Agaricus bisporus chitosan modified drug-loaded liposome and preparation method thereof

Abstract

The invention discloses an agaricus bisporus chitosan modified drug-loaded liposome and a preparation method thereof, and belongs to the technical field of nano-drug delivery. The preparation method specifically comprises the following steps: (1) preparing agaricus bisporus chitosan; (2) preparing a liposome lipid phase; (3) preparing a drug-loaded liposome suspension; and (4) agaricus bisporus chitosan modification. According to the preparation method, agaricus bisporus chitosan is extracted from agaricus bisporus sporocarp, soybean phospholipid and cholesterol are used as membrane materials to prepare the nano-liposome loaded with metformin hydrochloride, finally, the surface of the liposome is modified with the agaricus bisporus chitosan, and the obtained nano-liposome is uniform in particle size, good in dispersity and high in encapsulation efficiency and stability; meanwhile, cell experiments show that the agaricus bisporus chitosan nano-liposome has remarkable hypoglycemic activity and low cytotoxicity, is a drug loading system with excellent comprehensive performance, fully embodies the unique value of the agaricus bisporus chitosan in particle size regulation and control of the nano-liposome, and provides a carrier basis with a stable structure for efficient delivery of metformin hydrochloride.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine delivery technology, and more specifically to a mushroom chitosan-modified drug-loaded liposome and its preparation method. Background Technology

[0002] Metformin hydrochloride is a first-line hypoglycemic drug that works by inhibiting hepatic gluconeogenesis and improving insulin sensitivity, and is widely used in the treatment of type 2 diabetes. However, existing metformin hydrochloride formulations have significant drawbacks: low bioavailability (approximately 50%-60%), incomplete absorption in the gastrointestinal tract after oral administration; strong gastrointestinal irritation, easily causing adverse reactions such as nausea and diarrhea; and large fluctuations in blood drug concentration, requiring frequent dosing to maintain an effective therapeutic concentration.

[0003] Nanoliposomes, as drug delivery carriers, offer advantages such as strong targeting, sustained and controlled release, and reduced drug irritation, making them an important means of improving the efficacy of small molecule drug administration. However, conventional metformin hydrochloride liposomes still have shortcomings: the liposome bilayer has poor stability, is easily and rapidly cleared by the reticuloendothelial system in vivo, and has a short circulation time; the surface lacks specific modification, resulting in insufficient targeting and difficulty in accurately enriching the drug in diseased tissues; and the encapsulation efficiency is low, leading to easy drug leakage and affecting therapeutic efficacy.

[0004] In the prior art, CN202510313435.X discloses a PLGA nanoparticle material loaded with metformin hydrochloride and its preparation method. Although it can reduce drug irritation, the liposome encapsulation efficiency and stability are insufficient. CN101264331B discloses a chitosan-coated insulin liposome technology, but it uses chitosan, and its bioavailability and sustained-release effect need to be improved.

[0005] Therefore, improving the bioavailability and stability of metformin hydrochloride formulations is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a drug-loaded liposome modified with mushroom chitosan and its preparation method, so as to solve the problems of low bioavailability and poor stability of metformin hydrochloride preparations in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing chitosan-modified drug-loaded liposomes from Agaricus bisporus, specifically including the following steps: (1) Preparation of mushroom chitosan After washing and drying the fruiting bodies of Agaricus bisporus, the powder is pulverized into ultrafine powder, sieved, degreased, decolorized, deacetylated, and then freeze-dried under vacuum to obtain Agaricus bisporus chitosan for later use. (2) Preparation of liposome lipid phase Soybean phospholipids and cholesterol are dissolved in an organic solvent, stirred until dissolved, and then evaporated by rotary evaporation to form a uniform lipid film. (3) Preparation of drug-loaded liposome suspension Metformin hydrochloride was dissolved in PBS solution to obtain a metformin hydrochloride solution. The metformin hydrochloride solution was added to a lipid membrane, hydrated, and ultrasonically emulsified to obtain a liposome suspension. (4) Chitosan modification of Agaricus bisporus Dissolve button mushroom chitosan in acetic acid solution to obtain button mushroom chitosan solution, add to liposome suspension under stirring, centrifuge, add PBS to resuspend, and obtain button mushroom chitosan modified drug-loaded liposomes.

[0008] This invention uses mushroom chitosan, phospholipids, and cholesterol as carrier materials, and metformin hydrochloride as a model drug to prepare nanoliposomes through thin-film dispersion-ultrasound method. After surface modification with mushroom chitosan, nanoliposomes with high stability, high encapsulation efficiency, and good safety are obtained.

