Glycyrrhetinic acid albumin nanoparticles, preparation method and application

Abstract

The invention discloses glycyrrhetinic acid albumin nanoparticles. The glycyrrhetinic acid albumin nanoparticles are in a uniform spherical shape; the nanoparticles comprise glycyrrhetinic acid and albumin; the particle size of the nanoparticles is smaller than or equal to 200 nm, the encapsulation efficiency is 90%-95.10%, the drug loading capacity is 30.56%-63.40%, and glycyrrhetinic acid released by the nanoparticles within 24 hours is 60%-70%. The invention further discloses a preparation method of the glycyrrhetinic acid albumin nanoparticles, the nanoparticles obtained through the method are small in particle size and uniform in distribution, and the stability and safety of the nanoparticles are improved along with increase of the proportion of albumin; wherein the albumin-rich nanoparticles show dual-mode pharmacological activity in experiments for preventing and treating acute liver injury.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to glycyrrhetinic acid albumin nanoparticles, a preparation method and an application thereof. Background Art

[0002] Acute liver injury (ALI) is a serious, life-threatening disease. One of the hallmarks of ALI is Kupffer cell hyperactivation, accompanied by the release of large amounts of reactive oxygen species and inflammatory factors, leading to hepatocellular damage, elevated hepatosplenic index, and liver enzyme levels, further exacerbating liver dysfunction. Glycyrrhizic acid analogs have been widely used in the treatment of acute and chronic liver injury due to their broad-spectrum hepatoprotective, anti-inflammatory, and antioxidant properties. For example, the commercially available diammonium glycyrrhizinate injection and magnesium isoglycyrrhizinate injection have been shown to effectively reduce alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and reverse liver damage in patients with ALI. Unfortunately, glycyrrhizic acid analogs must undergo hydrolysis and metabolic conversion to pharmacologically active glycyrrhetinic acid (GA) to exert their efficacy. In acute liver injury, this metabolic activation process can delay pharmacological action and weaken therapeutic efficacy. Although glycyrrhetinic acid is the primary active component of glycyrrhizic acid, its poor water solubility and rapid clearance mean that it can only be used clinically as a prodrug.

[0003] Nano-delivery systems are widely used to overcome the defects of water-insoluble drugs. Their unique advantages include improving drug stability, extending half-life and targeted drug delivery. It is worth noting that the liver contains a large number of reticuloendothelial cells, such as Kupffer cells and unique endothelial cell fenestrations (about 100-200nm), which provide natural targets for liver phagocytosis and nanoparticle enrichment. Among various nanocarriers, human serum albumin (HSA) is the most abundant protein in human plasma and is considered to be a safe and reliable natural drug delivery carrier due to its low immunogenicity, biocompatibility and biodegradability. In particular, (Albumin-bound paclitaxel) is the first albumin nanoformulation approved by the U.S. Food and Drug Administration (FDA), which is a milestone in the field of nanomedicine. It avoids the use of toxic solubilizers and broadens the clinical indications of paclitaxel. One key factor in the success was rigorous drug screening, which required hydrophobic drugs with high binding affinity for HSA. Paclitaxel, for example, has a binding rate of up to 95% to HSA. Similarly, studies have shown that glycyrrhetinic acid can bind to plasma proteins at a rate of up to 98%. Furthermore, the liver provides a site for albumin synthesis, which facilitates the specific binding of albumin nanoparticles to hepatocytes.

[0004] CN 104758251A discloses a method for preparing liver-targeted curcumin-loaded nanoparticles based on albumin as a carrier. The drug synthesis and nanoparticle preparation processes described in this preparation method are relatively complicated, and the organic reagents and cross-linking agents used in the preparation method are highly toxic and difficult to remove. To solve the above problems, a preparation method and application of glycyrrhetinic acid albumin nanoparticles are proposed. Summary of the Invention

[0005] To address the problems of the prior art, the present invention improves the water solubility of glycyrrhetinic acid based on its high affinity for plasma proteins, allowing for stable delivery to the lesion site of the liver to prevent and treat acute liver injury. The present invention aims to provide glycyrrhetinic acid albumin nanoparticles prepared through non-covalent interactions, comprehensively screen for nanoparticle formulations with high encapsulation efficiency, high drug loading, stability, and high safety, and investigate their in vitro and in vivo fate and therapeutic effects on acute liver injury. Experimental results demonstrate that the albumin nanoformulation exhibits excellent stability and safety, significantly improves drug bioavailability and liver accumulation, prolongs drug circulation time in vivo, and exhibits bimodal pharmacological activity, demonstrating excellent preventive and therapeutic effects in an LPS / D-GalN-induced acute liver injury model.

[0006] To achieve the above object, the present invention provides a glycyrrhetinic acid albumin nanoparticles, wherein the nanoparticles are uniformly spherical;

[0007] The nanoparticles include glycyrrhetinic acid and albumin;

[0008] The particle size of the nanoparticles is less than or equal to 200 nm, the encapsulation rate is 90% to 95.1%, and the drug loading amount is 30.56% to 63.40%.

