Preparation method of immunoglobulin composite liposome nanoparticles with controlled release characteristic

Abstract

The invention discloses a preparation method of immunoglobulin composite liposome nanoparticles with a controlled release characteristic, and belongs to the field of food nutrition and functional factors. Cholesterol and sitosterol are creatively used as auxiliary supports of a lecithin liposome skeleton, the stability of an immunoglobulin liposome nanoparticle structure is improved, the slow release property in the simulation effect process is improved, and a pH response type hydrogel film formed based on electrostatic crosslinking is prepared from calcium alginate and chitosan. A more stable double-layer shell-core structure is formed, so that the stability of embedded immune globulin molecules in gastric juice is further improved, and fixed-point slow release in small intestines is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food nutrition and functional factors, and particularly relates to a preparation method of immunoglobulin complex liposome nanoparticles with controlled release characteristics. BACKGROUND

[0002] Bovine milk immunoglobulin has multiple biological activities such as regulating immune function of the body and enhancing disease resistance, but its bioavailability is relatively low in the complex environment of the gastrointestinal tract, and its stability is poor, which limits its wide application as a nutritional fortifier or functional additive in the field of food and the like. Therefore, it is of great significance to develop a delivery method that can effectively protect bovine milk immunoglobulin and improve its stability and bioavailability.

[0003] At present, as a new delivery strategy, the delivery carrier can effectively improve the water solubility, stability and bioavailability of food-derived active substances by using natural food macromolecules such as polysaccharides, proteins and lipids as wall materials to construct a steady-state delivery carrier and embedding food-derived active substances. Among them, the lipid nanoparticle, as a new drug delivery system, has the advantages of high physical stability and slow drug leakage of polymer nanoparticles, as well as the advantages of low toxicity and large-scale production of liposomes and emulsions, and can be used to encapsulate hydrophilic and lipophilic drugs. Through process adjustment and surface modification, it can control its targeting to specific tissues and has good development prospects.

[0004] However, the existing reports about liposomes as carriers of active substances are mostly focused on small molecule substances such as curcumin, anthocyanins, zeaxanthin and antibacterial peptides, such as the technical solution in patent CN 119745800 A. There are few studies on using liposomes as delivery carriers for macromolecular proteins, mainly because the steric hindrance effect of macromolecular substances can easily lead to the disintegration of liposomes, affecting the embedding efficiency and the stability of the delivered substances.

[0005] In addition, patent CN 120168430 A discloses a lipid nanoparticle for improving liver targeting of drugs and a preparation method and application thereof, wherein the lipid nanoparticle comprises PC type phospholipid and active ingredients, and the active ingredients are nucleic acid drugs; patent CN 120112281 A discloses a lipid composition and a method for delivering therapeutic agents, wherein it is pointed out that the lipid nanoparticle preferably contains a sterol. The above two patents both involve the content of lipid nanoparticles, but the objects embedded are not bovine milk immunoglobulin, and the preferred schemes involved are not suitable for the embedding of bovine milk immunoglobulin. SUMMARY

[0006] TECHNICAL PROBLEM

[0007] Encapsulation of bovine milk immunoglobulins is necessary to improve their stability in the gastrointestinal tract and broaden their applications. However, current technologies lack research on encapsulation of bovine milk immunoglobulins, and existing lipid nanoparticle technologies are not directly applicable to them. Therefore, it is necessary to explore a delivery method that can effectively protect bovine milk immunoglobulins and improve their stability and bioavailability.

[0008] Technical content

[0009] To address the aforementioned problems in existing technologies, this invention provides a method for preparing immunoglobulin liposome nanoparticles with controlled-release properties. It creatively proposes using cholesterol and sitosterol as auxiliary supports for the lecithin liposome backbone, improving the stability of the immunoglobulin liposome nanoparticle structure and enhancing the sustained-release properties during simulation. Furthermore, a pH-responsive hydrogel membrane based on electrostatic cross-linking is prepared using calcium alginate and chitosan, forming a more stable bilayer shell-core structure, further improving the stability of the encapsulated immunoglobulin molecules in gastric juice and achieving targeted, slow release in the small intestine.

