Bionic nano adjuvant and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a bionic nano adjuvant and application thereof. The preparation method comprises the following steps: taking selenium element modified mesoporous nano silicon dioxide as a carrier, loading fatty acid synthase small interfering RNA (FASN siRNA) and an antiviral drug entecavir (ETV) on the carrier to obtain a carrier loaded with a drug, then coating the carrier loaded with the drug by using a liver cancer cell membrane to obtain a cell membrane-carrier, and preparing the liver cancer cell membrane-carrier. And finally, coupling a phosphatidylinositol proteoglycan 3 (GPC3) antibody with the cell membrane of the cell membrane-carrier to obtain the bionic nano adjuvant. The bionic nano adjuvant provided by the invention has targeting property and relatively good biocompatibility, can deliver anti-tumor drugs into hepatocytes in a targeting manner, can also reduce active oxygen and lipid levels in a tumor microenvironment, and has application value in the fields of treatment of chronic hepatitis B and the like.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a bionic nano adjuvant and application thereof. Background Art

[0002] The main cause of liver cancer in my country is hepatitis B virus (HBV) infection. The current first-line treatments for liver cancer include radiotherapy, chemotherapy, immunotherapy, targeted therapy, etc. Among them, the objective response rate of immunotherapy such as atezolizumab and bevacizumab is only 30%. Therefore, it is of great significance to propose new and effective treatment plans for its special tumor microenvironment to benefit the survival of liver cancer patients.

[0003] In the HBV-related liver cancer tumor microenvironment, immune tolerance and chronic inflammation are important factors that promote the progression of hepatocellular carcinoma and non-response to immunotherapy. On the one hand, HBV promotes immune tolerance of innate immune cells by changing the metabolic form. HBV can upregulate key enzymes for fatty acid synthesis (acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN)), thereby enhancing the de novo synthesis of fatty acids and lipid accumulation, providing support for the construction of new cell membranes and energy metabolism for tumor cells, and negatively regulating the IFN signaling pathway. On the other hand, HBV promotes immune escape by upregulating ROS. The level of glycolysis metabolism in HBV-infected liver cancer cells increases significantly, producing more reactive oxygen species (ROS), resulting in the unresponsiveness of the RIG-I signaling pathway in monocytes such as dendritic cells and macrophages, inhibiting the antigen presentation process, and then leading to dysfunction of immune cells such as T cells, dendritic cells and natural killer (NK) cells, promoting the recruitment and activation of immunosuppressive cells in the tumor microenvironment, such as regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSCs), and reducing the ability of the immune system to kill tumor cells.

[0004] Clinical studies have reported that immunotherapy shows more significant therapeutic effects in patients with HBV-related liver cancer than in other liver cancer patients, suggesting that immunotherapy has great development value in HBV-related liver cancer. Entecavir (ETV) is an antiviral drug used to treat chronic hepatitis B (CHB). It can effectively inhibit HBV mRNA reverse transcription and DNA replication by competitively binding to HBV polymerase with deoxyguanosine triphosphate. Studies have shown that entecavir can upregulate the expression of PD-L1 in tumor cells, and PD1 / PD-L1 immune checkpoint therapy can effectively reduce the risk of immune escape. However, as a nucleoside analog, entecavir has relatively limited water solubility, and 62% to 73% is cleared by the kidneys after administration, which brings a heavy metabolic burden to the kidneys.

[0005] Targeted nanocarriers can deliver drugs directly to the site of action, increase the water solubility and local drug concentration of drugs, and reduce the impact on other tissues. In recent years, nano-drug delivery technology has provided an effective means to improve drug efficacy. However, due to the complexity of tumors, the targeting effects of anti-tumor bionic nanoadjuvants may be significantly different in different tumor types, different locations, and different tumor development stages. The currently developed bionic nanoadjuvants for liver cancer treatment have poor targeting, poor efficacy, and treatment limitations. Summary of the invention

[0006] In view of the technical problems existing in the above-mentioned prior art, the object of the present invention is to provide a biomimetic nanoadjuvant and its application. The biomimetic nanoadjuvant provided by the present invention has high efficiency, high biocompatibility and targeting, which not only realizes the precise targeted delivery of drugs to liver cancer cells, but also relieves the inherent immunosuppression mediated by HBV.

