A double Pickering emulsion for targeted delivery of polyunsaturated fatty acids, its preparation method and application

Abstract

This invention relates to the field of nutritional intervention technology, and more particularly to a double Pickering emulsion for targeted delivery of polyunsaturated fatty acids, its preparation method, and its application. The invention first prepares folic acid-modified soy protein isolate-chitosan electrostatic composite particles as solid particle emulsifiers to stably deliver polyunsaturated fatty acids in a double Pickering emulsion. The folic acid-modified soy protein isolate-chitosan electrostatic composite particles utilize the amphiphilic nature of soy protein isolate and the steric hindrance effect of chitosan to construct a dense, three-dimensional rigid physical barrier at the oil-water interface, significantly reducing the oxidative degradation rate of polyunsaturated fatty acids, ensuring their storage stability, and achieving long-term stability. Simultaneously, by leveraging the active targeting mediated by folic acid receptors and the pH-responsive properties of chitosan, targeted and precise controlled release and delivery of polyunsaturated fatty acids in the colon are achieved, providing a solid technical foundation for precise nutritional intervention in colon cancer.

Description

Technical Field

[0001] This invention relates to the field of nutritional intervention technology, and in particular to a double Pickering emulsion for targeted delivery of polyunsaturated fatty acids, its preparation method, and its application. Background Technology

[0002] Polyunsaturated fatty acids (such as alpha-linolenic acid, docosahexaenoic acid (DHA), or eicosapentaenoic acid (EPA)) have important physiological functions such as anti-inflammation, regulation of lipid metabolism, and inhibition of cancer cell proliferation, making them excellent raw materials for nutritional intervention. However, oils rich in polyunsaturated fatty acids generally suffer from problems such as easy oxidation, poor stability, poor water solubility, and low delivery efficiency, which limit their application in the field of precision nutrition.

[0003] Pickering emulsions are emulsion systems formed by using solid particles instead of traditional surfactants to stabilize the oil-water interface. They offer advantages such as high stability, no surfactant residue, and good biocompatibility, making them an important carrier for the encapsulation and delivery of functional oils. Currently, commonly used stabilizers in Pickering emulsions include proteins, polysaccharides, and inorganic particles. Among these, protein particles have broad application prospects in the food industry due to their wide availability, rich nutritional content, and biodegradability.

[0004] Currently, there are studies on using proteins or polysaccharides to prepare Pickering emulsions to encapsulate oils rich in ω-polyunsaturated fatty acids. However, these studies have the following shortcomings: First, the interfacial activity of stabilizers prepared from single proteins or polysaccharides is limited, resulting in insufficient emulsion stability and difficulty in meeting the requirements for processing and long-term storage. Second, there is a lack of targeting, making it difficult to accurately deliver the physiologically active components of polyunsaturated fatty acid oils, thus reducing their effectiveness in nutritional intervention. Third, mature processing and application methods have not been developed, making it difficult to scale up the production of the prepared Pickering emulsions. Summary of the Invention

[0005] In view of this, the present invention provides a double Pickering emulsion for targeted delivery of polyunsaturated fatty acids, its preparation method and application. The double Pickering emulsion for targeted delivery of polyunsaturated fatty acids provided by the present invention has high interfacial activity, has the ability to target and deliver polyunsaturated fatty acids, and meets the requirements for large-scale production.

[0006] This invention provides a targeted delivery of polyunsaturated fatty acids in a dual Pickering emulsion, which is a W1 / O / W2 type dual Pickering emulsion, comprising, from the inside out, an inner aqueous phase (W1), an oil phase (O), and an outer aqueous phase (W2); the oil phase comprises oil containing polyunsaturated fatty acids; folic acid-modified soy protein isolate-chitosan electrostatic composite particles are distributed at the oil-water interface of the inner aqueous phase and the oil phase; folic acid-modified soy protein isolate-chitosan electrostatic composite particles are also distributed at the oil-water interface of the oil phase and the outer aqueous phase.

[0007] Preferably, the oil containing polyunsaturated fatty acids includes one or more of flaxseed oil, perilla seed oil, sesame oil, safflower oil, and walnut oil.

[0008] Preferably, the preparation method of the folic acid-modified soy protein isolate-chitosan electrostatic composite particles includes the following steps: (1) Mix soy protein isolate and phosphate buffered saline solution (PBS solution, pH 7.4, molar concentration 0.1M) to obtain soy protein isolate solution; (2) Folic acid, dimethyl sulfoxide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were mixed and activated to obtain an activated folic acid solution; (3) The activated folic acid solution and the soy protein isolate solution are mixed (referred to as the first mixture) and subjected to an amidation reaction to obtain folic acid modified soy protein isolate; (4) The folic acid modified soy protein isolate is mixed with chitosan, glacial acetic acid and water (referred to as the second mixture) to obtain the folic acid modified soy protein isolate-chitosan electrostatic composite particles.

