Preparation method and application of targeted delivery system of metal polyphenol nano system

Abstract

The invention discloses a preparation method and application of a targeted delivery system of a metal polyphenol nano system. The invention discloses a targeted delivery system of a metal polyphenol nano system, and a preparation method of the targeted delivery system comprises the following steps: taking the metal polyphenol nano system formed based on epigallocatechin gallate and iron ions as a carrier, and loading fucoxanthin to prepare fucoxanthin nano particles with active oxygen response characteristics; then, after chondroitin sulfate capable of targeting macrophages and (5-carboxyl amyl) triphenyl phosphorus bromide capable of targeting mitochondria are coupled, the fucoxanthin delivery system with the active oxygen response characteristic and the targeting characteristic is further prepared through a double emulsion anti-solvent evaporation method. The system can realize release of fucoxanthin under stimulation of active oxygen, and realizes aggregation of a targeted delivery system in mitochondria of a specific inflammation site. The carrying system can effectively improve or relieve the inflammatory bowel disease, and the raw materials used in the carrying system are low in cost, the preparation conditions are mild, and large-scale production is easy.

Description

Technical Field

[0001] The present invention relates to the field of bionanotechnology, and in particular to a preparation method and application of a targeted delivery system of a metal polyphenol nanosystem. Background Art

[0002] Ulcerative colitis (UC) is a chronic, recurrent gastrointestinal inflammatory disease characterized by impaired intestinal barrier function, mucosal inflammation, excessive production of reactive oxygen species (ROS), and intestinal microbial dysbiosis. Its incidence has been increasing in recent years. Currently, the drugs used to treat UC are mainly based on amino acid salicylates, immunosuppressants, and biological therapeutics. However, these strategies cannot completely cure UC, and long-term medication use can cause serious side effects and financial burdens for patients. Therefore, utilizing functional food components to deliver them to the colon via an oral colonic delivery system to alleviate inflammatory symptoms is a safer and more effective strategy for treating colitis. Fucoxanthin is a carotenoid that is widely found in marine phytoplankton such as brown algae, diatoms and microalgae. The unique epoxy and allene structures in fucoxanthin give it significant health benefits in terms of antioxidant, anti-inflammatory, anti-diabetic and immune regulation. However, fucoxanthin has poor water solubility and stability and is easily affected by environmental factors such as pH, oxygen, ultraviolet rays and temperature, resulting in low bioavailability in the body, limiting its application in industries such as food, health products and medical treatment, and making it difficult to be effectively delivered to specific parts to exert its effect. Therefore, it is crucial to improve the water solubility and stability of fucoxanthin and enhance its bioavailability. At present, the protection, delivery and controlled release of functional factors are mainly achieved by constructing a carrier system. Common carrier systems mainly include micro / nanoemulsions, nanoparticles, nanoliposomes, microcapsules and hydrogels. However, conventional carrier systems cannot achieve targeted, concentrated, responsive and controlled release of functional factors, and the biological activity of functional factors cannot be fully exerted. In addition to the stable protection and delivery of functional factors, one of the main challenges is how to achieve responsive and controlled release of functional factors at the targeted action site. The targeting function is usually achieved by introducing specific ligands into the carrier system. The ligands can target the carrier system to a specific site by recognizing the corresponding receptors. The responsive controlled release function is usually achieved by introducing responsive groups into the carrier system. The responsive groups can sense changes in the external environment and change their own physical or chemical properties accordingly, thereby achieving precise control of the release of functional factors. By studying the interaction and stabilization mechanism between functional factors and carriers, and based on the release and absorption mechanism of functional factors after they are taken into the body, specific responsive groups and specific ligands are further introduced into the carrier to construct a targeted delivery system with responsive controllable characteristics, thereby achieving targeted delivery and responsive controlled release of functional factors, which is of great significance for accurately improving the bioavailability of functional factors. Metal polyphenol nanoparticles are a class of nanocomposites formed by the self-assembly of metal ions and natural polyphenol molecules through coordination. In recent years, they have attracted widespread attention in the biomedical field due to their excellent antioxidant and anti-inflammatory properties and good biocompatibility. These nanomaterials have shown promising application prospects in tumor therapy, anti-inflammatory intervention, and drug delivery. Their biological functions primarily stem from the inherent physiological activities of polyphenols. Polyphenols can inhibit the activity of key inflammatory metabolic enzymes, such as cyclooxygenase and lipoxygenase, effectively reducing the production of proinflammatory mediators and reactive oxygen species, thereby exerting antioxidant and anti-inflammatory effects. Furthermore, polyphenols can modulate various inflammatory signaling pathways, particularly by blocking the activation of the nuclear factor κB (NF-κB) pathway, further inhibiting the expression of inflammatory factors such as tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), thereby enhancing the overall anti-inflammatory effect. Therefore, the delivery of fucoxanthin using metal polyphenol nanoparticles can achieve a synergistic anti-inflammatory effect with fucoxanthin, significantly enhancing the antioxidant and anti-inflammatory activities of the entire system and thus improving therapeutic efficacy. Summary of the Invention

