be dispersed in water to obtain anti-inflammatory nanomicelles. The preparation method is simple and conducive to obtaining anti-inflammatory nanomicelles in industrial production.

[0025] In some specific examples, the preparation method of the above-mentioned anti-inflammatory nanomicelles includes: placing the phosphazene-based amphiphilic conjugate in water, sonicating it, and then allowing it to stand to obtain anti-inflammatory nanomicelles; wherein the sonication power is 40W to 100W, the sonication time is 5min to 30min, and / or the standing time is 5min to 60min.

[0026] It should be noted that, in a further preferred embodiment, the phosphazene-based amphiphilic conjugate is placed in water and then subjected to ultrasonic treatment. Ultrasonic treatment results in more uniform dispersion of the phosphazene-based amphiphilic conjugate and more stable anti-inflammatory nanomicelles. The ultrasonic power can be 40W–100W (e.g., 50W, 60W, 70W, 80W, or 90W), and the ultrasonic treatment time can be 5min–30min (e.g., 10min, 15min, 20min, or 25min). Additionally, the settling time can be 5min–60min (e.g., 10min, 20min, 30min, 40min, or 50min).

[0027] In some specific examples, the concentration of the phosphazene amphiphilic conjugate in water in the above-mentioned anti-inflammatory nanomicelles is ≥12.3 μg / mL.

[0028] It should be noted that the phosphazene amphiphilic conjugate in this invention can form a micelle structure when the concentration in water is ≥12.3 μg / mL.

[0029] Fourthly, in another aspect, the present invention provides a method for preparing a phosphazene-based amphiphilic conjugate, the method comprising: (1) reacting hexachlorophosphazene with Tpl to obtain intermediate 1; (2) reacting intermediate 1 with methoxy polyethylene glycol amino to obtain intermediate 2; (3) reacting intermediate 2 with propargylamine to obtain intermediate 3; (4) reacting intermediate 3 with azidoluminol to obtain the phosphazene-based amphiphilic conjugate; wherein the structural formulas of intermediate 1, intermediate 2 and intermediate 3 are respectively shown below: Intermediate 1: ; Intermediate 2: ; Intermediate 3: ; Where n is an integer from 10 to 50.

[0030] It should be noted that the structure of luminol azide is as follows: Furthermore, the phosphazene-based amphiphilic conjugates of this invention can be prepared according to methods known in the art based on the presented structure, or according to the methods listed above. Moreover, the preparation process of the phosphazene-based amphiphilic conjugates of this invention is based on stepwise nucleophilic substitution of hexachlorophosphazene and alkyne-azide click reaction; the synthesis method is simple, the structure is precise, and the combination and ratio of functional modules can be flexibly adjusted.

[0031] In some specific examples, the above preparation method includes: (a) dissolving hexachlorophosphazene in anhydrous dichloromethane, adding triethylamine, and adding dropwise a solution of Tpl in anhydrous dichloromethane to obtain intermediate 1; (b) dissolving intermediate 1 in anhydrous tetrahydrofuran, adding triethylamine, and adding dropwise a solution of methoxy polyethylene glycol amino in anhydrous tetrahydrofuran to obtain intermediate 2; (c) dissolving intermediate 2, triethylamine, and propargylamine in anhydrous tetrahydrofuran to obtain intermediate 3; and (d) dissolving intermediate 3 and luminol azide in a dimethyl sulfoxide / water mixed solvent, using copper sulfate and sodium ascorbate as click catalysts to obtain a phosphazene-based amphiphilic conjugate.

[0032] It should be noted that in the above preparation method, hexachlorophosphazene (HCCP) is the core precursor of the phosphazene-based skeleton, which connects various functional groups through nucleophilic substitution reactions of chlorine atoms; Tpl provides specific functional groups (such as hydroxyl, amino, etc.), which react with hexachlorophosphazene to form intermediate 1; azido-modified luminol provides an azide group, which undergoes a click reaction with the alkynyl group, while introducing the luminescent function of luminol, ultimately forming a phosphazene-based amphiphilic conjugate. In addition, triethylamine in this invention acts as an acid-binding agent, absorbing HCl (a byproduct of the nucleophilic substitution reaction) generated in the reaction, preventing HCl from interfering with the reaction or destroying the product, and can also adjust the alkalinity of the reaction system.