[0009] Preferably, the molar ratio of folic acid to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1.2~1.5; the molar ratio of folic acid to N-hydroxysuccinimide is 1:1.2~1.5; the molar ratio of folic acid to soy protein isolate is 1:30~50; the mass ratio of folic acid-modified soy protein isolate to the total mass of chitosan, glacial acetic acid and water is 1:0.5~2; and the mass ratio of chitosan to the total volume of glacial acetic acid and water is (0.8~1.8) mg:1 mL.

[0010] Preferably, the amidation reaction is carried out under light-protected conditions; the amidation reaction is carried out at room temperature for 4-6 hours; and the amidation reaction is carried out under stirring conditions.

[0011] Preferably, the folic acid-modified soy protein isolate-chitosan electrostatic composite particles are the sole stabilizer or emulsifier for targeted delivery of polyunsaturated fatty acids in a double Pickering emulsion.

[0012] Preferably, the mass ratio of the folic acid-modified soy protein isolate-chitosan electrostatic composite particles to the oil phase is 0.1~5:40~60; the volume ratio of the inner aqueous phase to the outer aqueous phase is 1:4.5~5.5; and the mass ratio of the total mass of the inner aqueous phase and the outer aqueous phase to the mass of the oil phase is 35~60:40~60.

[0013] This invention also provides a method for preparing the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids as described above, comprising the following steps: The oil phase, inner aqueous phase, outer aqueous phase, and folic acid-modified soy protein isolate-chitosan electrostatic composite particles were mixed and sheared to obtain the targeted delivery of polyunsaturated fatty acids in the double Pickering emulsion.

[0014] The present invention also provides the application of the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids described in the above-described scheme or the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids obtained by the preparation method described in the above-described scheme in the preparation of cell-targeting carriers or colon-targeting carriers.

[0015] Preferably, the cell-targeting carrier or colon-targeting carrier is loaded with one or more of water-soluble and lipid-soluble active substances.

[0016] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention first prepares folic acid-modified soy protein isolate-chitosan electrostatic composite particles as solid particle emulsifiers to stabilize and target the delivery of polyunsaturated fatty acids in a double Pickering emulsion. Soy protein isolate, a commonly used food-grade protein, is modified to enhance its interfacial activity, making it suitable for use as a Pickering emulsion stabilizer. Folic acid, as a cancer cell targeting ligand, can specifically bind to the folic acid receptor highly expressed on the surface of colon cancer cells, enabling targeted delivery of functional components to colon cancer cells and improving the efficiency of nutritional intervention. Chitosan, a natural polysaccharide, possesses good biocompatibility, antibacterial properties, and cationic characteristics. It can combine with anionic proteins through electrostatic interactions, further enhancing the stability and targeting performance of the folic acid-modified soy protein isolate-chitosan electrostatic composite particles.

[0017] Folic acid-modified soy protein isolate-chitosan electrostatic composite particles utilize the amphiphilicity of soy protein isolate and the steric hindrance effect of chitosan to construct a dense, three-dimensional rigid physical barrier at the oil-water interface, significantly reducing the oxidative degradation rate of polyunsaturated fatty acids, ensuring their storage stability, and achieving long-term stability. At the same time, by leveraging the active targeting mediated by folic acid receptors (specific binding of folic acid ligands to receptors on the surface of colon cancer cells) and the pH-responsive properties of chitosan, targeted and precise controlled release and delivery of polyunsaturated fatty acids in the colon are achieved, providing a solid technical foundation for precise nutritional intervention in colon cancer.

[0018] The double Pickering emulsion for targeted delivery of polyunsaturated fatty acids provided by this invention belongs to the W1 / O / W2 type double Pickering emulsion. The emulsion exhibits uniform particle size, high stability, and shows no stratification or oxidative deterioration after storage at room temperature for more than 3 months. It demonstrates strong targeting, effectively protecting the polyunsaturated fatty acids in the oil from oxidation, and achieving precise delivery to the colon. This solves the problems of easy oxidation, poor dispersion, poor stability, poor water solubility, and low targeted delivery efficiency of polyunsaturated fatty acid oils, promoting the high-value utilization and industrialization of polyunsaturated fatty acid oils. Furthermore, the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids provided by this invention can not only target and deliver fat-soluble active ingredients such as polyunsaturated fatty acids, but also target and deliver water-soluble active ingredients, and simultaneously target and deliver both fat-soluble and water-soluble active ingredients, showing broad application prospects.

