Preparation method and application of a targeted delivery system of metal polyphenol nanosystem

Abstract

The application discloses a preparation method and application of a targeted delivery system of a metal polyphenol nano system. The application discloses a targeted delivery system of a metal polyphenol nano system, and a preparation method thereof is as follows: taking a metal polyphenol nano system formed on the basis of epigallocatechin gallate and iron ions as a carrier, loading fucoxanthin to prepare fucoxanthin nanoparticles with active oxygen response characteristics; then coupling chondroitin sulfate which can target macrophages and (5-carboxypentyl) triphenylphosphonium bromide which can target mitochondria, and further preparing the fucoxanthin delivery system with active oxygen response characteristics and targeting characteristics through a double emulsion anti-solvent evaporation method. The system can realize the release of fucoxanthin under the stimulation of active oxygen, and realize the aggregation of the targeted delivery system in the mitochondria of a specific inflammatory site. The carrying system can effectively improve or relieve inflammatory bowel disease, and raw materials used in the application are low in cost, the preparation condition is mild, and the application is easy to scale production.

Description

Technical Field

[0001] This invention relates to the field of bionanotechnology, specifically to a method for preparing and applying a targeted delivery system of metal polyphenol nanosystems. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, relapsing inflammatory gastrointestinal disease characterized by impaired intestinal barrier function, mucosal inflammation, excessive production of reactive oxygen species (ROS), and gut microbiota dysbiosis. Its incidence has been increasing in recent years. Currently, medications used to treat UC are mainly based on amino acid salicylates, immunosuppressants, and biological agents. However, these strategies cannot completely cure UC, and long-term medication use can cause serious side effects and financial burden on patients. Therefore, utilizing functional food components delivered orally to the colon via a colonic delivery system to alleviate inflammatory symptoms is a safer and more effective strategy for managing colitis.

[0003] Fucoxanthin is a carotenoid widely found in marine phytoplankton such as brown algae, diatoms, and microalgae. The unique epoxy and propylene structures in fucoxanthin give it significant health benefits in areas such as antioxidation, anti-inflammation, anti-diabetic activity, and immune regulation. However, fucoxanthin has poor water solubility and stability, and is easily affected by environmental factors such as pH, oxygen, ultraviolet radiation, and temperature, resulting in low bioavailability and limiting its application in the food, health supplement, and medical industries. It is difficult to effectively deliver fucoxanthin to specific sites to exert its effects. Therefore, improving the water solubility and stability of fucoxanthin and enhancing its bioavailability is crucial.

[0004] Currently, the protection, delivery, and controlled release of functional factors are mainly achieved by constructing delivery systems. Common delivery system forms include micro / nanoemulsions, nanoparticles, nanoliposomes, microcapsules, and hydrogels. However, conventional delivery systems cannot achieve targeted, concentrated, and controllable release of functional factors, and the biological activity of these factors cannot be fully realized. Besides the stable and protective delivery of functional factors, one of the main challenges is how to achieve responsive and controllable release of functional factors at their target sites. Targeted function is usually achieved by introducing specific ligands into the delivery system. The ligands can recognize corresponding receptors and target the delivery system to specific sites. Response-controlled release is usually achieved by introducing responsive groups into the delivery system. These responsive groups can sense changes in the external environment and accordingly change their physical or chemical properties, thereby achieving precise control over 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 ingestion, specific responsive groups and specific ligands are further introduced into the carrier to construct a targeted delivery system with controllable response characteristics, thereby achieving targeted delivery and controllable release of functional factors. This is of great significance for precisely improving the bioavailability of functional factors.

[0005] 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 properties, anti-inflammatory activity, and good biocompatibility. These nanomaterials have shown promising applications in tumor therapy, anti-inflammatory intervention, and drug delivery. Their biological functions mainly derive from the physiological activities inherent in polyphenols themselves. Polyphenols can effectively reduce the generation of pro-inflammatory mediators and reactive oxygen species by inhibiting the activity of key inflammatory metabolic enzymes such as cyclooxygenase and lipoxygenase, thereby exerting antioxidant and anti-inflammatory effects. Simultaneously, polyphenols can regulate multiple inflammation-related signaling pathways, especially 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), thus enhancing the overall anti-inflammatory effect. Therefore, using metal polyphenol nanoparticles to deliver fucoxanthin can achieve a synergistic anti-inflammatory effect with fucoxanthin, significantly enhancing the overall antioxidant and anti-inflammatory activity of the system, thereby improving therapeutic efficacy. Summary of the Invention

