Electrically-induced auxiliary prepared nutrition delivery system as well as preparation method and application thereof
The core-shell structure nanoparticles prepared by electrically induction assisted antisolvent coprecipitation method solve the problem of poor stability and solubility of existing nanoparticles in the gastrointestinal environment, and achieve efficient delivery and sustained release of biologically active substances.
Patent Information
- Application Number
- CN202510306440.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-14
- Publication Date
- 2025-07-25
AI Technical Summary
Existing oral nanoparticles have poor solubility, poor stability and high environmental sensitivity, and it is difficult to effectively deliver biologically active substances in a harsh gastrointestinal environment.
The electro-induced assisted antisolvent coprecipitation method is used to utilize food-borne proteins such as zein, soy protein, gliadin or casein as amphiphilic shell proteins, combined with chitosan as a modification, and to induce nanoparticles to form core-shell structures through current, enhancing the stability and bioavailability of the nanoparticles.
The prepared nanoparticles have high stability and sustained release properties in the gastrointestinal environment, which improves the encapsulation rate of biologically active substances and cell uptake efficiency, and achieves effective delivery in harsh environments.
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Figure CN120361244A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of the preparation of functional nanomaterials, and particularly to a nutrition delivery system prepared by electro-induced assistance, a preparation method thereof, and an application thereof. Background Art
[0002] Oral nanoparticles play a crucial role in nutrition delivery, especially for people who need to take drugs or nutritional products for a long time. In recent years, there has been an endless stream of research on oral nanoparticles based on polypeptides or proteins, because oral nanoparticles based on polypeptides or proteins often have higher biocompatibility. Moreover, some polypeptides or proteins used to synthesize nanoparticles themselves have certain nutritional activities and can play certain advantages in the prevention and treatment of diseases. However, the oral polypeptide / protein nanoparticles on the market have disadvantages such as poor solubility, poor stability, environmental sensitivity, and difficulty in the harsh gastrointestinal environment. Therefore, in order to improve the bioavailability of active substances, designing and constructing a nano-nutrition delivery carrier with good stability and high bioavailability has become an important topic in the nano-nutrition delivery system.
[0003] Previous studies usually modified proteins alone and then assembled them with other modifiers and embedded substances. Although it can also improve the stability of the nanoparticle system to a certain extent, the process is complex. The present invention uses food-derived proteins as raw materials and constructs a nano-nutrition delivery system by electro-induced assisted antisolvent co-precipitation method. Summary of the Invention
[0004] The purpose of the present invention is to provide a preparation method of a nutrition delivery system prepared by electro-induced assistance for the technical defects existing in the prior art. Another purpose of the present invention is to provide a nutrition delivery system prepared by electro-induced assistance. The present invention also provides an application of a nutrition delivery system prepared by electro-induced assistance in the preparation of sustained-release drugs or nutritional agents.
[0005] The technical solution adopted to achieve the purpose of the present invention is as follows:
[0006] A preparation method of a nutrition delivery system prepared by electro-induced assistance, comprising the following steps:
[0007] Step 1, dissolve an amphiphilic shell-forming protein and a lipophilic embedding substance in solvent A respectively, then mix and stir well to dissolve, centrifuge and take the supernatant to obtain an embedding substance / amphiphilic shell-forming protein solution;
[0008] Step 2, under stirring conditions, the inclusion complex / amphiphilic shell-forming protein solution obtained in Step 1 is dropped into solvent B or chitosan solution C, where the chitosan solution is obtained by dissolving chitosan in solvent C. During the dropping process, a continuous current of 40 V and 100 mA is passed through. The inclusion complex / amphiphilic shell-forming protein precipitates in solvent B or solvent C, and stirring forms an electro-induced assisted-prepared amphiphilic shell-forming protein nanoparticles solution loaded with the inclusion complex or an amphiphilic shell-forming protein-chitosan nanoparticles solution loaded with the inclusion complex;
[0009] Step 3, the electro-induced assisted-prepared amphiphilic shell-forming protein nanoparticles solution loaded with the inclusion complex or the amphiphilic shell-forming protein-chitosan nanoparticles solution prepared in Step 2 is evaporated under negative pressure and the pH value is adjusted to obtain electro-induced assisted-prepared amphiphilic shell-forming protein-based nanoparticles loaded with the inclusion complex or amphiphilic shell-forming protein-based-chitosan nanoparticles. The amphiphilic shell-forming protein nanoparticles loaded with the inclusion complex have a core-shell structure, with the inclusion complex as the core and the amphiphilic shell-forming protein as the shell; the amphiphilic shell-forming protein-chitosan nanoparticles loaded with the inclusion complex also have a core-shell structure, with the inclusion complex as the core and the amphiphilic shell-forming protein modified by chitosan as the shell.
