Application of β-glucan in the preparation of liver-targeted drugs for the treatment of liver cancer; liver-targeted drugs

Abstract

The application relates to the field of nano drug loading technology, and discloses application of beta-glucan in preparation of liver-targeted drugs for treating liver cancer and the liver-targeted drugs, wherein the beta-glucan is derived from black fungus, and a chemical structural formula of the beta-glucan is that two beta-(1, 6)-glucose residues are arranged on the side chain of every three beta-(1, 3)-glucose main chains. The application provides a new use of black fungus beta-glucan in preparation of liver-targeted drugs for treating liver cancer. The black fungus beta-glucan has the abilities of liver targeting, enzymatic release and activation of macrophages, so that the black fungus beta-glucan can be used as a carrier of the liver-targeted drugs, the drugs can be stably delivered to the liver, and the release of the drugs is realized under the action of liver enzymes in the liver, so that the liver cancer (hepatocellular carcinoma and intrahepatic cholangiocarcinoma, etc.) can be effectively treated, the toxic side effect is small, and the drug carrier can have a synergistic effect with the drugs loaded thereon, so that the intrahepatic cholangiocarcinoma can be better treated.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine delivery technology, specifically to the application of β-glucan in the preparation of liver-targeted drugs for the treatment of liver cancer, and liver-targeted drugs. Background Technology

[0002] Currently used drugs for treating liver cancer, such as triptolide, docetaxel, and paclitaxel, have poor water solubility, low bioavailability, and systemic toxic side effects, thus limiting their application in treating liver cancer-related diseases. Taking triptolide as an example, it is one of the five most promising natural products for anti-cancer treatment. It can downregulate the NF-κB signaling pathway to delay the occurrence of hepatocellular carcinoma and delay hepatic steatosis and carcinogenesis by inhibiting fatty acid synthase expression and AKT / ERK phosphorylation, making it an effective candidate drug for clinical treatment of liver cancer. However, triptolide has poor water solubility, low bioavailability, and produces systemic toxic side effects; furthermore, decreased immunity after drug treatment can affect subsequent disease recurrence.

[0003] β-glucan is a polysaccharide widely found in fungal cell walls. It has great potential as a therapeutic agent and adjuvant. However, natural β-glucan is difficult to target the liver and often requires structural modification, which limits its application in the treatment of liver cancer. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems of high toxicity, poor water solubility, and inability to target the liver in existing anti-liver cancer drugs, and to provide an application of β-glucan in the preparation of liver-targeted drugs for the treatment of liver cancer, and a liver-targeted drug.

[0005] To achieve the above objectives, this invention provides an application of β-glucan in the preparation of liver-targeted drugs for treating liver cancer. The β-glucan is derived from black fungus, and its chemical structure has a main chain with two β-(1,3)-glucose residues as side chains for every three β-(1,6)-glucose residues. Preferably, the molecular weight of the β-glucan is 1.98 × 10⁻⁶. 6 ~2.40×10 6 g / mol.

[0006] Preferably, this specifically refers to the use of β-glucan as a carrier in the preparation of liver-targeted drugs for the treatment of liver cancer.

[0007] Preferably, the liver-targeting drug comprises a carrier and an anti-liver cancer drug loaded on the carrier, wherein the carrier contains the β-glucan.

[0008] Preferably, the anti-liver cancer drug is selected from at least one of triptolide, docetaxel, and paclitaxel.

[0009] Preferably, the drug loading of the liver-targeting drug is 38-42%;

[0010] Preferably, the particle size of the liver-targeting drug is 400–600 nm.

[0011] Preferably, the preparation process of the liver-targeted drug includes the following steps:

[0012] (1) Mix β-glucan with dimethyl sulfoxide to obtain a mixture A with a β-glucan concentration of 0.5-1.5 mg / mL;

[0013] (2) Mix the anti-liver cancer drug with dimethyl sulfoxide to obtain mixture B;

[0014] (3) Mix the mixture A and the mixture B, then dialyze with water for 2 to 3 days, and then freeze dry.

[0015] Preferably, in step (2), the concentration of the anti-liver cancer drug in the mixture B is 0.5–1.5 mg / mL;

[0016] Preferably, in step (3), the mass ratio of the anti-liver cancer drug in mixture B to the β-glucan in mixture A is 1:0.5-2.

[0017] Preferably, the liver cancer is intrahepatic cholangiocarcinoma.

[0018] A second aspect of the present invention provides a liver-targeted drug for treating liver cancer, the liver-targeted drug comprising a carrier and an anti-liver cancer drug loaded on the carrier;

[0019] The carrier is a β-glucan nanotube. β-glucan is derived from black fungus. Its chemical structure is that every three β-(1,3)-glucose main chains have two β-(1,6)-glucose residues as side chains.

[0020] Preferably, the anti-liver cancer drug is selected from at least one of triptolide, docetaxel, and paclitaxel.

