A small intestine targeted adhesive gel microsphere delivery system and its preparation method and application

Abstract

The application discloses a small intestine targeted gel microsphere delivery system. The system is composed of a zein / A-type gelatin / OSA starch nanogel particle inner core loaded with functional factors and a whey protein isolate / polyphenol / oxidized starch gel microsphere shell. The functional factor encapsulation rate of the delivery system is 59-98%, the leakage amount of the functional factor in the stomach and the small intestine ranges from 7.05 to 21.98%, the adhesion thickening of the unit volume small intestine targeted gel microsphere to the small intestine mucus layer is 1.15-4.31 nm, and the nanogel particle penetration efficiency Papp of the small intestine mucus and cell transport is 3.63*10 ‑6 ~5.64*10 ‑5 cm / s. The application improves the small intestine targeted delivery efficiency of the microsphere to the functional factors by regulating the type and degree of the intermolecular interaction of the components, and can be applied to the small intestine targeted absorption drugs related to the regulation of intestinal chronic inflammation and the enhancement of immunity.

Description

Technical Field

[0001] This invention belongs to the field of small intestine targeted controlled release delivery and intestinal transport and absorption technology of functional factors, and specifically relates to a method for preparing a small intestine targeted gel microsphere controlled release delivery system and its application in effectively overcoming mucus and cell layer barriers and improving the bioavailability of functional factors. Background Technology

[0002] Functional factors, such as polysaccharides, polyphenols, and vitamins, have attracted much attention due to their various bioactive functions, including anti-inflammatory, antioxidant, anti-tumor, and immune-enhancing effects. These functional factors can improve the body's sub-health state by influencing the metabolic cycle of body cells, regulating the secretion of anti-inflammatory factors and neurotransmitters, and modulating cellular immune responses. (YAN Z, ZHANG T, LIANG Y, ET AL. Dietary Bioactive Lipids: A Review on Absorption, Metabolism, and Health Properties[J]. Journal of Agricultural and Food Chemistry, 2021, 69(32): 8929-8943.)

[0003] However, during the gastrointestinal tract's transport, digestion, absorption, and utilization process, multiple physiological barriers severely restrict the bioavailability of functional factors in the small intestine, the core absorption site. First, functional factors face the influence of harsh pH environments and various digestive enzymes in different parts of the gastrointestinal tract. Second, the continuous secretion, shedding, transport, and renewal of the small intestinal mucus layer, along with its complex gel network structure and negative charge characteristics, pose significant challenges to the continuous release of functional factors from the mucus layer and their penetration into the intestinal epithelial cell layer. Finally, functional factors need to be absorbed by the small intestinal epithelial cells and undergo complex endocytosis and exocytosis to reach the capillary network of the lamina propria before entering the human circulatory system for efficient utilization. (SHUANG L, ZHAO D, LIU X, et al. The stomach, small intestine, and colon-specific gastrointestinal tractdelivery systems for bioactive nutrients[J]. Advances in Colloid and Interface Science, 2025, 341103503.; BAO, C.; LIU, B.; LI, B., et al., Enhanced Transport of Shape and Rigidity-Tuned alpha-Lactalbumin Nanotubes across Intestinal Mucus and Cellular Barriers. Nano Letters, 2020, 20 (2), 1352-1361.) Therefore, it is necessary to adopt a suitable encapsulation method to effectively encapsulate functional factors, and then target and deliver them to the small intestine. Through a certain mechanism, they are continuously released in the small intestinal mucus, and then efficiently penetrate the small intestinal mucus layer and be transported and absorbed by intestinal epithelial cells across the membrane, so as to meet the application requirements of efficient targeted controlled release delivery and absorption of functional factors in the small intestine.

[0004] Currently, there is limited research on constructing controlled-release delivery systems that load functional factors to enhance small intestinal controlled release, mucus penetration, and cell transport. Yang Liqun et al. (Patent publication CN201811006436.6: A small intestinal-targeted absorption and biodegradable glycogen derivative, its preparation method, and its tea polysaccharide-loaded nanoparticles) prepared small intestinal-targeted absorption and biodegradable glycogen derivative-loaded tea polysaccharide nanoparticles, which can promote the small intestinal absorption efficiency of tea polysaccharides. However, its small intestinal targeted delivery characteristics are weak, and its mucus penetration and cell transport efficiency still have certain defects. Wang et al. (WANG, Y.; ZHAO, Y.; CUI, Y., et al., Overcoming multiple gastrointestinal barriers by bilayer-modified hollow mesoporous silica nanocarriers. Acta Biomaterialia, 2018, 65, 405-416.) constructed nanoparticles for the delivery of paclitaxel and quercetin by modifying mesoporous silica with CPPs-targeted peptides and embedding it with hydrophilic succinate casein. However, its small intestine-targeted controlled release capability was weak and there were certain safety risks. Yang Fang et al. (Patent publication CN202210720629.8: A chestnut shell polyphenol sustained-release material for small intestine-targeted release and its preparation and application) prepared a chestnut shell polyphenol sustained-release material for small intestine-targeted release, which improved the release amount of chestnut shell polyphenols at the small intestine absorption site. However, its mucus penetration and transmembrane transport and absorption capabilities of small intestinal epithelial cells were weak. Therefore, the design and construction of delivery systems for achieving targeted controlled release of functional factors in the small intestine and enhancing the penetration of intestinal mucus and transport and absorption by epithelial cells still face bottlenecks such as weak targeting ability and low intestinal transport and absorption efficiency. Summary of the Invention

[0005] To address the issues of weak small intestine targeting capability and low small intestine transport and absorption efficiency in existing delivery systems, the present invention aims to provide a small intestine-targeting adhesive gel microsphere delivery system, which consists of a starch and protein composite assembled nanoparticle core loaded with functional factors and a whey protein isolate / polyphenol / oxidized starch gel microsphere shell.

[0006] Another objective of this invention is to provide a method for preparing the aforementioned small intestinal targeted adhesion gel microsphere delivery system. The gel microsphere delivery system of this invention mainly involves preparing nanoparticles loaded with functional factors from zein / type A gelatin / OSA starch using a microfluidic method, followed by encapsulation with whey protein isolate / polyphenols / oxidized starch to form small intestinal targeted gel microspheres. By controlling the proportions of the components in the delivery system and synergistically regulating the processing conditions, gel microspheres with different small intestinal targeted adhesion control, small intestinal mucus penetration, and cell transport capabilities are obtained. Furthermore, the functional factors released from the gel microspheres are transported by small intestinal epithelial cells to regulate the body's immune metabolism.

[0007] Another objective of this invention is to provide the application of the above-mentioned small intestine targeted adhesion gel microsphere delivery system in the preparation of small intestine targeted absorption drugs.

[0008] The objective of this invention is achieved through the following solution:

[0009] A small intestine-targeted adhesion gel microsphere delivery system comprises a starch and protein composite nanoparticle core loaded with functional factors and a whey protein isolate / polyphenol / oxidized starch gel microsphere shell. The mass ratio of the nanoparticle core to the gel microsphere shell is 1:(0.5~2). The nanoparticle core is composed of functional factors, zein, type A gelatin, and OSA starch in a mass ratio of 1:(1~20):(1~50):(2~50). The gel microsphere shell is composed of whey protein isolate, polyphenols, and oxidized starch in a mass ratio of 1:(0.01~0.10):(0.10~0.50).

[0010] A method for preparing the above-mentioned small intestinal targeted adhesion gel microsphere delivery system includes the following steps:

[0011] (1) Functional factors and zein are co-dissolved in an ethanol-water solution to form solution A, which is then introduced into a microfluidic chip along with water. Then, a heat-treated A-type gelatin aqueous solution is introduced into the microfluidic chip. Finally, a heat-treated OSA starch solution is introduced into the microfluidic chip. After a constant-temperature reaction, a starch and protein composite nanoparticle solution loaded with functional factors is prepared.