[0010] In the above technical solution, it is characterized in that in Step 1, the inclusion complex is curcumin, resveratrol or quercetin, the amphiphilic shell-forming protein is zein, soy protein, gliadin or casein, and the mass ratio of the amphiphilic shell-forming protein to the inclusion complex is 19:1 to 21:1. Zein, soy protein, gliadin and casein all have amphiphilicity and can be assembled into nanoparticles by the anti-solvent self-assembly method.
[0011] In the above technical solution, in Step 1, the solvent A is an ethanol-aqueous solution, the centrifugation rate is 10000 - 12000 rpm, the centrifugation temperature is 4 - 8 °C, and the centrifugation time is 20 - 30 min.
[0012] In the above technical solution, in Step 2, the solvent B is ultrapure water, the mass ratio of the amphiphilic shell-forming protein to chitosan is 5.5:1 to 6.5:1, the molecular weight of chitosan is 30 - 50 kDa, the solvent C is an acetic acid-aqueous solution, the concentration of the amphiphilic shell-forming protein in the amphiphilic shell-forming protein nanoparticles solution loaded with the inclusion complex is 1.0 mg / mL, the concentration of the amphiphilic shell-forming protein in the amphiphilic shell-forming protein-chitosan nanoparticles solution loaded with the inclusion complex is 1.0 mg / mL, and the concentration of chitosan is 0.1 - 0.2 mg / mL.
[0013] In the above technical solution, in Step 2, the dropping rate of the inclusion complex / amphiphilic shell-forming protein solution is 2 - 4 drops / second.
[0014] In the above technical solution, the stirring rates in Step 1 and Step 2 are both 600 - 800 rpm, and the stirring time for each step is 45 - 60 min.
[0015] In the above technical solution, in Step 3, the evaporation temperature is 40 - 50 °C, the pressure is -0.1 - -0.2 Mpa, and the pH value is adjusted to 3.5 - 4.0.
[0016] Another aspect of the present invention provides a nutraceutical delivery system prepared by electro - induced assistance.
[0017] In the above technical solution, the average particle size of the nutraceutical delivery system prepared by electro - induced assistance is 100.0 - 150.0 nm, the PDI is distributed between 0.011 - 0.008, the zeta potential is 39 - 50 mV, and the encapsulation efficiency is 81.0 - 88.0%.
[0018] Another aspect of the present invention provides the application of a nutraceutical delivery system prepared by electro - induced assistance in the preparation of sustained - release drugs or nutraceuticals.
[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0020] 1. The raw materials used in the present invention are all food - derived materials, having biocompatibility and biosafety. The amphiphilic shell - forming protein used is a food protein, having unique film - forming properties. With the evaporation of the solvent, self - assembly occurs to form nanoparticles. On the basis of using amphiphilic shell - forming protein, considering the characteristics of gastrointestinal digestion and cell uptake, the positively charged and hydrophobic substance chitosan is selected as another modifier. The protein / polysaccharide soluble complex has strong foaming and emulsifying properties and can be used as a carrier material for encapsulating and protecting bioactive ingredients, realizing the sustained - release effect of nanoparticles and higher bioaccessibility. The encapsulated resveratrol, quercetin, and curcumin are all lipophilic compounds and can be precipitated when back - dropped into the solvent and thus encapsulated.
[0021] 2. The method used in the present invention is an electro - induced assisted antisolvent co - precipitation process. The encapsulated / amphiphilic shell - forming protein solution is directly dropped into ultrapure water or chitosan solution, and a continuous current is passed during the dropping process to prepare a nutraceutical delivery system prepared by electro - induced assistance (i.e., amphiphilic shell - forming protein - based nanoparticles loaded with encapsulated substances or amphiphilic shell - forming protein - based - chitosan nanoparticles). There is no need to separately process the protein, and the operation is more convenient.
[0022] 3. The present invention uses an electroinduction method to assist in the preparation of amphiphilic shell protein-based nanoparticles loaded with embedded substances. The amphiphilic shell protein-based - chitosan nanoparticles have the characteristics of being simple, efficient, having mild reaction conditions, and no toxic side effects. Charged particles can migrate directionally under the action of an electric field, and at the same time, magnetic stirring is carried out on the nanoparticle solution to increase the collision probability between nanoparticles and enhance the electrostatic interaction force between nanoparticles, thereby obtaining nanoparticles with smaller particle sizes, more surface charges, and better stability. At the same time, the encapsulation efficiency can also be improved.