[0007] The first aspect of the present invention provides a bionic nanoadjuvant, which is prepared by the following steps: After the surface of mesoporous nano-silica is coated with selenium, fatty acid synthase small interfering RNA and antiviral drug entecavir are loaded to obtain a drug-loaded carrier; The drug-loaded carrier is coated with the liver cancer cell membrane to obtain a cell membrane-carrier; The liver cancer specific antibody is coupled to the cell membrane of the cell membrane-carrier to obtain the bionic nano adjuvant.

[0008] Furthermore, the mass ratio of the vector to the fatty acid synthase small interfering RNA is 1:0.06-0.15.

[0009] Preferably, the mass ratio of the vector to the fatty acid synthase small interfering RNA is 1:0.07.

[0010] Furthermore, the mass ratio of the carrier to entecavir is 1:0.2-0.25.

[0011] Preferably, the mass ratio of the carrier to entecavir is 1:0.25.

[0012] Furthermore, the selenium element is nano-selenium.

[0013] Furthermore, the liver cancer cell membrane is a HepG2 cell membrane.

[0014] Furthermore, the liver cancer-specific antibody is glypican 3.

[0015] The second aspect of the present invention provides a use of the above-mentioned bionic nano adjuvant in the preparation of a drug for preventing or treating liver cancer.

[0016] Furthermore, the bionic nanoadjuvant is used to prepare a drug for reducing the level of reactive oxygen or lipid in the tumor microenvironment.

[0017] In summary, compared with the prior art, the present invention has the following advantages and effects: (1) The present invention provides a biomimetic nanoadjuvant, which is obtained by using selenium-modified mesoporous nanosilica (M@Se) as a carrier, loading fatty acid synthase small interfering RNA and antiviral drug entecavir, and targeting and modifying the liver cancer cell membrane through coupling antibodies. The M@Se carrier retains the antioxidant property of selenium and obtains better dispersibility and stability. The biomimetic nanoadjuvant provided by the present invention can protect the drug from degradation on the one hand, and can enter the cell by endocytosis on the other hand, antagonize and offset the effect of active drug excretion by the cell, and increase the intracellular drug concentration. At the same time, it is coated with the liver cancer cell membrane and modified with the phosphatidylinositol protein glycan 3 (GPC3) antibody that is specifically highly expressed on the surface of liver cancer, so as to further enhance the targeting and penetration ability of the nanocarrier to the target cells and increase the drug delivery efficiency.

[0018] (2) The application principle of the bionic nanoadjuvant provided by the present invention is as follows: ① When the bionic nanoadjuvant reaches the tumor site, the targeting molecule GPC3 antibody modified on the surface of the bionic nanocarrier enables the bionic nanoadjuvant to specifically bind to the surface of liver cancer cells, reduce the abnormally elevated level of reactive oxygen in the tumor microenvironment and release the drug.

[0019] ②Fatty acid synthase small interfering RNA (FASN siRNA) and entecavir are gradually released from the biomimetic nanoadjuvant and enter the liver cancer cells. FASN siRNA will block lipid synthesis in liver cancer cells, inhibit tumor proliferation, promote the release of type I interferon, and stimulate antigen presenting cells to activate downstream anti-tumor immune response.

[0020] ③ Enable the activated antigen-presenting cells in the body to quickly recognize the viral stimulation signals, secrete inflammatory cytokines and type I interferon, stimulate the production and maturation of T cells, and infiltrate and kill tumor tissues, thereby inhibiting tumor growth.

[0021] (3) The biological value of nano-selenium in tumors is very important. In liver tissue, the specific mechanisms of action of selenium include: enhancing immune system function, improving antioxidant capacity, and inhibiting viral mutation. Selenium is the only nutrient directly related to viral infection. When the virus is lacking in selenium, it is very easy to mutate, thereby causing more serious damage to the body. Compared with traditional selenium intake, nano-selenium has higher safety and bioavailability. However, single nano-selenium is easy to aggregate and is not suitable for transportation or storage. The present invention prepares nano-selenium materials through redox reactions, and improves the stability of nano-selenium by hybridizing selenium with mesoporous nano-silica. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The synthetic flow chart of biomimetic nanoadjuvants.

[0023] Figure 2 The figure shows the experimental results of the loading capacity of entecavir (ETV) on the carrier M@Se; Figure A is the full-wavelength spectrum of entecavir at different concentrations; Figure B is the standard curve of the relationship between entecavir concentration and absorbance; Figure C is the experimental result of the loading capacity of M@Se for ETV.

