Preparation method of adriamycin-loaded ginseng selenium polysaccharide nano-carrier with immunoregulation and liver cancer cell inhibition functions

Abstract

The invention discloses a preparation method of a doxorubicin-loaded ginseng selenium polysaccharide nano-carrier with immunoregulation and liver cancer cell inhibition functions, and aims to solve the problems of toxic injury of tissues and organs and inhibition of an immune system caused by an antitumor drug doxorubicin. According to the invention, deoxycholic acid is grafted on the ginseng selenium polysaccharide, so that the ginseng selenium polysaccharide is hydrophobized and forms an amphiphilic polysaccharide polymer, and the limitations of large molecular weight, low bioavailability and the like of the ginseng selenium polysaccharide are overcome. The self-assembled ginseng selenium polysaccharide nanoparticles are used as carriers for delivering antitumor drugs, so that the self-assembled ginseng selenium polysaccharide nanoparticles have an immunoregulation effect and can effectively improve side effects such as immunosuppression caused by the antitumor drugs; and the carrier also has a pH sensitive response release characteristic, so that the targeting effect of the medicine in a tumor area is enhanced, and the innovative carrier has a good clinical application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of immune preparations, and in particular relates to a method for preparing a ginseng selenium polysaccharide nanocarrier loaded with doxorubicin that has the functions of immunomodulation and inhibition of liver cancer cells. Background Art

[0002] Doxorubicin (DOX), as an anthracycline chemotherapy drug, is a first-line anti-tumor drug currently in clinical use and has been approved by the US Food and Drug Administration for the treatment of gastric cancer, breast cancer, thyroid cancer, and ovarian cancer. However, the side effects of long-term use of DOX limit the scope and dosage of its disease treatment. Long-term use of DOX may cause damage to tissues, organs, and the immune system, especially to the heart, liver, and kidneys. In order to reduce the side effects of DOX and expand its therapeutic range and dosage, DOX is loaded into a new type of ginseng selenium polysaccharide nanocarrier with immunomodulatory effects to achieve a pH-responsive release effect in the tumor area, which can significantly reduce its toxic side effects and improve the safety and effectiveness of DOX in clinical treatment.

[0003] Ginseng polysaccharides have the effect of promoting the proliferation and differentiation of human immune cells. In clinical applications, they are mainly used to enhance immune function, fight cancer, reduce the side effects of chemotherapy, and curb tumor spread and regeneration. By grafting inorganic selenium with ginseng polysaccharides to synthesize ginseng selenium polysaccharides, the antioxidant, anti-tumor, and immune-enhancing biological activities of ginseng polysaccharides and selenium can be combined at the same time, making them synergistic and having higher pharmacological activity.

[0004] Polysaccharide self-assembled nanoparticles are an efficient and low-toxic drug delivery system that can aggregate in water to form a stable structure through non-covalent bonds between substances. The rich functional groups of polysaccharides make them easy to modify, and their good biocompatibility and biodegradability also make them an ideal raw material for preparing nanoparticles.

[0005] Although the direct combination of ginseng selenium polysaccharide and DOX can reduce side effects such as immunosuppression, due to the lack of specific recognition ability of DOX for tumor cells, it shows a wide systemic distribution in the body, resulting in low accumulation efficiency of the drug in tumor tissues. At the same time, it is widely distributed in normal tissues such as the heart, bone marrow, and gastrointestinal tract, causing severe dose-limiting toxicities such as cardiotoxicity and myelosuppression. This non-selective distribution not only reduces the anti-tumor efficacy of the drug but also exacerbates the systemic toxic reaction, limiting its clinical application dose and treatment course. Meanwhile, the poor targeting of DOX may also lead to insufficient drug concentration in the tumor microenvironment, potentially inducing drug resistance in tumor cells and further weakening the long-term treatment effect. Cardiotoxicity is the most significant dose-limiting toxicity of DOX, mainly caused by free radical generation, iron ion-mediated oxidative stress, and mitochondrial dysfunction, resulting in irreversible myocardial damage and heart failure, severely limiting the cumulative dose and long-term use of the drug. In addition, DOX acts widely on rapidly proliferating cells by intercalating into DNA and inhibiting topoisomerase II, causing myelosuppression, manifested as a decrease in white blood cells, red blood cells, and platelets, further increasing the risks of infection, anemia, and bleeding. DOX exerts toxic effects on rapidly proliferating immune cells (such as T cells, B cells, macrophages, and dendritic cells) by intercalating into DNA and inhibiting topoisomerase II, resulting in a decrease in their numbers and impaired functions, thus weakening the body's adaptive immune response and antigen presentation ability, leading to immunosuppression of the immune system. DOX also further exacerbates the immunosuppressive state in the tumor microenvironment by inducing tumor cells to release immunosuppressive factors (such as TGF-β and IL-10), promoting the expansion of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), and inhibiting the anti-tumor activities of effector T cells and natural killer cells (NK cells). This immunosuppressive effect not only limits the therapeutic effect of DOX but also may increase the risk of tumor recurrence and metastasis. Therefore, the immunosuppressive effect of DOX highlights its duality in anti-tumor treatment: on the one hand, it directly kills tumor cells, and on the other hand, it weakens the body's immune defense ability. And the above problems all need to be overcome by combining immunotherapy or developing a new delivery system. Summary of the Invention

[0006] The present invention aims to solve the problems of toxic damage to tissues and organs caused by the anti-tumor drug doxorubicin and immunosuppression, and provides a preparation method of a ginseng selenium polysaccharide nanocarrier loaded with doxorubicin with immunomodulatory and hepatoma cell inhibitory effects.

[0007] The preparation method of the ginseng selenium polysaccharide nanocarrier loaded with doxorubicin with immunomodulatory and hepatoma cell inhibitory effects of the present invention is carried out according to the following steps:

[0008] I. Extraction, separation, and purification of ginseng polysaccharide:

[0009] Using ginseng as raw material, crude ginseng polysaccharide was extracted by water extraction and alcohol precipitation method. Then, protein in the crude ginseng polysaccharide was removed by Sevage method to obtain refined ginseng polysaccharide. Further purification of the refined ginseng polysaccharide was carried out by column chromatography to obtain ginseng polysaccharide;

[0010] Second, use HNO 3 -Na 2 SeO 3 functional group method to synthesize ginseng selenized polysaccharide:

[0011] First, mix ginseng polysaccharide and HNO 3 solution, then add sodium selenite, and add BaCl 2 as a catalyst, stir well; react in a constant temperature oscillator; after the reaction is completed, cool the mixture to room temperature, and adjust the pH value to 5 - 6 with sodium bicarbonate solution; then add an appropriate amount of Na 2 SO 4 to remove BaCl 2 , centrifuge to collect the supernatant; load the supernatant into a dialysis bag, dialyze with distilled water, then concentrate, collect and freeze-dry to obtain ginseng selenized polysaccharide;