[0021] This invention provides a novel use of β-glucan derived from black fungus in the preparation of liver-targeted drugs for treating liver cancer. This black fungus β-glucan possesses liver-targeting, enzymatic release (drug release in the presence of hepatic drug-metabolizing enzymes), and macrophage activation capabilities. Using black fungus β-glucan as a carrier for liver-targeted drugs for treating liver cancer allows for stable drug delivery to the liver under normal blood conditions and drug release within the hepatic enzyme environment. Therefore, liver-targeted drugs prepared using black fungus β-glucan as a drug carrier can achieve targeted drug release to the liver, effectively treating liver cancer (hepatocellular carcinoma and intrahepatic cholangiocarcinoma, etc.) with minimal toxic side effects. Furthermore, this carrier can act as an immunomodulator to activate macrophages, synergistically enhancing the drug's effect and achieving highly effective anti-intrahepatic cholangiocarcinoma activity. Attached Figure Description

[0022] Figure 1 These are scanning electron microscope images of triptolide (Cel), black fungus β-glucan (BFP), and the liver-targeting drug (BFP-Cel) prepared in Example 1;

[0023] Figure 2 The graph shows the effects of triptolide (Cel), black fungus β-glucan (BFP), and the liver-targeting drug (BFP-Cel) prepared in Example 1 on the survival rate of normal hepatocytes.

[0024] Figure 3 The fluorescence distribution of isolated tissues from normal mice 4 hours after injection of the liver-targeting drug prepared in Example 1 and black fungus β-glucan (BFP);

[0025] Figure 4 This is a drug release curve of the liver-targeting drug prepared in Example 1 and commercially available triptolide in an in vitro simulated liver and normal blood environment;

[0026] Figure 5 This describes the change in molecular weight of black fungus β-glucan (BFP) over time in an in vitro simulated liver environment.

[0027] Figure 6 The results show the cell viability of isolated macrophages after 48 hours of stimulation with different concentrations of black fungus β-glucan (BFP).

[0028] Figure 7 This describes the polarization of RAW264.7 macrophages from M0 to M1 induced by black fungus β-glucan (BFP);

[0029] Figure 8 These are H&E staining images of liver and kidney sections of tumor-bearing mice after administration of triptolide (Cel), black fungus β-glucan (BFP), and the liver-targeting drug (BFP-Cel) prepared in Example 1.

[0030] Figure 9 The concentrations of ALT and AST in the serum of tumor-bearing mice after administration of triptolide (Cel), black fungus β-glucan (BFP), and the liver-targeting drug (BFP-Cel) prepared in Example 1;

[0031] Figure 10 The concentrations of CRE and BUN in the serum of tumor-bearing mice after administration of triptolide (Cel), black fungus β-glucan (BFP), and the liver-targeting drug (BFP-Cel) prepared in Example 1;

[0032] Figure 11 These are immunohistochemical staining images of tumor-bearing mice after administration of triptolide (Cel), black fungus β-glucan (BFP), and the liver-targeting drug (BFP-Cel) prepared in Example 1. Detailed Implementation

[0033] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0034] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0035] This invention provides the application of β-glucan in the preparation of liver-targeted drugs for the treatment of liver cancer, wherein the β-glucan is derived from black fungus, and its chemical structure has a main chain with two β-(1,3)-glucose residues as side chains for every three β-(1,6)-glucose residues.

[0036] Specifically, the β-glucan has the structure shown in (I).

[0037]

[0038] In one specific embodiment, the molecular weight of the β-glucan is 1.98 × 10⁻⁶. 6 ~2.40×10 6 g / mol, preferably 2.16 × 10 g / mol. 6 ~2.29×10 6 g / mol.

[0039] Structure determines the properties and functions of a substance, and the structure and chain conformation of natural polysaccharides inevitably affect their biological activity. Polysaccharides with the same chemical structure but different origins exhibit varying biological activities, which is closely related to differences in their chain conformations. The β-glucan described in this invention is derived from black fungus, and its chemical structure has a main chain with two β-(1,3)-glucose residues as side chains for every three β-(1,6)-glucose residues. Research has revealed that this black fungus β-glucan possesses high drug loading capacity and good water solubility, while also exhibiting liver-targeting capabilities, drug release in the presence of liver drug-metabolizing enzymes, and the ability to regulate the immune microenvironment (activate macrophages). Therefore, this invention proposes a novel application of β-glucan in the preparation of liver-targeted drugs for the treatment of liver cancer.

[0040] In one specific implementation, black fungus β-glucan is used as a drug carrier for liver-targeted drugs, stably delivering the drug to the liver and releasing it under the action of liver drug-metabolizing enzymes, thereby achieving targeted drug release in the liver, effectively treating liver cancer (hepatocellular carcinoma and intrahepatic cholangiocarcinoma, etc.) with few toxic side effects. At the same time, the drug carrier can activate macrophages, which have a synergistic effect with the drug they carry, thus better treating intrahepatic cholangiocarcinoma.

[0041] In another embodiment, the black fungus β-glucan can also act as a drug itself, targeting the liver and activating macrophages to achieve a highly effective anti-intrahepatic bile duct carcinoma effect.

[0042] In a preferred embodiment, β-glucan is specifically used as a carrier in the preparation of liver-targeted drugs for treating liver cancer, thereby enabling the prepared drug to effectively treat intrahepatic cholangiocarcinoma.