[0033] In some specific examples, the above preparation method satisfies one or more of conditions (i) to (iv): (i) In step (a): the molar ratio of hexachlorophosphazene, Tpl and triethylamine is 1:(0.5-1.2):(1-2.4); and / or the total amount of anhydrous dichloromethane used in the two applications is 10 mL / g to 20 mL / g based on the total mass of hexachlorophosphazene and Tpl, respectively; specifically, the molar ratio of hexachlorophosphazene, Tpl and triethylamine can also be 1:(0.5-1.2):(1-2.4), such as 1:0.8:1.5, 1:1:1.8 or 1:1:2.4, etc.; in addition, the two applications of dichloromethane are 10 mL / g to 20 mL / g based on the mass of hexachlorophosphazene and Tpl, such as 13 mL / g, 15 mL / g or 17 mL / g, etc. (ii) In step (b): the molar ratio of intermediate 1, methoxy polyethylene glycol amino and triethylamine is 1:(0.8-1):(1.6-2); and / or the total amount of anhydrous tetrahydrofuran used in the two steps is 10 mL / g to 20 mL / g based on the total mass of intermediate 1 and methoxy polyethylene glycol amino, respectively; specifically, the molar ratio of intermediate 1, methoxy polyethylene glycol amino and triethylamine can be 1:(0.8-1):(1.6-2), such as 1:0.8:2, 1:1:2 or 1:0.9:1.7, etc.; in addition, the total amount of anhydrous tetrahydrofuran used in the two steps can be 10 mL / g to 20 mL / g based on the mass of intermediate 1 and methoxy polyethylene glycol amino, such as 13 mL / g, 15 mL / g or 17 mL / g, etc. (iii) In step (c): the molar ratio of intermediate 2, propargylamine, and triethylamine is 1:(4-6):(8-10); and / or the amount of anhydrous tetrahydrofuran is related to the total mass of intermediate 2 and propargylamine by 10 mL / g to 20 mL / g; specifically, the molar ratio of intermediate 2, propargylamine, and triethylamine can be 1:(4-6):(8-10), such as 1:5:9, 1:5:8, or 1:6:8, etc.; in addition, the amount of anhydrous tetrahydrofuran is related to the total mass of intermediate 2 and propargylamine by 10 mL / g to 20 mL / g, such as 13 mL / g, 15 mL / g, or 17 mL / g, etc. (iv) In step (d): the molar ratio of intermediate 3, luminol azidide, copper sulfate, and sodium ascorbate is 1:(4-5):(1-2):(2-4); the amount of dimethyl sulfoxide / water mixed solvent used is related to the total mass of intermediate 3 and luminol azidide by 10 mL / g to 20 mL / g; and / or the volume ratio of dimethyl sulfoxide to water in the mixed solvent is 3:(1-3); specifically, the molar ratio of intermediate 3, luminol azidide, copper sulfate, and sodium ascorbate can be 1:(4-5):(1-2):(2-4), such as 1:4:1:3, 1:5:1:3, or 1:5:2:3, etc.; in addition, the amount of dimethyl sulfoxide / water mixed solvent used can be related to the total mass of intermediate 3 and luminol azidide by 10 mL / g to 20 mL / g. mL / g, such as 13 mL / g, 15 mL / g or 17 mL / g; in addition, the volume ratio of dimethyl sulfoxide to water in the mixed solvent is 3:(1 to 3), such as 3:1, 3:2 or 3:3, preferably 3:2.

[0034] It should be noted that the preparation method in this invention can satisfy any one of the above conditions (i) to (iv), and preferably all of the above conditions are satisfied at the same time.

[0035] Fifthly, embodiments of the present invention provide a pharmaceutical preparation comprising the above-mentioned anti-inflammatory nanomicelles and an effective amount of the active pharmaceutical ingredient.

[0036] It should be noted that the anti-inflammatory nanomicelles in this invention can serve as a carrier to carry pharmaceutical active ingredients in preparation of pharmaceutical formulations. These pharmaceutical active ingredients are all known in the art as therapeutic active ingredients. Furthermore, the dosage form of the pharmaceutical formulation is also known in the art, such as lyophilized powder, injections, aerosols, gels, or creams.

[0037] Sixthly, embodiments of the present invention provide the application of the above-mentioned anti-inflammatory nanomicelles in regulating the inflammatory microenvironment and / or scavenging reactive oxygen species.

[0038] It should be noted that the anti-inflammatory nanomicelles in this invention have excellent anti-inflammatory effects and can regulate the inflammatory microenvironment and / or scavenge reactive oxygen species.

