Flammulina velutipes polysaccharide nano-liposome as well as preparation method and application thereof

Abstract

The invention discloses flammulina velutipes polysaccharide nano-liposome as well as a preparation method and application thereof, and belongs to the technical field of biological functional materials. The preparation method specifically comprises the following steps: (1) sequentially performing hot water extraction, deproteinization, alcohol precipitation, dialysis and freeze-drying on flammulina velutipes raw material powder; (2) dissolving in a phosphoric acid buffer solution; (3) mixing and dissolving phospholipid, cationic lipid and a membrane regulator in an organic solvent; (4) mixing and ultrasonic treatment; and (5) carrying out reduced-pressure rotary evaporation, hydration with a phosphate buffer solution, ultrasonic treatment and extrusion. According to the flammulina velutipes polysaccharide nano-liposome and the preparation method thereof, cholesterol is replaced by phytosterol ester to prepare the flammulina velutipes polysaccharide nano-liposome, flammulina velutipes polysaccharide (FVP) is encapsulated in a water core in the liposome, the safety is higher, and the encapsulation efficiency, the storage stability and the immunocompetence of a system are improved. In-vitro experiments prove that the phytosterol ester lipidosome has the effect of promoting macrophage proliferation and nitric oxide generation, and the effect is obviously better than that of the traditional cholesterol lipidosome under the same condition.

Description

Technical Field

[0001] This invention relates to the field of biofunctional materials technology, and more specifically to a enoki mushroom polysaccharide nanoliposome, its preparation method, and its application. Background Technology

[0002] Flammulina velutipes polysaccharide (FVP) is the core active ingredient of flammulina velutipes, possessing significant immunomodulatory functions. It can enhance the body's immunity by activating macrophages, regulating T / B lymphocyte subsets, and improving gut microbiota. It also exhibits antioxidant and anti-inflammatory effects, showing great potential for application in functional foods and biomedicine. However, as a biomacromolecule, FVP is easily degraded by gastrointestinal enzymes after oral administration, and its poor intestinal mucosal permeability leads to low bioavailability, severely limiting its actual immune activity.

[0003] Nanoliposomes are nanovesicles composed of phospholipid bilayers that can simultaneously encapsulate hydrophilic and hydrophobic components. They exhibit high biocompatibility, low toxicity, and protect the load from enzymatic degradation, making them ideal carriers for improving the oral bioavailability of free radical vasoactive (FVP). In traditional liposome preparation, cholesterol is the core component regulating membrane rigidity and stability, but it has two major drawbacks: first, its biosafety is questionable, with excessive intake directly related to cardiovascular diseases such as hyperlipidemia and atherosclerosis; second, the membrane structure is not well-suited for lipid-soluble components, resulting in low encapsulation efficiency and easy leakage of water-soluble FVPs, failing to meet the requirements for stable delivery.

[0004] In summary, existing technologies have limitations in improving liposome performance: surface modification and other methods are difficult to balance with safety, and traditional cholesterol replacement studies have failed to simultaneously improve encapsulation efficiency and stability.

[0005] Therefore, how to prepare a nanoliposome of enoki mushroom polysaccharide with higher encapsulation efficiency, significantly enhanced stability, and better safety is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a flamingo mushroom polysaccharide nanoliposome, its preparation method and application, so as to solve the problems of low bioavailability caused by poor stability and encapsulation difficulties of flamingo mushroom polysaccharide (FVP) in the prior art, as well as the potential safety risks of traditional liposomes that depend on cholesterol.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing Flammulina velutipes polysaccharide nanoliposomes specifically includes the following steps: (1) The raw material powder of Enoki mushroom was subjected to hot water extraction, protein removal, alcohol precipitation, dialysis and freeze drying in sequence to obtain Enoki mushroom polysaccharide; (2) Dissolve the enoki mushroom polysaccharide in phosphate buffer solution to obtain the enoki mushroom polysaccharide solution for later use; (3) Phospholipids, cationic lipids and membrane regulators are mixed and dissolved in an organic solvent to obtain the liposome organic phase; (4) Add the enoki mushroom polysaccharide solution to the liposome organic phase and sonicate to obtain enoki mushroom polysaccharide liposome emulsion; (5) The enoki mushroom polysaccharide liposome emulsion was subjected to vacuum rotary evaporation, phosphate buffer hydration, sonication, and extrusion in sequence to obtain enoki mushroom polysaccharide nanoliposomes.

[0008] This invention involves dissolving the structural phospholipid dipalmitoyl phosphatidylcholine (DPPC), the cationic lipid 2,3-dioleoyl-propyl-trimethylammonium chloride (DOTAP), and the functionalized membrane regulator phytosterol ester (SE) in an organic phase to form a liposome organic phase. This phase is then mixed with an aqueous solution of enoki mushroom polysaccharide (FVP), subjected to ice bath sonication, and hydrated by vacuum rotary evaporation. This allows the lipids to self-assemble into a phospholipid bilayer structure encapsulating FVP within an inner water core. Finally, controlled extrusion is performed above the phospholipid phase transition temperature to obtain enoki mushroom polysaccharide nanoliposomes with a particle size of 80-130 nm, a Zeta potential ≥18 mV, and an encapsulation efficiency higher than 60%.

[0009] This invention constructs a novel nanoliposome with high encapsulation efficiency, improved stability, and excellent biocompatibility by completely replacing cholesterol with phytosterol esters (SE) and synergistic self-assembly with DPPC and DOTAP. This achieves functionalized nanoliposomes with improved encapsulation efficiency, increased stability, enhanced safety, and improved immune activity. This system provides a new solution for enhancing the application value of Flammulina velutipes polysaccharide nanocarriers.