[0043] The present invention does not limit the specific method for extracting β-glucan from black fungus, as long as the structure and molecular weight described above are met. In a specific embodiment, the β-glucan is extracted from black fungus by water extraction and alcohol precipitation. Further, the β-glucan is prepared according to the following steps: the fruiting body of black fungus is crushed, refluxed with ethyl acetate and acetone for 4 hours respectively, and then dried in an oven at 60°C. Subsequently, the black fungus fragments are soaked in a 70% ethanol aqueous solution, stirred for 24 hours, filtered and the filtrate is discarded. The swollen black fungus residue is immersed in a 0.9% NaCl aqueous solution, stirred at 85°C for 4 hours, and then stirred overnight at room temperature. The supernatant is collected, and then decolorized, deproteinized, dialyzed, and freeze-dried to obtain crude polysaccharide. The crude polysaccharide is redissolved in water, reprecipitated with ethanol, the precipitate is collected, redissolved in water, and freeze-dried to obtain black fungus polysaccharide. The obtained black fungus polysaccharide was chemically characterized. Size exclusion chromatography, methylation analysis, infrared spectroscopy, and nuclear magnetic resonance spectroscopy confirmed that the black fungus polysaccharide was a homogeneous β-glucan. Its chemical structure showed that every three β-(1,3)-glucose residues in the main chain had two β-(1,6)-glucose residues as side chains. The molecular weight of the β-glucan was further determined to be 1.98 × 10⁻⁶. 6 ~2.40×10 6 g / mol.

[0044] In a preferred embodiment, the liver-targeting drug includes a carrier and an anti-liver cancer drug loaded on the carrier, wherein the carrier contains the β-glucan.

[0045] Experiments have shown that dissolving the β-glucan from *Auricularia auricula-judae* in water at room temperature, and controlling the concentration of β-glucan in the solution to be between 0.1 and 1.5 mg / mL, can form β-glucan nanotubes. Utilizing the property that β-glucan from *Auricularia auricula-judae* can self-assemble into nanotubes in water, it can be used as a drug carrier. Through denaturation / renaturation, drugs can be encapsulated, thereby producing liver-targeted drugs. Therefore, in one specific embodiment, the liver-targeted drug comprises β-glucan nanotubes and an anti-liver cancer drug, wherein the anti-liver cancer drug is encapsulated in the hydrophobic cavity of the β-glucan nanotubes; wherein the β-glucan nanotubes are self-assembled from β-glucan.

[0046] In this invention, black fungus β-glucan has good biocompatibility and can stably load hydrophobic small molecule drugs for treating liver cancer (such as triptolide, docetaxel, paclitaxel, etc.). At the same time, black fungus β-glucan itself has the functions of liver targeting, enzymatic release, and regulation of the immune microenvironment. Therefore, using black fungus β-glucan as a carrier can ensure that the loaded drugs can exist stably under various physiological conditions, achieve targeted drug release to the liver, improve drug stability, and reduce dosage and toxic side effects.

[0047] This invention does not limit the specific type of the anti-liver cancer drug, as long as it can exert an anti-liver cancer effect. In a preferred embodiment, the anti-liver cancer drug is selected from at least one of triptolide, docetaxel, and paclitaxel. While all of these drugs can treat liver cancer, they suffer from problems such as low water solubility, high toxicity, poor targeting, and low bioavailability. This invention improves the safety, stability, and anti-liver cancer activity of these drugs by loading them onto β-glucan.

[0048] Celastrol (Cel) is a quinone methyl pentacyclic triterpenoid compound with significant antitumor activity, inhibiting more than 65% of tumor types, such as liver cancer, cervical cancer, gastric cancer, human breast cancer, and colorectal cancer. Of particular note, celastrol is an effective candidate for treating liver cancer. Therefore, in a more preferred embodiment, the anti-liver cancer drug is celastrol.

[0049] In a preferred embodiment, the liver-targeting drug comprises β-glucan nanotubes and triptolide, wherein the triptolide is encapsulated in the hydrophobic cavity of the β-glucan nanotubes.

[0050] In this invention, the liver-targeting drug has a drug loading of 38-42%, which is extremely high and results in good therapeutic effects.

[0051] In this invention, the particle size of the liver-targeting drug is 400-600 nm, preferably 450-500 nm.

[0052] In a preferred embodiment, the preparation process of the liver-targeting drug includes the following steps:

[0053] (1) Mix β-glucan with dimethyl sulfoxide to obtain a mixture A with a β-glucan concentration of 0.5-1.5 mg / mL;

[0054] (2) Mix the anti-liver cancer drug with dimethyl sulfoxide to obtain mixture B;

[0055] (3) Mix the mixture A and the mixture B, then dialyze with water for 2 to 3 days, and then freeze dry.

[0056] In this invention, the liver-targeting drug constructed by the self-assembly of black fungus β-glucan does not contain any other excipients with potential safety hazards. At the same time, it can improve drug absorption and reduce drug toxicity and side effects. Therefore, this invention solves the potential toxicity of traditional nanocarriers and provides a new approach for nanotechnology in drug delivery. In addition, the preparation process of this liver-targeting drug is simple and suitable for industrial production.

[0057] The present invention does not limit the specific parameters of mixing in step (1), as long as β-glucan can be fully dissolved in dimethyl sulfoxide.

[0058] The present invention does not limit the specific parameters of mixing in step (2), as long as the anti-liver cancer drug can be fully dissolved in dimethyl sulfoxide.

[0059] In a preferred embodiment, in step (2), the concentration of the anti-liver cancer drug in the mixture B is 0.5 to 1.5 mg / mL.

