Galactosyl fluorinated chitosan-EGCG (epigallocatechin gallate) nanoparticles and application thereof

Abstract

The invention discloses galactosyl fluorinated chitosan-EGCG (epigallocatechin gallate) nanoparticles and application thereof, and belongs to the field of steady-state delivery of functional foods. The preparation method comprises the following steps: firstly, synthesizing galactosyl modified fluorinated chitosan by utilizing an amidation reaction between lactobionic acid carboxyl and fluorinated chitosan amino, and successfully preparing the tea polyphenol EGCG-loaded nano steady-state delivery carrier through an ionic gel method. The carrier can utilize the fluorine effect of the fluorinated group on the surface of the carrier and the interaction of the galactose-SGLT1 receptor to enhance the intestinal targeted delivery of EGCG (epigallocatechin gallate). The preparation method is simple to operate, the prepared galactosyl-modified fluorinated chitosan nanoparticle delivery carrier has high loading rate on EGCG (epigallocatechin gallate), the bioavailability of EGCG can be improved, and the galactosyl-modified fluorinated chitosan nanoparticle delivery carrier is of great significance in promoting development and application of functional food.

Description

Technical Field

[0001] The present invention belongs to the technical field of food functional factor stabilization processing, and specifically relates to galactosyl fluorinated chitosan-EGCG nanoparticles and applications thereof. Background Art

[0002] Epigallocatechin gallate (EGCG) is a tea polyphenol found in high concentrations in green tea. The main bottlenecks facing EGCG's application are its poor stability and low intestinal absorption efficiency. EGCG is easily oxidized in neutral or alkaline environments, leading to degradation. Furthermore, EGCG's inefficient intestinal absorption makes it difficult to reach effective concentrations, resulting in low nutritional value. Therefore, developing EGCG delivery vehicles to improve their stability and bioavailability is a major challenge.

[0003] Chitosan, the only naturally occurring cationic polysaccharide, has been shown to possess advantages such as non-toxicity, biodegradability, and biocompatibility. However, due to the high concentration of amino groups in its structure, it dissolves rapidly in the gastric cavity, but cannot protect the nutrients it carries during passage through the stomach and small intestine, limiting its application as a food-grade carrier. Modified chitosan derivatives not only retain the original physicochemical properties and biological functions of chitosan but also possess new and improved properties such as increased water solubility over a wide pH range. In recent years, they have been widely synthesized and used to construct nutrient delivery systems. To further expand the application of chitosan-based carriers, chitosan and its derivatives are often combined with other biopolymers to develop delivery systems with a range of improved properties.

[0004] Sodium-glucose co-transporter 1 (SGLT1) is a protein receptor primarily expressed on the membranes of small intestinal epithelial cells that mediates the intracellular transport of galactosyl-containing molecules. Modifying polymer surfaces with fluorinated fatty chains can enhance their affinity for cell membranes, making them more easily permeable and facilitating their escape from endosomes. Therefore, constructing galactosyl-modified fluorinated chitosan nanoparticle delivery vehicles is an important strategy for achieving efficient intestinal absorption mediated by the SGLT1 pathway. Summary of the Invention

[0005] The purpose of the present invention is to provide a galactosyl fluorinated chitosan-EGCG nanoparticle and application thereof.

[0006] To achieve the above purpose and other related purposes, the present invention provides a technical solution: a galactosyl fluorinated chitosan-EGCG nanoparticle, the fluorination modification degree is 15%-30%; the galactosyl modification degree is 15%-25%.

[0007] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: the preparation method of the galactosyl fluorinated chitosan-EGCG nanoparticles is characterized by comprising the following steps:

[0008] Step 1: covalently bond the carboxyl group of heptafluorobutyric acid to the amino group of chitosan to prepare fluorinated chitosan;

[0009] Step 2: synthesizing galactosyl-modified fluorinated chitosan by amidation reaction between lactobionic acid and amino groups of fluorinated chitosan;

[0010] Step 3: Prepare galactosyl-modified fluorinated chitosan nanoparticles loaded with tea polyphenol EGCG by sodium tripolyphosphate ion cross-linking method.

[0011] The preferred technical solution is: the loading amount of the tea polyphenol EGCG is 5%-15%.

[0012] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: the use of the galactosyl fluorinated chitosan-EGCG nanoparticles in improving the bioavailability of tea polyphenol EGCG.

[0013] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: use of the galactosyl fluorinated chitosan-EGCG nanoparticles in the preparation of a drug for alleviating ulcerative colitis.

[0014] Due to the use of the above technical solution, the present invention has the following advantages compared with the prior art:

[0015] 1. The galactosyl fluorinated chitosan-EGCG nanoparticles of the present invention have a high encapsulation rate for tea polyphenol EGCG, the particle carrier has a uniform spherical morphology, and still exhibits good particle stability after storage at 25°C for 15 days.

[0016] 2. The interaction between galactose and SGLT1 receptor can enhance the uptake efficiency of intestinal cells for EGCG-loaded galactosyl fluorinated chitosan nanoparticles.

[0017] 3. Galactosyl fluorinated chitosan-EGCG nanoparticles can improve the permeation efficiency of EGCG in the intestine.

