Anti-liver tumor composition as well as preparation method and application thereof

Abstract

The invention discloses an anti-liver-tumor composition and application thereof. The anti-liver-tumor composition comprises norcantharidin solid lipid nanoparticles and astragalus polysaccharide, the norcantharidin solid lipid nanoparticles comprise at least one of a norcantharidin solid lipid nanoparticle NCTD-SLN, a norcantharidin solid lipid nanoparticle At-PEG2000-NCTD-SLNs, and a norcantharidin solid lipid nanoparticle At-PEG2000-ss-NCTD-SLNs, and the norcantharidin solid lipid nanoparticle comprises at least one of a norcantharidin solid lipid nanoparticle NCTD-SLN, a norcantharidin solid lipid nanoparticle At-PEG2000-NCTD-SLNs, a norcantharidin solid lipid nanoparticle At-PEG2000-NCTD-SLNs The norcantharidin solid lipid nanoparticles prepared by a certain preparation method have low toxicity in liver and kidney in vivo, have good biological safety, and have a good anti-liver tumor effect by combining the norcantharidin solid lipid nanoparticles with the astragalus polysaccharide according to a proper concentration ratio of the norcantharidin solid lipid nanoparticles and the astragalus polysaccharide.

Description

Technical Field

[0001] The invention belongs to the technical field of targeted drug research, and specifically relates to an anti-liver tumor composition, a preparation method and use thereof. Background Art

[0002] At present, the clinical treatment of hepatocellular carcinoma (HCC) is still mainly surgical treatment and chemoradiotherapy. Although chemoradiotherapy technology has made significant progress in recent decades, it has not significantly improved the long-term survival prospects of cancer patients. Some auxiliary treatment plans have been developed to improve the treatment effect. Based on the theory of seven emotions in traditional Chinese medicine, the establishment of a two-drug combination therapy is considered to be an effective solution to improve patient survival rate.

[0003] NCTD has a clear therapeutic effect on primary liver cancer, but its hepatotoxicity and renal toxicity seriously affect its clinical application. After being made into nanoparticles, it can not only improve liver targeting, but also reduce the dosage of NCTD to achieve the same therapeutic effect. In addition, APS613-1 aptamer can accurately target the GPC3 receptor on HepG2 liver cancer cells and improve the uptake capacity; astragalus polysaccharide APS can improve immunity and have a weak anti-tumor effect. At present, there is no relevant research on how to use solid lipid nanoparticles of NCTD combined with astragalus polysaccharide for anti-liver tumor. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an anti-liver tumor composition, a preparation method and use thereof. The anti-liver tumor composition has low liver and kidney toxicity in vivo, good biosafety and good anti-liver tumor effect.

[0005] The content of the present invention includes: An anti-liver tumor composition comprises norcanthracen solid lipid nanoparticles and astragalus polysaccharide, wherein the concentration of the norcanthracen solid lipid nanoparticles is 1.0-2.0 μg / mL, the concentration of astragalus polysaccharide is 300-900 μg / mL, and the concentration ratio of astragalus polysaccharide to norcanthracen solid lipid nanoparticles ranges from 150 to 900.

[0006] Furthermore, the solid lipid nanoparticles of norcanthracen include solid lipid nanoparticles of norcanthracen NCTD-SLNs, solid lipid nanoparticles of norcanthracen Apt-PEG 2000 -NCTD-SLNs and norcanthracen solid lipid nanoparticles Apt-PEG 2000 -ss-NCTD-SLNs at least one of the three; the NCTD-SLNs of the demethylcanthate comprises demethylcanthate and solid lipid nanoparticles; the Apt-PEG solid lipid nanoparticles of the demethylcanthate 2000-NCTD-SLNs or norcanthracen solid lipid nanoparticles Apt-PEG 2000 -ss-NCTD-SLNs include norcanthracen, solid lipid nanoparticles, long circulating chains and aptamers.

[0007] Furthermore, the solid lipid nanoparticles are soybean lecithin, and the long circulating chain is Mal-PEG 2000 -ss-DSPE or Mal-PEG 2000 -DSPE was purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0008] Furthermore, the aptamer is APS613-1, which was purchased from Shanghai Sangon Biotechnology Co., Ltd.