[0060] In a preferred embodiment, in step (3), the mass ratio of the anti-hepatocellular carcinoma drug in mixture B to the β-glucan in mixture A is 1:0.5-2.

[0061] The present invention does not limit the specific parameters of mixing in step (3), as long as the mixture A and the mixture B are fully mixed. In a specific embodiment, the mixing conditions in step (3) include: temperature of 25 to 37°C, time of 8 to 12 hours, and rotation speed of 300 to 500 r / min.

[0062] In this invention, the liver-targeted drug, using black fungus β-glucan as a carrier and loading anti-liver cancer drugs onto it, can achieve targeted release of anti-liver cancer drugs into the liver, thereby effectively treating intrahepatic cholangiocarcinoma. Simultaneously, black fungus β-glucan can activate macrophages and further regulate the immune microenvironment, thus synergistically enhancing the effects of anti-liver cancer drugs and further improving the treatment of intrahepatic cholangiocarcinoma. Therefore, in a preferred embodiment, the liver cancer specifically refers to intrahepatic cholangiocarcinoma.

[0063] The present invention also proposes a liver-targeted drug for treating liver cancer, the liver-targeted drug comprising a carrier and an anti-liver cancer drug loaded on the carrier;

[0064] The carrier is a β-glucan nanotube. β-glucan is derived from black fungus. Its chemical structure is that every three β-(1,3)-glucose main chains have two β-(1,6)-glucose residues as side chains.

[0065] In a preferred embodiment, the anti-liver cancer drug is selected from at least one of triptolide, docetaxel, and paclitaxel.

[0066] In a preferred embodiment, the preparation process of the liver-targeting drug includes the following steps:

[0067] β-glucan was mixed with dimethyl sulfoxide to obtain mixture A with a β-glucan concentration of 0.5–1.5 mg / mL; an anti-hepatocellular carcinoma drug was mixed with dimethyl sulfoxide to obtain mixture B; mixture A and mixture B were mixed, then dialyzed with water for 2–3 days, and then lyophilized.

[0068] In a preferred embodiment, the concentration of the anti-liver cancer drug in the mixture B is 0.5–1.5 mg / mL.

[0069] In a preferred embodiment, the mass ratio of the anti-hepatocellular carcinoma drug in mixture B to the β-glucan in mixture A is 1:0.5-2.

[0070] In a preferred embodiment, the liver-targeting drug is specifically used to treat intrahepatic cholangiocarcinoma.

[0071] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available.

[0072] The sources of some of the raw materials used in the following embodiments and test examples are as follows:

[0073] Dimethyl sulfoxide (DMSO) and anhydrous ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0074] The intrahepatic cholangiocarcinoma HUCCT1 cells were derived from the Shanghai Institute of Biochemistry and Cell Biology.

[0075] Thiazole blue dye (MTT) was purchased from Shanghai Maclean Biotechnology Co., Ltd.

[0076] Tripterygium wilfordii (Cel) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0077] The experimental mice were provided by Spiford (Beijing) Biotechnology Co., Ltd., and were 5-week-old healthy female FVB mice.

[0078] The chemical structure of black fungus β-glucan (BFP) has a main chain with two β-(1,3)-glucose residues as side chains for every three β-(1,6)-glucose residues, and a molecular weight of 2.16 × 10⁻⁶. 6 g / mol.

[0079] In the following examples, room temperature refers to 25±5℃.

[0080] Example 1

[0081] This embodiment is used to illustrate the liver-targeting drug and its preparation method according to the present invention.

[0082] (1) Weigh 5mg of black fungus β-glucan and add it to 5ml of dimethyl sulfoxide. Stir at 400r / min for 6h to completely dissolve it and obtain mixture A;

[0083] (2) Weigh 5mg of triptolide powder, add it to 5ml of dimethyl sulfoxide, and sonicate for 10min to completely dissolve it to obtain mixture B;

[0084] (3) Mixing solution A and mixing solution B at room temperature and stirring at 400 r / min for 12 h to obtain intermediate solution. Dialyze the intermediate solution in ultrapure water (MW3000) for 3 days and freeze dry to obtain tripterygium nanoparticle preparation, i.e. liver-targeted drug, denoted as BFP-Cel.

[0085] Example 2

[0086] This embodiment is used to illustrate the liver-targeting drug and its preparation method according to the present invention.

[0087] (1) Weigh 10mg of black fungus β-glucan and add it to 5ml of dimethyl sulfoxide. Stir at 400r / min for 6h to completely dissolve it and obtain mixture A;

[0088] (2) Weigh 5mg of triptolide powder, add it to 5ml of dimethyl sulfoxide, and sonicate for 10min to completely dissolve it to obtain mixture B;

[0089] (3) Mixing solution A and mixing solution B at room temperature and stirring at 400 r / min for 12 h to obtain intermediate solution. Dialyze the intermediate solution in ultrapure water (MW3000) for 3 days and freeze dry to obtain tripterygium oleoresin-loaded nano-formulation, i.e. liver-targeted drug.

[0090] Example 3

[0091] This embodiment is used to illustrate the liver-targeting drug and its preparation method according to the present invention.