[0006] The technical problem to be solved is that fucoxanthin has poor water solubility and stability, low oral bioavailability, and limited application. This invention uses a metal polyphenol nanoparticle system as a carrier to prepare fucoxanthin nanoparticles with ROS-responsive properties, improving the water solubility and stability of fucoxanthin. Then, chondroitin sulfate, which can target macrophages, and triphenylphosphine bromide, which can target mitochondria, are coupled together, and a fucoxanthin delivery system is prepared via a double emulsion antisolvent evaporation method. This promotes the targeted accumulation of fucoxanthin in macrophages at sites of intestinal inflammation and enables its release in mitochondria in response to ROS stimulation, enhancing the antioxidant effect and bioavailability of fucoxanthin.

[0007] Technical solution: A method for preparing a targeted delivery system of metal polyphenol nanosystems, comprising the following steps:

[0008] S1. Fucoxanthin was dissolved in anhydrous ethanol, and the resulting fucoxanthin ethanol solution was added to 3-(N-morpholino)propane sulfonic acid buffer. Then, EGCG solution and FeCl3 solution were added sequentially, and the mixture was stirred continuously to obtain a fucoxanthin nanoparticle dispersion.

[0009] S2. Dissolve chondroitin sulfate in water, add 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in sequence and stir until homogeneous, then add cystamine dihydrochloride and stir continuously. Dialyze in distilled water and freeze dry to obtain chondroitin sulfate-cystamine dihydrochloride.

[0010] S3. Dissolve (5-carboxypentyl)triphenylphosphine bromide in 2-(N-morpholine) ethanesulfonic acid buffer, add 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, stir until homogeneous, then add chondroitin sulfate-cystamine dihydrochloride, stir to react, dialyze in distilled water, and freeze-dry to obtain chondroitin sulfate-cystamine dihydrochloride-triphenylphosphine bromide;

[0011] S4. The fucoxanthin nanoparticle dispersion was dropped into an ethanol solution as the organic phase. The organic phase was then dropped into a chondroitin sulfate-cystamine dihydrochloride-triphenylphosphine bromide aqueous solution under stirring. The ethanol was removed by vacuum rotary evaporation to obtain the targeted delivery system.

[0012] 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; the continuous stirring time is 0.5-1 h.

[0013] 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).

[0014] 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 cutoff for dialysis is less than 1000 Da.

[0015] 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-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 5:(1-3).

[0016] Furthermore, in step S3, the concentration of 2-(N-morpholine) ethanesulfonic acid buffer is 0.1M, the reaction conditions are: reaction in the dark at room temperature for 12-36 hours, stirring speed is 600-1000 rpm; dialysis is performed for 48-96 hours, and the molecular weight cutoff of the dialysis is less than 1000 Da.

[0017] Furthermore, in step S3, the mass ratio of (5-carboxypentyl)triphenylphosphine bromide to chondroitin sulfate-cystamine dihydrochloride is (1-3):1; the mass ratio of 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 5:(1-3).

[0018] 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 vacuum rotary evaporation temperature is 40-45℃.

[0019] The delivery system based on the metal polyphenol system prepared by the above method is used in the preparation of colitis drugs for controlled release under reactive oxygen species stimulation.

[0020] Furthermore, it is used to alleviate colon tissue damage and inflammation.

[0021] Beneficial effects:

[0022] 1. This invention couples chondroitin sulfate, which can target macrophages, with triphenylphosphine bromide, which can target mitochondria, and then prepares a fucoxanthin delivery system via a double emulsion antisolvent evaporation method. The prepared targeted delivery system has functional properties such as stimulating release, targeted delivery, and effectively relieving colon inflammation, and can be used in the fields of health products and functional foods.

[0023] 2. This invention uses a metal polyphenol nanosystem as a carrier to prepare fucoxanthin nanoparticles with ROS-responsive characteristics. The prepared targeted delivery system is spherical and has advantages such as good stability, negative surface charge, and high encapsulation efficiency. It significantly enhances the antioxidant activity of fucoxanthin, enables the controllable release of fucoxanthin under ROS stimulation, and achieves targeted delivery and aggregation in intestinal inflammation sites and mitochondria, which can effectively alleviate colon inflammation.