[0012] (2) The composite assembled nanoparticle solution prepared in step (1) is thoroughly mixed with whey protein isolate. The mixed solution is then added to vegetable oil for shearing and mixing. Then, a heat-treated polyphenol solution is added for reaction. After the reaction is complete, a heat-treated oxidized starch solution is added for reaction. After the reaction is complete, the gel microspheres are allowed to stand and precipitate. All vegetable oil is removed by washing with organic solvent and then dried to obtain the small intestine targeted adhesion gel microsphere delivery system.

[0013] The functional factor mentioned in step (1) is one of curcumin, chlorogenic acid and quercetin; the molecular weight of the zein mentioned in step (1) is 20~50 kDa; the volume fraction of ethanol in the aqueous ethanol solution is 25-95%; the mass concentration of the functional factor in solution A is 1~30%.

[0014] Preferably, the molecular weight of the type A gelatin mentioned in step (1) is 35~75 kDa, and the heat-treated type A gelatin aqueous solution is obtained by heating at 50-80℃ for 0.5-2 hours, with a concentration of 8~40 mg / mL; the degree of substitution of the OSA starch mentioned in step (1) is 0.01~0.03, and the molecular weight is 2.43×10 5 ~9.51×10 5 The heat-treated OSA starch aqueous solution is obtained by heating at 75-95°C for 5-30 min, and has a concentration of 16-48 mg / mL.

[0015] Preferably, in step (1), the ratio of the flow rates of solution A, water, heat-treated type A gelatin solution, and heat-treated OSA starch solution into the microfluidic chip is 1:1-3:2-3:6-9;

[0016] More preferably, in step (1), the rates at which solution A, water, heat-treated type A gelatin solution, and heat-treated OSA starch solution are introduced into the microfluidic chip are 9 μL / min, 9 μL / min, 25 μL / min, and 80 μL / min, respectively.

[0017] The isothermal reaction mentioned in step (1) refers to a constant temperature reaction at 30-60℃ for 0.5-2 hours.

[0018] The whey protein isolate described in step (2) has a molecular weight of 20-100 kDa; the degree of oxidized starch substitution is 0.01-0.03, and the molecular weight is 3.60 × 10⁻⁶. 5 ~8.02×10 5 g / mol; the polyphenol is at least one of tannic acid and epigallocatechin gallate.

[0019] The total mass concentration of nanoparticles and whey protein isolate in the mixture after mixing the composite assembled nanoparticle solution with whey protein isolate in step (2) is 15-35%.

[0020] The vegetable oil mentioned in step (2) is at least one of corn oil, soybean oil, and peanut oil.

[0021] The amount of vegetable oil used in step (2) is such that the volume ratio of vegetable oil to the mixture is 5-30:1.

[0022] The shearing and mixing mentioned in step (2) refers to stirring and shearing at 65-85℃ for 10-30 minutes, then cooling to room temperature and maintaining stirring and shearing for 20 minutes, wherein the shearing speed is 10000 ~ 16000 r / min.

[0023] Preferably, the polyphenol solution after heat treatment in step (2) is an aqueous solution or aqueous dispersion with a mass concentration of 1 to 20% after being heated at 95 °C for 10 min.

[0024] Preferably, the oxidized starch solution after heat treatment in step (2) is an aqueous suspension with a mass concentration of 1 to 5% after being heated at 95 °C for 10 min.

[0025] The reaction described in step (2) with the addition of the heat-treated polyphenol solution refers to stirring at 1500 rpm for 6-12 hours at 20-40℃; the reaction described in step (2) with the addition of the heat-treated oxidized starch solution refers to stirring at 1500 rpm for 6-12 hours at 20-40℃.

[0026] The organic solvent mentioned in step (2) is at least one of n-hexane, diethyl ether, and acetone.

[0027] Preferably, the prepared nanoparticles have a core size ranging from 75 to 450 nm, a surface contact angle ranging from 61.80 to 72.07°, and a surface potential ranging from -0.20 to -4.78 mV; the small intestine-targeted gel microsphere delivery system has a size ranging from 15 to 45 μm and a surface potential ranging from -15.64 to -24.68 mV; the encapsulation efficiency of functional factors is 59% to 98%, and the leakage of functional factors in the stomach and small intestine ranges from 7.05% to 21.98%; the adhesion and thickening of the small intestine-targeted gel microspheres per unit volume with the small intestinal mucus layer is 1.15 to 4.31 nm; and the nanoparticle penetration efficiency (Papp) for small intestinal mucus and transcellular transport is 3.63 × 10⁻⁶. -6 ~5.64×10 -5 cm / s.

[0028] The aforementioned small intestine-targeted gel microsphere controlled-release delivery system possesses the characteristics of small intestine-targeted controlled release and transport encapsulation of functional factors, which can promote the development and application of drugs for small intestine-targeted absorption.

[0029] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0030] (1) This invention utilizes zein / type A gelatin / OSA starch to prepare nanoparticles loaded with functional factors, and then uses whey protein isolate / polyphenol / oxidized starch to prepare small intestine targeted gel microspheres. The product has good small intestine targeted adhesion, mucus penetration and transmembrane transport and absorption characteristics. The process is simple, safe and non-toxic.

[0031] (2) The small intestine targeted gel microspheres prepared in this invention can increase the adhesion and controlled release ability of functional factors by adjusting the interaction ability between the small intestine targeted gel microspheres and the small intestine mucus layer, thereby increasing the adhesion thickness of the small intestine targeted gel microspheres to the small intestine mucus layer by 4.31 nm per unit volume.

[0032] (3) The small intestinal targeted gel microspheres prepared in this invention effectively improve the small intestinal mucus penetration and transcellular transport efficiency of functional substances by adjusting the characteristics of starch and protein composite assembly of nanoparticles, achieving an efficiency Papp of 5.64 × 10⁻⁶. -5 cm / s.

[0033] (4) The small intestine-targeting gel microspheres prepared in this invention can regulate the body's immune metabolism by efficiently delivering functional factors for absorption and transport. They can be applied to the development of small intestine-targeting absorption drugs that regulate chronic intestinal inflammation and enhance immunity. Attached Figure Description

[0034] Figure 1 The effects of different component ratios and processing conditions on the particle size of the delivery system in Examples 1-12 are shown.

[0035] Figure 2 The effects of different component ratios and processing conditions on the small intestinal adhesion properties of the delivery system in Examples 1-12 are shown.

[0036] Figure 3 The effects of different component ratios and processing conditions in Examples 1-12 on the efficiency of small intestinal mucus penetration and transcellular transport in the delivery system.

[0037] Figure 4 This is a schematic diagram illustrating the working principle of the microfluidic chip for preparing nanoparticles with functional loading factors according to the present invention. Figure 4 The components represented by each number are as follows:

[0038] 1. First phase sample inlet; 2. Second phase sample inlet; 3. First shear port; 4. Third phase sample inlet; 5. Second shear port; 6. Fourth phase sample inlet; 7. Third shear port; 8. Constant temperature reaction zone; 9. Outlet.

[0039] Figure 5The images shown are laser confocal microscopy images from Examples 4, 5, and 9. Nanoparticles carrying functional factors were fluorescently labeled with Cy5 dye (640 nm / 670 nm), and the small intestine-targeting adhesion gel microsphere assembly materials—whey protein isolate and oxidized starch—were labeled with rhodamine B isothiocyanate (550 nm / 580 nm). Detailed Implementation

[0040] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0041] Particle size testing method: Dynamic light scattering was used to measure the particle size of the samples. At 37℃, dispersions of different materials at 1 mg / mL were prepared, and particle size was measured at different pH values ​​(7).