[0018] 4. Galactosyl fluorinated chitosan-EGCG nanoparticles can effectively alleviate weight loss, bloody diarrhea and shortened colon length in mice with ulcerative colitis.

[0019] 5. Based on the targeted recognition effect between SGLT1 receptor and galactose, the present invention constructs EGCG-loaded galactosyl fluorinated chitosan nanoparticles, realizes the efficient intestinal delivery of tea polyphenol EGCG, and provides a certain research basis for the development of related functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the synthesis of fluorinated chitosan.

[0021] Figure 2 Schematic diagram of the synthesis of galactosyl fluorinated chitosan.

[0022] Figure 3 Diagram of mouse model construction and intervention pattern.

[0023] Figure 4 Chitosan modified with different ligands 1 H NMR spectrum.

[0024] Figure 5 Fluorinated chitosan and galactosyl fluorinated chitosan 19 F NMR spectroscopy.

[0025] Figure 6 This is a scanning electron micrograph of galactosylfluorinated chitosan-EGCG nanoparticles.

[0026] Figure 7 Storage stability analysis of galactosylfluorinated chitosan-EGCG nanoparticles (25°C).

[0027] Figure 8 The uptake capacity of HEK 293T-SGLT1 cells for galactosylfluorinated chitosan nanoparticles.

[0028] Figure 9 The uptake capacity of galactosyl fluorinated chitosan nanoparticles by small intestine tissue in mice 8 hours after oral administration.

[0029] Figure 10 Effects of galactosylfluorinated chitosan-EGCG nanoparticles on body weight changes in mice with ulcerative colitis.

[0030] Figure 11 Effects of galactosylfluorinated chitosan-EGCG nanoparticles on the disease activity index of mice with ulcerative colitis.

[0031] Figure 12 Figure 3. Effects of galactosyl fluorinated chitosan-EGCG nanoparticles on colon length in mice with ulcerative colitis. A: Diagram of the colon in each group of mice; B: Statistics of colon length in each group of mice.

[0032] Figure 13This is the H&E staining image of the colon tissue of mice after intervention with galactosyl fluorinated chitosan-EGCG nanoparticles in ulcerative colitis.

[0033] Figure 14 This is the Alcian blue staining of the colon of mice with ulcerative colitis after intervention with galactosylfluorinated chitosan-EGCG nanoparticles. DETAILED DESCRIPTION

[0034] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in these embodiments.

[0035] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification, so that people familiar with this technology can understand and read them. They are not used to limit the limiting conditions that can be implemented in the present invention, so they have no technical significance. The modification of any structure, change in proportional relationship or adjustment of size are provided below. The following examples are provided to better understand the present invention, but are not intended to limit the present invention. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional consumables and biochemical reagent stores.

[0036]

[0037] Example 1: Galactosyl fluorinated chitosan-EGCG nanoparticles and their applications

[0038] Chemical Synthesis of Fluorinated Chitosan (FCS)

[0039] 36.5 μL of heptafluorobutyric acid was taken and dissolved in an appropriate amount of anhydrous dimethyl sulfoxide, and 1.5 times the reaction amount of EDC-HCl and NHS was added, and stirred in the dark for 1 hour. The activated heptafluorobutyric acid solution was slowly added dropwise to 10 mL of chitosan acetic acid aqueous solution (20 mg / mL, pH 6.0) under rapid stirring, and stirred in the dark for 12 hours; after the reaction was completed, the reacted liquid was placed in a dialysis bag (Mw=3500Da) and dialyzed for 2 days, and the dialysate was ultrapure water; after the dialysis was completed, it was placed in a -80°C refrigerator for pre-freezing, and then vacuum freeze-dried to obtain fluorinated chitosan (FCS). The schematic diagram of the synthesis of fluorinated chitosan is shown as follows: Figure 1 shown.

[0040] Synthesis of galactosylfluorinated chitosan (FGC)

[0041] Weigh 0.24g of lactobionic acid in a beaker, dissolve it in 5mL of tetramethylethylenediamine / HCl buffer (pH 4.7), add 1.5 times the reaction amount of EDC-HCl and NHS, and stir in the dark for 1h. Slowly add the above-activated lactobionic acid solution to 22mL of fluorinated chitosan hydrochloric acid aqueous solution (10mg / mL, pH 5.0) under rapid stirring, and react at room temperature for 72h. The obtained product is dialyzed with a dialysis bag (Mw=3500Da) for 3 days. After the dialysis is completed, it is placed in a -80℃ refrigerator for pre-freezing, and then vacuum freeze-dried to obtain galactosyl fluorinated chitosan (FGC). The schematic diagram of the synthesis of galactosyl fluorinated chitosan is shown as follows Figure 2 shown.

[0042] Chitosan amino substitution degree detection:

[0043] The amino substitution degree of chitosan was determined by ninhydrin colorimetry. The specific steps are as follows:

[0044] (1) Preparation of 0.40 g / L chitosan standard solution: Accurately weigh 0.0400 g of chitosan powder, dissolve it in 0.50 mol / L acetic acid aqueous solution, and dilute to volume in a 100 mL volumetric flask for later use.

[0045] (2) Preparation of ninhydrin reagent: Weigh 85 mg of ninhydrin and 15 mg of reduced ninhydrin and dissolve them in 10 mL of ethylene glycol methyl ether.