[0009] Another object of the present invention is to provide a method for preparing the above-mentioned anti-liver tumor composition, wherein the method for preparing the norcantharidin solid lipid nanoparticles comprises the following steps: weighing glyceryl monostearate, soybean lecithin and norcantharidin, dissolving them in methanol to form an organic phase, taking a poloxamer aqueous solution to form an aqueous phase, heating the two phases at the same time, slowly injecting the aqueous phase into the organic phase under a water bath, stirring to volatilize the methanol, ultrasonicating while hot, cooling and solidifying, and filtering to obtain the norcantharidin solid lipid nanoparticles NCTD-SLNs.

[0010] Furthermore, the preparation method of the norcanthracen solid lipid nanoparticles further comprises the following steps: adding a long circulating chain Mal-PEG to the norcanthracen solid lipid nanoparticles NCTD-SLNs; 2000 -DSPE or Mal-PEG 2000 -ss-DSPE, forming Mal-PEG 2000 -NCTD-SLNs or Mal-PEG 2000 -ss-NCTD-SLN; the aptamer APS613-1 was combined with the Mal-PEG 2000 -NCTD-SLNs or Mal-PEG 2000 -ss-NCTD-SLN was incubated in a nitrogen environment at 4°C in the dark for 24 h to obtain the cantharicin solid lipid nanoparticles Apt-PEG 2000 -NCTD-SLNs or norcanthracen solid lipid nanoparticles Apt-PEG 2000 -ss-NCTD-SLNs.

[0011] Furthermore, the mass ratio of the APS613-1 to the NCTD-SLNs is 1:150.

[0012] Furthermore, the mass ratio of poloxamer to soybean lecithin is 2:1.

[0013] Furthermore, the ultrasonication time is 10 min, and the heating temperature is 65°C.

[0014] Another object of the present invention is to provide a use of the above anti-liver tumor composition for preparing anti-liver tumor drugs.

[0015] Beneficial effects of the present invention: (1) The solid lipid nanoparticles of demethylcantharitin in the anti-liver tumor composition of the present application have a clear and transparent appearance, a light blue opalescence, a relatively uniform particle size, a high encapsulation rate, and good stability; (2) The demethylcanthracen solid lipid nanoparticles in the anti-liver tumor composition of the present application have no obvious tissue toxicity in vivo, have relatively small toxic and side effects, and have a certain degree of biosafety; (3) The combined drug index CI of the two drugs of the present application, Apt-PEG solid lipid nanoparticles, was between 0.9 and 1.1, and the two drugs showed an additive effect. CI>1.10 was an antagonistic effect, and CI<0.9 was a synergistic effect. In addition, it was found that the concentration of the Apt-PEG group in the preparation was too low, and the antagonistic effect between the two drugs was significant. The tumor inhibition rate of the combined drug was mainly related to the Apt-PEG 2000 -ss-NCTD-SLNs tumor inhibition rate, Apt-PEG 2000 -ss-NCTD-SLNs played a dominant role in inhibiting the proliferation of HepG2 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effect of NCTD culture time on the survival rate of HepG2 cells (n=6).

[0017] Figure 2 The effect of polysaccharide culture time on the survival rate of HepG2 cells (n=6).

[0018] Figure 3 The effect of each NCTD solid lipid nanoparticle on the survival rate of HepG2 cells (n=6).

[0019] Figure 4 This is a bar graph of the IC50 of each NCTD solid lipid nanoparticle on HepG2 cells (n=6), where A-NCTD stock solution, B-NCTD-SLNs, C-Apt-PEG2000-NCTD-SLNs, D-Apt-PEG2000-ss-NCTD-SLNs, and E-cisplatin.

[0020] Figure 5 is the tumor growth curve after treatment with different drugs (n=5).

[0021] Figure 6are tumors in different dosing groups (n=5), where A represents normal saline, B represents NCTD-SLNs, C represents Apt-PEG2000-NCTD-SLNs, D represents Apt-PEG2000-ss-NCTD-SLNs, E represents Apt-PEG2000-ss-NCTD-SLNs+APS, and F represents cisplatin.