[0012] The volume percentage of the HNO 3 solution is 0.3% - 0.7%;

[0013] The ratio of the mass of the ginseng polysaccharide to the volume of the HNO 3 solution is 0.8 - 1.2 mg: 100 mL;

[0014] The ratio of the mass of the sodium selenite to the volume of the HNO 3 solution is 520 - 530 mg: 50 mL;

[0015] The ratio of the mass of the BaCl 2 to the volume of the HNO 3 solution is 0.3 - 0.7 g: 50 mL;

[0016] The temperature for the reaction in the constant temperature oscillator is 68 - 73 °C, and the time is 6 - 7 h;

[0017] Third, preparation of ginseng selenized polysaccharide - deoxycholic acid amphiphilic polymer:

[0018] Weigh deoxycholic acid, 4 - dimethylaminopyridine and 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide, dissolve them in dimethyl sulfoxide, and react for 1 hour at room temperature using a magnetic stirrer to obtain an activation reaction solution;

[0019] Weigh the ginseng selenized polysaccharide and dissolve it in 1 mL of dimethyl sulfoxide to obtain a ginseng selenized polysaccharide solution. Then, gradually add the activation reaction solution dropwise and carry out the grafting reaction at room temperature for 48 hours. After the reaction, add anhydrous ethanol, let it stand for layering, and then centrifuge to obtain a precipitate. Wash the precipitate, redissolve it in deionized water, and then perform dialysis treatment with distilled water. Collect the solution and perform freeze-drying to obtain the ginseng selenized polysaccharide-deoxycholic acid amphiphilic polymer;

[0020] The molar ratio of the deoxycholic acid, 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1:1.2; the mass fraction of deoxycholic acid in the activation reaction solution is 30-70%;

[0021] The volume ratio of the activation reaction solution to the ginseng selenized polysaccharide solution is 0.8-1.2:1;

[0022] The content of ginseng selenized polysaccharide in the ginseng selenized polysaccharide solution is 0.008-0.012 g / L;

[0023] IV. Preparation of ginseng selenium polysaccharide nanoparticles by dialysis method:

[0024] Weigh the ginseng selenized polysaccharide-deoxycholic acid amphiphilic polymer and dissolve it in dimethyl sulfoxide to obtain a mixed solution with a concentration of 1 mg / mL. Then, use distilled water dialysis to remove dimethyl sulfoxide, followed by ultrasonic treatment for 10 min. Finally, dilute the liquid with double-distilled water to 5 times the volume of the initial mixed solution to obtain the ginseng selenium polysaccharide nanoparticles;

[0025] V. Preparation of doxorubicin-loaded ginseng selenium polysaccharide nanoparticles:

[0026] Dissolve doxorubicin and the ginseng selenium polysaccharide nanoparticles in 1 mL of dimethyl sulfoxide to obtain a mixed solution. Then, use distilled water dialysis to remove dimethyl sulfoxide, followed by ultrasonic treatment for 10 min. Finally, dilute the liquid with double-distilled water to 5 times the volume of the initial mixed solution to obtain the doxorubicin-loaded ginseng selenium polysaccharide nanoparticles;

[0027] The mass ratio of the ginseng selenium polysaccharide nanoparticles to doxorubicin is 18-22:1;

[0028] The ratio of the mass of the ginseng selenium polysaccharide nanoparticles to the volume of dimethyl sulfoxide is 8-12 mg:1 mL.

[0029] The beneficial effects of the present invention are:

[0030] 1. The doxorubicin-loaded ginseng selenium polysaccharide nanoparticles (DOX / SGD NPs) of the present invention can stimulate the phagocytosis of macrophages and the secretion of NO, improve the immunosuppressive effect of DOX, and thus enhance the immune activity of macrophages in the tumor microenvironment. The synergistic effect of the drug carrier significantly improves the inhibitory effect of DOX on the proliferation of liver cancer cells, demonstrating its potential in anti-liver cancer treatment. By grafting deoxycholic acid onto ginseng selenium polysaccharide, the hydrophilic ginseng selenium polysaccharide is hydrophobized to form an amphiphilic polysaccharide polymer, overcoming the limitations of large molecular weight and low bioavailability of ginseng selenium polysaccharide. Using self-assembled ginseng selenium polysaccharide nanoparticles as a carrier for anti-tumor drug delivery, it not only has an immunomodulatory effect and can effectively improve the side effects such as immunosuppression caused by anti-tumor drugs, but also has pH-sensitive responsive release characteristics, thereby enhancing the targeting effect of the drug in the tumor area. This innovative carrier has good clinical application prospects.

[0031] 2. The present invention simplifies the extraction and separation process of ginseng homogeneous polysaccharide GPS-Ⅰa using a DEAE-52 cellulose anion exchange column and a Sephadex G-100 gel permeation chromatography column, and has a high total sugar content. The constructed ginseng selenium polysaccharide-deoxycholic acid amphiphilic polymer micelle (Se-GPS-DOCA) based on ginseng selenium polysaccharide exhibits a low critical micelle concentration (CMC), enhances the stability of the micelle, can effectively protect the drug and accurately deliver it to the liver cancer tissue. The ginseng selenium polysaccharide-deoxycholic acid amphiphilic polymer micelle can accelerate the release of DOX in the acidic environment of the tumor, increase the drug concentration, while maintaining the structural stability in the blood circulation and reducing the potential toxicity. DOX / SGD NPs did not cause obvious hemolysis at high concentrations, indicating its safety in intravenous administration.

[0032] 3. The present invention uses ginseng selenium polysaccharide nanoparticles as an active ingredient for in vitro immunomodulation and inhibition of liver cancer cells, and applies it to the preparation of a drug delivery carrier for the anti-tumor drug DOX; loading DOX into the nano-carrier to achieve co-delivery of the two can effectively improve the problems of immunosuppression and poor targeting. Brief Description of the Drawings

[0033] Figure 1 The infrared spectrum of Se-GPS obtained in Example 1;

[0034] Figure 2 The XRD spectrum of Se-GPS obtained in Example 1;

[0035] Figure 3 The infrared spectrum of Se-GPS-DOCA obtained in Example 1;

[0036] Figure 4 The XRD spectrum of Se-GPS-DOCA obtained in Example 1;

[0037] Figure 5 The critical micelle concentration result graph of SGD NPS obtained in Example 1;

[0038] Figure 6 The appearance graph of DOX / SGD NPS obtained in Example 1;

[0039] Figure 7 The potential result graph of DOX / SGD NPS obtained in Example 1;

[0040] Figure 8 The stability result graph of DOX / SGD NPS obtained in Example 1;

[0041] Figure 9 The XRD spectrum of DOX / SGD NPS obtained in Example 1;

[0042] Figure 10 The TEM graph (300 nm) of DOX / SGD NPS obtained in Example 1;

[0043] Figure 11 The SEM graph (1 μm) of DOX / SGD NPS obtained in Example 1;

[0044] Figure 12 The in vitro release result graph of DOX / SGD NPS obtained in Example 1;

[0045] Figure 13 The hemolytic evaluation graph of DOX / SGD NPS obtained in Example 1;

[0046] Figure 14 The ability graph of DOX / SGD NPS obtained in Example 1 to phagocytize RAW264.7 cells;

[0047] Figure 15 The influence graph of DOX / SGD NPS obtained in Example 1 on the secretion of NO by RAW264.7;

[0048] Figure 16 The influence graph of DOX / SGD NPS obtained in Example 1 on the survival rate of hepatoma cells HepG2; Detailed implementation manners

[0049] The technical solution of the present invention is not limited to the following listed specific implementation manners, and also includes any reasonable combination among the specific implementation manners.