[0009] Optionally, the nanoparticles release 60% to 70% of glycyrrhetinic acid within 24 hours.

[0010] Optionally, the particle size of the nanoparticles is 130 to 145 nm.

[0011] The present invention also provides a method for preparing glycyrrhetinic acid albumin nanoparticles, which specifically comprises the following steps:

[0012] 1) dissolving glycyrrhetinic acid in an organic solvent to obtain a glycyrrhetinic acid solution;

[0013] 2) dispersing the albumin solution in water to obtain a diluted albumin solution;

[0014] 3) slowly adding the glycyrrhetinic acid solution obtained in step 1) dropwise to the diluted albumin solution in step 2), and rotary evaporating to obtain glycyrrhetinic acid albumin nanoparticles;

[0015] Optionally, the organic solvent in step 1) is at least one of dimethyl sulfoxide, dimethylformamide, and methanol.

[0016] Optionally, the organic solvent in step 1) is methanol.

[0017] Optionally, the concentration of the albumin solution in step 2) is 40-200 mg / mL.

[0018] Optionally, the concentration of glycyrrhetinic acid in the glycyrrhetinic acid solution in step 1) is 9-27 mg / mL.

[0019] Optionally, the concentration of glycyrrhetinic acid in the glycyrrhetinic acid solution in step 1) is 9-18 mg / mL.

[0020] The concentration of the diluted albumin solution in step 2) is 0.1 to 9 mg / mL;

[0021] In step 3), the volume ratio of the glycyrrhetinic acid solution obtained in step 1) to the diluted albumin solution in step 2) is 1:4-30.

[0022] Preferably, in step 3), the volume ratio of the glycyrrhetinic acid solution obtained in step 1) to the diluted albumin solution in step 2) is 1:7-9.

[0023] Optionally, the albumin in the albumin solution in step 2) is selected from at least one of human serum albumin and bovine serum albumin.

[0024] Optionally, the albumin in the albumin solution in step 2) is human serum albumin with a molecular weight of 66 KDa.

[0025] Optionally, the glycyrrhetinic acid albumin nanoparticles obtained in step 3) have a particle size of 130 to 145 nm, a polydispersity index (PDI) of 0 to 0.2, an encapsulation efficiency of 90% to 95.10%, and a drug loading of 30.56% to 63.40%.

[0026] Optionally, the glycyrrhizic acid albumin nanoparticles are used in the prevention / treatment of acute liver injury, acute and chronic hepatitis, liver cancer, as well as in drug delivery systems and drug delivery.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The results of Examples 1 to 3 of the present invention show that when the mass ratio of glycyrrhetinic acid to albumin is regulated, glycyrrhetinic acid molecules and albumin can spontaneously assemble in water to form nanoparticles with a particle size between 130 and 145 nm and a polydispersity coefficient of less than 0.2, which is conducive to liver-targeted accumulation through the reticuloendothelial phagocytic system and the unique fenestration structure of liver sinusoidal endothelial cells. The negative surface charge of the nanoparticles (-20 mV) is conducive to effectively preventing the aggregation of nanoparticles through charge repulsion, thereby improving the stability of the nanoparticles. At the same time, the prepared glycyrrhetinic acid albumin nanoparticles have a high encapsulation efficiency (greater than 90%) and a drug loading of 30.56% to 63.40%.

[0029] (2) The stability and safety of the glycyrrhetinic acid albumin nanoparticles obtained by the technical solution of the present invention increase with the increase of the proportion of albumin, and the albumin-rich nanoparticles have good stability and pharmacokinetic behavior; in the prevention and treatment of acute liver injury experiments, they exhibited dual-mode pharmacological activity and time-dependently improved the survival rate of mice, highlighting the powerful therapeutic potential of glycyrrhetinic acid albumin nanoparticles for acute liver injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Transmission electron microscopy images of glycyrrhetinic acid albumin nanoparticles prepared in Examples 1 to 3 of the present application;

[0031] Figure 2 This is a graph showing the particle size changes of the glycyrrhetinic acid albumin nanoparticles prepared in Examples 1 to 3 of the present application when placed at 4°C, at room temperature, and on a shaker at 37°C;

[0032] Figure 3 This is a graph showing the release curve of glycyrrhetinic acid from glycyrrhetinic acid albumin nanoparticles prepared in Examples 1 to 3 of the present application in PBS containing 1% sodium lauryl sulfate;

[0033] Figure 4 This is a graph showing the survival curve of mouse mononuclear macrophages (RAW 264.7 cells) in the glycyrrhetinic acid albumin nanoparticles and glycyrrhetinic acid solution prepared in Examples 1 to 3 of the present application;

[0034] Figure 5 This is a graph showing the survival curve of normal mouse liver cells (AML 12 cells) in the glycyrrhetinic acid albumin nanoparticles and glycyrrhetinic acid solution prepared in Examples 1 to 3 of the present application;

[0035] Figure 6Graph showing changes in intracellular nitric oxide concentration in the prevention mode of an in vitro anti-inflammatory experiment using glycyrrhetinic acid albumin nanoparticles and glycyrrhetinic acid solution prepared in Examples 1 to 3 of the present application (ns: P ≥ 0.05*: P < 0.05**: P < 0.01***: P < 0.001****: P < 0.0001 (both two-sided t-tests));