[0039] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0040] Preparation Examples In this embodiment of the invention, the synthetic route of anti-inflammatory nanomicelles (PL4T1NM) is as follows: Figure 1 As shown, it is obtained by stepwise affinity substitution reaction and alkyne-azido group click reaction, and the specific steps are as follows: (1) Hexachlorophosphazene (HCCP) (12 mmol) and triethylamine (20 mmol) were dissolved in 50 mL of anhydrous dichloromethane. 20 mL of anhydrous dichloromethane solution of Tpl (10 mmol) was added dropwise under -20ºC ice-cold ethanol conditions. The mixture was stirred and reacted for 3 h at this temperature. The intermediate 1 (HCCP(Tpl)1) was obtained by silica gel column chromatography. (2) Dissolve HCCP(Tpl)1 (1.8 mmol) in 20 mL of anhydrous tetrahydrofuran, add triethylamine (3 mmol), and then add 15 mL of anhydrous tetrahydrofuran solution of methoxy polyethylene glycol amino (1.5 mmol, average molecular weight 2000) dropwise at -0ºC. After reacting for 2 h, separate and purify by silica gel column chromatography to obtain intermediate 2 (PT1). (3) PT1 (0.82 mmol), triethylamine (7.38 mmol) and propargylamine (3.69 mmol) were dissolved in 10 mL of anhydrous tetrahydrofuran and stirred at 65ºC for 24 h under nitrogen protection. The intermediate 3 (PP4T1) was obtained by silica gel column chromatography. (4) PP4T1 (0.56 mmol) and luminol azide (2.35 mmol) were dissolved in 20 mL of dimethyl sulfoxide / water mixed solvent (volume ratio 3:2). Copper sulfate (0.94 mmol) and sodium ascorbate (1.88 mmol) were used as click reaction catalysts. After stirring at 25ºC for 12 h, the solvent was removed by freeze drying. The phosphazene amphiphilic conjugate (PL4T1) was obtained by silica gel column chromatography.

[0041] (4) Place 10 mg of PL4T1 in 1 mL of water, sonicate it with 60 W power for 10 min, and let it stand for 30 min to obtain anti-inflammatory nanomicelles (PL4T1NM). The anti-inflammatory nanomicelles can self-assemble into micelle nanoparticles of 60 nm-80 nm in aqueous solution.

[0042] Characterization test The 1H NMR spectrum of the phosphazene-based amphiphilic conjugate (PL4T1) prepared in Example 1 is as follows: Figure 2 As shown.

[0043] The infrared spectra of the phosphazene-based amphiphilic conjugate (PL4T1) prepared in Example 1, as well as hexachlorophosphazene (HCCP), intermediate 1 (HCCP(Tpl)1), intermediate 2 (PT1), and intermediate 3 (PP4T1) are shown below. Figure 3 As shown.

[0044] Transmission electron microscopy (TEM) images and particle size distribution diagrams of anti-inflammatory nanomicelles (PL4T1NM) prepared from the phosphazene-based amphiphilic conjugate (PL4T1) in Example 1 in aqueous dispersion are shown below. Figure 4 As shown.

[0045] The critical micelle concentration curve of the phosphazene-based amphiphilic conjugate (PL4T1) prepared in Example 1 was determined by pyrene fluorescence method. Specifically, 10 μL of 6.0 × 10⁻⁶ phosphate ether was used. -5 A 2 mL glass bottle containing 1 mol / L pyrene-acetone solution was placed in a fume hood to evaporate the acetone. Then, 1 mL of the sample solution was added, resulting in a final pyrene-acetone concentration of 6.0 × 10⁻⁶. -7 mol / L; then place in a 50℃ oven for equilibration for 10h, and then cool at room temperature for 10h; the excitation spectrum of the sample was determined using a fluorescence spectrometer, with the excitation and divergence slit widths both being 2.5nm, the scanning speed being 240nm / min, the emission wavelength being set to 390nm, and the scanning range being 300nm~360nm.

[0046] Test results as follows Figure 5 As shown, the results indicate that the anti-inflammatory nanomicelles (PL4T1NM) prepared by the phosphazene amphiphilic conjugate (PL4T1) have high stability and can form micelle structures at concentrations above 12.3 μg / mL.