[0042] Surface contact angle test method: The sample material is prepared into a circular thin film with d=1 mm and placed on a glass slide. The interface contact angle is measured by a video optical contact angle meter. A drop of water (3 μL) is slowly dropped onto the surface of the thin film by a stainless steel syringe. After equilibration for 30 s, the image is captured by a high-speed camera. The droplet profile is fitted by the variable weight least squares method using the binary image edge feature method. The interface contact angle is calculated by the Young-Laplace differential equation iterative method.

[0043] Surface potential testing method: Under the conditions of 37℃ and pH=7, 1 mg / mL dispersions of different materials were prepared. A He-Ne 633 nm laser was used as the light source, the measurement angle was set to 173°, the equilibration time was 120 s, and each sample was scanned 3 times to obtain the Zeta-potential value of the sample.

[0044] Functional factor encapsulation efficiency test method: After preparing the gel microsphere delivery system loaded with functional factors, collect all supernatants during the preparation process and determine the amount of free functional factors in the supernatant.

[0045]

[0046] Methods for testing the leakage of functional factors in the stomach and small intestine: The controlled release performance of functional factors in simulated oral cavity, stomach and small intestine was determined by using an in vitro method that simulates the physiological environment of the digestive tract. Specific methods: (1) Simulated oral digestion stage: 100 mg sample was mixed with 10 mL of simulated saliva containing 6% mucin (SSF, catalog number MSS0511, Shanghai Weikun Biotechnology Co., Ltd.), pH was adjusted to 6.80, and digested for 2 min under shaking at 100 rpm; (2) Simulated gastric digestion stage: 10 mL of simulated gastric juice containing 4000 U / mL pepsin (SGF, catalog number MSG0250, Shanghai Weikun Biotechnology Co., Ltd.) with pH 1.20 was mixed with the sample (10 mL) after simulated oral digestion. The pH value in the stomach simulated its pH environment in the fasting state, i.e., it was adjusted to 1.20, and digested under shaking at 37℃ and 100 rpm for 2 h; (3) Simulated small intestinal digestion stage: the sample (20 mL) after digestion by simulated gastric juice was adjusted to pH = 6.80. Subsequently, 20 mL of simulated small intestinal fluid consisting of pH 6.80 containing 200 U / mL trypsin solution, 20 mM bile salt solution, and buffer solution (6.8 mM KCl, 0.8 mM KH2PO4, 38.4 mM NaCl, 0.33 mM MgCl2(H2O)6, 85 mM NaHCO3, and 0.6 CaCl2(H2O)2) was added under continuous stirring. Digestion was carried out at 100 rpm for 6 h with continuous shaking. Samples were taken after the entire simulated digestion process, and the supernatant was collected by centrifugation at 3000 rpm for 5 min. The content of functional factors in the supernatant was measured to calculate the leakage of functional factors in the stomach and small intestine.

[0047] Method for measuring the adhesion thickening of small intestinal targeted gel microspheres to the small intestinal mucus layer per unit volume: The adhesion thickening of small intestinal targeted gel microspheres to the small intestinal mucus layer per unit volume after simulated gastric digestion was measured using a Quartz Crystal Microbalance with Dissipation (QCM-D). After cleaning the gold-plated sensor, the shifts in resonant frequency ΔF and dissipation ΔD were recorded at 25°C and a flow rate of 150 μL / min. Different samples were prepared using PBS buffer (pH 6.8). The experiment included the following steps: (1) Rinse the sensor with buffer (pH=6.8) to achieve a stable baseline; form a mucin layer with 1 mg / mL mucin solution (pH=6.8); (2) Rinse the sensor with buffer to remove unbound mucin molecules; (3) Add 0.6 mg / mL dispersion of different samples (pH=6.8) to the mucin layer at a flow rate of 150 μL / min; (4) Rinse with buffer (pH=6.8) to remove unbound nanoparticles, and calculate the adhesion and thickening of small intestinal targeted gel microspheres to the small intestinal mucus layer per unit volume according to the viscoelastic model.

[0048] Methods for assessing the efficiency of functional factors penetrating intestinal mucus and transcellular transport: The Transwell assay was used in Caco-2 and HT29-MTX-E12 cell models to determine the efficiency of functional factors penetrating intestinal mucus and transcellular transport. Cells in logarithmic growth phase (Caco-2 and HT29-MTX-E12 cells) were selected and resuspended in fresh complete culture medium to form 2 × 10⁶ cells. 5 Cells / mL cell suspension (9:1 ratio). Add 0.2 mL of cell suspension to the upper chamber of a 24-well polycarbonate Transwell membrane (0.4 μm, 0.33 cm⁻¹). 2 (Filter membrane surface area), add 0.6 mL of culture medium to the lower chamber of the Transwell chamber, change the culture medium every two days, and monitor the change of cell resistance before changing the medium. Culture for 21 days under standard culture conditions of 37°C, 95% humidity and 5% carbon dioxide, and select cells with resistance > 300 Ω·cm. 2The Transwell chamber was used as a Caco-2 / HT29-MTX-E12 co-culture cell model for subsequent experiments. The efficiency of transmembrane transport of cells was determined using the constructed Caco-2 / HT29-MTX-E12 cell co-culture model. 0.2 mL of Cy5 fluorescently labeled composite assembled nanoparticles (0.2 mg / mL, based on zein concentration) was added to the upper chamber and incubated with the co-cultured cells. 0.6 mL of DMEM medium was added to the lower chamber. At different incubation time points (0, 1, 2, 4, 6, and 8 h), 0.2 mL of solution was taken from the lower chamber and replenished with the corresponding volume of DMEM medium. Fluorescence intensity was measured using flow cytometry, and the sample concentration passing through the membrane per unit time was calculated. The transmembrane transport efficiency of intestinal epithelial cells was expressed as the apparent permeability coefficient (Papp, cm / s), calculated using the following formula:

[0049]

[0050] Where, d Q / d t denoted as , where is the slope of the straight line representing the change in concentration in the lower chamber over time; A is the membrane surface area of ​​the Transwell (0.33 cm²). 2 C0 represents the initial concentration of the fluorescently labeled nanoparticles.

[0051] Example 1

[0052] (1) Curcumin and 20 kDa zein were dissolved in 75% ethanol water at a mass ratio of 1:1 to prepare a 30% mass concentration solution (calculated as curcumin). This solution, along with deionized water, was simultaneously introduced into the first and second phase sample loading ports of the microfluidic chip at a rate of 9 μL / min. Then, at a mass ratio of functional factor to type A gelatin of 1:1, an 8 mg / mL 75 kDa type A gelatin solution was heated at 50°C for 2 h and introduced into the third phase sample loading port at a rate of 25 μL / min. Finally, at a mass ratio of functional factor to OSA starch of 1:2, 16 mg / mL type A gelatin with a degree of substitution of 0.01 and a molecular weight of 2.43 × 10⁻⁶ was added. 5 After heating the OSA starch solution at 75 °C for 10 min, it was added to the fourth phase injection port at a rate of 80 μL / min. After reacting at a constant temperature of 40 °C for 0.5 h, a starch-protein composite nanoparticle solution loaded with functional factors was prepared.