[0003] Technical problems to be solved: Fucoxanthin has poor water solubility and stability, low oral utilization rate, and limited application. The present invention uses a metal polyphenol nanosystem as a carrier to prepare fucoxanthin nanoparticles with ROS-responsive properties, thereby improving the water solubility and stability of fucoxanthin. Chondroitin sulfate, which can target macrophages, and triphenylphosphine bromide, which can target mitochondria, are then coupled, and a fucoxanthin delivery system is prepared by a double emulsion anti-solvent evaporation method. This promotes the targeted enrichment of fucoxanthin in macrophages at sites of intestinal inflammation, and enables its release in response to ROS stimulation in mitochondria, thereby enhancing the antioxidant effect and bioavailability of fucoxanthin.

[0004] Technical solution: A method for preparing a targeted delivery system of a metal polyphenol nanosystem, comprising the following steps: S1. Dissolve fucoxanthin in anhydrous ethanol, add the obtained fucoxanthin ethanol solution to 3-(N-morpholino)propane sulfonic acid buffer, then add EGCG solution and FeCl3 solution in sequence, and stir continuously to obtain a fucoxanthin nanoparticle dispersion; S2. Chondroitin sulfate was dissolved in water, 3-(3-dimethylamino-propyl)-1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added sequentially and stirred, and then cystamine dihydrochloride was added with continuous stirring, dialyzed in distilled water, and freeze-dried to obtain chondroitin sulfate - cystamine dihydrochloride; S3. (5-carboxypentyl) triphenylphosphonium bromide was dissolved in 2-(N-morpholino)ethanesulfonic acid buffer, 3-(3-dimethylamino-propyl)-1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added, stirred evenly, and then chondroitin sulfate-cystamine dihydrochloride was added. The mixture was stirred for reaction, dialyzed in distilled water, and freeze-dried to obtain chondroitin sulfate-cystamine dihydrochloride-triphenylphosphonium bromide; S4. The fucoxanthin nanoparticle dispersion is dropped into an ethanol solution as an organic phase, and the organic phase is then dropped into a stirred chondroitin sulfate-cystamine dihydrochloride-triphenylphosphine bromide aqueous solution. The ethanol is removed by vacuum rotary evaporation to obtain a targeted delivery system. Furthermore, in step S1, the concentration of the fucoxanthin ethanol solution is 3-7 mg / mL; the concentration of the 3-(N-morpholino)propanesulfonic acid buffer is 8-10 mM, and the pH is 7.4; and the continuous stirring time is 0.5-1 h. Furthermore, in step S1, the concentration of the EGCG solution is 8-10 mg / mL, the concentration of the FeCl3 solution is 8-10 mg / mL; and the mass ratio of fucoxanthin, EGCG and FeCl3 is 1:(1-2):(1-6). Furthermore, in step S2, the stirring time is 2-5 hours, the continuous stirring time is 12-36 hours, the dialysis time is 48-96 hours, and the molecular weight of the dialysis cutoff is less than 1000Da. Furthermore, in step S2, the concentration of chondroitin sulfate dissolved in water is 12-15 mg / mL; the mass ratio of chondroitin sulfate to cystamine dihydrochloride is 1:(1-3); and the mass ratio of 3-(3-dimethylamino-propyl)-1-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 5:(1-3). Furthermore, in step S3, the concentration of the 2-(N-morpholino)ethanesulfonic acid buffer is 0.1 M, the stirring reaction conditions are 12-36 hours at room temperature in a dark environment, the stirring speed is 600-1000 rpm, and the dialysis is carried out for 48-96 hours, and the molecular weight of the dialyzed residue is less than 1000 Da. Furthermore, in step S3, the mass ratio of (5-carboxypentyl)triphenylphosphine bromide to the chondroitin sulfate-cystamine dihydrochloride is (1-3):1; the mass ratio of 3-(3-dimethylamino-propyl)-1-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 5:(1-3). Furthermore, in step S4, the dripping rate is 4-6 mL / h, the concentration of the chondroitin sulfate-cystamine dihydrochloride-triphenylphosphine bromide aqueous solution is 1.5-2.5 mg / mL; and the temperature of the vacuum rotary evaporation is 40-45°C. The metal polyphenol system-based delivery system prepared by the above preparation method is used in the preparation of colitis drugs for controlled release under the stimulation of active oxygen. Further, it is used to relieve colon tissue damage and inflammation. Beneficial effects: 1. The present invention couples chondroitin sulfate, which can target macrophages, and triphenylphosphine bromide, which can target mitochondria, and then prepares a fucoxanthin delivery system through a double emulsion anti-solvent evaporation method. The prepared targeted delivery system has functional properties such as stimulated release, targeted delivery, and effective relief of colon inflammation, and can be used in the fields of health products and functional foods. 2. The present invention uses a metal polyphenol nanosystem as a carrier to prepare fucoxanthin nanoparticles with ROS-responsive properties. The prepared targeted delivery system is spherical and has the advantages of good stability, negative surface charge, and high encapsulation efficiency. It significantly enhances the antioxidant activity of fucoxanthin, enables controlled release of fucoxanthin under ROS stimulation, and achieves targeted delivery and aggregation at sites of intestinal inflammation and within mitochondria, effectively alleviating colon inflammation. 3. Although the dual-targeted delivery system constructed in the prior art has improved stability and bioavailability to a certain extent, thereby enhancing its efficacy in the treatment of chronic colitis, its delivery carrier itself does not have anti-inflammatory function, and the therapeutic effect mainly depends on the active ingredient delivered. In contrast, the targeted delivery system based on the metal polyphenol nanosystem constructed in the present invention introduces EGCG, which not only stabilizes the structure as a carrier component, but also has significant antioxidant and anti-inflammatory activities. This component can work synergistically with fucoxanthin, further enhancing the biological activity of the overall system on the basis of improving the stability and delivery efficiency of fucoxanthin, thereby significantly improving the therapeutic effect. 