Preparation method and application of delivery system for regulating intestinal microecology based on resveratrol loaded on ginger exosome

Abstract

The invention discloses a preparation method and application of a delivery system for regulating and controlling intestinal microecology based on resveratrol loaded on ginger exosome, and relates to the technical field of biomedicine. The turmeric nano-vesicles are extracted from turmeric plant species through an ultracentrifugation method, and resveratrol is loaded into the turmeric nano-vesicles through an ultrasonic method. The turmeric nano-vesicle loaded with resveratrol combines the characteristics of resveratrol and turmeric nano-vesicles, is stable in structure, can be efficiently enriched at a colitis disease part, reduces pro-inflammatory factors at the inflammation part, removes peroxides at the ulcerative colitis inflammation part, and has a remarkable treatment effect on ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a preparation method and application of a hybrid vesicle orally loaded with STA-5326. Background Art

[0002] Inflammatory bowel disease (IBD) is a digestive disorder characterized by chronic, recurrent intestinal inflammation. The two main categories of IBD are Crohn's disease (CD) and ulcerative colitis (UC). Although the exact cause of IBD is not yet clear, it is generally believed to result from a combination of factors such as genetic susceptibility, immune system dysregulation, environmental influences, and intestinal microbiota imbalance. Current medical treatments for IBD include aminosalicylates, corticosteroids, immunosuppressants, biologics (such as anti-tumor necrosis factor agents), and emerging small molecule targeted therapies. However, there are still challenges in terms of drug tolerance and treatment efficacy, highlighting the need for therapies that can improve the treatment effect of IBD patients while reducing side effects.

[0003] Turmeric contains the active ingredient curcumin, which has attracted significant attention in the medical field due to its diverse biological activities. Curcumin is used for the prevention and treatment of various diseases by virtue of its potent anti-inflammatory, anti-tumor, and neuroprotective properties. Turmeric-derived nanovesicles (TNV), similar to extracellular vesicles, generally have a diameter between 20 and 150 nanometers and have many advantages over traditional drug carriers, such as low toxicity, minimal immunogenicity, excellent biocompatibility, and biodegradability. They can also directly deliver therapeutic agents to the lesion area through targeted biomolecular action, thereby enhancing the treatment effect. However, relevant literature shows that TNV can mainly reduce the intestinal inflammatory factors induced by dextran sulfate (DSS) in the ulcerative colitis model, but cannot effectively reduce the accumulation of peroxides in the intestine.

[0004] Resveratrol (RSV), a polyphenolic compound present in many plants, has attracted attention due to its biological activities and potential health benefits, particularly its antioxidant and free radical scavenging properties. Studies have shown that RSV scavenges free radicals through multiple mechanisms, including directly binding to them to stabilize these molecules and reduce cell damage. In addition, the anti-inflammatory, anti-tumor, and neuroprotective effects exhibited by RSV are closely related to its antioxidant ability. Moreover, significant gastrointestinal side effects may occur when RSV is taken orally, which poses a problem for patients with ulcerative colitis. Summary of the Invention

[0005] The objective of the present invention is to provide a preparation method and application of TNV loaded with RSV, so as to solve the problems existing in the above-mentioned prior art. The present invention attempts to use pulsed ultrasound to load RSV into TNV, thereby minimizing the gastrointestinal reactions associated with RSV, while synergistically enhancing the anti-inflammatory and free radical scavenging characteristics of combination therapy, and ultimately improving the efficacy in treating ulcerative colitis-related diseases.

[0006] Based on this, the present invention provides the following solutions:

[0007] The present invention provides a TNV loaded with RSV, and the TNV is prepared by gradient centrifugation.

[0008] The present invention also provides a preparation method of the above-mentioned TNV loaded with RSV, wherein RSV is loaded into TNV by ultrasonic treatment to obtain the TNV loaded with RSV.

[0009] The present invention also provides the application of the above-mentioned TNV loaded with RSV in the treatment of ulcerative colitis.

[0010] The present invention discloses the following technical effects:

[0011] 1) In the present invention, TNV can better stay in the intestinal inflammation site in ulcerative colitis for a long time, enabling the TNV loaded with RSV to exert its function for a longer time.

[0012] 2) The method for loading RSV in the present invention is simple, efficient, and easy to operate, and the formed structure is stable and uniform.