[0036] Figure 7 Graph showing changes in intracellular nitric oxide concentration in the treatment mode of an in vitro anti-inflammatory experiment using glycyrrhetinic acid albumin nanoparticles and glycyrrhetinic acid solution prepared in Examples 1 to 3 of the present application (ns: P ≥ 0.05*: P < 0.05**: P < 0.01***: P < 0.001****: P < 0.0001 (both two-sided t-tests));

[0037] Figure 8 This is a mass spectrum of sodium glycyrrhetinic acid (GA-Na) prepared in Example 7 of the present application;

[0038] Figure 9 The H NMR spectrum of sodium glycyrrhetinic acid (GA-Na) prepared in Example 7 of the present application ( 1 H NMR) spectra;

[0039] Figure 10 This is a purity chart of sodium glycyrrhetinic acid (GA-Na) prepared in Example 7 of the present application;

[0040] Figure 11 The blood concentration-time curves of glycyrrhetinic acid in the in vivo pharmacokinetic experiments of the glycyrrhetinic acid albumin nanoparticles prepared in Examples 1 to 3 of the present application and the sodium glycyrrhetinic acid solution prepared in Example 7 are shown;

[0041] Figure 12 Graph showing changes in liver and spleen index in the prevention mode in an in vivo anti-hepatitis experiment of the glycyrrhetinic acid albumin nanoparticles prepared in Example 3 of the present application and the sodium glycyrrhetinic acid solution prepared in Example 7 (ns: P ≥ 0.05*: P < 0.05**: P < 0.01***: P < 0.001****: P < 0.0001 (all two-sided t-tests));

[0042] Figure 13 Graph showing changes in serum aspartate aminotransferase and alanine aminotransferase levels in the prevention mode of an in vivo anti-hepatitis experiment of the glycyrrhetinic acid albumin nanoparticles prepared in Example 3 of the present application and the sodium glycyrrhetinic acid solution prepared in Example 7 (ns: P ≥ 0.05*: P < 0.05**: P < 0.01***: P < 0.001****: P < 0.0001 (all two-sided t-tests));

[0043] Figure 14Graph showing changes in liver and spleen index under treatment mode in an in vivo anti-hepatitis experiment for the glycyrrhetinic acid albumin nanoparticles prepared in Example 3 of the present application and the sodium glycyrrhetinic acid solution prepared in Example 7 (ns: P ≥ 0.05*: P < 0.05**: P < 0.01***: P < 0.001****: P < 0.0001 (all two-sided t-tests));

[0044] Figure 15 Graph showing changes in serum aspartate aminotransferase and alanine aminotransferase levels under treatment mode in an in vivo anti-hepatitis experiment of glycyrrhetinic acid albumin nanoparticles prepared in Example 3 of the present application and the sodium glycyrrhetinic acid solution prepared in Example 7 (ns: P≥0.05*: P<0.05**: P<0.01***: P<0.001****: P<0.0001 (all two-sided t-tests)). DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to specific examples, but the present invention is not limited thereto in any way.

[0046] The experimental methods described in the following examples, unless otherwise specified, are conventional methods; the reagents and materials, unless otherwise specified, can be obtained from commercial sources;

[0047] PBS solution (phosphate buffered saline) and SDS (sodium dodecyl sulfate) were purchased from Shandong Yuwang Reagent Co., Ltd.; LPS (lipopolysaccharide) was purchased from Sigma-Aldrich; Giess reagent I and Giess reagent II were from a nitric oxide detection kit purchased from Beyotime Biotechnology Co., Ltd.; MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Dalian Meilun Biotechnology Co., Ltd.; mouse mononuclear macrophages (RAW 264.7 cells) and mouse normal hepatocytes (AML 12 cells) were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee; Kunming (KM) mice were obtained from Liaoning Changsheng Biotechnology Co., Ltd.; aspartate aminotransferase (AST) and alanine aminotransferase (ALT) kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0048] The morphology of the glycyrrhetinic acid albumin nanoparticles prepared in Examples 1 to 3 was tested using a transmission electron microscope (JEM1200EX);

[0049] The particle size, surface charge and storage stability of the glycyrrhetinic acid albumin nanoparticles prepared in Examples 1 to 3 were tested using a Malvern particle size analyzer (Nano-ZS90);

[0050] The encapsulation efficiency and drug loading of the glycyrrhetinic acid albumin nanoparticles prepared in Examples 1 to 3 were tested using an ultracentrifuge (CS120FNX) and a high-performance liquid chromatograph (Chromaster);

[0051] The in vitro release, pharmacokinetic behavior, and in vivo distribution of the glycyrrhetinic acid albumin nanoparticles prepared in Examples 1 to 3 were analyzed using a high performance liquid chromatography (Chromaster);

[0052] The cellular safety and in vitro anti-inflammatory effect of the glycyrrhetinic acid albumin nanoparticles prepared in Examples 1 to 3 on mouse mononuclear macrophages (RAW264.7 cells) and mouse normal liver cells (AML 12 cells) were tested using a multifunctional microplate reader (Varioskan LUX);

[0053] The molecular weight of sodium glycyrrhetinate was tested using a Fourier transform ion cyclotron resonance mass spectrometer (solariX);

[0054] The molecular structure of sodium glycyrrhetinic acid was tested using a nuclear magnetic resonance spectrometer (Bruker ARX400).

