A nano-liposome embedded food source functional polypeptide, and a preparation method and application thereof
By using nanoliposome encapsulation technology and leveraging the specific recognition mechanism of β-glucan with the Dectin-1 receptor on the surface of microfolded cells, the problem of precise delivery of food-derived functional peptides in the gastrointestinal environment was solved, achieving efficient transport to specific regions of the small intestine and improving the stability and functional activity of the peptides.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- OCEAN UNIV OF CHINA
- Filing Date
- 2025-09-03
- Publication Date
- 2026-06-12
AI Technical Summary
Food-derived functional peptides are easily hydrolyzed by enzymes in the gastrointestinal environment, making it difficult to deliver them precisely to specific absorption sites in the intestine, thus affecting their functional activity.
Using nanoliposome encapsulation technology, food-derived functional peptides are encapsulated in nanoliposomes formed by lecithin, cholesterol, and DSPE-PEG-β-glucan conjugates. Precise delivery is achieved using microfluidic assembly technology. Through the specific recognition mechanism of β-glucan and Dectin-1 receptor on the surface of microfolded cells, absorption in specific areas of the intestine is realized.
It significantly improves the stability and bioavailability of food-derived functional peptides, enables efficient transport to specific areas of the small intestine, protects peptides from digestive enzyme degradation, and enhances the functional activity of peptides.
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Figure CN120815059B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of targeted delivery technology, and in particular to a nanoliposome-encapsulated food-derived functional polypeptide, its preparation method, and its application. Background Technology
[0002] Food-derived functional peptides are food-derived functional factors prepared from food-derived proteins through enzymatic hydrolysis, separation, and purification. Based on their relative molecular mass distribution, they can be divided into food-derived oligopeptides (molecular weight less than 1000 Da) and food-derived polypeptides (molecular weight greater than 1000 Da). According to their functional characteristics, they can be classified into antihypertensive peptides, antioxidant peptides, immunomodulatory peptides, and antimicrobial peptides, etc. These peptides, after entering the human body through ingestion, can fully exert their various physiological activities. However, in the gastrointestinal environment, the presence of various gastrointestinal hydrolytic enzymes and the difficulty in precisely delivering peptides to specific absorption sites in the intestine severely limit the effective exertion of their functional activities.
[0003] The intestine, as the primary site of nutrient absorption and transport, has different parts with specific absorption functions and target regions. Therefore, achieving precise targeted delivery of different types of bioactive substances is a crucial prerequisite for their full absorption, transport, and biological activity. The small intestine is the main region for peptide absorption and a preferred target site for functional peptide delivery. Microfold cells (Mcells) are a type of specialized epithelial cell distributed in the small intestine, playing a vital role in the absorption and transport of proteins and peptides. Due to the lack of an intestinal mucus covering their surface and their highly efficient endocytic capacity, microfold cells can mediate the transmembrane transport of peptides, thereby achieving efficient absorption of functional peptides. This characteristic provides ideal conditions for receptor recognition and the absorption and transport of bioactive substances in the small intestine. Summary of the Invention
[0004] The purpose of this invention is to provide a method for preparing and applying food-derived functional peptides encapsulated in nanoliposomes, in order to solve the problem that food-derived functional peptides are easily hydrolyzed by enzymes in the gastrointestinal environment and are difficult to deliver precisely to specific absorption sites in the intestine.
[0005] To achieve the above objectives, the present invention provides a method for encapsulating food-derived functional peptides in nanoliposomes, comprising the following preparation process:
[0006] S1. Mix β-glucan, dimethyl sulfoxide, 1,1-carbonyldiimidazole and carboxyl-functionalized phospholipid-polyethylene glycol, react to obtain a reaction solution, and dialyze the reaction solution to obtain DSPE-PEG-β-glucan conjugate.
[0007] S2. Mix lecithin, cholesterol, DSPE-PEG-β-glucan conjugate and ethanol, and sonicate to obtain a liposome solution. Mix the food-derived functional peptides and phosphate buffer to obtain a food-derived functional peptide solution.
[0008] S3. Inject the liposome solution into the lipid phase channel of the microfluidic chip, and inject the food-derived functional peptide solution into the aqueous phase channel of the microfluidic chip. The nanoliposome-encapsulated complex is formed by microfluidic assembly. The nanoliposome-encapsulated complex is diluted and dialyzed to obtain the food-derived functional peptide encapsulated in nanoliposomes.
[0009] In this invention, the specific mixing steps in S1 are as follows: first, β-glucan and dimethyl sulfoxide are mixed and magnetically stirred to obtain a mixed solution. Then, under light-protected conditions, 1,1-carbonyldiimidazole is added to the mixed solution and stirred. Next, carboxyl-functionalized phospholipid-polyethylene glycol is added and mixed. The magnetic stirring temperature is 18-25℃, the magnetic stirring speed is 300-500 rpm, the magnetic stirring time is 3-5 hours, and the stirring time is 30-40 minutes.