[0009] Further, in step (1) above, the defatting, decolorization, and deacetylation are specifically as follows: add 5% sodium hydroxide solution at a mass-volume ratio of 1g:(10-30)mL, heat in a water bath at 70-90℃ for 2-6h to remove minerals, centrifuge to collect the precipitate, and wash with distilled water until neutral; add 3% hydrogen peroxide solution to the precipitate at a mass-volume ratio of 1g:(10-30)mL, stir in the dark at 20-40℃ for 1-3h to decolorize, centrifuge to collect the precipitate, and wash until neutral; add 50% sodium hydroxide solution to the decolorized precipitate at a mass-volume ratio of 1g:(10-30)mL, heat in a water bath at 140-160℃ for 1-5h to deacetylate, centrifuge to collect the precipitate, and wash until neutral.

[0010] The further beneficial effects of the above-mentioned approach are that chitosan, a natural cationic polysaccharide, possesses excellent biocompatibility, biodegradability, and mucosal adhesion. It can modify the surface of liposomes through electrostatic interactions, thereby improving liposome stability and targeting. As a widely available natural chitosan, Agaricus bisporus chitosan, compared to chitosan derived from crustaceans, has advantages such as more uniform molecular weight distribution, higher bioactivity, and lower toxicity.

[0011] Furthermore, in step (2) above, the mass ratio of soybean phospholipids to cholesterol is (3-5):1.

[0012] Furthermore, in step (2) above, the temperature of rotary evaporation is 20-40℃, the rotation speed is 30-70rpm, and the time is 20-40min.

[0013] The further beneficial effect of the above method is that rotary evaporation removes organic solvents and forms a uniform lipid film.

[0014] Furthermore, in step (3) above, the mass ratio of metformin hydrochloride to soybean lecithin is (0.5-2):1.

[0015] Furthermore, in step (3) above, the hydration temperature is 30-50℃ and the time is 0.5-2h.

[0016] Furthermore, in step (3) above, ultrasonic emulsification is performed using an ice bath probe with a power of 200-400W and a time of 5-20min.

[0017] Furthermore, in step (4) above, the concentration of the button mushroom chitosan solution is 0.1-1 mg / mL; the volume ratio of the liposome suspension to the button mushroom chitosan solution is (0.5-2):1.

[0018] This invention also claims protection for a *Agaricus bisporus* chitosan-modified drug-loaded liposome prepared by the above-mentioned preparation method, with a particle size of 110-160 nm, a polydispersity index (PDI) of 0.1-0.2, and a zeta potential of 0-2 mV; after 21 days of refrigeration at 4°C, the particle size showed no significant change, the encapsulation efficiency changed by approximately 5%-10%, the glucose consumption in an insulin-resistant HepG2 cell model was ≥3.5 mmol / L, and the cell survival rate after HepG2 cell treatment was ≥75%, indicating that *Agaricus bisporus* chitosan nanoliposomes can improve the stability of metformin hydrochloride and have significant in vitro hypoglycemic effects and low cytotoxicity.

[0019] This invention also claims the use of a drug-loaded liposome modified with Agaricus bisporus chitosan prepared by the above method in the preparation of metformin hydrochloride formulations.

[0020] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: (1) The degree of deacetylation and characteristic peaks of major functional groups of the chitosan extracted from Agaricus bisporus in this invention match those of crustacean chitosan, and the preparation process is more green and environmentally friendly than traditional shrimp and crab shell-derived chitosan. (2) In this invention, chitosan from Agaricus bisporus is used to modify metformin hydrochloride liposomes. Chitosan is adsorbed onto the surface of the liposomes through electrostatic interaction, forming a stable protective layer and significantly improving the physical stability of the liposomes.

[0021] (3) The preparation method of the present invention is simple and mild, the process parameters are easy to control, the encapsulation rate is high, and it is suitable for industrial production, providing a new solution for the clinical application of metformin hydrochloride.