[0047] Performance testing (I) Evaluation of the total antioxidant capacity of anti-inflammatory nanomicelles (PL4T1 NM) 4 mg of PL4T1NM prepared in Example 1 was dissolved in 1 mL of deionized water to prepare a 4 mg / mL PL4T1NM aqueous solution. This solution was then diluted with water to obtain aqueous solutions with concentrations of 4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL. The total antioxidant capacity of the samples was then determined using a total antioxidant capacity assay kit (ABTS method, Beyotime), with Trolox as the standard sample. The antioxidant capacity of the samples was expressed as Trolox-equivalent antioxidant capacity (TEAC). The results are as follows: Figure 6 As shown, the results indicate that the anti-inflammatory nanomicelles have high antioxidant capacity, with the inhibition rate of 1g sample being the same as that of 0.4mM Trolox.

[0048] (II) Evaluation of the free radical scavenging ability of anti-inflammatory nanomicelles (PL4T1 NM) Prepare 100 μg / mL DPPH methanol solution and store it in the dark at low temperature. Prepare PL4T1NM methanol solutions of 4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL and 0.03125 mg / mL respectively. Take 100 μL of PL4T1NM methanol solution and add it to a 96-well plate. Use 100 μL of methanol as a blank sample. Set up 3 replicates for each sample. Except for the blank sample, add 100 μL of DPPH methanol solution to each well using a pipette. Measure the OD value at 520 nm in a microplate reader (TECAN M200Pro) at the set time points and calculate the DPPH clearance percentage of different concentrations of PL4T1.

[0049] In addition, the PL4T1 NM prepared in Example 1 was used to prepare aqueous solutions with concentrations of 4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL and 0.03125 mg / mL, respectively. The hydroxyl radical scavenging ability was then tested according to the instructions of the hydroxyl radical assay kit (Nanjing Jiancheng).

[0050] The percentage of DPPH scavenging and the percentage of hydroxyl radical scavenging of PL4T1 at different concentrations are as follows: Figure 7As shown, the results indicate that the anti-inflammatory nanomicelles exhibit rapid and powerful DPPH free radical scavenging performance. At 10 min, the scavenging rate of PL4T1NM at a concentration of 2 mg / mL reached 96%, and the concentration-dependent curve of hydroxyl radical scavenging also shows that 1 mg of PL4T1NM scavenges 30 U of hydroxyl radicals.

[0051] (III) Evaluation of the hydrogen peroxide scavenging capacity of anti-inflammatory nanomicelles (PL4T1 NM) 51 μL of 30% (w / v) hydrogen peroxide was diluted to 50 mL with deionized water to obtain an approximately 10 mM hydrogen peroxide aqueous solution. Additionally, PL4T1 NM aqueous solutions with concentrations of 8 mg / mL, 4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL were prepared using the PL4T1 NM prepared in Example 1. The hydrogen peroxide aqueous solution and the PL4T1 NM solutions of different concentrations were mixed at a volume ratio of 1:1 and incubated in a shaker at 37°C for 2 hours to obtain different mixtures. Each mixture was diluted 100 times with deionized water, and the residual hydrogen peroxide content was determined using a hydrogen peroxide detection kit (Beyotime). The hydrogen peroxide content in the control sample was used as the initial concentration value of hydrogen peroxide. Each sample was measured three times, and the hydrogen peroxide scavenging capacity of each sample at different concentrations was calculated.

[0052] The hydrogen peroxide scavenging curves of different concentrations of PL4T1 NM are as follows: Figure 8 As shown, the results indicate that the anti-inflammatory nanomicelles exhibit good hydrogen peroxide scavenging ability, with 1 mg PL4T1 NM scavenging 2.3 μmol of hydrogen peroxide.

[0053] (iv) Evaluation of the superoxide anion scavenging ability of anti-inflammatory nanomicelles (PL4T1 NM) The PL4T1NM prepared in Example 1 was prepared into aqueous solutions of 8 mg / mL, 4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL and 0.0625 mg / mL, respectively. The superoxide anion scavenging ability was then tested according to the instructions of the superoxide anion inhibition and generation assay kit (Nanjing Jiancheng).

[0054] Superoxide anion scavenging curves of PL4T1NM aqueous solutions at different concentrations are shown below. Figure 9 As shown, the results indicate that the anti-inflammatory nanomicelles exhibit a certain superoxide anion scavenging ability, with 1 mg PL4T1NM scavenging 0.23 U of superoxide anions.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A phosphazene-based amphiphilic conjugate, the structural formula of which is shown below: in: R is n is an integer from 10 to 50; R1 is ; R2 is .