[0024] 3. While existing dual-target delivery systems improve stability and bioavailability to some extent, thus enhancing their efficacy in treating chronic colitis, the delivery carrier itself lacks anti-inflammatory function, and the therapeutic effect primarily depends on the delivered active ingredient. In contrast, the targeted delivery system based on a metal polyphenol nanoparticle system constructed in this invention introduces EGCG, which not only acts as a carrier component to stabilize the structure but also possesses significant antioxidant and anti-inflammatory activity. This component can synergistically work with fucoxanthin, enhancing the stability and delivery efficiency of fucoxanthin while further strengthening the overall bioactivity of the system, thereby significantly improving the therapeutic effect.

[0025] 4. The method for preparing the cell membrane / mitochondrial targeted delivery system of the present invention is simple and mild, uses inexpensive raw materials, has mild encapsulation conditions, and is easy to scale up for production. Attached Figure Description

[0026] Figure 1 This is a transmission electron microscope image of the targeted delivery system prepared in Example 1;

[0027] Figure 2 This is a transmission electron microscope image of the targeted delivery system prepared in Comparative Example 1;

[0028] Figure 3 The figures show the in vitro release of the targeted delivery system under ROS conditions. Figure A is Example 1, and Figure B is Comparative Example 2.

[0029] Figure 4 This is a graph showing the effect 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;

[0030] Figure 5 Figure A shows the mitochondrial distribution in RAW264.7 macrophages after fluorescent labeling in Example 1. Figure B shows the distribution of mitochondria in RAW264.7 macrophages using the hydrophobic fluorescent probe coumarin 6 (C6) instead of the targeted delivery system in Example 1. Figure C is an overlay of the distribution of mitochondria labeled with the red fluorescent probe and the targeted delivery system in Example 1 in RAW264.7 macrophages.

[0031] Figure 6 Figure A shows the intracellular distribution of the fluorescently labeled mitochondria in RAW264.7 macrophages after comparison example 1. Figure B shows the distribution of mitochondria in RAW264.7 macrophages after the red fluorescent probe is labeled. Figure C shows the distribution of mitochondria in RAW264.7 macrophages after the hydrophobic fluorescent probe coumarin 6 (C6) replaces the targeting delivery system of comparison example 1. Figure C is an overlay of the distribution of mitochondria labeled with the red fluorescent probe and the targeting delivery system of comparison example 1 in RAW264.7 macrophages.

[0032] Figure 7 Figure A shows the intracellular distribution of the fluorescently labeled mitochondria in RAW264.7 macrophages, labeled with the red fluorescent probe. Figure B shows the distribution of mitochondria in RAW264.7 macrophages using the hydrophobic fluorescent probe coumarin 6 (C6) instead of the targeted delivery system in Comparative Example 2. Figure C is an overlay of the distribution of mitochondria labeled with the red fluorescent probe and the targeted delivery system in Comparative Example 2 in RAW264.7 macrophages.

[0033] Figure 8 The figure shows the distribution of the hydrophobic fluorescent probe coumarin 6 (C6) in the intestinal tissue of mice with colitis induced by 3% sodium dextran sulfate, using the targeted delivery system instead of Example 1.

[0034] Figure 9 The distribution of the hydrophobic fluorescent probe coumarin 6 (C6) in the intestinal tissue of mice with colitis induced by 3% sodium dextran sulfate in the comparative example 1 is shown in the figure.

[0035] Figure 10 The figure shows the distribution of the hydrophobic fluorescent probe coumarin 6 (C6) in the intestinal tissue of mice with colitis induced by 3% dextran sulfate sodium salt in the targeted delivery system of the actual comparison example 2. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are illustrative of the present invention, but the present invention is not limited to the following embodiments:

[0037] Example 1

[0038] A method for preparing a targeted delivery system of a metal polyphenol nanosystem includes the following steps:

[0039] S1. Fucoxanthin was dissolved in ethanol to obtain a 5 mg / mL fucoxanthin ethanol solution. Under stirring, the fucoxanthin ethanol solution was added to 3-(N-morpholino)propane sulfonate buffer (MOPS) (10 mM, pH = 7.4), followed by the sequential addition of EGCG solution (10 mg / mL) and FeCl3 solution (10 mg / mL). The mixture was continuously stirred at 600 rpm for 0.5 h at room temperature to obtain a fucoxanthin nanoparticle dispersion. The mass ratio of fucoxanthin, EGCG, and FeCl3 was 1:1:1.