[0010] In this invention, the molecular weight of β-glucan is 2400 kDa and the molecular weight of carboxyl-functionalized phospholipid-polyethylene glycol is 5000 Da.
[0011] In this invention, 1,1-carbonyldiimidazole is used as an activator, which can effectively activate the active hydroxyl sites in β-glucan after stirring for 30-40 minutes.
[0012] In this invention, the mass-to-volume ratio of β-glucan to dimethyl sulfoxide in S1 is 100 mg: 10-20 mL; the mass ratio of β-glucan, 1,1-carbonyldiimidazole, carboxyl-functionalized phospholipids to polyethylene glycol is 100: 3-4: 80-120.
[0013] In this invention, the reaction temperature in S1 is 70-90℃, and the reaction time is 12-18h.
[0014] In this invention, the molecular weight cutoff for dialysis in S1 is 8000-14000 Da, the dialysis solution is water, the dialysis temperature is 3-4℃, the dialysis time is 70-72h, and the dialysis solution is changed every 8h during the dialysis process.
[0015] In this invention, dialysis is used to remove unreacted monomers, byproducts, and dimethyl sulfoxide.
[0016] In this invention, after dialysis, the dialysis product is freeze-dried to obtain the DSPE-PEG-β-glucan conjugate. The freeze-drying temperature is not limited and any temperature known to those skilled in the art can be used; the freeze-drying time is 12-36 hours.
[0017] In this invention, the mass ratio of lecithin, cholesterol, DSPE-PEG-β-glucan conjugate, and food-derived functional peptides in S2 is 4-5:1:1:1-1.2.
[0018] In this invention, the specific process of mixing in S2 is as follows: first, lecithin, cholesterol, and DSPE-PEG-β-glucan conjugate are mixed, and then ethanol is added.
[0019] In this invention, the amount of ethanol used is not limited, but it is used only as a solvent.
[0020] In this invention, the amount of phosphate buffer is not limited, and it is used only as a solvent.
[0021] In this invention, the ACE inhibitory peptide includes egg white-derived peptide RADHPFL or egg white-derived peptide YAEERYPIL.
[0022] In this invention, the ultrasonic power in S2 is 200-400W, the ultrasonic time is 5-8min, and the ultrasonic method is to continue ultrasonication after a 3s interval after every 5s of ultrasonication.
[0023] In this invention, the flow rate of the liposome solution in the lipid phase channel of S3 is 1-1.5 mL / min, and the flow rate of the food-derived functional peptide solution in the aqueous phase channel is 3-3.5 mL / min.
[0024] In this invention, the volume concentration of ethanol in the nanoliposome-embedded complex diluted in S3 is less than 1%.
[0025] In this invention, the specific process of dialysis in S3 is as follows: the diluted product is placed in a dialysis bag and dialysis is performed.
[0026] In this invention, the dialysis temperature in S3 is 3-4℃, the dialysis time is 20-24h, and the molecular weight cutoff for dialysis is 10-15Da.
[0027] The present invention also provides a nanoliposome-encapsulated food-derived functional peptide prepared by the above-described method of encapsulating food-derived functional peptides in nanoliposomes.
[0028] This invention also provides the application of the above-mentioned nanoliposome-encapsulated food-derived functional peptides in the intestinal-targeted delivery of functional food factors.
[0029] The present invention has the following beneficial effects:
[0030] This invention provides a method for encapsulating food-derived functional peptides in nanoliposomes, comprising the following preparation steps: S1, mixing β-glucan, dimethyl sulfoxide, 1,1-carbonyldiimidazole, and carboxyl-functionalized phospholipid-polyethylene glycol, reacting to obtain a reaction solution, and dialyzing the reaction solution to obtain a DSPE-PEG-β-glucan conjugate; S2, mixing lecithin, cholesterol, the DSPE-PEG-β-glucan conjugate, and ethanol, and sonicating to obtain a liposome solution, adding the food-derived functional peptide to phosphate buffer to obtain a food-derived functional peptide solution; S3, injecting the liposome solution into the lipid phase channel of a microfluidic chip, injecting the food-derived functional peptide solution into the aqueous phase channel of the microfluidic chip, forming a nanoliposome-encapsulated complex through microfluidic assembly, diluting and dialyzing the nanoliposome-encapsulated complex to obtain the nanoliposome-encapsulated food-derived functional peptide. The preparation method provided by this invention uses microfluidic assembly technology to encapsulate food-derived functional peptides in nanoliposomes formed by lecithin, cholesterol, and DSPE-PEG-β-glucan conjugates. This method has the characteristics of high encapsulation efficiency, stable structure, and uniform distribution, which significantly improves the stability and bioavailability of food-derived functional peptides.
[0031] In this invention, lecithin, cholesterol, and carboxyl-functionalized phospholipid-polyethylene glycol are used as liposomes. All three have structures similar to cell membranes, which can effectively cross the cell membrane barrier. At the same time, they also protect food-derived functional peptides and prevent them from being degraded by digestive enzymes, thus achieving efficient transport of food-derived functional peptides in the gastrointestinal environment.