[0023] 4. The electroinduction treatment and chitosan modification of the present invention endow the surface of the nanoparticles with a large amount of positive charge, that is, there is a large electrostatic repulsion between the nanoparticles to resist the aggregation and precipitation of the nanoparticles caused by the intrusion of salt ions in the environment, improving the stability of the nanoparticles and the tolerance to the gastrointestinal environment. The prepared electroinduction-assisted nutrient delivery system can cope with the harsh gastrointestinal environment and has a high positive charge at the same time, achieving efficient cell uptake. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 It is the microscopic morphology diagram of the electroinduction-assisted nutrient delivery systems of Examples 1, 2 and Comparative Examples 1, 2 of the present invention.
[0025] Figure 2 It is the Fourier transform infrared spectroscopy diagram of the electroinduction-assisted nutrient delivery systems of Examples 1, 2 and Comparative Examples 1, 2 of the present invention.
[0026] Figure 3 It is the gastrointestinal stability and curcumin sustained release of the electroinduction-assisted nutrient delivery systems of Examples 1, 2 and Comparative Examples 1, 2 of the present invention.
[0027] Figure 4 It is the in vitro Iec-6 cell uptake of the electroinduction-assisted nutrient delivery systems of Examples 1, 2 and Comparative Examples 1, 2 of the present invention. DETAILED DESCRIPTION OF THE INVENTION
[0028] The following further describes the present invention in detail with specific examples. It should be understood that the specific examples described herein are only used to explain the present invention and are not used to limit the present invention.
[0029] Example 1
[0030] A preparation method of zein-based nanoparticles loaded with embedded substances, comprising the following steps:
[0031] Step 1, at normal temperature and pressure, weigh 1000 mg of zein and 50 mg of curcumin, and dissolve them in 50 mL of 70% (v / v) ethanol-aqueous solution respectively to obtain a zein solution and a curcumin solution. Mix the two solutions with a magnetic stirrer and stir at 600 rpm for 60 min until the solution is uniformly transparent. Then centrifuge at 10000 rpm for 20 min to remove the insoluble part and obtain a curcumin / zein solution with a protein concentration of 10 mg / mL.
[0032] Step 2, under magnetic stirring, use a syringe to drop the curcumin / zein solution obtained in Step 1 into ultrapure water. During the dropping process, insert a conductive glass into the solution and connect it to a continuous current of 40 V and 100 mA. After the dropping is completed, the curcumin / zein solution precipitates in the ultrapure water. Disconnect the power supply and stir for 45 min to form a curcumin-loaded zein nanoparticle solution. The concentration of zein in the curcumin-loaded zein nanoparticle solution is 1 mg / mL.
[0033] Step 3, remove the excess ethanol from the curcumin-loaded zein nanoparticle solution prepared in Step 2 in a rotary evaporator (45 °C, -0.1 MPa), and adjust the solution to pH = 3.8 ± 0.1 to obtain zein-based nanoparticles loaded with the embedded substance prepared by electro-induced assistance.
[0034] Example 2
[0035] A method for preparing zein-based-chitosan nanoparticles loaded with an embedded substance, comprising the following steps:
[0036] Step 1, at normal temperature and pressure, weigh 1000 mg of zein and 50 mg of curcumin, and dissolve them in 50 mL of 70% (v / v) ethanol-aqueous solution respectively to obtain a zein solution and a curcumin solution. Mix the two solutions with a magnetic stirrer and stir at 600 rpm for 60 min until the solution is uniformly transparent. Then centrifuge at 10000 rpm for 20 min to remove the insoluble part and obtain a curcumin / zein solution with a protein concentration of 10 mg / mL.
[0037] Step 2, under magnetic stirring, use a syringe to drop the curcumin / zein solution obtained in Step 1 into the chitosan solution. During the dropping process, insert a conductive glass into the solution and connect it to a continuous current of 40 V and 100 mA. After the dropping is completed, disconnect the power supply, and the curcumin / zein solution precipitates in the acetic acid aqueous solution. Stir for 45 min to form a curcumin-loaded zein-chitosan nanoparticle solution. The concentration of zein in the curcumin-loaded zein-chitosan nanoparticle solution is 1 mg / mL, and the concentration of chitosan is 0.2 mg / mL.
[0038] The preparation method of the chitosan solution is as follows: at normal temperature and pressure, 17 mg of chitosan powder is weighed and dissolved in 90 mL of 0.1% (v / v) acetic acid - aqueous solution; under the condition of water bath heating at 80 - 90 °C, it is stirred for 60 min by a magnetic stirrer until the solution is uniform and transparent.