[0010] The specific objectives of this invention include: (1) Completely replace cholesterol with phytosterol esters (SE) to eliminate the safety hazards of traditional liposomes from the source of membrane material, while utilizing the physiological activity of SE to improve the biocompatibility of the carrier; (2) By synergistic self-assembly of SE with DPPC and DOTAP, a dense and ordered lipid bilayer is constructed, which increases the encapsulation rate of enoki mushroom polysaccharide to more than 60%, thus solving the problems of low encapsulation rate and poor stability. (3) Enhance the immune synergistic effect of the carrier system, so that the activation ability and immunomodulatory activity of the obtained nanoliposomes on macrophages are superior to those of traditional cholesterol liposomes.

[0011] Furthermore, in step (2) above, the concentration of the enoki mushroom polysaccharide solution is 2-10 mg / mL.

[0012] Furthermore, in step (3) above, the phospholipid is dipalmitoylphosphatidylcholine (DPPC), the cationic lipid is 2,3-dioleoyl-propyltrimethylammonium chloride (DOTAP), and the membrane regulator is phytosterol ester (SE), with a mass ratio of 20:(1-3):(2-8).

[0013] The further beneficial effects of the above-mentioned method are that phytosterol esters (SE) are structurally similar to cholesterol, can be embedded in liposome membranes and regulate membrane properties, and their esterified form has better lipid solubility and bioavailability than free sterols. They can also lower LDL cholesterol, have higher safety, and are an ideal substitute for cholesterol.

[0014] This invention uses phytosterol esters (SE) to replace cholesterol, fundamentally optimizing the membrane structure and successfully preparing enoki mushroom polysaccharide (FVP) nanoliposomes with higher encapsulation efficiency, significantly enhanced stability, and superior safety, providing a more reliable carrier solution for its application.

[0015] This invention constructs a composite membrane material using DPPC as the structural basis, DOTAP as the cationic functional component, and SE as a safe membrane modifier. Subsequently, a reverse evaporation method is used to drive the membrane material and the aqueous phase of enoki mushroom polysaccharide to undergo synergistic self-assembly, efficiently encapsulating FVP within the aqueous phase of the liposomes. Finally, through key process steps such as ice bath temperature-controlled ultrasound, gentle rotary evaporation for membrane formation, and controlled extrusion above the phospholipid phase transition temperature, the structure and properties of the liposomes are systematically regulated to form stable enoki mushroom polysaccharide nanoliposomes.

[0016] Furthermore, in step (4) above, the mass ratio of the enoki mushroom polysaccharide solution to the liposome organic phase is 1:(2-10).

[0017] Furthermore, in step (4) above, the conditions for ultrasound are ice water bath, power of 200-400 W, time of 5-10 min, 2.0 s on, 2.0 s off.

[0018] Furthermore, in step (5) above, the vacuum degree of the rotary evaporator is 0.03-0.08 MPa, the water bath temperature is 40-60℃, and the rotation speed is 30-60 rpm.

[0019] Furthermore, in step (5) above, the conditions for ultrasound are ice water bath, power of 200-400 W, time of 10-30 min, 2.0 s on, 2.0 s off.

[0020] Furthermore, in step (5) above, a polycarbonate film with a pore size of 100-200 nm is extruded at a temperature of 40-60℃.

[0021] The present invention also claims protection for a kind of enoki mushroom polysaccharide nanoliposome prepared by the above preparation method, which has a particle size of 80-130 nm, a zeta potential of 18-20 mV, a PDI≤0.3, and a polysaccharide encapsulation efficiency ≥60%.

[0022] The present invention also claims protection for the use of the enoki mushroom polysaccharide nanoliposomes prepared by the above preparation method in the preparation of foods, pharmaceuticals and cosmetics containing enoki mushroom polysaccharides.

[0023] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: (1) Encapsulation efficiency is significantly improved The introduction of phytosterol esters optimized the encapsulation capacity of the lipid bilayer for water-soluble polysaccharides, increasing the encapsulation efficiency of *Flammulina velutipes* polysaccharide nanoliposomes to over 60%, with a maximum of 71.64%, which is about 30% higher than that of traditional cholesterol liposomes. (2) Storage stability is significantly improved Under the condition of storage at 4℃ in the dark for 14 days, the particle size of SE-based nanoliposomes increased by less than 15%, and the zeta potential decayed by less than 5%, showing superior physical stability and charge retention compared to the cholesterol system. (3) Better cell safety Cell viability assays showed that phytosterol ester liposomes did not exhibit cytotoxicity and maintained high cell viability, demonstrating good biocompatibility.

[0024] (4) Enhanced immune activation capacity Compared to traditional cholesterol liposomes, phytosterol liposomes can more effectively promote the release of nitric oxide (NO) from macrophages, indicating that they can better support the immunomodulatory activity of enoki mushroom polysaccharides while delivering them.

[0025] In summary, this invention utilizes a phospholipid bilayer composed of the structural phospholipid dipalmitoylphosphatidylcholine (DPPC), the cationic lipid 2,3-dioleoyl-propyl-trimethylammonium chloride (DOTAP), and phytosterol esters (SE) as functionalized membrane modulators. These esters are then used to synergistically encapsulate *Flammulina velutipes* polysaccharide (FVP) in an aqueous inner phase via reverse evaporation. The resulting nanoliposomes have a particle size of 80-130 nm, a Zeta potential ≥18 mV, and an encapsulation efficiency of over 60% for FVP. This invention uses phytosterol esters to replace cholesterol in the preparation of *Flammulina velutipes* polysaccharide nanoliposomes. The *Flammulina velutipes* polysaccharide (FVP) is encapsulated within an aqueous core in the liposomes, resulting in higher safety and improved encapsulation efficiency, storage stability, and immunomodulatory activity. In vitro experiments have demonstrated that phytosterol ester liposomes significantly promote macrophage proliferation and nitric oxide production compared to traditional cholesterol liposomes under the same conditions. Attached Figure Description