[0032] This invention modifies the surface of liposomes (lecithin, cholesterol, and carboxyl-functionalized phospholipids-polyethylene glycol) with β-glucan, and utilizes the specific recognition mechanism of β-glucan with the Dectin-1 receptor on the surface of microfolded cells to precisely deliver food-derived functional peptides to the absorption sites in specific regions of the intestine.
[0033] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description
[0034] Figure 1 The infrared spectra of β-Glucan, DSPE-PEG-COOH, and DSPE-β-Glucan in Example 1 of this invention are shown.
[0035] in, Figure 2Figure (a) shows the particle size distribution. Figure (a) shows the particle size distribution of LipNPs (1), RADHPFL@LipNPs (2), YAEERYPIL@LipNPs (3), βLipNPs (4), RADHPFL@βLipNPs (5), and YAEERYPIL@βLipNPs (6). Figure 2 (b) in the diagram is the Zeta potential diagram. Figure 2 Figure (c) is a transmission electron microscope image. Figure (c) (1) is a transmission electron microscope image of LipNPs. Figure (c) (2) is a transmission electron microscope image of RADHPFL@LipNPs. Figure (c) (3) is a transmission electron microscope image of YAEERYPIL@LipNPs. Figure (c) (4) is a transmission electron microscope image of βLipNPs. Figure (c) (5) is a transmission electron microscope image of RADHPFL@βLipNPs. Figure (c) (6) is a transmission electron microscope image of YAEERYPIL@βLipNPs.
[0036] Figure 3 This is a graph showing the encapsulation efficiency test results of the present invention;
[0037] in, Figure 3 Figure (a) shows the encapsulation efficiency test results of RADHPFL@βLipNPs and RADHPFL@LipNPs. Figure 3 (b) in the figure shows the encapsulation efficiency test results of YAEERYPIL@βLipNPs and YAEERYPIL@LipNPs;
[0038] Figure 4 This is a graph showing the changes in ACE inhibition rate before and after gastrointestinal digestion according to the present invention.
[0039] in, Figure 4 (a) shows the changes in ACE inhibition rate in the RADHPFL@βLipNPs group, Control R group, and RADHPFL group. Figure 4 (b) in the figure shows the changes in ACE inhibition rate in the YAEERYPIL@βLipNPs group, ControlY group, and YAEERYPIL group;
[0040] Figure 5 This is a graph showing the cytotoxicity analysis results of the present invention;
[0041] in, Figure 5(a) in the figure is a bar chart showing the cell viability of the RADHPFL@βLipNPs group, the RADHPFL@LipNPs group, and the RADHPFL group. Figure 5 (b) in the figure is a bar chart of cell viability in the YAEERYPIL@βLipNPs group, YAEERYPIL@LipNPs group, and YAEERYPIL group;
[0042] Figure 6 This is a graph showing the migration analysis results of the ACE inhibitory peptide of this invention;
[0043] in, Figure 6 (a) in the diagram is a validation diagram for establishing a transepithelial cell model. Figure 6 (b) in the figure shows the migration efficiency results for the RADHPFL@βLipNPs group, RADHPFL@LipNPs group, YAEERYPIL@βLipNPs group, and YAEERYPIL@LipNPs group.
[0044] Figure 7 Figure showing the results of liposome-targeted capture analysis of RAW264.7 macrophages;
[0045] in, Figure 7 (a) in the diagram shows the competitive binding of FITC-labeled liposomes to RAW264.7 macrophages via the Dectin-1 receptor. Figure 7 Image (b) shows confocal laser scanning microscopy images of the βLipNPs group, LipNPs group, and βLipNPs+Laminarin group in RAW264.7 cells. Figure 7 (c) in the figure represents the average fluorescence intensity of the βLipNPs group, the LipNPs group, and the βLipNPs+Laminarin group. Detailed Implementation
[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. The features mentioned above or in the specific examples mentioned in this invention can be combined arbitrarily, and these specific embodiments are only used to illustrate the invention and are not intended to limit the scope of the invention.
[0047] In the following examples, the egg white peptides RADHPFL and YAEERYPIL were purchased from Sangon Biotech (Shanghai) Co., Ltd.
[0048] Lecithin, cholesterol, carboxyl-functionalized phospholipids-polyethylene glycol, and β-glucan were all purchased from Aladdin Biochemical Technology Co., Ltd., Shanghai, China.
[0049] Example 1
[0050] S1. Mix 100 mg of β-glucan with 20 mL of dimethyl sulfoxide and stir magnetically at 500 rpm for 4 h at 25 °C to obtain a mixed solution. Under light-protected conditions, add 3.9 mg of 1,1'-carbonyldiimidazole to the mixed solution and stir for 30 min. Then add 100 mg of carboxyl-functionalized phospholipid-polyethylene glycol (DSPE-PEG-COOH) and mix. React at 80 °C for 16 h to obtain a reaction solution. Place the reaction solution in an MD77 dialysis bag with a molecular weight cutoff of 8000-14000 Da and use water as the dialysis solution. Dialyze at 4 °C for 72 h, changing the dialysis solution every 8 h. After obtaining the dialysis product, freeze-dry the dialysis product for 24 h to obtain the DSPE-PEG-β-glucan conjugate (DSPE-β-Glucan).
