A curcumin and anthocyanin co-delivery preparation based on functionalized probiotic extracellular vesicles and a preparation method thereof

Abstract

This invention presents a method for co-encapsulating and targeting curcumin and anthocyanins based on functionalized probiotic extracellular vesicles, achieving highly efficient oral targeted delivery of curcumin and anthocyanins. This method uses probiotic-derived extracellular vesicles as the basic carrier, both of which exhibit good biocompatibility, digestive stability, and loading capacity. Secondly, to enhance carrier function, this invention synthesizes EGCp with an amphiphilic structure and embeds it into the vesicle membrane through self-assembly, thereby endowing the carrier EGC with inherent antioxidant and anti-inflammatory activities. Furthermore, to endow the carrier system with highly efficient targeting capability against inflammatory cells, the surface of the engineered vesicles is modified with octenyl succinic anhydride grafted with hyaluronic acid to obtain a functional formulation capable of co-delivering curcumin and anthocyanins. EGCp and OSA-HA work synergistically to construct a co-delivery system that can simultaneously and efficiently load lipophilic curcumin and hydrophilic anthocyanins and actively target inflammatory sites.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, specifically relating to a curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles and its preparation method. Background Technology

[0002] Curcumin is a natural low-molecular-weight polyphenol extracted from turmeric, possessing various biological activities such as antibacterial, antioxidant, and anti-inflammatory properties. However, curcumin's poor water solubility and low chemical stability under light and physiological pH conditions severely limit its absorption and bioavailability in vivo. Anthocyanins are a class of water-soluble flavonoids widely found in berries such as blueberries and blackberries, also exhibiting various biological effects including antioxidant, anti-inflammatory, and blood glucose and lipid regulation. However, their stability is easily affected by temperature, light, pH, and digestion processes, resulting in low bioavailability. Therefore, developing an effective oral delivery system that can simultaneously improve the stability, antioxidant capacity, and bioavailability of both curcumin and anthocyanins has significant application value.

[0003] Currently, studies have employed delivery systems such as nanoliposomes, polysaccharides, and protein-polyphenol complexes to encapsulate curcumin or anthocyanins. While these methods can improve stability and bioactivity to some extent, they still have limitations such as complex preparation processes, potential cytotoxicity of synthetic materials, and limited encapsulation efficiency. In recent years, extracellular vesicles, as a naturally derived nanocarrier, have shown great potential in the field of drug delivery. Compared with synthetic liposomes, extracellular vesicles have excellent biocompatibility and low immunogenicity. However, most extracellular vesicles currently used for delivering active ingredients are derived from mammalian cells, resulting in high production costs and low yields. In addition, extracellular vesicles derived from milk are also widely used for oral delivery of active ingredients, but often face challenges such as complex separation steps and low efficiency. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles and its preparation method. The method first selects and isolates two types of probiotic-derived extracellular vesicles as basic carriers: one is outer membrane vesicles (OMVs) derived from the Gram-negative bacterium *Escherichia coli* Nissle 1917, and the other is extracellular vesicles (EVs) derived from the Gram-positive bacterium *Lactobacillus plantarum*. These two natural vesicles possess good biocompatibility, gastrointestinal digestibility, and loading capacity. Secondly, to enhance the function of these two vesicle carriers, this invention synthesizes EGCp. This compound is obtained by selectively acylating food-grade tea polyphenol EGC (epigallocatechin gallate), possessing an amphiphilic structure similar to phospholipids. EGCp is self-assembled into the membrane structure of OMVs / EVs, aiming to utilize the inherent antioxidant and anti-inflammatory activities of tea polyphenols to enhance the bioactivity of the carrier itself. Finally, to achieve active targeting of inflammatory cells, this invention further modifies the above-mentioned functionalized OMVs / EVs using OSA-HA. Through the synergistic effect of EGCp and OSA-HA, an intelligent co-delivery system capable of simultaneously and efficiently transporting lipophilic curcumin and hydrophilic anthocyanins, and possessing inflammation-targeting capabilities, is finally constructed.

[0005] A method for preparing a curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles includes the following steps:

[0006] (1) EGC is subjected to ortho-phenolic hydroxyl acylation, palmitoyl chloride esterification and protecting group removal reaction to obtain EGCp;

[0007] (2) Octenyl succinic anhydride was grafted with hyaluronic acid in an alkaline buffer system and then purified by dialysis to obtain OSA-HA;

[0008] (3) Add curcumin solution, EGCp solution, anthocyanin solution, OSA-HA solution and probiotic-derived extracellular vesicle suspension to acetic acid buffer in sequence, then sonicate, and then centrifuge and ultrafiltration to purify the product to obtain the curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles.