[0039] Step 3: The curcumin - loaded / zein - chitosan sodium nanoparticles solution prepared in Step 2 is placed in a rotary evaporator (45 °C, -0.1 MPa) to remove the excess ethanol, and the solution is adjusted to pH = 3.8 ± 0.1 to obtain zein - based - chitosan nanoparticles loaded with the encapsulated substance prepared by electro - induced assistance.
[0040] In each example of the present invention, the conditions of electro - induction are a continuous current of 40 V and 100 mA. This is because when the fixed current is 100 mA, when the set treatment voltage is less than 40 V, the number of charges provided additionally by the electro - induction treatment is limited, resulting in larger particle size and lower potential of the prepared nanoparticles. When the treatment voltage is higher than 40 V, the power is too large, causing partial denaturation and aggregation of zein, resulting in a lower encapsulation rate of the nanoparticles. In each example of the present invention, zein can also be replaced by soy protein, gliadin or casein. Zein, soy protein, gliadin and casein all have amphiphilicity and can be assembled into nanoparticles by the anti - solvent self - assembly method. In each example of the present invention, curcumin can also be replaced by resveratrol or quercetin. The encapsulated substances resveratrol, quercetin and curcumin are all lipophilic compounds and can be precipitated and encapsulated when back - dropped into the solvent. After replacement, similar effects to those of Example 1 and Example 2 can be achieved.
[0041] Comparative Example 1
[0042] A preparation method of zein - based nanoparticles loaded with an encapsulated substance includes the following steps:
[0043] Step 1: At normal temperature and pressure, 1000 mg of zein and 50 mg of curcumin are weighed and dissolved in 50 mL of 70% (v / v) ethanol - aqueous solution respectively to obtain a zein solution and a curcumin solution. The two solutions are mixed by a magnetic stirrer and stirred at 600 rpm for 60 min until the solution is uniform and transparent. Subsequently, it is centrifuged at 10000 rpm for 20 min to remove the insoluble part to obtain a curcumin / zein solution with a protein concentration of 10 mg / mL.
[0044] Step 2: Under magnetic stirring, use a syringe to drop the curcumin / zein solution obtained in Step 1 into ultrapure water, stir for 45 min to form a curcumin-loaded zein nanoparticle solution, wherein the concentration of zein in the curcumin-loaded zein nanoparticle solution is 1 mg / mL.
[0045] Step 3: Remove the excess ethanol from the curcumin-loaded zein nanoparticle solution prepared in Step 2 in a rotary evaporator (45 °C, -0.1 MPa), adjust the solution to pH = 3.8 ± 0.1 to obtain zein-based-chitosan nanoparticles loaded with the inclusion compound.
[0046] Comparative Example 2
[0047] A method for preparing zein-based-chitosan nanoparticles loaded with an inclusion compound, comprising the following steps:
[0048] Step 1: At normal temperature and pressure, weigh 1000 mg of zein and 50 mg of curcumin, dissolve them in 50 mL of 70% (v / v) ethanol-aqueous solution respectively to obtain a zein solution and a curcumin solution. Mix the two solutions with a magnetic stirrer, stir at 600 rpm for 60 min until the solution is uniformly transparent, and then centrifuge at 10000 rpm for 20 min to remove the insoluble part to obtain a curcumin / zein solution with a protein concentration of 10 mg / mL.
[0049] Step 2: Under magnetic stirring, use a syringe to drop the curcumin / zein solution obtained in Step 1 into the chitosan solution, stir for 45 min to form a curcumin-loaded zein-chitosan nanoparticle solution, wherein the concentration of zein in the curcumin-loaded zein-chitosan nanoparticle solution is 1 mg / mL, and the concentration of chitosan is 0.17 mg / mL.
[0050] The preparation method of the chitosan solution is as follows: At normal temperature and pressure, weigh 17 mg of chitosan powder, dissolve it in 90 mL of 0.1% (v / v) acetic acid-aqueous solution; under heating conditions, stir with a magnetic stirrer for 60 min until the solution is uniformly transparent.
[0051] Step 3: Remove the excess ethanol from the curcumin-loaded zein-chitosan nanoparticle solution prepared in Step 2 in a rotary evaporator (45 °C, -0.1 MPa), adjust the solution to pH = 3.8 ± 0.1 to obtain zein-based-chitosan nanoparticles loaded with the inclusion compound.