[0092] (1) Weigh 2.5 mg of black fungus β-glucan and add it to 5 ml of dimethyl sulfoxide. Stir at 400 r / min for 6 h to completely dissolve it and obtain mixture A;

[0093] (2) Weigh 5mg of triptolide powder, add it to 5ml of dimethyl sulfoxide, and sonicate for 10min to completely dissolve it to obtain mixture B;

[0094] (3) Mixing solution A and mixing solution B at room temperature and stirring at 400 r / min for 12 h to obtain intermediate solution. Dialyze the intermediate solution in ultrapure water (MW3000) for 3 days and freeze dry to obtain tripterygium oleoresin-loaded nano-formulation, i.e. liver-targeted drug.

[0095] Test Example 1

[0096] 1. Take 1 mg of black fungus β-glucan (BFP), triptolide (Cel), and the liver-targeting drug (BFP-Cel) prepared in Example 1. Under an accelerating voltage of 20.0 kV, observe the microstructure of Cel, BFP, and BFP-Cel using scanning electron microscopy. The specific method is as follows: First, place the sample in a high-vacuum sputtering instrument to deposit a carbon film, then place it in an ion sputtering instrument to deposit a conductive film (platinum). Scan the sample at different magnifications and observe its morphology in solution using scanning electron microscopy. The results are as follows. Figure 1 As shown.

[0097] Depend on Figure 1 It can be seen that, in the dry state, free triptolide has a sheet-like structure, pure black fungus β-glucan has a tubular hollow nanofiber morphology, and the liver-targeting drug BFP-Cel has a smooth surface and a dense structure, still maintaining the integrity of the polysaccharide structure. Triptorelide has been successfully encapsulated in the hydrophobic cavity of the polysaccharide.

[0098] Test Example 2

[0099] 1. The liver-targeting drugs prepared in Examples 1-3 were tested for drug loading and encapsulation efficiency.

[0100] Accurately weigh the Cel reference solution and dilute it with DMSO to prepare a series of standard solutions with concentrations of 3.75 μg / ml, 6.25 μg / ml, 12.5 μg / ml, 25 μg / ml, 30 μg / ml, and 50 μg / ml. Measure the absorbance at 504.5 nm using a UV spectrophotometer and plot the standard curve.

[0101] The liver-targeting drugs prepared in Examples 1-3 were dissolved in DMSO, and the absorbance was measured using a UV-Vis spectrophotometer. The content of Cel in the liver-targeting drugs was determined by substituting the absorbance into a standard curve. The drug loading (DLC) and encapsulation efficiency (DLE) were calculated using the following formula:

[0102] Encapsulation efficiency (%) = [WCel content in the nano-formulation / Wtotal mass of Cel added] × 100%

[0103] Drug loading rate (%) = [Cel content in W nano-formulation / W total drug mass] × 100%

[0104] Drug loading capacity (DLC) of Cel is defined as the percentage of loaded Cel relative to the total weight of drug-loaded nanoparticles. Encapsulation efficiency (DLE) of Cel is defined as the percentage of loaded Cel relative to the initial drug dosage.

[0105] Test results: The test results of the standard curve of triptolide are shown in Table 1 below.

[0106] Table 1

[0107] Concentration (μg / mL) absorbance 3.75 0.108 6.25 0.217 12.5 0.352 25 0.693 50 1.469 100 2.985

[0108] As shown in Table 1, with absorbance (y) as the ordinate and Cel concentration x (μg / mL) as the abscissa, the standard curve is y = 0.0298x - 0.0107 (r = 0.9994, n = 3). Calculations show that the liver-targeting drugs prepared in Examples 1-3 have a drug loading of 38-42% and an encapsulation efficiency of 85-89%.

[0109] 2. Dissolve the liver-targeting drugs prepared in Examples 1-3 in water to form a liver-targeting drug solution. Take 1 mL of the liver-targeting drug solution and dilute it 5 times with water. Use a Malven particle size analyzer to determine the particle size, potential and polydispersity index (PDI) of the solution. The results are shown in Table 1.

[0110]

[0111]

[0112] When the mass ratio of BFP to Cel is 2:1, 1:1 and 1:2, the liver-targeting drug solution is in a negatively charged state. When the mass ratio of BFP to Cel is 1:1, the negative charge of the formulation is enhanced and the particle size is the smallest.

[0113] Test Example 3

[0114] The safety of the liver-targeted drug (BFP-Cel) prepared in Example 1 was evaluated.

[0115] The MTT assay was used to investigate the toxicity of different concentrations of BFP, Cel and BFP-Cel prepared in Example 1 to L02 cells.

[0116] The specific method includes: taking L02 cells (normal human liver cells) in the logarithmic growth phase, and then... 4 Cells were evenly seeded in 96-well plates at a density of 1 cell / well. A blank control group, a control group, and a drug-treated group were set up. The drug-treated group consisted of blank carrier BFP, BFP-Cel, and free drug Cel, prepared at different concentrations using complete culture medium (free drug Cel concentration ranged from 0 to 4 μg / mL). -1The concentration of Cel in BFP-Cel was the same as that of free drug Cel; the concentration of BFP was the same as that of BFP in BFP-Cel. After adding the cells and incubating for 24 hours, 20 μL of culture medium and MTT solution were added to each well. After incubation for 4 hours, the absorbance of the cell solution at 570 nm was measured using a microplate reader to determine the cell viability.