[0019] This invention employs a one-step high-speed shearing method to prepare a double Pickering emulsion for targeted delivery of polyunsaturated fatty acids. All raw materials used are food-grade, exhibiting good biocompatibility, safety, and non-toxicity. The preparation process is green and environmentally friendly, leaving no harmful residues, meeting food safety requirements, and has a wide range of applications. The double Pickering emulsion for targeted delivery of polyunsaturated fatty acids provided by this invention is suitable for application in the preparation of colonic nutritional intervention products or colonic drug delivery products, such as precision nutritional intervention foods for colon cancer, functional foods, and formulated foods. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of this invention, the accompanying drawings used in the embodiments of this invention or in the prior art are briefly described below. For those skilled in the art, other drawings can be derived from the following drawings without creative effort, and all such drawings are within the protection scope of this invention.

[0021] Figure 1 The cumulative release curves of free fatty acids from the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids in test example 1 in different simulated digestive solutions are shown. Figure 2 To test the in vitro proliferation inhibition curves of Caco-2 colon cancer cells by different treatment groups in Example 2; Figure 3 The images shown are scanning electron microscope (SEM) images of soy protein isolate, folic acid-modified soy protein isolate, and folic acid-modified soy protein isolate-chitosan electrostatic composite particles from Example 1, at a magnification of 1000x. Among them, a is a scanning electron microscope image of soy protein isolate, b is a scanning electron microscope image of folic acid-modified soy protein isolate, and c is a scanning electron microscope image of folic acid-modified soy protein isolate-chitosan electrostatic composite particles. Figure 4 An optical microscope image of the targeted delivery of polyunsaturated fatty acids double Pickering emulsion prepared in Example 2, magnified 400 times; Figure 5 Particle size distribution diagram of the targeted delivery of polyunsaturated fatty acids double Pickering emulsion prepared in Example 2; Figure 6 The stability comparison diagram of the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids prepared in Example 2 before and after storage at 4°C in the dark for 50 days is shown. Detailed Implementation

[0022] This invention provides a targeted delivery of polyunsaturated fatty acids in a dual Pickering emulsion, which is a W1 / O / W2 type dual Pickering emulsion, comprising, from the inside out, an inner aqueous phase (W1), an oil phase (O), and an outer aqueous phase (W2); the oil phase comprises oil containing polyunsaturated fatty acids; folic acid-modified soy protein isolate-chitosan electrostatic composite particles are distributed at the oil-water interface of the inner aqueous phase and the oil phase; folic acid-modified soy protein isolate-chitosan electrostatic composite particles are also distributed at the oil-water interface of the oil phase and the outer aqueous phase.

[0023] In this invention, the water in the internal aqueous phase is preferably deionized water.

[0024] In this invention, the oil containing polyunsaturated fatty acids preferably includes one or more of flaxseed oil, perilla seed oil, sesame oil, safflower oil, and walnut oil, and more preferably flaxseed oil.

[0025] In this invention, the preparation method of the folic acid-modified soy protein isolate-chitosan electrostatic composite particles preferably includes the following steps: (1) Mix soy protein isolate and phosphate buffered saline solution (PBS solution, pH 7.4, molar concentration 0.1M) to obtain soy protein isolate solution; (2) Folic acid, dimethyl sulfoxide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were mixed and activated to obtain an activated folic acid solution; (3) The activated folic acid solution and the soy protein isolate solution are mixed (referred to as the first mixture) and subjected to an amidation reaction to obtain folic acid modified soy protein isolate; (4) The folic acid modified soy protein isolate is mixed with chitosan, glacial acetic acid and water (referred to as the second mixture) to obtain the folic acid modified soy protein isolate-chitosan electrostatic composite particles.

[0026] In this invention, the mass concentration of soy protein isolate in the soy protein isolate solution is preferably 1.5~3.5 mg / mL, more preferably 2~3 mg / mL, and even more preferably 2.5 mg / mL.

[0027] In this invention, the molar ratio of folic acid to the volume of dimethyl sulfoxide is preferably (10~20) mmol:1L, more preferably (13~17) mmol:1L, and even more preferably 15 mmol:1L.

[0028] In this invention, the molar ratio of folic acid and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is preferably 1:1.2 to 1.5, more preferably 1:1.3 to 1.4.