[0040] S2. Chondroitin sulfate was dissolved in distilled water to prepare a solution with a concentration of 14.5 mg / mL. 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added, and the mixture was stirred at room temperature for 3 h to activate the carboxyl groups on the chondroitin sulfate. Cystamine dihydrochloride was then added and stirred for 24 h. The above mixed solution was dialyzed in distilled water for 72 h. 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-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide was 5:1.

[0041] S3. Dissolve (5-carboxypentyl)triphenylphosphine bromide in 2-(N-morpholine) ethanesulfonic acid buffer to prepare a 0.1M solution. Then add 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. Stir at 800 rpm for 3 h at room temperature to activate the carboxyl group on (5-carboxypentyl)triphenylphosphine bromide. Then add chondroitin sulfate-cystamine dihydrochloride aqueous solution and react at 800 rpm for 24 h at room temperature in the dark. Then dialyze in distilled water for 72 h, collect the solution in the dialysis bag, freeze-dry to obtain chondroitin sulfate-cystamine dihydrochloride-triphenylphosphine bromide. The mass ratio of (5-carboxypentyl)triphenylphosphine bromide to chondroitin sulfate-cystamine dihydrochloride is 1:1; the mass ratio of 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 5:1.

[0042] S4. The fucoxanthin nanoparticle dispersion was loaded into a 5 mL syringe and uniformly dripped into an ethanol solution at a flow rate of 5 mL / h to form the organic phase. The resulting mixed solution was then loaded back into a 5 mL syringe and uniformly dripped into a 2 mg / mL chondroitin sulfate-cystamine dihydrochloride-triphenylphosphine bromide aqueous solution under stirring at 1000 rpm at a flow rate of 5 mL / h. The ethanol was removed by vacuum rotary evaporation at 43 °C to obtain the targeted delivery system.

[0043] Comparative Example 1

[0044] The difference between this comparative example and Example 1 is that triphenylphosphine bromide, which can target mitochondria in macrophages, was not grafted onto chondroitin sulfate-cystamine dihydrochloride, as detailed below:

[0045] S1. Fucoxanthin was dissolved in ethanol to obtain a 5 mg / mL fucoxanthin ethanol solution. Under stirring, the fucoxanthin ethanol solution was added to 3-(N-morpholino)propane sulfonate buffer (MOPS) (10 mM, pH = 7.4), followed by the sequential addition of EGCG solution (10 mg / mL) and FeCl3 solution (10 mg / mL). The mixture was continuously stirred at 600 rpm for 0.5 h at room temperature to obtain a fucoxanthin nanoparticle dispersion. The mass ratio of fucoxanthin, EGCG, and FeCl3 was 1:1:1.

[0046] S2. Chondroitin sulfate was dissolved in distilled water to prepare a solution with a concentration of 14.5 mg / mL. 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added, and the mixture was stirred at room temperature for 3 h to activate the carboxyl groups on the chondroitin sulfate. Cystamine dihydrochloride was then added and stirred for 24 h. The above mixed solution was dialyzed in distilled water for 72 h. 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-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide was 5:1.

[0047] S3. The fucoxanthin nanoparticle dispersion was loaded into a 5 mL syringe and uniformly dripped into an ethanol solution at a flow rate of 5 mL / h to form the organic phase. The resulting mixed solution was then loaded back into a 5 mL syringe and uniformly dripped into a 2 mg / mL chondroitin sulfate-cystamine dihydrochloride aqueous solution under stirring at 1000 rpm at a flow rate of 5 mL / h. The ethanol was removed by vacuum rotary evaporation at 43 °C to obtain the targeted delivery system.