[0053] (2) Prepare an aqueous solution with a total mass concentration of 15% by mixing the nanoparticles obtained in step (1) and whey protein isolate of 20 kDa at a mass ratio of 2.22:1. Add the solution dropwise to corn oil at a water-oil ratio of 20:1 at 10000 r / min. Stir at 75°C for 20 min, then cool to room temperature and stir for another 20 min. Then, add a 1% tannic acid solution heated at 95°C for 10 min at a mass ratio of whey protein isolate to tannic acid of 1:0.01 and react at 25°C for 9 h. Then, add a 1% tannic acid solution with a degree of substitution of 0.01 and a molecular weight of 3.60 × 10⁻⁶ at a mass ratio of whey albumin to oxidized starch of 1:0.10 and a mass concentration of 1% tannic acid solution heated at 95°C for 10 min. 5 A solution of oxidized starch at a concentration of g / mol was reacted at 25 °C for 12 h to achieve a mass ratio of nanoparticle core to gel microsphere shell of 1:0.5. After the reaction, the gel microspheres were allowed to settle and precipitate. After washing with n-hexane to remove excess corn oil, the microspheres were dried to obtain a small intestine-targeted adhesion gel microsphere delivery system.

[0054] The nanoparticles were tested and found to have a core size of 75 nm, a surface contact angle of 69.85°, and a surface potential of -0.20 mV. The small intestine-targeted gel microsphere delivery system had a size of 17 μm and a surface potential of -16.75 mV. The encapsulation efficiency of functional factors was 65%, and the leakage of functional factors in the stomach and small intestine was 23.7%. The adhesion and thickening of the small intestine-targeted gel microspheres to the small intestinal mucus layer per unit volume was 3.45 nm. The nanoparticle penetration efficiency (Papp) for small intestinal mucus and transcellular transport was 2.25 × 10⁻⁶. -5 cm / s.

[0055] Example 2

[0056] (1) Curcumin and 50 kDa zein were mixed in 75% ethanol water at a mass ratio of 1:5 to prepare a 30% mass concentration solution (calculated as curcumin). The solution was then introduced into the first and second phase sample loading ports of the microfluidic chip at a rate of 9 μL / min, along with water. Then, at a mass ratio of 1:5 between the functional factor and type A gelatin, an 8 mg / mL 35 kDa type A gelatin solution was heated at 60℃ for 0.5 h and introduced into the third phase sample loading port at a rate of 25 μL / min. Finally, at a mass ratio of 1:5 between the functional factor and OSA starch, a 16 mg / mL solution with a degree of substitution of 0.02 and a molecular weight of 4.45 × 10⁻⁵ was added. 5 After heating the OSA starch solution at 95 °C for 5 min, it was added to the fourth phase injection port at a rate of 80 μL / min. After reacting at 30 °C for 2 h, a starch-protein composite nanoparticle solution loaded with functional factors was prepared.

[0057] (2) Prepare an aqueous solution with a total mass concentration of 35% by mixing the nanoparticles obtained in step (1) and 60 kDa whey protein isolate at a mass ratio of 1.2:1. Add the solution dropwise to soybean oil at a water-oil ratio of 5:1 at 16000 r / min. Stir at 65°C for 30 min and then cool to room temperature while stirring for 20 min. Then, add a 1% tannic acid solution heated at 95°C for 10 min at a mass ratio of whey protein isolate to tannic acid of 1:0.10 and react at 20°C for 12 h. Then, add a 5% tannic acid solution with a degree of substitution of 0.01 and a molecular weight of 3.60 × 10⁻⁶, heated at 95°C for 10 min at a mass ratio of whey albumin to oxidized starch of 1:0.10. 5 A solution of oxidized starch at a concentration of g / mol was reacted at 20°C for 12 h to achieve a mass ratio of 1:1 between the core of the nanoparticles and the shell of the gel microspheres. After the reaction, the gel microspheres were allowed to settle and precipitate. After washing with n-hexane to remove excess corn oil, the microspheres were dried to obtain a small intestine-targeted adhesion gel microsphere delivery system.

[0058] The nanoparticles were tested and found to have a core size of 230 nm, a surface contact angle of 65.37°, and a surface potential of -1.15 mV. The small intestine-targeted gel microsphere delivery system had a size of 22 μm and a surface potential of -18.63 mV. The encapsulation efficiency of functional factors was 71%, and the leakage of functional factors in the stomach and small intestine was 15.52%. The adhesion and thickening of the small intestine-targeted gel microspheres to the small intestinal mucus layer per unit volume was 4.03 nm. The nanoparticle penetration efficiency (Papp) for small intestinal mucus and transcellular transport was 7.75 × 10⁻⁶. -6 cm / s.

[0059] Example 3

[0060] (1) Curcumin and 20 kDa zein were mixed in 75% ethanol water at a mass ratio of 1:1 to prepare a 1% mass concentration solution (calculated as curcumin). The solution was then introduced into the first and second phase sample ports of the microfluidic chip at a rate of 9 μL / min, along with water. Then, a 16 mg / mL 35 kDa type A gelatin solution was heated at 80℃ for 1 h and introduced into the third phase sample port at a rate of 25 μL / min, with a mass ratio of functional factor to type A gelatin of 1:1. Finally, a 16 mg / mL type A gelatin solution with a degree of substitution of 0.02 and a molecular weight of 4.45 × 10⁻⁵ was prepared into a solution with a mass ratio of functional factor to OSA starch of 1:25. 5 After heating the OSA starch solution at 95 °C for 10 min, it was added to the fourth phase injection port at a rate of 80 μL / min. After reacting at a constant temperature of 60 °C for 0.5 h, a starch-protein composite nanoparticle solution loaded with functional factors was prepared.

[0061] (2) Prepare an aqueous solution with a total mass concentration of 15% by mixing the nanoparticles obtained in step (1) and 40 kDa whey protein isolate at a mass ratio of 0.9:1. Add the solution dropwise to peanut oil at a mass ratio of 30:1 at 16000 r / min. Stir at 75°C for 20 min and then cool to room temperature while stirring for another 20 min. Then, add a 1% epigallocatechin gallate solution heated at 95°C for 10 min at a mass ratio of 1:0.05 to whey protein isolate and epigallocatechin gallate. React at 40°C for 6 h. Then, add a 1% solution with a degree of substitution of 0.03 and a molecular weight of 8.02 × 10⁻⁶ to whey albumin and oxidized starch at a mass ratio of 1:0.30 to whey albumin and oxidized starch heated at 95°C for 10 min at a mass concentration of 1%. 5 A solution of oxidized starch at a concentration of g / mol was reacted at 40℃ for 6 hours to achieve a mass ratio of nanoparticle core to gel microsphere shell of 1:1.5. After the reaction, the gel microspheres were allowed to settle and precipitate. After washing with acetone to remove excess corn oil, the microspheres were dried to obtain a small intestine-targeted adhesion gel microsphere delivery system.

[0062] The nanoparticles were found to have a core size of 115 nm, a surface contact angle of 63.39°, and a surface potential of -4.13 mV. The small intestine-targeted gel microsphere delivery system had a size of 31 μm and a surface potential of -20.19 mV. The encapsulation efficiency of functional factors was 61%, and the leakage of functional factors in the stomach and small intestine was 10.06%. The adhesion and thickening of the small intestine-targeted gel microspheres to the small intestinal mucus layer per unit volume was 3.97 nm. The nanoparticle penetration efficiency (Papp) for small intestinal mucus and transcellular transport was 1.16 × 10⁻⁶. -5 cm / s.