[0013] 3) The TNV loaded with RSV in the present invention can reduce the contents of peroxides and pro-inflammatory factors in the intestinal inflammation site in ulcerative colitis. Brief Description of the Drawings

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0015] Figure 1 It is the morphology and particle size statistics of TNV in the present invention; wherein: A is the transmission electron micrograph of TNV; B is the particle size distribution diagram of TNV.

[0016] Figure 2 It is the drug loading amount and loading capacity analysis of TNV@RSV.

[0017] Figure 3Particle size statistical chart of TNV@RSV within 7 days.

[0018] Figure 4 Analysis of characteristic peak of ESR spectrum of RSV and TNV@RSV.

[0019] Figure 5 Analysis of the uptake ability of macrophages to TNV and TNV@RSV.

[0020] Figure 6 Evaluation of the uptake ability of macrophages to TNV@RSV under different conditions.

[0021] Figure 7 Analysis of the ability of TNV@RSV to scavenge peroxides.

[0022] Figure 8 Analysis of the ability of TNV@RSV to reduce inflammatory factors.

[0023] Figure 9 Analysis of the enrichment of different components in healthy mice and model mice in vivo.

[0024] Figure 10 Analysis of the enrichment of different components in healthy mice and model mice in ex vivo organs.

[0025] Figure 11 Schematic diagram of the experiment for treating acute ulcerative colitis in mice. The groups are blank control PBS group, DSS model group, TNV, RSV, and TNV@RSV treatment groups.

[0026] Figure 12 Colon images of different administration groups.

[0027] Figure 13 Statistics of colon length, analysis of body weight and DAI changes within 9 days for different administration groups. Detailed implementation mode

[0028] Now, various exemplary implementation modes of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation modes of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing specific implementation modes and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0030] Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention has been described only in terms of preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0031] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which will be apparent to those skilled in the art. Other embodiments obtained from the description of the present invention will be apparent to those skilled in the art. The description of the present invention and the examples are merely exemplary.

[0032] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0033] Example 1 Preparation and Characterization of TNV

[0034] 1. Preparation of TNV

[0035] 1.1 Physically decompose turmeric rhizomes using a juicer to collect the supernatant.

[0036] 1.2 Adopt gradient centrifugation method, centrifuge at 1000g for 10 min, 2000g for 20 min, 3000g for 30 min, 10000g for 60 min, 150000g for 90 min, resuspend the precipitate with PBS to obtain TNV.

[0037] 1.3 After suspension in PBS, quantitatively analyze TNV using the Micro BCA Protein Assay Kit.

[0038] 2. Transmission Electron Microscopy Observation

[0039] Take TNV and TNV@RSV resuspended in PBS, respectively take 10 μL and spot it on the copper grid, precipitate for 5 min, suck off the excess liquid with filter paper, then negatively stain with uranyl acetate for 30 s, suck off the excess liquid with filter paper, air dry naturally for 10 min, and observe the morphology of the vesicles on a transmission electron microscope at 120 kV. The results are as Figure 1 shown in A.

[0040] 3. Measurement of Particle Size

[0041] The particle size of the vesicles was measured using a dynamic light scattering particle size analyzer: Take 1 mL of TNV with a concentration greater than 200 μg / mL, blow it evenly and put it into the detection cell of the dynamic light scattering particle size analyzer for on-machine detection. The results are as Figure 1 shown in B. TNV is evenly distributed at 100 nm.

[0042] Preparation and Characterization of TNV@RSV in Example 2

[0043] 1. Preparation of TNV@RSV

[0044] 1.1 Using the TNV prepared in Example 1, prepare 3 TNV solutions with the same protein concentration (1 mg / mL), add 100 μL to each ep tube, and label them as tubes 1-3 respectively. Add solutions containing 50 μg, 100 μg, and 200 μg of RSV to each tube.

[0045] 1.2 Pulse ultrasonic treatment of the mixed solution using an ultrasonic cleaner. The specific operation is as follows: Insert the ep tube into the floating buoy to make it suspended in the ultrasonic cleaner. Adjust the ultrasonic power to 40 kHz and the temperature to 4 °C. Perform pulsed ultrasound with on / off for 3 min each, repeat 5 times, and then take it out.

[0046] 1.3 After the ultrasound is completed, put the ep tube into a water bath thermostat, adjust the temperature to 37 °C, and incubate for 1 h to restore the membrane stability.

[0047] 1.4 After the water bath thermostat incubation is completed, transfer the mixed solution of the drug and hybrid exosomes to a 100 kD ultrafiltration tube, centrifuge at 4,500 g for 15 min, and ultrafilter the excess drug molecules.