2. The intermediate, whose structure is shown below: Intermediate 1: ;or Intermediate 2: ;or Intermediate 3: ; in, n is an integer between 10 and 50.

3. Anti-inflammatory nanomicelles, characterized in that, The phosphazene-based amphiphilic conjugate of claim 1 is obtained by dispersing it in water.

4. The anti-inflammatory nanomicelles according to claim 3, characterized in that, The preparation method of anti-inflammatory nanomicelles includes: placing a phosphazene-based amphiphilic conjugate in water, sonicating it, and then allowing it to stand to obtain anti-inflammatory nanomicelles; wherein, The ultrasonic power is 40W to 100W, and the ultrasonic treatment time is 5min to 30min; and / or The settling time is 5 to 60 minutes.

5. The anti-inflammatory nanomicelles according to claim 4, characterized in that, The concentration of the phosphazene amphiphilic conjugate in water is ≥12.3 μg / mL.

6. The method for preparing the phosphazene-based amphiphilic conjugate according to claim 1, characterized in that, Preparation methods include: (1) React hexachlorophosphazene and Tpl to obtain intermediate 1; (2) Intermediate 1 and methoxy polyethylene glycol amino were reacted to obtain intermediate 2; (3) Intermediate 2 is reacted with propargylamine to obtain intermediate 3; (4) Intermediate 3 was reacted with azidolated luminol to obtain a phosphazene amphiphilic conjugate; The structural formulas of intermediate 1, intermediate 2, and intermediate 3 are shown below: Intermediate 1: ; Intermediate 2: ; Intermediate 3: ; Where n is an integer from 10 to 50.

7. The preparation method according to claim 6, characterized in that, Preparation methods include: (a) Dissolve hexachlorophosphazene in anhydrous dichloromethane, add triethylamine, and add dropwise a solution of Tpl in anhydrous dichloromethane to obtain intermediate 1. (b) Dissolve intermediate 1 in anhydrous tetrahydrofuran, add triethylamine, and add dropwise an anhydrous tetrahydrofuran solution of methoxy polyethylene glycol amino to obtain intermediate 2. (c) Intermediate 2, triethylamine and propargylamine are dissolved in anhydrous tetrahydrofuran and reacted to obtain intermediate 3; (d) Intermediate 3 and luminol azide were dissolved in a dimethyl sulfoxide / water mixed solvent, and copper sulfate and sodium ascorbate were used as click reaction catalysts to obtain a phosphazene amphiphilic conjugate.

8. The preparation method according to claim 7, characterized in that, The preparation method satisfies one or more of conditions (i) to (iv): (i) In step (a): The molar ratio of hexachlorophosphazene, Tpl, and triethylamine is 1:(0.5–1.2):(1–2.4); and / or The total amount of anhydrous dichloromethane used in the two applications is 10 mL / g to 20 mL / g, respectively, based on the total mass of hexachlorophosphazene and Tpl. (ii) In step (b): The molar ratio of intermediate 1, methoxy polyethylene glycol amino, and triethylamine is 1:(0.8–1):(1.6–2); and / or The total amount of anhydrous tetrahydrofuran used in the two applications was 10 mL / g to 20 mL / g, respectively, based on the total mass of intermediate 1 and methoxy polyethylene glycol amino. (iii) In step (c): The molar ratio of intermediate 2, propargylamine, and triethylamine is 1:(4-6):(8-10); and / or The amount of anhydrous tetrahydrofuran used is related to the total mass of intermediate 2 and propargylamine by 10 mL / g to 20 mL / g. (iv) In step (d): The molar ratio of intermediate 3, luminol azidide, copper sulfate and sodium ascorbate is 1:(4-5):(1-2):(2-4); The amount of dimethyl sulfoxide / water mixed solvent used is related to the total mass of intermediate 3 and luminol azide by 10 mL / g to 20 mL / g; and / or The volume ratio of dimethyl sulfoxide to water in the mixed solvent is 3:(1-3).

9. A pharmaceutical preparation, characterized in that, It includes the anti-inflammatory nanomicelles as described in any one of claims 3 to 5 and an effective amount of the pharmaceutically active ingredient.

10. The application of the anti-inflammatory nanomicelles described in claim 1 in regulating the inflammatory microenvironment and / or scavenging reactive oxygen species.

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