[0024] Figure 3 Figure 2 is the experimental result of the loading capacity of the carrier M@Se for fatty acid synthase small interfering RNA (FASN siRNA); Figure A is the result of the gel electrophoresis experiment, and the bands from left to right are the electrophoresis graphs under the conditions of M@Se and FASN siRNA mixing mass ratios of 0:1, 1:1, 2:1, 5:1, 10:1 and 15:1; Figure B is the quantitative result of the fluorescence intensity of the electrophoresis bands in Figure A.

[0025] Figure 4 The particle size characterization results of unloaded MSN, carrier M@Se, drug-loaded carrier si / ETV-M@Se and biomimetic nanoadjuvant si / ETV-M@SeMA.

[0026] Figure 5 These are the experimental results of bionic nanoadjuvant stability verification; Figure A and Figure B are the results of bionic nanoadjuvant M@SeMA without drug loading placed at room temperature for 0h and 1h, respectively.

[0027] Figure 6 This is a graph showing the modification efficiency test results of surface antibodies.

[0028] Figure 7 These are the test results of bionic nanoadjuvants using a transmission electron microscope; Figure A is an electron microscope image of the bionic nanoadjuvant, and Figure B is a particle size distribution diagram of the bionic nanoadjuvant.

[0029] Figure 8 Figure 1 shows the results of the cytotoxicity test of bionic nano-adjuvants. Figure A shows the effects of different doses of bionic nano-adjuvants on cell activity, and Figure B shows the effects of the action time of bionic nano-adjuvants on cell activity. ** indicates p <0.01.

[0030] Fig. 9 Figure 1 is the result of the test of the inhibitory ability of bionic nano-adjuvant on lipid synthesis; Figure A, Figure B and Figure C are the fluorescence microscopy images of the control group, lipid over-synthesis group and lipid over-synthesis group treated with bionic nano-adjuvant, respectively; Figure D is the statistical data of the fluorescence signals in Figures A to C; ns indicates no significant difference compared with the control group, *** indicates p<0.001.

[0031] Fig.10 These are the test results of the bionic nano-adjuvant's ability to scavenge ROS; the samples in Figures A, B, C, and D are the control group, the bionic nano-preparation treatment group, the ROS positive control group, and the ROS-added bionic nano-adjuvant treatment group, respectively.

[0032] Fig.11 These are the results of H&E staining of mouse tissues. From top to bottom, each row of tissues are heart, liver, spleen, lung and kidney. From left to right, each column is the control group, free drug group (FASN siRNA+ETV), bionic nanoadjuvant group loaded with only fatty acid synthase small interfering RNA (FASN siRNA), bionic nanoadjuvant group loaded with only entecavir (ETV), and bionic nanoadjuvant group loaded with both FASN siRNA and ETV.

[0033] Fig.12 Figure A is the mouse tumor growth curve, and Figure B is the mouse survival curve; Ctrl represents the untreated control group, si+ETV represents the free drug group (FASN siRNA+ETV), si-M@SeMA represents the bionic nanoadjuvant administration group loaded with only FASN siRNA, ETV-M@SeMA represents the bionic nanoadjuvant administration group loaded with only ETV, and si / ETV-M@SeMA represents the bionic nanoadjuvant administration group loaded with both FASN siRNA and ETV; * represents p <0.05,** indicates p <0.01, *** indicates p <0.001. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0035] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0036] The accumulation of lipids and reactive oxygen species in the tumor microenvironment limits the responsiveness of immune cells, thereby affecting the effectiveness of tumor immunotherapy. Entecavir is an antiviral drug used to treat chronic hepatitis B (CHB), but its water solubility is relatively limited. After administration, 62% to 73% is cleared by the kidneys, which places a heavy metabolic burden on the kidneys. Targeted nanocarriers can deliver drugs directly to the site of action, increase the water solubility and local drug concentration of the drug, and reduce the impact on other tissues. However, the bionic nanoadjuvants currently developed for the treatment of liver cancer have problems such as poor targeting, poor efficacy, and treatment limitations.