[0051] S2. Mix 0.0122g of lecithin, 0.0028g of cholesterol, and 0.0028g of DSPE-PEG-β-glucan conjugate, then add 5mL of ethanol and sonicate at 300W for 5min (sonication mode: 5s sonication followed by 3s interval) to obtain a liposome solution. Add 0.0028g of egg white peptide RADHPFL to 2mL of phosphate buffer (pH 7.4) to obtain a food-derived functional peptide solution.
[0052] S3. Using a dual-channel injection pump, liposome solution and food-derived functional peptide solution were injected into the lipid phase channel and aqueous phase channel of the microfluidic chip, respectively. The flow rate of liposome solution in the lipid phase channel was set to 1 mL / min and the flow rate of food-derived functional peptide solution in the aqueous phase channel was set to 3 mL / min. The nanoliposome-encapsulated complex was assembled by microfluidic assembly and then diluted with phosphate buffer to a volume concentration of 0.8% of ethanol in the nanoliposome-encapsulated complex. The diluted product was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed at 4°C for 24 h to obtain nanoliposome-encapsulated food-derived functional peptide, denoted as RADHPFL@βLipNPs.
[0053] Example 2
[0054] S1. Mix 100 mg of β-glucan with 20 mL of dimethyl sulfoxide and stir magnetically at 500 rpm for 4 h at 25 °C to obtain a mixed solution. Under light-protected conditions, add 3.9 mg of 1,1'-carbonyldiimidazole to the mixed solution and stir for 30 min. Then add 100 mg of carboxyl-functionalized phospholipid-polyethylene glycol (DSPE-PEG-COOH) and mix. React at 80 °C for 16 h to obtain a reaction solution. Place the reaction solution in an MD77 dialysis bag with a molecular weight cutoff of 8000-14000 Da and use water as the dialysis solution. Dialyze at 4 °C for 72 h, changing the dialysis solution every 8 h. After obtaining the dialysis product, freeze-dry the dialysis product for 24 h to obtain the DSPE-PEG-β-glucan conjugate (DSPE-β-Glucan).
[0055] S2. Mix 0.0122g of lecithin, 0.0028g of cholesterol, and 0.0028g of DSPE-PEG-β-glucan conjugate, then add 5mL of ethanol. Sonicate for 5min at 300W (sonication mode: 5s sonication followed by 3s interval) to obtain a liposome solution. Add 0.00308g of egg white peptide YAEERYPIL to 2mL of phosphate buffer (pH 7.4) to obtain a food-derived functional peptide solution.
[0056] S3. Using a dual-channel injection pump, liposome solution and food-derived functional peptide solution were injected into the lipid phase channel and aqueous phase channel of the microfluidic chip, respectively. The flow rate of liposome solution in the lipid phase channel was set to 1 mL / min and the flow rate of food-derived functional peptide solution in the aqueous phase channel was set to 3 mL / min. The nanoliposome-encapsulated complex was assembled by microfluidic technology. Then, it was diluted with phosphate buffer to a volume concentration of ethanol in the nanoliposome-encapsulated complex of 0.8%. The diluted product was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed at 4°C for 24 h to obtain nanoliposome-encapsulated food-derived functional peptide, denoted as YAEERYPIL@βLipNPs.
[0057] Comparative Example 1
[0058] S1. Mix 0.0122g of lecithin, 0.0028g of cholesterol, and 0.0028g of carboxyfunctionalized phospholipid-polyethylene glycol, then add 5mL of ethanol and sonicate at 300W for 5min (sonication method: 5s sonication followed by 3s interval) to obtain liposome solution.
[0059] S2. Using a dual-channel injection pump, inject liposome solution and 2 mL of phosphate buffer (PBS, pH 7.4) into the lipid phase channel and aqueous phase channel of the microfluidic chip, respectively. Set the flow rate of liposome solution in the lipid phase channel to 1 mL / min and the flow rate of phosphate buffer in the aqueous phase channel to 3 mL / min. Assemble liposomes through microfluidic technology. Dilute with phosphate buffer to a volume concentration of 0.8% ethanol in the liposomes to obtain a diluent. Transfer the diluent to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyze at 4°C for 24 h to obtain blank liposomes, denoted as LipNPs.