[0052] Comparative Example 3
[0053] A method for preparing a zein nanoparticle solution loaded with an embedded substance, comprising the following steps:
[0054] Step 1, at normal temperature and pressure, weigh 1000 mg of zein and 50 mg of curcumin, and dissolve them in 50 mL of 70% (v / v) ethanol-aqueous solution respectively to obtain a zein solution and a curcumin solution. Insert conductive glass into the zein solution and the curcumin solution respectively, connect a continuous current of 40 V and 100 mA, and the energization time is the dropping time for preparing nanoparticles by the electro-induced assisted anti-solvent co-precipitation method in Examples 1 and 2. Then mix the two solutions through a magnetic stirrer, stir at 600 rpm for 60 min until the solution is uniformly transparent, and then centrifuge at 10000 rpm for 20 min to remove the insoluble part to obtain a curcumin / zein solution with a protein concentration of 10 mg / mL.
[0055] Step 2, under magnetic stirring, use a syringe to drop the curcumin / zein solution obtained in Step 1 into ultrapure water, stir for 45 min to form a curcumin / zein nanoparticle solution loaded with curcumin, and the concentration of zein in the curcumin / zein nanoparticle solution loaded with curcumin is 1 mg / mL.
[0056] Step 3, remove the excess ethanol from the curcumin / zein nanoparticle solution prepared in Step 2 in a rotary evaporator (45 °C, -0.1 MPa), and adjust the solution to pH = 3.8 ± 0.1 to obtain zein-based nanoparticles loaded with the embedded substance.
[0057] Comparative Example 4
[0058] A method for preparing zein-based-chitosan nanoparticles loaded with an embedded substance, comprising the following steps:
[0059] Step 1, at normal temperature and pressure, weigh 1000 mg of zein and 50 mg of curcumin, and dissolve them in 50 mL of 70% (v / v) ethanol-aqueous solution respectively to obtain a zein solution and a curcumin solution. Insert conductive glass into the solutions respectively, connect a continuous current of 40 V and 100 mA, and the energization time is the dropping time for preparing nanoparticles by the electro-induced assisted anti-solvent co-precipitation method in Examples 1 and 2. Then mix the two solutions through a magnetic stirrer, stir at 600 rpm for 60 min until the solution is uniformly transparent, and then centrifuge at 10000 rpm for 20 min to remove the insoluble part to obtain a curcumin / zein solution with a protein concentration of 10 mg / mL;
[0060] Step 2, under magnetic stirring, use a syringe to drop the curcumin / zein solution obtained in Step 1 into the chitosan solution, stir for 45 min to form a curcumin / zein-chitosan nanoparticle solution, wherein the zein concentration in the curcumin / zein-chitosan nanoparticle solution is 1 mg / mL and the chitosan concentration is 0.17 mg / mL.
[0061] The preparation method of the chitosan solution is as follows: at normal temperature and pressure, weigh 17 mg of chitosan powder and dissolve it in 90 mL of 0.1% (v / v) acetic acid-aqueous solution; under the condition of water bath heating at 80-90 °C, stir with a magnetic stirrer for 60 min until the solution is uniform and transparent, then insert a conductive glass into the solution and connect a continuous current of 40 V and 100 mA, and the energization time is the dropping time for preparing nanoparticles by the electro-induced assisted antisolvent co-precipitation method in Examples 1 and 2.
[0062] Step 3, remove the excess ethanol from the curcumin / zein-chitosan nanoparticle solution prepared in Step 2 in a rotary evaporator (45 °C, -0.1 MPa), and adjust the solution to pH = 3.8 ± 0.1 to obtain zein-based-chitosan nanoparticles loaded with the inclusion.
[0063] In order to ensure that the protein concentrations of the samples in each example are the same, use ultrapure water to make up the volume of the zein-based nanoparticles obtained in Example 1, Comparative Example 1, and Comparative Example 3 and the zein-based-chitosan nanoparticles in Example 2, Comparative Example 2, and Comparative Example 4 before rotary evaporation. Analyze the zein-based-chitosan nanoparticle solution and the zein-based-chitosan nanoparticle solution obtained after supplementing with ultrapure water as follows:
[0064] Determination of average particle size, dispersity index, zeta potential, encapsulation efficiency, microscopic morphology, and infrared spectrum: After diluting the zein-based nanoparticle solutions obtained in Example 1, Comparative Example 1, and Comparative Example 3 and the zein-based-chitosan nanoparticle solutions in Example 2, Comparative Example 2, and Comparative Example 4 to appropriate concentrations after supplementing the volume with ultrapure water, use a nano laser particle size analyzer and a zeta potential analyzer to measure the particle size distribution, polydispersity index, and zeta potential of the samples, and measure each sample in parallel 6 times and take the average value. According to the different solubilities of curcumin and zein in ethanol-aqueous solutions with different concentrations, the freeze-drying and reconstitution method is used to determine the encapsulation efficiency of the nanoparticle samples. Use a field emission electron microscope to observe the microscopic morphology of the nanoparticles. Use a Fourier transform infrared spectrometer to analyze the infrared spectra of each substance in the samples to judge the changes in the surface functional groups of the samples.
