Liposome co-loaded with soybean meal polypeptide and baicalein as well as preparation method and application of liposome

Abstract

The invention provides a liposome co-loaded with soybean meal polypeptide and baicalein as well as a preparation method and application thereof. The liposome co-loaded with soybean meal polypeptide and baicalein comprises a liposome, baicalein entrapped on a phospholipid layer of a hydrophobic layer of the liposome, a hydrophilic core and soybean meal polypeptide on the surface of the liposome. Firstly, a liposome membrane material and baicalein are assembled through a reverse evaporation method, then baicalein liposome and soybean meal polypeptide are assembled through an ultrasonic dispersion method, and the liposome co-loaded with soybean meal polypeptide and baicalein is prepared. According to the invention, a liposome drug loading technology is adopted, so that the two drugs can reach a pharmacological site sufficiently on the premise of ensuring the stability and the safety, and the synergistic effect of the two drugs in vivo is realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a liposome co-loaded with soybean meal polypeptide and baicalin, its preparation method and application. Background Technology

[0002] Soybean meal contains 40%-50% protein, and soybean meal peptides (molecular weight <1000 Da) can be prepared through enzymatic hydrolysis. These peptides are rich in essential amino acids such as glutamic acid and arginine, and possess unique biological activities. Studies have shown that soybean meal peptides have physiological functions such as antioxidation, blood pressure reduction, and immunomodulation. They can also enhance immunity by activating the TLR4 / NF-κB pathway and inhibit tumor cell growth. However, peptides suffer from problems such as gastrointestinal enzymatic hydrolysis, short half-life, and poor membrane permeability, which severely limit their bioavailability.

[0003] Baicalein, the core active ingredient of the traditional Chinese medicine Scutellaria baicalensis, is a flavonoid compound with multiple effects, including anti-inflammatory (inhibition of the COX-2 / PGE2 pathway) and antioxidant (activation of the Nrf2 / ARE pathway). However, its extremely low water solubility (0.03 mg / mL), rapid metabolism (half-life <1 hour), and chemical instability (easily degraded by light) limit its clinical application.

[0004] Single active ingredients in disease treatment are often limited by their singular target, short duration of action, and accumulation of adverse reactions. Baicalein and soybean meal peptides, derived from natural plants and plant byproducts respectively, both possess good biocompatibility and multi-target activity. Baicalein primarily exerts its anti-inflammatory, antioxidant, and anti-tumor effects by inhibiting inflammatory signaling pathways such as NF-κB, COX-2, and PGE2; however, its low solubility, rapid metabolism, and short half-life in vivo limit its bioavailability. Soybean meal peptides, rich in hydrophilic low-molecular-weight active peptides, possess antioxidant, immunomodulatory, and cell-protective effects. They can enhance the body's antioxidant and immune defenses by activating Nrf2 / ARE and TLR4-related pathways, but they also suffer from easy degradation by gastrointestinal enzymes and low absorption rates.

[0005] This system utilizes the amphiphilic structure of liposomes to encapsulate hydrophilic peptides in the aqueous phase or anchor them on the surface to avoid gastrointestinal enzymatic degradation and prolong in vivo circulation time; hydrophobic baicalin is embedded in the lipid bilayer to improve solubility and stability. The two work synergistically to achieve multiple functions: (1) Antioxidant synergy - peptides directly scavenge free radicals, and baicalin upregulates SOD and GSH-Px activity; (2) Anti-inflammatory-immune synergy - baicalin inhibits NF-κB pathway activation and reduces TNF-α and IL-6 levels, while soybean meal peptides promote macrophage polarization towards M2 type and restore immune balance; (3) Antitumor enhancement - baicalin induces tumor cell apoptosis, and peptides inhibit angiogenesis and enhance immune response.

[0006] This system combines the multi-target effects of natural products with the controllable release characteristics of nanocarriers, and can be used to regulate lipid metabolism disorders and inflammation-related diseases, such as hyperlipidemia, atherosclerosis and chronic inflammatory states. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing and applying liposomes co-loaded with soybean meal polypeptides and baicalin. By using liposomes to co-load drugs, the problem of insufficient drug utilization can be solved.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A liposome co-loaded with soybean meal peptides and baicalin includes a liposome, baicalin loaded in a hydrophobic phospholipid layer of the liposome, a hydrophilic core, and soybean meal peptides on the surface of the liposome.

[0009] The liposomes are modified with negatively charged polyethylene glycol on their surface.

[0010] The liposomes have a particle size of 155nm-170nm and a dispersion index (PDI) of 0.20-0.21.