[0037] The invention provides a bionic nano adjuvant and application thereof. The present invention firstly uses selenium-modified mesoporous nano-silica (M@Se) as a carrier, loads fatty acid synthase small interfering RNA (FASN siRNA) and the antiviral drug entecavir (ETV), and obtains the drug-loaded carrier si / ETV-M@Se, then coats si / ETV-M@Se with liver cancer cell membrane to obtain cell membrane-carrier si / ETV-M@SeM, and finally couples phosphatidylinositol proteoglycan 3 (GPC3) antibody with si / ETV-M@SeM to obtain a bionic nano-adjuvant si / ETV-M@SeMA; then the hydrated particle size, stability, antibody modification efficiency, dispersibility, uniformity and biocompatibility of the bionic nano-adjuvant are characterized; then the safe concentration range of the bionic nano-adjuvant is explored, and it is verified at the cellular level that the bionic nano-adjuvant can significantly downregulate the intracellular lipid accumulation level and reactive oxygen level, and at the in vivo level it is verified that the bionic nano-adjuvant has good biocompatibility, can significantly inhibit the tumor progression of liver cancer mice, and effectively prolong the life span of mice. The specifically targeted biomimetic nanoadjuvant designed for the hepatitis B (HBV) positive tumor microenvironment of the present invention has important clinical value in relieving the patient's immune escape and enhancing the immunotherapy effect.

[0038] Example 1: Construction of biomimetic nanoadjuvant si / ETV-M@SeMA The construction flow chart of biomimetic nanoadjuvant si / ETV-M@SeMA is shown in Figure 1 As shown, the specific preparation steps are as follows: 1. Preparation of carrier M@Se Firstly, mesoporous nano-silica (MSN) was synthesized by the ammonium fluoride method. Selenous acid and ascorbic acid were added to MSN. Selenous acid was reduced by ascorbic acid to obtain nano-selenium. Nano-selenium was then synthesized into a selenium (Se) shell on the surface of MSN to obtain stable M@Se (mesoporoussilica nanoparticles@selenium, M@Se).

[0039] 2. Preparation of drug-loaded carrier si / ETV-M@Se 1 mg of M@Se was dissolved in 50 mL of ethanol, and then 0.25 mg of ETV and 0.67 mg of FASN siRNA were added, mixed, incubated overnight on a shaker at 4 °C at 40 rpm, and centrifuged at 8000 rpm for 10 min to obtain si / ETV-M@Se.

[0040] At the same time, the present invention explores the loading capacity of M@Se for ETV and FASN siRNA, as follows: (1) Loading capacity of M@Se on ETV M@Se was dissolved in ethanol, and different concentrations of free ETV dissolved in DMSO were added and mixed. The mixture was incubated overnight on a shaker at 4 °C at 40 rpm, and centrifuged at 8000 g for 10 min to collect the precipitate.

[0041] The masses of ETV and M@Se in each experimental group were 1mg+10mg, 1mg+5mg, 1mg+2mg, 1mg+1mg, 1mg+0.5mg, and 1mg+0.25mg, respectively.

[0042] By scanning the full wavelength of ETV solutions with different concentrations, Figure 2 As shown in Figure A, there is a maximum absorption peak at a wavelength of 256.4nm, and then a standard curve of the relationship between ETV concentration and absorbance is obtained at a scanning wavelength of 256.4nm, as shown in Figure 1. Figure 2 As shown in Figure B, the standard curve is Y=9.419*X+1.696, where X represents the ETV concentration and Y represents the absorbance. Using this standard curve, the mass of the eluted ETV can be calculated by the absorbance and volume of the eluent. The experimental results are shown in Figure 2 As shown in Figure C, the maximum loading amount of ETV for 1 mg of M@Se is 0.25 mg.

[0043] (2) Loading capacity of M@Se for FASN siRNA 1 mg, 2 mg, 5 mg, 10 mg and 15 mg of M@Se were mixed evenly with 1 mg of FASN siRNA, and the control group was taken without M@Se. The mixture was incubated overnight on a shaker at 4 ℃ and 40 rpm. The unloaded siRNA was washed away by centrifugation at 8000 g for 10 min to obtain a precipitate, which was the test material.

[0044] The gel retardation experiment was used to detect the loading amount of M@Se on FASN siRNA. The experimental results are as follows: Figure 3 As shown, from Figure 3 It can be seen that by gradually increasing the mass of M@Se in a certain mass of FASN siRNA, it was found that M@Se weighing 15 mg can stably load 1 mg of siRNA.

