Dioscorea polysaccharide liposome nano immunopotentiating adjuvant, application and preparation method

Abstract

The application discloses a kind of yam polysaccharide liposome nano immunopotentiating adjuvant, application and preparation method, based on liposome self-assembly mechanism, preparation is completed by adjusting flow rate and flow rate ratio control particle size through microfluidic device, the microfluidic device includes product outlet, solution mixing channel and several injection ports, the injection port and product outlet are all equipped with the catheter connected with external equipment;Raw materials for preparing the liposome include yam polysaccharide, egg yolk lecithin and cholesterol.By the size design of channel in preparation process, flow rate ratio and the control of total flow rate, the particle size of blank liposome prepared can be realized particle size regulation in 50nm-250nm range, distribution coefficient is less than 0.2, with single dispersibility.Yam polysaccharide liposome prepared has encapsulation efficiency of 79.02%, average particle size 154.2nm, PDI is 0.083, product is uniform and stable, with sustained-release effect, in vitro cell experiment shows that it can promote the proliferation of mouse spleen lymphocytes, with immunopotentiating effect.

Description

Technical Field

[0001] This invention relates to the field of immunology and pharmaceutical preparation technology, and in particular to a yam polysaccharide liposome nano-immunoenhancing adjuvant, its application and preparation method. Background Technology

[0002] Microfluidics is an emerging technology that can be applied to manipulate micro- and nano-scale self-assembled systems to achieve precise control over particle size. The microchannels in microfluidic chips can reach the micrometer level, achieving ideal diffusion and mixing effects for fluids. Microfluidic methods offer the ability to continuously optimize and produce uniform nanoparticles, overcoming many limitations of traditional mass production methods.

[0003] Liposomes are artificially synthesized, closed, spherical vesicles that mimic cell membranes. They can carry various hydrophilic, hydrophobic, and amphoteric substances, which can be encapsulated within the aqueous phase of the liposome, inserted into the lipid bilayer, or adsorbed or attached to the liposome surface, serving as both cell membrane mimics and drug carriers. As a drug carrier, the size of liposomes is related to their distribution location and duration in vivo; different drugs produce varying therapeutic effects at different locations within the body. Furthermore, the particle size of liposomes also affects their stability and encapsulation efficiency.

[0004] Liposomes produced by extruding liposomes through polycarbonate membranes can yield liposomes with specified pore sizes and acceptable PDI (≤0.2), making it the most acceptable and reproducible process for manufacturing liposomes with known defined characteristics. However, extrusion is also a laborious and time-consuming process. In liposome production, microfluidics, through the use of intersecting microchannels, enables high-level mixing of fluids within the channels. This not only replaces the lipid hydration and extrusion steps in the liposome production process but also allows for flexible control of nanoscale particle sizes.

[0005] Yam is a well-known edible and medicinal plant in China. Yam polysaccharides are an important component of yam, mainly composed of mannose, xylose, arabinose, glucose, and galactose. Yam polysaccharides have been shown to possess various biological activities, such as immunomodulatory, antioxidant, and antitumor activities.

[0006] Because polysaccharides have unique structural characteristics—such as the type of monosaccharide, linkage sites, configuration of monosaccharides and glycosidic bonds, and the number of repeating units—these vary depending on the type of polysaccharide extracted. Therefore, existing liposome preparation methods offer limited guidance for the preparation of yam polysaccharide liposome nanoparticles. Furthermore, preparing polysaccharide liposomes with high encapsulation efficiency remains a recognized challenge. Obtaining yam polysaccharide liposome nanoparticles with both high drug encapsulation efficiency and small particle size is even more difficult.

[0007] This invention discloses a yam polysaccharide liposome nano-immunoenhancing adjuvant, its application, and its preparation method. It is the first time that yam polysaccharide liposomes have been prepared using a microfluidic chip, achieving the controllable preparation of yam polysaccharide liposomes with high encapsulation efficiency. Summary of the Invention

[0008] The purpose of this invention is to provide a microfluidic-based yam polysaccharide liposome nano-immunoenhancing adjuvant, its application, and its preparation method, achieving controllable particle size and high monodispersity.

[0009] To achieve the above-mentioned objectives, the technical solution adopted by this invention is: a method for preparing a yam polysaccharide liposome nano-immunoenhancing adjuvant, based on the liposome self-assembly mechanism, using a microfluidic device to adjust the flow rate and flow rate ratio to control the particle size to complete the preparation. The microfluidic device includes a product outlet, a solution mixing channel, and several injection ports, and both the injection ports and the product outlet are provided with conduits for connection to external equipment; the raw materials for preparing the liposomes include yam polysaccharide, egg yolk lecithin, and cholesterol.