4. The method of the present invention for preparing the cell membrane / mitochondria targeted delivery system is simple and mild, uses low-cost raw materials, has mild embedding conditions, and is easy to scale up production. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a transmission electron micrograph of the targeted delivery system prepared in Example 1; Figure 2 This is a transmission electron micrograph of the targeted delivery system prepared in Comparative Example 1; Figure 3 Figure 1 is a graph showing the in vitro release of the targeted delivery system under ROS conditions, Figure A is Example 1, and Figure B is Comparative Example 2; Figure 4 This figure shows the effects of the targeted delivery systems prepared in Example 1 and Comparative Example 1 on the colon length of mice with colitis induced by 4% dextran sulfate sodium salt; Figure 5 Figure 1 is the distribution of the cells after fluorescent labeling in Example 1. Figure A shows the distribution of mitochondria in RAW264.7 macrophages labeled with a red fluorescent probe. Figure B shows the distribution of mitochondria in RAW264.7 macrophages using the hydrophobic fluorescent probe coumarin (C6) instead of the targeted delivery system in Example 1. Figure C is an overlay of the distribution of mitochondria labeled with a red fluorescent probe and the targeted delivery system in Example 1 in RAW264.7 macrophages. Figure 6 Figure 1 is the distribution of the fluorescent labeling of Comparative Example 1 in cells. Figure A shows the distribution of mitochondria in RAW264.7 macrophages labeled with a red fluorescent probe. Figure B shows the distribution of mitochondria in RAW264.7 macrophages using the hydrophobic fluorescent probe coumarin (C6) instead of the targeted delivery system of Comparative Example 1. Figure C shows the superimposed distribution of mitochondria labeled with a red fluorescent probe and the targeted delivery system of Comparative Example 1 in RAW264.7 macrophages. Figure 7Figure 2 is the distribution of the steps in the cells after fluorescent labeling of Comparative Example 2. Figure A is a diagram of the mitochondria in RAW264.7 macrophages labeled with a red fluorescent probe. Figure B is a diagram of the distribution of mitochondria in RAW264.7 macrophages using the hydrophobic fluorescent probe coumarin (Coumarin 6, C6) instead of the targeted delivery system of Comparative Example 2. Figure C is an overlay of the distribution of mitochondria labeled with a red fluorescent probe and the targeted delivery system of Comparative Example 2 in RAW264.7 macrophages. Figure 8 This is a diagram showing the distribution of the hydrophobic fluorescent probe coumarin (Coumarin 6, C6) in the intestinal tissue of mice with colitis induced by 3% dextran sulfate sodium salt, instead of the targeted delivery system of Example 1; Figure 9 This is a diagram showing the distribution of the hydrophobic fluorescent probe coumarin (Coumarin 6, C6) in the intestinal tissue of mice with colitis induced by 3% dextran sulfate sodium salt, replacing the targeted delivery system of Comparative Example 1; Figure 10 This is a diagram showing the distribution of the hydrophobic fluorescent probe coumarin (Coumarin 6, C6) in the intestinal tissue of mice with colitis induced by 3% dextran sulfate sodium salt, in which the targeted delivery system of comparative example 2 is used instead. DETAILED DESCRIPTION The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are intended to explain the present invention, but the present invention is not limited to the following embodiments: Example 1 A method for preparing a targeted delivery system of a metal polyphenol nanosystem comprises the following steps: S1. Dissolve fucoxanthin in ethanol to obtain a 5 mg / mL fucoxanthin ethanol solution. Under stirring, add the fucoxanthin ethanol solution to 3-(N-morpholino)propanesulfonic acid buffer (MOPS) (10 mM, pH = 7.4), then add EGCG solution (10 mg / mL) and FeCl3 solution (10 mg / mL) in sequence. Stir continuously at 600 rpm for 0.5 h at room temperature to obtain a fucoxanthin nanoparticle dispersion. The mass ratio of fucoxanthin, EGCG, and FeCl3 is 1:1:1. S2. Chondroitin sulfate was dissolved in distilled water to prepare a solution with a concentration of 14.5 mg / mL. 3-(3-dimethylamino-propyl)-1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added and stirred at room temperature for 3 hours to activate the carboxyl groups on the chondroitin sulfate. Cystamine dihydrochloride was then added and stirred for 24 hours. The mixed solution was dialyzed in distilled water for 72 hours, and the solution in the dialysis bag was collected and freeze-dried to obtain chondroitin sulfate-cystamine dihydrochloride. The mass ratio of chondroitin sulfate to cystamine dihydrochloride was 1:1; the mass ratio of 3-(3-dimethylamino-propyl)-1-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide was 5:1. S3. (5-carboxypentyl)triphenylphosphonium bromide is dissolved in 2-(N-morpholino)ethanesulfonic acid buffer to prepare a solution with a concentration of 0.1M, and then 3-(3-dimethylamino-propyl)-1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added, and the carboxyl group on (5-carboxypentyl)triphenylphosphonium bromide is activated by stirring at 800 rpm at room temperature for 3 h. Then, a chondroitin sulfate-cystamine dihydrochloride aqueous solution is added, and the reaction is stirred at 800 rpm at room temperature in the dark for 24 h; then, the solution is dialyzed in distilled water for 72 h, the solution in the dialysis bag is collected, and lyophilized to obtain chondroitin sulfate-cystamine dihydrochloride-triphenylphosphonium bromide. The mass ratio of (5-carboxypentyl)triphenylphosphonium bromide to the chondroitin sulfate-cystamine dihydrochloride is 1:1; the mass ratio of the 3-(3-dimethylamino-propyl)-1-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 5:1. S4. The