[0011] A method for preparing liposomes co-loaded with soybean meal polypeptides and baicalin, comprising the following steps: Step 1: Soybean meal peptides are obtained by enzymatic hydrolysis of soybean meal using alkaline protease and dissolved in phosphate buffer solution, preferably at pH 6.5-7.0; Step 2: Add liposome membrane material and baicalein solution dissolved in organic solvent to the buffer solution in step 1), stir and evaporate to remove organic solvent; Step 3: Treat the residual liquid from Step 2 with ultrasound. The ultrasound power is preferably 250W to 350W and the treatment time is preferably 20 to 30 minutes. Then, separate the liposomes and suspend them in phosphate buffer to prepare a liposome suspension injection. Step 4: The liposome suspension injection from Step 3 is extruded through an extruder and then filtered through a 0.22μm microporous membrane to obtain drug-loaded liposomes.

[0012] The mass ratio of baicalein to liposome membrane material is 0.5–3:10–20, and the mass ratio of soybean meal peptides to baicalein is 1:5–10.

[0013] The mass ratio of baicalin to liposome membrane material is 1:15; The liposome membrane material described in step 2 includes hydrogenated soybean lecithin, cholesterol, and distearate phosphatidylethanolamine-polyethylene glycol 2000.

[0014] The molar ratio of hydrogenated soybean lecithin, cholesterol, and distearate phosphatidylethanolamine-polyethylene glycol 2000 is 1–3.5:0.5–1:0.2–0.6.

[0015] The molar ratio of hydrogenated soybean lecithin, cholesterol, and distearate phosphatidylethanolamine-polyethylene glycol 2000 is 2:1:0.4.

[0016] The above-mentioned applications of liposomes in lowering blood lipid levels and alleviating inflammatory responses.

[0017] Compared with existing technologies, the present invention has the following beneficial effects: The present invention provides a liposome capable of simultaneously encapsulating two drugs, soybean meal peptides and baicalein, successfully achieving the co-delivery of these two drugs. While ensuring stability and safety, the liposome allows both drugs to reach the pharmacological site in sufficient quantities, thereby achieving a synergistic effect of the two drugs in vivo. The liposomes of the present invention, co-encapsulating soybean meal peptides and baicalein, exhibit excellent synergistic antioxidant and anti-inflammatory effects in vivo, and can be used to develop drugs for the treatment of lipid metabolism disorders and inflammation-related diseases, such as hyperlipidemia, atherosclerosis, and chronic inflammatory diseases. Detailed Implementation

[0018] The specific embodiments of the present invention will be further described in detail below with reference to examples. These examples are only used to more clearly illustrate the technical solutions of the present invention, so that those skilled in the art can better understand and utilize the present invention, and are not intended to limit the scope of protection of the present invention.

[0019] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment to implement them according to conventional conditions or conditions recommended by the manufacturer.

[0020] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0021] In this embodiment, the liposomes co-loaded with soybean meal peptides and baicalin include liposomes, baicalin loaded in the hydrophobic phospholipid layer of the liposome, a hydrophilic core, and soybean meal peptides on the surface of the liposomes.

[0022] The liposomes are modified with negatively charged polyethylene glycol on their surface. This polyethylene glycol modification makes the surface potential of the liposomes more electrically neutral, which is beneficial to the stability of the nanoparticles in vivo and enables a longer cycle time.

[0023] The present invention provides a method for preparing liposomes co-loaded with soybean meal peptides and baicalein. First, liposome membrane material and baicalein are assembled by reverse evaporation. Then, baicalein liposomes are assembled with soybean meal peptides by ultrasonic dispersion to obtain liposomes co-loaded with soybean meal peptides and baicalein.

[0024] Example 1: A method for preparing liposomes co-loaded with soybean meal polypeptides and baicalin, comprising the following steps: Step 1: Use alkaline protease to enzymatically hydrolyze and separate soybean meal protein to obtain soybean meal peptides, and dissolve them in phosphate buffer solution; Step 2: Add liposome membrane material and baicalein solution dissolved in organic solvent to the buffer solution of Step 1, stir and evaporate to remove organic solvent; Step 3: Treat the residual liquid from Step 2 with ultrasound at a power of 300W for 25 minutes, then separate the liposomes and suspend them in phosphate buffer to prepare a liposome suspension injection. Step 4: The liposome suspension injection from Step 3 is extruded through an extruder and then filtered through a 0.22μm microporous membrane to obtain drug-loaded liposomes.

[0025] Preferably, the mass ratio of baicalein to liposome membrane material is 1:15, and the mass ratio of soybean meal peptides to baicalein is 1:8.