[0117] The formula for calculating the cell proliferation inhibition rate (cell viability) is as follows:

[0118]

[0119] Test results are as follows Figure 2 As shown.

[0120] Depend on Figure 2 It can be seen that after incubation with normal hepatocytes L02 cells for 24 hours, BFP has no toxic effect on L02 cells, and the cell survival rate is still greater than 80% within 24 hours. Moreover, the toxicity of BFP-Cel to normal hepatocytes is lower than that of free Cel, indicating that the coating of Cel with BFP can effectively reduce the toxicity of Cel to normal hepatocytes, proving that BFP-Cel has good in vivo safety.

[0121] Test Example 4

[0122] The liver-targeting properties of black fungus β-glucan (BFP) and liver-targeting drugs were evaluated.

[0123] Fluorescent labeling of BFP was performed. 100 mg of BFP was weighed and dissolved thoroughly in 20 mL of DMSO. 14 mg of FITC, 40 μL of pyridine, and 8 μL of dibutyltin dilaurate were added. The mixture was reacted in a high-pressure, light-protected reaction tube at 100 °C for 4 h. The product was then precipitated with four volumes of ethanol and centrifuged at 3000 rpm / min for 15 min at room temperature. The precipitate was washed three times with ethanol, dialyzed against ultrapure water for three days, and then freeze-dried to obtain a yellow flocculent substance, named FITC-BFP.

[0124] Nile red is a substance that emits red fluorescence and, like Cel, is hydrophobic. Since Cel does not have a fluorescence absorption peak, Nile red dye is used instead of Cel in formulation preparation. An appropriate amount of BFP is weighed and completely dissolved in DMSO solution to a concentration of 1 mg / ml; then, a certain amount of Nile red is added dropwise and completely dissolved in DMSO to a concentration of 1 mg / ml. The solution is then transferred to a dialysis bag (MW 8000-14000) for dialyzing, and freeze-dried to obtain BFP-Red-Nile.

[0125] The prepared FITC-BFP and BFP-Red-Nile were intravenously injected into mice at a concentration of 2 mg / kg. After 4 hours, isolated mouse organs were harvested. In vivo imaging was used to investigate the liver-targeting behavior of the BFP and BFP-Cel formulations in normal mice. The results are as follows: Figure 3 As shown (where, Figure 3 A represents the test results of FITC-BFP. Figure 3 B represents the test result of BFP-Red-Nile.

[0126] Depend on Figure 3 It can be seen that after 4 hours, the fluorescence signal of FITC-BFP was mainly distributed in the liver, indicating that BFP can be used as a liver-targeting carrier to deliver drugs. The BFP-Nile-red prepared by using Nile red dye instead of Cel showed that the fluorescence signal was mainly distributed in the liver and spleen, proving that BFP loaded with Cel can be successfully transported to the liver and accumulated in the liver, thus achieving the purpose of liver targeting.

[0127] Test Example 5

[0128] The liver-targeting drug prepared in Example 1 was evaluated for its enzymatic release function.

[0129] Preparation of Cel solution: Accurately weigh the Cel reference standard, dissolve it in 1 mL of anhydrous ethanol, then add 500 μL of polyoxyethylene castor oil, and then add pure water to prepare a free Cel solution with a Cel concentration of 200 μg / mL.

[0130] Preparation of BFP-Cel solution: Dissolve the liver-targeting drug obtained in Example 1 in water to prepare a BFP-Cel solution with a concentration of 1 mg / ml.

[0131] 1. Release of liver-targeted drugs and triptolide in in vitro simulated liver and normal blood environments.

[0132] Simulate normal blood environment: Accurately transfer 2 mL of free Cel solution and 2 mL of BFP-Cel solution into dialysis bags (MWCO = 8000-14000), tie both ends tightly, and then place them into a release medium consisting of 20 mL of PBS (pH = 7.4).

[0133] Simulate the liver's enzymatic (epoxide hydrolase) environment: Accurately transfer 2 mL of BFP-Cel solution into a release medium consisting of 20 mL of PBS (pH = 7.4) containing 0.5 U / mL epoxide hydrolase.

[0134] The drug-added release medium was shaken at 75 rpm and the drug release behavior was observed at 37°C. At 2 h, 4 h, 8 h, 10 h, 24 h, 48 h, and 72 h, 2 mL of release medium was aspirated, and the same volume and temperature of release medium were simultaneously added to maintain a constant dialysis environment. The drug content in the release medium was determined (using the supernatant obtained after centrifugation following enzyme inactivation at 95°C), the cumulative release percentage was calculated, and a release curve was plotted. The test results are shown below. Figure 4 As shown.

[0135] Depend on Figure 4 It can be seen that under normal physiological conditions (pH=7.4), free Cel is almost completely released within 10 hours, exhibiting a rapid release characteristic; while BFP-Cel exhibits a sustained-release characteristic, indicating that after Cel is loaded with BFP, it can be safely transported in the blood and maintain a slow release for a long time. After the addition of enzymes to simulate the liver environment, the release rate of the drug in BFP-Cel is significantly accelerated, confirming that BFP-Cel can achieve enzyme-controlled release in the liver.

[0136] 2. Changes in the molecular weight of β-glucan from black fungus over time in an in vitro simulated liver environment.