[0022] In summary, this invention extracts button mushroom chitosan from the fruiting body of *Agaricus bisporus*, uses soybean lecithin and cholesterol as membrane materials to prepare metformin hydrochloride-loaded nanoliposomes, and finally modifies the surface of the liposomes with button mushroom chitosan. The resulting nanoliposomes have uniform particle size, good dispersibility, and high encapsulation efficiency and stability. Meanwhile, cell experiments show that the metformin hydrochloride-loaded liposomes modified with button mushroom chitosan exhibit significant hypoglycemic activity and low cytotoxicity, making them a superior drug delivery system with excellent overall performance. This fully demonstrates the unique value of button mushroom chitosan in the particle size regulation of nanoliposomes, providing a structurally stable carrier basis for the efficient delivery of metformin hydrochloride. Attached Figure Description

[0023] Figure 1 The graph shows the particle size change of liposome samples stored at 4°C for 0-21 days. Figure 2 The graph shows the change in encapsulation efficiency of liposome samples stored at 4°C for 0-21 days. Figure 3 The survival rate of HepG2 cells after treatment with chitosan and liposome samples; Figure 4 The glucose consumption of insulin-resistant HepG2 cells by chitosan and liposome samples; Figure 5 Fourier transform infrared spectra of chitosan and liposome samples. Detailed Implementation

[0024] 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.

[0025] Example 1 The preparation method of chitosan-modified drug-loaded liposomes from Agaricus bisporus includes the following steps: (1) Preparation of mushroom chitosan After washing and drying the fruiting bodies of Agaricus bisporus, the powder was ultra-finely pulverized and passed through a 100-mesh sieve. A 5% sodium hydroxide solution was added at a mass-to-volume ratio of 1 g:10 mL, and the mixture was heated in an 80°C water bath for 2 hours to remove minerals. The precipitate was collected by centrifugation and washed with distilled water until neutral. A 3% hydrogen peroxide solution was added to the precipitate at a mass-to-volume ratio of 1 g:10 mL, and the mixture was stirred at 30°C in the dark for 1 hour to decolorize. The precipitate was collected by centrifugation and washed until neutral. A 50% sodium hydroxide solution was added to the decolorized precipitate at a mass-to-volume ratio of 1 g:10 mL, and the mixture was heated in a 150°C water bath for 1 hour to deacetylate. The precipitate was collected by centrifugation and washed until neutral. The mixture was then freeze-dried under vacuum to obtain Agaricus bisporus chitosan for later use. (2) Preparation of liposome lipid phase Weigh soybean lecithin and cholesterol in a 3:1 mass ratio, dissolve them in chloroform, and vortex to dissolve them to obtain a lipid phase. Place the lipid phase in a rotary evaporator and evaporate it at 20°C and 30 rpm for 20 min to remove the chloroform and form a uniform lipid film on the inner wall of a round-bottom flask. (3) Preparation of drug-loaded liposome suspension Metformin hydrochloride was weighed according to a phospholipid to metformin hydrochloride mass ratio of 0.5:1, dissolved in PBS buffer, and stirred to obtain a metformin hydrochloride solution. The metformin hydrochloride solution was added to a round-bottom flask and hydrated at 30°C for 0.5 h with magnetic stirring during the process to obtain a crude liposome suspension. The crude liposome suspension was then sonicated in an ice bath with a 200W probe for 5 min to obtain a liposome suspension. (4) Chitosan modification of Agaricus bisporus Dissolve button mushroom chitosan in 1% acetic acid at a mass-to-volume ratio of 0.01 g: 10 mL to obtain a 0.1% button mushroom chitosan solution. Slowly add the liposome suspension to the button mushroom chitosan solution at a volume ratio of 2:1. After vortexing and homogenization, centrifuge and discard the supernatant. Resuspend in PBS solution to obtain button mushroom chitosan-modified drug-loaded liposomes.

[0026] Example 2 The preparation method of chitosan-modified drug-loaded liposomes from Agaricus bisporus includes the following steps: (1) Preparation of mushroom chitosan After washing and drying the fruiting bodies of Agaricus bisporus, the powder was ultra-finely pulverized and passed through a 100-mesh sieve. A 5% sodium hydroxide solution was added at a mass-to-volume ratio of 1 g:20 mL, and the mixture was heated in an 80°C water bath for 4 hours to remove minerals. The precipitate was collected by centrifugation and washed with distilled water until neutral. A 3% hydrogen peroxide solution was added to the precipitate at a mass-to-volume ratio of 1 g:20 mL, and the mixture was stirred at 30°C in the dark for 2 hours to decolorize. The precipitate was collected by centrifugation and washed until neutral. A 50% sodium hydroxide solution was added to the decolorized precipitate at a mass-to-volume ratio of 1 g:20 mL, and the mixture was heated in a 150°C water bath for 3 hours to deacetylate. The precipitate was collected by centrifugation and washed until neutral. The mixture was then freeze-dried under vacuum to obtain Agaricus bisporus chitosan for later use. (2) Preparation of liposome lipid phase Soybean lecithin and cholesterol were weighed in a mass ratio of 4:1, dissolved in chloroform, and vortexed to obtain a lipid phase. The lipid phase was placed in a rotary evaporator and evaporated at 30°C and 50 rpm for 30 min to remove chloroform and form a uniform lipid film on the inner wall of a round-bottom flask. (3) Preparation of drug-loaded liposome suspension Metformin hydrochloride was weighed according to a phospholipid to metformin hydrochloride mass ratio of 1:1, dissolved in PBS buffer, and stirred to obtain a metformin hydrochloride solution. The metformin hydrochloride solution was added to a round-bottom flask and hydrated at 40°C for 1 hour with magnetic stirring during the process to obtain a crude liposome suspension. The crude liposome suspension was then sonicated in an ice bath with a 300W probe for 10 minutes to obtain a liposome suspension. (4) Chitosan modification of Agaricus bisporus Dissolve button mushroom chitosan in 1% acetic acid at a mass-to-volume ratio of 0.05 g: 10 mL to obtain a 0.5% button mushroom chitosan solution. Slowly add the liposome suspension to the button mushroom chitosan solution at a volume ratio of 1:1. After vortexing and homogenization, centrifuge and discard the supernatant. Resuspend in PBS solution to obtain button mushroom chitosan-modified drug-loaded liposomes.