[0060] Comparative Example 2
[0061] S1. Mix 100 mg of β-glucan with 20 mL of dimethyl sulfoxide and stir magnetically at 500 rpm for 4 h at 25 °C to obtain a mixed solution. Under light-protected conditions, add 3.9 mg of 1,1'-carbonyldiimidazole to the mixed solution and stir for 30 min. Then add 100 mg of carboxyl-functionalized phospholipid-polyethylene glycol (DSPE-PEG-COOH) and mix. React at 80 °C for 16 h to obtain a reaction solution. Place the reaction solution in an MD77 dialysis bag with a molecular weight cutoff of 8000-14000 Da and use water as the dialysis solution. Dialyze at 4 °C for 72 h, changing the dialysis solution every 8 h. After obtaining the dialysis product, freeze-dry the dialysis product for 24 h to obtain the DSPE-PEG-β-glucan conjugate (DSPE-β-Glucan).
[0062] S2. Mix 0.0122g of lecithin, 0.0028g of cholesterol, and 0.0028g of DSPE-PEG-β-glucan conjugate, add 5mL of ethanol, and sonicate for 5min at 300W (sonication mode: 5s sonication followed by 3s interval) to obtain liposome solution.
[0063] S3. Using a dual-channel injection pump, liposome solution and phosphate buffer (pH 7.4) were injected into the lipid phase channel and aqueous phase channel of the microfluidic chip, respectively. The flow rate of the liposome solution in the lipid phase channel was set to 1 mL / min and the flow rate of the phosphate buffer in the aqueous phase channel was set to 3 mL / min. Liposomes were assembled by microfluidic assembly and diluted with phosphate buffer to a volume concentration of 0.8% of ethanol in the liposomes. The diluted product was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed at 4°C for 24 h to obtain β-glucan-modified blank liposomes, denoted as βLipNPs.
[0064] Comparative Example 3
[0065] S1. Mix 0.0122g of lecithin, 0.0028g of cholesterol, and 0.0028g of carboxyfunctionalized phospholipid-polyethylene glycol, then add 5mL of ethanol. Sonicate for 5min at 300W (sonication mode: 5s sonication followed by 3s interval) to obtain a liposome solution. Add 0.0028g of egg white peptide RADHPFL to 2mL of phosphate buffer (pH 7.4) to obtain a food-derived functional peptide solution.
[0066] S2. Using a dual-channel injection pump, liposome solution and food-derived functional peptide solution were injected into the lipid phase channel and aqueous phase channel of the microfluidic chip, respectively. The flow rate of liposome solution in the lipid phase channel was set to 1 mL / min and the flow rate of food-derived functional peptide solution in the aqueous phase channel was set to 3 mL / min. The nanoliposome-encapsulated complex was assembled by microfluidic technology. Then, it was diluted with phosphate buffer to a volume concentration of ethanol in the nanoliposome-encapsulated complex of 0.8%. The diluted product was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed at 4°C for 24 h to obtain nanoliposome-encapsulated food-derived functional peptide, denoted as RADHPFL@LipNPs.
[0067] Comparative Example 4
[0068] S1. Mix 0.0122g of lecithin, 0.0028g of cholesterol, and 0.0028g of carboxyl-functionalized phospholipid-polyethylene glycol, then add 5mL of ethanol. Sonicate for 5min at 300W (sonication mode: 5s sonication followed by 3s interval) to obtain a liposome solution. Add 0.00308g of egg white peptide YAEERYPIL to 2mL of phosphate buffer (pH 7.4) to obtain a food-derived functional peptide solution.
[0069] S2. Using a dual-channel injection pump, liposome solution and food-derived functional peptide solution were injected into the lipid phase channel and aqueous phase channel of the microfluidic chip, respectively. The flow rate of liposome solution in the lipid phase channel was set to 1 mL / min and the flow rate of food-derived functional peptide solution in the aqueous phase channel was set to 3 mL / min. The nanoliposome-encapsulated complex was assembled by microfluidic technology. Then, it was diluted with phosphate buffer to a volume concentration of ethanol in the nanoliposome-encapsulated complex of 0.8%. The diluted product was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed at 4°C for 24 h to obtain nanoliposome-encapsulated food-derived functional peptide, denoted as YAEERYPIL@LipNPs.
[0070] Characterization and detection:
[0071] Infrared spectroscopy was performed on carboxyl-functionalized phospholipid-polyethylene glycol (DSPE-PEG-COOH), β-glucan, and the DSPE-PEG-β-glucan conjugate (DSPE-β-Glucan) prepared in Example 1. The specific procedure included:
[0072] Take 1 mg each of DSPE-PEG-COOH, β-Glucan, and DSPE-β-Glucan, and grind them separately with 100 mg of dry potassium bromide in an agate mortar. Press the mixture into transparent thin tablets using a tablet press, place them in the sample holder, and then insert them into the fixed position in the instrument's sample chamber for detection. The results are as follows: Figure 1 As shown. From Figure 1 It can be seen that at 2800cm -1 The strong absorption bands appearing nearby are attributed to the stretching vibration of the CH bonds in DSPE-PEG-COOH, and the 1662 cm⁻¹ band. -1 The characteristic peaks in the vicinity correspond to the NH2 stretching vibration mode, while those in the 1000-1150 cm⁻¹ range... -1 The broad spectral bands in the range originate from the characteristic vibrations of the COO bonds within the β-glucan ring structure; the systematic appearance of the above peaks indicates that DSPE-PEG-COOH and β-glucan have successfully formed a DSPE-PEG-β-glucan conjugate (DSPE-β-Glucan) through chemical bonding.