[0050] Specific implementation manner 1: The preparation method of the doxorubicin-loaded ginseng selenium polysaccharide nanocarrier with immunomodulatory and hepatoma cell inhibitory effects in this implementation manner is carried out according to the following steps:

[0051] I. Extraction, separation and purification of ginseng polysaccharide:

[0052] Using ginseng as raw material, crude ginseng polysaccharide was obtained by water extraction and alcohol precipitation method. Then, protein in the crude ginseng polysaccharide was removed by Sevage method to obtain refined ginseng polysaccharide. Further purification of the refined ginseng polysaccharide was carried out by column chromatography to obtain ginseng polysaccharide;

[0053] II. Synthesis of ginseng selenized polysaccharide by HNO 3 -Na 2 SeO 3 functional group method:

[0054] Firstly, ginseng polysaccharide was mixed with HNO 3 solution. Subsequently, sodium selenite was added, and BaCl 2 was added as a catalyst and stirred evenly; the reaction was carried out in a constant temperature oscillator; after the reaction was completed, the mixture was cooled to room temperature, and the pH value was adjusted to 5 - 6 with sodium bicarbonate solution; subsequently, an appropriate amount of Na 2 SO 4 was added to remove BaCl 2 , and the supernatant was collected by centrifugation; the supernatant was loaded into a dialysis bag, dialyzed with distilled water, concentrated, collected and freeze-dried to obtain ginseng selenized polysaccharide;

[0055] The volume percentage of the HNO 3 solution is 0.3% - 0.7%;

[0056] The ratio of the mass of the ginseng polysaccharide to the volume of the HNO 3 solution is 0.8 - 1.2 mg: 100 mL;

[0057] The ratio of the mass of the sodium selenite to the volume of the HNO 3 solution is 520 - 530 mg: 50 mL;

[0058] The ratio of the mass of the BaCl 2 to the volume of the HNO 3 solution is 0.3 - 0.7 g: 50 mL;

[0059] The temperature for the reaction in the constant temperature oscillator is 68 - 73 °C, and the time is 6 - 7 h;

[0060] III. Preparation of ginseng selenized polysaccharide - deoxycholic acid amphiphilic polymer:

[0061] Weigh deoxycholic acid, 4 - dimethylaminopyridine and 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide, dissolve them in dimethyl sulfoxide, and react for 1 hour at room temperature using a magnetic stirrer to obtain an activated reaction solution;

[0062] Weigh the ginseng selenium polysaccharide and dissolve it in 1 mL of dimethyl sulfoxide to obtain a ginseng selenium polysaccharide solution. Then, dropwise add the activation reaction solution and carry out the grafting reaction at room temperature for 48 hours. After the reaction is completed, add anhydrous ethanol, let it stand for layering, and then centrifuge to obtain a precipitate. Wash the precipitate, redissolve it in deionized water, and then perform dialysis treatment with distilled water. Collect the solution and perform freeze-drying to obtain the ginseng selenium polysaccharide-deoxycholic acid amphiphilic polymer;

[0063] The molar ratio of the deoxycholic acid, 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1:1.2; the mass fraction of deoxycholic acid in the activation reaction solution is 30-70%;

[0064] The volume ratio of the activation reaction solution to the ginseng selenium polysaccharide solution is 0.8-1.2:1;

[0065] The content of ginseng selenium polysaccharide in the ginseng selenium polysaccharide solution is 0.008-0.012 g / L;

[0066] IV. Prepare the ginseng selenium polysaccharide nanocarrier by dialysis method:

[0067] Weigh the ginseng selenium polysaccharide-deoxycholic acid amphiphilic polymer and dissolve it in dimethyl sulfoxide to obtain a mixed solution with a concentration of 1 mg / mL. Then, use distilled water dialysis to remove dimethyl sulfoxide, followed by ultrasonic treatment for 10 min. Finally, dilute the liquid with double-distilled water to 5 times the volume of the initial mixed solution to obtain the ginseng selenium polysaccharide nanocarrier;

[0068] V. Prepare the doxorubicin-loaded ginseng selenium polysaccharide nanoparticles:

[0069] Dissolve doxorubicin and the ginseng selenium polysaccharide nanocarrier in 1 mL of dimethyl sulfoxide to obtain a mixed solution. Then, use distilled water dialysis to remove dimethyl sulfoxide, followed by ultrasonic treatment for 10 min. Finally, dilute the liquid with double-distilled water to 5 times the volume of the initial mixed solution to obtain the doxorubicin-loaded ginseng selenium polysaccharide nanoparticles;

[0070] The mass ratio of the ginseng selenium polysaccharide nanocarrier to doxorubicin is 18-22:1;

[0071] The ratio of the mass of the ginseng selenium polysaccharide nanocarrier to the volume of dimethyl sulfoxide is 8-12 mg:1 mL.

[0072] This embodiment has the following beneficial effects:

[0073] 1. The DOX / SGD NPs of this embodiment can stimulate the phagocytosis of macrophages and the secretion of NO, improve the immunosuppressive effect of DOX, and further enhance the immune activity of macrophages in the tumor microenvironment. The synergistic effect of the drug carrier significantly improves the inhibitory effect of DOX on the proliferation of liver cancer cells, demonstrating its potential in anti-liver cancer treatment. In this embodiment, deoxycholic acid is grafted onto ginseng selenium polysaccharide to hydrophobize the hydrophilic ginseng selenium polysaccharide and form an amphiphilic polysaccharide polymer, overcoming the limitations of large molecular weight and low bioavailability of ginseng selenium polysaccharide. Using self-assembled ginseng selenium polysaccharide nanoparticles as a carrier for anti-tumor drug delivery not only has an immunomodulatory effect and can effectively improve the side effects such as immunosuppression caused by anti-tumor drugs, but also has pH-sensitive responsive release characteristics, thereby enhancing the targeting effect of the drug in the tumor area. This innovative carrier has good clinical application prospects.