[0065] Verification of gastrointestinal stability and sustained release performance of nanoparticles: In this invention, the INFOGEST 2.0 model was used to simulate the behavior of nanoparticles during in vitro digestion. INFOGEST 2.0 is an improved digestion model designed to more accurately simulate the complex environment of the human digestive system. The following are the specific steps of the experimental method:
[0066] Prepare simulated digestive fluids, including saliva, gastric juice, and intestinal juice, and the components of these fluids are formulated according to the standards of the INFOGEST 2.0 model.
[0067] According to the steps specified by the INFOGEST 2.0 model, first add the saliva simulation solution to the test tube, keep it at 37°C, and gently stir to ensure uniform mixing.
[0068] Gradually add the gastric juice simulation solution, adjust the pH to 3.0, and incubate at 37°C for 2 h to simulate the gastric digestion process. After digestion is completed, immediately neutralize the gastric juice, add the intestinal juice simulation solution, and adjust the pH to 7.0. Continue to incubate at 37°C for 4 h to simulate the intestinal digestion process.
[0069] Measure the particle size, potential, and curcumin release amount of the nanoparticles after digestion at each stage, respectively.
[0070] Verification of the cell uptake ability of nanoparticles: The uptake of nanoparticles by Iec-6 cells was observed using CLSM. When the confluence of Iec-6 cells reached 80%, the original culture medium was discarded, the cells were washed, and the culture medium containing nanoparticles was added. After 6 h, the original culture medium was discarded, the cell layer was washed, and 4% paraformaldehyde solution was added under light-proof conditions. After 15 min, the cells were washed and the cell nuclei were stained with DAPI solution. After 15 min, observation was carried out under the water lens of CLSM. To quantify the cell uptake of nanoparticles, Iec-6 cells were inoculated in a six-well plate at a certain density and incubated adherently. When the cells grew to 80%, the original culture medium was removed, the cells were washed, and the culture medium containing nanoparticles was added for incubation. After 6 h, the cells were washed with pre-cooled PBS, and RIPA solution (200 μL / well) was added to lyse the cells under ice bath conditions. After 30 min, the lysed cell suspension was collected and centrifuged (20000 rpm, 4°C, 20 min). A certain amount of DMSO was added to the supernatant, and the fluorescence intensity of curcumin in the solution was measured to determine the amount of nanoparticles in the cells.
[0071] Result analysis:
[0072] 1. Particle size, PDI index, zeta potential, and encapsulation efficiency of nanoparticles:
[0073] As shown in Table 1, the average particle sizes of Examples 1-2 were 101.67±3.53 nm and 150.03±2.53 nm, respectively. The particle size of Example 2 was larger than that of Example 1 because chitosan was introduced on the surface of the nanoparticles in Example 2, so the nanoparticles would become larger. The nanoparticles also carried more positive charges, and the electrostatic repulsion between the nanoparticles was stronger, so the system was in a relatively stable state. The average particle sizes of Examples 1 and 2 were significantly lower than those of Comparative Examples 1 and 2. The PDI of Examples 1-2 was distributed between 0.011 and 0.008, indicating good uniformity. The encapsulation efficiency of Example 1 was 81.09%, significantly higher than that of Comparative Example 1 and Comparative Example 3; the encapsulation efficiency of Example 2 was 88.17%, significantly higher than that of Comparative Example 2 (64.19%) and Comparative Example 4 (63.82%). Comparing Examples 1-2 and Comparative Examples 3-4, it can be seen that after individually electroinducing each component and then preparing nanoparticles by the anti-solvent co-precipitation method, the effect achieved by simultaneously performing electroinduction and the anti-solvent co-precipitation method cannot be achieved.
[0074] Table 1 Particle size, PDI, zeta potential and encapsulation efficiency of different nanoparticles
[0075]
[0076] Note: The values in the table are the average standard deviation (n = 6). The superscripts a, b, c, d, e in the table represent whether there are significant differences in the same performance data of different examples or comparative examples. When the superscript letters are the same, it means there is no significant difference. When the superscript letters are different, it means there is a significant difference. Taking the average particle size as an example, the superscripts of Comparative Example 1, Comparative Example 3 and Example 2 are all b, indicating that there is no significant difference in the average particle sizes of the three. The superscripts of Comparative Example 1 and Comparative Example 2 are different, indicating that there is a significant difference in the average particle sizes of Comparative Example 1 and Comparative Example 2.