[0029] In this invention, the molar ratio of folic acid to N-hydroxysuccinimide is preferably 1:1.2 to 1.5, more preferably 1:1.3 to 1.4.

[0030] In this invention, the activation time is preferably 1.5 hours.

[0031] In this invention, the molar ratio of folic acid to soy protein isolate is preferably 1:30~50, more preferably 1:35~45, and even more preferably 1:40.

[0032] In this invention, the first mixing is preferably: adding an activated folic acid solution dropwise to a soy protein isolate solution; the dropwise addition rate is preferably 0.8 mL / min.

[0033] In this invention, the amidation reaction is preferably carried out under light-protected conditions; the temperature of the amidation reaction is preferably room temperature, and the reaction time is preferably 4-6 hours, more preferably 5 hours; the amidation reaction is preferably carried out under stirring conditions. This invention achieves graft modification of soy protein isolate through the above-described amidation reaction.

[0034] In this invention, the amidation reaction preferably further includes sequential purification and drying of the resulting product; the purification is preferably dialysis purification; the parameters for the dialysis purification preferably include: a molecular weight cutoff of 8000 Da in the dialysis bag, dialysis in deionized water for 60 hours, with the deionized water being replaced every 6 hours; the drying is preferably freeze-drying; the freeze-drying temperature is preferably -60°C, and the holding time is preferably 36~48 hours, more preferably 40~42 hours. This invention removes unreacted impurities through purification.

[0035] In this invention, the mass ratio of the folic acid-modified soy protein isolate to the total mass of chitosan, glacial acetic acid and water is preferably 1:0.5~2, more preferably 1:1~1.5, and even more preferably 1:1.2.

[0036] In this invention, the volume ratio of glacial acetic acid to water is preferably 0.5~2:100, more preferably 1~1.8:100, and even more preferably 1.3~1.5:100.

[0037] In this invention, the mass ratio of chitosan to the total volume of glacial acetic acid and water is preferably (0.8~1.8) mg:1 mL, more preferably (1~1.5) mg:1 mL, and even more preferably 1.2 mg:1 mL.

[0038] In this invention, the second mixing is preferably stirring; the stirring speed is preferably 200~500 rpm, more preferably 300~400 rpm; the second mixing time is preferably 8~12 hours, more preferably 10 hours.

[0039] In this invention, the folic acid-modified soy protein isolate-chitosan electrostatic composite particles are preferably the sole stabilizer or emulsifier for targeted delivery of polyunsaturated fatty acids in a double Pickering emulsion.

[0040] In this invention, the mass ratio of the folic acid-modified soy protein isolate-chitosan electrostatic composite particles to the oil phase is preferably 0.1~5:40~60, more preferably 0.5~5:45~55, and even more preferably 1.5~3:50.

[0041] In this invention, the water in the external aqueous phase is preferably deionized water.

[0042] In this invention, the volume ratio of the inner water phase to the outer water phase is preferably 1:4.5 to 5.5, and more preferably 1:5.

[0043] In this invention, the mass ratio of the total mass of the inner water phase and the outer water phase to the mass of the oil phase is preferably 35~60:40~60, more preferably 40~50:45~55, and even more preferably 48~50:48~50.

[0044] This invention also provides a method for preparing the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids as described above, comprising the following steps: The oil phase, inner aqueous phase, outer aqueous phase, and folic acid-modified soy protein isolate-chitosan electrostatic composite particles were mixed and sheared to obtain the targeted delivery of polyunsaturated fatty acids in the double Pickering emulsion.

[0045] In this invention, the shearing speed of the shearing process is preferably 10,000~22,000 r / min, more preferably 14,000~20,000 r / min, and even more preferably 17,000 r / min; the time is preferably 2~10 minutes, more preferably 4~7 minutes; the deviation of the shearing speed of the shearing process is preferably ≤±500 rpm.

[0046] In this invention, the emulsion temperature during the shearing process is preferably no more than 50°C, and more preferably 30-45°C. This invention controls the above temperature to prevent oxidation and deterioration of oil phase and other substances. The prepared double Pickering emulsion for targeted delivery of polyunsaturated fatty acids needs to be stored in a sealed container to avoid light and high temperatures.

[0047] The present invention provides a double Pickering emulsion for targeted delivery of polyunsaturated fatty acids, which simulates gastrointestinal release performance in vitro and has pH-responsive colon-targeted release characteristics.

[0048] The present invention also provides the application of the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids described in the above-described scheme or the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids obtained by the preparation method described in the above-described scheme in the preparation of cell-targeting carriers or colon-targeting carriers.