[0010] Preferably, the injection port includes an aqueous solution inlet and a lipid solution inlet, both of which are connected to an external micro-injection pump via conduits; the product flows into an external receiving device through a conduit at the product outlet.

[0011] Preferably, the aqueous phase solution inlet is a yam polysaccharide buffer solution inlet, and the lipid phase solution inlet is a lipid ethanol solution inlet.

[0012] Preferably, the solution mixing channel includes a fluid focusing mixing unit and a straight shearing channel with baffles.

[0013] Preferably, the microfluidic device is a lollipop-shaped microfluidic chip, which is made of polydimethylsiloxane (PDMS) and has a lipid phase solution inlet between two adjacent aqueous solution inlets. The microfluidic chip is treated with a plasma machine to bond and seal the chip channels, eliminating the need for fixtures and allowing for reuse.

[0014] Preferably, there are two aqueous solution inlets and one lipid solution inlet, with the lipid solution inlet located between the two aqueous solution inlets.

[0015] Preferably, the conduit is a polytetrafluoroethylene tube with an inner diameter of 0.3–0.8 mm and an outer diameter of 0.9–1.4 mm.

[0016] Preferably, the width of the injection port, product outlet, and mixing channel ranges from 50μm to 300μm, the channel height ranges from 50μm to 200μm, and the length of the straight shearing channel ranges from 5mm to 20mm.

[0017] Preferably, the specific steps include:

[0018] Step 1: Take egg yolk lecithin and cholesterol, add them to anhydrous ethanol, and sonicate to dissolve them to prepare a lipid phase solution;

[0019] Step 2: Add yam polysaccharide to phosphate buffer, sonicate to dissolve, and filter through a 0.45μm organic nylon membrane to obtain an aqueous solution;

[0020] Step 3: Add the aqueous phase solution and lipid phase solution to the external micro-injection pump, fix them on the micro-injection pump, connect them to the injection port through the catheter, and set the flow rate.

[0021] Step 4: The aqueous phase solution and the lipid phase solution merge in the fluid focusing mixing unit. Ethanol diffuses into the buffer phase, and the ethanol concentration decreases. Lipid molecules self-assemble and encapsulate the yam polysaccharide aqueous phase to form crude yam polysaccharide liposomes. Under the action of shear force in the straight shear channel, the particle size is further reduced. The product flows into the receiving device through the conduit on the product outlet.

[0022] Step 5: Collect the product, remove ethanol by rotary evaporation, and bring the volume up to the original solution volume with phosphate buffer solution to obtain the target yam polysaccharide liposome nano-formulation, which is then stored at low temperature.

[0023] Preferably, in step one, the mass concentration ratio of egg yolk lecithin to cholesterol is 1:1 to 10:1, and the concentration of egg yolk lecithin in anhydrous ethanol is 5 mg / mL to 15 mg / mL.

[0024] Preferably, the mass concentration ratio of egg yolk lecithin to cholesterol includes, but is not limited to, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, and 10:1, and is preferably 5:1 to 8:1.

[0025] Preferably, the concentration of the egg yolk lecithin in anhydrous ethanol includes, but is not limited to, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, and 15 mg / mL, and is more preferably 5-10 mg / mL.

[0026] Preferably, in step two, the concentration of yam polysaccharide in phosphate buffer is 0.02 mg / mL to 2 mg / mL.

[0027] Preferably, in step two, the concentration of yam polysaccharide in phosphate buffer includes, but is not limited to, 0.02 mg / mL, 0.05 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, and 2 mg / mL, and is preferably 0.2 mg / mL to 1 mg / mL.

[0028] Preferably, in step three, the particle size, dispersibility, and encapsulation efficiency of yam polysaccharide liposomes can be controlled by adjusting the flow rates of the lipid phase and the aqueous phase; the sum of the flow rates of the yam polysaccharide buffer phase and the lipid phase is 400 μL / min to 1000 μL / min, and the flow rate ratio is 3:1 to 9:1.

[0029] Preferably, in step three, the sum of the flow rates of the yam polysaccharide buffer phase and the lipid phase includes, but is not limited to, 400 μL / min, 450 μL / min, 500 μL / min, 550 μL / min, 600 μL / min, 650 μL / min, 700 μL / min, 750 μL / min, 800 μL / min, 850 μL / min, 900 μL / min, 950 μL / min, and 1000 μL / min; more preferably, it is 600 μL / min to 900 μL / min.