[0009] In step (1) above:

[0010] Specifically, the preparation process of EGCp (EGC palmitate) is as follows:

[0011] 1.1. EGC, propionic anhydride and triethylamine were subjected to ortho-phenolic hydroxyl acylation in anhydrous acetonitrile. After the reaction was completed, the protected product was obtained.

[0012] 1.2 Dissolve the protected product in anhydrous dichloromethane, and add palmitoyl chloride and pyridine to carry out the hydroxylation reaction of fatty alcohols;

[0013] 1.3. Hydroxylamine was added to the esterified system to remove the propionyl protecting group. After the reaction was completed, EGC palmitate was obtained.

[0014] In step 1.1 above:

[0015] Preferably, the molar ratio of EGC to propionic anhydride is 1:(5~10).

[0016] Preferably, the molar ratio of EGC to triethylamine is 1:(5~10).

[0017] Preferably, the molar volume ratio of EGC to anhydrous acetonitrile is 0.08~0.12 mol / L.

[0018] Preferably, the reaction temperature is room temperature and the reaction time is 4 h.

[0019] As a preferred method, after the reaction is completed, the reaction mixture is extracted with ethyl acetate, washed twice with deionized water, dehydrated and dried with anhydrous magnesium sulfate, filtered, and then the ethyl acetate is removed by rotary evaporation to obtain the EGC protected product.

[0020] In step 1.2 above:

[0021] Preferably, the molar ratio of the protected product to palmitoyl chloride is 1:(2~4).

[0022] Preferably, the molar ratio of the protected product to pyridine is 1:(1~4).

[0023] Preferably, the molar volume ratio of the protected product to anhydrous dichloromethane is 0.08~0.12 mol / L.

[0024] Preferably, the reaction temperature is room temperature and the reaction time is 0.5 h.

[0025] Preferably, after the esterification reaction is completed, 95% ethanol is added to the system to react and remove excess palmitoyl chloride, and the system after removing excess palmitoyl chloride is used in step 1.3.

[0026] As a further preferred option, the volume ratio of added ethanol to anhydrous dichloromethane is 1:1.

[0027] As a further preferred option, the reaction temperature after adding ethanol is room temperature, and the reaction time is 2 h.

[0028] Preferably, pyridine and palmitoyl chloride are added dropwise to the solution of the protected product.

[0029] In step 1.3 above:

[0030] Preferably, hydroxylamine is added in the form of an aqueous solution with a concentration of 50 wt.%.

[0031] Preferably, the molar ratio of the protected product to hydroxylamine is 1:(10~15).

[0032] Preferably, the reaction temperature for removing the propionyl protecting group is room temperature, and the reaction time is 2 h.

[0033] As a preferred option, the following post-treatment is performed after the deprotection reaction is completed:

[0034] After extraction with ethyl acetate, the reaction solution was washed twice each with 1 mol / L hydrochloric acid solution and deionized water, and then dehydrated and dried with anhydrous magnesium sulfate. The organic phase was then collected by filtration and rotary evaporation. The resulting solid was washed with petroleum ether, filtered, and dried to obtain EGC palmitate.

[0035] In step (2) above:

[0036] Preferably, the molar ratio of hyaluronic acid (HA) to octenyl succinic anhydride (OSA) is 1:(90~120). More preferably, it is 1:100.

[0037] Preferably, the molecular weight of hyaluronic acid is 100~200 kDa.

[0038] Preferably, the pH range of the alkaline buffer system is 8.0 to 9.5. More preferably, it is 8.5.

[0039] Preferably, the alkaline buffer system is a 2 mol / L NaHCO3 solution.

[0040] Preferably, during the grafting reaction, a 0.5 mol / L NaOH solution is used to adjust the pH of the NaHCO3 buffer solution.

[0041] Preferably, octenyl succinic anhydride is added dropwise.

[0042] Preferably, the grafting reaction time is 8–24 h, and the reaction temperature is room temperature. More preferably, it is 12 h.

[0043] As a preferred method, the dialysis purification process is as follows:

[0044] The reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 8-12 kDa, and dialyzed with deionized water at room temperature for 20-30 h, with the water changed every 4 h. After the dialysis was completed, the dialysate was freeze-dried to obtain the OSA-HA.