[0022] Figure 7 From left to right are the HE staining images, PCNA detection images and TUNEL detection images of tumors in Saline, NCTD-SLNs, Apt-PEG2000-NCTD-SLNs, Apt-PEG2000-ss-NCTD-SLNs, Apt-PEG2000-ss-NCTD-SLNs+APS, and Cisplatin groups (from left to right), with a scale of 200 μm.

[0023] Figure 8 This is the body weight change curve of nude mice after treatment with different preparations (n=5).

[0024] Fig. 9 These are H&E staining images of the heart, liver, spleen, lung, and kidney after treatment with different preparations. The scale bar is 200 μm.

[0025] Fig.10 The hepatotoxicity (ALT, AST) and nephrotoxicity (BUN, CREA) in serum of different preparation groups were evaluated (n=5).

[0026] Fig.11 It is a diagram for establishing dialysis equilibrium.

[0027] Fig.12 It is the response surface and contour plot of the interaction between two factors.

[0028] Fig.13 This is the result of agarose gel electrophoresis experiment, from left to right: (free Apt, Apt-PEG2000-NCTD-SLNs, mal-PEG2000-NCTD-SLNs).

[0029] Fig.14 This is the result of agarose gel electrophoresis experiment, from left to right: (free Apt, Apt-PEG2000-NCTD-SLNs 1:50, 1:100, 1:150, 1:200).

[0030] Fig.15 These are the appearance pictures of differently modified NCTD-SLNs (from left to right: NCTD-SLNs, Apt-PEG2000-NCTD-SLNs, Apt-PEG2000-ss-NCTD-SLNs).

[0031] Fig.16 These are transmission electron micrographs of differently modified NCTD-SLNs (A: NCTD-SLNs B: Apt-PEG2000-ss-NCTD-SLNs).

[0032] Fig.17 This is the particle size distribution diagram of differently modified NCTD-SLNs (A: NCTD-SLNs B: Apt-PEG2000- NCTD-SLNs C: Apt-PEG2000-ss-NCTD-SLNs).

[0033] Fig.18 Zeta potential diagram of differently modified NCTD-SLNs (A: NCTD-SLNsB: Apt-PEG2000- NCTD-SLNsC: Apt-PEG2000-ss-NCTD-SLNs).

[0034] Fig.19 This is a diagram of the hemolysis experiment results (A: Astragalus polysaccharide B: Apt-PEG2000-ss-NCTD-SLNs). DETAILED DESCRIPTION

[0035] Example 1 Preparation of norcantharidin solid lipid nanoparticles NCTD-SLNs: accurately weigh the prescribed amount of glyceryl monostearate 75 mg, soybean lecithin 50 mg and norcantharidin 6 mg, dissolve in 5 mL methanol to form an organic phase, take 100 mg of poloxamer and add 15 mL of distilled water to form an aqueous phase, heat the two phases to 65 °C at the same time, slowly inject the aqueous phase into the organic phase under 1000 r / min magnetic stirring in a 65 °C water bath, stir at constant temperature for 1 h to evaporate the methanol, place in an ultrasonic cell crusher for 10 min while hot, cool and solidify at room temperature, and filter through a 0.22 μm microporous filter membrane to obtain NCTD-SLNs; Preparation of Norcantharidin Solid Lipid Nanoparticles Apt-PEG 2000 -NCTD-SLNs or Apt-PEG 2000 -ss-NCTD-SLNs: Add 9 mg of long-chain Mal-PEG2000-DSPE or mal-PEG2000-ss-DSPE to form Mal-PEG 2000 -NCTD-SLNs or Mal-PEG 2000 -ss-NCTD-SLN; the aptamer APS613-1 was added at a weight ratio of 1:150 between the aptamer and SLNs, and the Mal-PEG 2000 -NCTD-SLNs or Mal-PEG 2000-ss-NCTD-SLN was incubated in a nitrogen atmosphere at 4°C for 24 h in the dark to obtain Apt-PEG 2000 -NCTD-SLNs or Apt-PEG 2000 -ss-NCTD-SLNs.