[0030] Preferably, in step three, the flow rate ratio of the yam polysaccharide buffer phase to the lipid phase includes, but is not limited to, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, and 9:1, and is preferably 5:1 to 8:1.

[0031] Preferably, in step four, the mixing mode at the fluid focal point is that the lipid phase encapsulates the aqueous phase, which is then enveloped by the outer aqueous phase.

[0032] Preferably, in step five, the single collection volume of the product is 10 mL to 20 mL, the rotary evaporation speed is 30 r / min to 60 r / min, the rotary evaporation temperature is 30 °C to 50 °C, and the rotary evaporation time is 5 min to 10 min.

[0033] Preferably, in step five, the single collection volume of the product includes, but is not limited to, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, and 20 mL.

[0034] Preferably, in step five, the rotary evaporation speed includes, but is not limited to, 30 r / min, 35 r / min, 40 r / min, 45 r / min, 50 r / min, 55 r / min, and 60 r / min, and is preferably 35 r / min to 45 r / min.

[0035] Preferably, in step five, the rotary evaporation temperature includes, but is not limited to, 30°C, 32°C, 35°C, 38°C, 40°C, 42°C, 45°C, 48°C, and 50°C, with 40°C to 45°C being the most preferred.

[0036] Preferably, in step five, the rotary evaporation time includes, but is not limited to, 5 min, 6 min, 7 min, 8 min, 9 min, and 10 min, and is preferably 6 min to 8 min.

[0037] Preferably, in step five, the storage temperature is 2–5°C, and more preferably 4°C.

[0038] As described above, the preparation method of this application can achieve particle size regulation of yam polysaccharide liposomes in the range of 50 nm to 250 nm, with a distribution coefficient of less than 0.2.

[0039] This application also claims protection for a yam polysaccharide liposome nano-immunoenhancing adjuvant, prepared using the preparation method described above.

[0040] This application also claims the use of a yam polysaccharide liposome nano-immunoenhancing adjuvant as described above in immune-enhancing drugs and vaccine adjuvants.

[0041] The yam polysaccharide liposomes prepared by the method described above have uniform particle size and good dispersibility. When dialyzed in phosphate buffer at 37°C, the liposomes slow down the release of yam polysaccharides, exhibiting a sustained-release effect.

[0042] In the above study, the in vitro effect of yam polysaccharide liposomes on the proliferation of mouse spleen lymphocytes was investigated. The concentration range that alone stimulated lymphocytes showed a significant proliferation effect was selected. In combination with ConA / LPS, it promoted the proliferation of T / B cells. It was found that compared with other blank groups and control groups, yam polysaccharide liposomes could significantly promote lymphocyte proliferation at various concentrations, indicating that it has a sustained immune-enhancing function.

[0043] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0044] 1. By combining microfluidic technology and utilizing channel shear force, the prepared yam polysaccharide liposomes exhibit controllable particle size and a good distribution coefficient (PDI < 0.2). The process is simple, reproducible, and beneficial for the production of yam polysaccharide liposome nano-immunoenhancing adjuvants. The encapsulation efficiency of yam polysaccharide liposomes can reach 79.02%, with an average particle size of 154.2 nm and a PDI of 0.083. The product is homogeneous, stable, and exhibits a sustained-release effect.

[0045] 2. The preparation method of this invention is simple to operate, and the prepared yam polysaccharide liposomes have excellent comprehensive properties. In vitro cell experiments show that they can promote the proliferation of mouse spleen lymphocytes and have an immune-enhancing effect, providing a choice for vaccine adjuvants. Attached Figure Description

[0046] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, some of the drawings in the following description are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0047] Figure 1 This is a schematic diagram of a microfluidic device.

[0048] Figure 2 Figure showing the particle size of blank liposomes prepared with different flow rate ratios of aqueous and lipid phases and total flow rate.

[0049] Figure 3 Distribution coefficients of blank liposomes prepared with different flow rate ratios of aqueous and lipid phases and total flow rate.

[0050] Figure 4 Transmission electron microscopy image of the yam polysaccharide liposomes prepared in this invention.

[0051] Figure 5 The diagram shows the in vitro release results of the yam polysaccharide liposomes and yam polysaccharides prepared in this invention.

[0052] Figure 6 The results of yam polysaccharide liposomes stimulating lymphocyte proliferation alone.

[0053] Figure 7 The results of T lymphocyte proliferation stimulated by yam polysaccharide liposomes in conjunction with concanavalin A protein.

[0054] Figure 8 The results of yam polysaccharide liposomes synergistically stimulating B lymphocyte proliferation.