[0046] (3) Accurately pipette 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of chitosan standard solution into 15 mL centrifuge tubes, add 1 mL each of 3 mol / L pH 5.0 sodium acetate buffer and ninhydrin reagent, respectively, and make up to 3.0 mL with ultrapure water. Mix well, heat in a boiling water bath for 10 min, cool to room temperature, dilute to 10 mL with 60% ethanol solution, mix well, let stand for 20 min, and measure the absorbance at 570 nm to prepare a standard curve.

[0047] Fluorinated chitosan and galactosyl fluorinated chitosan were used to replace the chitosan standard solution. The absorbance was determined using the above method, and the degree of substitution was calculated based on the standard curve.

[0048] NMR spectroscopy:

[0049] Weigh 10 mg of chitosan, fluorinated chitosan and galactosyl fluorinated chitosan respectively, dissolve them in 0.50 mL of heavy water solution containing 2% deuterated acetic acid, and record their 1 H NMR spectrum.

[0050] Weigh 10 mg of fluorinated chitosan and galactosyl fluorinated chitosan, dissolve them in 0.50 mL of a heavy water solution containing 2% deuterated acetic acid, and record their19 F NMR spectrum.

[0051] Preparation of galactosyl fluorinated chitosan-EGCG nanoparticles:

[0052] An accurate amount of galactosyl fluorinated chitosan was dissolved in 1% (v / v) acetic acid. EGCG was then added dropwise to the 1.50 mg / mL fluorinated chitosan solution at a 15:1 fluorinated chitosan to EGCG mass ratio. Under magnetic stirring at 600 rpm, a 0.80 mg / mL sodium tripolyphosphate (TPP) solution was added dropwise to the fluorinated chitosan solution at a 4:1 fluorinated chitosan to sodium tripolyphosphate (TPP) mass ratio. After stirring for 30 minutes, galactosyl fluorinated chitosan-EGCG nanoparticles (EGCG@FGC) were obtained.

[0053] Particle size distribution and Zeta potential determination:

[0054] The particle size distribution and surface potential of the nanoparticles were measured using a dynamic light scattering analyzer (Malvern laser particle size analyzer). The nanoparticle solution was added to a square particle size cup and a U-shaped potential cup. The test temperature was 25°C, and each sample was measured in triplicate.

[0055] Determination of EGCG loading rate of nanoparticles:

[0056] The EGCG-loaded nanoparticle solution was placed in a 10 KDa ultrafiltration tube and centrifuged at 3000 g for 20 min. The filtrate was collected and the EGCG content in the filtrate was determined by HPLC. The EGCG loading efficiency (EE) of the nanoparticles was calculated according to the following formula:

[0057]

[0058] The chromatographic conditions for HPLC determination of EGCG content are as follows:

[0059] Mobile phase A: Add 90 mL of acetonitrile, 20 mL of acetic acid, and 2 mL of EDTA-2Na solution to a 1000 mL volumetric flask, dilute to the mark with water, shake well, and pass the solution through a 0.45 μm membrane.

[0060] Mobile phase B: Add 800 mL of acetonitrile, 20 mL of acetic acid, and 2 mL of EDTA-2Na solution to a 1000 mL volumetric flask, dilute to the mark with water, shake well, and pass the solution through a 0.45 μm membrane.

[0061] Gradient elution program: 100% phase A for 10 min; 100% phase A → 68% phase A, 32% phase B within 15 min; 68% phase A, 32% phase B for 10 min; → 100% phase A;

[0062] Flow rate: 1.0 mL / min; Detection wavelength: 278 nm;

[0063] Injection volume: 10 μL; column temperature: 35°C;

[0064] Chromatographic column: Shimnex CS C18 (250×4.6 mm, 5 μm, Shimadzu, Japan).

[0065] Scanning electron microscopy observation:

[0066] The surface morphology of chitosan nanoparticles was observed using a scanning electron microscope. The sample to be tested was attached to a sample plate with conductive adhesive, and then gold was sputtered onto the sample using an ion sputtering instrument (gold plating time was 30 seconds). The sample was then observed under a scanning electron microscope.

[0067] Storage stability of nanoparticles:

[0068] The nanoparticle solutions were stored at 25°C for 0, 3, 6, 9, 12 and 15 days, respectively. The changes in particle size distribution and surface potential of EGCG@CS, EGCG@FCS and EGCG@FGC after storage for different time periods were determined by Malvern laser particle size analyzer.

[0069] In vitro cellular uptake of nanoparticles:

[0070] (1) Preparation of coumarin-6 loaded chitosan nanoparticles: Under 600 rpm magnetic stirring, 0.20 mg / mL coumarin-6 solution was added dropwise to 1.50 mg / mL solutions of different chitosan-based compounds. After the reaction for 15 min, an appropriate amount of 0.80 mg / mL TPP solution was added to the mixed solution. The mixture was reacted at room temperature in the dark for 45 min to obtain chitosan-coumarin-6 nanoparticles.

[0071] (2) Ultrafiltration concentration: The chitosan-coumarin 6 nanoparticles after reaction were ultrafiltered through a 10 kDa ultrafiltration tube to remove free coumarin 6, and the solution was concentrated to 5 mg / mL.