[0074] 2. This embodiment simplifies the extraction and separation process of ginseng homogeneous polysaccharide GPS-Ⅰa using a DEAE-52 cellulose anion exchange column and a Sephadex G-100 gel permeation chromatography column, and has a high total sugar content. The constructed ginseng selenium polysaccharide-deoxycholic acid amphiphilic polymer micelle (Se-GPS-DOCA) based on ginseng selenium polysaccharide exhibits a low critical micelle concentration (CMC), enhances the stability of the micelle, can effectively protect the drug and precisely deliver it to the liver cancer tissue. The ginseng selenium polysaccharide-deoxycholic acid amphiphilic polymer micelle can accelerate the release of DOX in the acidic environment of the tumor, increase the drug concentration, while maintaining the structural stability in the blood circulation and reducing potential toxicity. DOX / SGD NPs did not cause obvious hemolysis at high concentrations, indicating its safety in intravenous administration.

[0075] 3. This embodiment uses ginseng selenium polysaccharide nanoparticles as an active ingredient for in vitro immunomodulation and inhibition of liver cancer cells, and applies it to the preparation of an anti-tumor drug DOX drug delivery carrier; loading doxorubicin into the nano-carrier to achieve co-delivery of the two can effectively improve the problems of immunosuppression and poor targeting.

[0076] Specific Embodiment 2: The difference between this embodiment and Specific Embodiment 1 is that in the process of extracting ginseng crude polysaccharide by the water extraction and alcohol precipitation method in Step 1, the ratio of the mass of ginseng to the volume of distilled water is 0.8 - 1.2 g: 20 mL.

[0077] Specific Embodiment 3: The difference between this embodiment and Specific Embodiment 1 or 2 is that in the process of removing proteins from ginseng crude polysaccharide using the Sevage method in Step 1: the ratio of the mass of ginseng crude polysaccharide to the volume of distilled water used is 0.8 - 1.2 g: 3 mL, and the mass ratio of ginseng crude polysaccharide to Sevage reagent is 0.8 - 1.2: 4, and the protein removal is repeated 1 - 6 times.

[0078] Embodiment 4: The difference between this embodiment and any one of Embodiments 1 to 3 is that during the further purification of ginseng polysaccharide by column chromatography in Step 1, DEAE-52 cellulose column chromatography and Sephadex G-100 dextran gel column chromatography are successively used.

[0079] Embodiment 5: The difference between this embodiment and any one of Embodiments 1 to 4 is that during the purification by DEAE-52 cellulose column chromatography, a mixed solution is prepared according to the ratio of the mass of refined ginseng polysaccharide to the volume of distilled water of 0.8 - 1.2 g: 10 mL. The mixed solution is filtered through a 0.45 μm filter membrane to obtain a refined ginseng polysaccharide solution. The refined ginseng polysaccharide solution is added into the DEAE-52 cellulose column, with a flow rate of 48 mL / h. Elution is carried out using a 0.2 mol / L NaCl solution. The eluate is collected using centrifuge tubes, 4 mL is collected in each tube. The absorbance value of the eluate in different centrifuge tubes is detected by the phenol-sulfuric acid method. The eluate of the main absorption peak is collected and freeze-dried to obtain a crude polysaccharide product.

[0080] Embodiment 6: The difference between this embodiment and any one of Embodiments 1 to 5 is that during the purification by Sephadex G-100 gel permeation chromatography column, a mixed solution is prepared according to the ratio of the mass of the crude polysaccharide product to the volume of ultrapure water of 0.8 - 1.2 g: 10 mL. The mixed solution is filtered through a 0.45 μm filter membrane to obtain a crude polysaccharide product solution. The crude polysaccharide product solution is added into the Sephadex G-100 gel permeation chromatography column, and elution is carried out using pure water, with a flow rate of 20 mL / h. The eluate is collected using centrifuge tubes, 2 mL is collected in each tube. The sugar content of each component is determined by the phenol-sulfuric acid method. The part where the sugar content reaches the peak in the elution curve is collected, that is, the eluate corresponding to the single symmetric peak.

[0081] Embodiment 7: The difference between this embodiment and any one of Embodiments 1 to 6 is that the mass fraction of the sodium bicarbonate solution in Step 2 is 5.4 - 5.8%.

[0082] Embodiment 8: The difference between this embodiment and any one of Embodiments 1 to 7 is that when washing the precipitate in Step 3, absolute ethanol and ether are respectively used for cleaning, and the cleaning is repeated three times.

[0083] Embodiment 9: The difference between this embodiment and any one of Embodiments 1 to 8 is that in Step 2, the volume percentage of the HNO 3 solution is 0.3% - 0.7%;

[0084] In Step 2, the ratio of the mass of GPS-Ia to the volume of the HNO 3 solution is 0.8 - 1.2 mg: 100 mL;

[0085] In step two, the mass ratio of sodium selenite to the volume of HNO 3 solution is 520 - 530 mg: 50 mL;

[0086] In step two, the mass ratio of BaCl 2 to the volume of HNO 3 solution is 0.3 - 0.7 g: 50 mL;

[0087] In step two, the reaction temperature in the constant temperature oscillator is 68 - 73 °C, and the reaction time is 6 - 7 h.

[0088] Specific embodiment ten: The difference between this embodiment and one of embodiments one to nine is that: when washing the precipitate in step three, anhydrous ethanol and ether are used for cleaning respectively, and the cleaning is repeated three times;

[0089] In step three, the molar ratio of deoxycholic acid, 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1:1.2; the mass fraction of deoxycholic acid in the activation reaction solution is 30 - 70%;

[0090] In step three, the volume ratio of the activation reaction solution to the Se-GPS solution is 0.8 - 1.2:1;

[0091] In step three, the content of Se-GPS in the Se-GPS solution is 0.008 - 0.012 g / L.

[0092] Example 1

[0093] The preparation method of the immunomodulatory and doxorubicin-loaded hepatoma cell-inhibiting ginseng selenium polysaccharide nanocarrier of the present invention is carried out according to the following steps:

[0094] I. Extraction, separation, and purification of ginseng polysaccharide:

[0095] Using ginseng as the raw material, crude ginseng polysaccharide is extracted by the water extraction and alcohol precipitation method, then the protein in the crude ginseng polysaccharide is removed by the Sevage method to obtain refined ginseng polysaccharide, and then column chromatography is selected to further purify the refined ginseng polysaccharide to obtain ginseng polysaccharide (GPS-Ia); the yield of the single-component ginseng polysaccharide GPS-Ia is 3.22 ± 0.05%, and the polysaccharide content of GPS-Ia is 95.75% ± 1.54% after testing and analysis.