[0055] Among them, 001 is the inlet A of the yam polysaccharide phosphate buffer solution; 003 is the inlet B of the yam polysaccharide phosphate buffer solution; 002 is the lipid phase inlet; and 004 is the product outlet. Detailed Implementation

[0056] 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.

[0057] Comparative Example 1

[0058] This comparative example involves the preparation of blank liposomes, and the specific steps include:

[0059] Egg yolk lecithin and cholesterol were dissolved in anhydrous ethanol by sonication to prepare the lipid phase solution; phosphate buffer (pH=7.4) was used as the aqueous phase solution.

[0060] Take two 30mL syringes, draw up the aqueous solution, fix them on a multichannel microinfusion pump, and connect them to the pump via tubing. Figure 1 The microfluidic chip device shown is connected to the yam polysaccharide phosphate buffer solution inlet A001 and B003. A 10 mL syringe is used to draw up the lipid phase solution, which is fixed on a multichannel microinjection pump and connected to the lipid phase inlet 002 of the microfluidic chip via a catheter. The total flow rate is set to 400 μL / min, and the injection rates of the yam polysaccharide phosphate buffer solution inlets A001 and B003 are kept consistent, with a flow rate ratio of 3:1 to the lipid phase inlet 002. The product flows out through the catheter at the product outlet 004 of the microfluidic chip and is collected in a 15 mL centrifuge tube. Ethanol is removed by rotary evaporation at 40 °C for 5 min, and the volume is adjusted to the original solution volume with phosphate buffer solution to obtain the target blank liposome (BL), which is then stored at 4 °C.

[0061] Comparative Example 2

[0062] This comparative example involves the particle size control of blank liposomes. The preparation method of blank liposomes is the same as that of comparative example 1, wherein the flow rate ratios are 3:1, 5:1, 7:1, and 9:1, and the total flow rate is 400 μL / min, 600 μL / min, 800 μL / min, and 1000 μL / min, respectively. The effects of different flow rate ratios and total flow rates on the particle size and distribution coefficient of blank liposomes are investigated.

[0063] The effects of different flow rate ratios and total flow rates on the particle size of blank liposomes are as follows: Figure 2Under these flow rate ratio and total flow rate conditions, blank liposomes can be prepared with controllable particle size in the range of 50 nm to 250 nm, and the particle size tends to decrease as the total flow rate and flow rate ratio increase.

[0064] The effects of different flow rate ratios and total flow rates on the particle size of blank liposomes are as follows: Figure 3 The distribution coefficient of the blank liposomes under both the flow rate ratio and the total flow rate conditions was less than 0.2, indicating that the blank liposomes prepared under these conditions had good monodispersity.

[0065] Example 1

[0066] This embodiment relates to a method for preparing a yam polysaccharide liposome nano-immunoenhancing adjuvant. Based on the liposome self-assembly mechanism, the preparation is completed by adjusting the flow rate and flow rate ratio through a microfluidic device to control the particle size. The microfluidic device includes a product outlet, a solution mixing channel, and several injection ports. Both the injection ports and the product outlet are equipped with conduits for connection to external devices. The raw materials for preparing the liposomes include yam polysaccharide, egg yolk lecithin, and cholesterol.

[0067] Preferably, the injection port includes an aqueous solution inlet and a lipid solution inlet, both of which are connected to an external micro-injection pump via conduits; the product flows into an external receiving device through a conduit at the product outlet.

[0068] Preferably, the aqueous phase solution inlet is a yam polysaccharide buffer solution inlet, and the lipid phase solution inlet is a lipid ethanol solution inlet.

[0069] Preferably, the solution mixing channel includes a fluid focusing mixing unit and a straight shearing channel with baffles.

[0070] Preferably, the microfluidic device is a lollipop-shaped microfluidic chip, which is made of polydimethylsiloxane (PDMS) and has a lipid phase solution inlet between two adjacent aqueous solution inlets. The microfluidic chip is treated with a plasma machine to bond and seal the chip channels, eliminating the need for fixtures and allowing for reuse.

[0071] Preferably, there are two aqueous solution inlets and one lipid solution inlet, with the lipid solution inlet located between the two aqueous solution inlets.

[0072] Preferably, the conduit is a polytetrafluoroethylene tube with an inner diameter of 0.3–0.8 mm and an outer diameter of 0.9–1.4 mm.

[0073] Preferably, the width of the injection port, product outlet, and mixing channel ranges from 50μm to 300μm, the channel height ranges from 50μm to 200μm, and the length of the straight shearing channel ranges from 5mm to 20mm.