[0026] Preferably, the liposome membrane material in step 2 includes hydrogenated soybean lecithin, cholesterol, and distearate phosphatidylethanolamine-polyethylene glycol 2000.

[0027] Preferably, the molar ratio of hydrogenated soybean lecithin, cholesterol, and distearate phosphatidylethanolamine-polyethylene glycol 2000 is 2:1:0.4.

[0028] Example 2: Detection of antioxidant activity of soybean meal peptides and baicalin in combination.

[0029] Liposomes co-loaded with soybean meal polypeptide and baicalin were prepared according to the method in Example 1, and then baicalin reagent and soybean meal polypeptide reagent with the same drug dosage as the liposomes were prepared.

[0030] Normal clean-grade male Kunming mice (weighing approximately 20g) were randomly divided into 6 groups, with 20 mice in each group: basal diet group (control group), high-fat diet model group (model group), model + baicalein group, model group + soybean meal peptide group, model + baicalein + soybean meal peptide group, and model group + co-loaded liposome group.

[0031] Mice were acclimatized to their environment for three days before model initiation. The control group was fed a normal basal diet daily with free access to water, while the other groups were fed a high-fat diet for model initiation with free access to water. The experimental groups were administered the corresponding formulations according to the grouping protocol. All drugs were administered intraperitoneally at 100 mg / kg. -1 ·d -1 (Based on total drug dosage), the mice were administered the drug continuously for 3 weeks. The control group and the model group were injected intraperitoneally with the same volume of physiological saline. After the last administration, the mice were fasted for 12 hours, and about 1 mL of blood was collected from the abdominal aorta under anesthesia. The blood was centrifuged at 3000 r / min for 10 minutes, and about 0.5 mL of serum was collected to determine the activity levels of T-AOC (total antioxidant capacity), SOD (superoxide dismutase), CAT (catalase), GSH-PX (glutathione peroxidase), and MDA (malondialdehyde).

[0032] Table 1. Contents of T-AOC, SOD, CAT, GSH-PX, and MDA under each experimental condition. Group MDA (μmol / L) SOD (U / mg) CAT (U / mg) GSH-PX (U / mg) T-AOC (U / mg) control group 10.50±0.70 9.50±0.45 10.40±0.47 10.10±0.48 9.20±0.33 High-fat diet model group 15.20±0.43 6.80±0.56 7.50±0.45 6.70±0.71 6.30±0.76 Model + Baicalein Group 12.80±0.73 8.10±0.39 8.60±0.51 8.00±0.73 7.80±0.59 Model + soybean meal peptide group 12.40±0.49 8.30±0.60 8.90±0.51 8.20±0.74 8.00±0.51 Model + Baicalein + Soybean Meal Peptide Group 11.20±0.62 8.90±0.80 9.50±0.48 9.00±0.40 8.80±0.76 Model + Co-loaded Liposomes <![CDATA[10.80±0.48 ab ]]> <![CDATA[9.20±0.45 ab ]]> <![CDATA[9.80±0.54 ab ]]> <![CDATA[9.40±0.52 ab ]]> <![CDATA[9.00±0.54 ab ]]> Note: The contents of T-AOC (total antioxidant capacity), SOD (superoxide dismutase), CAT (catalase), GSH-PX (glutathione peroxidase), and MDA (malondialdehyde) were determined using commercial reagent kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions. Data for each group are expressed as mean ± SD (n=6). Groups showing significant differences compared to the high-fat diet model group are marked 'a' (P<0.05), and groups showing significant differences compared to the "model + baicalin + soybean meal polypeptide group" are marked 'b' (P<0.05).

[0033] The results are shown in Table 1. Compared with the normal control group, the activities of T-AOC, SOD, CAT, and GSH-PX in the serum of mice in the model group were significantly decreased (P<0.05), while the MDA content was significantly increased (P<0.05), indicating that the oxidative stress model induced by the high-fat diet was successfully established. After intervention with different drugs, the antioxidant indicators in the serum were improved, and the trends were as follows: 1. All four drug treatment groups could increase the activities of T-AOC, SOD, CAT, and GSH-PX to varying degrees, while reducing the MDA level, indicating that drug treatment can effectively alleviate oxidative stress damage; 2. The baicalein group and the soybean meal peptide group had similar effects in improving the activity of antioxidant enzymes, and the combined drug treatment group (baicalein + soybean meal peptide group) further enhanced the antioxidant capacity compared with the single drug group; 3. The soybean meal peptide and baicalein co-loaded liposome group showed the most significant effect, with the activities of T-AOC, SOD, CAT, and GSH-PX approaching normal levels, and the MDA content decreasing to the lowest level (P<0.05), showing a significant synergistic antioxidant effect.