[0063] Example 4

[0064] (1) Curcumin and 50 kDa zein were mixed in 75% ethanol water at a mass ratio of 1:20 to prepare a 30% mass concentration solution (calculated as curcumin). The solution was then introduced into the first and second phase sample ports of the microfluidic chip at a rate of 9 μL / min, along with water. Then, at a mass ratio of 1:50 between the functional factor and type A gelatin, an 8 mg / mL 75 kDa type A gelatin solution was heated at 60℃ for 2 h and introduced into the third phase sample port at a rate of 25 μL / min. Finally, at a mass ratio of 1:50 between the functional factor and OSA starch, a 48 mg / mL solution with a degree of substitution of 0.03 and a molecular weight of 9.51 × 10⁻⁶ was prepared. 5 After heating the OSA starch solution at 75 °C for 30 min, it was added to the fourth phase injection port at a rate of 80 μL / min. After reacting at a constant temperature of 40 °C for 1 h, a starch-protein composite nanoparticle solution loaded with functional factors was prepared.

[0065] (2) Prepare an aqueous solution with a total mass concentration of 35% by mixing the nanoparticles obtained in step (1) and 100 kDa whey protein isolate at a mass ratio of 0.755:1. Add the solution dropwise to corn oil at a water-oil ratio of 20:1 at 10000 r / min. Stir at 85°C for 10 min and then cool to room temperature while stirring for 20 min. Then, add a 10% tannic acid solution heated at 95°C for 10 min at a mass ratio of whey protein isolate to tannic acid of 1:0.01 and react at 25°C for 9 h. Finally, add a 1% tannic acid solution with a degree of substitution of 0.03 and a molecular weight of 8.02 × 10⁻⁶, heated at 95°C for 10 min at a mass ratio of whey albumin to oxidized starch of 1:0.50. 5 A solution of oxidized starch at a concentration of g / mol was reacted at 25°C for 12 h to achieve a mass ratio of nanoparticle core to gel microsphere shell of 1:2. After the reaction, the gel microspheres were allowed to settle and precipitate. After washing with n-hexane to remove excess corn oil, the microspheres were dried to obtain a small intestine-targeted adhesion gel microsphere delivery system.

[0066] The nanoparticles were tested and found to have a core size of 450 nm, a surface contact angle of 61.80°, and a surface potential of -4.78 mV. The small intestine-targeted gel microsphere delivery system had a size of 45 μm and a surface potential of -24.68 mV. The encapsulation efficiency of functional factors was 98%, and the leakage of functional factors in the stomach and small intestine was 7.05%. The adhesion and thickening of the small intestine-targeted gel microspheres to the small intestinal mucus layer per unit volume was 1.15 nm. The nanoparticle penetration efficiency (Papp) for small intestinal mucus and transcellular transport was 3.63 × 10⁻⁶. -6 cm / s.

[0067] Example 5

[0068] (1) Chlorogenic acid and 50 kDa zein were mixed in 25% ethanol water at a mass ratio of 1:20 to prepare a 30% mass concentration solution (calculated as chlorogenic acid). The solution was then introduced into the first and second phase sample ports of the microfluidic chip at a rate of 9 μL / min, along with water. Then, a 40 mg / mL 75 kDa type A gelatin solution was heated at 60℃ for 1 h and introduced into the third phase sample port at a rate of 25 μL / min, with a mass ratio of functional factor to type A gelatin of 1:30. Finally, a 16 mg / mL solution of type A gelatin with a degree of substitution of 0.03 and a molecular weight of 9.51 × 10⁻⁶ was prepared in 25% ethanol water at a mass ratio of functional factor to OSA starch of 1:50. 5 After heating the OSA starch solution at 95 °C for 10 min, it was added to the fourth phase injection port at a rate of 80 μL / min. After reacting at a constant temperature of 40 °C for 1 h, a starch-protein composite nanoparticle solution loaded with functional factors was prepared.

[0069] (2) Prepare an aqueous solution with a total mass concentration of 15% by mixing the nanoparticles obtained in step (1) and 100 kDa whey protein isolate at a mass ratio of 1:1. Add the solution dropwise to soybean oil at a water-oil ratio of 20:1 at 10000 r / min. Stir at 75°C for 20 min and then cool to room temperature while stirring for 20 min. Then, add a 1% tannic acid solution heated at 95°C for 10 min at a mass ratio of whey protein isolate to tannic acid of 1:0.01 and react at 20°C for 12 h. Then, add a 5% tannic acid solution with a degree of substitution of 0.01 and a molecular weight of 3.60 × 10⁻⁶, heated at 95°C for 10 min at a mass ratio of whey albumin to oxidized starch of 1:0.50. 5 A solution of oxidized starch at a concentration of g / mol was reacted at 25°C for 6 hours to achieve a mass ratio of 1:1.5 between the core of the nanoparticles and the shell of the gel microspheres. After the reaction, the gel microspheres were allowed to settle and precipitate. After washing with n-hexane to remove excess corn oil, the microspheres were dried to obtain a small intestine-targeted adhesion gel microsphere delivery system.

[0070] The nanoparticles were found to have a core size of 343 nm, a surface contact angle of 62.73°, and a surface potential of -3.47 mV. The small intestine-targeted gel microsphere delivery system had a size of 39 μm and a surface potential of -17.15 mV. The encapsulation efficiency of functional factors was 95%, and the leakage of functional factors in the stomach and small intestine was 12.19%. The adhesion and thickening of the small intestine-targeted gel microspheres to the small intestinal mucus layer per unit volume was 1.98 nm. The nanoparticle penetration efficiency (Papp) for small intestinal mucus and transcellular transport was 5.54 × 10⁻⁶. -6 cm / s.

[0071] Example 6

[0072] (1) Chlorogenic acid and 20 kDa zein were mixed in 25% ethanol water at a mass ratio of 1:1 to prepare a 1% mass concentration solution (calculated as chlorogenic acid). The solution was then introduced into the first and second phase sample ports of the microfluidic chip at a rate of 9 μL / min, along with water. Then, at a mass ratio of 1:10 between the functional factor and type A gelatin, an 8 mg / mL 50 kDa type A gelatin solution was heated at 60℃ for 0.5 h and introduced into the third phase sample port at a rate of 25 μL / min. Finally, at a mass ratio of 1:10 between the functional factor and OSA starch, a 16 mg / mL solution with a degree of substitution of 0.02 and a molecular weight of 4.45 × 10⁻⁶ was prepared. 5 After heating the OSA starch solution at 95 °C for 10 min, it was added to the fourth phase injection port at a rate of 80 μL / min. After reacting at a constant temperature of 40 °C for 1 h, a starch-protein composite nanoparticle solution loaded with functional factors was prepared.

[0073] (2) Prepare a 15% aqueous solution by mixing the nanoparticles obtained in step (1) with 100 kDa whey protein isolate at a mass ratio of 1.4:1. Add the solution dropwise to corn oil at a water-oil ratio of 20:1 at 10000 r / min. Stir at 75°C for 20 min and then cool to room temperature while stirring for another 20 min. Then, add a 1% tannic acid solution heated at 95°C for 10 min at a mass ratio of whey protein isolate to tannic acid of 1:0.10 and react at 25°C for 9 h. Finally, add a 1% tannic acid solution with a degree of substitution of 0.01 and a molecular weight of 3.60 × 10⁻⁶, heated at 95°C for 10 min at a mass ratio of whey albumin to oxidized starch of 1:0.30. 5 A solution of oxidized starch (g / mol) was reacted at 40℃ for 6 h to achieve a mass ratio of 1:1 between the nanoparticle core and the gel microsphere shell. After the reaction, the gel microspheres were allowed to settle and precipitate. After washing with ether to remove excess corn oil, the microspheres were dried to obtain a small intestine-targeted adhesion gel microsphere delivery system.

[0074] The nanoparticles were tested and found to have a core size of 135 nm, a surface contact angle of 64.63°, and a surface potential of -2.75 mV. The small intestine-targeted gel microsphere delivery system had a size of 28 μm and a surface potential of -18.95 mV. The encapsulation efficiency of functional factors was 63%, and the leakage of functional factors in the stomach and small intestine was 11.45%. The adhesion and thickening of the small intestine-targeted gel microspheres to the small intestinal mucus layer per unit volume was 3.35 nm. The nanoparticle penetration efficiency (Papp) for small intestinal mucus and transcellular transport was 3.25 × 10⁻⁶. -5 cm / s.