[0045] In step (3) above:

[0046] Preferably, for every 1.0 × 10⁻⁶ ppm added to the acetate buffer... 9 ~1.0×10 10 For each extracellular vesicle, add 80-120 μg of curcumin, 80-120 μg of anthocyanins, 80-120 μg of EGCp, and 30-50 μg of OSA-HA. More preferably, for every 1.0 × 10⁻⁶ extracellular vesicles added... 9 ~1.0×10 10 Each extracellular vesicle contains 100 μg of curcumin, 100 μg of anthocyanin, 100 μg of EGCp, and 40 μg of OSA-HA. A further preferred formulation is 4 × 10⁻⁶ vesicles per vesicle. 9 Each extracellular vesicle was supplemented with 100 μg of curcumin, 100 μg of anthocyanin, 100 μg of EGCp, and 40 μg of OSA-HA.

[0047] Preferably, in the acetate buffer, curcumin solution and EGCp solution are added first, followed by anthocyanin solution, OSA-HA solution and extracellular vesicle suspension in sequence.

[0048] Preferably, the probiotic extracellular vesicles are selected from at least one of the outer membrane vesicles derived from Escherichia coli Nissle 1917 and extracellular vesicles derived from Lactobacillus plantarum.

[0049] Preferably, the particle concentration of the extracellular vesicle suspension is 1.0 × 10⁻⁶. 9 ~1.0×10 10 Cells / mL. More preferably 4.0 × 10⁻⁶. 9 per mL.

[0050] Preferably, the concentrations of the curcumin solution, EGCp solution, anthocyanin solution, and OSA-HA solution are each independently selected from 3 to 6 mg / mL. More preferably, the concentrations of the curcumin solution, EGCp solution, anthocyanin solution, and OSA-HA solution are all 4 mg / mL.

[0051] Preferably, the solvents for both the curcumin solution and the EGCp solution are aqueous ethanol solutions with a volume concentration of 65-80%. More preferably, the solvents for both the curcumin solution and the EGCp solution are aqueous ethanol solutions with a volume concentration of 70%.

[0052] Preferably, the solvent for the anthocyanin solution is an acetate buffer solution with a pH of 4.0 to 5.5. More preferably, it is an acetate buffer solution with a pH of 5.0.

[0053] Preferably, the solvent for the OSA-HA solution is deionized water.

[0054] Preferably, the dispersion medium for the extracellular vesicle suspension is PBS buffer with a pH of 7.2 to 7.4.

[0055] Preferably, the pH of the acetate buffer solution is 4.0 to 5.5. More preferably, it is 5.0.

[0056] Preferably, the ultrasonic treatment is performed in an ice bath using an intermittent ultrasonic mode. During this process, the hydrophobic palmitoyl chain of EGCp and the hydrophobic octenyl succinic acid side chain of OSA-HA synergistically insert into the phospholipid bilayer of extracellular vesicles, achieving functional modification of the extracellular vesicles; simultaneously, curcumin is embedded in the membrane structure of the extracellular vesicles, while anthocyanins are embedded in the hydrophilic lumen of the extracellular vesicles.

[0057] As a further preferred embodiment, the ice bath temperature is 0℃, the ultrasonic working time is 20~40 s, the interval time is 20~40 s, the number of cycles is 5~8, and the ultrasonic amplitude is 25~40% of the rated amplitude. As an even more preferred embodiment, the ultrasonic working time is 30 s, the interval time is 30 s, the number of cycles is 6, and the ultrasonic amplitude is 30% of the rated amplitude.

[0058] Preferably, the centrifugation speed is 10,000~15,000 × g, and the centrifugation time is 5~15 min. The purpose of this centrifugation step is to remove free curcumin precipitate. More preferably, the centrifugation speed is 12,000 × g, and the centrifugation time is 10 min.

[0059] Preferably, ultrafiltration purification is performed using an ultrafiltration tube with a molecular weight cutoff of 30-100 kDa for ultrafiltration concentration, and the cutoff fraction is collected.

[0060] Specifically:

[0061] The supernatant obtained after centrifugation to remove free curcumin was concentrated by ultrafiltration using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30-100 kDa to remove free anthocyanins, EGCp, and OSA-HA. A 50 kDa ultrafiltration centrifuge tube was further preferred.