[0096] During the extraction of crude ginseng polysaccharide by the water extraction and alcohol precipitation method, the mass ratio of ginseng to the volume of distilled water is 0.8 - 1.2 g: 20 mL;

[0097] During the process of removing proteins from crude ginseng polysaccharides using the Sevage method: the mass ratio of crude ginseng polysaccharides to the volume of distilled water used is 0.8 - 1.2 g : 3 mL, and the mass ratio of crude ginseng polysaccharides to Sevage reagent is 0.8 - 1.2 : 4. The protein removal process is repeated 1 - 6 times.

[0098] During the further purification of ginseng polysaccharides by column chromatography, DEAE - 52 cellulose column chromatography and Sephadex G - 100 dextran gel column chromatography are used successively.

[0099] During the purification by DEAE - 52 cellulose column chromatography, a mixture is prepared according to the mass ratio of refined ginseng polysaccharides to the volume of distilled water of 0.8 - 1.2 g : 10 mL. The mixture is filtered through a 0.45 μm filter membrane to obtain a refined ginseng polysaccharide solution. The refined ginseng polysaccharide solution is added into the DEAE - 52 cellulose column at a flow rate of 48 mL / h, and eluted with a 0.2 mol / L NaCl solution. The eluate is collected in centrifuge tubes, 4 mL per tube. The absorbance values of the eluate in different centrifuge tubes are detected by the phenol - sulfuric acid method. The eluate of the main absorption peak is collected and freeze - dried to obtain a crude polysaccharide product.

[0100] During the purification by Sephadex G - 100 gel permeation chromatography column, a mixture is prepared according to the mass ratio of the crude polysaccharide product to the volume of ultrapure water of 0.8 - 1.2 g : 10 mL. The mixture is filtered through a 0.45 μm filter membrane to obtain a crude polysaccharide product solution. The crude polysaccharide product solution is added into the Sephadex G - 100 gel permeation chromatography column and eluted with pure water at a flow rate of 20 mL / h. The eluate is collected in centrifuge tubes, 2 mL per tube. The sugar content of each component is determined by the phenol - sulfuric acid method. The part with the sugar content reaching the peak in the elution curve is collected, that is, the eluate corresponding to the single symmetric peak.

[0101] II. Synthesis of ginseng - selenized polysaccharides by the HNO 3 -Na 2 SeO 3 functional group method:

[0102] First, mix GPS - Ⅰa and HNO 3 solution, then add sodium selenite, and add BaCl 2 as a catalyst, and stir well. React in a constant - temperature oscillator. After the reaction is completed, cool the mixture to room temperature and adjust the pH value to 5 - 6 with sodium bicarbonate solution. Then add an appropriate amount of Na 2 SO 4 to remove BaCl 2, centrifugally collect the supernatant; load the supernatant into a dialysis bag, dialyze with distilled water, then perform concentration collection and freeze-drying to obtain selenium-enriched ginseng polysaccharide (Se-GPS); through test analysis, the selenium content in Se-GPS is 4.21 ± 0.25 mg / g.

[0103] The volume percentage of the said HNO 3 solution is 0.3 - 0.7%; the mass ratio of GPS-Ia to the volume of the HNO 3 solution is 0.8 - 1.2 mg: 100 mL; the mass ratio of sodium selenite to the volume of the HNO 3 solution is 520 - 530 mg: 50 mL; the mass ratio of BaCl 2 to the volume of the HNO 3 solution is 0.3 - 0.7 g: 50 mL;

[0104] The temperature for the reaction in the said constant temperature oscillator is 68 - 73 °C, and the time is 6 - 7 h;

[0105] III. Preparation of selenium-enriched ginseng polysaccharide-deoxycholic acid amphiphilic polymer:

[0106] Weigh deoxycholic acid, 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide, dissolve them in dimethyl sulfoxide, and react for 1 hour at room temperature using a magnetic stirrer to obtain an activation reaction solution;

[0107] Weigh Se-GPS and dissolve it in 1 mL of dimethyl sulfoxide to obtain a Se-GPS solution, then dropwise add the activation reaction solution and carry out a grafting reaction at room temperature for 48 hours; after the reaction, add absolute ethanol, let it stand for stratification and then centrifuge to obtain a precipitate, wash the precipitate, redissolve it in deionized water after washing, then perform dialysis treatment with distilled water, collect the solution and carry out freeze-drying to obtain selenium-enriched ginseng polysaccharide-deoxycholic acid amphiphilic polymer (Se-GPS-DOCA);

[0108] When washing the precipitate, it is washed with absolute ethanol and ether respectively, and the washing is repeated three times;

[0109] The molar ratio of deoxycholic acid, 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide is 1:1:1.2; the mass fraction of deoxycholic acid in the activation reaction solution is 30 - 70%;

[0110] The volume ratio of the activation reaction solution to the Se-GPS solution is 0.8 - 1.2:1;

[0111] The content of Se-GPS in the said Se-GPS solution is 0.008 - 0.012 g / L;

[0112] IV. Preparation of ginseng selenium polysaccharide nanocarriers by dialysis method:

[0113] Weigh Se-GPS-DOCA and dissolve it in dimethyl sulfoxide to obtain a mixed solution with a concentration of 1 mg / mL. Then, use distilled water for dialysis to remove dimethyl sulfoxide, followed by sonication for 10 min. Finally, dilute the liquid with double-distilled water to 5 times the volume of the initial mixed solution to obtain ginseng selenium polysaccharide nanocarriers (SGD NPs); through test analysis, the critical micelle concentration CMC value of SGD NPS is 0.14 mg / mL; the particle size is 78.07 ± 1.59 nm, and the PDI is 15.8 ± 1.0%.

[0114] V. Preparation of doxorubicin-loaded ginseng selenium polysaccharide nanoparticles:

[0115] Dissolve 5 mg of doxorubicin (DOX) and SGD NPs in 1 mL of dimethyl sulfoxide to obtain a mixed solution. Then, use distilled water for dialysis to remove dimethyl sulfoxide, followed by sonication for 10 min. Finally, dilute the liquid with double-distilled water to 5 times the volume of the initial mixed solution to obtain DOX / SGD NPs;

[0116] The mass ratio of the said SGD NPs to DOX is 18 - 22:1;

[0117] The ratio of the mass of the said SGD NPs to the volume of dimethyl sulfoxide is 8 - 12 mg:1 mL;

[0118] The DOX / SGD NPS obtained in Step V of Example 1, through test analysis, the particle size of DOX / SGD NPs is (112.49 ± 1.89) nm, the PDI is (25.1 ± 0.7)%; the Zeta potential is -18.31 mV.