[0036] Example 2 (in vitro experiment) (1) Training time assessment HepG2 cells in the logarithmic growth phase were taken and inoculated in a 96-well plate at a density of 5000-8000 cells / well. After the cells adhered to the wall, 100 μL of drug solution diluted in different culture media (the concentration of the stock solution of demethylcantharidin was 5, 10, 20, 40, 80, 100 μg / mL, and the concentration of polysaccharides was 100, 300, 600, 900, 1200, 1500 μg / mL) was added to each well. 6 wells were placed in a constant temperature incubator for 24, 48, and 72 hours. After that, the culture medium in the well was aspirated, and 100 μL of 10% CCK8 solution was added to each well. The culture was continued in a constant temperature incubator for 1 hour. After the culture was completed, the absorbance value (OD value) of each well was detected at 450 nm using an enzyme marker. The larger the OD value, the more surviving cells there were. The inhibition rate was calculated according to the following formula. The results are shown in Table 1 and Table 2. Figure 1 , Figure 2 .

[0037]

[0038] OD 对照 is the absorbance value of cells without drug, OD 药物 is the absorbance value of cells plus drugs, OD 空白 is the absorbance value without cells or drugs.

[0039] The NCTD-statistical histogram shows that the inhibition rate of NCTD on HepG2 cells is time-concentration dependent, that is, with the continuous increase of time and concentration, it is obvious that the cell survival rate gradually decreases. Among them, the killing effect of NCTD on HepG2 cells for 48h is significantly higher than that for 24h, and the cell survival rate for 72h is not significantly higher than that for 48h, and the IC 50 The difference is not big. Considering the time cost and the fact that the culture medium may not meet the requirements of cell growth if the culture time is too long, the combined drug intervention is followed by culture for 48 hours.

[0040] The inhibitory effect of APS on HepG2 cells did not change much with the extension of culture time, and the inhibition rate did not change significantly with the increase of dose, and there was no obvious regularity. Considering the combination with norcantharidin, 48h was selected as the culture time for investigation.

[0041] Table 1 Effect of NCTD culture time on the survival rate (%) of HepG2 cells

[0042] Table 2 Effect of APS culture time on HepG2 cell survival rate (%)

[0043] (2) Effects of different modified NCTD solid lipid nanoparticles on HepG2 cell cytotoxicity HepG2 cells in the logarithmic growth phase were taken, and 0.0492 mg / mL of NCTD-SLNs were added, with 6 replicates in each group, and the results are shown in Table 3. From the data in the table, it can be seen that if NCTD-SLNs are added according to the method of the stock solution of NCTD-SLNs, the inhibition rate of 10 μg / mL has reached more than 90%, and the trend of the results is not good. Therefore, on this basis and combined with the results of the preliminary experiment, the concentrations of NCTD-SLNs were re-selected to be 0.5, 1, 2, 4, 6, and 8 μg / mL to repeat the experiment; at the same time, the positive control group (cisplatin) was designed to select concentrations of 0.1, 0.5, 1, 2, 4, and 6 μg / mL for the experiment, with 6 parallel wells, and the results are shown in Tables 3, 4, and 5. Figure 3 and Figure 4 , by IC 50 The results showed that the IC 50 Compared with the IC of NCTD stock solution 50 The difference was much smaller, with a very significant difference, indicating that the inhibition rate of HepG2 cells was significantly improved after being made into nanoparticles. 2000 -NCTD-SLNs, Apt-PEG 2000 -ss-NCTD-SLNs (P<0.01), which proves that targeted molecular modification enhances the inhibitory effect on liver cancer cells. This may be because the aptamer can bind to the receptors on the cell surface, increasing the cell's uptake of the drug. However, there is no significant difference between the Apt-PEG2000-NCTD-SLNs and Apt-PEG2000-ss-NCTD-SLNs groups. This may be because the redox effect cannot be reflected at the cellular level and needs to be further verified in animals.