[0062] As a further preferred option, the centrifugation conditions for ultrafiltration concentration are centrifugation at 10,000 × g for 5 min and centrifugation temperature of 4℃.

[0063] As a preferred embodiment, a method for preparing a curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles includes the following steps:

[0064] (1) Synthesize EGCp, which is prepared by steps including phenolic hydroxyl protection, palmitoyl chloride esterification and deprotection;

[0065] (2) Synthesize OSA-HA by grafting octenyl succinic anhydride with hyaluronic acid in alkaline NaHCO3 buffer and then purifying by dialysis.

[0066] (3) Prepare ethanol solutions of curcumin and EGCp, anthocyanin acetate buffer solution, and OSA-HA aqueous solution respectively;

[0067] (4) In the acetic acid buffer system, the curcumin solution prepared in step (3) and EGCp solution, anthocyanin solution, OSA-HA solution and extracellular vesicle suspension are added in sequence to form a mixed system;

[0068] (5) The mixture system described in step (4) is subjected to ultrasonic treatment. During this process, the hydrophobic palmitoyl chain of EGCp and the hydrophobic octenyl succinic acid side chain of OSA-HA are synergistically inserted into the phospholipid bilayer of the vesicle to achieve functional modification of the vesicle. At the same time, curcumin is embedded in the vesicle membrane structure, and anthocyanins are embedded in the hydrophilic inner cavity of the vesicle.

[0069] (6) Centrifuge the sonicated system of step (5) to remove free curcumin, and then use an ultrafiltration centrifuge tube to centrifuge and ultrafiltration purify the obtained supernatant to remove free anthocyanins, EGCp and OSA-HA. The resulting residue is the curcumin and anthocyanin co-delivery formulation of the functionalized probiotic extracellular vesicles.

[0070] This invention relates to a curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles, used to achieve highly efficient oral targeted delivery of curcumin and anthocyanins. The method of this invention first uses natural extracellular vesicles derived from probiotics as the basic carrier, which possesses good biocompatibility, digestive stability, and loading capacity. Secondly, to enhance the carrier's function, this invention synthesizes EGCp with an amphiphilic structure and embeds it into the vesicle membrane through self-assembly, thereby endowing the carrier EGC with inherent antioxidant and anti-inflammatory activities. Further, to endow the carrier system with highly efficient targeting capability against inflammatory cells, the surface of the above-mentioned functionalized vesicles is modified by grafting hyaluronic acid with octenyl succinic anhydride, thus obtaining a functional formulation capable of co-delivering curcumin and anthocyanins. EGCp and OSA-HA work synergistically to construct an intelligent co-delivery system that can simultaneously and efficiently load lipophilic curcumin and hydrophilic anthocyanins and actively target inflammatory sites.

[0071] This invention also provides a curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles, prepared by any of the methods described above. This co-delivery formulation can be used as an oral targeted therapy for inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease). Through the active targeting of OSA-HA to inflammatory sites (such as those with high CD44 receptor expression) and the enhanced antioxidant and anti-inflammatory activity of EGCp, this system can synergistically deliver curcumin and anthocyanins to the intestinal inflammatory area, achieving highly effective local treatment and reducing systemic side effects. Furthermore, this formulation can be used to develop functional foods or special medical purpose formulations with intestinal health regulation functions. Utilizing its good biocompatibility, digestive stability, and inflammation-targeting ability, it can effectively protect and target the delivery of curcumin and anthocyanins to specific sites in the intestine, exerting multiple health benefits including anti-inflammatory, antioxidant, and intestinal flora regulation.

[0072] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0073] (1) Innovative carrier source, combining biocompatibility and production feasibility: This invention abandons the traditional mammalian cell-derived EVs (high production cost and low yield) or synthetic nanomaterials (potential cytotoxicity), and innovatively selects extracellular vesicles derived from probiotics (Escherichia coli Nissle 1917 and Lactobacillus plantarum) as carriers. This type of carrier itself has good biocompatibility and low immunogenicity, and can be produced on a large scale and at low cost through fermentation.

[0074] (2) Functional modification of the carrier simultaneously achieves functional enhancement and active targeting: Unlike traditional simple embedding, this invention proposes a coherent vesicle functionalization strategy. First, by directionally inserting the synthesized amphiphilic molecule EGCp into the vesicle phospholipid bilayer, the carrier is membrane engineered to endow it with the inherent antioxidant and anti-inflammatory activities of tea polyphenols; further, OSA-HA is used to modify the surface of the functionalized vesicles so that they can specifically recognize the CD44 receptor highly expressed at the site of inflammation, thereby achieving active targeting of inflammatory cells and significantly improving drug accumulation and efficacy at the lesion site.