[0048] Comparative Example 2

[0049] The difference between this comparative example and Example 1 is that triphenylphosphine 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, as detailed below:

[0050] 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. Then, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added and stirred at room temperature for 1 h. Subsequently, fucoxanthin was added and stirred in the dark at room temperature for 24 h. The above mixed solution was dialyzed in dimethyl sulfoxide for 24 h and dialyzed in distilled water for 48 h. The solution in the dialysis bag (dialysis molecular weight 500 Da) was collected and freeze-dried at -50 °C under vacuum of 1 Pa for 48 h 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.

[0051] S2. Synthesis of chondroitin sulfate-cystamine dihydrochloride: Chondroitin sulfate was dissolved in distilled water to prepare a solution with a concentration of 50 mg / mL. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added, and the mixture was stirred at room temperature for 2 h. Then, cystamine dihydrochloride was added and stirred for 24 h. The above mixed solution was dialyzed in distilled water for 48 h. The solution in the dialysis bag (dialysis molecular weight 500 Da) was collected and freeze-dried to obtain chondroitin sulfate-cystamine dihydrochloride. The mass ratio of chondroitin sulfate to cystamine dihydrochloride was 100:1, and the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide was 5:2.

[0052] S3. Synthesis of cell membrane-targeting chondroitin sulfate-cystamine dihydrochloride-EGCG: EGCG was dissolved in an ethanol solution, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added. The mixture was stirred at 1200 rpm for 1 h under ice-water bath conditions. Subsequently, an aqueous solution of chondroitin sulfate-cystamine dihydrochloride was added to the above mixture, and the mixture was stirred at 1200 rpm for 4 h at 80 °C. Then, it was cooled to room temperature and magnetically stirred at 1200 rpm for 24 h. Finally, the above mixture was dialyzed in distilled water for 48 h. The solution in the dialysis bag (dialysis molecular weight 500 Da) was collected and freeze-dried to obtain the chondroitin sulfate-cystamine dihydrochloride-EGCG polymer. 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.

[0053] S4. Preparation of Nanoparticles: A (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 uniformly added dropwise at a flow rate of 5 mL / h to a 25 mg / mL chondroitin sulfate-cystamine dihydrochloride-EGCG aqueous solution under magnetic stirring at 1200 rpm. The resulting mixed solution was then refilled into the syringe and uniformly added dropwise at a flow rate of 5 mL / h to ethanol under magnetic stirring at 1200 rpm. The ethanol was then removed by rotary evaporation at 45 °C to obtain dual-targeted nanoparticles.

[0054] Performance testing:

[0055] 1. Microstructure observation

[0056] The microstructure of the functional factor delivery system was observed using transmission electron microscopy: 20 μL of the functional factor delivery system dispersion was dropped onto a copper mesh support, excess liquid was absorbed with filter paper, and the sample was air-dried for a period of time. Subsequently, a small amount of 1% uranium acetate solution was added for negative staining, and finally, observation was performed at an accelerating voltage of 80 kV.

[0057] Figure 1 The image shows a transmission electron microscope (TEM) image of the functional factor delivery system prepared in Example 1. This delivery system exhibits a uniformly dispersed core-shell spherical structure. Figure 2 The image shows a transmission electron microscope (TEM) image of the functional factor delivery system prepared for Comparative Example 1. As can be seen from the image, the prepared targeted delivery system is a uniformly dispersed spherical structure, which also has a core-shell structure.

[0058] 2. Basic physical indicators

[0059] Encapsulation efficiency was determined by high-performance liquid chromatography (HPLC): A certain amount of the functional factor delivery system was accurately weighed and dissolved in anhydrous ethanol, sonicated for 30 min, and then centrifuged at 5000g for 5 min at 4℃. Subsequently, the concentration of encapsulated fucoxanthin in the supernatant was determined by HPLC, and the content of encapsulated fucoxanthin in the supernatant was calculated according to the known fucoxanthin standard curve. The specific conditions for HPLC analysis were as follows: a C18 column was used for chromatographic analysis; mobile phase A was ultrapure water, mobile phase B was chromatographically pure methanol, the flow rate was 0.7 mL / min, the column operating temperature was 35℃, the injection volume was 10 μL, and the UV detection wavelength was 450 nm. The encapsulation efficiency was calculated using the following formula:

[0060] EE (%) = (Encapsulated fucoxanthin content / Added fucoxanthin content) × 100%

[0061] The particle size and potential of the functional factor delivery system were determined using a laser particle size analyzer: the dispersion of the functional factor delivery system was diluted 100 times with distilled water and added to the sample cell. The particle size and potential were measured using a nanoparticle size and zeta potential analyzer at 25°C. Each sample was measured three times independently, and the average value was taken to ensure the accuracy of the results.