[0119] Figure 1 It is the infrared spectrogram of Se-GPS obtained in Example 1; among them, the absorption peak at 3421.2 cm -1 is the O-H stretching vibration peak of ginseng polysaccharide, and the absorption peak at 2916.4 cm -1 is the C-H stretching vibration peak of the methylene group of ginseng polysaccharide. The absorption peak at 1652.7 cm -1 is the O-H bending vibration absorption peak of ginseng polysaccharide, and at 1413.3 cm -1 , 1373.4 cm -1 , 1241.4 cm -1 , 1154.7 cm -1 , 1078.8 cm -1 , 1015.1 cm -1The absorption peaks at etc. are the C-N or C=O stretching vibration peaks in ginseng polysaccharide. After adding sodium selenite, the product retains the characteristic peaks of the above-mentioned ginseng polysaccharide, indicating that the main structure of ginseng polysaccharide is retained in the product. At the same time, new absorption peaks appear at 1047.3 cm -1 and 670.6 cm -1 . The absorption peak at 1047.3 cm -1 is the O-Se-O stretching vibration peak, and the absorption peak at 670.6 cm -1 is the Se-O-C stretching vibration peak. In summary, it is not difficult to judge that selenium has been successfully modified on ginseng polysaccharide, and the possible form is selenate ester.

[0120] Figure 2 is the XRD pattern of Se-GPS obtained in Example 1. Both ginseng polysaccharide and selenium-enriched ginseng polysaccharide have C characteristic peaks near 20°. Among them, compared with ginseng polysaccharide, a new characteristic peak appears near 42.5° in the selenium-enriched polysaccharide, and this peak coincides with the characteristic peak of selenium element. Combining with the infrared spectrum, it further proves that ginseng polysaccharide has been successfully selenium-enriched.

[0121] Figure 3 is the infrared spectrum of Se-GPS-DOCA obtained in Example 1. The infrared characteristic absorption peaks of Se-GPS appear in the spectrum. Among them, 3400.5 cm -1 is the O-H stretching vibration peak on Se-GPS, and the peak at 1151.4 cm -1 is the characteristic absorption peak of the β-glycosidic bond of Se-GPS. The vibration absorption peak at 1239.1 cm -1 is the stretching vibration absorption peak of C-O-C on Se-GPS, and the peak at 672.9 cm -1 is the Se-O-C absorption peak of Se-GPS. In addition to the above characteristic absorption peaks of Se-GPS, a C=O stretching vibration peak of ester carbonyl appears at 1744.8 cm -1 , and a strong symmetric stretching vibration peak of methylene C-H appears at 2858.1 cm -1 . This fully shows that the carboxyl group on deoxycholic acid has undergone esterification with the hydroxyl group on the sugar, indicating that deoxycholic acid has been grafted onto the hydroxyl group of Se-GPS, and Se-GPS-DOCA is successfully prepared.

[0122] Figure 4XRD pattern of Se-GPS-DOCA obtained in Example 1; Se-GPS shows a blunt peak near 22°, while DOCA shows multiple sharp absorption peaks in the range of 10° - 25°. Different from Se-GPS and DOCA, Se-GPS-DOCA shows a blunt peak near 30°, and the blunt peak near 45° becomes smaller compared to Se-GPS, indicating a change in its crystal form. Combining with the infrared spectrum, it further demonstrates the successful synthesis of Se-GPS-DOCA.

[0123] Figure 5 Critical micelle concentration result graph of SGD NPS obtained in Example 1; Based on the amphiphilic polymer micelles of ginseng selenized polysaccharide, the amphiphilic copolymer Se-GPS-DOCA is self-assembled from the hydrophilic segment Se-GPS and the hydrophobic segment DOCA in water to form core-shell structured nanoparticles. It has a low CMC value (0.14 mg / mL), and the CMC value of this micelle is much lower than 2.3 mg / mL of the low molecular weight surfactant SDS. This indicates that it has good stability and can ensure a complete nanostructure even at a low concentration, which is beneficial for protecting the loaded drug and preventing the micelle from dissociating during in vivo circulation and accurately delivering it to the liver cancer tissue.

[0124] Figure 6 Appearance graph of DOX / SGD NPS obtained in Example 1; Blank SGDNPs (a) is a clear and transparent liquid, and DOX / SGDNPs are clear light orange-red liquids in both pH 7.4 (b) and pH 5.0 (c) buffer solutions. In contrast, DOX / SGD NPs are slightly turbid in the medium of pH 5.0, which may be due to the breakage of the hydrophilic layer of the micelle, resulting in the precipitation of the drug. When the solution medium is pH 5.0 buffer solution, the particle size of DOX / SGD NPs is (112.49 ± 1.89) nm, and the PDI is (25.1 ± 0.7)%; when the solution medium is pH 7.4 buffer solution, the particle size of DOX / SGD micelles is (103.5 ± 3.9) nm, and the PDI is (21.16 ± 0.8)%. The blank particle size is (78.07 ± 1.59) nm, and the PDI is (15.8 ± 1.0)%. After drug loading, the particle size increases slightly, which is due to the fact that DOX is loaded into the hydrophobic core of SGD NPs through van der Waals force interaction, resulting in an increase in volume.

[0125] Figure 7Potential result graph of DOX / SGD NPS obtained in Example 1; the Zeta potential of DOX / SGD NPs is -18.31 mV. This is because the ginseng polysaccharide purified in this study is an acidic polysaccharide with a negative charge and a relatively large absolute value. It can be preliminarily determined that the micelle has good stability. In addition, since cell membranes usually carry a negative charge, negatively charged DOX / SGD NPs are not easily recognized and phagocytosed by macrophages, thus prolonging the in vivo circulation time.

[0126] Figure 8 Stability result graph of DOX / SGD NPS obtained in Example 1; the stability results show that the drug loading and particle size of DOX / SGDNPs in PBS (pH 7.4) solution (in the dark) do not change significantly within 30 days, indicating that DOX / SGD is relatively stable within 30 days.

[0127] Figure 9 XRD spectrum of DOX / SGD NPS obtained in Example 1; there are a large number of sharp peaks of DOX in the range of 10° - 40°. These peaks can also be observed in the physical mixture powder of free DOX and Se-GPS-DOCA. It shows that the crystal state of DOX in the physical mixture has not changed. However, when DOX is encapsulated into SGD NPs, these sharp crystallization peaks disappear, and its peak shape is similar to that of SGD NPs, which is significantly different from the physical mixture of DOX and Se-GPS-DOCA. This indicates that DOX in DOX / SGD NPs is not in a crystal state or a molecular diffusion state. The transformation of the drug from a crystal to an amorphous drug will contribute to faster dissolution and release of the drug because the crystal lattice energy is lower and the thermodynamic energy is higher. Therefore, it can be concluded from the analysis of the XRD pattern that DOX is successfully loaded into SGD NPs rather than in a simple physical mixture form.

[0128] Figure 10 and Figure 11 TEM image (300 nm) and SEM image (1 μm) of DOX / SGD NPS obtained in Example 1; TEM and SEM results show that the morphology of the nanoparticles is spherical, well-dispersed, without aggregation, and the particle size is about 100 nm.