[0055] Example 1: Preparation of glycyrrhetinic acid-albumin nanoparticles (glycyrrhetinic acid to albumin mass ratio of 1:0.5)

[0056] 7.2 mg of glycyrrhetinic acid was accurately weighed and dissolved in 800 μL of methanol to obtain a glycyrrhetinic acid solution; 90 μL of a 40 mg / mL human serum albumin solution was pipetted into 7.110 mL of water to form a diluted albumin aqueous solution; the glycyrrhetinic acid solution was slowly added dropwise to 7.2 mL of the diluted albumin aqueous solution under stirring, and glycyrrhetinic acid albumin nanoparticles with uniform particle size spontaneously formed under high-speed stirring. The methanol in the preparation was removed by a rotary evaporator to obtain glycyrrhetinic acid albumin nanoparticles with a glycyrrhetinic acid to albumin mass ratio of 1:0.5 (1:0.5GA@HSA nanoparticles). The particle size, polydispersity coefficient, surface charge, encapsulation efficiency and drug loading of the glycyrrhetinic acid albumin nanoparticles are shown in Table 1; the particle size and morphology of the glycyrrhetinic acid albumin nanoparticles are shown in Table 1. Figure 1 As shown, the test results show that the prepared albumin nanoparticles are uniform spherical.

[0057] Storage stability test: The albumin nanoparticles prepared in this example were placed at 4°C for 28 days, at room temperature for 7 days, and on a shaker at 37°C for 12 hours. The results of the storage stability test are shown in Figure 2. Figure 2 As shown, the results show that the particle size of the albumin nanoparticles obtained in this example did not change significantly when placed in a 4°C, room temperature, or 37°C shaking environment.

[0058] Example 2: Preparation of glycyrrhetinic acid-albumin nanoparticles (glycyrrhetinic acid to albumin mass ratio of 1:1)

[0059] 7.2 mg of glycyrrhetinic acid was accurately weighed and dissolved in 800 μL of methanol to obtain a glycyrrhetinic acid solution; 180 μL of a 40 mg / mL human serum albumin solution was transferred to 7.020 mL of water to form a diluted albumin aqueous solution; the glycyrrhetinic acid solution was slowly added dropwise to 7.2 mL of the diluted albumin aqueous solution under stirring, and glycyrrhetinic acid albumin nanoparticles with uniform particle size were spontaneously formed under high-speed stirring. The methanol in the preparation was removed by a rotary evaporator to obtain glycyrrhetinic acid albumin nanoparticles with a glycyrrhetinic acid to albumin mass ratio of 1:1 (1:1 GA@HSA nanoparticles). The particle size, polydispersity coefficient, surface charge, encapsulation efficiency and drug loading of the glycyrrhetinic acid albumin nanoparticles are shown in Table 1; the particle size and morphology of the glycyrrhetinic acid albumin nanoparticles are shown in Table 1. Figure 1 As shown, the test results show that the prepared albumin nanoparticles are uniform spherical.

[0060] Storage stability test: The albumin nanoparticles prepared in this example were placed at 4°C for 28 days, at room temperature for 7 days, and on a shaker at 37°C for 12 hours. The results of the storage stability test are shown in Figure 2. Figure 2 As shown, the results show that there is no significant change in particle size when standing at 4°C, standing at room temperature, or in a shaking environment at 37°C.

[0061] Example 3: Preparation of glycyrrhetinic acid-albumin nanoparticles (glycyrrhetinic acid to albumin mass ratio of 1:2)

[0062] 7.2 mg of glycyrrhetinic acid was accurately weighed and dissolved in 800 μL of methanol to obtain a glycyrrhetinic acid solution; 360 μL of a 40 mg / mL human serum albumin solution was transferred to 6.840 mL of water to form a diluted albumin aqueous solution; the methanol solution was slowly added dropwise to 7.2 mL of the diluted albumin aqueous solution under stirring, and glycyrrhetinic acid albumin nanoparticles with uniform particle size spontaneously formed under high-speed stirring. The methanol in the preparation was removed by a rotary evaporator to obtain glycyrrhetinic acid albumin nanoparticles with a glycyrrhetinic acid to albumin mass ratio of 1:2 (1:2GA@HSA nanoparticles). The particle size, polydispersity coefficient, surface charge, encapsulation efficiency and drug loading of the glycyrrhetinic acid albumin nanoparticles are shown in Table 1; the particle size and morphology of the albumin nanoparticles are shown in Table 1. Figure 1 As shown, the test results show that the prepared albumin nanoparticles are uniform spherical.