[0072] (3) Cell plating: HEK293T-SGLT1 cells were digested with 1 mL of trypsin for 0.5 min. 2 mL of complete culture medium was added to terminate the digestion. The cells were then pipetted and the cell suspension was centrifuged at 300 g for 3 min. The supernatant was discarded and the cells were resuspended in 3 mL of complete culture medium. 50 μL of the cell suspension was transferred to a laser confocal microplate dish. 1 mL of complete culture medium was added to the dish and pipetted evenly. The dish was then placed in a 37°C CO2 cell culture incubator and cultured overnight.

[0073] (4) Sample addition: 10 μL of coumarin-6 loaded nanoparticles were added to the confocal culture dish containing HEK 293T-SGLT1 cells, and the dish was placed in a 37°C CO2 cell culture incubator for 2 hours.

[0074] (5) Staining: Discard the culture medium and wash the cells twice with PBS. After the cells are fixed, add 100 μL of DAPI staining solution to the confocal dish and stain in the dark at 37°C for 10 min. After staining, wash the cells twice with PBS to remove excess staining solution.

[0075] (6) After washing, 100 μL of PBS was added to the laser confocal microscope, and the endocytosis of the nanoparticles by HEK293T-SGLT1 cells was observed using a laser confocal microscope.

[0076] Adhesion and penetration of chitosan nanoparticles in the intestine

[0077] (1) Preparation of coumarin-6 loaded chitosan nanoparticles;

[0078] (2) Sampling: Each mouse was gavaged with 300 μL of the solution. After fasting, the mice were gavaged and, 8 hours later, the mice with the corresponding numbers were killed. The abdomen of the mouse was cut open, the intestine was removed, and placed in a 15 cm culture dish containing 10 mL of cold sterile PBS. The distal part of the intestine was carefully grasped with the help of forceps, and then flushed with 5 mL of cold sterile PBS using a needle connected to a 5 mL syringe to remove feces. The cleaned intestine was placed in a new PBS culture dish. The intestinal tissue was fixed in 4% paraformaldehyde overnight.

[0079] (3) OCT embedding: squeeze an appropriate amount of OCT embedding medium into the large end of a rubber-tipped dropper; use clean tweezers to place the tissue in the middle of the OCT container, and then freeze the container in liquid nitrogen to form a block; after the frozen block is formed, it can be placed in a constant temperature box microtome for frozen sectioning;

[0080] (4) Fixation: Take the OCT tissue block out of the large end of the glue-tipped dropper, apply a layer of OCT embedding glue on the sample holder, place the OCT block containing the tissue on it, and place it in the microtome for freezing and fixation;

[0081] (5) Sectioning: When sectioning, the position and angle of the anti-roll plate should be appropriate, and the blade and tissue should be kept cool; gently attach the tissue slices with a positively charged glass slide; wash with PBS to remove the residual OCT on the slices; add 1 drop of mounting medium containing DAPI, cover with a coverslip, and observe under a laser confocal microscope.

[0082] Construction of mouse ulcerative colitis model and experimental grouping design:

[0083] Experimental Animals: SPF-grade C57BL / 6J mice (male, 7 weeks old, weighing 20 ± 2 g) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. and housed in an SPF-grade animal room at the Experimental Animal Center of Anhui Agricultural University. The room maintained a constant temperature (22 ± 1°C) and humidity (50 ± 5%). The lighting cycle varied day and night (8:00 am–8:00 pm), accurately simulating a 12-h day and night. The mice had free access to food and water. All experimental and operational procedures complied with relevant animal ethics regulations and were approved by the Animal Experimentation Ethics Committee of Anhui Agricultural University.

[0084] Experimental grouping and material collection: Before the experiment began, mice were acclimatized in the breeding room for 1 week and randomly divided into groups according to their weight, with 10 mice in each group and 5 mice in each cage. DSS induction method: During the experiment, the control group drank distilled water all the time, while the other groups drank 3% DSS solution freely for 7 days to induce an acute ulcerative colitis model. After the model was successfully established, the DSS solution was replaced with distilled water. Starting on the 7th day, the mice were gavaged once a day for one week at a dose of 5 mg / kg. All C57BL / 6J male mice were kept for 2 weeks. The weight and feces of the mice were recorded at the same time every day, and the changes in the mice were recorded and plotted. Figure 3 After the experiment, plasma, liver, kidney, spleen, colon tissue, colon contents, etc. were collected for subsequent experimental analysis.

[0085] Disease Activity Index Score and Colon Length Measurement:

[0086] The weight, fecal formation and fecal bleeding of the mice were scored according to the table below to calculate the disease activity index (DAI).

[0087] DAI scoring criteria:

[0088]

[0089] Measurement of mouse colon length: The length of the mouse colon after dissection was measured with a ruler, and the average value of each group was calculated, and then relevant statistical analysis was performed.