[0010] The technical solution of the present invention is as follows:

[0011] The first objective of this invention is to provide a method for preparing immunoglobulin complex liposome nanoparticles with controlled-release properties, comprising the following steps:

[0012] (1) Immunoglobulin dissolution: Prepare an immunoglobulin solution by mixing immunoglobulin powder with water;

[0013] (2) Preparation of liposome membrane: Egg yolk lecithin, cholesterol and sitosterol were dissolved in a solvent and dispersed evenly. After all the solvent was evaporated, a lipid membrane was obtained.

[0014] (3) Hydration treatment: Mix the lipid membrane prepared in step (2) with the immunoglobulin solution and stir to promote hydration;

[0015] (4) Ultrasonic-assisted dispersion: After the hydration in step (3) is complete, water is added, and then ultrasonic treatment is performed at low temperature to obtain a liposome dispersion.

[0016] (5) Hydrogel membrane formation: Sodium alginate and chitosan are added to the liposome dispersion and stirred to disperse evenly to obtain a mixture. The mixture is then dropped into calcium chloride solution to form hydrogel beads. After standing and soaking for a period of time, the hydrogel beads are removed.

[0017] (6) Freeze-drying: The hydrogel beads obtained in step (5) are freeze-dried to obtain immunoglobulin complex liposome nanoparticles.

[0018] Furthermore, in step (1), the mass concentration of immunoglobulin in the immunoglobulin solution is 1-3%.

[0019] Furthermore, in step (2), the mass ratio of egg yolk lecithin to cholesterol is 8:0.8 to 1.2.

[0020] Furthermore, in step (2), the mass ratio of egg yolk lecithin to sitosterol is 8:0.8 to 1.2.

[0021] Specifically, in step (2), the mass ratio of egg yolk lecithin to sitosterol and cholesterol is 8:1:1.

[0022] Furthermore, in step (2), the solvents are chloroform and methanol, with a volume ratio of 1:2 to 1:1.

[0023] Furthermore, in step (2), rotary evaporation or heating is used to evaporate all the solvent.

[0024] Furthermore, in step (3), the mass ratio of the lipid membrane to the immunoglobulin in the immunoglobulin solution is 5:2-10:1.

[0025] Preferably, in step (3), the mass ratio of the lipid membrane to the immunoglobulin in the immunoglobulin solution is 15:2 to 6.

[0026] More preferably, in step (3), the mass ratio of the lipid membrane to the immunoglobulin in the immunoglobulin solution is 15:2.5 to 3.5.

[0027] Specifically, the mass ratio of the lipid membrane to the immunoglobulin in the immunoglobulin solution in step (3) is 15:3.

[0028] Furthermore, in step (3), the hydration process is carried out at a temperature of 30-50 degrees Celsius for 20-40 minutes.

[0029] Preferably, the hydration process temperature in step (3) is 35-40 degrees Celsius.

[0030] Specifically, the hydration process temperature in step (3) can be 37 degrees Celsius.

[0031] Furthermore, the volume of water added in step (4) is 1 to 15 times the volume of the immunoglobulin solution in step (3).

[0032] Furthermore, the low temperature in step (4) is 0 to 5°C.

[0033] Furthermore, the ultrasonic treatment time in step (4) is 5 to 10 minutes.

[0034] Furthermore, in step (5), the amount of sodium alginate added is 0.2 to 0.5% of the mass of the liposome dispersion.

[0035] Furthermore, in step (5), the amount of chitosan added is 0.2 to 0.5% of the mass of the liposome dispersion.

[0036] Preferably, in step (5), the amount of sodium alginate added is 0.25 to 0.3% of the mass of the liposome dispersion.

[0037] Preferably, the amount of chitosan added in step (5) is 0.25 to 0.3% of the mass of the liposome dispersion.

[0038] Furthermore, in step (5), the calcium chloride solution has a calcium chloride mass concentration of 0.5% to 1%.

[0039] Furthermore, the soaking time in step (5) is 2 to 5 hours.

[0040] The present invention provides immunoglobulin complex liposome nanoparticles prepared according to the above method.

[0041] The application of immunoglobulin complex liposome nanoparticles provided by this invention in the food field.

[0042] Compared with the prior art, the present invention achieves the following beneficial effects:

[0043] (1) Optimization and innovation of liposome skeleton: This invention is the first to combine cholesterol and sitosterol as auxiliary support for lecithin liposome skeleton, which significantly enhances the stability of liposome nanoparticle structure, effectively resists the steric hindrance effect of macromolecular proteins, improves the encapsulation efficiency of immunoglobulins, and enables more immunoglobulins to be effectively delivered.