[0027] Example 3 The preparation method of chitosan-modified drug-loaded liposomes from Agaricus bisporus includes the following steps: (1) Preparation of mushroom chitosan After washing and drying the fruiting bodies of Agaricus bisporus, the powder was ultra-finely pulverized and passed through a 100-mesh sieve. A 5% sodium hydroxide solution was added at a mass-to-volume ratio of 1 g:30 mL, and the mixture was heated in an 80°C water bath for 6 hours to remove minerals. The precipitate was collected by centrifugation and washed with distilled water until neutral. A 3% hydrogen peroxide solution was added to the precipitate at a mass-to-volume ratio of 1 g:30 mL, and the mixture was stirred at 30°C in the dark for 3 hours to decolorize. The precipitate was collected by centrifugation and washed until neutral. A 50% sodium hydroxide solution was added to the decolorized precipitate at a mass-to-volume ratio of 1 g:30 mL, and the mixture was heated in a 150°C water bath for 5 hours to deacetylate. The precipitate was collected by centrifugation and washed until neutral. The mixture was then freeze-dried under vacuum to obtain Agaricus bisporus chitosan for later use. (2) Preparation of liposome lipid phase Weigh soybean lecithin and cholesterol in a 5:1 ratio, dissolve them in chloroform, and vortex to dissolve them to obtain the lipid phase. Place the lipid phase in a rotary evaporator and evaporate it at 40°C and 70 rpm for 40 min to remove the chloroform and form a uniform lipid film on the inner wall of a round-bottom flask. (3) Preparation of drug-loaded liposome suspension Metformin hydrochloride was weighed according to a phospholipid to metformin hydrochloride mass ratio of 2:1, dissolved in PBS buffer, and stirred to obtain a metformin hydrochloride solution. The metformin hydrochloride solution was added to a round-bottom flask and hydrated at 50°C for 1 hour with magnetic stirring during the process to obtain a crude liposome suspension. The crude liposome suspension was then sonicated in an ice bath with a 400W probe for 20 minutes to obtain a liposome suspension. (4) Chitosan modification of Agaricus bisporus Dissolve button mushroom chitosan in 1% acetic acid at a mass-to-volume ratio of 0.1 g: 10 mL to obtain a 1% button mushroom chitosan solution. Slowly add the liposome suspension to the button mushroom chitosan solution at a volume ratio of 0.5:1. After vortexing and homogenization, centrifuge and discard the supernatant. Resuspend in PBS solution to obtain button mushroom chitosan-modified drug-loaded liposomes.

[0028] Comparative Example 1 The method for preparing drug-loaded liposomes without chitosan modification specifically includes the following steps: (1) Preparation of liposome lipid phase Soybean lecithin and cholesterol were weighed in a 3:1 mass ratio, dissolved in chloroform, and vortexed to obtain a lipid phase. The lipid phase was placed in a rotary evaporator and evaporated at 30°C and 50 rpm for 30 min to remove chloroform and form a uniform lipid film on the inner wall of a round-bottom flask. (2) Preparation of drug-loaded liposome suspension Metformin hydrochloride was weighed at a mass ratio of phospholipid to metformin hydrochloride of 1:1, dissolved in PBS buffer, and stirred to obtain a metformin hydrochloride solution. The metformin hydrochloride solution was added to a round-bottom flask and hydrated at 40°C for 1 hour with magnetic stirring during the process to obtain a crude liposome suspension. The crude liposome suspension was then sonicated in an ice bath with a 300W probe for 10 minutes to obtain unmodified chitosan-loaded liposomes.