[0074] Preferably, the specific steps include:

[0075] Step 1: Take egg yolk lecithin and cholesterol, add them to anhydrous ethanol, and sonicate to dissolve them to prepare a lipid phase solution;

[0076] Step 2: Add yam polysaccharide to phosphate buffer, sonicate to dissolve, and filter through a 0.45μm organic nylon membrane to obtain an aqueous solution;

[0077] Step 3: Add the aqueous phase solution and lipid phase solution to the external micro-injection pump, fix them on the micro-injection pump, connect them to the injection port through the catheter, and set the flow rate.

[0078] Step 4: The aqueous phase solution and the lipid phase solution merge in the fluid focusing mixing unit. Ethanol diffuses into the buffer phase, and the ethanol concentration decreases. Lipid molecules self-assemble and encapsulate the yam polysaccharide aqueous phase to form crude yam polysaccharide liposomes. Under the action of shear force in the straight shear channel, the particle size is further reduced. The product flows into the receiving device through the conduit on the product outlet.

[0079] Step 5: Collect the product, remove ethanol by rotary evaporation, and bring the volume up to the original solution volume with phosphate buffer solution to obtain the target yam polysaccharide liposome nano-formulation, which is then stored at low temperature.

[0080] Preferably, in step one, the mass concentration ratio of egg yolk lecithin to cholesterol is 1:1 to 10:1, and the concentration of egg yolk lecithin in anhydrous ethanol is 5 mg / mL to 15 mg / mL.

[0081] Preferably, the mass concentration ratio of egg yolk lecithin to cholesterol includes, but is not limited to, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, and 10:1, and is preferably 5:1 to 8:1.

[0082] Preferably, the concentration of the egg yolk lecithin in anhydrous ethanol includes, but is not limited to, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, and 15 mg / mL, and is more preferably 5-10 mg / mL.

[0083] Preferably, in step two, the concentration of yam polysaccharide in phosphate buffer is 0.02 mg / mL to 2 mg / mL.

[0084] Preferably, in step two, the concentration of yam polysaccharide in phosphate buffer includes, but is not limited to, 0.02 mg / mL, 0.05 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, and 2 mg / mL, and is preferably 0.2 mg / mL to 1 mg / mL.

[0085] Preferably, in step three, the particle size, dispersibility, and encapsulation efficiency of yam polysaccharide liposomes can be controlled by adjusting the flow rates of the lipid phase and the aqueous phase; the sum of the flow rates of the yam polysaccharide buffer phase and the lipid phase is 400 μL / min to 1000 μL / min, and the flow rate ratio is 3:1 to 9:1.

[0086] Preferably, in step three, the sum of the flow rates of the yam polysaccharide buffer phase and the lipid phase includes, but is not limited to, 400 μL / min, 450 μL / min, 500 μL / min, 550 μL / min, 600 μL / min, 650 μL / min, 700 μL / min, 750 μL / min, 800 μL / min, 850 μL / min, 900 μL / min, 950 μL / min, and 1000 μL / min; more preferably, it is 600 μL / min to 900 μL / min.

[0087] Preferably, in step three, the flow rate ratio of the yam polysaccharide buffer phase to the lipid phase includes, but is not limited to, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, and 9:1, and is preferably 5:1 to 8:1.

[0088] Preferably, in step four, the mixing mode at the fluid focal point is that the lipid phase encapsulates the aqueous phase, which is then enveloped by the outer aqueous phase.

[0089] Preferably, in step five, the single collection volume of the product is 10 mL to 20 mL, the rotary evaporation speed is 30 r / min to 60 r / min, the rotary evaporation temperature is 30 °C to 50 °C, and the rotary evaporation time is 5 min to 10 min.

[0090] Preferably, in step five, the single collection volume of the product includes, but is not limited to, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, and 20 mL.

[0091] Preferably, in step five, the rotary evaporation speed includes, but is not limited to, 30 r / min, 35 r / min, 40 r / min, 45 r / min, 50 r / min, 55 r / min, and 60 r / min, and is preferably 35 r / min to 45 r / min.

[0092] Preferably, in step five, the rotary evaporation temperature includes, but is not limited to, 30°C, 32°C, 35°C, 38°C, 40°C, 42°C, 45°C, 48°C, and 50°C, with 40°C to 45°C being the most preferred.

[0093] Preferably, in step five, the rotary evaporation time includes, but is not limited to, 5 min, 6 min, 7 min, 8 min, 9 min, and 10 min, and is preferably 6 min to 8 min.

[0094] Preferably, in step five, the storage temperature is 2–5°C, and more preferably 4°C.

[0095] As described above, the preparation method of this application can achieve particle size regulation of yam polysaccharide liposomes in the range of 50 nm to 250 nm, with a distribution coefficient of less than 0.2.