[0073] The particle size distribution, polydispersity index, and zeta potential of RADHPFL@βLipNPs, YAEERYPIL@βLipNPs, LipNPs, βLipNPs, RADHPFL@LipNPs, and YAEERYPIL@LipNPs were determined under a constant temperature of 25℃ using Dynamic Light Scattering (DLS) and observed using transmission electron microscopy. The specific procedure is as follows:
[0074] Take 1 mL of each of RADHPFL@βLipNPs, YAEERYPIL@βLipNPs, LipNPs, βLipNPs, RADHPFL@LipNPs, and YAEERYPIL@LipNPs and place them in a Malvern particle size analyzer for particle size determination. Then, add the samples to an insertion cell for Zeta potential measurement. The refractive index and absorbance index of the liposomes were 1.433 and 0.010, respectively, while the refractive index and viscosity of the phosphate buffer were 1.33 and 1.0, respectively.
[0075] Take 10 μL of each of the following: RADHPFL@βLipNPs, YAEERYPIL@βLipNPs, LipNPs, βLipNPs, RADHPFL@LipNPs, and YAEERYPIL@LipNPs. Add each sample dropwise onto a 200-mesh copper grid coated with a carbon film. Let the samples stand for 1 hour until dry. Then, stain the grid with 2% phosphotungstic acid and let it stand for 2 minutes. Finally, dry the grid for 1 hour and observe it using a transmission electron microscope.
[0076] The above test results are as follows Figure 2 As shown, from Figure 2 It can be seen that the particle size of RADHPFL@βLipNPs and YAEERYPIL@βLipNPs is in the range of 100-150 nm, and the PDI is between 0.1 and 0.3, exhibiting a significant Tyndall effect. The potential is around -20 mV, showing good stability. Furthermore, transmission electron microscopy revealed that RADHPFL@βLipNPs, YAEERYPIL@βLipNPs, LipNPs, βLipNPs, RADHPFL@LipNPs, and YAEERYPIL@LipNPs all exhibit a distinct spherical structure and good dispersibility. The spherical structure remained unchanged after β-glucan modification and liposome encapsulation of ACE inhibitory peptides (egg white-derived peptides RADHPFL or YAEERYPIL).
[0077] Determination of encapsulation efficiency:
[0078] The encapsulation efficiency of RADHPFL@βLipNPs, YAEERYPIL@βLipNPs, RADHPFL@LipNPs, and YAEERYPIL@LipNPs was determined by high performance liquid chromatography (HPLC), specifically including:
[0079] High-performance liquid chromatography (HPLC) separation was performed using a C18 reversed-phase analytical column (250 × 4.6 mm). The mobile phase consisted of solvent A (0.1% TFA dissolved in ultrapure water) and solvent B (0.1% TFA dissolved in acetonitrile). The column temperature was set to 30 °C, the sample injection volume to 30 μL, the flow rate to 1 mL / min, and the absorbance detector wavelength to 214 nm. The total peptide concentration was calculated based on a pre-established ACE inhibitory peptide standard curve. Separately, 1 mL of sample was placed in a 3 kDa molecular weight cutoff ultrafiltration centrifuge tube (Millipore Amicon uLtra) and centrifuged at 7500 × g for 10 min at 4 °C. The filtrate was collected and the free peptide concentration was determined under the same conditions. Finally, the encapsulation efficiency was calculated using the following formula:
[0080]
[0081] The encapsulation efficiency results are as follows Figure 3 As shown, from Figure 3 As can be seen, the encapsulation rate can reach over 75% using the preparation method of this invention.
[0082] Changes in ACE inhibition rate before and after gastrointestinal digestion:
[0083] RADHPFL@βLipNPs, YAEERYPIL@βLipNPs, RADHPFL, and YAEERYPIL were mixed with pepsin (protein:substrate mass ratio of 1:50 w / w, 3400 U), and the pH was adjusted to 2.0 with HCl solution (0.1 mol / L) to simulate the gastric juice environment. The mixture was incubated at 37°C for 2 h (simulating the digestion process by gastric juice). Then, the pH was adjusted to 7.5 with NaOH (1 mol / L) to simulate the intestinal juice, and trypsin was added. The mixture was incubated at 37°C for 2 h (simulating the digestion process by intestinal juice). Finally, the trypsin was inactivated by heating at 95°C for 10 min, and samples were taken for analysis.
[0084] The sampling and testing process includes: taking 1 mL of the liquid after digestion in the gastric and intestinal environments, respectively, and then mixing it with 4 mL of 0.3% Triton-X-100. The liposome membrane is lysed by vortexing, and the mixture is centrifuged to obtain the supernatant.