fucoxanthin nanoparticle dispersion was placed in a 5 mL syringe and evenly dripped into the ethanol solution at a flow rate of 5 mL / h as the organic phase; the obtained mixed solution was re-placed into a 5 mL syringe and evenly dripped into a 2 mg / mL chondroitin sulfate-cystamine dihydrochloride-triphenylphosphine bromide aqueous solution in a stirring state of 1000 rpm at a flow rate of 5 mL / h. The ethanol was removed by vacuum rotary evaporation at 43 ° C to obtain a targeted delivery system. Comparative Example 1 The difference between this comparative example and Example 1 is that triphenylphosphonium bromide that can target mitochondria in macrophages is not grafted onto chondroitin sulfate-cystamine dihydrochloride, specifically as follows: S1. Dissolve fucoxanthin in ethanol to obtain a 5 mg / mL fucoxanthin ethanol solution. Under stirring, add the fucoxanthin ethanol solution to 3-(N-morpholino)propanesulfonic acid buffer (MOPS) (10 mM, pH = 7.4), then add EGCG solution (10 mg / mL) and FeCl3 solution (10 mg / mL) in sequence. Stir continuously at 600 rpm for 0.5 h at room temperature to obtain a fucoxanthin nanoparticle dispersion. The mass ratio of fucoxanthin, EGCG, and FeCl3 is 1:1:1. S2. Chondroitin sulfate was dissolved in distilled water to prepare a solution with a concentration of 14.5 mg / mL. 3-(3-dimethylamino-propyl)-1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added and stirred at room temperature for 3 hours to activate the carboxyl groups on the chondroitin sulfate. Cystamine dihydrochloride was then added and stirred for 24 hours. The mixed solution was dialyzed in distilled water for 72 hours, and the solution in the dialysis bag was collected and freeze-dried to obtain chondroitin sulfate-cystamine dihydrochloride. The mass ratio of chondroitin sulfate to cystamine dihydrochloride was 1:1; the mass ratio of 3-(3-dimethylamino-propyl)-1-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide was 5:1. S3. The fucoxanthin nanoparticle dispersion was placed in a 5 mL syringe and evenly dripped into the ethanol solution at a flow rate of 5 mL / h as the organic phase; the obtained mixed solution was re-placed into a 5 mL syringe and evenly dripped into a 2 mg / mL chondroitin sulfate-cystamine dihydrochloride aqueous solution in a stirring state of 1000 rpm at a flow rate of 5 mL / h. The ethanol was removed by vacuum rotary evaporation at 43 ° C to obtain a targeted delivery system. Comparative Example 2 The difference between this comparative example and Example 1 is that triphenylphosphonium bromide, which can target mitochondria in macrophages, is directly grafted onto fucoxanthin, and chondroitin sulfate-cystamine dihydrochloride-EGCG polymer is used as the wall material, specifically as follows: S1. Synthesis of mitochondrial-targeted (5-carboxypentyl)triphenylphosphine bromide-modified fucoxanthin polymer: (5-carboxypentyl)triphenylphosphine bromide was dissolved in dimethyl sulfoxide to prepare a solution with a concentration of 20 mg / mL, and then N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added and stirred at room temperature for 1 hour. Subsequently, fucoxanthin was added and stirred in the dark at room temperature for 24 hours. The above mixed solution was dialyzed in dimethyl sulfoxide for 24 hours and in distilled water for 48 hours. The solution in the dialysis bag (dialysis molecular weight 500Da) was collected and freeze-dried at a vacuum degree of 1Pa and -50°C for 48 hours to obtain (5-carboxypentyl)triphenylphosphine bromide-modified fucoxanthin. The mass ratio of (5-carboxypentyl)triphenylphosphine bromide to fucoxanthin was 1:5, and the mass ratio of N,N'-dicyclohexylcarbodiimide to 4-dimethylaminopyridine was 2:1; S2. Synthesis of chondroitin sulfate-cystamine dihydrochloride: Chondroitin sulfate is dissolved in distilled water to a concentration of 50 mg / mL, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added, and stirred at room temperature for 2 hours, followed by addition of cystamine dihydrochloride and stirring for 24 hours. The above mixed solution is dialyzed in distilled water for 48 hours, and the solution in the dialysis bag (dialysis molecular weight 500Da) is collected and freeze-dried to obtain chondroitin sulfate-cystamine dihydrochloride. Wherein, the mass ratio of chondroitin sulfate to cystamine dihydrochloride is 100: 1, and the mass ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 5: 2; S3. Synthesis of cell membrane-targeted chondroitin sulfate-cystamine dihydrochloride-EGCG: EGCG was dissolved in an ethanol solution, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide were added, and the mixture was stirred at 1200 rpm for 1 hour in an ice-water bath. Subsequently, the chondroitin sulfate-cystamine dihydrochloride aqueous solution was added to the above mixture, stirred at 1200 rpm at 80°C for 4 hours, and then cooled to room temperature and magnetically stirred at 1200 rpm for 24 hours. Finally, the above mixed solution was dialyzed in distilled water for 48 hours, and the solution in the dialysis bag (dialysis molecular weight 500Da) was collected and freeze-dried to obtain chondroitin sulfate-cystamine dihydrochloride-EGCG polymer. Among them, the mass ratio of EGCG to chondroitin sulfate-cystamine dihydrochloride was 71:50, and the mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide was 197:115; S4. Preparation of nanoparticles: (5-carboxypentyl) triphenylphosphine bromide modified fucoxanthin polymer was dispersed in ethanol and loaded into a 5 mL syringe as the organic phase. The solution was evenly dripped into a 25 mg / mL chondroitin sulfate-cystamine dihydrochloride-EGCG aqueous solution at a flow rate of 5 mL / h under magnetic stirring at 1200 rpm. The obtained mixed solution was re-loaded into the syringe and evenly dripped into ethanol at a flow rate of 5 mL / h under magnetic stirring at 1200 rpm. The ethanol was then removed by rotary evaporation at 45 ° C to obtain dual-targeted nanoparticles. Performance measurement: 1. Microstructure observation The microstructure of the functional factor delivery system was observed using transmission electron microscopy: 20 μL of the functional factor delivery system dispersion was dripped onto a copper mesh support. Excess liquid was removed with filter paper, and the sample was air-dried for a period of time. Subsequently, a small amount of 1% uranyl acetate solution was added for negative staining, and finally, the sample was observed at an accelerating voltage of 80 kV. Figure 1This is a transmission electron micrograph of the functional factor delivery system prepared in Example 1. The delivery system is a uniformly dispersed core-shell spherical structure. Figure 2 This is a transmission electron micrograph of the functional factor delivery system prepared in comparative example 1. It can be seen from the figure that the prepared targeted delivery system is a uniformly dispersed spherical structure and also has a core-shell structure. 