[0044] (2) Innovative double-shell core structure: This invention ingeniously combines sodium alginate and chitosan through electrostatic cross-linking to form a pH-responsive hydrogel membrane, which is then combined with liposomes to construct a unique double-shell core structure. This structure gives the delivery system better protective performance, ensuring that immunoglobulins are stable in harsh environments such as gastric juice, and enabling targeted and slow release in the small intestine, prolonging their action time in the intestine, improving bioavailability, and better exerting the physiological functions of immunoglobulins such as regulating immunity. Attached Figure Description

[0045] Figure 1 The particle size of the IgG sustained-release carriers prepared for comparison data analysis of Examples 1, 3 and Comparative Example 3 is compared.

[0046] Figure 2 The particle size of the IgG sustained-release carriers prepared in Examples 1, 3, 4, and 5 is compared.

[0047] Figure 3The particle size of the IgG sustained-release carriers prepared in Examples 1, 4, 1, and 2 is compared.

[0048] Figure 4 The sustained-release carriers prepared in Example 1 and Comparative Example 6 simulate the IgG release curves during in vitro digestion. Detailed Implementation

[0049] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0050] Source of raw materials

[0051] Immunoglobulin powder (IgG) is extracted from commercially available milk. The extraction method involves purifying IgG using a cation exchange resin, followed by freeze-drying to obtain immunoglobulin powder with a purity of 80% and a molecular weight of 150 kDa, making it a large protein molecule. The extraction method is described in the preparation method of patent 2025106884235, specifically as follows:

[0052] (1) Raw material pretreatment: ordinary commercial milk is selected as raw material, and impurities and fat are removed by filtration and centrifugation to obtain skim milk;

[0053] (2) Acidification precipitation: Adjust the pH of skim milk to 4.5, let it stand for 1 hour to precipitate, separate and remove the precipitate, and collect the supernatant;

[0054] (3) Ceramic microfiltration: The supernatant collected in step (2) is subjected to ceramic membrane microfiltration to remove residual casein micelles and fat droplets. The ceramic membrane used has a pore size of 30 nm to obtain the permeate.

[0055] (4) Ultrafiltration combined with nanofiltration separation: The permeate collected in step (3) is concentrated by ultrafiltration membrane with a molecular weight cutoff of 30 kDa to obtain permeate 1 and retentate 1; then permeate 1 is further concentrated and separated by nanofiltration membrane with a molecular weight cutoff of 1000 Da. After the concentration factor reaches 15-20 times, the concentration is stopped to obtain retentate 2 and permeate 2.

[0056] (5) Selective adsorption by ion exchange resin: The retentate 1 obtained in step (4) is adsorbed by ion exchange resin. The adsorption pH is 5.0. In this pH range, both immunoglobulins and lactoferrin are positively charged and can be selectively adsorbed on the ion exchange resin. The concentration of the sample buffer salt is 0.05 mol / L and the adsorption time is 40 min. The cation exchange resin used is Mitsubishi DIAION SK1B strong acid cation exchange resin.

[0057] (6) Ion exchange resin elution: The ion exchange resin adsorbed in step (5) is eluted: The specific elution process is to first use a 0.5% NaCl solution with a mass fraction of 3 times the resin volume to remove impurity proteins with weak adsorption characteristics on the surface, and then use a mixed solution of 4% NaCl and 0.05% sodium acetate to elute. The eluent is collected and the temperature is maintained at 25℃ during the elution process.

[0058] (7) Mix the permeate 2 collected in step (4) and the eluent collected in step (6), and then freeze-dry to obtain immunoglobulin powder.

[0059] Example 1

[0060] (1) Immunoglobulin dissolution: Take immunoglobulin powder and dissolve it in water to obtain an immunoglobulin solution with a concentration of 2wt%.

[0061] (2) Preparation of liposome membrane: Egg yolk lecithin, cholesterol, and sitosterol were taken, with a total mass of 100 mg. The ratio of the three materials was 70% lecithin, 30% cholesterol and sitosterol, and a mass ratio of 1:1 for cholesterol and sitosterol. The above membrane materials were dissolved in chloroform and methanol (volume ratio of chloroform and methanol was 1:2). After being evenly dispersed, the organic solvent was completely evaporated in a rotary evaporation at 50 degrees Celsius, so that lecithin, cholesterol, and sitosterol formed a lipid membrane.