[0075] (3) Highly efficient co-encapsulation platform to overcome the differences in physicochemical properties of various active ingredients: This invention utilizes the unique structure of functionalized vesicles to successfully construct a co-delivery platform that can simultaneously and efficiently load lipophilic curcumin and hydrophilic anthocyanins. This platform perfectly solves the industry problem of poor compatibility and low encapsulation rate of two very different active ingredients in a single system, achieving synergistic effects.

[0076] (4) The preparation process is simple and controllable, which is conducive to standardized production: The core preparation steps of this invention are based on sequential mixing and ultrasonic-induced self-assembly. The process is mild and does not require complex chemical coupling or harsh conditions. The purification steps use conventional centrifugation and ultrafiltration, which are easy to scale up and control in terms of quality. The entire process route is simple, efficient and reproducible. Attached Figure Description

[0077] Figure 1 The 1H NMR spectrum of the EGC palmitate obtained in Example 1;

[0078] Figure 2 The mass spectrum of the EGC palmitate obtained in Example 1;

[0079] Figure 3 This is a transmission electron microscope image of the co-delivery vector based on two extracellular vesicles in Example 1. Detailed Implementation

[0080] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0081] In the following examples, OMVs / EVs are extracted using the following method:

[0082] Single colonies of *EcN* and *Lactobacillus plantarum*, preserved by streaking, were inoculated separately into their respective suitable liquid media (LB medium for *EcN* and MRS medium for *Lactobacillus plantarum*) and cultured overnight at 37°C with shaking. 200 μL of each bacterial culture was then transferred to 1600 mL of the corresponding fresh medium and cultured overnight at 37°C with shaking. Subsequent extraction procedures were the same: after culturing, the bacterial cells were removed by centrifugation at 4°C. The resulting supernatant was filtered through a 0.45 μm sterile filter and concentrated using a 100 kDa ultrafiltration centrifuge tube. Finally, extracellular vesicles (OMVs from *EcN* and EVs from *Lactobacillus plantarum*) were collected by ultracentrifugation at 4°C (180,000 × g, 30 min). The collected vesicles were washed with PBS, resuspended in PBS, and stored at -80°C for later use.

[0083] Example 1

[0084] (1) Synthesis of EGCp

[0085] Propionic anhydride (131.5 μL, 1.0 mmol) and triethylamine (138.6 μL, 1.0 mmol) were added dropwise to anhydrous acetonitrile (2 mL) containing dissolved EGC (61.2 mg, 0.2 mmol). The resulting mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction mixture was extracted with ethyl acetate, washed twice with deionized water, and then dehydrated and dried over anhydrous magnesium sulfate. After filtration, ethyl acetate was removed by rotary evaporation to obtain the protected EGC product.

[0086] Pyridine (24.2 μL, 0.3 mmol) was added dropwise to anhydrous dichloromethane (2 mL) containing the EGC protected product (117.2 mg, 0.2 mmol), followed by slow dropwise addition of palmitoyl chloride (125.8 μL, 0.4 mmol). The resulting mixture was stirred at room temperature for 30 min to obtain the intermediate esterified product. To remove excess palmitoyl chloride, 95% ethanol (2 mL) was added to the reaction system, and the reaction was continued for 2 h.

[0087] Hydroxylamine aqueous solution (50 wt.%, 122.5 μL, 2.0 mmol) was slowly added to the above reaction system, and the reaction was stirred at room temperature for 2 h. The reaction solution was extracted with ethyl acetate, washed twice each with 1 mol / L hydrochloric acid solution and deionized water, and then dehydrated and dried with anhydrous magnesium sulfate. The organic phase was collected by filtration and rotary evaporation. The resulting solid product was suspended in petroleum ether, filtered, and dried to obtain EGCp.

[0088] The proton NMR spectrum of the prepared EGCp (see) Figure 1 A significant saturated fatty chain signal was observed in the 0.8–1.4 ppm range, confirming the successful attachment of the palmitoyl chain to the EGC molecule. (Mass spectrometry [see image]) Figure 2 This further confirms the structural characteristics ([2M-H]). - 1087.18).