[0077] 2. Microscopic morphology of nanoparticles:
[0078] As Figure 1 shown, the shape of Comparative Example 1 was irregular and the uniformity was poor. After modifying it with chitosan, the shapes of some particles became more regular and presented as uniform spheres. The morphologies of Examples 1-2 showed a more uniform and smooth form because the presence of electroinduction increased the electrostatic repulsion between the nanoparticles, thereby improving the uniformity and stability of the nanoparticles.
[0079] 3. Infrared spectrum of nanoparticles:
[0080] Referring to Figure 2 a and b in -1 , the characteristic peak of zein at 3415.46 cm -1This is attributed to the hydrogen bonding between zein and curcumin; zein is located at 3415.46 cm -1 The characteristic peak at is shifted to 3423.41 cm in Example 2. -1 This is attributed to the hydrogen bonding between zein and chitosan. Chitosan is at 1420.24 cm due to the deprotonation of the amino group. -1 Characteristic peaks appeared, and the characteristic peaks of curcumin disappeared in both Example 1-2 and Comparative Example 1-2, indicating that curcumin was successfully embedded.
[0081] 4. Verification of gastrointestinal stability and sustained release performance of nanoparticles:
[0082] like Figure 3 As shown in a and b, after digesting Examples 1-2 and Comparative Examples 1-2, the particle size and potential at each period were measured. By comparing different Examples and Comparative Examples at the same period, it can be found that Example 2 has the best gastrointestinal digestion stability. The fluctuation of the particle size of Example 2 during the entire digestion period is the smallest, and the absolute value of the zeta potential is the highest, followed by Example 1. The largest fluctuation is in Comparative Example 1. This is because the surface of the nanoparticles of Comparative Example 1, which has not been subjected to electrical induction treatment and chitosan modification, has less positive charge and is difficult to resist the harsh gastrointestinal environment due to the lack of the protective effect of chitosan. The gastrointestinal digestion stability results of Example 2 show that the electrical induction treatment and chitosan modification give the nanoparticles a larger positive charge on the surface, that is, the nanoparticles have a larger electrostatic repulsion to resist the aggregation and precipitation of the nanoparticles due to the invasion of salt ions in the environment, thereby improving the stability of the nanoparticles and the tolerance to the gastrointestinal environment.
[0083] like Figure 3As shown in c, after digesting Examples 1-2 and Comparative Examples 1-2 respectively, the release amount of curcumin at the end of each period was measured. Specifically, Example 2 showed a positive effect on the sustained-release effect of curcumin due to both electro-induced treatment and chitosan modification. Especially in the 2h simulated gastric juice digestion period, the release amount of curcumin in Comparative Example 1 was as high as 83.68%, while that of Example 2 was only 36.37%. This significant difference can be mainly attributed to two factors. First, due to the auxiliary effect of electro-induced treatment, the structure of Example 2 was denser, which improved the dispersion stability of the whole system, effectively resisted the neutralization of the system charge by a large amount of salt ions in the gastrointestinal tract, and thus avoided the destruction and aggregation of the nanoparticle structure. Second, zein has been proven to be hydrolyzed by pepsin during simulated gastric digestion, and the modification of chitosan effectively protected zein and reduced its decomposition degree in the stomach. When the simulated digestion process entered the 4h intestinal digestion stage, the final release amount of curcumin was measured, and the results showed that the release amount of curcumin in the nanoparticles of Examples 1-2 and Comparative Examples 1-2 reached about 90%. It should be noted that in the whole digestion process of Example 2, the highest amount of Cur was released in the small intestine stage, indicating that electro-induced treatment and chitosan modification successfully achieved the purpose of slow release of nanoparticles in the intestine.
[0084] 5. Determination of the in vitro cellular uptake rate of nanoparticles:
[0085] The uptake of Cur by Iec-6 cells is shown in Figure 4 a and b. Among them, after incubating with cells in Comparative Example 1 for 6h, only weak fluorescence peaks were shown in the CLSM images. In contrast, a large amount of yellow fluorescence corresponding to curcumin was observed in the nuclei and cytoplasm of Iec-6 cells treated in Comparative Example 2, proving that chitosan modification helps nanoparticles enter cells. Research shows that the highly negatively charged heparan sulfate proteoglycan on the cell surface plays an important role in cellular uptake, so cationic polymers can enhance the uptake by cells through electrostatic interaction. When electro-induced assistance and chitosan modification were adopted, the uptake of nanoparticles changed significantly. For example, enhanced yellow fluorescence could be observed in the cells of Example 2, indicating that electro-induced treatment and the introduction of chitosan have a positive effect on improving cellular uptake.