[0063] Storage stability test: The albumin nanoparticles prepared in this example were placed at 4°C for 28 days, at room temperature for 7 days, and on a shaker at 37°C for 12 hours. The results of the storage stability test are shown in Figure 2. Figure 2As shown, the results showed that there was no significant change in particle size at 4°C, room temperature, and 37°C shaking environments.

[0064] Table 1. Particle size, polydispersity coefficient, surface charge, encapsulation efficiency, and drug loading of glycyrrhetinic acid albumin nanoparticles

[0065]

[0066] The results of Examples 1-3 demonstrate that, when the mass ratio of glycyrrhetinic acid to albumin is adjusted, glycyrrhetinic acid molecules can spontaneously assemble in water to form nanoparticles with a particle size between 130 and 145 nm and a polydispersity coefficient less than 0.2, facilitating targeted liver accumulation via the reticuloendothelial phagocytic system and the unique fenestration structure of hepatic sinusoidal endothelial cells. The negative surface charge (-20 mV) of the nanoparticles effectively prevents nanoparticle aggregation through charge repulsion, thereby improving nanoparticle stability. Furthermore, the prepared glycyrrhetinic acid-albumin nanoparticles exhibit a high encapsulation efficiency (greater than 90%) and a drug loading of 30.56% to 63.40%.

[0067] Example 4: In vitro drug release behavior of glycyrrhetinic acid albumin nanoparticles

[0068] First, a PBS solution (pH 7.4) containing 1% sodium dodecyl sulfate (SDS) was prepared as a release medium to investigate the in vitro release of glycyrrhetinic acid albumin nanoparticles. 200 nmol of glycyrrhetinic acid albumin nanoparticles prepared in Examples 1 to 3 were respectively placed in a dialysis bag with a molecular cutoff of 8000-14000 Da, and the dialysis bag was placed in a container containing 25 mL of release medium and mixed. The above system was placed in a constant temperature oscillator (37°C, 100 rpm / min) and oscillated for 0 to 24 hours. At a preset time point (0 to 24 h), 500 μL of the above solution was drawn, and 500 μL of release medium was added at the same time. The concentration of glycyrrhetinic acid released by the glycyrrhetinic acid albumin nanoparticles at the preset time point was determined by high performance liquid chromatography to obtain the glycyrrhetinic acid release rate. The test results are shown in FIG. Figure 3 As shown, the results showed that in the PBS release medium containing 1% SDS, the glycyrrhetinic acid solution exhibited a burst release, reaching 100% release. However, the glycyrrhetinic acid albumin nanoparticles obtained in Implementations 1-3 slowed the burst release of glycyrrhetinic acid and exhibited a slow, sustained release process. The albumin nanoparticles released approximately 60%-70% of the glycyrrhetinic acid within 24 hours, and the release rate of glycyrrhetinic acid from the nanoparticles increased with increasing albumin content. The albumin-mediated release gradient suggests that albumin-rich nanoparticles may have improved safety, pharmacokinetics, and prolonged blood circulation time.

[0069] Example 5: In vitro cell safety experiment of glycyrrhetinic acid albumin nanoparticles

[0070] The MTT assay was used to investigate the cytotoxicity of glycyrrhetinic acid albumin nanoparticles on mouse mononuclear macrophages (RAW 264.7 cells) and normal mouse hepatocytes (AML 12 cells). RAW 264.7 cells (20,000 cells / well) or AML 12 cells (8,000 cells / well) were seeded into 96-well plates and incubated in a 37°C, 5% CO2 incubator for 12 or 24 hours. After the cells adhered, the culture medium was discarded and 100 μL of fresh culture medium diluted with glycyrrhetinic acid solution (0.5% DMSO) at concentrations of 5, 10, 20, 50, 100, 200, 500, and 1000 μmol / L was added, along with three ratios of glycyrrhetinic acid albumin nanoparticles prepared in Examples 1-3 (1:0.5, 1:1, and 1:2). Three parallel wells were set for each concentration. The wells to which only blank cell culture medium was added served as the control group, and the wells to which only PBS solution was added and no cells were used as zeroing wells. 24 hours after drug addition, the 96-well plate was removed, 100 μL of MTT solution (1 mg / mL) was added to each well, and the plate was incubated in an incubator for 4 hours before being shaken off. The 96-well plate was inverted on filter paper to fully absorb the residual liquid, and 150 μL of DMSO was added to each well and shaken on an oscillator for 15 minutes to dissolve the blue-purple formazan crystals. The absorbance value of each well after zeroing was measured at 490 nm or 570 nm using a microplate reader and the survival rate of RAW 264.7 cells and AML 12 cells was calculated. The cell survival rate results are shown in Table 1. Figure 4 、 5 As shown in the figure, the results showed that compared with glycyrrhetinic acid solution, glycyrrhetinic acid albumin nanoparticles had better safety against two normal cells. This is because it takes a certain amount of time for glycyrrhetinic acid to be released from albumin nanoparticles. Among the three ratios of albumin nanoparticles, 1:2GA@HSA nanoparticles had better cell safety. This was because as the albumin ratio increased, the drug release rate gradually slowed down. The safety of 1:2GA@HSA nanoparticles on normal cells was better than that of 1:1GA@HSA nanoparticles and 1:0.5GA@HSA nanoparticles. The half-maximal inhibitory concentration (IC50) was calculated by GraphPadPrism 9.0 software. 50 value), IC 50 The calculation results of the values are shown in Table 2. The calculation results show that 1:2GA@HSA nanoparticles have the best safety for normal cells.