[0026] Figure 1 The bar chart shows the encapsulation efficiency of the *Flammulina velutipes* polysaccharide nanoliposomes prepared in Examples 1-4 and Comparative Examples 1-2. Figure 2 The particle size stability diagram of the *Flammulina velutipes* polysaccharide nanoliposomes prepared in Examples 1-4 and Comparative Examples 1-2 at 4°C is shown. Figure 3 The graph shows the zeta potential changes of the *Flammulina velutipes* polysaccharide nanoliposomes prepared in Examples 1-4 and Comparative Examples 1-2 at 4°C. Figure 4 RAW 264.7 cell viability graphs of the *Flammulina velutipes* polysaccharide nanoliposomes prepared in Examples 1-4 and Comparative Examples 1-2; Figure 5 The NO production levels in RAW 264.7 cells of the *Flammulina velutipes* polysaccharide nanoliposomes prepared in Examples 1-4 and Comparative Examples 1-2 are shown. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] In the following examples, dipalmitoylphosphatidylcholine (DPPC) and 2,3-dioleoyl-propyltrimethylammonium chloride (DOTAP) were purchased from Aivito (Shanghai) Pharmaceutical Technology Co., Ltd., and phytosterol esters (SE) were purchased from Xi'an Guobang Industrial Co., Ltd.

[0029] Example 1 The preparation method of Flammulina velutipes polysaccharide nanoliposomes specifically includes the following steps: (1) Take dried enoki mushrooms, pulverize them using an ultra-micro pulverizer and pass them through a 200-mesh sieve to obtain enoki mushroom ultra-micro powder; add 95% ethanol at a mass-to-volume ratio of 1:6.5 (g / mL), heat to reflux reaction at 81℃, reflux reaction once for a total of 4.5 h, remove impurities such as lipids and pigments, vacuum filter to collect the precipitate, dry to obtain enoki mushroom decolorizing powder; add enoki mushroom decolorizing powder to pure water at a mass-to-volume ratio of 1:31 (g / mL), and hot water extract at 86℃ for 2.8 h; after extraction, centrifuge at 8500 rpm for 14 min, collect the supernatant; add Sevage reagent (chloroform: n-butanol = 4.5:1) to the supernatant, the volume ratio of supernatant to Sevage reagent is 4.5:1, shake vigorously for 22 min. After centrifugation, the lower protein precipitate was removed. This deproteinization process was repeated 5 times until no obvious protein layer was observed. 95% ethanol was slowly added to the supernatant after deproteinization to bring the ethanol volume fraction to 82%. The mixture was allowed to stand at 4°C for 25 h for alcohol precipitation. Subsequently, the mixture was centrifuged at 8500 rpm for 18 min, the precipitate was collected, dissolved in a small amount of deionized water, and transferred to a dialysis bag (molecular weight cutoff 3500 Da). Dialysis was performed using deionized water as the dialysis medium for 46 h. After dialysis, the liquid in the dialysis bag was freeze-dried in a freeze dryer to obtain enoki mushroom polysaccharide powder. (2) Dissolve the enoki mushroom polysaccharide powder in a phosphate buffer solution (pH=7.4) to prepare an enoki mushroom polysaccharide solution with a concentration of 2 mg / mL, which is used as the aqueous phase of the liposomes; (3) Weigh 100 mg of DPPC, 5 mg of DOTAP and 10 mg of SE in a mass ratio of 20:1:2. Mix the three together and add 5 mL of chloroform. Vortex until completely dissolved to obtain a homogeneous and transparent liposome organic phase. (4) The above aqueous phase is slowly added to the organic phase at a volume ratio of organic phase:water phase = 2:1. At the same time, it is placed in an ice-water bath and ultrasonically emulsified at a power of 200 W for 5 min (2.0 s on, 2.0 s off) to obtain a milky white emulsion. (5) Transfer the emulsion to a rotary evaporator, set the vacuum degree to 0.04 MPa, the water bath temperature to 40℃, and the rotation speed to 30 rpm, and remove the organic reagents by rotary evaporation under reduced pressure until a white gel forms on the inner wall of the flask; add 5 mL of phosphate buffer solution to the flask to hydrate it, so that the white gel completely detaches from the inner wall of the flask; then place the hydrated system in an ice-water bath and sonicate it at 200 W power for 10 min (on for 2.0 s, off for 2.0 s) to obtain a crude liposome suspension; place the crude liposome suspension in an extruder and extrude it 11 times each through polycarbonate membranes with 200 nm and 100 nm pore sizes at 40℃, and collect the final liquid, which is the uniform particle size of the enoki mushroom polysaccharide nanoliposomes.