[0137] Approximately 2 ml of BFP solution (1 mg / ml) was mixed with 2 ml of epoxide hydrolase solution (0.5 U / ml). The mixture was degraded at 37°C for 30 min, 4 h, and 24 h, and samples were taken after each incubation. After standing, the samples were inactivated by boiling water for 10 min, and the supernatant was collected and freeze-dried. The control group was incubated with PBS buffer instead of epoxide hydrolase, following the same in vitro incubation method. The molecular weight of the freeze-dried polysaccharide samples was analyzed using a size exclusion chromatograph (LC-20, SHIMADZU, Japan) combined with a multi-angle laser spectroscopy spectrometer (DAWNHELEOS II λ0 = 633 nm, Wyatt Technology Co. Ltd., USA) and a differential detector. Chromatographic columns (SB-806HQ and SB-804HQ columns, 7.8 mm × 300 mm, Shodex, Japan) and guard columns (OHpak SB-G, Shodex, Japan) were used. The above polysaccharide sample was dissolved in 0.9% NaCl solution at a concentration of 1 mg / mL, and optically cleaned by filtering with a 0.45 μm filter before injection. The column temperature was 25℃, the flow rate was 0.5 mL / min, and the injection volume was 0.5 mL. The change in molecular weight of BFP before and after degradation was determined.

[0138] The results show ( Figure 5 After being treated in an in vitro environment simulating liver drug-metabolizing enzymes, the molecular weight of BFP tends to decrease over time.

[0139] Furthermore, test results showed that the molecular weight increased from 1.418 × 10⁻⁶. 6 Reduced to 1.121×10 6 .

[0140] The above results indicate that β-glucan BFP from black fungus may be gradually broken down by hepatic drug-metabolizing enzymes present in the liver.

[0141] Test Example 6

[0142] The effects of black fungus β-glucan (BFP) on in vitro immune cell proliferation and polarization were evaluated.

[0143] (1) The MTT assay was used to investigate the proliferative effect of different concentrations of BFP on RAW264.7 cells. RAW264.7 cells in the logarithmic growth phase were used, and BFP was added at a concentration of 1×10⁻⁶. 4 Cells were evenly seeded in 96-well plates at a density of 1 cell / well; after treatment with 0–200 g / mL BFP for 48 h, cell viability was determined by MTT assay. The results are shown below. Figure 6 As shown.

[0144] (2) Take RAW264.7 cells in the logarithmic growth phase and adjust the cell density to 1.0 × 10⁻⁶. 6 Add 2 mL of BFP solution to each well of a 6-well plate, and after the cells have adhered, add 200 μg / mL of BFP solution. Perform three replicates per group. Collect cells after 24 hours of culture. Stain cell surface and intracellular markers according to antibody staining procedures, and analyze by flow cytometry. Test results are shown below. Figure 7 As shown.

[0145] The antibody staining steps are as follows:

[0146] Take 100 μL of cells / tube (approximately 1 × 10⁶ cells) into a flow cytometry tube; add 5 μL of CD86 antibody and mix well, incubate at 4°C in the dark for 30 min to stain the cell surface marker; after incubation, add 2 mL of PBS (containing 1% BSA) to resuspend the cells, centrifuge at 1000 rpm for 5 min, and discard the supernatant; add 500 μL of PBS to resuspend the cells and detect them by flow cytometry.

[0147] Depend on Figure 6 It can be seen that BFP can significantly promote the proliferation of macrophages RAW.264.7.

[0148] Depend on Figure 7 It can be seen that the expression of CD86, a surface molecule of M1 polarization in RAW264.7 macrophages, increased significantly after BFP induction, indicating that BFP can intervene in the immune cell microenvironment and induce macrophages to polarize from M0 to M1.

[0149] Test Example 7

[0150] The liver-targeting drug prepared in Example 1 was evaluated for its toxicity reduction effect using pharmacodynamics.

[0151] (1) Two mL of saline solution containing AKT, Yap, and SB plasmids was rapidly injected into the tail vein of wild-type FVB mice. The mass ratio of AKT, Yap, and SB in the saline solution was 12.5:12.5:1. Subsequently, the transfected mice were randomly divided into four groups (n=5): the model group (AKT-Yap), the free cel group, the BFP-Cel group, and the BFP group; they were fed a standard diet for 3 weeks. Untransfected mice were assigned to the WT group (n=4) and fed the same diet. Starting from week 4 post-transfection, transfected mice were administered the drugs via tail vein injection. Cel, BFP-Cel, and BFP were dissolved in 0.9% NaCl aqueous solution before injection. Specifically, the model group received 0.2 mL of 0.9% NaCl aqueous solution; the free Cel group received 2 mg / kg Cel; the BFP-Cel group had the same Cel content in the lyophilized and reconstituted aqueous solution as the free Cel group, with a dose of 2 mg / kg; and the BFP group had the content of black fungus polysaccharides in the aqueous solution calculated based on the drug loading, with a dose of 3.4 mg / kg. Injections were administered every other day, and the weight of all mice was recorded the day before each injection. This continued for 4 weeks.

[0152] (2) Sample collection and tissue preparation: At the end of the treatment period, all animals were fasted overnight and weighed. Liver, lung, heart, spleen, and kidney tissues were collected and weighed; blood samples were collected and centrifuged at 3000 rpm for 8 minutes at 4°C to separate serum for biochemical analysis. The liver was washed and weighed.