[0044] Table 3 Effects of various NCTD solid lipid nanoparticles on the survival rate of HepG2 cells (n=6)

[0045] Table 4 Effects of various NCTD solid lipid nanoparticles on the survival rate of HepG2 cells (n=6)

[0046] Table 5 Effect of cisplatin on the survival rate of HepG2 cells (n=6)

[0047] (3) Study on the combined administration of Apt-PEG2000-ss-NCTD-SLNs and astragalus polysaccharide (APS) Compusyn software is based on the mathematical model established by Chou Talalay, which can evaluate the combined effect of two drugs using the median effect principle and combination index theorem. 2000 -ss-NCTD-SLNs combined with APS have a synergistic inhibitory effect on the cell activity of HepG2 cells. Three concentrations of the drug mixture (Apt-PEG 2000 -ss-NCTD-SLNs 0.5, 1, 2μg / mL and APS 300, 600, 900μg / mL), CCK8 experiment was performed according to the experimental method under 4.1, and the combination index (CI) after the combination of different concentrations of drugs was calculated using Compusyn software. CI < 0.9, synergistic effect; 0.9 < CI < 1.10, additive effect, CI > 1.10, antagonistic effect. The results are shown in Table 6. When the two drugs are combined at different concentrations, the CI is between 0.9 and 1.1 in most cases, indicating that the two drugs show an additive effect, and a few drug ratios show synergistic and antagonistic effects. At the same time, it was found that the concentration of the norcantharidin group in the preparation was too low, and the antagonistic effect between the two drugs was significant. The tumor inhibition rate of the combined drug use was mainly related to Apt-PEG 2000 -ss-NCTD-SLNs tumor inhibition rate, Apt-PEG 2000 -ss-NCTD-SLNs played a dominant role in inhibiting the proliferation of HepG2 cells.

[0048] Table 6 Apt-PEG 2000 -Combination index (CI) of ss-NCTD-SLNs combined with APS on HepG2

[0049] FA* indicates the inhibition rate of combined drug use on HepG2 cells CCK8 is a kit for detecting cell proliferation and cytotoxicity developed by Dojindo Chemical Research Institute of Japan. It is an alternative to the MTT method. Compared with the MTT method, this method has high sensitivity, reliable data and good reproducibility. It has been widely used in activity detection of some biologically active factors, large-scale anti-tumor drug screening, cell proliferation tests, cytotoxicity tests and drug sensitivity tests.

[0050] HepG2 cells are a commonly used cell line for studying liver tumors, and GPC3 protein is highly expressed on the cell membrane. In this study, CCK8 assay was used to investigate the effects of NCTD-SLNs and Apt-PEG 2000 -NCTD-SLNs, Apt-PEG 2000 The cell proliferation inhibition rate of -ss-NCTD-SLNs was time- and dose-dependent in each group. Compared with the NCTD stock solution, the IC 50 Significantly reduced Apt-PEG 2000 -NCTD-SLNs, Apt-PEG 2000 -ss-NCTD-SLNs has a stronger inhibitory effect on tumor cells than NCTD-SLNs (P < 0.01), which may be due to the modification of the targeting molecule increasing the cell uptake rate of NCTD, but there is no significant difference between the targeting preparations with or without disulfide bond modification. It is speculated that it is difficult to form a tumor microenvironment in in vitro cell experiments, so the advantage of disulfide bonds cannot be reflected, and it needs to be further verified in in vivo experiments. APS alone has a small inhibitory effect on HepG2 cells, and there is no obvious pattern. At the same time, after Apt-PEG2000-ss-NCTD-SLNs and APS were combined, most drug concentrations showed an additive effect, and a few drug ratios showed synergistic and antagonistic effects. The inhibition rate of each group on HepG2 cells mainly depends on Apt-PEG2000-ss-NCTD-SLNs. Therefore, it can be concluded that Apt-PEG2000-ss-NCTD-SLNs plays a leading role in inhibiting the proliferation of HepG2 cells.