[0030] Example 2 The preparation method of Flammulina velutipes polysaccharide nanoliposomes specifically includes the following steps: (1) Take dried enoki mushrooms, pulverize them using an ultra-micro pulverizer and pass them through a 200-mesh sieve to obtain enoki mushroom ultra-micro powder; add 95% ethanol at a mass-to-volume ratio of 1:6 (g / mL), heat to reflux reaction at 80℃, reflux reaction once, for a total of 4 h, remove impurities such as lipids and pigments, vacuum filter to collect the precipitate, dry to obtain enoki mushroom decolorizing powder; add enoki mushroom decolorizing powder to pure water at a mass-to-volume ratio of 1:30 (g / mL), and hot water extract at 85℃ for 2.5 h; after extraction, centrifuge at 8000 rpm for 15 min, collect the supernatant; add Sevage reagent (chloroform: n-butanol = 5:1) to the supernatant, the volume ratio of supernatant to Sevage reagent is 4:1, shake vigorously for 20 minutes. Centrifuge after min to remove the lower protein precipitate. Repeat this deproteinization process 5 times until no obvious protein layer is visible. Slowly add 95% ethanol to the supernatant after deproteinization to make the ethanol volume fraction of the system reach 80%. Let it stand at 4℃ for 24 h for alcohol precipitation. Then centrifuge at 8000 rpm for 20 min, collect the precipitate, dissolve it with a small amount of deionized water and transfer it to a dialysis bag (molecular weight cutoff 3500 Da). Dialyze with deionized water as the dialysis medium for 48 h. After dialysis, place the liquid in the dialysis bag in a freeze dryer to freeze dry and obtain enoki mushroom polysaccharide powder. (2) Dissolve the enoki mushroom polysaccharide powder in a phosphate buffer solution (pH=7.4) to prepare an enoki mushroom polysaccharide solution with a concentration of 2.5 mg / mL, which is used as the aqueous phase of the liposomes; (3) Weigh 100 mg of DPPC, 15 mg of DOTAP and 40 mg of SE in a mass ratio of 20:3:8. Mix the three together and add 10 mL of chloroform. Vortex until completely dissolved to obtain a homogeneous and transparent liposome organic phase. (4) The above aqueous phase was slowly added to the organic phase at a volume ratio of organic phase: aqueous phase = 4:1, and the mixture was placed in an ice-water bath and ultrasonically emulsified at a power of 300 W for 8 min to obtain a milky white emulsion. (5) Transfer the emulsion to a rotary evaporator, set the vacuum degree to 0.05 MPa, the water bath temperature to 40℃, and the rotation speed to 45 rpm, and remove the organic reagents by rotary evaporation under reduced pressure until a white gel forms on the inner wall of the flask; add 10 mL of phosphate buffer solution with pH=7.4 to the flask to hydrate it, so that the white gel completely detaches from the inner wall of the flask; then place the hydrated system in an ice-water bath and sonicate it at 300 W power for 12 min (on for 2.0 s, off for 2.0 s) to obtain a crude liposome suspension; place the crude liposome suspension in an extruder and extrude it 11 times each through polycarbonate membranes with pore sizes of 200 nm and 100 nm at 55℃, and collect the final liquid, which is the uniform particle size of the enoki mushroom polysaccharide nanoliposomes.

[0031] Example 3 The preparation method of Flammulina velutipes polysaccharide nanoliposomes specifically includes the following steps: (1) Take dried enoki mushrooms, pulverize them using an ultra-micro pulverizer and pass them through a 200-mesh sieve to obtain enoki mushroom ultra-micro powder; add 95% ethanol at a mass-to-volume ratio of 1:6 (g / mL), heat to reflux reaction at 80℃, reflux reaction once, for a total of 4 h, remove impurities such as lipids and pigments, filter to collect the precipitate, dry to obtain enoki mushroom decolorizing powder; add enoki mushroom decolorizing powder to pure water at a mass-to-volume ratio of 1:32 (g / mL), and extract with hot water at 85℃ for 3 h; after extraction, centrifuge at 8000 rpm for 15 min, collect the supernatant; add Sevage reagent (chloroform: n-butanol = 5:1) to the supernatant, the volume ratio of supernatant to Sevage reagent is 4:1, shake vigorously for 20 minutes. Centrifuge after min to remove the lower protein precipitate. Repeat this deproteinization process 5 times until no obvious protein layer is visible. Slowly add 95% ethanol to the supernatant after deproteinization to make the ethanol volume fraction of the system reach 80%. Let it stand at 4℃ for 24 h for alcohol precipitation. Then centrifuge at 8000 rpm for 20 min, collect the precipitate, dissolve it with a small amount of deionized water and transfer it to a dialysis bag (molecular weight cutoff 3500 Da). Dialyze with deionized water as the dialysis medium for 48 h. After dialysis, place the liquid in the dialysis bag in a freeze dryer to freeze dry and obtain enoki mushroom polysaccharide powder. (2) Dissolve the enoki mushroom polysaccharide powder in a phosphate buffer solution (pH=7.4) to prepare an enoki mushroom polysaccharide solution with a concentration of 6 mg / mL, which is used as the aqueous phase of the liposomes; (3) Weigh 100 mg of DPPC, 5 mg of DOTAP and 15 mg of SE in a mass ratio of 20:1:3. Mix the three and add 12 mL of chloroform. Vortex until completely dissolved to obtain a homogeneous and transparent liposome organic phase. (4) The above aqueous phase was slowly added to the organic phase at a volume ratio of organic phase:water phase = 6:1, and the mixture was placed in an ice-water bath and ultrasonically emulsified at a power of 350 W for 8 min to obtain an emulsion. (5) Transfer the emulsion to a rotary evaporator, set the vacuum degree to 0.07 MPa, the water bath temperature to 45℃, and the rotation speed to 50 rpm, and remove the organic reagents by rotary evaporation under reduced pressure for 25 min until a milky white gel forms on the inner wall of the flask; add 15 mL of phosphate buffer solution to the flask to hydrate it, so that the gel completely detaches from the inner wall of the flask; then place the hydrated system in an ice-water bath and sonicate it at 350 W power for 25 min (on for 2.0 s, off for 2.0 s) to obtain a crude liposome suspension; place the crude liposome suspension in an extruder and extrude it 11 times through a polycarbonate membrane with a pore size of 200 nm at 57℃, and collect the final liquid, which is the uniform particle size of the enoki mushroom polysaccharide nanoliposomes.