[0096] This embodiment also relates to a yam polysaccharide liposome nano-immunoenhancing adjuvant, which is prepared using the preparation method described above.

[0097] This embodiment also relates to the application of a yam polysaccharide liposome nano-immunoenhancing adjuvant as described above in immune-enhancing drugs and vaccine adjuvants.

[0098] The encapsulation efficiency of the yam polysaccharide liposomes prepared in this embodiment was determined by a combination of protamine sulfate method and phenol-sulfuric acid method. Specific steps included:

[0099] Isolation of free yam polysaccharides: 0.5 mL of liposomes was transferred to a 5 mL centrifuge tube, and 0.5 mL of protamine sulfate (10 mg / mL) was added. After thorough mixing, the mixture was allowed to stand for 3 min. Then, 3 mL of phosphate buffer was added, and the mixture was centrifuged at 4000 rpm for 30 min at room temperature. The supernatant was collected, and the polysaccharide content (i.e., the amount of free yam polysaccharides) was determined. 0.5 mL of n-propanol was added to the precipitate to dissolve and break the emulsion, and 3 mL of phosphate buffer was added. The polysaccharide content (i.e., the amount of encapsulated yam polysaccharides) was then determined.

[0100] Determination of polysaccharide content: Accurately pipette 200 μL of sample into a 2 mL stoppered plastic centrifuge tube, add 100 μL of 6% redistilled phenol solution and 500 μL of concentrated sulfuric acid, shake quickly and let stand at room temperature for 15 min. Accurately pipette 200 μL of each reaction solution into a 96-well microplate, repeating each sample in triplicate. Using distilled water as a blank, measure the OD value at 490 nm using a microplate reader, and substitute the values ​​into the standard curve to determine the polysaccharide content.

[0101] Encapsulation ratio = (C in / C all )×100%, where C in The content of polysaccharides encapsulated in liposomes; C all It is the sum of the content of encapsulated polysaccharides and free polysaccharides in liposomes.

[0102] Example 2

[0103] This embodiment is based on the above embodiment 1, and the similarities with the above embodiment 1 will not be repeated.

[0104] In this embodiment, egg yolk lecithin and cholesterol were added to anhydrous ethanol, wherein the concentration of egg yolk lecithin in anhydrous ethanol was 10 mg / mL, and the mass concentration ratio of egg yolk lecithin to cholesterol was 1.7:1. The mixture was dissolved by sonication to form a lipid phase solution. Yam polysaccharide was added to phosphate buffer (pH=7.4) and dissolved by sonication. The solution was then filtered through a 0.45 μm organic nylon membrane to form a yam polysaccharide buffer solution as an aqueous phase solution.

[0105] Take two 30mL syringes, draw up the aqueous solution, fix them on the multichannel microinfusion pump, and inject the solution through the catheters as follows: Figure 1 The microfluidic chip device shown is connected to the yam polysaccharide phosphate buffer solution inlet A001 and B003. A 10 mL syringe is used to draw up the lipid phase solution, which is fixed on a multi-channel microinjection pump and connected to the lipid phase inlet 002 of the microfluidic chip device via a catheter. The total flow rate is set to 800 μL / min, and the injection rates of the yam polysaccharide phosphate buffer solution inlets A001 and B003 are kept consistent, with a flow rate ratio of 6:1 to the lipid phase inlet 002. The product flows out through the catheter at the product outlet 004 of the microfluidic chip and is collected in a 15 mL centrifuge tube. Ethanol is removed by rotary evaporation at 40 °C for 6 min, and the volume is adjusted to the original solution volume with phosphate buffer solution to obtain the target yam polysaccharide liposome (YPL), which is then stored at 4 °C.

[0106] The encapsulation efficiency of the obtained liposomes was 79.02%. The average particle size of the yam polysaccharide liposome nanoparticles was 154.2 nm, with a PDI of 0.083, indicating a monodisperse structure, as determined by a Malvern particle size analyzer. Transmission electron microscopy revealed... Figure 4 As shown, the yam liposomes are uniform and round, approximately spherical, and a distinct double-layer structure is visible in the figure.

[0107] Example 3

[0108] This embodiment is based on the above embodiment 1 or 2, and the similarities with the above embodiments will not be repeated.