2. Basic physical indicators The encapsulation efficiency was determined by high performance liquid chromatography: a certain amount of functional factor delivery system was accurately weighed and dissolved in anhydrous ethanol. After ultrasonic treatment for 30 minutes, the mixture was centrifuged at 5000g for 5 minutes at 4°C. Subsequently, the concentration of encapsulated fucoxanthin in the supernatant was determined by high performance liquid chromatography, and the content of encapsulated fucoxanthin in the supernatant was calculated based on the known fucoxanthin standard curve. The specific conditions for high performance liquid chromatography analysis are as follows: chromatographic analysis was performed using a C18 column, mobile phase A was ultrapure water, mobile phase B was chromatographically pure methanol, the flow rate was 0.7 mL / min, the operating temperature of the column was 35°C, the injection volume was 10 μL, and the UV detection wavelength was 450 nm. The encapsulation efficiency calculation formula is: EE (%) = encapsulated fucoxanthin content / added fucoxanthin content × 100% The particle size and potential of the functional factor delivery system were measured using a laser particle size analyzer: the functional factor delivery system dispersion was diluted 100-fold with distilled water and added to the sample cell. The particle size and zeta potential were measured using a nanoparticle size analyzer and a zeta potential analyzer at 25°C. Three independent measurements were performed for each sample, and the average was used to ensure the accuracy of the results. Table 1 shows the basic index characterization of the functional factor delivery system prepared in the examples and comparative examples. The encapsulation efficiency of fucoxanthin in the functional factor delivery system in the examples and comparative examples was determined by high performance liquid chromatography. The encapsulation efficiency of fucoxanthin in the functional factor delivery system prepared in Example 1 was 85.08%, and the encapsulation efficiency of fucoxanthin in the functional factor delivery system prepared in Comparative Example 1 was 90.45%. However, the encapsulation efficiency of Comparative Example 2 was lower than 70% due to the change of the wall material. The hydrodynamic diameter and potential of the functional factor delivery system in the examples and comparative examples were then determined by a laser particle size analyzer. The particle size of the functional factor delivery system prepared in Example 1 was 195.08 nm and -35.57 ± 0.79 mV. The particle size of Comparative Example 2 was much larger than that of Example 1. It is possible that the difference in preparation method and wall material resulted in larger particles and reduced stability. Table 1 3. In vitro release of ROS under different concentrations A certain amount of the functional factor delivery system was dissolved in different PBS buffer solutions (pH 7.4, pH 7.4 containing 0.1, 1, and 10 mM H2O2, or pH 6.0, pH 6.0 containing 0.1, 1, and 10 mM H2O2) and incubated at 37°C and 100 rpm. At specific time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, and 48 hours), the samples were separated by centrifugation, and the supernatant was collected to measure the concentration of fucoxanthin. The functional factor delivery system pellet was then resuspended in the same fresh buffer and incubated again to observe the release characteristics. Figure 3 A shows the in vitro release of the functional factor delivery system prepared in Example 1 under conditions of different ROS concentrations. In PBS buffer with the same pH, the rate of release of fucoxanthin from the delivery system in the group containing H2O2 was significantly faster than that in the PBS group, and as the concentration of added H2O2 increased, the release rate of fucoxanthin increased significantly, and the release rate increased over time. Under conditions of H2O2 concentration of 10mM (pH 7.4), after incubation for 48 hours, the release rate of fucoxanthin can reach 47.71%. These results indicate that the delivery system has ROS response characteristics, which enables the delivery system to smoothly release fucoxanthin when entering an inflamed intestinal environment containing high concentrations of ROS, thereby exerting an anti-inflammatory effect. Figure 3 B is the in vitro release of comparative example 2 under different ROS concentrations. It can be seen that its ROS response characteristics are poor and the sustained release effect is not good. 4. Effect of delivery system pretreatment on colon length in mice with 3% dextran sulfate sodium salt-induced colitis The dextran sodium sulfate-induced mouse colitis model was used to evaluate the intervention effect of the functional factor delivery system on intestinal damage in mice. In short, after 30 C57BL / 6J mice were adaptively raised for one week, the mice were randomly divided into 5 groups according to body weight: a healthy control group, a dextran sodium sulfate (DSS)-induced group, a comparative example group 1, a vehicle group, and an example group 1. During the entire 3-week experimental period, the healthy control group and the DSS group mice were gavaged with normal saline, and the other groups of mice began