[0129] Figure 12Figure showing the in vitro release results of DOX / SGD NPS obtained in Example 1; within 36 h, the release amount of DOX reached over 60% under the condition of pH 5.0. Under acidic conditions, the solubility of DOX increased, the ester bonds were broken, and the interaction between the nanoparticles and DOX weakened, resulting in the rapid release of DOX. While under the condition of pH 7.4, the release of DOX was less than 30%. This phenomenon may be attributed to the hydrophobic interaction between DOX and the nanoparticles, reflecting the stability of DOX / SGD NPs under normal physiological pH conditions. Based on the above experimental phenomena, it can be reasonably inferred that the micelles are in a relatively stable state (pH 7.4) in normal human cell tissue fluid. When the micelles are in a weakly acidic environment, the release rate of DOX accelerates. The reason for this phenomenon is that the ester bonds break in the acidic environment, the core structure of the micelles is disordered, and the hydrophobic DOCA block extends towards the hydrophilic outer shell of the micelles, causing the micelles to dissociate and release DOX.

[0130] Figure 13 Figure showing the hemolytic evaluation of DOX / SGD NPS obtained in Example 1; no obvious hemolysis phenomenon occurred in DOX / SGD micelles at various concentrations. When the concentration was as high as 2 mg / mL, the hemolysis rate was still less than 2%, indicating that DOX / SGD has good blood compatibility and the safety of intravenous administration.

[0131] Figure 14 Figure showing the ability of DOX / SGD NPS obtained in Example 1 to phagocytose RAW264.7 cells; in the experiment, the control group was only given 100 μL of culture medium without adding any drugs or treatment substances, which was used to evaluate the basic phagocytic ability of RAW264.7 cells without intervention. Compared with the control group, the phagocytosis of macrophages was inhibited in the DOX (89.28%) and DOX / SGD NPs (94.37%) groups; the phagocytosis of macrophages was enhanced in the Se-GPS (152.83%) and SGD NPs (159.09%) groups. Compared with Se-GPS, the phagocytic ability of SGD NPs on macrophages increased significantly (P < 0.01), mainly because it has a smaller particle size and can more significantly stimulate the activity of macrophages. DOX has an immunosuppressive effect, and after loading it into SGD NPs to form DOX / SGD NPs, the immunosuppressive effect was significantly reduced (P < 0.05).

[0132] Figure 15Effect diagram of DOX / SGD NPS obtained in Example 1 on the secretion of NO by RAW264.7; in the experiment, the blank control group was only given 100 μL of culture medium without adding any drugs or treatment substances, which was used to evaluate the ability of RAW264.7 cells to secrete NO without intervention. Compared with the untreated macrophages as the blank control group, both Se-GPS (13.90 μM) and SGD NPs (14.32 μM) significantly promoted the secretion of NO content, and compared with Se-GPS, SGD NPs had a stronger effect on the secretion of NO by macrophages (P < 0.01), proving that nanonizing macromolecular polysaccharides can significantly enhance the immune effect of Se-GPS. And ginseng selenized polysaccharide has a stronger immune-stimulating activity on macrophages, indicating that selenized polysaccharide has better immunomodulatory activity. Compared with the blank control group (5.84 μM), the NO secretion in the DOX (3.96 μM) group was significantly reduced, indicating its immunosuppressive effect. After loading it into micelles to prepare DOX / SGD NPs (4.41 μM), the NO secretion increased and the immunosuppressive effect was significantly reduced (P < 0.01).

[0133] Figure 16 Effect diagram of DOX / SGD NPS obtained in Example 1 on the survival rate of hepatocellular carcinoma cells HepG2; DOX / SGDNPs inhibited the growth of HepG2 cells more significantly than SGD NPs (40.68%), indicating that after preparing Se-GPS into SGD NPs micelles, the tumor suppression effect was significantly increased (P < 0.001). Compared with free DOX (63.75%) and Se-GPS (29.02%), DOX / SGD NPs (68.38%) showed a higher inhibition rate. It shows that loading DOX into SGD NPs can significantly improve the inhibitory effect of DOX on the proliferation of hepatocellular carcinoma cells HepG2 (P < 0.01), which may be due to the synergistic effect of DOX and ginseng selenized polysaccharide.

Claims

1. A method for preparing a ginseng selenium polysaccharide nanocarrier loaded with doxorubicin that has the functions of immunomodulation and inhibition of liver cancer cells, characterized in that: The preparation method of the ginseng selenium polysaccharide nanocarrier loaded with doxorubicin having the functions of immunomodulation and inhibition of liver cancer cells is carried out according to the following steps:

1. Extraction, separation and purification of ginseng polysaccharides: Ginseng is used as a raw material, and a water extraction and alcohol precipitation method is used to extract ginseng crude polysaccharide, and then the protein in the ginseng crude polysaccharide is removed by a Sevage method to obtain ginseng refined polysaccharide, and then column chromatography is used to further purify the ginseng refined polysaccharide to obtain ginseng polysaccharide; 2. Synthesis of ginseng selenized polysaccharide using HNO3-Na2SeO3 functional group method: First, ginseng polysaccharide and HNO3 solution are mixed, then sodium selenite is added, and BaCl2 is added as a catalyst, and the mixture is stirred evenly; the reaction is carried out in a constant temperature oscillator; after the reaction is completed, the mixture is cooled to room temperature, and the pH value is adjusted to 5-6 with a sodium bicarbonate solution; then an appropriate amount of Na2SO4 is added to remove BaCl2, and the supernatant is collected by centrifugation; the supernatant is placed in a dialysis bag, dialyzed with distilled water, concentrated, collected and freeze-dried to obtain ginseng selenized polysaccharide; The volume percentage of the HNO3 solution is 0.3%-0.7%; The ratio of the mass of the ginseng polysaccharide to the volume of the HNO3 solution is 0.8-1.2 mg: 100 mL; The ratio of the mass of sodium selenite to the volume of HNO3 solution is 520-530 mg:50 mL; The ratio of the mass of BaCl2 to the volume of HNO3 solution is 0.3-0.7 g:50 mL; The temperature of the reaction in the constant temperature oscillator is 68-73°C and the time is 6-7h; 3. Preparation of Ginseng Selenated Polysaccharide-Deoxycholic Acid Amphiphilic Polymer: Weigh deoxycholic acid, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, dissolve in dimethyl sulfoxide, and react at room temperature for 1 hour using a magnetic stirrer to obtain an activation reaction solution; Weigh ginseng selenide polysaccharide and dissolve it in 1 mL of dimethyl sulfoxide to obtain a ginseng selenide polysaccharide solution, then add the activation reaction solution dropwise, and carry out grafting reaction at room temperature for 48 hours; after the reaction is completed, add anhydrous ethanol, stand for stratification, and then centrifuge to obtain a precipitate, wash the precipitate, redissolve it in deionized water, and then dialyze it with distilled water, collect the solution and freeze-dry it to obtain a ginseng selenide polysaccharide-deoxycholic acid amphiphilic polymer; The molar ratio of deoxycholic acid, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1:1.2; the mass fraction of deoxycholic acid in the activation reaction solution is 30-70%; The volume ratio of the activation reaction solution to the ginseng selenized polysaccharide solution is 0.8-1.2:1; The content of ginseng selenide polysaccharide in the ginseng selenide polysaccharide solution is 0.008-0.012 g / L; 4. Preparation of ginseng selenium polysaccharide nanocarriers by dialysis method: Weigh ginseng selenium polysaccharide-deoxycholic acid amphiphilic polymer and dissolve it in dimethyl sulfoxide to obtain a mixed solution with a concentration of 1 mg / mL, then dialyze with distilled water to remove dimethyl sulfoxide, then ultrasonicate for 10 minutes, and finally dilute the liquid with double distilled water to 5 times the volume of the initial mixed solution to obtain ginseng selenium polysaccharide nanocarrier; 5. Preparation of Ginseng Selenium Polysaccharide Nanoparticles Loaded with Doxorubicin: Doxorubicin and ginseng selenium polysaccharide nanocarriers were dissolved in 1 mL of dimethyl sulfoxide to obtain a mixed solution, which was then dialyzed with distilled water to remove dimethyl sulfoxide, followed by ultrasonication for 10 minutes, and finally the liquid was diluted with double distilled water to 5 times the volume of the initial mixed solution to obtain ginseng selenium polysaccharide nanoparticles loaded with doxorubicin; The mass ratio of the ginseng selenium polysaccharide nanocarrier to doxorubicin is 18-22:1; The ratio of the mass of the ginseng selenium polysaccharide nanocarrier to the volume of dimethyl sulfoxide is 8-12 mg:1 mL.