[0032] Example 4 The preparation method of Flammulina velutipes polysaccharide nanoliposomes specifically includes the following steps: (1) Take dried enoki mushrooms, pulverize them using an ultra-micro pulverizer and pass them through a 200-mesh sieve to obtain enoki mushroom ultra-micro powder; add 95% ethanol at a mass-to-volume ratio of 1:5 (g / mL), heat to reflux reaction at 78℃, reflux reaction once for a total of 3 h, remove impurities such as lipids and pigments, vacuum filter to collect the precipitate, dry to obtain enoki mushroom decolorizing powder; add enoki mushroom decolorizing powder to pure water at a mass-to-volume ratio of 1:28 (g / mL), and hot water extract at 83℃ for 2 h; after extraction, centrifuge at 7000 rpm for 12 min, collect the supernatant; add Sevage reagent (chloroform: n-butanol = 4:1) to the supernatant, the volume ratio of supernatant to Sevage reagent is 3:1, shake vigorously for 15 min and centrifuge to remove the lower protein precipitate, repeat this deproteinization operation 4 times until there is no obvious protein layer; slowly add 95% ethanol to the supernatant after deproteinization. Ethanol was used to make the system ethanol volume fraction reach 75%, and the mixture was allowed to stand at 4℃ for 20 h for alcohol precipitation. Then, it was centrifuged at 7000 rpm for 15 min, the precipitate was collected, dissolved in a small amount of deionized water and transferred to a dialysis bag (molecular weight cutoff 3500 Da). Dialysis was performed with deionized water for 40 h. After dialysis, the liquid in the dialysis bag was placed in a freeze dryer for freeze drying to obtain enoki mushroom polysaccharide powder. (2) Dissolve the enoki mushroom polysaccharide powder in a phosphate buffer solution (pH=7.4) to prepare an enoki mushroom polysaccharide solution with a concentration of 10 mg / mL, which is used as the aqueous phase of the liposomes; (3) Weigh 100 mg of DPPC, 5 mg of DOTAP and 10 mg of SE in a mass ratio of 20:1:2. Mix the three and add 20 mL of chloroform. Vortex until completely dissolved to obtain a homogeneous and transparent liposome organic phase. (4) The above aqueous phase is slowly added dropwise to the organic phase at a volume ratio of organic phase: aqueous phase = 10:1. At the same time, the mixture is placed in an ice-water bath and ultrasonically emulsified at a power of 400 W for 10 min (2.0 s on, 2.0 s off) to obtain a milky white emulsion. (5) Transfer the emulsion to a rotary evaporator, set the vacuum degree to 0.08 MPa, the water bath temperature to 60℃, and the rotation speed to 30 rpm, and remove the organic reagents by rotary evaporation under reduced pressure until a white gel forms on the inner wall of the flask; add 20 mL of phosphate buffer solution to the flask to hydrate it, so that the white gel completely detaches from the inner wall of the flask; then place the hydrated system in an ice-water bath and sonicate it at 400 W power for 10 min (on for 2.0 s, off for 2.0 s) to obtain a crude liposome suspension; place the crude liposome suspension in an extruder and extrude it 11 times through a polycarbonate membrane with a pore size of 200 nm at 40℃, and collect the final liquid, which is the uniform particle size of the enoki mushroom polysaccharide nanoliposomes.

[0033] Comparative Example 1 The preparation method of Flammulina velutipes polysaccharide nanoliposomes specifically includes the following steps: (1) Take dried enoki mushrooms, pulverize them using an ultra-micro pulverizer and pass them through a 200-mesh sieve to obtain enoki mushroom ultra-micro powder; add 95% ethanol at a mass-to-volume ratio of 1:6 (g / mL), heat to reflux reaction at 80℃, reflux reaction once, for a total of 4 h, remove impurities such as lipids and pigments, vacuum filter to collect the precipitate, dry to obtain enoki mushroom decolorizing powder; add enoki mushroom decolorizing powder to pure water at a mass-to-volume ratio of 1:30 (g / mL), and hot water extract at 85℃ for 2.5 h; after extraction, centrifuge at 8000 rpm for 15 min, collect the supernatant; add Sevage reagent (chloroform: n-butanol = 5:1) to the supernatant, the volume ratio of supernatant to Sevage reagent is 4:1, shake vigorously for 20 minutes. Centrifuge after min to remove the lower protein precipitate. Repeat this deproteinization process 5 times until no obvious protein layer is visible. Slowly add 95% ethanol to the supernatant after deproteinization to make the ethanol volume fraction of the system reach 80%. Let it stand at 4℃ for 24 h for alcohol precipitation. Then centrifuge at 8000 rpm for 20 min, collect the precipitate, dissolve it with a small amount of deionized water and transfer it to a dialysis bag (molecular weight cutoff 3500 Da). Dialyze with deionized water as the dialysis medium for 48 h. After dialysis, place the liquid in the dialysis bag in a freeze dryer to freeze dry and obtain enoki mushroom polysaccharide powder. (2) Dissolve the enoki mushroom polysaccharide powder in a phosphate buffer solution (pH=7.4) to prepare an enoki mushroom polysaccharide solution with a concentration of 2.5 mg / mL, which is used as the aqueous phase of the liposomes; (3) Weigh 100 mg of DPPC, 15 mg of DOTAP and 40 mg of cholesterol in a mass ratio of 20:3:8. Mix the three together and add 10 mL of chloroform. Vortex until completely dissolved to obtain a homogeneous and transparent liposome organic phase. (4) The above aqueous phase was slowly added to the organic phase at a volume ratio of organic phase: aqueous phase = 4:1. At the same time, the mixture was placed in an ice-water bath and ultrasonically emulsified at a power of 300 W for 8 min to obtain a milky white emulsion. (5) Transfer the emulsion to a rotary evaporator, set the vacuum degree to 0.05 MPa, the water bath temperature to 40℃, and the rotation speed to 45 rpm, and remove the organic reagents by rotary evaporation under reduced pressure until a white gel forms on the inner wall of the flask; add 10 mL of phosphate buffer solution with pH=7.4 to the flask to hydrate it, so that the white gel completely detaches from the inner wall of the flask; then place the hydrated system in an ice-water bath and sonicate it at 300 W power for 12 min (on for 2.0 s, off for 2.0 s) to obtain a crude liposome suspension; place the crude liposome suspension in an extruder and extrude it 11 times each through polycarbonate membranes with 200 nm and 100 nm pore sizes at 55℃, and collect the final liquid, which is the enoki mushroom polysaccharide nanoliposome.