to be gavaged with different samples at a dose of 200 μL / day. After 14 consecutive days of gavage, except for the healthy control group, the other groups were given 3.0% DSS solution. At the same time, different samples were continued to be gavaged every day, and the weight, diarrhea, bloody stools and other symptoms of the mice were regularly monitored. Figure 4The effects of pretreatment with the targeted delivery system prepared in Example 1 and Comparative Example 1 on the colon length of mice with 3% DSS-induced colitis were shown. The results showed that the colon length of the DSS-treated group was significantly shorter than that of the healthy control group, indicating that the colon tissue had suffered DSS-induced damage. However, intervention with Comparative Example 1 and Example 1 significantly improved the shortening of colon length. Compared to Comparative Example 1, the intervention effect of Example 1 was more pronounced. This demonstrates that the delivery system can effectively alleviate the shortening of colon length in mice with DSS-induced colitis. 5. Distribution of targeted delivery systems in cells or animal organs The hydrophobic fluorescent probe coumarin (Coumarin 6, C6) was used instead of the fucoxanthin in the examples and comparative examples to prepare a C6-loaded fluorescently labeled delivery system. The distribution of the targeted delivery system in cells or animal organs was observed to investigate the targeting of the prepared delivery system. like Figure 5 As shown, the embodiment has a relatively complete structure and strong fluorescence, and the green fluorescence overlaps with the mitochondrial fluorescence labeled with the mitochondrial red fluorescent probe ( Figure 5 C), indicating that the delivery system modified with (5-carboxypentyl)triphenylphosphonium bromide has a better accumulation effect in mitochondria. Figure 6 It can be seen that the green fluorescence intensity of the fluorescent-labeled targeted delivery system prepared in Comparative Example 1 is relatively weak in mitochondria ( Figure 6 A), but can have a certain overlap with the mitochondrial fluorescence labeled with mitochondrial red fluorescent probe ( Figure 6 C), indicating that the delivery system can accumulate in mitochondria. Figure 7 As shown, the fluorescence of Comparative Example 2 due to its incomplete structure is also significantly weaker, indicating that its delivery effect is poor. 6. Targeting ability of the tracer delivery system to the mouse intestine After 12 hours of oral administration of the prepared fluorescent delivery system to mice, the fluorescent images of the intestines of the healthy group and the DSS group were recorded. Six-week-old male C57 mice (SPF, 20±0.75g) were randomly divided into three groups: a healthy group, a comparative example group 1, and an example group 1. They were placed in an environment of 22±2°C and a relative humidity of 60±5%, and adapted to a cycle of 12h light and 12h dark for 7 days. Afterwards, the mice in the DSS group drank 3.0% (w / v) DSS aqueous solution freely for 7 days to establish a colitis mouse model. Afterwards, the three groups of mice were given the fluorescent delivery system respectively. After 12 hours of digestion and metabolism, the mice were killed and the mouse intestines were collected for analysis. At the same time, the IVISSpectrum small animal in vivo fluorescence imager was used to detect the distribution of fluorescent signals in the mouse intestine. Figure 8 The stronger fluorescence signal was also observed in mice with colitis induced by dextran sulfate sodium salt, and the fluorescence intensity was significantly higher than that of Figure 9 and Figure 10 , indicating that the fluorescent delivery system prepared in Example 1 accumulated more in the inflamed colon tissue, while the fluorescent delivery system prepared in the comparative example accumulated less in the inflamed colon tissue. In particular, the fluorescence intensity of Comparative Example 2 was significantly lower than that of Example 1 and Comparative Example 1. This may be related to the overexpression of CD44 receptors in the inflammatory site. The fluorescent delivery system prepared in Example 1 showed an enhanced accumulation effect in the inflamed colon tissue due to the introduction of chondroitin sulfate and triphenylphosphine bromide. Chondroitin sulfate has a strong affinity for the CD44 receptors expressed in the inflammatory site, and triphenylphosphine bromide can further promote the targeting of the fluorescent delivery system to mitochondrial tissue. The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a targeted delivery system of a metal polyphenol nanosystem, characterized in that: The following steps are involved: S1. Dissolve fucoxanthin in anhydrous ethanol, add the obtained fucoxanthin ethanol solution to 3-(N-morpholino)propane sulfonic acid buffer, then add EGCG solution and FeCl3 solution in sequence, and stir continuously to obtain a fucoxanthin nanoparticle dispersion; S2. Chondroitin sulfate was dissolved in water, 3-(3-dimethylamino-propyl)-1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added sequentially and stirred, and then cystamine dihydrochloride was added with continuous stirring, dialyzed in distilled water, and freeze-dried to obtain chondroitin sulfate - cystamine dihydrochloride; S3. (5-carboxypentyl) triphenylphosphonium bromide was dissolved in 2-(N-morpholino)ethanesulfonic acid buffer, 3-(3-dimethylamino-propyl)-1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added, stirred evenly, and then chondroitin sulfate-cystamine dihydrochloride was added. The mixture was stirred for reaction, dialyzed in distilled water, and freeze-dried to obtain chondroitin sulfate-cystamine dihydrochloride-triphenylphosphonium bromide; S4. The fucoxanthin nanoparticle dispersion is dropped into an ethanol solution as an organic phase, and the organic phase is then dropped into a stirred chondroitin sulfate-cystamine dihydrochloride-triphenylphosphine bromide aqueous solution. The ethanol is removed by vacuum rotary evaporation to obtain a targeted delivery system.