[0062] Table 1 shows the basic performance characteristics of the functional factor delivery systems prepared in the examples and comparative examples. The encapsulation efficiency of fucoxanthin in the functional factor delivery systems of the examples and comparative examples was determined by high-performance liquid chromatography (HPLC). The encapsulation efficiency of fucoxanthin in the functional factor delivery system prepared in Example 1 was 85.08%, and that in the functional factor delivery system prepared in Comparative Example 1 was 90.45%. However, the encapsulation efficiency of Comparative Example 2 was less than 70% due to the change in the wall material. Next, the hydrodynamic diameter and potential of the functional factor delivery systems in the examples and comparative examples were 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 the potential was -35.57 ± 0.79 mV. The particle size of Comparative Example 2 was much larger than that of Example 1, possibly due to differences in the preparation method and wall material, resulting in larger particles and reduced stability.

[0063] Table 1

[0064]

[0065]

[0066] 3. In vitro release under different concentrations of ROS

[0067] 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. Samples were separated by centrifugation at specific time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, and 48 h), and the supernatant was collected to measure the concentration of fucoxanthin. The functional factor delivery system precipitate was then resuspended in the same fresh buffer solution and incubated further to observe the release characteristics.

[0068] Figure 3A shows the in vitro release of the functional factor delivery system prepared in Example 1 under different ROS concentrations. In PBS buffer at the same pH, fucoxanthin was released from the delivery system at a significantly faster rate in the H2O2-containing group than in the PBS group. Furthermore, the release rate of fucoxanthin increased significantly with increasing H2O2 concentration, and this increase continued over time. At an H2O2 concentration of 10 mM (pH 7.4), the release rate of fucoxanthin reached 47.71% after 48 hours of incubation. These results indicate that the delivery system possesses ROS-responsive characteristics, enabling it to successfully release fucoxanthin upon entering a highly ROS-laden inflamed intestinal environment, thereby exerting an anti-inflammatory effect. Figure 3 B shows the in vitro release under different ROS concentrations in Comparative Example 2. It can be seen that its ROS response characteristics are poor and the sustained-release effect is not good.

[0069] 4. Effect of delivery system pretreatment on colon length in mice with 3% dextran sulfate-induced colitis.

[0070] The intervention effect of a functional factor delivery system on intestinal injury in mice was evaluated using a dextran sulfate sodium (DSS)-induced colitis model. In short, 30 C57BL / 6J mice were acclimatized for one week and then randomly divided into 5 groups according to body weight: a healthy control group, a DSS-induced group, a comparative group, a carrier group, and an example group. During the entire 3-week experimental period, the healthy control group and the DSS group were administered physiological saline by gavage, while the other groups were administered different samples by gavage at a dose of 200 μL / day. After 14 days of continuous gavage, except for the healthy control group, the other groups were given 3.0% DSS solution. Meanwhile, different samples were continued to be administered by gavage daily, and the mice's body weight, diarrhea, and bloody stools were monitored regularly. Figure 4 The effect of pretreatment with the targeted delivery system prepared for Example 1 and Comparative Example 1 on colon length in mice with 3% DSS-induced colitis was investigated. The results showed that the colon length in the DSS-treated group was significantly shorter than that in the healthy control group, indicating that the colonic tissue suffered DSS-induced damage. 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 significant. This demonstrates that the delivery system can effectively alleviate the shortening of colon length in DSS-induced colitis mice.

[0071] 5. Distribution of targeted delivery systems in cells or animal organs

[0072] A fluorescently labeled delivery system loaded with C6 was prepared by replacing fucoxanthin in the examples and comparative examples with the hydrophobic fluorescent probe coumarin 6 (C6). The distribution of the targeted delivery system in cells or animal organs was observed, and the targeting ability of the prepared delivery system was examined.