[0153] (3) The collected liver and kidney tissues were fixed with tissue fixative and stored at 4°C. After 24 hours, the tissues were transferred to 75% alcohol, dehydrated using an automated tissue dehydrator, embedded in paraffin wax using a paraffin embedding machine, and sectioned using a microtome. The sections were developed in a 37°C water bath, retrieved, drained, and subjected to routine HE staining. The tissues were observed under a microscope, and the test results were as follows: Figure 8 As shown.

[0154] (4) The concentrations of ALT and AST in the collected serum were determined using a commercially available ALT / AST microplate assay kit, following the kit instructions. Further evaluation of the effects of Cel, BFP-Cel, and BFP on liver tissue during the 4-week treatment period was conducted, and the test results are as follows: Figure 9 As shown.

[0155] (5) The concentrations of creatinine (CRE) and blood urea nitrogen (BUN) in the collected serum were determined using a commercially available BUN / CRE microplate assay kit to further evaluate the effects of Cel, BFP-Cel, and BFP on renal tissue during the 4-week treatment period. The test results are as follows: Figure 10 As shown.

[0156] Depend on Figure 8 It can be seen that, after administration, BFP-Cel significantly reduces liver and kidney toxicity compared to free Cel.

[0157] Depend on Figure 9 It can be seen that, compared with the free cel group, the serum ALT and AST levels in the BFP-Cel group were significantly decreased, indicating that BFP-Cel can alleviate cel-induced liver damage and reduce its hepatotoxicity.

[0158] Depend on Figure 10 It can be seen that, compared with the free cel group, the serum CRE and BUN levels in the BFP-Cel group were significantly reduced, indicating that BFP-Cel can effectively alleviate cel-induced kidney damage.

[0159] Test Example 8

[0160] The pharmacodynamics of the liver-targeting drug prepared in Example 1 were evaluated to assess its synergistic effect.

[0161] The mouse liver tissue collected in Test 7 was embedded in paraffin, and the samples were sectioned at a thickness of 4 μm. Three paraffin-embedded tissue sections were then subjected to immunohistochemical staining.

[0162] Test result processing:

[0163] (1) Prepare the primary antibody with PBS according to the antibody dilution ratio, and add it evenly to the liver tissue. Incubate overnight at 4°C. The secondary antibody is a working solution of rabbit / mouse secondary antibody labeled with horseradish peroxidase (HRP). Incubate at room temperature for 10 min. Use DAB kit for color development. Brownish-yellow cells are positive cells. All sections are counterstained with hematoxylin.

[0164] (2) Images were acquired under the same conditions and from different fields of view using the Cell Sens image acquisition system. Changes in the expression levels of proliferation markers (PCNA, KI67, CK-19) in the liver tissue of mice in different formulation groups after drug administration were observed. The test results are as follows: Figure 11 As shown.

[0165] Depend on Figure 11 It can be seen that the inhibition of intrahepatic cholangiocarcinoma by Cel after being loaded with BFP is significantly enhanced, indicating that Cel and BFP can play a synergistic role.

[0166] It is understandable that the preparation principle of Examples 2-3 is similar to that of Example 1. Therefore, the liver-targeting drugs prepared in Examples 2-3 can also deliver triptolide to the liver and release the drug in the liver drug-enzyme environment, with less toxic side effects.

[0167] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. Use of β-glucan in the preparation of a carrier for a liver-targeted drug for the treatment of liver cancer, wherein, The β-glucan is derived from Auricularia auricula, and has a chemical structural formula of two β-(1, 6)-glucose residues of side chains on every three β-(1, 3)-glucose main chains of a main chain, and a molecular weight of 1.98×10 6 g / mol. 6 2.40×10 6 g / mol. The liver-targeted drug comprises a carrier and an anti-liver cancer drug loaded on the carrier, and the anti-liver cancer drug is tripterine. The liver-targeted drug prepared by using the β-glucan as the carrier can realize site-specific targeting drug release of the liver.

2. Use according to claim 1, characterized in that, The drug loading amount of the liver-targeted drug is 38-42%.

3. Use according to claim 2, characterized in that, The particle size of the liver-targeted drug is 400-600 nm.

4. Use according to any one of claims 1 to 3, characterized in that, The preparation process of the liver-targeted drug comprises the following steps: (1) mixing β-glucan and dimethyl sulfoxide to obtain a mixed solution A with a β-glucan concentration of 0.5-1.5 mg / mL; (2) mixing an anti-liver cancer drug and dimethyl sulfoxide to obtain a mixed solution B; (3) mixing the mixed solution A and the mixed solution B, then performing water dialysis for 2-3 days, and then freeze-drying.

5. Use according to claim 4, characterized in that, In step (2), the concentration of the anti-liver cancer drug in the mixed solution B is 0.5-1.5 mg / mL.

6. Use according to claim 5, characterized in that, In step (3), the mass ratio of the amount of the anti-liver cancer drug in the mixed solution B to the amount of the β-glucan in the mixed solution A is 1:0.5-2.

7. The use according to claim 1, characterized in that, The liver cancer is intrahepatic cholangiocarcinoma.

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