[0029] Comparative Example 2 The method for preparing chitosan-modified drug-loaded liposomes from crustaceans specifically includes the following steps: (1) Preparation of liposome lipid phase Soybean lecithin and cholesterol were weighed in a mass ratio of 4:1, dissolved in chloroform, and vortexed to obtain a lipid phase. The lipid phase was placed in a rotary evaporator and evaporated at 30°C and 50 rpm for 30 min to remove chloroform and form a uniform lipid film on the inner wall of a round-bottom flask. (2) Preparation of drug-loaded liposome suspension Metformin hydrochloride was weighed according to a phospholipid to metformin hydrochloride mass ratio of 1:1, dissolved in PBS buffer, and stirred to obtain a metformin hydrochloride solution. The metformin hydrochloride solution was added to a round-bottom flask and hydrated at 40°C for 1 hour with magnetic stirring during the process to obtain a crude liposome suspension. The crude liposome suspension was then sonicated in an ice bath with a 300W probe for 10 minutes to obtain a liposome suspension. (3) Chitosan modification of Agaricus bisporus Chitosan (commercially available, degree of deacetylation 90%, molecular weight 30kDa) was dissolved in 1% acetic acid at a mass-to-volume ratio of 0.05g:10mL to obtain a 0.5% chitosan solution. The liposome suspension was slowly added dropwise to the chitosan solution at a volume ratio of 1:1. After vortexing and homogenization, the supernatant was discarded by centrifugation, and the suspension was resuspended in PBS solution to obtain chitosan-modified drug-loaded liposomes.

[0030] Performance testing The performance of the Agaricus bisporus chitosan-modified drug-loaded liposomes (A, B, C) prepared in Examples 1-3, the unmodified drug-loaded liposomes prepared in Comparative Example 1, and the chitosan-modified drug-loaded liposomes (D) prepared in Comparative Example 2 were tested. The specific test items and methods are as follows: 1. Particle size, PDI, and Zeta potential Particle size, PDI, and Zeta potential were determined using a Malvern particle size analyzer, with each sample measured in triplicate. The results are shown in Table 1.

[0031] Table 1. Particle size, PDI, and Zeta potential of metformin hydrochloride liposomes in Examples 1-3 and Comparative Examples 1-2

[0032] Note: ae indicates that there are significant differences in the physical properties of the five liposome samples.

[0033] Particle size reflects the dispersion size of nanoliposomes. The smaller the particle size and the more uniform the distribution, the better it is for targeted delivery in vivo and improved bioavailability. PDI (polydispersity index) reflects the uniformity of particle size distribution. PDI≤0.3 indicates good dispersibility. Zeta potential reflects colloidal stability. The higher the positive potential, the longer the liposomes circulate in vivo and the easier it is to interact with negatively charged biological membranes (such as intestinal mucosa).

[0034] As shown in Table 1, compared with the unmodified chitosan-loaded liposomes of Comparative Example 1, the chitosan-modified liposomes A, B, and C of Examples 1-3 and liposome D of Comparative Example 2 showed significant differences in particle size, PDI, and Zeta potential, indicating that chitosan modification can effectively regulate the physicochemical properties of liposomes. Among them, the performance of chitosan-modified liposomes B and C of Examples 2-3 was superior to that of liposome D of Comparative Example 2 (prepared by chitosan polymerization of crustaceans), demonstrating the unique advantages of Agaricus bisporus chitosan-modified nanoliposomes.

[0035] Comparative Example 1 showed the smallest particle size of unmodified chitosan-loaded liposomes. After chitosan modification, the particle sizes of liposomes A, B, C, and D all changed. Among them, the particle size of chitosan-modified liposome B was slightly larger than that of the other modified samples, but it was still in the nanoscale (100-200 nm), which is beneficial for passive targeting. In contrast, the particle size of liposome D in Comparative Example 2 was significantly larger than that of chitosan-modified liposomes B and C, indicating that Agaricus bisporus chitosan has a greater advantage in particle size regulation.

[0036] The PDI of the unmodified chitosan-loaded liposomes in Comparative Example 1 was around 0.23. After chitosan modification, the PDI of liposome B dropped to around 0.10, indicating that the chitosan-modified drug-loaded liposomes from Agaricus bisporus have better dispersibility and more uniform particle size distribution.