[0073] like Figure 5 As shown, the embodiment has a relatively complete structure and strong fluorescence, with the green fluorescence overlapping with the mitochondrial fluorescence labeled by the red fluorescent probe. Figure 5 C) indicates that the delivery system modified with (5-carboxypentyl)triphenylphosphine bromide exhibits good accumulation within mitochondria. Observation Figure 6 It can be seen that the fluorescently labeled targeted delivery system prepared in Comparative Example 1 exhibits relatively weak green fluorescence intensity within the mitochondria. Figure 6 A), but it can overlap to some extent with the mitochondrial fluorescence labeled by the mitochondrial red fluorescent probe (A). Figure 6 C) indicates that the delivery system can aggregate within mitochondria. For example... Figure 7 As shown, Comparative Example 2, with its incomplete structure, also exhibited significantly weaker fluorescence, indicating poor delivery performance.

[0074] 6. Targeting ability of the tracer delivery system in the mouse intestine

[0075] Twelve hours after oral administration of the prepared fluorescent delivery system to mice, fluorescence 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 control group, and an example group. They were placed in an environment of 22±2℃ and 60±5% relative humidity, acclimatizing for 7 days with a 12-hour light-12-hour dark cycle. Subsequently, the DSS group mice were given free access to 3.0% (w / v) DSS aqueous solution for 7 days to establish a mouse model of colitis. The three groups of mice were then administered the fluorescent delivery system. Twelve hours after digestion and metabolism, the mice were sacrificed, and their intestines were collected for analysis. Simultaneously, the distribution of fluorescence signals in the mouse intestines was detected using an IVIS Spectrum small animal in vivo fluorescence imaging system.

[0076] Figure 8 A stronger fluorescence signal, also observed in mice with dextran sulfate sodium induced colitis, was significantly higher than that observed in mice with dextran sulfate sodium induced colitis. Figure 9 and Figure 10The results indicate 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 very little 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 at 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 receptor expressed at the inflammatory site, and triphenylphosphine bromide can further promote the targeting of the fluorescent delivery system to mitochondrial tissue.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for preparing a targeted delivery system of a metal polyphenol nano system, characterized by, Includes the following steps: S1. Fucoxanthin was dissolved in anhydrous ethanol, and the resulting fucoxanthin ethanol solution was added to 3-(N-morpholino)propane sulfonic acid buffer. Then, EGCG solution and FeCl3 solution were added sequentially, and the mixture was stirred continuously to obtain a fucoxanthin nanoparticle dispersion. S2. Dissolve chondroitin sulfate in water, add 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in sequence and stir until homogeneous, then add cystamine dihydrochloride and stir continuously. Dialyze in distilled water and freeze dry to obtain chondroitin sulfate-cystamine dihydrochloride. S3. Dissolve (5-carboxypentyl)triphenylphosphine bromide in 2-(N-morpholine) ethanesulfonic acid buffer, add 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, stir until homogeneous, then add chondroitin sulfate-cystamine dihydrochloride, stir to react, dialyze in distilled water, and freeze-dry to obtain chondroitin sulfate-cystamine dihydrochloride-triphenylphosphine bromide; S4. The fucoxanthin nanoparticle dispersion was dropped into an ethanol solution as the organic phase. The organic phase was then dropped into a chondroitin sulfate-cystamine dihydrochloride-triphenylphosphine bromide aqueous solution under stirring. The ethanol was removed by vacuum rotary evaporation to obtain the 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; 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 EGCG solution is 8-10 mg / mL, the concentration of FeCl3 solution is 8-10 mg / mL, and 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 cutoff for dialysis is less than 1000 Da; 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-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 5:(1-3).

5. 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 2-(N-morpholine) ethanesulfonic acid buffer is 0.1M, the reaction conditions are: reaction in the dark at room temperature for 12-36 hours, stirring speed is 600-1000 rpm; dialysis is performed for 48-96 hours, and the molecular weight cutoff of the dialysis is less than 1000 Da.

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 mass ratio of (5-carboxypentyl)triphenylphosphine bromide to chondroitin sulfate-cystamine dihydrochloride is (1-3):1; the mass ratio of 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 5:(1-3).

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

8. The application of the metal polyphenol-based delivery system prepared by the method according to any one of claims 1-7 in the preparation of colitis drugs, characterized in that, For controlled release under reactive oxygen species stimulation.

9. The application according to claim 8, characterized in that: It is used to relieve damage and inflammation of the colon tissue.

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