[0071] Table 2. Cellular safety of glycyrrhetinic acid solution and glycyrrhetinic acid albumin nanoparticles

[0072]

[0073] Example 6: In vitro anti-inflammatory experiment of glycyrrhetinic acid albumin nanoparticles

[0074] The in vitro anti-inflammatory effect of glycyrrhetinic acid albumin nanoparticles was investigated by inducing a RAW264.7 cell inflammation model with LPS (1 μg / mL) and monitoring the intracellular nitric oxide content. RAW264.7 cells (20,000 cells / well) were seeded into 96-well plates and incubated overnight in a 37°C, 5% CO2 incubator. After the cells adhered, the culture medium was discarded and different concentrations of glycyrrhetinic acid solution and glycyrrhetinic acid albumin nanoparticles prepared in Examples 1-3 were administered. One hour later, 1 μg / mL LPS was added to induce cell inflammation and incubated with the drug for 24 hours. This served as the prevention group, with 3 parallel wells set for each concentration. The same conditions were used to plate the cells. After the cells adhered, the culture medium was discarded and 1 μg / mL LPS was added to induce cell inflammation. After 24 hours, the culture medium was discarded again and different concentrations of glycyrrhetinic acid solution and GA@HSA nanoparticles prepared in Examples 1-3 were added and incubated for another 24 hours. This served as the treatment group, with 3 parallel wells set for each concentration. The wells with only blank cell culture medium were used as negative control groups, the wells with only PBS and no cells were used as zero-adjustment wells, and the blank cell culture medium was treated with 1 μg / mL LPS as the positive control group for inflammation. After incubation, 50 μL of cell supernatant was aspirated from each well and mixed with 50 μL of Giess reagent I and II. The nitric oxide level was detected at 540 nm using a multifunctional microplate reader. The nitric oxide concentration test results are shown in Figure 2. Figure 6 、 7 As shown in the results, the drug administration in both prevention and treatment modes showed a dose-dependent inhibition of the increase in nitric oxide concentration in RAW 264.7 cells, and compared with the positive control group, it was able to reduce the intracellular nitric oxide level by more than 50%. Among them, glycyrrhetinic acid solution can more effectively reduce the concentration of nitric oxide, but high concentrations of glycyrrhetinic acid solution will kill macrophages, especially in the treatment mode. When the inhibitory effect on nitric oxide reached 50%, the concentration of glycyrrhetinic acid solution was significantly higher than IC 50 , which can cause a certain degree of damage to cells and pose a safety risk. However, nanoparticles with a high albumin ratio (1:1GA@HSA and 1:2GA@HSA nanoparticles) can have a long-lasting anti-inflammatory effect while ensuring cell safety due to their improved stability and sustained intracellular drug release behavior.

[0075] Example 7: Synthesis of Sodium Glycyrrhetinate (GA-Na)

[0076] Weigh 1.3g (2.76mmol) of glycyrrhetinic acid and 0.16g (4mmol) of sodium hydroxide into a 250mL eggplant-shaped flask, add 50mL of ethanol until the solid is completely dissolved, and heat under reflux at 85°C for 2 hours. After the reaction is completed, cool to room temperature, use a vacuum rotary evaporator to remove anhydrous ethanol until 5-10mL remains, stop the rotary evaporation and transfer to a 25mL eggplant-shaped flask, and place in a -20°C refrigerator for crystallization. After the product is completely crystallized, filter under reduced pressure to obtain the product sodium glycyrrhetinic acid (GA-Na). The mass spectrum and nuclear magnetic resonance of the product sodium glycyrrhetinic acid (GA-Na) are shown as follows: Figure 8 、 9 As shown, the mass spectrum results show that the molecular weight of the product is 493.38, and the nuclear magnetic resonance test results are as follows:

[0077] 1 H NMR(600MHz,DMSO-d6)δ5.45(s,1H,-COCH),4.40(s,1H,-CHOH),3.01(dd,1H,- CH OH),2.66-2.51(m,1H,-CO CH C-), 2.30(d,1H,-CHC CH CH2-),2.04(td,,2H,-CH2CH2CCOOH),1.89-1.71(m,4H,-CHCH2CH2-),1.71-1.25(m,10H,-CH2 CH 2C-),1.23-1.06(m,2H,-CH3C CH 2CH2-),1.15-1.02(m,3H,-CH3CCOOH),1.02(d,6H,-CC(CH3)CH2-,-CHC(CH3)CH2-),0.95-0.93(m,1H,-CH3C CH CH2-), 0.91-0.62(m,12H,-C (CH 3)2,-C CH 3)

[0078] The purity of the product sodium glycyrrhetinate (GA-Na) was tested by high performance liquid chromatography. Figure 10 As shown, the purity is 98.61%.