[0075] Example 7

[0076] (1) Chlorogenic acid and 50 kDa zein were mixed in 25% ethanol water at a mass ratio of 1:20 to prepare a 30% mass concentration solution (calculated as chlorogenic acid). The solution was then introduced into the first and second phase sample ports of the microfluidic chip at a rate of 9 μL / min, along with water. Then, at a mass ratio of 1:30 between the functional factor and type A gelatin, an 8 mg / mL 50 kDa type A gelatin solution was heated at 60℃ for 1 h and introduced into the third phase sample port at a rate of 25 μL / min. Finally, at a mass ratio of 1:50 between the functional factor and OSA starch, 48 mg / mL type A gelatin with a degree of substitution of 0.02 and a molecular weight of 4.45 × 10⁻⁶ was added. 5 After heating the OSA starch solution at 95 °C for 10 min, it was added to the fourth phase injection port at a rate of 80 μL / min. After reacting at 50 °C for 1 h, a starch-protein composite nanoparticle solution loaded with functional factors was prepared.

[0077] (2) Prepare an aqueous solution with a total mass concentration of 35% by mixing the nanoparticles obtained in step (1) and 20 kDa whey protein isolate at a mass ratio of 2.3:1. Add the solution dropwise to corn oil at a water-oil ratio of 5:1 at 16000 r / min. Stir at 65°C for 30 min and then cool to room temperature and stir for 20 min. Then, add a 10% epigallocatechin gallate solution heated at 95°C for 10 min at a mass ratio of whey protein isolate to epigallocatechin gallate of 1:0.05 and react at 25°C for 9 h. Then, add a 1% solution of epigallocatechin gallate with a mass concentration of 0.01 and a molecular weight of 3.60 × 10⁻⁶, heated at 95°C for 10 min at a mass ratio of whey albumin to oxidized starch of 1:0.10. 5 A solution of oxidized starch at a concentration of g / mol was reacted at 25°C for 12 h to achieve a mass ratio of nanoparticle core to gel microsphere shell of 1:0.5. After the reaction, the gel microspheres were allowed to settle and precipitate. After washing with n-hexane to remove excess corn oil, the microspheres were dried to obtain a small intestine-targeted adhesion gel microsphere delivery system.

[0078] The nanoparticles were found to have a core size of 392 nm, a surface contact angle of 62.33°, and a surface potential of -3.95 mV. The small intestine-targeted gel microsphere delivery system had a size of 19 μm and a surface potential of -17.67 mV. The encapsulation efficiency of functional factors was 79%, and the leakage of functional factors in the stomach and small intestine was 14.45%. The adhesion and thickening of the small intestine-targeted gel microspheres to the small intestinal mucus layer per unit volume was 2.95 nm. The nanoparticle penetration efficiency (Papp) for small intestinal mucus penetration and transcellular transport was 4.11 × 10⁻⁶. -6 cm / s.

[0079] Example 8

[0080] (1) Chlorogenic acid and 20 kDa zein were mixed in 25% ethanol water at a mass ratio of 1:10 to prepare a 30% mass concentration solution (calculated as chlorogenic acid). The solution was then introduced into the first and second phase sample ports of the microfluidic chip at a rate of 9 μL / min, along with water. Then, a 32 mg / mL 35 kDa type A gelatin solution was heated at 60℃ for 1 h and introduced into the third phase sample port at a rate of 25 μL / min, with a mass ratio of functional factor to type A gelatin of 1:10. Finally, a 16 mg / mL solution with a degree of substitution of 0.03 and a molecular weight of 9.51 × 10⁻⁶ was prepared into a solution with a mass ratio of functional factor to OSA starch of 1:10. 5 After heating the OSA starch solution at 95 °C for 5 min, it was added to the fourth phase injection port at a rate of 80 μL / min. After reacting at a constant temperature of 40 °C for 1 h, a starch-protein composite nanoparticle solution loaded with functional factors was prepared.

[0081] (2) Prepare an aqueous solution with a total mass concentration of 15% by mixing the nanoparticles obtained in step (1) and 40 kDa whey protein isolate at a mass ratio of 1.35:1. Add the solution dropwise to corn oil at a water-oil ratio of 20:1 at 10000 r / min. Stir at 75°C for 20 min and then cool to room temperature while stirring for another 20 min. Then, add a 1% tannic acid solution heated at 95°C for 10 min at a mass ratio of whey protein isolate to tannic acid of 1:0.05 and react at 25°C for 9 h. Finally, add a 1% tannic acid solution with a degree of substitution of 0.03 and a molecular weight of 8.02 × 10⁻⁶, heated at 95°C for 10 min at a mass ratio of whey albumin to oxidized starch of 1:0.30. 5 A solution of oxidized starch at a concentration of g / mol was reacted at 25°C for 12 h to achieve a mass ratio of 1:1 between the nanoparticle core and the gel microsphere shell. After the reaction, the gel microspheres were allowed to settle and precipitate. After washing with n-hexane to remove excess corn oil, the microspheres were dried to obtain a small intestine-targeted adhesion gel microsphere delivery system.

[0082] The nanoparticles were found to have a core size of 198 nm, a surface contact angle of 67.48°, and a surface potential of -2.01 mV. The small intestine-targeted gel microsphere delivery system had a size of 30 μm and a surface potential of -21.85 mV. The encapsulation efficiency of functional factors was 71%, and the leakage of functional factors in the stomach and small intestine was 13.35%. The adhesion and thickening of the small intestine-targeted gel microspheres to the small intestinal mucus layer per unit volume was 2.35 nm. The nanoparticle penetration efficiency (Papp) for small intestinal mucus and transcellular transport was 9.33 × 10⁻⁶. -6 cm / s.

[0083] Example 9

[0084] (1) Quercetin and 20 kDa zein were mixed in 95% ethanol water at a mass ratio of 1:1 to prepare a 1% mass concentration solution (calculated as quercetin). The solution was then introduced into the first and second phase sample ports of the microfluidic chip at a rate of 9 μL / min, along with water. Then, at a mass ratio of 1:1 between the functional factor and type A gelatin, an 8 mg / mL 35 kDa type A gelatin solution was heated at 70℃ for 1 h and introduced into the third phase sample port at a rate of 25 μL / min. Finally, at a mass ratio of 1:2 between the functional factor and OSA starch, 16 mg / mL type A gelatin with a degree of substitution of 0.02 and a molecular weight of 4.45 × 10⁻⁶ was added. 5 After heating the OSA starch solution at 95 °C for 10 min, it was added to the fourth phase injection port at a rate of 80 μL / min. After reacting at a constant temperature of 40 °C for 1 h, a starch-protein composite nanoparticle solution loaded with functional factors was prepared.

[0085] (2) Prepare a 35% aqueous solution by mixing the nanoparticles obtained in step (1) with 20 kDa whey protein isolate at a mass ratio of 0.8:1. Add the solution dropwise to corn oil at a water-oil ratio of 20:1 at 16000 r / min. Stir at 75°C for 20 min, then cool to room temperature and stir for another 20 min. Then, add a 20% tannic acid solution heated at 95°C for 10 min at a mass ratio of whey protein isolate to tannic acid of 1:0.10. React at 25°C for 9 h. Finally, add a 5% solution of whey protein albumin and oxidized starch at a mass ratio of 1:0.50, heated at 95°C for 10 min. The solution has a degree of substitution of 0.03 and a molecular weight of 8.02 × 10⁻⁶. 5 A solution of oxidized starch (g / mol) was reacted at 25°C for 12 h to achieve a mass ratio of nanoparticle core to gel microsphere shell of 1:2. After the reaction, the gel microspheres were allowed to settle and precipitate. After washing with n-hexane to remove excess corn oil, the microspheres were dried to obtain a small intestine-targeted adhesion gel microsphere delivery system.