[0037] Regarding zeta potential, the zeta potential of the unmodified chitosan-loaded liposomes in Comparative Example 1 was negative. After modification with Agaricus bisporus chitosan, the zeta potential of several liposomes showed an increasing trend. The increase in zeta potential indicates that Agaricus bisporus chitosan endows the liposomes with stronger colloidal stability and facilitates their interaction with biological membranes, which plays a key role in prolonging in vivo circulation time and improving drug bioavailability.

[0038] 2. In vitro stability The liposomes were refrigerated at 4°C, and the particle size and encapsulation efficiency were measured at 0, 7, 14, and 21 days.

[0039] Free drug and liposomes were separated by high-speed centrifugation. The liposomes were placed in a refrigerated centrifuge and centrifuged at 4℃ and 10000 rpm for 30 min. The supernatant was collected, and the absorbance at 233 nm was measured to calculate the encapsulation efficiency of metformin hydrochloride. Results are as follows: Figure 1 and Figure 2 As shown.

[0040] Depend on Figure 1It was found that the particle size of unmodified chitosan-loaded liposomes gradually increased with storage time, increasing by approximately 17.3% after 21 days. After chitosan modification, the particle size changes of liposomes A, B, C, and D showed significant differences. Among them, chitosan-modified liposome A showed the slowest particle size increase, increasing by only about 4.6% after 21 days, demonstrating excellent particle size stability. Although the initial particle size of chitosan-modified liposome B (159.45 nm) was slightly larger than other modified samples, it was still within the 100-200 nm nanometer range (beneficial for passive targeting), and increased by only about 1.8% after 21 days, showing outstanding particle size stability. Chitosan-modified liposome C increased by about 6.5%. Chitosan-modified liposome D showed the most significant particle size increase, increasing by about 4.5% after 21 days, and its particle size was significantly larger than that of Agaricus bisporus chitosan-modified liposomes B and C. Combined with PDI data (the PDI of chitosan-modified liposomes B was as low as 0.10), this further demonstrates that the chitosan-modified drug-loaded liposomes from Agaricus bisporus have better dispersibility and more uniform particle size distribution.

[0041] A higher encapsulation efficiency and a smaller decrease in concentration during storage indicate a stronger ability of liposomes to encapsulate the drug and less drug leakage. Figure 2 It was found that the initial encapsulation efficiency of unmodified chitosan-loaded liposomes was only 75.4%, and it decreased to 52.2% after 21 days, a decrease of 30.8%, indicating severe drug leakage. After chitosan modification, the encapsulation efficiency of all groups of liposomes was significantly improved. The Agaricus bisporus chitosan-modified liposome B had the highest initial encapsulation efficiency (91.2%), which remained at 83.1% after 21 days, a decrease of only 8.9%. The encapsulation efficiency of liposomes A and C decreased by 8.6% and 10.2% respectively, showing good stability. The initial encapsulation efficiency of crustacean chitosan-modified liposome D was 89.13%, which decreased to 82.4% after 21 days, a decrease of 7.6%, and the encapsulation efficiency at all time points was significantly lower than that of Agaricus bisporus chitosan-modified liposome B, indicating that Agaricus bisporus chitosan has a better effect on drug encapsulation and leakage inhibition.

[0042] 3. Cytotoxicity Chitosan (chic acid from Agaricus bisporus in Example 2 and chitosan from crustaceans in Comparative Example 2) and liposome samples were administered to HepG2 cells at certain concentrations, and the cytotoxicity of the samples was detected by the CCK-8 assay. The results are as follows: Figure 3 As shown.

[0043] The cytotoxicity of each group of samples was assessed by detecting cell viability (using the blank control group as a baseline; higher viability indicated lower cytotoxicity and better biocompatibility). Figure 3The results showed that the cell survival rate in the CON group (blank control group) was close to 100%, representing the baseline activity of HepG2 cells under normal growth conditions; the cell survival rate in the CS group (chitosan group) was approximately 86%, slightly lower than the blank control group but still maintained at a high level, indicating that chitosan has weak toxicity to HepG2 cells and good biocompatibility; the cell survival rate in the ACS group (button mushroom chitosan group) was approximately 76%, slightly lower than the CS group, but still within the low-toxicity safety range of above 75%, indicating that button mushroom chitosan itself has low cytotoxicity; the cell survival rate in the MET group (metformin hydrochloride technical grade group) was approximately 72%, further decreasing compared to the ACS group, reflecting that metformin hydrochloride technical grade has certain inherent cytotoxicity to HepG2 cells; the cell survival rate in the MET-LP group (unmodified chitosan-loaded liposomes) rebounded to around 87%, significantly higher than that of the MET group. The study suggests that encapsulation with liposomes can effectively reduce the cytotoxicity of metformin hydrochloride and improve its biocompatibility.