[0109] This embodiment relates to the in vitro release study of yam polysaccharide liposomes, specifically including:

[0110] 3 mL of yam polysaccharide liposomes (YPL) and 3 mL of yam polysaccharide buffer (YP) were placed in separate dialysis bags and sealed. PBS (pH 7.4) was selected as the release medium. 30 mL of PBS was placed in a 50 mL centrifuge tube, which was then placed in the sealed dialysis bag. The tube was sealed and placed in a shaker at 37°C. Samples were taken at 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 24 h. 1 mL of the release medium was taken, and 1 mL of PBS was added to the release medium. The polysaccharide content was determined using the phenol-sulfuric acid method, and the cumulative drug release rate was calculated.

[0111] Depend on Figure 5 The in vitro release results show that YP achieved a cumulative drug release percentage of 93.39% in the first 8 hours, indicating almost complete release. In contrast, YPL achieved a cumulative drug release percentage of 42.84% in the first 8 hours and 76.41% after 24 hours, showing a gradual increase in the cumulative drug release percentage over time. Comparing the two curves reveals that YPL has a significantly better sustained-release effect than YP. This is attributed to the unique surface and small-size effects of the liposomes' nanoscale particle size, which slows down drug release.

[0112] Example 4

[0113] This embodiment is based on the above embodiment 1 or 2, and the similarities with the above embodiments will not be repeated.

[0114] This embodiment relates to the effect of yam polysaccharide liposomes on the proliferation of mouse spleen lymphocytes.

[0115] Using yam polysaccharide liposomes (YPL) prepared under optimal conditions as the research object, and yam polysaccharide buffer (YP) and blank liposomes (BP) as controls, we explored their effects on the proliferation of mouse spleen lymphocytes in vitro.

[0116] (1) Effect of YPL alone on the proliferation of mouse spleen lymphocytes

[0117] Mouse spleen lymphocytes were harvested and the cell density was adjusted to 5 x 10⁻⁶ cells / mL. 6YPL was serially diluted nine times with mouse spleen lymphocyte complete medium, and 100 μL of each concentration was added to each well of a 96-well cell culture plate. Four replicates of each concentration were performed. Mouse spleen lymphocyte complete medium was added as a cell control (BC). The plates were incubated at 37°C and 5% CO2 for 48 h. Then, 100 μL of mouse spleen lymphocyte complete medium containing 10% CCK8 was added to each well, and the plates were incubated for 60 min. The plates were then removed, and the OD values ​​of each well were measured at 450 nm using a microplate reader.

[0118] Depend on Figure 6 As shown, the A450 values ​​of YPL at concentrations ranging from 3.90615 μg / mL to 1000 μg / mL were all higher than those of the cell control group, indicating that YPL promoted the proliferation of mouse splenic lymphocytes. The A450 values ​​were significantly different from the control group at concentrations ranging from 7.8125 μg / mL to 500 μg / mL (P>0.05); and YPL significantly promoted the proliferation of mouse splenic lymphocytes at concentrations ranging from 31.25 μg / mL to 250 μg / mL.

[0119] (2) Effect of YPL synergistic with concanavalin A (ConA) on T lymphocyte proliferation

[0120] Mouse spleen lymphocytes were harvested and the cell density was adjusted to 5 x 10⁻⁶ cells / mL. 6 80 μL of each cell culture medium was added to each well of a 96-well cell culture plate. 20 μL of ConA (final concentration 10 μg / mL) was added to each well to stimulate T lymphocyte transformation and proliferation. Subsequently, 100 μL of different concentrations of YPL, YP, and BL were added, with each concentration replicated in 4 wells. A ConA control group was set up by adding complete medium containing mouse spleen lymphocytes, and a cell control group (BC) without ConA was also set up. After continuous culture in a cell culture incubator at 37°C and 5% CO2 for 48 h, 20 μL of CK8 was added to each well. After standing in the incubator for 60 min, the plates were removed and the OD value of each well at 450 nm was detected using a microplate reader.

[0121] like Figure 7 As shown, within the concentration range of 31.25 μg / mL to 250 μg / mL, the absorbance value of the YPL group first increased slowly and then decreased slowly with the increase of concentration, and the A450 value reached the highest at a concentration of 125 μg / mL. In addition, the A450 value of the YPL group was significantly higher than that of the YP group, indicating that within this concentration range, YPL can better synergistically promote the proliferation of mouse splenic T lymphocytes with ConA than YP.