[0086] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. A preparation method of a nutrition delivery system prepared by electro-induced assistance, characterized in that, It includes the following steps: Step 1: Dissolve the amphiphilic shell-forming protein and the lipophilic inclusion in solvent A respectively, then mix and stir well to dissolve, centrifuge and take the supernatant to obtain an inclusion / amphiphilic shell-forming protein solution; Step 2: Under stirring conditions, drop the inclusion / amphiphilic shell-forming protein solution obtained in Step 1 into solvent B or a C solution of chitosan, where the chitosan solution is obtained by dissolving chitosan in solvent C. During the dropping process, apply a continuous current of 40V and 100mA. The inclusion / amphiphilic shell-forming protein precipitates in solvent B or solvent C, and stir to form an electro-induced assisted prepared amphiphilic shell-forming protein nanoparticles solution loaded with the inclusion or an amphiphilic shell-forming protein-chitosan nanoparticles solution loaded with the inclusion; Step 3: Subject the electro-induced assisted prepared amphiphilic shell-forming protein nanoparticles solution loaded with the inclusion or the amphiphilic shell-forming protein-chitosan nanoparticles solution loaded with the inclusion prepared in Step 2 to negative pressure evaporation, adjust the pH value to obtain electro-induced assisted prepared amphiphilic shell-forming protein-based nanoparticles or amphiphilic shell-forming protein-based-chitosan nanoparticles. The amphiphilic shell-forming protein nanoparticles loaded with the inclusion are of a core-shell structure, with the inclusion as the core and the amphiphilic shell-forming protein as the shell; the amphiphilic shell-forming protein-chitosan nanoparticles loaded with the inclusion are also of a core-shell structure, with the inclusion as the core and the amphiphilic shell-forming protein modified by chitosan as the shell.
2. The preparation method of the nutraceutical delivery system prepared by electro-induced assistance according to claim 1, wherein, In Step 1, the inclusion is curcumin, resveratrol or quercetin, the amphiphilic shell-forming protein is zein, soy protein, gliadin or casein, and the mass ratio of the amphiphilic shell-forming protein to the inclusion is 19:1 to 21:
1.
3. The preparation method of the electro-induced assisted prepared nutrient delivery system according to claim 1, characterized in that, In Step 1, solvent A is an ethanol-aqueous solution, the centrifugation rate is 10000 - 12000rpm, the centrifugation temperature is 4 - 8°C, and the centrifugation time is 20 - 30min.
4. The preparation method of the electro-induced assisted prepared nutrient delivery system according to claim 1, characterized in that, In Step 2, solvent B is ultrapure water, the mass ratio of the amphiphilic shell-forming protein to chitosan is 5.5:1 to 6.5:1, the molecular weight of chitosan is 30 - 50kDa, solvent C is an acetic acid-aqueous solution, the concentration of the amphiphilic shell-forming protein in the amphiphilic shell-forming protein nanoparticles solution loaded with the inclusion is 1.0mg / mL, the concentration of the amphiphilic shell-forming protein in the amphiphilic shell-forming protein-chitosan nanoparticles solution loaded with the inclusion is 1.0mg / mL, and the concentration of chitosan is 0.1 - 0.2mg / mL.
5. The preparation method of the electro-induced assisted prepared nutrient delivery system according to claim 1, characterized in that, In Step 2, the dropping rate of the inclusion / amphiphilic shell-forming protein solution is 2 - 4 drops / second.
6. The preparation method of the electro-induced assisted prepared nutrient delivery system according to claim 1, characterized in that, In Steps 1 and 2, the stirring rate is 600 - 800rpm, and the stirring time for each step is 45 - 60min.
7. The preparation method of the electro-induced assisted prepared nutrient delivery system according to claim 1, characterized in that, In Step 3, the evaporation temperature is 40 - 50°C, the pressure is -0.1 to -0.2Mpa, and the pH value is adjusted to 3.5 - 4.
0.
8. An electro-induced assisted prepared nutrient delivery system prepared by the preparation method according to any one of claims 1 - 7.
9. The nutraceutical delivery system prepared by electro-induced assistance according to claim 8, wherein, The average particle size of the electro-induced assisted prepared nutrient delivery system is 100.0 - 150.0 nm, the PDI is distributed between 0.011 - 0.008, the zeta potential is 39 - 50 mV, and the encapsulation efficiency is 81.0 - 88.0%.
10. Use of an electro-induced assisted prepared nutrient delivery system as described in claim 8 in the preparation of a sustained-release drug or nutrient.