[0044] The cytotoxicity of the drug-loaded liposomes modified with Agaricus bisporus chitosan (ACS) (ACS-MET-LP) showed a clear concentration-dependent effect: the cell survival rate of the 0.1% ACS-MET-LP group (i.e., chitosan-modified liposome A) was about 73%, which was similar to the activity level of the metformin hydrochloride original drug group; the cell survival rate of the 0.5% ACS-MET-LP group (i.e., chitosan-modified liposome B) increased to 76%, which was consistent with the survival level of the Agaricus bisporus chitosan group alone; and the cell survival rate of the 1% ACS-MET-LP group (i.e., chitosan-modified liposome C) further increased to 81%, which was close to the activity level of the crustacean chitosan group. This indicates that as the concentration of Agaricus bisporus chitosan modification increases, the cytotoxicity of the drug-loaded liposomes gradually decreases and the biocompatibility gradually increases.

[0045] 4. In vitro hypoglycemic activity A HepG2 insulin resistance model was constructed. Glucose concentration was measured 18 hours after drug administration using a glucose oxidase assay, and glucose consumption was calculated. Results are as follows: Figure 4 As shown.

[0046] The hypoglycemic activity of each group of samples in improving insulin resistance was evaluated by detecting glucose consumption in the cell culture system. The results showed that: Figure 4The glucose consumption of the Model group (insulin resistance model group) and the CS group (chitosan group) was only 1.3 mmol / L, indicating that the insulin resistance model was successfully constructed and that chitosan alone had no effect on improving glucose consumption in this model. The glucose consumption of the MET-LP group (unmodified chitosan-loaded liposomes) was about 2.3 mmol / L, only slightly higher than that of the model group, suggesting that simple liposome encapsulation weakens the hypoglycemic activity of metformin hydrochloride. The glucose consumption of the MET group (formula metformin hydrochloride) increased to 3.5 mmol / L, reflecting the basic hypoglycemic activity of the positive drug, but the effect was weaker than that of the ACS group (button mushroom chitosan group), which had a glucose consumption of 4.4 mmol / L. This proves that button mushroom chitosan itself has a significant activity in improving insulin resistance in HepG2 cells, and the effect is better than that of the formula metformin hydrochloride.

[0047] The hypoglycemic effect of the drug-loaded liposome modified with Agaricus bisporus chitosan (ACS-MET-LP) was concentration-dependent: the glucose consumption of the 0.1% ACS-MET-LP group was 3.9 mmol / L, which was better than that of the MET-LP group; the glucose consumption of the 0.5% ACS-MET-LP group increased to 4.7 mmol / L, which was close to the activity of the ACS group alone; the glucose consumption of the 1% ACS-MET-LP group reached 4.9 mmol / L, which was the highest among all groups. This indicates that 1% Agaricus bisporus chitosan modification can maximize the hypoglycemic activity of the drug-loaded liposome, and its effect on improving insulin resistance in HepG2 cells is slightly better than that of Agaricus bisporus chitosan alone.

[0048] 5. Fourier transform infrared spectrum To further verify the differences between the Agaricus bisporus chitosan sample and crustacean chitosan samples, whether the nanoliposome modification was successful, and whether the drug was successfully encapsulated, infrared spectroscopy was performed on the above substances. The results are as follows: Figure 5 As shown.

[0049] Depend on Figure 5 It can be seen that at 3300 cm -1 Approximately (OH / NH₃), 2800-3000 cm⁻¹ -1 (CH2 / CH3), 1600 cm -1 Left and right (amide I band, C=O), 1000 cm -1The main functional group characteristic peaks of the left and right (COC) chitosan sample (ACS) showed a high degree of matching with the standard (CS), with no obvious impurity peaks or missing characteristic peaks, indicating that its chemical structure was consistent with the standard and its quality was good; the metformin hydrochloride liposomes (MET-LP) showed high values ​​at 3285.7, 1635.3, 1376.2, and 1021.5 cm⁻¹. -1 Compared with blank liposomes, the characteristic peaks at the isowavenumbers showed peak position shifts, intensity changes, or the appearance of new peaks, indicating that the drug interacts with the liposomes. Furthermore, the infrared peaks of the drug-loaded liposomes (MET-LP) matched the characteristics of the MET guanidine group, while those of the blank liposomes did not appear, indicating that the drug was successfully encapsulated in the liposomes.