[0045] These results verified the successful loading of ETV and siRNA by M@Se.

[0046] 3. Preparation of cell membrane-carrier si / ETV-M@SeM In order to make the carrier have a certain tumor targeting property, the inventors further coated the prepared drug-loaded carrier si / ETV-M@Se with liver cancer cell membranes. The specific steps are as follows: ① Culture HepG2 cells (purchased from Shanghai Fuheng Biotechnology Co., Ltd.) for 10 8 After 3 s, the cells were collected, digested with 2 mM EDTA solution, rinsed with PBS, centrifuged at 500 g for 10 min, and the precipitate was collected.

[0047] ② Wash the collected precipitate three times with 500 mL of PBS solution, then soak it in 5 mL of hypotonic buffer (20 mMTris-HCl pH = 7.5, 10 mM KCl, 2 mM MgCl2 and 1 tablet of EDTA-free mini protease inhibitor / 10 mL) for 30 min, coarsely grind it 35 times and finely grind it 35 times with a grinder, collect the grinding product, centrifuge it at 4000 rpm for 10 min, and collect the supernatant.

[0048] ③ Add 5 mL of hypotonic buffer to the precipitate after centrifugation in step ②, grind it coarsely 55 times and finely 55 times with a grinder again, centrifuge it at 4000 rpm for 10 min, and collect the supernatant.

[0049] ④ Combine the supernatants from step ② and step ③, centrifuge at 150,000 g for 90 min using an ultracentrifuge, remove the supernatant, add 1 mL LTE buffer to the precipitate, sonicate for 5 min, and extrude repeatedly 20 times using an Avanti miniextruder and a 400 nm filter to obtain the prepared cell membrane.

[0050] ⑤ Mix the prepared cell membrane with si / ETV-M@Se, ultrasonicate for 5 min, and shake at 4°C overnight. Then wash away the excess cell membrane with Q water to obtain the cell membrane-carrier si / ETV-M@SeM.

[0051] 4. Preparation of biomimetic nanocarrier si / ETV-M@SeMA To achieve the targeting of bionic nanoadjuvants to liver cancer cells, the inventors coupled phosphatidylinositol proteoglycan-3 (GPC3) antibody with si / ETV-M@SeM.

[0052] Aminated distearate phosphatidylethanolamine polyethylene glycol 5k (DSPE-PEG5k-NH2) was purchased from Xi'an Ruixi Biological; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and hydroxysuccinimide (NHS) were purchased from Aladdin Chemical Reagent Co., Ltd.

[0053] 2.2 mg of EDC and 1.4 mg of NHS were added to the si / ETV-M@SeM solution coated with DSPE-PEG5k-NH2 modified cell membrane, stirred at room temperature for 4 h, and then 15 μg of GPC3 antibody (purchased from Sewell Biotechnology) was added and stirred at room temperature overnight to obtain the biomimetic nanoadjuvant si / ETV-M@SeMA.

[0054] Example 2: Characterization of biomimetic nanoadjuvants 1. Characterization of hydrated particle size The biomimetic nanoadjuvant was characterized by a nanoparticle size Zeta potential analyzer to detect the hydrated particle size of the nanoadjuvant.

[0055] The results are as follows Figure 4 As shown, the average hydrated particle size of the unloaded mesoporous nanosilica (MSN) is 95.17 nm, the average hydrated particle size of the carrier M@Se is 109.33 nm, the average hydrated particle size of the drug-loaded carrier si / ETV-M@Se is 133.96 nm, and the average hydrated particle size of the bionic nanoadjuvant si / ETV-M@SeMA is 145.44 nm, showing a gradually increasing hydrodynamic size.

[0056] 2. Stability test MSN, single-element nanoselenium (SeNPs) and biomimetic nanoadjuvant (M@SeMA) without drug loading were placed at room temperature for 0 h and then observed. Figure 5 As shown in Figure A, MSNs are bluish white, SeNPs are red, and M@SeMA is light red from Se. After being placed at room temperature for 1 h, Figure 5 As shown in Figure B, SeNPs aggregate more severely, while MSNs and M@SeMA are still uniformly distributed without obvious aggregation, indicating that the M@SeMA provided by the present invention has good stability.