[0090] Mouse colon hematoxylin & eosin (H&E) staining and tissue evaluation:

[0091] (1) Tissue dehydration: After immersion and fixation in 4% paraformaldehyde solution for 24 h, the mouse colon tissue was taken, cleaned, and cut into appropriate pieces. Then, it was wrapped with gauze and placed in an embedding box and rinsed with running water for several hours or overnight to wash away the fixative. The washed tissue was then dehydrated step by step with 70-100% alcohol (alcohol stepwise dehydration steps: 70% ethanol soaking for 3 h → 80% ethanol soaking for 1 h → 90% ethanol soaking for 30 min → 95% ethanol I for 30 min → 95% ethanol II for 30 min → 100% ethanol I for 30 min → 100% ethanol II for 20 min);

[0092] (2) Transparent treatment: soak in 1 / 2 xylene for 20 min (the volume ratio of anhydrous ethanol to xylene is 1:1) → soak in xylene I for 20 min → soak in xylene II for 10 min;

[0093] (3) Melt the wax block to remove xylene: Place the transparent tissue in wax tanks and soak it: wax tank I (60℃) for 1 hour → wax tank II (60℃) for 1 hour → wax tank III (60℃) for 1 hour;

[0094] (4) Embedding: Fold the embedding box and embed the tissue in a paraffin embedding machine. Cool on ice and then trim the sections.

[0095] (5) Sectioning and spreading: Place the fixed and trimmed paraffin block on the clamping table of the microtome, fix the microtome knife on the knife clamp, with the blade facing up and the blade about 15 degrees to the paraffin slice, adjust the thickness regulator to the required slice thickness, usually 6-8μm, and start slicing. Then put the cut wax slice into a water bath at 42℃ and spread it. After it is fully spread, take another clean glass slide to pick up the spread slice; after cutting, place it on a clean glass slide, and then bake it in a 42℃ baking machine for 120 minutes;

[0096] (6) Dewaxing and rehydration: Place the cut tissue sections in an oven (60°C, 1 hour) to melt the paraffin. Soak the slides in xylene I for 10 minutes → xylene II for 5 minutes → xylene III for 5 minutes. Then soak the slides in 100-70% alcohol in a step-by-step manner (100% ethanol I for 5 minutes → 100% ethanol II for 5 minutes → 95% ethanol I for 5 minutes → 95% ethanol II for 5 minutes → 80% ethanol for 5 minutes → 70% ethanol for 5 minutes → water). Place the slides in distilled water before staining.

[0097] (7) HE kit staining: Soak in hematoxylin dye for 2 minutes, at which point the entire tissue section turns purple, and then wash thoroughly with water for 1 minute; then soak in differentiation solution for 10 seconds, and the tissue section will be differentiated into purple-red by the acidic differentiation solution; then wash with water for 7 minutes to wash off the excess dye, because the pH value of water is higher than that of the differentiation solution and the tissue section turns blue again, and rinse until the tissue section turns light blue; finally soak in eosin dye for 1 minute and 30 seconds, wash with water several times to remove the excess dye, and then dehydrate quickly;

[0098] (8) Dehydration: Dehydration by immersion in 85-100% alcohol in a step-by-step manner (85% ethanol soaking for 20 seconds → 95% ethanol soaking for 30 seconds → 100% ethanol I soaking for 1 minute → 100% ethanol II soaking for 1 minute → xylene I soaking for 1 minute → xylene II soaking for 1 minute → xylene III soaking for 1 minute);

[0099] (9) Sealing: Ultrasonicate the neutral resin in advance, add a drop of neutral resin sealing agent, and cover with a cover glass;

[0100] (10) Observation: After the slides are completely air-dried, they are observed and recorded under an optical microscope at a magnification of 40 / 100.

[0101] Alcian blue staining of colonic mucin:

[0102] The distribution of mucin in the colon tissue of mice in each experimental group was observed using Alcian blue staining. The specific steps are as follows:

[0103] The steps of paraffin embedding, dewaxing and rehydration, dehydration, and sealing of mouse colon were the same as described above.

[0104] The specific steps of Alcian blue staining include:

[0105] (1) Dewax and rehydrate the colon tissue slides;

[0106] (2) Soak the rehydrated slides in Alcian blue acidified solution for 3 minutes;

[0107] (3) Then place in Alcian staining solution for 30 minutes and rinse with running water;

[0108] (4) Then, place the slide in Nuclear Fast Red staining solution for 5 minutes and rinse with running water for 1 minute to remove excess staining solution.

[0109] (5) Finally, dehydrate with gradient ethanol, make transparent with xylene, and seal with neutral gum.

[0110] Data analysis and processing methods:

[0111] Data were analyzed and plotted using GraphPad Prism 9. *p < 0.05 indicated a significant difference, and **p < 0.01 and ***p < 0.001 indicated an extremely significant difference.

[0112] Determination of amino substitution degree:

[0113] The absorbance (y) was linearly regressed against the chitosan concentration (x). In the range of 0-0.40 mg / mL, a good linear curve was obtained, and the regression equation was: y = 0.4299x + 0.0738, R 2 =0.9953. Where: y is the absorbance of the solution; x is the concentration of chitosan in the solution.

[0114] Fluorinated chitosan and galactosyl fluorinated chitosan solutions were used instead of chitosan standard solution, and the absorbance was measured at 570 nm. According to the standard curve, the amino substitution degree of fluorinated chitosan was calculated to be 20.72±0.51%. Based on the fluorinated chitosan, galactosyl was grafted onto chitosan, and the galactosyl substitution degree of galactosyl fluorinated chitosan was calculated to be 19.53±0.30%.