[0050] In summary, the chitosan-modified drug-loaded liposomes from *A. bisporus* (especially A and B) significantly outperformed liposomes D prepared from crustacean chitosan and the group without chitosan modification in terms of particle size stability and encapsulation efficiency. They also exhibited significant hypoglycemic activity and low cytotoxicity, making them a superior drug delivery system in terms of overall performance. This fully demonstrates the unique value of *A. bisporus* chitosan in the field of nanoliposomes, providing a structurally stable carrier basis for the efficient delivery of metformin hydrochloride.

[0051] 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 Agaricus bisporus chitosan-modified drug-loaded liposomes, characterized in that, Specifically comprising the following steps: (1) Preparation of Agaricus bisporus chitosan After the Agaricus bisporus fruiting body is washed, dried, and ultra-finely pulverized, it is sieved, defatted, decolorized, and deacetylated, and vacuum freeze-dried to obtain Agaricus bisporus chitosan, which is prepared for use; (2) Preparation of a lipid phase of liposomes Soybean phospholipid and cholesterol are dissolved in an organic solvent, stirred and dissolved, and rotary evaporated to form a uniform lipid film; (3) Preparation of a drug-loaded liposome suspension Metformin hydrochloride is dissolved in a PBS solution to obtain a metformin hydrochloride solution, which is added to the lipid film, hydrated, and ultrasonically emulsified to obtain a liposome suspension; (4) Agaricus bisporus chitosan modification The Agaricus bisporus chitosan is dissolved in an acetic acid solution to obtain an Agaricus bisporus chitosan solution, which is added to the liposome suspension under stirring, centrifuged, and resuspended in PBS to obtain the Agaricus bisporus chitosan-modified drug-loaded liposomes.

2. The preparation method of Agaricus bisporus chitosan modified drug-loaded liposomes according to claim 1, characterized in that, In step (1), the defatting, decolorizing, and deacetylation are specifically as follows: 5% sodium hydroxide solution is added at a mass-volume ratio of 1g:(10-30)mL, heated at 70-90°C in a water bath for 2-6h, the mineral substances are removed, the precipitate is centrifuged and washed with distilled water until neutral; 3% hydrogen peroxide solution is added to the precipitate at a mass-volume ratio of 1g:(10-30)mL, stirred in the dark at 20-40°C for 1-3h, decolorized, the precipitate is centrifuged and washed until neutral; 50% sodium hydroxide solution is added to the decolorized precipitate at a mass-volume ratio of 1g:(10-30)mL, heated at 140-160°C in a water bath for 1-5h, deacetylated, the precipitate is centrifuged and washed until neutral.

3. The method for preparing Agaricus bisporus chitosan-modified drug-loaded liposomes according to claim 1, characterized in that, In step (2), the mass ratio of soybean phospholipid to cholesterol is (3-5):

1.

4. The method for preparing a chitosan-modified drug-loaded liposome from *Agaricus bisporus* according to claim 1, characterized in that, In step (2), the rotary evaporation is performed at a temperature of 20-40°C, a rotation speed of 30-70rpm, and for a time of 20-40min.

5. The method for preparing a chitosan-modified drug-loaded liposome from *Agaricus bisporus* according to claim 1, characterized in that, In step (3), the mass ratio of metformin hydrochloride to soybean phospholipid is (0.5-2):

1.

6. The method for preparing a chitosan-modified drug-loaded liposome from *Agaricus bisporus* according to claim 1, characterized in that, In step (3), the hydration is performed at a temperature of 30-50°C for a time of 0.5-2h.

7. The method according to claim 1, wherein the method is characterized by the steps of: In step (3), the ultrasonic emulsification is performed using an ice bath probe at a power of 200-400W for a time of 5-20min.

8. The method for preparing a chitosan-modified drug-loaded liposome from *Agaricus bisporus* according to claim 1, characterized in that, In step (4), the concentration of the Agaricus bisporus chitosan solution is 0.1-1mg / mL; and the volume ratio of the liposome suspension to the Agaricus bisporus chitosan solution is (0.5-2):

1.

9. The Agaricus bisporus chitosan-modified liposome drug carrier prepared by the method according to any one of claims 1-8, characterized in that, The particle size is 110-160nm, the PDI is 0.1-0.2, and the Zeta potential is 0-2mv.

10. Use of the Agaricus bisporus chitosan-modified drug-loaded liposomes prepared by the preparation method of any one of claims 1-8 in the preparation of a metformin hydrochloride preparation.

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