[0049] In this invention, the target cells of the cell-targeting vector are preferably Caco-2 colon cancer cells.

[0050] The double Pickering emulsion for targeted delivery of polyunsaturated fatty acids provided by this invention can be used to prepare colonic nutrition intervention products or colonic drug delivery carriers. Its stable system structure enables the encapsulation and precise targeted delivery of substances.

[0051] In this invention, the loading object of the cell-targeting carrier or colon-targeting carrier is preferably one or more of water-soluble active ingredients and lipid-soluble active ingredients; the water-soluble active ingredient is preferably epigallocatechin gallate (EGCG); the lipid-soluble active ingredient is preferably one or more of curcumin and polyunsaturated fatty acids.

[0052] To further illustrate the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments.

[0053] Example 1: This embodiment prepared folic acid-modified soy protein isolate-chitosan electrostatic composite particles, and the specific steps are as follows: S1. Preparation of soy protein isolate solution: Soy protein isolate was dissolved in phosphate buffer solution with a pH of 7.4 and a molar concentration of 0.1M to prepare a soy protein isolate solution with a mass concentration of 2.0 mg / mL. S2. Activation of folic acid: Folic acid was dissolved in dimethyl sulfoxide to prepare a folic acid solution with a concentration of 15 mmol / L. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added to the solution, wherein the molar ratio of folic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide was 1:1.2:1.2. The solution was activated for 1.5 h to obtain an activated folic acid solution. S3. Grafting modification and purification of soy protein isolate: The activated folic acid solution prepared in step S2 was added dropwise to the soy protein isolate solution at a rate of 0.8 mL / min. The molar ratio of folic acid to soy protein isolate was 1:40. After the addition was complete, the mixture was stirred at room temperature in the dark for 5 hours to carry out the amidation reaction. After the reaction was completed, the reaction solution was purified by dialysis. The molecular weight cutoff of the dialysis bag was 8000 Da. Dialysis was performed in deionized water for 60 hours, with the deionized water being replaced every 6 hours to remove unreacted impurities. After dialysis, the mixture was freeze-dried at -60℃ for 36 hours to obtain folic acid-modified soy protein isolate powder. S4. Preparation of chitosan solution: Take chitosan and dissolve it in 1% (v / v) glacial acetic acid solution to prepare a chitosan solution with a mass concentration of 1.0 mg / mL. S5. Preparation of folic acid modified soy protein isolate-chitosan electrostatic composite particle solution: The folic acid modified soy protein isolate powder obtained in step S3 was added to the chitosan solution obtained in step S4 at a mass ratio of 1:1. The mixture was stirred at 500 rpm for 8 hours to obtain a folic acid modified soy protein isolate-chitosan electrostatic composite particle solution. The solution was then freeze-dried at -60℃ for 36 hours to obtain folic acid modified soy protein isolate-chitosan electrostatic composite particles.

[0054] Example 2: This embodiment prepared a double Pickering emulsion for targeted delivery of polyunsaturated fatty acids, specifically a double Pickering emulsion for targeted delivery of flaxseed oil. The specific steps are as follows: Based on mass parts, take 48 parts of flaxseed oil, 0.5 parts of folic acid modified soy protein isolate-chitosan electrostatic composite particles prepared in Example 1, and 48.5 parts of deionized water (inner aqueous phase and outer aqueous phase combined). One-step preparation: 200 mL of flaxseed oil was used as the oil phase, 50 mL of deionized water as the inner aqueous phase, and 250 mL of deionized water as the outer aqueous phase. The oil phase, inner aqueous phase, and outer aqueous phase were added together into a container, along with 5 g of folic acid-modified soy protein isolate-chitosan electrostatic composite particles. The resulting mixture was placed in a constant-temperature water bath, with the system temperature controlled to not exceed 50°C. Emulsification was performed by shearing at 18000 rpm for 5 min, with a shearing speed deviation ≤ ±500 rpm. After shearing, the resulting emulsion was placed in a 4°C refrigerator and allowed to stand for 16 h. A stable and uniform double Pickering emulsion for targeted delivery of polyunsaturated fatty acids was obtained. Testing showed that the initial emulsion of the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids prepared in this embodiment was uniformly dispersed, without stratification or precipitation.

[0055] System testing: The targeted delivery polyunsaturated fatty acid double Pickering emulsion prepared in this embodiment consists only of an inner aqueous phase, a linseed oil phase, an outer aqueous phase, and folic acid-modified soy protein isolate-chitosan electrostatic composite particles, without any other substances. Testing showed that the targeted delivery polyunsaturated fatty acid double Pickering emulsion was a milky white, uniformly dispersed liquid, without stratification, floating oil, or sedimentation, with a particle size distribution of approximately 1 μm. After storage at 4°C in the dark for 50 days, no demulsification, stratification, or oxidative deterioration occurred, demonstrating excellent system stability.