[0062] (3) Hydration treatment: Take 100mg of the lipid membrane prepared in step (2) and 1mL of 2wt% immunoglobulin solution. The mass ratio of lipid membrane to IgG powder in the hydration treatment is 5:1. Stir and mix to promote hydration. The hydration temperature is 37 degrees and the time is 20 minutes.

[0063] (4) Ultrasonic-assisted dispersion: After complete hydration, slowly add 8 mL of water and sonicate under ice bath for 5 min to prepare liposome dispersion loaded with immunoglobulin.

[0064] (5) Hydrogel film formation: Sodium alginate and chitosan are added to the liposome dispersion obtained in step (4). The total amount of sodium alginate and chitosan added is 0.5% of the mass of the liposome dispersion. The mass ratio between sodium alginate and chitosan is 1:1. After stirring and dispersing evenly, the above solution is added dropwise to a calcium chloride solution with a mass concentration of 0.5% to form hydrogel beads with multi-level structural characteristics. After soaking in the calcium chloride solution for 4 hours, the excess solution is filtered out by sieve.

[0065] (6) Freeze-drying: The hydrogel beads formed above are dried to obtain liposome nanoparticles with a complete spherical structure, which are referred to as IgG sustained-release carriers.

[0066] Example 2

[0067] Unlike Example 1, the mass ratio of lipid membrane to IgG solution in step (3) hydration process was adjusted to 15:2, specifically: 150mg lipid membrane and 1mL 2wt% immunoglobulin solution were stirred and mixed to promote hydration.

[0068] Example 3

[0069] Unlike Example 1, the hydration temperature in step (3) is adjusted to 50 degrees.

[0070] Example 4

[0071] Unlike Example 1, the proportion of egg yolk lecithin in step (2) is adjusted to 80%, the total proportion of cholesterol and sitosterol is 20%, and the mass ratio of cholesterol and sitosterol is 1:1.

[0072] Comparative Example 1

[0073] Unlike Example 1, the proportion of egg yolk lecithin in step (2) is adjusted to 100%, and the total proportion of cholesterol and sitosterol is 0%.

[0074] Comparative Example 2

[0075] Unlike Example 1, the proportion of egg yolk lecithin in step (2) is adjusted to 90%, the total proportion of cholesterol and sitosterol is 10%, and the mass ratio of cholesterol and sitosterol is 1:1.

[0076] Comparative Example 3

[0077] Unlike Example 1, the hydration temperature in step (3) is adjusted to 20 degrees.

[0078] Comparative Example 4

[0079] Unlike Example 1, the mass ratio of lipid membrane to IgG solution in step (3) hydration process was adjusted to 5:2, specifically: 50 mg lipid membrane and 1 mL 2 wt% immunoglobulin solution were stirred and mixed to promote hydration.

[0080] Comparative Example 5

[0081] Unlike Example 1, the mass ratio of lipid membrane to IgG solution in step (3) hydration process was adjusted to 10:1. Specifically, 200 mg of lipid membrane and 1 mL of 2 wt% immunoglobulin solution were stirred and mixed to promote hydration.

[0082] Comparative Example 6

[0083] Unlike Example 1, chitosan in step (5) is removed from the hydrogel membrane, and sodium alginate is used as the hydrogel membrane material.

[0084] Comparative Example 7

[0085] Unlike Example 1, cholesterol in step (2) is omitted, i.e., egg yolk lecithin accounts for 80% and sitosterol accounts for 20%.

[0086] Comparative Example 8

[0087] Unlike Example 1, sitosterol is omitted in step (2), i.e., egg yolk lecithin accounts for 80% and cholesterol accounts for 20%.

[0088] Comparative Example 9

[0089] Unlike Example 1, the mass ratio of cholesterol to sitosterol in step (2) is adjusted so that the proportion of egg yolk lecithin is 80%, the proportion of cholesterol and sitosterol is 20%, and the mass ratio of cholesterol to sitosterol is 2:1.