2. The method for preparing a targeted delivery system of a metal polyphenol nanosystem according to claim 1, characterized in that: In step S1, the concentration of the fucoxanthin ethanol solution is 3-7 mg / mL; the concentration of the 3-(N-morpholino)propanesulfonic acid buffer is 8-10 mM, and the pH is 7.4; and the continuous stirring time is 0.5-1 h.

3. The method for preparing a targeted delivery system of a metal polyphenol nanosystem according to claim 1, characterized in that: In step S1, the concentration of the EGCG solution is 8-10 mg / mL, and the concentration of the FeCl3 solution is 8-10 mg / mL; the mass ratio of fucoxanthin, EGCG and FeCl3 is 1:(1-2):(1-6).

4. The method for preparing a targeted delivery system of a metal polyphenol nanosystem according to claim 1, characterized in that: In step S2, the stirring time is 2-5 hours, the continuous stirring time is 12-36 hours, the dialysis time is 48-96 hours, and the molecular weight of the dialysis cutoff is less than 1000 Da.

5. The method for preparing a targeted delivery system of a metal polyphenol nanosystem according to claim 1, characterized in that: In step S2, the concentration of chondroitin sulfate dissolved in water is 12-15 mg / mL; the mass ratio of chondroitin sulfate to cystamine dihydrochloride is 1:(1-3); and the mass ratio of 3-(3-dimethylamino-propyl)-1-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 5:(1-3).