[0056] Test Example 1: The targeted delivery of polyunsaturated fatty acids through the double Pickering emulsion prepared in Example 2 was subjected to an in vitro simulated gastrointestinal release behavior test, as detailed below: 1) Material preparation: Example 2 prepared a double Pickering emulsion for targeted delivery of polyunsaturated fatty acids, simulated gastric juice (SGF, pH 1.2), simulated intestinal juice (SIF, pH 6.8), simulated colonic juice (SCF, pH 7.4), dialysis bags, PBS buffer, free fatty acid (FFA) kit, UV-Vis spectrophotometer, and constant temperature water bath shaker.

[0057] 2) Testing method: Preparation of simulated digestive fluids: Simulated gastric juice (SGF, pH 1.2): contains pepsin; simulated intestinal juice (SIF, pH 6.8): contains pancreatic enzymes; simulated colonic juice (SCF, pH 7.4): uses a phosphate buffer system.

[0058] Take 5 mL of the targeted delivery of polyunsaturated fatty acids double Pickering emulsion prepared in Example 2, put it into a dialysis bag, place the dialysis bag in a centrifuge tube containing 50 mL of simulated digestion solution, and place it in a constant temperature water bath shaker at 37°C and 100 rpm for a light-protected shaking release experiment.

[0059] Samples were taken at regular intervals of 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, and 12h. After each sampling, an equal volume of fresh simulated digestion fluid at the same temperature was added. The free fatty acid (FFA) kit was used for color development, and the absorbance was measured by a UV-Vis spectrophotometer to calculate the cumulative release rate of free fatty acids.

[0060] 3) Test results: like Figure 1As shown, in a simulated gastric fluid environment (pH 1.2), the cumulative release rate of FFA within the first 2 hours was less than 30%, indicating a slow release rate. This suggests that the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids is structurally stable in the gastric environment and can effectively protect the encapsulated flaxseed oil from premature release. After entering a simulated intestinal fluid environment (pH 6.8), the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids still maintained a low release rate, with a cumulative release rate of less than 50% after 4 hours. When the system entered a simulated colonic fluid environment (pH 7.4), the release rate of the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids significantly accelerated, with a cumulative release rate of over 90% within 12 hours, exhibiting obvious colon-targeted release characteristics.

[0061] The above results indicate that the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids prepared in this invention has good pH responsiveness and colon-targeted release performance, enabling precise release of flaxseed oil in the colon and making it suitable as a nutritional intervention carrier for colon cancer.

[0062] Test Example 2: The in vitro cytotoxicity and targeting validation of the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids prepared in Example 2 are as follows: Using Caco-2 colon cancer cells as a model, curcumin (Cur) and epigallocatechin gallate (EGCG) were used as active inhibitory components. The in vitro antitumor activity of the drug-loaded emulsion was evaluated using the CCK-8 assay, and the folic acid receptor-mediated targeting effect was verified by folic acid competitive inhibition experiments.

[0063] 1) Materials: Example 2 includes the preparation of a targeted delivery of polyunsaturated fatty acids using a double Pickering emulsion, folic acid, colon cancer cells (Caco-2), DMEM high-glucose medium, fetal bovine serum, 0.25% (w / v) trypsin, a CCK-8 cytotoxicity assay kit, a CO2 cell incubator, and an ELISA reader.

[0064] 2) Testing method: Cell culture: Caco-2 cells were seeded in DMEM medium containing 10% (v / v) fetal bovine serum and cultured in a cell culture incubator at 37°C and 5% (v / v) CO2. When the cell confluence reached 80%–90%, the cells were digested with 0.25% trypsin to prepare a single-cell suspension, and the cell density was adjusted to 5 × 10⁶ cells / year. 3 Cells / well were seeded in 96-well plates and cultured for 24 hours to allow them to adhere.