[0122] (3) Effects of YPL synergistic with lipopolysaccharide (LPS) on B lymphocyte proliferation

[0123] Mouse spleen lymphocytes were harvested and the cell density was adjusted to 5 x 10⁻⁶ cells / mL. 6 80 μL of LPS (final concentration 10 μg / mL) was added to each well of a 96-well cell culture plate, followed by 20 μL of LPS in each well to stimulate B lymphocyte transformation and proliferation. Then, 100 μL of different concentrations of YPL, YP, and BL were added, with each concentration repeated in 4 wells. A control group was set up with complete medium containing mouse spleen lymphocytes as LPS, and a cell control group (BC) without LPS was also set up. After continuous culture in a cell culture incubator at 37°C and 5% CO2 for 48 h, 20 μL of LCK8 was added to each well. After standing in the incubator for 60 min, the plate was removed and the OD value of each well at 450 nm was detected using a microplate reader.

[0124] like Figure 8 As shown, within the concentration range of 31.25 μg / mL to 250 μg / mL, the absorbance value of the YPL group first increased slowly and then decreased slowly with increasing concentration, reaching its highest value at a concentration of 125 μg / mL. Within this concentration range, the A450 values ​​of the YPL group were significantly higher than those of the YP group, indicating that within this concentration range, YPL synergistically promotes the proliferation of mouse splenic B lymphocytes better than YP with LPS.

[0125] Based on the results of the in vitro lymphocyte proliferation stimulation experiment using yam polysaccharide liposomes, it can be concluded that yam polysaccharide liposomes can stimulate the proliferation of T and B lymphocytes, both alone and in synergy with ConA and LPS, and the results show that both have a proliferation-promoting effect. This indicates that the yam polysaccharide liposomes involved in this invention can enhance the immune activity of yam polysaccharides and can be used as an immune enhancer or as a vaccine adjuvant to improve the therapeutic effect of animal vaccines.

[0126] 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 method for preparing a yam polysaccharide liposome nano-immune-enhancing adjuvant, characterized in that, Based on the self-assembly mechanism of liposomes, the particle size is controlled by adjusting the flow rate and flow rate ratio through a microfluidic device. The microfluidic device includes a product outlet, a solution mixing channel and several injection ports. Both the injection ports and the product outlet are equipped with conduits for connection to external devices. The raw materials for preparing the liposomes include yam polysaccharide, egg yolk lecithin, and cholesterol; The injection port includes an aqueous solution inlet and a lipid solution inlet, both of which are connected to an external micro-injection pump via conduits; the product flows into an external receiving device through a conduit at the product outlet. The solution mixing channel includes a fluid focusing mixing unit and a straight shearing channel with baffles; Specifically, the steps include the following: Step 1: Take egg yolk lecithin and cholesterol, add them to anhydrous ethanol, and sonicate to dissolve them to prepare a lipid phase solution; Step 2: Add yam polysaccharide to phosphate buffer, sonicate to dissolve, and filter through a 0.45μm organic nylon membrane to obtain an aqueous solution; Step 3: Add the aqueous phase solution and lipid phase solution to the external micro-injection pump, fix them on the micro-injection pump, connect them to the injection port through the catheter, and set the flow rate. Step 4: The aqueous phase solution and the lipid phase solution merge in the fluid focusing mixing unit. Ethanol diffuses into the buffer phase, and the ethanol concentration decreases. Lipid molecules self-assemble and encapsulate the yam polysaccharide aqueous phase to form crude yam polysaccharide liposomes. Under the action of shear force in the straight shear channel, the particle size is further reduced. The product flows into the receiving device through the conduit on the product outlet. Step 5: Collect the product, remove ethanol by rotary evaporation, and bring the volume up to the original solution volume with phosphate buffer solution to obtain the target yam polysaccharide liposome nano-formulation, which is then stored at low temperature. In step three, the particle size, dispersibility, and encapsulation efficiency of yam polysaccharide liposomes are controlled by adjusting the flow rates of the lipid phase and the aqueous phase; the sum of the flow rates of the yam polysaccharide buffer phase and the lipid phase is 800 μL / min, and the flow rate ratio is 6:

1. In step one, the mass concentration ratio of egg yolk lecithin to cholesterol is 1:1 to 10:1, and the concentration of egg yolk lecithin in anhydrous ethanol is 5 mg / mL to 15 mg / mL. In step two, the concentration of yam polysaccharide in phosphate buffer is 0.02 mg / mL to 2 mg / mL; In step five, the single collection volume of product is 10 mL to 20 mL, the rotary evaporation speed is 30 r / min to 60 r / min, the rotary evaporation temperature is 30℃ to 50℃, and the rotary evaporation time is 5 min to 10 min.

2. A yam polysaccharide liposome nano-immune-enhancing adjuvant, characterized in that: It is prepared by the preparation method described in claim 1.

3. The application of the yam polysaccharide liposome nano-immunoenhancing adjuvant as described in claim 2 in the preparation of immunoenhancing drugs and vaccine adjuvants.

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