[0034] Example 3: Stability test of liposomes co-loaded with soybean meal peptides and baicalin: To verify the stability of drug-loaded liposomes in vivo, the liposomes were dispersed in PBS + 0.5% BSA (simulated peritoneal fluid) solution and co-incubated at 37°C. Particle size distribution and zeta potential were measured daily using a Malvern dynamic light scattering particle size analyzer, with each sample measured three times for three consecutive days. Changes in hydrated particle size, PDI, zeta potential, and encapsulation efficiency were monitored.

[0035] Table 2. Changes in hydrated particle size, PDI, Zeta potential, and encapsulation efficiency in PBS + 0.1–0.5% BSA (simulated peritoneal fluid). DAY Encapsulation rate (%) Hydrated particle size (nm) PDI Zeta potential (mV) 1 91.65±1.20 155.37±2.84 0.208±0.006 14.52±0.15 2 88.98±1.45 160.95±3.02 0.204±0.005 14.68±0.12 3 83.78±1.87 168.55±2.96 0.208±0.007 14.68±0.13 The results, shown in Table 2, indicate that the particle size and dispersion of the drug-loaded liposomes remained stable over three days, exhibiting stable surface charges and no significant aggregation. This demonstrates the good stability of the liposomes co-loaded with soybean meal peptides and baicalin. The stability of the liposomes, especially in simulated gastrointestinal fluids, lays the foundation for subsequent oral administration.

[0036] This invention is not limited to the above-described embodiments. Any modifications, improvements, or substitutions that can be conceived by those skilled in the art without departing from the essential content of this invention fall within the scope of this invention.

Claims

1. A liposome co-loaded with soybean meal polypeptides and baicalin, characterized in that: It includes liposomes, baicalin loaded in the hydrophobic phospholipid layer of the liposomes, the hydrophilic core, and soybean meal peptides on the surface of the liposomes.

2. The liposome co-loaded with soybean meal polypeptide and baicalin according to claim 1, characterized in that: The liposomes are modified with negatively charged polyethylene glycol on their surface.

3. The liposomes co-loaded with soybean meal polypeptides and baicalin according to claim 2, characterized in that: The liposomes have a particle size of 155nm-170nm and a dispersion index (PDI) of 0.20-0.

21.

4. A method for preparing liposomes co-loaded with soybean meal polypeptides and baicalin, used to prepare liposomes co-loaded with soybean meal polypeptides and baicalin as described in any one of claims 1 to 3, characterized in that: Includes the following steps: Step 1: Use alkaline protease to enzymatically hydrolyze and separate soybean meal protein to obtain soybean meal peptides, and dissolve them in phosphate buffer solution, preferably at pH 6.5-7.0; Step 2: Add liposome membrane material and baicalein solution dissolved in organic solvent to the buffer solution of Step 1, stir and evaporate to remove organic solvent; Step 3: The residual liquid from Step 2 is treated with ultrasound, and then the liposomes are separated and suspended in phosphate buffer to prepare a liposome suspension injection. Step 4: The liposome suspension injection from Step 3 is extruded through an extruder and then filtered through a 0.22μm microporous membrane to obtain drug-loaded liposomes.

5. The method for preparing liposomes co-loaded with soybean meal polypeptides and baicalin according to claim 4, characterized in that: The mass ratio of baicalein to liposome membrane material is 0.5–3:10–20, and the mass ratio of soybean meal peptides to baicalein is 1:5–10.

6. The method for preparing liposomes co-loaded with soybean meal polypeptides and baicalin according to claim 5, characterized in that: The mass ratio of baicalin to liposome membrane material is 1:

15.

7. The method for preparing liposomes co-loaded with soybean meal polypeptides and baicalin according to claim 4, characterized in that: The liposome membrane material described in step 2 includes hydrogenated soybean lecithin, cholesterol, and distearate phosphatidylethanolamine-polyethylene glycol 2000.

8. The method for preparing liposomes co-loaded with soybean meal polypeptides and baicalin according to claim 7, characterized in that: The molar ratio of hydrogenated soybean lecithin, cholesterol, and distearate phosphatidylethanolamine-polyethylene glycol 2000 is 1~3.5:0.5~1:0.2~0.

6.

9. The method for preparing liposomes co-loaded with soybean meal polypeptides and baicalin according to claim 8, characterized in that: The molar ratio of hydrogenated soybean lecithin, cholesterol, and distearate phosphatidylethanolamine-polyethylene glycol 2000 is 2:1:0.

4.

10. The application of liposomes according to claims 1 to 9 in lowering blood lipid levels and alleviating inflammatory responses.