[0086] The nanoparticles were tested and found to have a core size of 92 nm, a surface contact angle of 63.27°, and a surface potential of -3.35 mV. The small intestine-targeted gel microsphere delivery system had a size of 42 μm and a surface potential of -23.52 mV. The encapsulation efficiency of functional factors was 59%, and the leakage of functional factors in the stomach and small intestine was 7.05%. The adhesion and thickening of the small intestine-targeted gel microspheres to the small intestinal mucus layer per unit volume was 4.31 nm. The nanoparticle penetration efficiency (Papp) for small intestinal mucus and transcellular transport was 5.64 × 10⁻⁶. -5 cm / s.

[0087] Example 10

[0088] (1) Quercetin and 20 kDa zein were mixed in 95% ethanol water at a mass ratio of 1:10 to prepare a 30% mass concentration solution (calculated as quercetin). The solution was then introduced into the first and second phase sample ports of the microfluidic chip at a rate of 9 μL / min, along with water. Then, a 40 mg / mL 75 kDa type A gelatin solution was heated at 60℃ for 1 h and introduced into the third phase sample port at a rate of 25 μL / min, with a mass ratio of functional factor to type A gelatin of 1:10. Finally, a 16 mg / mL solution of type A gelatin with a degree of substitution of 0.03 and a molecular weight of 9.51 × 10⁻⁶ was prepared into a solution with a mass ratio of functional factor to OSA starch of 1:20. 5 After heating the OSA starch solution at 75 °C for 30 min, it was added to the fourth phase injection port at a rate of 80 μL / min. After reacting at a constant temperature of 40 °C for 1 h, a starch-protein composite nanoparticle solution loaded with functional factors was prepared.

[0089] (2) Prepare a 15% aqueous solution by mixing the nanoparticles obtained in step (1) with 60 kDa whey protein isolate at a mass ratio of 1.07:1. Add the solution dropwise to corn oil at a water-oil ratio of 20:1 at 10000 r / min. Stir at 75°C for 20 min, then cool to room temperature and stir for another 20 min. Then, add a 1% epigallocatechin gallate solution heated at 95°C for 10 min at a mass ratio of 1:0.10 to whey protein isolate and epigallocatechin gallate. React at 25°C for 9 h. Then, add a 1% solution of epigallocatechin gallate with a degree of substitution of 0.03 and a molecular weight of 8.02 × 10⁻⁶ to whey albumin and oxidized starch at a mass ratio of 1:0.50 to whey albumin and oxidized starch heated at 95°C for 10 min at a mass concentration of 1%. 5 A solution of oxidized starch at a concentration of g / mol was reacted at 25°C for 12 h to achieve a mass ratio of nanoparticle core to gel microsphere shell of 1:1.5. After the reaction, the gel microspheres were allowed to settle and precipitate. After washing with acetone to remove excess corn oil, the microspheres were dried to obtain a small intestine-targeted adhesion gel microsphere delivery system.

[0090] The nanoparticles were tested and found to have a core size of 109 nm, a surface contact angle of 62.28°, and a surface potential of -3.11 mV. The small intestine-targeted gel microsphere delivery system had a size of 35 μm and a surface potential of -22.39 mV. The encapsulation efficiency of functional factors was 77%, and the leakage of functional factors in the stomach and small intestine was 8.67%. The adhesion and thickening of the small intestine-targeted gel microspheres to the small intestinal mucus layer per unit volume was 3.75 nm. The nanoparticle penetration efficiency (Papp) for small intestinal mucus penetration and transcellular transport was 3.67 × 10⁻⁶. -5 cm / s.

[0091] Example 11

[0092] (1) Quercetin and 50 kDa zein were mixed in 95% ethanol water at a mass ratio of 1:10 to prepare a 30% mass concentration solution (calculated as quercetin). The solution was then introduced into the first and second phase sample ports of the microfluidic chip at a rate of 9 μL / min, along with water. Then, at a mass ratio of 1:10 between the functional factor and type A gelatin, an 8 mg / mL 35 kDa type A gelatin solution was heated at 60℃ for 1 h and introduced into the third phase sample port at a rate of 25 μL / min. Finally, at a mass ratio of 1:30 between the functional factor and OSA starch, 48 mg / mL type A gelatin with a degree of substitution of 0.02 and a molecular weight of 4.45 × 10⁻⁶ was added. 5 After heating the OSA starch solution at 95 °C for 10 min, it was added to the fourth phase injection port at a rate of 80 μL / min. After reacting at a constant temperature of 40 °C for 1 h, a starch-protein composite nanoparticle solution loaded with functional factors was prepared.

[0093] (2) Prepare a 35% aqueous solution by mixing the nanoparticles obtained in step (1) with 20 kDa whey protein isolate at a mass ratio of 1.11:1. Add the solution dropwise to corn oil at a water-oil ratio of 20:1 at 10000 r / min. Stir at 75°C for 20 min and then cool to room temperature while stirring for 20 min. Then, add a 1% epigallocatechin gallate solution heated at 95°C for 10 min at a mass ratio of 1:0.01 to whey protein isolate and epigallocatechin gallate. React at 25°C for 12 h. Then, add a 5% solution of epigallocatechin gallate with a degree of substitution of 0.01 and a molecular weight of 3.60 × 10⁻⁶ at a mass ratio of 1:0.10 to whey albumin and oxidized starch heated at 95°C for 10 min. 5 A solution of oxidized starch at a concentration of g / mol was reacted at 25°C for 12 h to achieve a mass ratio of 1:1 between the nanoparticle core and the gel microsphere shell. After the reaction, the gel microspheres were allowed to settle and precipitate. After washing with n-hexane to remove excess corn oil, the microspheres were dried to obtain a small intestine-targeted adhesion gel microsphere delivery system.

[0094] The nanoparticles were tested and found to have a core size of 210 nm, a surface contact angle of 63.51°, and a surface potential of -3.95 mV. The small intestine-targeted gel microsphere delivery system had a size of 27 μm and a surface potential of -16.67 mV. The encapsulation efficiency of functional factors was 81%, and the leakage of functional factors in the stomach and small intestine was 20.19%. The adhesion and thickening of the small intestine-targeted gel microspheres to the small intestinal mucus layer per unit volume was 2.15 nm. The nanoparticle penetration efficiency (Papp) for small intestinal mucus and transcellular transport was 8.74 × 10⁻⁶. -6 cm / s.

[0095] Example 12

[0096] (1) Quercetin and 50 kDa zein were mixed in 95% ethanol water at a mass ratio of 1:20 to prepare a 30% mass concentration solution (calculated as quercetin). The solution was then introduced into the first and second phase sample ports of the microfluidic chip at a rate of 9 μL / min, along with water. Then, at a mass ratio of 1:20 between the functional factor and type A gelatin, an 8 mg / mL 75 kDa type A gelatin solution was heated at 60℃ for 1 h and introduced into the third phase sample port at a rate of 25 μL / min. Finally, at a mass ratio of 1:30 between the functional factor and OSA starch, a 16 mg / mL solution with a degree of substitution of 0.03 and a molecular weight of 9.51 × 10⁻⁶ was prepared. 5 After heating the OSA starch solution at 95 °C for 10 min, it was added to the fourth phase injection port at a rate of 80 μL / min. After reacting at a constant temperature of 40 °C for 1 h, a starch-protein composite nanoparticle solution loaded with functional factors was prepared.