[0115] Amino substitution degree of fluorinated chitosan and galactosyl fluorinated chitosan

[0116] Sample name Abbreviation Fluorination substitution degree Galactosyl substitution degree Fluorinated chitosan FCS 20.72±0.51 / Galactosyl fluorinated chitosan FGC 20.72±0.51 19.53±0.30

[0117] Note: Data are expressed as mean ± standard deviation (n = 3).

[0118] NMR Characterization of Galactosyl Fluorinated Chitosan

[0119] pass 1 The structures of chitosan, fluorinated chitosan and galactosyl fluorinated chitosan were analyzed by H NMR. Figure 4 As shown, it shows that the galactose group has been successfully grafted onto the chitosan molecule.

[0120] The reaction mechanism of chitosan and heptafluorobutyric acid to synthesize fluorinated chitosan is as follows Figure 1 As shown. EDC-HCl first reacts with the carboxyl group (-COOH) of heptafluorobutyric acid to generate an unstable intermediate through nucleophilic addition. The intermediate reacts with NHS to generate NHS ester with higher activity. Finally, the NHS-activated ester reacts with the primary amino group (-NH2) of chitosan to form an amide bond, thereby obtaining fluorinated chitosan (F7CS). On this basis, we prepared galactosyl fluorinated chitosan (FGC) by the condensation reaction between the carboxyl group (-COOH) of lactobionic acid and the primary amino group (-NH2) on fluorinated chitosan. By 19 F NMR confirmed the successful modification of the fluorinated groups ( Figure 5 ).

[0121] Characterization of the physical and chemical properties of nanoparticles:

[0122] Construction and characterization of EGCG@FGC nanoparticles:

[0123] Fluorine, the most electronegative element, significantly influences the conformation and physicochemical properties of organic compounds. Furthermore, incorporation of fluorine atoms into ligands can enhance ligand-ligand interactions, making it a useful tool for optimizing the activity of drugs or naturally occurring active small molecules. As shown in the table below, the addition of fluorinated ligands reduced the average particle size of EGCG nanoparticles from 240.30 nm to 213.57 nm, the PDI value from 0.206 to 0.120, the potential from 33.31 mV to 28.24 mV, and the loading rate from 89.13% to 91.48%. Introducing galactosyl ligands into fluorinated chitosan reduced the average nanoparticle size from 213.57 nm to 192.73 nm, while maintaining no significant changes in the PDI value or loading rate. These results indicate that the interaction between galactosyl fluorinated chitosan and EGCG is enhanced, resulting in a gradual decrease in nanoparticle size and a more uniform size distribution as the loading rate increases.

[0124] Characterization of EGCG nanoparticles loaded with chitosan modified with different ligands:

[0125]

[0126] Next, we observed the surface morphology of EGCG@CS, EGCG@FCS, and EGCG@FGC nanoparticles by scanning electron microscopy. Figure 6 The results showed that EGCG@CS, EGCG@FCS and EGCG@FGC nanoparticles all had a uniform spherical morphology.

[0127] Storage stability of EGCG@FGC nanoparticles:

[0128] Since EGCG has poor stability in solution when exposed to the environment, using chitosan nanoparticles to load EGCG can enhance its stability while maintaining its natural structure. Therefore, the stability of nanoparticles is also crucial to the stability of EGCG. Therefore, we conducted a storage stability experiment on EGCG@FGC. At 25°C, the nanoparticle solution was stored for 0, 3, 6, 9, 12 and 15 days respectively. The particle size potential changes of EGCG@FGC after storage for different time periods were measured using a Malvern laser particle size analyzer. Figure 7 As shown in the figure, after 15 days of storage, the average particle size, PDI and surface potential of EGCG@FGC nanoparticles did not change significantly, showing good stability.

[0129] Uptake of galactosylfluorinated chitosan nanoparticles by HEK293T-SGLT1 cells:

[0130] Coumarin-6 can be used as a fluorescent probe for in vivo tracking, cellular uptake, and mechanism studies of microparticle drug delivery systems. This example uses coumarin-6 as a model drug in a chitosan nanoparticle delivery system to explore the uptake capacity of HEK293T-SGLT1 cells and the small intestine for galactosylfluorinated chitosan nanoparticles. Laser confocal microscopy was used to obtain images of HEK293T-SGLT1 cells taking up nanoparticles loaded with coumarin-6. Figure 8 As shown in the figure, after 2 h of incubation, the intensity of green fluorescence in the cytoplasm of the Coumarin-6@FGC group was significantly higher than that of the Coumarin-6@CS and Coumarin-6@FCS groups, indicating that HEK293T-SGLT1 cells had a high absorption capacity of Coumarin-6@FGC nanoparticles.