[0048] 2. Determination of the drug loading of TNV@RSV

[0049] The drug loading of RSV was measured using a UV spectrophotometer:

[0050] 2.1 Dissolve RSV in ethanol to prepare standard solutions with concentrations of 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL respectively.

[0051] 2.2 Determination of the drug loading

[0052] Put the filtrate of TNV@RSV after ultrafiltration in step 1.4 into a cuvette and detect its UV absorption peak value. The results show that when the mass ratio of RSV to TNV is 5:1, 5:2, 1:1, and 1:2, the drug loadings are 10%, 15%, 40%, and 35% respectively. As the drug concentration increases, the drug loading increases. When the mass ratio is 1:1, the maximum drug loading and encapsulation rate are achieved. Therefore, the subsequent experiments were carried out with the mass ratio of RSV to TNV being 1:1.

[0053] 3. TNV@RSV Stability Detection

[0054] Disperse TNV@RSV in PBS and measure its particle size every day. As Figure 3 shown, the particle size of TNV@RSV is 100 nm within 7 days, demonstrating good stability of TNV@RSV, which is beneficial for storage.

[0055] Example 3 Evaluation of the Free Radical Scavenging Ability of TNV@RSV

[0056] Generate hydroxyl radicals (·OH) by co-culturing 25 μL H2O2 (0.315 mM) with 70 μL FeCl2 (0.735 mM), and capture them with 100 μM 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Nitrogen radicals are generated by ultraviolet irradiation of S-nitroso-N-acetylpenicillamine (SNAP, 1.125 mM), and captured with 50 μM 2-diethoxyphosphoryl-2-methyl-1-oxo-3,4-dihydropyrrole-1-ium (DEPMPO). To evaluate the free radical scavenging ability, add TNV@RSV or RSV (equal amount of RSV) to the mixture, and analyze the resulting solution using an electron spin resonance (ESR) spectrometer (Bruker E500). The change in the amplitude of the characteristic peak of the ESR spectrum indicates the scavenging effect.

[0057] Example 4 Evaluation of Cell Uptake Ability

[0058] 1. Label TNV and TNV@RSV with DiI (10 μM) at 37 °C for 30 min. Remove free DiI using a NAP-5 chromatography column according to the manufacturer's instructions. To evaluate cell uptake behavior, seed Raw 264.7 at a density of 10 4 cells / well in a confocal dish and incubate for 24 h under normal (without LPS) or inflammatory (1 μg / mL LPS) conditions. Then add DiI-labeled TNV or TNV@RSV (25 μg / mL) to the cells and incubate for an additional 12 h. Fix the treated cells with 4% paraformaldehyde at room temperature for 15 min. After incubating with Hoechst 33342 for 10 min, image the cells using a confocal laser scanning microscope (CLSM).

[0059] 2. Label TNV and TNV@RSV with DiI (10 μM) at 37 °C for 30 min. Remove free DiI using a NAP-5 chromatography column according to the manufacturer's instructions. To evaluate cell uptake behavior, seed Raw 264.7 at a density of 10 5Cells were seeded in 24-well plates at a density of

[0060] Example 5 In vitro ROS Scavenging and Anti-inflammatory Capability of TNV@RSV

[0061] 1. RAW 264.7 cells were cultured in 24-well plates with a culture medium containing 100 μM H2O2 and 1 μg / mL LPS (simulating the oxidative stress and inflammatory environment of UC). TNV or TNV@RSV (25 μg / mL) was added to the cells and incubated for 2 hours. The cells were collected, stained with DCFH-DA for 30 minutes, and then analyzed by flow cytometry.

[0062] 2. RAW 264.7 cells were cultured in 24-well plates with a culture medium containing 100 μM H2O2 and 1 μg / mL LPS (simulating the oxidative stress and inflammatory environment of UC). TNV or TNV@RSV (25 μg / mL) was added to the cells and incubated for 2 hours. The cell supernatants were collected, and the levels of pro-inflammatory factors IL-6, IL-12, and MPO were detected using the corresponding ELISA kits according to the instructions.

[0063] Example 6 In vivo Distribution of TNV@RSV

[0064] 1. Establishment of a mouse ulcerative colitis model

[0065] 1.1 Six 8- to 9-week-old female C57BL / 6 mice were prepared.