[0079] Example 8: Pharmacokinetic Study of Glycyrrhetinic Acid Albumin Nanoparticles

[0080] Thirty healthy male SD rats weighing 180-220 g were randomly divided into five groups. They were fasted for 12 hours before administration and had free access to water. Four of the groups were injected via the tail vein with 40 mg / kg (equivalent dose of glycyrrhetinic acid) of the GA-Na solution synthesized in Example 7 and the glycyrrhetinic acid albumin nanoparticles prepared in Examples 1 to 3. Blood was collected at the specified time points (0 to 24 hours) and the drug concentration in plasma was determined by high performance liquid chromatography. The blood drug concentration test results are shown in Table 3 and Figure 11 As shown in the results, the test results show that after administration, the GA-Na solution is rapidly metabolized and cleared from the body, with a small area under the drug-time curve, a short half-life, and an average residence time of only 1.31 hours. In contrast, the three ratios of glycyrrhetinic acid albumin nanoparticles are slowly cleared from the body, significantly prolonging the blood circulation time of glycyrrhetinic acid. The t 1 / 2 The pharmacokinetic parameters of 1:2GA@HSA nanoparticles were superior to those of 1:1GA@HSA nanoparticles, followed by 1:0.5GA@HSA nanoparticles, which were attributed to the excellent stability of 1:2GA@HSA nanoparticles. These results indicate that albumin binding significantly prolongs the in vivo circulation time of glycyrrhetinic acid.

[0081] Table 3. Pharmacokinetic parameters of sodium glycyrrhetinate solution and glycyrrhetinic acid albumin nanoparticles

[0082]

[0083] Example 9: In vivo anti-hepatitis effect of glycyrrhetinic acid albumin nanoparticles

[0084] Kunming (KM) mice were randomly divided into 14 groups, with four mice in each group. Normal saline was used as the negative control group, and intraperitoneal injection of LPS / D-GalN (50μg / kg LPS, 800mg / kg D-GalN) was used as the positive control group for acute liver injury. Mice were given the GA-Na solution synthesized in Example 7 and the 1:2 GA@HSA nanoparticles prepared in Example 3 (glycyrrhetinic acid equivalent dose: 40mg / kg) before and after intraperitoneal injection of LPS / D-GalN, as a prevention group (4, 8, 12 hours) and a treatment group (0, 0.5, 1 hour). Mice were killed 8 hours after LPS / D-GalN injection, and the mouse serum, liver and spleen were collected for further analysis and evaluation of the in vivo anti-hepatitis effect of glycyrrhetinic acid albumin nanoparticles.

[0085] The evaluation results of the prevention group at 4, 8, and 12 hours were as follows Figure 12 、13 As shown, Figure 12 The results showed that compared with the negative control group, the liver and spleen index of the acute liver injury positive group increased significantly, and the preventive mode (administration 4, 8, and 12 hours before LPS / D-GalN injection) could significantly improve the increase in the liver and spleen index; among them, the glycyrrhetinic acid albumin nanoparticles and GA-Na solution groups had similar improvement effects, especially when administered 8 and 12 hours in advance, the liver and spleen index was no different from that of the negative control group. Figure 13 Results showed that the liver function indicators of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the acute liver injury-positive group increased significantly. After preventive administration, liver function indicators decreased significantly. The preventive effect of glycyrrhetinic acid albumin nanoparticles and GA-Na solution groups was comparable. The 1:2GA@HSA nanoparticle group showed a superior effect on downregulating AST after 8 hours of pretreatment, which was superior to GA-Na solution. At the same time, liver function indicators returned to normal ranges 8 and 12 hours before administration. This superior recovery ability from acute liver injury may be due to the prolonged blood circulation time and enhanced liver accumulation of 1:2GA@HSA nanoparticles.

[0086] The evaluation results of the treatment group at 0, 0.5 and 1 hours were as follows Figure 14 As shown, Figure 14 The results showed that the treatment regimen (administered 0, 0.5, and 1 hour after LPS / D-GalN injection) could improve the increased liver and spleen index to some extent, but the improvement gradually weakened with the delay of administration. Similarly, the improvement effect of glycyrrhetinic acid albumin nanoparticles and GA-Na solution was comparable. Figure 15 The results showed that after administration in the treatment mode, liver function indicators decreased significantly, and the therapeutic effects of glycyrrhizic acid albumin nanoparticles and GA-Na solution groups were comparable. As the administration time was delayed, the recovery efficiency of liver function indicators gradually weakened, indicating that the more timely the administration of treatment for acute liver injury, the better the therapeutic effect.

[0087] The results of evaluations at 4, 8, and 12 hours in the prevention group and 0, 0.5, and 1 hour in the treatment group showed that glycyrrhetinic acid albumin nanoparticles had a comparable effect on the recovery of acute liver injury as GA-Na solution. Furthermore, the prevention and treatment of acute liver injury exhibited a time-dependent effect, with both increased prevention time and timely drug treatment improving acute liver injury.