[0065] Experimental Grouping and Sample Processing: Cells were divided into the following 4 groups, with 3 replicates per group: ① FA-Targeted Cur+EGCG Emulsion: Different concentrations of Cur+EGCG co-encapsulated FA-targeted Cur+EGCG emulsion were added, with concentration gradients of 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, or 160 μM (based on the molar concentration of Cur); ② Free Cur+EGCG: Different concentrations of equimolar mixtures of free Cur and EGCG were added, with the same concentration gradient as above; ③ FA Competition Group: Cells were pre-incubated with excess free folic acid (final concentration 1 mM) for 1 h, and then FA-targeted Cur+EGCG emulsion with the same concentration as in group ① was added, with the same concentration gradient as above; ④ Blank FA-targeted Cur+EGCG emulsion carrier group (…). Emulsion): Add blank targeted delivery double pickerine emulsion at the same amount as the above-mentioned drug-loaded targeted delivery polyunsaturated fatty acid double pickerine emulsion group. Add each group of samples to 96-well plates and incubate with Caco-2 cells for 24 h.

[0066] CCK-8 cell viability assay: After incubation, add 10 μL of CCK-8 reagent to each well and incubate in the dark for 1-2 hours. Measure the absorbance (OD) of each well at 450 nm using a microplate reader. 450 The blank wells containing only culture medium and CCK-8 reagent were used as zeroing wells, and the cell wells without sample treatment were used as negative control wells. The calculation was performed using the following formula: .

[0067] 3) Test Results and Analysis: The curves showing the relative cell viability of each group as a function of Cur / EGCG concentration are as follows: Figure 2As shown, the cell viability of the double-pickerlin emulsion carrier group targeting polyunsaturated fatty acids remained above 95% within the concentration range of 1–160 μM, with no significant cytotoxicity, indicating that the double-pickerlin emulsion targeting polyunsaturated fatty acids has good biocompatibility. The inhibitory effect of the drug-loaded double-pickerlin emulsion group targeting polyunsaturated fatty acids was more significant than that of the free drug group, indicating that folic acid targeting delivery enhanced the inhibitory effect of Cur and EGCG on colon cancer cells. The cell viability of the folic acid competitive inhibition group was higher than that of the double-pickerlin emulsion group targeting polyunsaturated fatty acids at the same concentration, and the difference increased with increasing concentration, indicating that excess free folic acid competitively occupied the folic acid receptor on the cell surface, blocking the specific binding of the double-pickerlin emulsion targeting polyunsaturated fatty acids to the receptor, verifying the folic acid receptor-mediated active targeting delivery mechanism. Therefore, the double-pickerlin emulsion group targeting polyunsaturated fatty acids exhibited the strongest tumor cell inhibitory activity, and the competitive inhibition experiment verified the folic acid receptor-mediated targeting effect.

[0068] As can be seen from the test examples, the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids prepared in this invention has good biocompatibility and strong targeting, and can significantly enhance the inhibitory effect of Cur and EGCG on colon cancer cells, and can be used as a nutritional intervention carrier for colon cancer.

[0069] Test Example 3: Using scanning electron microscopy, the soy protein isolate, folic acid-modified soy protein isolate, and folic acid-modified soy protein isolate-chitosan electrostatic composite particles in Example 1 were observed, and the results are as follows: Figure 3 As shown. According to Figure 3 It can be seen that it shows the morphological and structural changes of soy protein isolate, folic acid modified soy protein isolate, and folic acid modified soy protein isolate-chitosan electrostatic composite particles.

[0070] Test Example 4: The targeted delivery of polyunsaturated fatty acids using a double Pickering emulsion prepared in Example 2 was observed using an optical microscope, and the results are as follows: Figure 4 As shown. According to Figure 4 It can be seen that the double Pickering emulsion particles, which show targeted delivery of polyunsaturated fatty acids, are uniformly dispersed and exhibit a typical W1 / O / W2 dual structure. The oil phase is the continuous phase, and the internal aqueous phase is uniformly dispersed in flaxseed oil.

[0071] Test Example 5: The particle size distribution of the targeted delivery polyunsaturated fatty acid double Pickering emulsion prepared in Example 2 was tested, and the results are as follows: Figure 5 As shown. According to Figure 5It can be seen that the double Pickering emulsion, which shows targeted delivery of polyunsaturated fatty acids, has a uniform particle size distribution with a particle size of about 1 μm.

[0072] Test Example 6: The stability of the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids prepared in Example 2 was tested. The double Pickering emulsion for targeted delivery of polyunsaturated fatty acids was stored at 4°C in the dark for 50 days, and the stability changes before and after storage were compared. The results are as follows: Figure 6 As shown. According to Figure 6 It can be seen that the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids showed no stratification, no demulsification, and no oxidative deterioration after storage, proving that the double Pickering emulsion for targeted delivery of polyunsaturated fatty acids prepared in this invention has excellent stability.