2. The method for preparing the ginseng selenium polysaccharide nanocarrier loaded with doxorubicin having immunomodulatory and liver cancer cell inhibition properties according to claim 1, characterized in that: In the process of extracting crude ginseng polysaccharides by water extraction and alcohol precipitation as described in step 1, the ratio of the mass of ginseng to the volume of distilled water is 0.8-1.2 g:20 mL.

3. The method for preparing the ginseng selenium polysaccharide nanocarrier loaded with doxorubicin having the functions of immunomodulation and inhibition of liver cancer cells according to claim 1, characterized in that: In the process of removing protein from ginseng crude polysaccharide by Sevage method as described in step 1: the ratio of the mass of ginseng crude polysaccharide to the volume of distilled water used is 0.8-1.2 g:3 mL, the mass ratio of ginseng crude polysaccharide to Sevage reagent is 0.8-1.2:4, and the protein removal is repeated 1-6 times.

4. The method for preparing the ginseng selenium polysaccharide nanocarrier loaded with doxorubicin having immunomodulatory and liver cancer cell inhibition properties according to claim 1, characterized in that: In the process of further purifying ginseng polysaccharide by column chromatography as described in step 1, DEAE-52 cellulose column chromatography and Sephadex G-100 polyacrylamide gel column chromatography are used in sequence.

5. The method for preparing the ginseng selenium polysaccharide nanocarrier loaded with doxorubicin having the functions of immunomodulation and inhibition of liver cancer cells according to claim 4, characterized in that: In the process of purification by DEAE-52 cellulose column chromatography, a mixed solution is prepared according to the ratio of the mass of ginseng refined polysaccharide to the volume of distilled water of 0.8-1.2g:10mL, and the mixed solution is filtered through a 0.45μm filter membrane to obtain a ginseng refined polysaccharide solution; the ginseng refined polysaccharide solution is added into the DEAE-52 cellulose column layer at a flow rate of 48mL / h, and eluted with a NaCl solution with a concentration of 0.2mol / L, and the eluate is collected in a centrifuge tube, with 4mL collected in each tube, and the absorbance value of the eluate in different centrifuge tubes is detected by a phenol-sulfuric acid method, and the main absorption peak eluate is collected, and freeze-dried to obtain the polysaccharide primary product.

6. The method for preparing the ginseng selenium polysaccharide nanocarrier loaded with doxorubicin having the functions of immunomodulation and inhibition of liver cancer cells according to claim 4, characterized in that: In the purification process using Sephadex G-100 gel permeation chromatography column, a mixed solution is prepared according to the ratio of the mass of the polysaccharide primary product to the volume of ultrapure water of 0.8-1.2 g: 10 mL, the mixed solution is filtered through a 0.45 μm filter membrane to obtain a polysaccharide primary product solution, the polysaccharide primary product solution is added to the Sephadex G-100 gel permeation chromatography column, eluted with pure water at a flow rate of 20 mL / h, the eluate is collected by a centrifuge tube, 2 mL is collected in each tube, and the sugar content of each component is determined by the phenol-sulfuric acid method; the part where the sugar content reaches the peak value in the elution curve, that is, the eluate corresponding to the single symmetrical peak is collected.

7. The method for preparing the ginseng selenium polysaccharide nanocarrier loaded with doxorubicin having the functions of immunomodulation and inhibition of liver cancer cells according to claim 1, characterized in that: Step 2: The mass fraction of the sodium bicarbonate solution is 5.4-5.8%.

8. The method for preparing the ginseng selenium polysaccharide nanocarrier loaded with doxorubicin having the functions of immunomodulation and inhibition of liver cancer cells according to claim 1, characterized in that: In step 3, the precipitate is washed with anhydrous ethanol and ether respectively, and the washing is repeated three times.

9. The method for preparing the ginseng selenium polysaccharide nanocarrier loaded with doxorubicin having the functions of immunomodulation and inhibition of liver cancer cells according to claim 1, characterized in that: The volume percentage of the HNO3 solution in step 2 is 0.3% to 0.7%; The ratio of the mass of GPS-Ⅰa and the volume of HNO3 solution in step 2 is 0.8-1.2 mg:100 mL; The ratio of the mass of sodium selenite to the volume of HNO3 solution in step 2 is 520-530 mg:50 mL; The ratio of the mass of BaCl2 to the volume of HNO3 solution in step 2 is 0.3-0.7 g:50 mL; The temperature of the reaction in the constant temperature oscillator in step 2 is 68-73° C. and the reaction time is 6-7 hours.

10. The method for preparing the ginseng selenium polysaccharide nanocarrier loaded with doxorubicin having the functions of immunomodulation and inhibition of liver cancer cells according to claim 1, characterized in that: In step 3, the precipitate is washed with anhydrous ethanol and ether respectively, and the washing is repeated three times; The molar ratio of deoxycholic acid, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in step 3 is 1:1:1.2; the mass fraction of deoxycholic acid in the activation reaction solution is 30-70%; The volume ratio of the activation reaction solution and the Se-GPS solution in step 3 is 0.8-1.2:1; The content of Se-GPS in the Se-GPS solution in step 3 is 0.008-0.012 g / L.

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