[0034] Comparative Example 2 The preparation method of Flammulina velutipes polysaccharide nanoliposomes specifically includes the following steps: (1) Take dried enoki mushrooms, pulverize them using an ultra-micro pulverizer and pass them through a 200-mesh sieve to obtain enoki mushroom ultra-micro powder; add 95% ethanol at a mass-to-volume ratio of 1:6 (g / mL), heat to reflux reaction at 80℃, reflux reaction once, for a total of 4 h, remove impurities such as lipids and pigments, filter to collect the precipitate, dry to obtain enoki mushroom decolorizing powder; add enoki mushroom decolorizing powder to pure water at a mass-to-volume ratio of 1:32 (g / mL), and extract with hot water at 85℃ for 3 h; after extraction, centrifuge at 8000 rpm for 15 min, collect the supernatant; add Sevage reagent (chloroform: n-butanol = 5:1) to the supernatant, the volume ratio of supernatant to Sevage reagent is 4:1, shake vigorously for 20 minutes. Centrifuge after min to remove the lower protein precipitate. Repeat this deproteinization process 5 times until no obvious protein layer is visible. Slowly add 95% ethanol to the supernatant after deproteinization to make the ethanol volume fraction of the system reach 80%. Let it stand at 4℃ for 24 h for alcohol precipitation. Then centrifuge at 8000 rpm for 20 min, collect the precipitate, dissolve it with a small amount of deionized water and transfer it to a dialysis bag (molecular weight cutoff 3500 Da). Dialyze with deionized water as the dialysis medium for 48 h. After dialysis, place the liquid in the dialysis bag in a freeze dryer to freeze dry and obtain enoki mushroom polysaccharide powder. (2) Dissolve the enoki mushroom polysaccharide powder in a phosphate buffer solution (pH=7.4) to prepare an enoki mushroom polysaccharide solution with a concentration of 6 mg / mL, which is used as the aqueous phase of the liposomes; (3) Weigh 100 mg of DPPC, 5 mg of DOTAP and 15 mg of cholesterol in a mass ratio of 20:1:3. Mix the three together and add 12 mL of chloroform. Vortex until completely dissolved to obtain a homogeneous and transparent liposome organic phase. (4) The above aqueous phase is slowly added to the organic phase at a volume ratio of organic phase: aqueous phase = 6:1. At the same time, the mixture is placed in an ice-water bath and ultrasonically emulsified at a power of 350 W for 8 min to obtain an emulsion. (5) Transfer the emulsion to a rotary evaporator, set the vacuum degree to 0.07 MPa, the water bath temperature to 45℃, and the rotation speed to 50 rpm, and remove the organic reagents by rotary evaporation under reduced pressure until a milky white gel forms on the inner wall of the flask; add 15 mL of phosphate buffer solution (pH=7.4) to the flask to hydrate it, so that the gel completely detaches from the inner wall of the flask; then place the hydrated system in an ice-water bath and sonicate it at 350W power for 25 min (on for 2.0 s, off for 2.0 s) to obtain a crude liposome suspension; place the crude liposome suspension in an extruder and extrude it 11 times through a polycarbonate membrane with a pore size of 200 nm at 57℃, and collect the final liquid, which is the enoki mushroom polysaccharide nanoliposome.

[0035] Performance testing The encapsulation efficiency, particle size, polydispersity index, zeta potential, and storage stability of *Flammulina velutipes* polysaccharide / phytosterol ester nanoliposomes A, B, C, and D prepared by the methods in Examples 1-4 and those of *Flammulina velutipes* polysaccharide / cholesterol nanoliposomes E and F prepared by Comparative Examples 1-2 were tested respectively. Furthermore, to further evaluate the in vitro safety and immunomodulatory activity of *Flammulina velutipes* polysaccharide nanoliposomes, the cell viability and NO production of RAW 264.7 macrophages from Examples 1-4 and Comparative Examples 1-2 were compared. The specific experimental procedures are as follows: 1. Determination of particle size, PDI, zeta potential, and encapsulation efficiency. Dynamic light scattering (DLS) was used, and the particle size distribution was analyzed using a Malvern Zetasizer Nano ZS90 nanoparticle size analyzer. The *Flammulina velutipes* polysaccharide nanoliposomes (AF) from Examples 1-4 and Comparative Examples 1-2 were diluted 10-fold with a phosphate buffer solution at pH 7.4, ultrasonically dispersed for 1 min, and then placed in quartz cuvettes. The detection temperature was set at 25°C, and the equilibration time was 2 min. Each sample was measured in triplicate, and the average value was used as the final particle size and PDI (polydispersity index) result.

[0036] The Zeta potential detection module of the same model of nanoparticle size analyzer was used. The sample was diluted with deionized water to a suitable concentration, added to the Zeta potential detection cell, and equilibrated at 25°C for 3 min. Each sample was measured in triplicate, and the average value was taken as the potential result.

[0037] Glucose was used as a standard to obtain a glucose standard sugar solution with a concentration of 0.1 mg / mL. 0, 40, 80, 120, 160, and 200 μL of the standard sugar solution were taken into dry test tubes, and water was added to a final volume of 200 μL to obtain standard sugar solutions with concentration gradients. 500 μL each of the liposome shell and the glycopeptide-encapsulated liposome solution were centrifuged at 12000 r / min for 15 min at 4℃. The supernatant was collected and diluted 5-fold with PBS, and 200 μL of each solution was taken into test tubes as the sample solution to be tested. 0.2 mL of 6% phenol and 1 mL of concentrated sulfuric acid were added to each test tube solution, and the mixture was quickly mixed thoroughly and allowed to stand in the dark for 30 min. 200 μL of the test tube solution was added to each well of a 96-well plate, with three parallel studies. The absorbance was measured at 490 nm using a microplate reader to plot a glucose standard curve. The concentration of free polysaccharides in the supernatant was calculated, and the encapsulation efficiency of the *Flammulina velutipes* polysaccharide nanoliposomes (AF) was then calculated.

[0038] The results are shown in Table 1 and Figure 1 As shown.