[0088] Example 10: Safety evaluation of glycyrrhetinic acid albumin nanoparticles

[0089] Kunming (KM) mice were randomly divided into 14 groups, with six mice in each group. Normal saline was used as the negative control group, and intraperitoneal injection of LPS / D-GalN (50μg / kg LPS, 800mg / kg D-GalN) was used as the positive control group for acute liver injury. Mice were given the GA-Na solution synthesized in Example 7 and the 1:2GA@HSA nanoparticles (glycyrrhetinic acid equivalent dose: 40mg / kg) prepared in Example 3 before and after intraperitoneal injection of LPS / D-GalN, as the prevention group (4, 8, 12 hours) and the treatment group (0, 0.5, 1 hour). The death of mice within 24 hours was observed and recorded. The specific survival rate results are shown in Tables 4 and 5.

[0090] The number of dead animals and the survival rate in the prevention group (4, 8, and 12 hours) can be seen from Table 4. Within 24 hours, the mortality rate of mice in the acute liver injury positive control group reached 100%. Drug treatment in the prevention mode can significantly improve the survival rate of mice. When administered 4 hours in advance, the survival rates of the glycyrrhetinic acid albumin nanoparticles and GA-Na solution groups are consistent. In particular, when administered 8 and 12 hours in advance, glycyrrhetinic acid albumin nanoparticles have a better ability to improve the survival rate of mice.

[0091] Table 5 shows the number of animals that died and the survival rates in the treatment groups (0, 0.5, and 1 hours). In this treatment mode, administering the drug within 0.5 hours after LPS / D-GalN injection increased mouse survival. These results are consistent with the in vivo anti-hepatitis results of glycyrrhetinic acid albumin nanoparticles, indicating that the prevention and treatment of acute liver injury exhibit time-dependent effects, with both increased prevention time and timely drug treatment improving mouse survival.

[0092] Table 4. Survival rates of GA-Na solution and 1:2 GA@HSA nanoparticles in the preventive mode

[0093]

[0094] Table 5. Survival rates of GA-Na solution and 1:2GA@HSA nanoparticles under treatment mode

[0095]

[0096] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A glycyrrhetinic acid albumin nanoparticle, characterized in that: The nanoparticles are uniformly spherical; The nanoparticles include glycyrrhetinic acid and albumin; The particle size of the nanoparticles is less than or equal to 200 nm, the encapsulation rate is 90% to 95.10%, and the drug loading amount is 30.56% to 63.40%.

2. The glycyrrhetinic acid albumin nanoparticles according to claim 1, characterized in that The nanoparticles release 60% to 70% of glycyrrhetinic acid within 24 hours; The mass ratio of glycyrrhetinic acid to albumin in the nanoparticles is 1:0.1-9; Preferably, the mass ratio of glycyrrhetinic acid to albumin in the nanoparticles is 1:0.5-2; The particle size of the nanoparticles is 130-145 nm.

3. The method for preparing glycyrrhetinic acid albumin nanoparticles according to any one of claims 1 or 2, characterized in that: At least the following steps are included: Step 1): dissolving glycyrrhetinic acid in an organic solvent to obtain a glycyrrhetinic acid solution; Step 2): dispersing the albumin solution in water to obtain a diluted albumin solution; Step 3): slowly add the glycyrrhetinic acid solution obtained in step 1) dropwise to the diluted albumin solution in step 2), and perform rotary evaporation to obtain glycyrrhetinic acid albumin nanoparticles.

4. The preparation method according to claim 3, characterized in that The organic solvent in step 1) is at least one of dimethyl sulfoxide, dimethylformamide, and methanol; Preferably, the organic solvent in step 1) is methanol.

5. The preparation method according to claim 3, characterized in that The concentration of the albumin solution in step 2) is 40 to 200 mg / mL.

6. The preparation method according to claim 3, characterized in that The concentration of glycyrrhetinic acid in the glycyrrhetinic acid solution in step 1) is 9-27 mg / mL.

7. The preparation method according to claim 6, characterized in that The concentration of glycyrrhetinic acid in the glycyrrhetinic acid solution in step 1) is 9-18 mg / mL.

8. The preparation method according to claim 3, characterized in that The concentration of the diluted albumin solution in step 2) is 0.1 to 9 mg / mL; In step 3), the volume ratio of the glycyrrhetinic acid solution obtained in step 1) to the diluted albumin solution in step 2) is 1:4-30; Preferably, in step 3), the volume ratio of the glycyrrhetinic acid solution obtained in step 1) to the diluted albumin solution in step 2) is 1:7-9.

9. The preparation method according to claim 3, characterized in that The albumin in the albumin solution in step 2) is selected from at least one of human serum albumin and bovine serum albumin; Preferably, the albumin in the albumin solution in step 2) is human serum albumin.

10. The glycyrrhetinic acid albumin nanoparticles according to any one of claims 1 or 2 and / or the glycyrrhetinic acid albumin nanoparticles prepared by the preparation method according to any one of claims 3 to 9 are used in the prevention / treatment of acute liver injury, acute and chronic hepatitis, liver cancer, as well as in drug delivery systems and drug delivery.

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