6. The method for preparing a targeted delivery system of a metal polyphenol nanosystem according to claim 1, characterized in that: In step S3, the concentration of the 2-(N-morpholino)ethanesulfonic acid buffer is 0.1 M, and the stirring reaction conditions are: the reaction is carried out in a dark environment at room temperature for 12-36 hours, the stirring speed is 600-1000 rpm, and the dialysis is carried out for 48-96 hours, and the molecular weight of the dialyzed residue is less than 1000 Da.

7. The method for preparing a targeted delivery system of a metal polyphenol nanosystem according to claim 1, characterized in that: In the step S3, the mass ratio of (5-carboxypentyl)triphenylphosphine bromide to the chondroitin sulfate-cystamine dihydrochloride is (1-3):1; the mass ratio of 3-(3-dimethylamino-propyl)-1-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 5:(1-3).

8. The method for preparing a targeted delivery system of a metal polyphenol nanosystem according to claim 1, characterized in that: The dripping speed in step S4 is 4-6 mL / h, the concentration of the chondroitin sulfate-cystamine dihydrochloride-triphenylphosphine bromide aqueous solution is 1.5-2.5 mg / mL; and the temperature of the vacuum rotary evaporation is 40-45°C.

9. Use of the delivery system based on the metal polyphenol system prepared by the preparation method according to any one of claims 1 to 8 in the preparation of colitis drugs, characterized in that: Used for controlled release under the stimulation of reactive oxygen species.

10. The use according to claim 9, characterized in that: Used to relieve colon tissue damage and inflammation.

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