[0039] Table 1. Properties of Flammulina aurea polysaccharide nanoliposomes from Examples 1-4 and Comparative Examples 1-2

[0040] According to Table 1 and Figure 1 It can be seen that the enoki mushroom polysaccharide / phytosterol ester (SE) nanoliposome AD is superior to the enoki mushroom polysaccharide / cholesterol nanoliposomes E and F in key physicochemical indicators.

[0041] Regarding particle size, the AD liposomes ranged from 82.35±0.22 nm to 129.53±0.38 nm, while the cholesterol samples E and F had particle sizes of 93.79±0.81 nm and 139.87±0.55 nm, respectively. The SE liposomes exhibited a smaller standard deviation in particle size data, indicating superior batch-to-batch homogeneity.

[0042] Regarding polydispersity index (PDI) and encapsulation performance, the PDI of sample AD ranged from 0.16±0.01 to 0.21±0.01, slightly higher than that of samples E (0.10±0.01) and F (0.11±0.02). The rigid steroidal core of cholesterol mainly plays a role in filling and stabilizing the membrane structure, which is conducive to the formation of a uniform and ordered membrane, hence the lower PDI. SE, due to the higher conformational flexibility introduced by its fatty acid side chains, can form a more dynamically adaptable lipid bilayer after co-assembly with DPPC and DOTAP. This structure can more effectively accommodate enoki mushroom polysaccharide (FVP) through local adjustments during the encapsulation process. Although it slightly increases the particle size distribution width, it directly leads to a significant improvement in encapsulation efficiency: the encapsulation efficiency of phytosterol ester sample AD ranged from 61.29±0.40% to 71.64±0.64%, significantly higher than that of cholesterol sample E (40.64±1.64%) and sample F (54.26±1.61%). This indicates that the introduction of SE optimizes the membrane's adaptability to water-soluble polysaccharides through its molecular flexibility, effectively solving the problem of low encapsulation efficiency.

[0043] Regarding stability, the zeta potential of sample AD ranged from 18.24±0.51 mV to 19.82±0.18 mV, both higher than that of cholesterol sample E (15.63±0.17 mV) and sample F (17.30±0.45 mV). The higher zeta potential helps enhance the electrostatic repulsion between liposomes, thereby improving their physical stability during storage and dispersion.

[0044] In summary, the experimental results show that using SE to replace cholesterol in the construction of nanoliposomes is not a simple substitution, but rather a synergistic effect with DPPC and DOTAP through its molecular properties, forming a functionalized membrane structure more suitable for encapsulating water-soluble polysaccharides. This structure significantly improves the encapsulation efficiency of *Flammulina velutipes* polysaccharides while maintaining good particle size uniformity and colloidal stability, providing a superior delivery system for subsequent applications.

[0045] 2. Stability determination at 4℃ Each sample was sealed and stored in a 4°C refrigerator away from light. Particle size, PDI and Zeta potential were measured on days 0, 3, 7 and 14.

[0046] like Figure 2 As shown, regarding particle size stability, the particle size increase of all phytosterol ester samples A, B, C, and D during storage was generally lower than that of cholesterol samples (E and F). Specifically, sample A had a particle size of 82.35 nm on day 0, which increased to approximately 93.62 nm by day 14, an increase of approximately 13.7%; sample B increased from approximately 90.04 nm to approximately 108.27 nm, an increase of approximately 20.2%; sample C increased from approximately 124.77 nm to approximately 137.90 nm, an increase of approximately 10.5%; and sample D increased from approximately 129.53 nm to approximately 143.01 nm, an increase of approximately 10.4%. In contrast, the particle size of cholesterol sample E increased from approximately 93.79 nm to approximately 108.30 nm, an increase of approximately 15.4%; and sample F increased from approximately 139.87 nm to approximately 177.77 nm, an increase of approximately 27.1%. The results showed that the particle size increase of phytosterol ester samples was generally lower than that of cholesterol samples during storage, especially the C and D samples, which showed the smallest increase, indicating that they have better physical structural stability.

[0047] like Figure 3 As shown, regarding the change in zeta potential, the potential decay of phytosterol ester samples was generally lower than that of cholesterol samples. The potential of sample A decreased from approximately 19.82 mV to approximately 19.25 mV, a decrease of approximately 2.9%; sample B decreased from approximately 19.17 mV to approximately 18.47 mV, a decrease of approximately 3.7%; sample C decreased from approximately 18.57 mV to approximately 18.00 mV, a decrease of approximately 3.1%; and sample D decreased from approximately 18.24 mV to approximately 17.79 mV, a decrease of approximately 2.5%. In contrast, the potential of cholesterol sample E decreased from approximately 15.63 mV to approximately 12.33 mV, a decrease of approximately 21.1%; and sample F decreased from approximately 17.30 mV to approximately 14.87 mV, a decrease of approximately 14.0%. This indicates that the surface charge of phytosterol ester liposomes remains more stable during storage, which helps maintain the electrostatic repulsion and colloidal dispersion of the system, and slows down the aggregation tendency.

[0048] In summary, the experimental data show that during the 14-day storage period, SE liposomes exhibited lower particle size increase and potential decay than cholesterol liposomes, indicating that they have relatively better physical stability and charge retention capacity, which is beneficial for the storage and transportation of formulations.