[0066] 1.2 UC model establishment: The mice were housed in an animal facility under specific pathogen-free conditions, and a mouse ulcerative colitis model was established by adding 3% DSS to the drinking water. The mice with ulcerative colitis were allowed to drink freely, and the drinking water was changed every 2 days. After 7 days, the water was changed to normal water without 3% DSS.

[0067] 2. Determination of targeting effect

[0068] TNV and TNV@RSV were stained with DiR and grouped into a blank Healthy group and a DSS group. The Healthy group was given TNV@RSV. The DSS mice were randomly divided into 2 groups, with 3 mice in each group, namely the TNV group and the TNV@RSV group, and the dosage was 20 mg / kg of protein concentration. In vivo fluorescence photographs were taken at 3H, 6H, 12H, and 24H after gavage. The results are as Figure 9As shown, at 24 hours, most of the drugs in the Healthy group had been metabolized, and there were still some drugs in the colon of the DSS group. The results are as Figure 10 shown. At 6 hours, the maximum amount of TNV@RSV was reached in the colon of DSS mice, which better demonstrated that TNV@RSV could be better enriched in the inflamed area.

[0069] Example 7 Therapeutic effect of TNV@RSV on UC model mice

[0070] 1. Treatment of ulcerative colitis model

[0071] 1.1 UC modeling: Mice were housed in an animal facility under specific pathogen-free conditions, and an ulcerative colitis mouse model was established by adding 3% DSS to the drinking water. The mice with ulcerative colitis were allowed to drink water freely, and the drinking water was changed every 2 days. After 7 days, the water was changed to normal water without 3% DSS.

[0072] 1.2 Mouse grouping: The mice were randomly divided into 5 groups with 6 mice in each group. The groups were blank control PBS group, DSS model group, RSV, TNV, and TNV@RSV treatment groups. The dosage was 20 mg / kg of protein concentration. The preparation methods of the oral drugs for each group were the same as those in Example 3.

[0073] 1.3 Drug administration treatment: Oral treatment was given to the mice on the 1st, 3rd, and 5th days after modeling.

[0074] 2. Recording of mouse colon length, body weight, and disease activity index score

[0075] During the treatment process, the body weight and disease activity index of the mice were measured and recorded every day. The DAI was determined by recording the body weight, stool consistency, and fecal occult blood every day. Combining the percentage of body weight loss in mice (weight unchanged is 0, 1 - 5 is 1 point, 5 - 10 is 2 points, 10 - 15 is 3 points, greater than 15 is 4 points), stool viscosity (normal is 0, loose stool is 2 points, diarrhea is 4 points), and stool bleeding (normal 0 points, occult blood positive is 2 points, overt bleeding is 4 points), a comprehensive score was made, and the total score of the 3 results was divided by 3 to obtain the statistical analysis result of the DAI value. On the 9th day, all the mice were euthanized, and the cecum and colon tissues were extracted for colon length statistics. Figure 13 As shown in A, after treatment with TNV@RSV, the colon length was closer to that of the PBS group. As Figure 13 shown in B, after treatment with TNV@RSV, the body weight increased relative to the DSS group; Figure 13 In C, the disease severity index decreased significantly in the later stage after treatment with TNV@RSV.

[0076] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A preparation method of turmeric vesicles loaded with resveratrol, characterized in that, Extract nanovesicles from turmeric rhizome tissue.

2. A loaded medicinal turmeric vesicle, characterized in that, Obtain turmeric nanovesicles from the extracted turmeric tissue by gradient centrifugation.

3. A drug-loaded turmeric nanovesicle, characterized in that, Load resveratrol into the turmeric nanovesicles to obtain drug-loaded turmeric nanovesicles.

4. Use of a drug-loaded turmeric nanovesicle in the preparation of a drug, characterized in that, The drug is used for the treatment of ulcerative colitis.

5. The preparation method according to claim 3, characterized in that, Load resveratrol into the nanovesicles of turmeric by sonication.

6. The drug-loaded turmeric nanovesicles according to claim 2, wherein The particle size of the vesicles is 100 - 200 nm.

7. The application according to claim 4, characterized in that, The drug-loaded turmeric nanovesicles can increase the length of the colon in ulcerative colitis.

8. The application according to claim 4, wherein The drug-loaded turmeric nanovesicles can reduce peroxides in the inflamed area of ulcerative colitis.

9. The application according to claim 4, characterized in that The drug-loaded turmeric nanovesicles can reduce inflammatory factors in the colon of ulcerative colitis.

10. The application according to claim 4, wherein The dosage form of the drug is an oral preparation.

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