[0090] Performance testing

[0091] (1) Comparative data analysis of the IgG sustained-release vectors prepared in Examples 1, 3 and 3: Encapsulation efficiency and particle size comparison

[0092] The formation of liposomes involves the hydration of phospholipids, forming a phospholipid bilayer. In thin-film dispersion, the addition of water causes the ordered phospholipid layer to detach from the wall and curl up to form a liposome. Therefore, hydration is a crucial step, and it must occur above the phase transition temperature of the phospholipid. Generally, the rotary evaporation temperature of lipids is 20-25°C, while liposomes with high Tc may reach around 45°C.

[0093] In the preparation process, liposomes were prepared by keeping other conditions constant and only changing the hydration temperature, controlling the water bath temperature at 20, 37, and 50°C. As shown in Table 1, the encapsulation efficiency of liposomes increased with increasing temperature, but decreased slightly when the water bath temperature reached 50°C. Higher water bath temperatures lead to faster evaporation of organic solvents, potentially resulting in an uneven phospholipid film, and high temperatures may also accelerate phospholipid oxidation; however, if the temperature is too low, the organic solvent may not be completely removed, and the phase transition temperature of egg yolk lecithin may not be reached, resulting in a lower encapsulation efficiency. Therefore, the hydration temperature range protected in this invention is 30-50°C.

[0094] Table 1. Comparison of IgG sustained-release vector encapsulation rates in Examples 1, 3, and Comparative Example 3.

[0095]

[0096] (2) Comparison of encapsulation efficiency and particle size of IgG sustained-release carriers prepared in Examples 1, 2, 4 and 5

[0097] During the preparation process, other conditions remained constant except for the proportion of IgG powder, and its effect on IgG liposome encapsulation was investigated. As shown in Table 2, when the proportion of IgG powder was small (membrane material:IgG powder = 10:1), the encapsulation efficiency was low. With increasing IgG concentration, the encapsulation efficiency of IgG liposomes slowly increased, reaching a maximum of 31.31% when the membrane material:IgG powder ratio was 5:1. However, when the membrane material:IgG powder ratio was 5:2, the encapsulation efficiency began to decrease because the IgG concentration exceeded the saturation limit of the liposome membrane. The particle size of all four groups of samples was less than 80 nm, and the PDI values ​​showed no significant difference, exhibiting a relatively stable distribution. In summary, membrane material:IgG powder ratios of 5:1 and 15:2 are more favorable for the preparation of IgG liposomes. The ratio of membrane material to IgG powder protected during the hydration step in this invention ranges from 5:1 to 15:2.

[0098] Table 2 Comparison of encapsulation efficiency of IgG sustained-release vectors prepared in Examples 1, 2, 4, and 5

[0099]

[0100] (3) Comparative data analysis of IgG sustained-release vectors prepared in Examples 1, 7, 8 and 9 showed encapsulation efficiency and loading.

[0101] During the preparation process, all other conditions remained unchanged, only the combination ratio of cholesterol and sitosterol was varied. Egg yolk lecithin accounted for 80%, and the two sterols together accounted for 20%, with the mass ratio of cholesterol to sitosterol being 1:1, 2:1, 0:1 (cholesterol omitted), and 1:0 (sitosterol omitted), namely Example 1, Comparative Example 9, Comparative Example 7, and Comparative Example 8. When cholesterol or sitosterol was added alone, the encapsulation efficiency was less than 25%. Adding cholesterol and sitosterol in combination significantly improved the encapsulation efficiency, reaching a maximum of 31.31%. This may indicate that cholesterol and sitosterol modify the membrane structure in different ways; cholesterol makes the lipid membrane denser, while sitosterol has one more ethyl group than cholesterol, increasing steric hindrance and reducing liposome membrane fluidity, which is beneficial for liposome stability. Combining the two not only reduces the amount of each individual component added but also has a synergistic effect, significantly improving the encapsulation efficiency. However, when the mass ratio of cholesterol to sitosterol was 2:1, the encapsulation efficiency was 27.38%, lower than 31.31%, indicating that the mass ratio of cholesterol and sitosterol in the combination affects the encapsulation effect, with a mass ratio of 1:1 being more effective. In summary, combining cholesterol and sitosterol is more beneficial for liposome preparation; liposomes prepared by adding cholesterol or sitosterol alone do not have high encapsulation efficiency. Therefore, the 1:1 ratio of cholesterol to sitosterol claimed in this invention is the optimal ratio for improving IgY encapsulation efficiency and is a key innovation of this invention.