[0131] Internalization of galactosylfluorinated chitosan nanoparticles in the small intestine:

[0132] In order to track the absorption of nanoparticles in the small intestine after oral administration, laser confocal microscopy was used to observe the internalization of coumarin 6-loaded nanoparticles in intestinal cells. Figure 9 As shown, 8 hours after oral administration, the absorption of coumarin 6 in the villus cavity increased. In addition, during the same period, the coumarin 6-loaded nanoparticle group exhibited stronger fluorescence intensity than the coumarin 6 alone group, while the fluorescence intensity of the Coumarin-6@CS and Coumarin-6@FCS groups was weaker than that of the Coumarin-6@FGC group during the same period, indicating that the small intestine has a higher absorption rate of Coumarin-6@FGC nanoparticles. This is attributed to the enhanced ability to overcome some oral barriers with the help of fluorinated groups and galactose groups. Among them, grafting fluorinated groups on the chitosan surface can enhance the affinity of chitosan with cell membranes, making it easier to pass through cell membranes; in addition, galactose surface modification gives the nanoparticles a high affinity and specificity for intestinal epithelial cells, which can promote their transport across the absorptive epithelium through cell action or carrier-mediated transport, thereby increasing the amount of Coumarin-6@FGC internalized into the epithelium.

[0133] Animal-level evaluation of EGCG@FGC nanoparticles for efficient oral absorption of EGCG:

[0134] EGCG@FGC nanoparticles alleviate body weight changes in mice with ulcerative colitis:

[0135] In order to investigate the protective effect of chitosan modified with different ligands on DSS-induced ulcerative colitis mice, mice were given 3% DSS for 7 days and weighed every day. When there was no significant difference in the amount of 3% DSS drinking water, the weight of mice in the CK group showed a steady upward trend, while the weight of mice in the model group decreased significantly compared with the CK group. Figure 10 As shown in the results, compared with the DSS group, EGCG, EGCG@CS, EGCG@FCS and EGCG@FGC groups could improve the weight loss of mice caused by ulcerative colitis, among which oral EGCG@FGC intervention significantly reduced the body weight of mice with DSS-induced ulcerative colitis.

[0136] Effects of EGCG@FGC nanoparticles on DAI changes in ulcerative colitis mice:

[0137] DAI is an important evaluation method for evaluating the severity of ulcerative colitis. It evaluates the degree of weight loss, stool formation and blood in the stool of mice, and takes the sum of the three indicators to obtain DAI. When mice were given 3% DSS to induce free drinking for 7 days, the weight, stool viscosity and fecal bleeding of the mice were recorded every day. It was found that compared with the CK group, the weight loss percentage of mice in the modeling group was greater than 15%, and there were thin liquid stools and gross blood in the stool. The comprehensive combination of the three indicators showed that the DAI was higher. Starting from the 7th day, the mice were gavage treated. Compared with the DSS group, the EGCG, EGCG@CS, EGCG@FCS and EGCG@FGC gavage groups could reduce the DAI scores. At the same time, the symptoms of thin liquid stools and gross blood in the stools of the three groups of mice, EGCG@CS, EGCG@FCS and EGCG@FGC, were also significantly improved. Among them, the DAI score of the EGCG@FGC group was significantly reduced, reflecting a better intervention effect ( Figure 11 ).

[0138] Effects of EGCG@FGC nanoparticle intervention on colon length in mice with ulcerative colitis:

[0139] like Figure 12As shown in the results, colon shortening is an important evaluation indicator of colitis in mice. By measuring the colon length of each group of mice, it was found that after the mice were given 3% DSS to induce free drinking for 7 days and after one week of treatment, the colon length of the DSS group was significantly reduced by 39.38% compared with the CK group; after oral EGCG intervention, the colon length in the EGCG group increased by 7.87% compared with the DSS group; the colon length of the EGCG@CS group, EGCG@FCS group and EGCG@FGC group increased by 19.69%, 31.89% and 49.61% respectively compared with the DSS group; compared with the EGCG group, the colon length of the EGCG@CS group, EGCG@FCS group and EGCG@FGC group increased by 10.95%, 22.26% and 38.69% respectively. Furthermore, the colon lengths of the EGCG@FCS and EGCG@FGC groups increased by 10.20% and 25.00%, respectively, compared to the EGCG@CS group. The colon length of the EGCG@FGC group also increased by 13.43% compared to the EGCG@FCS group. In summary, intervention with different nanoparticles significantly improved the symptoms of shortened colon length in DSS-induced ulcerative colitis mice, with EGCG@FGC nanoparticles demonstrating a superior intervention effect.

[0140] Effects of EGCG@FGC nanoparticles intervention on the pathological morphology of colon tissue in mice with ulcerative colitis:

[0141] The severity of colonic injury in mice after drug treatment was assessed by hematoxylin-eosin (H&E) staining of colon tissue. Figure 13 As shown, the colonic tissue of mice in the CK group showed no signs of inflammation, and the intestinal tissue morphology was intact. Compared with the CK group, the structural integrity of the colonic mucosa of mice in the DSS group was damaged, with a significant loss of glands, destruction of intestinal crypt structure, desquamation of intestinal epithelial cells, and a large number of inflammatory cell infiltrations. Compared with the colonic damage in the DSS group, the EGCG group had little improvement. The symptoms of colonic mucosal damage in the nanoparticle group were improved, with relief of glandular loss, mucosal ulceration, and inflammatory cell infiltration. In particular, the intestinal mucosa in the EGCG@FGC group was relatively intact after treatment, and inflammatory cell infiltration was significantly reduced.