[0097] (2) Prepare an aqueous solution with a total mass concentration of 15% by mixing the nanoparticles obtained in step (1) and 100 kDa whey protein isolate at a mass ratio of 2.22:1. Add the solution dropwise to corn oil at a water-oil ratio of 20:1 at 10000 r / min. Stir at 85°C for 30 min and then cool to room temperature and stir for 20 min. Then, add a 20% tannic acid solution heated at 95°C for 10 min at a mass ratio of whey protein isolate to tannic acid of 1:0.01 and react at 25°C for 6 h. Then, add a 1% solution of whey protein albumin and oxidized starch at a mass ratio of 1:0.10 and a mass concentration of 1% after heating at 95°C for 10 min. The solution has a degree of substitution of 0.01 and a molecular weight of 3.60 × 10⁻⁶. 5 A solution of oxidized starch at a concentration of g / mol was reacted at 25 °C for 12 h to achieve a mass ratio of nanoparticle core to gel microsphere shell of 1:0.5. After the reaction, the gel microspheres were allowed to settle and precipitate. After washing with n-hexane to remove excess corn oil, the microspheres were dried to obtain a small intestine-targeted adhesion gel microsphere delivery system.

[0098] The nanoparticles were found to have a core size of 260 nm, a surface contact angle of 72.07°, and a surface potential of -4.11 mV. The small intestine-targeted gel microsphere delivery system had a size of 15 μm and a surface potential of -15.64 mV. The encapsulation efficiency of functional factors was 75%, and the leakage rate of functional factors in the stomach and small intestine was 31.98%. The adhesion and thickening of the small intestine-targeted gel microspheres to the small intestinal mucus layer per unit volume was 1.75 nm. The nanoparticle penetration efficiency (Papp) for small intestinal mucus and transcellular transport was 5.92 × 10⁻⁶. -6 cm / s.

Claims

1. A small intestine-targeted adhesion gel microsphere delivery system, characterized in that: The small intestine-targeted adhesion gel microsphere delivery system comprises a starch and protein composite nanoparticle core loaded with biological functional factors and a whey protein isolate / polyphenol / oxidized starch gel microsphere shell. The mass ratio of the nanoparticle core to the gel microsphere shell is 1:(0.5~2). The nanoparticle core is composed of functional factors, zein, type A gelatin, and OSA starch in a mass ratio of 1:(1~20):(1~50):(2~50). The gel microsphere shell is composed of whey protein isolate / polyphenol / oxidized starch in a mass ratio of 1:(0.01~0.10):(0.10~0.50). The preparation method of the small intestine targeted adhesion gel microsphere delivery system includes the following steps: (1) Functional factors and zein are co-dissolved in an ethanol-water solution to form solution A, which is then introduced into a microfluidic chip along with water. Then, a heat-treated A-type gelatin aqueous solution is introduced into the microfluidic chip. Finally, a heat-treated OSA starch solution is introduced into the microfluidic chip. After a constant-temperature reaction, a starch and protein composite nanoparticle solution loaded with functional factors is prepared. (2) The composite assembled nanoparticle solution prepared in step (1) is thoroughly mixed with whey protein isolate. The mixed solution is then added to vegetable oil for shearing and mixing. Then, a heat-treated polyphenol solution is added for reaction. After the reaction is complete, a heat-treated oxidized starch solution is added for reaction. After the reaction is complete, the gel microspheres are allowed to stand and precipitate. After washing with organic solvent to remove all vegetable oil, the microspheres are dried to obtain the small intestine targeted adhesion gel microsphere delivery system. The polyphenol mentioned in step (2) is at least one of tannic acid and epigallocatechin gallate; the functional factor mentioned in step (1) is one of curcumin, chlorogenic acid and quercetin. The volume fraction of ethanol in the aqueous ethanol solution described in step (1) is 25-95%; the mass concentration of the functional factor in solution A is 1-30%. The heat-treated type A gelatin aqueous solution mentioned in step (1) is obtained by heating at 50-80℃ for 0.5-2h, with a concentration of 8-40 mg / mL; The heat-treated OSA starch aqueous solution mentioned in step (1) is obtained by heating at 75-95°C for 5-30 minutes, with a concentration of 16-48 mg / mL; In step (1), the ratio of the flow rates of solution A, water, heat-treated type A gelatin solution, and heat-treated OSA starch solution into the microfluidic chip is 1:1-3:2-3:6-9. The isothermal reaction mentioned in step (1) refers to a reaction at a constant temperature of 30-60℃ for 0.5-2 hours; The total mass concentration of nanoparticles and whey protein isolate in the mixture after mixing the composite assembled nanoparticle solution with whey protein isolate in step (2) is 15-35%; The amount of vegetable oil used in step (2) satisfies the following: the volume ratio of vegetable oil to the mixture is 5-30:1; The polyphenol solution after heat treatment mentioned in step (2) is an aqueous solution or aqueous dispersion with a mass concentration of 1 to 20% after being heated at 95 °C for 10 min; The oxidized starch solution after heat treatment mentioned in step (2) is an aqueous suspension with a mass concentration of 1-5% after being heated at 95 °C for 10 min; The shearing and mixing mentioned in step (2) refers to stirring and shearing at 65-85℃ for 10-30 minutes, then cooling to room temperature and maintaining stirring and shearing for 20 minutes, wherein the shearing speed is 10000 ~ 16000 r / min; The reaction described in step (2) with the addition of the heat-treated polyphenol solution refers to stirring at 1500 rpm for 6-12 hours at 20-40℃; the reaction described in step (2) with the addition of the heat-treated oxidized starch solution refers to stirring at 1500 rpm for 6-12 hours at 20-40℃.

2. The small intestine targeted adhesion gel microsphere delivery system according to claim 1, characterized in that: The nanoparticles have a core size ranging from 75 to 450 nm, a surface contact angle of 72.07 to 61.80°, and a surface potential ranging from -0.20 to -4.78 mV. The small intestine-targeted gel microsphere delivery system has a size ranging from 15 to 45 μm and a surface potential ranging from -15.64 to -24.68 mV. The encapsulation efficiency of functional factors is 59% to 98%, and the leakage of functional factors in the stomach and small intestine ranges from 7.05% to 21.98%. The adhesion and thickening of the small intestine-targeted gel microspheres per unit volume with the small intestine mucus layer is 1.15 to 4.31 nm. The nanoparticle penetration efficiency (Papp) for small intestine mucus and transcellular transport is 3.63 × 10⁻⁶. -6 ~5.64×10 -5 cm / s.

3. The small intestine targeted adhesion gel microsphere delivery system according to claim 1, characterized in that: The molecular weight of zein in step (1) is 20~50 kDa; The type A gelatin mentioned in step (1) has a molecular weight of 35~75 kDa, and the OSA starch mentioned in step (1) has a degree of substitution of 0.01~0.03 and a molecular weight of 2.43×10⁻⁶. 5 ~9.51×10 5 g / mol.

4. The small intestine targeted adhesion gel microsphere delivery system according to claim 1, characterized in that: The whey protein isolate described in step (2) has a molecular weight of 20-100 kDa; the degree of oxidized starch substitution is 0.01-0.03, and the molecular weight is 3.60 × 10⁻⁶. 5 ~8.02×10 5 g / mol; The vegetable oil mentioned in step (2) is at least one of corn oil, soybean oil, and peanut oil; The organic solvent mentioned in step (2) is at least one of n-hexane, diethyl ether, and acetone.

5. The use of the small intestine-targeted gel microsphere delivery system according to any one of claims 1-2 in the preparation of drugs for small intestine-targeted absorption.

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