[0102] Table 3 Comparison of encapsulation rates of IgG sustained-release vectors prepared in Example 1, Comparative Example 7, Comparative Example 8, and Comparative Example 9

[0103]

[0104]

[0105] (4) Encapsulation efficiency, particle size and surface potential of IgG sustained-release carriers prepared in Examples 1, 4, Comparative Examples 1 and 2

[0106] The presence of cholesterol and sitosterol in liposomes plays a crucial role in liposome stability and membrane fluidity. In the preparation process, by keeping other conditions constant and only changing the ratio of egg yolk lecithin to cholesterol + sitosterol, liposomes were prepared using ratios of 7:3, 8:2, 9:1, and 10:0 (w:w), as shown in Examples 1, 4, Comparative Example 1, and Comparative Example 2. As shown in Table 3, when the ratio of phospholipid to cholesterol + sitosterol increased from 7:3 to 8:2, the encapsulation efficiency initially increased from 29.15% to 43.21%. This is due to the beneficial effect of cholesterol on the more compact arrangement of phospholipid molecules, enhancing their orderliness within the liposome and preventing phospholipid loss, thereby improving the stability of the liposome membrane. However, with further increases in the ratio of phospholipid to cholesterol + sitosterol, the encapsulation efficiency dropped below 20%. This indicates that when the ratio of cholesterol + sitosterol in the phospholipid bilayer is too high, the liposome membrane becomes too rigid and prone to rupture, affecting the distribution of IgY between the bilayer membranes and leading to a decrease in encapsulation efficiency. Therefore, a lecithin:(cholesterol + sitosterol) ratio between 7:3 and 8:2 is more conducive to obtaining sustained-release carriers with high IgG loading rates. The liposome nanoparticles prepared under all conditions had diameters less than 100 nm and a PDI of approximately 0.25, indicating small particle size. Increasing the lecithin content is beneficial for preparing liposome nanoparticles with even smaller particle sizes. The overall system exhibits good homogeneity and stability. Therefore, in the liposome membrane preparation process protected by this invention, the lecithin content ranges from 70-80%, and the corresponding cholesterol + sitosterol content ranges from 20-30%.

[0107] Table 4 Comparison of encapsulation efficiency of IgG sustained-release vectors prepared in Examples 1, 4, Comparative Example 1, and Comparative Example 2

[0108]

[0109] like Figure 3 As shown, the liposomes prepared with an egg yolk lecithin:cholesterol ratio of 8:2 had the largest absolute zeta potential (-37.0 ± 1.1 mV) and the smallest PDI value. Therefore, considering all factors, an egg yolk lecithin:cholesterol ratio of 8:2 was chosen for preparing IgY liposomes.

[0110] (5) The IgG sustained-release carriers prepared in Example 1 and Comparative Example 6 exhibited similar release characteristics during simulated digestion.

[0111] Improving the physicochemical properties of liposomes is a prerequisite for the sustained release of IgG during digestion. To further demonstrate the necessity of using sodium alginate and chitosan to prepare a hydrogel membrane encapsulating liposomes in step (5) of this invention, the release characteristics of the sustained-release carriers prepared in Example 1 and Comparative Example 6 during simulated digestion were tested and compared with unencapsulated free IgG, as shown in Table 5. During simulated gastric digestion, 58.49% of the unencapsulated IgG was released within 2 hours, indicating that the protein would be suddenly degraded by gastric juice without the encapsulation of the sustained-release carrier. In the 2-5 hour stage, the cumulative release rate of IgY reached 100%, indicating that without the encapsulation of the carrier, IgY alone has no sustained-release effect and cannot achieve the purpose of long-term efficacy.

[0112] Table 5 shows the percentage of IgG degradation in the sustained-release carriers prepared in Example 1 and Comparative Example 6 during simulated in vitro digestion.