[0057] 3. Antibody modification efficiency detection The modification efficiency of the antibody on the surface of the biomimetic nanoadjuvant was verified using an antibody with green fluorescence, and the experimental results were photographed under the fluorescence field using a UV gel transilluminator.

[0058] The results are as follows Figure 6 As shown, compared with M@Se, the un-drug-loaded biomimetic nanoadjuvant M@SeMA modified with fluorescent antibodies can detect strong fluorescence intensity, indicating that the antibody has a considerable modification efficiency on the surface of the biomimetic nanoadjuvant.

[0059] 4. Dispersion and uniformity testing The biomimetic nanoadjuvant si / ETV-M@SeMA prepared in Example 1 was detected using a transmission electron microscope.

[0060] The results are as follows Figure 7 As shown, the biomimetic nanoadjuvant si / ETV-M@SeMA has good dispersibility, and a slightly lighter cell membrane coating can be seen around the dark M@Se core. Its particle size is 100-120nm, with an average particle size of 116.3 nm, and has good uniformity. This shows that the biomimetic nanoadjuvant si / ETV-M@SeMA provided by the present invention has uniform properties and certain stability.

[0061] 5. Biocompatibility testing 293T cells (purchased from Shanghai Fuheng Biotechnology Co., Ltd.) were treated with M@Se, and the cell counting kit-8 (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) was used according to the instructions to detect changes in cell activity.

[0062] The results are as follows Figure 8 As shown in the figure, 150 μg / mL M@Se had no significant cytotoxicity in 293T cells. There was no significant cytotoxicity within 72 h after treatment of cells with 150 mg / mL M@Se. The experimental results further confirmed the safe concentration range of M@Se.

[0063] Example 3: Evaluation of metabolic reprogramming and immunostimulatory effects of biomimetic nanoadjuvants at the cellular level HBV + Hepatoma cell culture: HepG2.2.25 (purchased from Shanghai Fuheng Biotechnology Co., Ltd.) was used as HBV + Hepatoma cell model. HepG2.2.25 contains the HBV genome and uses the HBV promoter. It is relatively close to liver cancer cells during / after in vitro infection and is suitable for HBV-related functional gene research.

[0064] 1. Detection of the ability to inhibit lipid synthesis The culture medium containing OA (Oleic Acid) was used to simulate the tumor microenvironment with high fatty acid levels. The bionic nanoadjuvant si / ETV-M@SeMA (100 μg / mL) was added to the cell culture medium to treat the liver cancer cell HepG2 for 24 hours. The fluorescent dye BODIPY 493 / 503 was used to characterize the fluorescence signal changes related to intracellular lipid accumulation. The images were taken with a ZEISS inverted fluorescence microscope and quantitatively characterized by ImageJ software.

[0065] The results are as follows Fig. 9 As shown, the bionic nanoadjuvant si / ETV-M@SeMA significantly downregulated the intracellular lipid accumulation level in HepG2 cells with high fatty acid levels, proving that the bionic nanoadjuvant si / ETV-M@SeMA can significantly inhibit the lipid synthesis ability of hepatocytes, indicating that it has potential lipid metabolism reprogramming ability in the tumor microenvironment in vivo.

[0066] 2. Detection of ROS scavenging ability HepG2 liver cancer cells were treated with M@Se (100 μg / mL) for 24 h. The fluorescent probe 2,7-dichlorofluorescein diacetate (DCFH-DA) was used to characterize the fluorescence signal changes of intracellular reactive oxygen species (ROS) levels. The ZEISS inverted fluorescence microscope and ZEN software were used to quantitatively characterize the reactive oxygen species levels.

[0067] The results are as follows Fig.10 As shown in Figure 2, M@Se significantly downregulated the intracellular ROS level in HepG2 cells cultured in vitro. The experimental results further demonstrated that M@Se has significant ROS scavenging ability, indicating that the biomimetic nanoadjuvant si / ETV-M@SeMA also has the potential to downregulate ROS levels in the tumor microenvironment in vivo.