[0051] Example 3 (In vivo anti-liver tumor experiment) BALB / C nude mice (half male and half female, 4-5 weeks old) were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. and kept in the Experimental Animal Management Center of Hunan University of Chinese Medicine under SPF conditions of 25±2℃ and 60±5% relative humidity. They were free to eat and drink water and were adaptively fed for 3 days before the experiment. All animal experiments were approved by the Experimental Animal Ethics Committee of Hunan University of Chinese Medicine. Construction of BALB / C nude mouse tumor model: After BALB / C nude mice were adaptively fed for 3 days, HepG2 cells in the logarithmic growth phase were obtained and digested with trypsin. The cells were resuspended in PBS and the cell concentration was adjusted to 1×10 7 Each mouse was inoculated with 1×10 6 When the tumor grows to 80 mm 3 The tumor volume was calculated as follows: 1 / 2 × long diameter × short diameter 2 .

[0052] (1) Tumor growth inhibition study The tumor-bearing nude mice were divided into 6 groups: model group (normal saline), positive control group (cisplatin 4 mg / kg), and 4 drug-treated groups (NCTD-SLNs, Apt-PEG 2000 -NCTD-SLNs, Apt-PEG 2000 -ss-NCTD-SLNs were 2mg / kg, Apt-PEG2000-ss-NCTD-SLNs 2mg / kg+APS10mg / kg), except for cisplatin intraperitoneal injection, the other preparations were injected by tail vein, once every 2 days, 7 times, the tumor volume was measured on the same day, and the tumor inhibition rate was calculated. The tumor inhibition rate formula was: (model group tumor volume-dosing group tumor volume) / model group tumor volume × 100%. The nude mice were killed on the second day after the last administration, the tumor was removed, and photos were taken.

[0053] (2) Tumor pathological analysis-H&E staining Hematoxylin-eosin staining is referred to as H&E staining. Hematoxylin staining solution is alkaline, which mainly makes the chromatin in the cell nucleus and the nucleic acid in the cytoplasm purple-blue; eosin is an acidic dye, which mainly makes the components in the cytoplasm and extracellular matrix red. H&E staining is the most commonly used technical method in pathology, so this experiment uses H&E staining to analyze tumor tissue sections; the specific steps are as follows: 1) Fixation: Place the tumor sample in 4% paraformaldehyde for fixation at room temperature for more than 24 hours; 2) Dehydration: Place the tissue in 75% ethanol for 4 hours, 85% ethanol for 2 hours, 90% ethanol for 1.5 hours, 95% ethanol for 1.5 hours, anhydrous ethanol for 30 minutes, anhydrous ethanol and xylene mixed solution for 15 minutes, xylene I for 15 minutes, and xylene II for 15 minutes. 3) Wax immersion: Place the dehydrated tissue in a 1 / 2 paraffin + 1 / 2 xylene solution, bake in a 40°C oven for 40 min, then soak in paraffin I at 65°C for 1.5 h, paraffin II at 65°C for 1.5 h, and paraffin III at 65°C for 1.5 h; 4) Embedding: Take out the tissue that has been thoroughly soaked in wax, take it out from the embedding frame after the wax block solidifies, and trim it into a regular block; 5) Slice: Place the trimmed wax block in a -20℃ freezer to cool, and then place the cooled wax block in a paraffin slicer to slice it, 4μm thick. Float the slices on 40℃ warm water on the slicer to flatten the tissue, pick up the tissue with a glass slide, and bake the slices in a 60℃ oven. After the water is dried and the wax is melted, take it out and store it at room temperature for later use; 6) Dewaxing and hydration: sequentially place the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 75% ethanol for 5 min, and hydrate in distilled water for later use; 7) Staining: Slices were stained with hematoxylin solution for 3-5 minutes, washed with running water, differentiated with 1% hydrochloric acid ethanol, washed with running water, blued with bluing solution, and rinsed with running water. Slices were dehydrated in 85% and 95% graded alcohol for 5 minutes each, and stained with eosin solution for 5 minutes; 8) Dehydration and sealing: The slices were placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, xylene II for 5 min to make them transparent, and then sealed with neutral gum; 9) Microscope examination, image acquisition and analysis.