[0085] The control groups were the RADHPFL group and the YAEERYPIL group, which were not digested by the gastric and intestinal fluid environments mentioned above, and were denoted as control group R (Control R) and control group Y (Control Y), respectively.
[0086] The ACE inhibition rates of the RADHPFL@βLipNPs group, RADHPFL group, YAEERYPIL@βLipNPs group, YAEERYPIL group, and control group were measured using the ACE kit WST. The results are as follows: Figure 4 As shown.
[0087] from Figure 4 It can be seen that, in the control group (without digestion by gastric and intestinal juices), the ACE inhibition rates were extremely high. However, after digestion by gastric and intestinal juices, the ACE inhibition rates of YAEERYPIL and RADHPFL (the YAEERYPIL and RADHPFL groups, respectively) were significantly reduced. After liposome encapsulation (the RADHPFL@βLipNPs and YAEERYPIL@βLipNPs groups), although digestion by gastric and intestinal juices was performed, their ACE inhibition rates were not significantly different from those of the control group. These results indicate that liposome encapsulation significantly improved the tolerance of the ACE-inhibiting peptides to the gastrointestinal environment, preserving their antihypertensive potential.
[0088] Cytotoxicity analysis:
[0089] Cytotoxicity was determined using the standard CCK-8 assay.
[0090] Caco-2 cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% non-essential amino acids, 100 μg / mL streptomycin, and 100 U / mL penicillin, and incubated at 37°C, 5% CO2, and 90% relative humidity. Caco-2 cells were then incubated at a rate of 1 × 10⁻⁶ cells / mL. 5 Cells were seeded at 100 μL per well in 96-well plates and cultured for 24 h. After cell adhesion, RADHPFL, YAEERYPIL, RADHPFL@βLipNPs, YAEERYPIL@βLipNPs, RADHPFL@LipNPs, and YAEERYPIL@LipNPs were added to the 96-well plates at the following concentrations (0 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL), respectively, with 6 replicates per group (the 0 mg / mL group was treated with an equal volume of PBS). After treatment, the plates were incubated in a cell culture incubator for 24 h. Then, the culture medium was aspirated, and 10 μL of CCK-8 solution was added to each well. The plates were incubated for 2 h, and the absorbance at 450 nm was measured using a microplate reader to determine cell viability. The results are shown below. Figure 5 As shown.
[0091] from Figure 5 It can be seen that at concentrations ≤0.5 mg / mL, the cell viability of RADHPFL, YAEERYPIL, RADHPFL@βLipNPs, YAEERYPIL@βLipNPs, RADHPFL@LipNPs, and YAEERYPIL@LipNPs showed no significant difference compared to 0 mg / mL, with cell viability all exceeding 90%. This indicates that at concentrations ≤0.5 mg / mL, the RADHPFL@βLipNPs and YAEERYPIL@βLipNPs provided by this invention have no significant toxicity.
[0092] Migration analysis of ACE inhibitory peptides in intestinal epithelium:
[0093] Two cell models were constructed: a co-culture of Caco-2 cells and Raji cells to simulate the monolayer membrane structure of M cells (microfolded cells) and the monolayer membrane structure of Caco-2 cells. First, Caco-2 cells were cultured at 2 × 10⁻⁶ cells per cell line. 5Caco-2 cells / mL were seeded onto Transwell plates and seeded with Raji cells on day 12 for further proliferation and differentiation. Cell proliferation and differentiation into a monolayer took 21 days. DMEM medium was changed every two days during the first week, and then daily with fresh medium. Transepithelial resistivity (TEER) of the Caco-2 cell monolayer was measured using a Millicell ERS meter and was found to be over 300 Ωcm. 2 This indicates that the monolayer is intact and can be used for subsequent research. For two different cell models, RADHPFL@βLipNPs, YAEERYPIL@βLipNPs, RADHPFL@LipNPs, and YAEERYPIL@LipNPs were added to the top layer, respectively, and incubated at 37°C for 2 hours. Then, 500 μL of the lower layer solution was taken and analyzed using high-performance liquid chromatography (HPLC). The results are as follows: Figure 6 As shown.
[0094] from Figure 6 It was found that, compared with the Caco-2 cell monolayer structure, the co-cultured M cell monolayer exhibited higher uptake of ACE repressor peptides embedded in liposomes. Furthermore, β-glucan modification further enhanced the uptake of ACE repressor peptides (RADHPFL@βLipNPs, YAEERYPIL@βLipNPs). These results indicate that the unique "microfold" structure of the M cells, after co-culture, provides a greater advantage in macromolecule uptake. The resulting M cell monolayer possesses stronger endocytic capacity than the Caco-2 model alone, thereby promoting efficient uptake of ACE repressor peptides.