[0049] 3. CCK-8 cell viability assay Logarithmically growing RAW 264.7 macrophages were seeded at 2.7 × 10⁴ cells / well in 96-well cell culture plates, with 200 μL of DMEM medium containing 10% fetal bovine serum added to each well. The plates were incubated at 37°C with 5% CO₂ for 24 h. The old medium was discarded. Positive control and experimental groups were treated with nanoliposomes containing 1 μg / mL lipopolysaccharide (LPS) and 200 μg / mL *Flammulina velutipes* polysaccharide, respectively (Examples 1-4, Comparative Examples 1-2). The control group received an equal volume of medium. Each group was divided into six replicates. After another 24 h of incubation, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated in the dark for 0.5 h. The absorbance (OD value) was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0050] Figure 4 The results showed that the *Flammulina velutipes* polysaccharide nanoliposomes AD were superior to *Flammulina velutipes* polysaccharide / cholesterol nanoliposomes E and F in terms of overall cell safety and biocompatibility. At the experimental concentration of 200 μg / mL, the macrophage survival rate in the phytosterol ester liposome treatment groups remained at a high level, significantly higher than that in the control group. In contrast, although the cell survival rates of the cholesterol liposome groups E and F were still higher than those of the control group, they were generally lower than those in the phytosterol ester group.

[0051] In summary, the phytosterol ester-based enoki mushroom polysaccharide nanoliposomes exhibited superior biocompatibility and potential proliferative activity in a macrophage model, providing a reliable cell safety basis for further research on their applications in immune regulation.

[0052] 4. NO generation determination RAW 264.7 cells were fed at a rate of 2.7 × 10⁻⁶. 4 Each sample was seeded into a 96-well plate. After 24 h of incubation, the culture medium was discarded. The positive control group and experimental group were inoculated with culture medium containing 1 μg / mL LPS and 200 μg / mL sample, respectively. The blank group was inoculated with culture medium without LPS and sample. Each group had 6 replicates. After 24 h of incubation, the supernatant of each well was collected, and the NO content was determined by the Griess reagent method: 50 μL of supernatant was mixed with 50 μL of Griess reagent A, and then 50 μL of Griess reagent B was added. After incubation for 10 min, the OD value was measured at 540 nm. A standard curve was plotted using sodium nitrite as a standard, and the NO production of the sample was calculated.

[0053] Figure 5The results showed that *Flammulina velutipes* polysaccharide nanoliposomes (AD) exhibited a stronger overall stimulatory effect in inducing NO production in RAW 264.7 macrophages compared to *Flammulina velutipes* polysaccharide / cholesterol nanoliposomes (E, F). In the phytosterol ester groups, group D produced the highest NO level, followed by group A, both significantly higher than cholesterol groups E and F. While groups B and C had lower NO levels, they were still comparable to or slightly higher than cholesterol group F. NO is a key marker molecule for macrophage activation, and its production level can reflect the immunostimulatory potential of the material.

[0054] The above results indicate that, under the experimental conditions, phytosterol ester liposomes (especially A and D) exhibited relatively stronger macrophage activation ability, providing experimental evidence for further investigation of their immunomodulatory function.

[0055] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of a Flammulina velutipes polysaccharide nano-liposome, characterized in that, Specifically comprising the following steps: (1) sequentially subjecting the raw material powder of Flammulina velutipes to hot water extraction, deproteinization, alcohol precipitation, dialysis, and freeze-drying to obtain Flammulina velutipes polysaccharide; (2) dissolving the Flammulina velutipes polysaccharide in a phosphate buffer solution to obtain a Flammulina velutipes polysaccharide solution for standby; (3) mixing and dissolving phospholipid, cationic lipid, and membrane regulator in an organic solvent to obtain a liposome organic phase; (4) adding the Flammulina velutipes polysaccharide solution to the liposome organic phase and subjecting to ultrasonication to obtain a Flammulina velutipes polysaccharide liposome emulsion; (5) sequentially subjecting the Flammulina velutipes polysaccharide liposome emulsion to reduced pressure rotary evaporation, phosphate buffer hydration, ultrasonication, and extrusion to obtain the Flammulina velutipes polysaccharide nanoliposome. 2.The preparation method of the Flammulina velutipes polysaccharide nano-liposome according to claim 1, characterized in that, In step (2), the concentration of the Flammulina velutipes polysaccharide solution is 2-10 mg / mL.

3. The preparation method of the Flammulina velutipes polysaccharide nano-liposome according to claim 1, characterized in that, In step (3), the phospholipid is dipalmitoyl phosphatidylcholine, the cationic lipid is 2,3-dioleoyl-propyl-trimethylammonium chloride, and the membrane regulator is phytosterol ester, with a mass ratio of 20:(1-3):(2-8). 4.The preparation method of the Flammulina velutipes polysaccharide nano-liposome according to claim 1, characterized in that, In step (4), the mass ratio of the Flammulina velutipes polysaccharide solution to the liposome organic phase is 1:(2-10).

5. The preparation method of the Flammulina velutipes polysaccharide nano-liposome according to claim 1, characterized in that, In step (4), the ultrasonication is performed in an ice water bath at a power of 200-400 W for 5-10 min, with 2.0 s on and 2.0 s off. 6.The preparation method of the Flammulina velutipes polysaccharide nano-liposome according to claim 1, characterized in that, In step (5), the reduced pressure rotary evaporation is performed at a vacuum degree of 0.03-0.08 MPa, a water bath temperature of 40-60℃, and a rotation speed of 30-60 rpm.

7. The preparation method of the Flammulina velutipes polysaccharide nano-liposome according to claim 1, characterized in that, In step (5), the ultrasonication is performed in an ice water bath at a power of 200-400 W for 10-30 min, with 2.0 s on and 2.0 s off. 8.The preparation method of the Flammulina velutipes polysaccharide nano-liposome according to claim 1, characterized in that, In step (5), the extrusion is performed using a polycarbonate membrane with a pore size of 100-200 nm at a temperature of 40-60℃.

9. The Flammulina velutipes polysaccharide nanoliposome prepared by the method according to any one of claims 1-8, characterized in that, The particle size is 80-130 nm, the Zeta potential is 18-20 mV, the PDI is ≤0.3, and the polysaccharide encapsulation rate is ≥60%.

10. Use of the Flammulina velutipes polysaccharide nanoliposome prepared by the preparation method of any one of claims 1-8 in the preparation of food, medicine, and cosmetics containing Flammulina velutipes polysaccharide.

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