[0068] Example 4: In vivo evaluation of metabolic reprogramming and tumor suppression effects of biomimetic nanoadjuvants in the tumor microenvironment Construction of Hepg2.2.15 liver cancer mouse tumor model: 1×10 5 HepG2.2.25 cells were evenly mixed in 50 μL of matrix gel and injected subcutaneously into 8-week-old male Balb / c nude mice. The tumor size was recorded with a vernier caliper, and the tumor volume was calculated as follows: tumor volume = 0.5 × tumor length × tumor width 2 When the tumor grows to 200 mm 3 The drug was administered when the volume of the mouse increased; that is, starting from the 15th day after tumor inoculation, the drug was administered once every 3 days, for a total of 7 times; the tumor inhibition ability of the bionic nanoadjuvant was continuously monitored and detected. After 60 days, the main organs of the mice were taken for H&E staining to detect whether the bionic nanoadjuvant had toxic side effects on organs other than the tumor.

[0069] The drug administration experiment was divided into 5 groups: Group 1 was a control group without treatment; Group 2 was a free administration group of FASN siRNA+ETV; Group 3 was an administration group of the bionic nanoadjuvant si-M@SeMA loaded only with fatty acid synthase small interfering RNA prepared according to the preparation method of Example 1; Group 4 was an administration group of the bionic nanoadjuvant ETV-M@SeMA loaded only with entecavir prepared according to the preparation method of Example 1; Group 5 was a bionic nanoadjuvant group prepared in Example 1; the dosage of each group was 5 mg / kg.

[0070] 1. Biocompatibility of biomimetic nanoadjuvants The main organs of mice were stained with hematoxylin and eosin (H&E) and immunohistochemically analyzed: after euthanasia, the heart, liver, spleen, lung, and kidney tissues were fixed with neutral formalin solution (purchased from Wuhan Saiweier Biotechnology Co., Ltd.); after dehydration, paraffin embedding, and sectioning. The tissue sections were 5 μm thick and 200 μm deep; the tissue sections of the main organs were stained with H&E and imaged with an optical microscope.

[0071] The results are as follows Fig.11 As shown, there was no abnormality in the slice structure of the mouse's major organ tissues, including heart, liver, spleen, lung and kidney, indicating that the nanoadjuvant has good biocompatibility.

[0072] 2. Detecting the inhibitory ability of biomimetic nanoadjuvants on tumors In order to monitor the tumor growth in mice, IVIS Spectrum Imaging System was used for in vivo imaging. Before imaging, mice were intraperitoneally injected with D-luciferin at a dose of 150 μg / g. Three minutes after the injection, the mice were anesthetized by inhalation of isoflurane. After the mice were completely unconscious, they were placed in the imaging system for imaging.

[0073] The results are as follows Fig.12 As shown in the figure, it can be seen from the mouse tumor growth curve and survival curve that the bionic nanoadjuvant si / ETV-M@SeMA significantly inhibits the tumor progression of liver cancer mice and can effectively prolong the life of mice.

[0074] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, once those skilled in the art know the basic inventive concept, they can make other changes and modifications to these embodiments, and these changes and modifications all fall within the scope of the present invention.

[0075] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. If these modifications and variations of the present invention fall within the scope of the equivalent technology of the present invention, the present invention is also intended to include these modifications and variations.

Claims

1. A biomimetic nanoadjuvant, characterized in that: The bionic nano adjuvant is prepared by the following steps: After the surface of mesoporous nano-silica is coated with selenium, fatty acid synthase small interfering RNA and antiviral drug entecavir are loaded to obtain a drug-loaded carrier; The drug-loaded carrier is coated with the liver cancer cell membrane to obtain a cell membrane-carrier; The liver cancer specific antibody is coupled to the cell membrane of the cell membrane-carrier to obtain the bionic nano adjuvant.

2. The biomimetic nanoadjuvant according to claim 1, characterized in that The mass ratio of the vector to the fatty acid synthase small interfering RNA is 1:0.06-0.

15.

3. The biomimetic nanoadjuvant according to claim 1, characterized in that The mass ratio of the carrier to entecavir is 1:0.2-0.

25.

4. The biomimetic nanoadjuvant according to claim 1, characterized in that The selenium element is nano-selenium.

5. The biomimetic nanoadjuvant according to claim 1, characterized in that The liver cancer cell membrane is a HepG2 cell membrane.

6. The biomimetic nanoadjuvant according to claim 1, characterized in that The liver cancer-specific antibody is glypican 3.

7. Use of the bionic nanoadjuvant according to claim 1 in preparing a drug for preventing or treating liver cancer.

8. The use according to claim 7, characterized in that: The bionic nano adjuvant is used to prepare a drug for reducing the level of reactive oxygen or lipid in the tumor microenvironment.

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