[0095] Analysis of liposome-targeted capture by RAW264.7 macrophages:
[0096] Fluorescent staining of the liposome shells (LipNPs and βLipNPs) was performed using fluorescein isothiocyanate (FITC) to obtain FITC-LipNPs (LipNPs group) and FITC-βLipNPs (βLipNPs group), respectively. In vitro experiments used mouse-derived Dectin-1 expressed protein. The targeting function of the vector was studied using the (RAW264.7) cell line. RAW264.7 cells were cultured in high-glucose medium (DMEM) at 37°C, 90% relative humidity, and 5% CO2. The inhibition group (β-LipNPs + Laminarin) was pre-blocked with the Dectin-1 receptor using the Dectin-1 inhibitor Laminarin (1 mg / mL). The fluorescently labeled vector was then co-cultured with RAW264.7 cells. Afterward, the cell culture medium was removed, washed three times with PBS, and immobilized in 4% paraformaldehyde at 25°C for 30 min. The nuclei were then stained with DAPI. Imaging was performed at 0 h, 2 h, 4 h, and 8 h using a laser confocal scanning microscope. The results are shown below. Figure 7 As shown.
[0097] from Figure 7 It was observed that over time, a distinct green fluorescence appeared around the blue cell nucleus, indicating that macrophages had phagocytosed the liposomes, and the number of fluorescence particles gradually increased. At 4 hours, the β-LipNPs group showed a more significant fluorescence intensity, indicating a stronger binding ability with macrophages. The fluorescence intensity was 1.44 times higher than that of the LipNPs group, demonstrating in vitro targeted binding ability. In contrast, the fluorescence intensity of the inhibition group (β-LipNPs + Laminarin) after blocking dectin-1 was significantly reduced, further demonstrating that the specific recognition of β-glucan and Dectin-1 is the main mechanism of nanoparticle endocytosis.
[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A method for encapsulating food-derived functional polypeptides in nanoliposomes, characterized in that, The preparation process includes the following: S1. Mix β-glucan, dimethyl sulfoxide, 1,1-carbonyldiimidazole and carboxyl-functionalized phospholipid-polyethylene glycol, react to obtain a reaction solution, and dialyze the reaction solution to obtain DSPE-PEG-β-glucan conjugate. S2. Mix lecithin, cholesterol, DSPE-PEG-β-glucan conjugate and ethanol, and sonicate to obtain a liposome solution. Add the food-derived functional peptides to phosphate buffer to obtain a food-derived functional peptide solution. S3. Inject the liposome solution into the lipid phase channel of the microfluidic chip, and inject the food-derived functional peptide solution into the aqueous phase channel of the microfluidic chip. The nanoliposome-encapsulated complex is formed by microfluidic assembly. The nanoliposome-encapsulated complex is diluted and dialyzed to obtain nanoliposome-encapsulated food-derived functional peptides. The mass ratio of lecithin, cholesterol, DSPE-PEG-β-glucan conjugate, and food-derived functional peptides in S2 is 4-5:1:1:1-1.
2. The food-derived functional peptide is an ACE inhibitory peptide, which is either the egg white-derived peptide RADHPFL or the egg white-derived peptide YAEERYPIL.
2. The method for encapsulating food-derived functional polypeptides in nanoliposomes according to claim 1, characterized in that, The mass-to-volume ratio of β-glucan to dimethyl sulfoxide in S1 is 100 mg: 10-20 mL; The mass ratio of β-glucan, 1,1-carbonyldiimidazole, carboxyl-functionalized phospholipids to polyethylene glycol is 100:3-4:80-120.
3. The method for encapsulating food-derived functional polypeptides in nanoliposomes according to claim 1, characterized in that, The reaction temperature in S1 is 70-90℃, and the reaction time is 12-18h.
4. The method for encapsulating food-derived functional polypeptides in nanoliposomes according to claim 1, characterized in that, The molecular weight cutoff for dialysis in S1 is 8000-14000 Da, the dialysis solution is water, the dialysis temperature is 3-4℃, and the dialysis time is 70-72 h.
5. The method for encapsulating food-derived functional polypeptides in nanoliposomes according to claim 1, characterized in that, The ultrasonic power in S2 is 200-400W, and the ultrasonic time is 5-8 minutes.
6. The method for encapsulating food-derived functional polypeptides in nanoliposomes according to claim 1, characterized in that, In S3, the flow rate of liposome solution in the lipid phase channel is 1-1.5 mL / min, and the flow rate of food-derived functional peptide solution in the aqueous phase channel is 3-3.5 mL / min.
7. The method for encapsulating food-derived functional polypeptides in nanoliposomes according to claim 1, characterized in that, The dialysis temperature in S3 is 3-4℃, the dialysis time is 20-24h, and the molecular weight cutoff for dialysis is 10-15 Da.
8. The nanoliposome-encapsulated food-derived functional peptide prepared by the method of nanoliposome encapsulation of food-derived functional peptides according to any one of claims 1-7.
9. The use of the nanoliposome-encapsulated food-derived functional peptides of claim 8 in the preparation of intestinal-targeted delivery drugs.