[0113]

[0114] Figure 4 The figures show the IgG release curves of the sustained-release carriers prepared in Example 1 and Comparative Example 6 during simulated in vitro digestion. As can be seen from the figures, the IgG release curves of lipid-encapsulated carriers are lower than those of unencapsulated IgG, especially during the gastric digestion phase (0-2h). The cumulative release curve of the IgG sustained-release carriers shows a trend of slow release followed by rapid release. In the first 2h, the release curve is relatively slow. The cumulative IgG release rate in the sustained-release carrier prepared in Comparative Example 6 is approximately 30%, while the cumulative IgG release rate in the sustained-release carrier prepared in Example 1 is approximately 20%. This indicates that the sodium alginate and chitosan composite hydrogel membrane is more beneficial for protecting IgG and controlling its release, inhibiting the burst release of this protein in the stomach, and achieving a targeted, slow release into the small intestine, effectively further promoting the long-term release of IgG and exerting its efficacy for a longer period. This may be because a single sodium alginate hydrogel membrane is easily affected by pH, causing instantaneous disintegration due to changes in intermolecular electrostatic interactions. In contrast, the cross-linking forces of the sodium alginate and chitosan composite hydrogel include complex forces such as hydrogen bonding, electrostatic interactions, and hydrophobic interactions, forming a denser and more stable hydrogel network, thereby more effectively ensuring the controlled release of IgG. This result fully demonstrates the effectiveness and necessity of constructing a composite hydrogel membrane using sodium alginate and chitosan as proposed in step (5) of this invention.

[0115] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing immunoglobulin complex liposome nanoparticles with controlled-release properties, characterized in that, Includes the following steps: (1) Immunoglobulin dissolution: Prepare an immunoglobulin solution by mixing immunoglobulin powder with water; (2) Preparation of liposome membrane: Egg yolk lecithin, cholesterol and sitosterol were dissolved in a solvent and dispersed evenly. After all the solvent was evaporated, a lipid membrane was obtained. The mass ratio of egg yolk lecithin to cholesterol was 8:0.8-1.

2. The mass ratio of egg yolk lecithin to sitosterol was 8:0.8-1.

2. (3) Hydration treatment: Mix the lipid membrane prepared in step (2) with the immunoglobulin solution and stir to promote hydration; the mass ratio of the lipid membrane to the immunoglobulin in the immunoglobulin solution is 5:2-10:1; the hydration process temperature is 30-50 degrees and the time is 20-40 minutes; (4) Ultrasonic-assisted dispersion: After the hydration in step (3) is complete, water is added, and then ultrasonic treatment is performed at low temperature to obtain a liposome dispersion. (5) Hydrogel membrane formation: Sodium alginate and chitosan were added to the liposome dispersion and stirred to disperse evenly to obtain a mixture. The mixture was then dropped into a calcium chloride solution to form hydrogel beads. After standing and soaking for a period of time, the hydrogel beads were removed. The amount of sodium alginate added was 0.2-0.5% of the mass of the liposome dispersion; the amount of chitosan added was 0.2-0.5% of the mass of the liposome dispersion. (6) Freeze-drying: The hydrogel beads obtained in step (5) are freeze-dried to obtain immunoglobulin complex liposome nanoparticles.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass concentration of immunoglobulins in the immunoglobulin solution is 1-3%.

3. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of the lipid membrane to the immunoglobulin in the immunoglobulin solution is 15:2-6.

4. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of immunoglobulins in the lipid membrane and the immunoglobulin solution is 15:2.5 to 3.

5.

5. The preparation method according to claim 1, characterized in that, The hydration process in step (3) is carried out at a temperature of 35-40 degrees Celsius.

6. The preparation method according to claim 1, characterized in that, In step (4), the volume of water added is 1 to 15 times the volume of the immunoglobulin solution in step (3); the low temperature is 0 to 5°C; and the ultrasonic treatment time is 5 to 10 minutes.

7. The preparation method according to claim 1, characterized in that, In step (5), the amount of sodium alginate added is 0.25-0.3% of the mass of the liposome dispersion; the amount of chitosan added is 0.25-0.3% of the mass of the liposome dispersion.

8. The preparation method according to claim 1, characterized in that, In step (5), the calcium chloride solution has a calcium chloride mass concentration of 0.5-1% and the soaking time is 2-5 hours.

9. An immunoglobulin complex liposome nanoparticle, characterized in that, The immunoglobulin complex liposome nanoparticles are prepared according to any one of claims 1 to 8.

10. The application of the immunoglobulin complex liposome nanoparticles as described in claim 9 in the food field.

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