[0089] (2) Synthesis of OSA-HA

[0090] Weigh 1.25 g of HA (molecular weight 100-200 kDa) and dissolve it in 50 mL of deionized water. Then, add NaHCO3 to this system to form a 2 mol / L carbonate buffer system. Simultaneously, adjust the pH to 8.5 with 0.5 mol / L NaOH solution and pre-stir at room temperature for 1 h. Add OSA slowly dropwise to the solution at a molar ratio of OSA to HA of 100:1 and stir at room temperature for 12 h. Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 8-12 kDa and dialyze with deionized water at room temperature for 24 h, changing the water every 4 h. Finally, freeze-dry the dialysate to obtain solid OSA-HA and store at -20℃.

[0091] (3) Functionalized OMVs / EVs co-encapsulated curcumin and anthocyanins

[0092] First, 70% ethanol stock solutions of curcumin, 70% ethanol stock solutions of EGCp, acetate buffer (pH 5.0) stock solutions of anthocyanins, and deionized water stock solutions of OSA-HA were prepared separately, with a concentration of 4 mg / mL for each stock solution.

[0093] Next, in two 815 μL portions of acetate buffer (pH 5.0), the following solutions were added sequentially: 25 μL curcumin stock solution (containing 100 μg) and EGCp stock solution (containing 100 μg), 25 μL anthocyanin stock solution (containing 100 μg), and 10 μL LOSA-HA stock solution (containing 40 μg). Finally, 100 μL of solution containing approximately 4.0 × 10⁻⁶ ppm was added. 9 A PBS suspension of 1 OMVs / EVs was gently mixed. The mixture was then placed in an ice bath at 0°C and sonicated to promote embedding and self-assembly. The amplitude was set to 30% of the rated amplitude, and sonication was performed in a pulse mode with 30 s on and 30 s off intervals for a total of 6 cycles. Finally, the sonicated mixture was centrifuged at 12,000 × g for 10 min at 4°C to discard the precipitate and collect the supernatant. The supernatant was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 50 kDa and centrifuged at 10,000 × g for 5 min at 4°C. The concentrate in the centrifuge tube was retained, which is the curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles, denoted as Cur-Ant-OMVs@EGCp@OSA-HA and Cur-Ant-EVs@EGCp@OSA-HA, respectively.

[0094] Characterization tests of functionalized OMVs / EVs co-delivery vectors:

[0095] To determine the content of encapsulated curcumin, the entire supernatant (approximately 1 mL) collected after ultrasonication and centrifugation was mixed with 9 mL of 95% ethanol and treated in a water bath ultrasonic bath (200 W, 25℃) for 5 min to destroy the vesicle structure and release the encapsulated curcumin into the solution. Subsequently, the solution was centrifuged at 5,000 × g for 10 min to remove insoluble impurities, and the absorbance of the clear supernatant was measured at a wavelength of 420 nm. Based on the measured concentration of encapsulated curcumin (calculated from the absorbance), the encapsulation efficiency (EE%) was calculated according to the following formula (1):

[0096] (1)

[0097] In the formula: W is the total mass of curcumin initially added;

[0098] W1 represents the mass of encapsulated curcumin measured from the supernatant.

[0099] The anthocyanin content was determined by pH differential method, with the molar extinction coefficient and molecular weight of cyanidin-3-O-glucoside serving as the standard conversion reference. The specific steps are as follows: Two samples of the ultrafiltration filtrate from step (3) were diluted with potassium chloride buffer (pH 1.0) and sodium acetate buffer (pH 4.5), respectively, and their absorbances at 520 nm and 700 nm were measured. The free anthocyanin content (C) in the sample was calculated using the following formulas (2) and (3):

[0100] (2)

[0101] (3)

[0102] in, This represents the difference in absorbance at 520 nm and 700 nm; and The values ​​represent the difference in absorbance after dilution with pH 1.0 buffer and pH 4.5 buffer, respectively.

[0103] M W The molecular weight of cyanidin-3-O-glucoside is 449.2 g / mol.

[0104] DF represents the dilution factor;

[0105] ε is the molar extinction coefficient of cyanidin-3-O-glucoside in pH 1.0 buffer, with a value of 26,900 L·mol⁻¹. -1 ·cm -1 ;

[0106] l is the optical path length (1 cm);

[0107] Based on the measured content of encapsulated anthocyanins, the encapsulation efficiency (EE%) is calculated according to the following formula (4):

[0108] EE% = (Total anthocyanin content - Free anthocyanin content) / Total anthocyanin content × 100% (4)

[0109] The total anthocyanin content is the initial anthocyanin feed amount.