[0142] Effects of EGCG@FGC nanoparticle intervention on colonic mucus changes in mice with ulcerative colitis:

[0143] Goblet cells can secrete mucus, which can lubricate and protect the intestinal epithelium. The mucin in the mucus can be stained blue by Alcian blue dye, which is used to judge the integrity of the colon mucus layer. Figure 14The images show the changes in mucus in the mouse colon tissue observed using Alcian blue staining at 40× and 100× magnifications. As can be seen, goblet cells in the DSS group were significantly reduced compared to the CK group, and their size varied, indicating that DSS treatment damaged the intestinal mucosal structure. The images also show that the nanoparticle group effectively alleviated colitis-induced goblet cell loss and intestinal mucosal damage. However, the EGCG@FGC group was significantly more effective in alleviating colitis than the EGCG@CS and EGCG@FCS groups.

[0144] EGCG has many excellent properties such as anti-tumor, anti-inflammatory, and antioxidant, but its low oral bioavailability and poor stability seriously hinder its efficiency in oral delivery. Therefore, we used nanoparticles formed by self-assembly of galactosyl fluorinated chitosan and sodium tripolyphosphate to construct a small intestine absorption delivery vehicle to achieve gastrointestinal stability, retention, and permeability. The oral delivery system of EGCG using galactosyl fluorinated chitosan nanoparticles was analyzed, and the alleviating effect of the galactosyl fluorinated chitosan-EGCG delivery system on ulcerative colitis was investigated using a mouse model. The main research results are as follows:

[0145] 1. Fluorinated chitosan was successfully synthesized by the amidation reaction between the carboxyl group of heptafluorobutyric acid and the amino group of chitosan. Based on this, galactosyl fluorinated chitosan was prepared by reacting with lactobionic acid. The fluorinated substitution degree of fluorinated chitosan was measured by ninhydrin reaction and was 20.72±0.51%. The galactosyl substitution degree of fluorinated chitosan was 19.53±0.30%. 19 FNMR and 1 H NMR analysis demonstrated that both fluorinated groups and galactosyl functional groups were successfully grafted onto the chitosan molecules.

[0146] EGCG-loaded galactosyl fluorinated chitosan nanoparticles (EGCG@FGC) were successfully prepared using an ionotropic gelation method. When the mass ratio of galactosyl fluorinated chitosan to EGCG was 15:1, the EGCG@FGC nanoparticles exhibited a particle size of 192.73±6.08 nm, a PDI of 0.122±0.005, a zeta potential of 25.89±0.47 mV, and a loading rate of 91.61±0.11%. Scanning electron microscopy revealed that the EGCG-loaded galactosyl fluorinated chitosan nanoparticles exhibited a uniform spherical morphology. Furthermore, the nanoparticles exhibited good stability after storage at 25°C for 15 days.

[0147] 3. SGLT1 receptor can mediate the uptake efficiency of EGCG@FGC nanoparticles by HEK293T-SGLT1 cells and significantly enhance the permeability of EGCG@FGC nanoparticles in the small intestinal mucosa.

[0148] 4. Using dextran sulfate sodium salt-induced ulcerative colitis in mice as an inflammatory model, the ability of EGCG-loaded galactosylfluorinated chitosan nanoparticles to alleviate ulcerative colitis was evaluated. The results showed that EGCG@FGC nanoparticles could alleviate weight loss, bloody diarrhea, and shortened colon length in mice with ulcerative colitis, with EGCG@FGC nanoparticles showing superior improvement compared to EGCG@CS and EGCG@FCS nanoparticles. Further analysis of colon tissue by hematoxylin & eosin staining and Alcian blue staining showed that EGCG@FGC nanoparticles could increase colonic crypt depth and mucin secretion in mice with ulcerative colitis, while reducing inflammatory cell infiltration, thereby alleviating the degree of intestinal mucosal damage.

[0149] In summary, the construction of EGCG-loaded galactosylfluorinated chitosan nanoparticle oral delivery system can improve the stability and bioavailability of EGCG in the gastrointestinal tract, thereby achieving effective anti-inflammatory activity.

[0150] The above description is only used to explain the preferred embodiments of the present invention and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A galactosyl fluorinated chitosan-EGCG nanoparticle, characterized by: The fluorination modification degree is 15%-30%; the galactosyl modification degree is 15%-25%.

2. The method for preparing galactosyl fluorinated chitosan-EGCG nanoparticles according to claim 1 or 2, characterized in that: The following steps are involved: Step 1: covalently bond the carboxyl group of heptafluorobutyric acid to the amino group of chitosan to prepare fluorinated chitosan; Step 2: synthesizing galactosyl-modified fluorinated chitosan by amidation reaction between lactobionic acid and amino groups of fluorinated chitosan; Step 3: Prepare galactosyl-modified fluorinated chitosan nanoparticles loaded with tea polyphenol EGCG by sodium tripolyphosphate ion cross-linking method.

3. The method for preparing galactosyl fluorinated chitosan-EGCG nanoparticles according to claim 3, wherein: The loading amount of the tea polyphenol EGCG is 5%-15%.

4. Use of the galactosylfluorinated chitosan-EGCG nanoparticles according to claim 1 or 2 in improving the bioavailability of tea polyphenol EGCG.

5. Use of the galactosyl fluorinated chitosan-EGCG nanoparticles according to claim 1 or 2 in the preparation of a medicament for alleviating ulcerative colitis.

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