[0073] The embodiments of the present invention have been described above; however, these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the above embodiments of the present invention without inventive effort are within the protection scope of the present invention.

Claims

1. A double Pickering emulsion for targeted delivery of polyunsaturated fatty acids, characterized in that, It is a W1 / O / W2 type double Pickerling emulsion, which consists of an inner aqueous phase, an oil phase, and an outer aqueous phase from the inside out; The oil phase includes oils containing polyunsaturated fatty acids; The oil-water interface between the internal aqueous phase and the oil phase contains folic acid-modified soy protein isolate-chitosan electrostatic composite particles. The oil-water interface between the oil phase and the external aqueous phase contains folic acid-modified soy protein isolate-chitosan electrostatic composite particles.

2. The dual Pickering emulsion for targeted delivery of polyunsaturated fatty acids according to claim 1, characterized in that, The oils containing polyunsaturated fatty acids include one or more of flaxseed oil, perilla seed oil, sesame oil, safflower oil, and walnut oil.

3. The targeted delivery of polyunsaturated fatty acids in a double Pickering emulsion according to claim 1, characterized in that, The preparation method of the folic acid-modified soy protein isolate-chitosan electrostatic composite particles includes the following steps: (1) Mix soy protein isolate and phosphate buffer solution, wherein the phosphate buffer solution has a pH of 7.4 and a molar concentration of 0.1M, to obtain soy protein isolate solution; (2) Folic acid, dimethyl sulfoxide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were mixed and activated to obtain an activated folic acid solution; (3) The activated folic acid solution and the soy protein isolate solution are mixed and subjected to an amidation reaction to obtain folic acid modified soy protein isolate; (4) The folic acid modified soy protein isolate is mixed with chitosan, glacial acetic acid and water to obtain the folic acid modified soy protein isolate-chitosan electrostatic composite particles.

4. The targeted delivery of polyunsaturated fatty acids in a double Pickering emulsion according to claim 3, characterized in that, The molar ratio of folic acid to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1.2~1.5; The molar ratio of folic acid to N-hydroxysuccinimide is 1:1.2~1.5; The molar ratio of folic acid to soy protein isolate is 1:30~50; The mass ratio of the folic acid-modified soy protein isolate to the total mass of chitosan, glacial acetic acid, and water is 1:0.5~2. The mass ratio of chitosan to the total volume of glacial acetic acid and water is (0.8~1.8) mg:1 mL.

5. The targeted delivery of polyunsaturated fatty acids in a double Pickering emulsion according to claim 3, characterized in that, The amidation reaction was carried out under light-protected conditions; The amidation reaction was carried out at room temperature for 4 to 6 hours. The amidation reaction was carried out under stirring conditions.

6. The targeted delivery of polyunsaturated fatty acids in a double Pickering emulsion according to claim 1, characterized in that, The folic acid-modified soy protein isolate-chitosan electrostatic composite particles are the sole stabilizer or emulsifier for targeted delivery of polyunsaturated fatty acids in dual Pickering emulsions.

7. The targeted delivery of polyunsaturated fatty acids in a double Pickering emulsion according to claim 1, characterized in that, The mass ratio of the folic acid-modified soy protein isolate-chitosan electrostatic composite particles to the oil phase is 0.1~5:40~60; The volume ratio of the internal water phase to the external water phase is 1:4.5~5.5; The mass ratio of the total mass of the internal water phase to the external water phase and the mass of the oil phase is 35~60:40~60.

8. A method for preparing a double Pickering emulsion for targeted delivery of polyunsaturated fatty acids, characterized in that, The targeted delivery polyunsaturated fatty acid double Pickering emulsion is the targeted delivery polyunsaturated fatty acid double Pickering emulsion according to any one of claims 1 to 7, comprising the following steps: The oil phase, inner aqueous phase, outer aqueous phase, and folic acid-modified soy protein isolate-chitosan electrostatic composite particles were mixed and sheared to obtain the targeted delivery of polyunsaturated fatty acids in the double Pickering emulsion.

9. The application of a double Pickering emulsion for targeted delivery of polyunsaturated fatty acids in the preparation of cell-targeting carriers or colon-targeting carriers, characterized in that, The targeted delivery polyunsaturated fatty acid double Pickering emulsion is the targeted delivery polyunsaturated fatty acid double Pickering emulsion according to any one of claims 1 to 7 or the targeted delivery polyunsaturated fatty acid double Pickering emulsion obtained by the preparation method according to claim 8.

10. The application according to claim 9, characterized in that, The cell-targeting or colon-targeting carrier is loaded with one or more of water-soluble and lipid-soluble active substances.

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