[0110] Encapsulation efficiency measurements showed that both probiotic-derived extracellular vesicles (OMVs) exhibited good encapsulation capabilities for curcumin and anthocyanins. Specifically, OMVs derived from *Escherichia coli* Nissle 1917 achieved an encapsulation rate of 41.6% for curcumin and 25.4% for anthocyanins; while EVs derived from *Lactobacillus plantarum* achieved an encapsulation rate of 35.9% for curcumin and 24.6% for anthocyanins. Although OMVs showed slightly higher encapsulation rates for both active ingredients than EVs, both methods effectively loaded both active ingredients. Figure 3 As shown, after probiotic-derived extracellular vesicles were functionalized with EGCp and OSA-HA and co-encapsulated with curcumin and anthocyanins, the resulting two formulations, Cur-Ant-OMVs@EGCp@OSA-HA and Cur-Ant-EVs@EGCp@OSA-HA, both maintained intact vesicle structures, exhibited well-dispersed spherical morphology, and had similar particle sizes.

Claims

1. A method for preparing a curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles, characterized in that, Includes the following steps: (1) EGC is subjected to ortho-phenolic hydroxyl acylation, palmitoyl chloride esterification and protecting group removal reaction to obtain EGCp; (2) Octenyl succinic anhydride was grafted with hyaluronic acid in an alkaline buffer system and then purified by dialysis to obtain OSA-HA; (3) Add curcumin solution, EGCp solution, anthocyanin solution, OSA-HA solution and probiotic-derived extracellular vesicle suspension to acetic acid buffer in sequence, then sonicate, and then centrifuge and ultrafiltration to purify the product to obtain the curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles.

2. The method for preparing a curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles according to claim 1, characterized in that, In step (2), the molar ratio of hyaluronic acid to octenyl succinic anhydride is 1:(90~120); The pH range of the alkaline buffer system is 8.0 to 9.

5.

3. The method for preparing a curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles according to claim 1, characterized in that, In step (2), the dialysis purification process is as follows: The reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 8-12 kDa, and dialyzed with deionized water at room temperature for 20-30 hours, with the water changed every 4 hours. After the dialysis was completed, the dialysate was freeze-dried to obtain the OSA-HA.

4. The method for preparing a curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles according to claim 1, characterized in that, In step (3), for every 1.0 × 10⁻⁶ ppm added to the acetate buffer... 9 ~1.0×10 10 For each extracellular vesicle, add 80-120 μg of curcumin, 80-120 μg of anthocyanins, 80-120 μg of EGCp, and 30-50 μg of OSA-HA.

5. The method for preparing a curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles according to claim 1, characterized in that, The probiotic extracellular vesicles are selected from at least one of the outer membrane vesicles derived from Escherichia coli Nissle 1917 and extracellular vesicles derived from Lactobacillus plantarum. The particle concentration of the extracellular vesicle suspension was 1.0 × 10⁻⁶. 9 ~1.0×10 10 per mL.

6. The method for preparing a curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles according to claim 1, characterized in that, In step (3), the concentrations of curcumin solution, EGCp solution, anthocyanin solution, and OSA-HA solution are each independently selected from 3 to 6 mg / mL.

7. The method for preparing a curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles according to claim 1, characterized in that, In step (3), the solvents for curcumin solution and EGCp solution are ethanol aqueous solution with a volume concentration of 65~80% respectively; The solvent for the anthocyanin solution is an acetate buffer solution with a pH of 4.0-5.5; The solvent for OSA-HA solution is deionized water; The extracellular vesicle suspension was dispersed in PBS buffer at pH 7.2–7.

4.

8. The method for preparing a curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles according to claim 1, characterized in that, In step (3), the pH value of the acetic acid buffer is 4.0~5.5; The ultrasonic treatment was performed in an ice bath using an intermittent ultrasonic mode.

9. The method for preparing a curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles according to claim 1, characterized in that, In step (3), the centrifugation speed is 10,000~15,000 × g, and the centrifugation time is 5~15 min; Ultrafiltration purification was performed using an ultrafiltration tube with a molecular weight cutoff of 30-100 kDa for ultrafiltration concentration, and the cutoff fraction was collected.

10. A curcumin and anthocyanin co-delivery formulation based on functionalized probiotic extracellular vesicles, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 9.

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