[0054] (3) PCNA detection of cell proliferation in nude mouse tumor tissue Principle of PCNA proliferation detection experiment: Detecting the expression of proliferating cell nuclear antigen (PCNA) in cells can be used as an indicator to evaluate the cell proliferation status; PCNA exists in the nuclei of dividing animal and plant cells in a ring structure and participates in DNA replication and damage repair; when tumor cells proliferate vigorously, the proliferation process is accompanied by the replication of a large amount of DNA, so PCNA is often used to detect the proliferation ability of tumor cells.

[0055] 1) Dewax the paraffin sections to water: sequentially place the sections in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and wash with distilled water.

[0056] 2) Antigen repair: Place the tissue sections in a repair box filled with citric acid antigen repair buffer (PH 6.0) in a microwave oven for antigen repair; boil over medium heat, then turn off the power for 10 minutes and boil over medium-low heat. After natural cooling, place the slides in PBS (PH 7.4) and wash on a decolorizing shaker for 3 times, 5 minutes each time.

[0057] 3) Serum blocking: Block with 3% goat serum at room temperature for 30 minutes.

[0058] 4) Add primary antibody (PCNA): Gently shake off the blocking solution, add primary antibody prepared in a certain proportion of PBS on the slices, and lay the slices flat in a humidified box and incubate at 4°C overnight.

[0059] 5) Add secondary antibody: Place the slide in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time; after the slices are slightly dried, add secondary antibody (CY3 goat anti-mouse) in the circle to cover the tissue and incubate at room temperature in the dark for 50 minutes.

[0060] 6) DAPI counterstaining of cell nuclei: Place the slide in PBS (PH7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time; after the slices are slightly dried, add DAPI staining solution in the circle and incubate at room temperature for 10 minutes away from light.

[0061] 7) Sealing: Place the slides in PBS (PH7.4) and wash them on a decolorizing shaker for 3 times, 5 minutes each time; after the sections are slightly dried, seal them with anti-fluorescence quenching sealing medium.

[0062] 8) Microscopic examination and photography: The sections were observed and images were collected under an upright fluorescence microscope.

[0063] (4) TUNEL detection of cell apoptosis in nude mouse tumor tissue Principle of TUNEL apoptosis assay: One of the basic characteristics of apoptosis is DNA breakage; broken DNA will expose 3 ， The -OH end can be catalyzed by terminal deoxynucleotidyl transferase (TdT) and dUTP labeled with fluorescein (FITC). The occurrence of apoptosis can be quickly detected by fluorescence microscopy. This method is called terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL).

[0064] 1) Dewax the paraffin sections to water: sequentially place the sections in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and wash with distilled water.

[0065] 2) Antigen repair: Place the tissue sections in a repair box filled with citric acid antigen repair buffer (PH6.0) in a microwave oven for antigen repair. Boil on medium heat and then turn off the power for 10 minutes and boil on medium-low heat. After natural cooling, place the slides in PBS (PH7.4) and wash on a decolorizing shaker for 3 times, each time for 5 minutes.

[0066] 3) Serum blocking: Block with 3% goat serum at room temperature for 30 minutes.

[0067] 4) Add reagents 1 and 2: Gently shake off the blocking solution, take an appropriate amount of reagent 1 (TdT) and reagent 2 (dUTP) in the tunel kit and mix them in a ratio of 1:10. Place the slices flat in a humidified box and incubate overnight at 4°C. Add a small amount of water to the box to maintain humidity.

[0068] 5) DAPI counterstaining of cell nuclei: Place the slide in PBS (PH7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add DAPI staining solution in the circle and incubate at room temperature for 10 minutes away from light.

[0069] 6) Sealing: Place the slides in PBS (PH7.4) and wash them on a decolorizing shaker for 3 times, 5 minutes each time. After the sections are slightly dried, seal them with anti-fluorescence quenching sealing medium.

[0070] 7) Microscopic examination and photography: The sections were observed and images were collected under an inverted fluorescence microscope.

[0071] (5) In vivo pharmacodynamic studies in animals In order to investigate the anti-tumor effects of different SLNs in vivo, a nude mouse subcutaneous anti-tumor model was established, the tumor growth after administration was recorded, and the tumor inhibition rate was calculated. The results are shown in Tables 7, 8, Figure 5 and Figure 6 The results showed that the growth of tumors was inhibited to varying degrees after administration of each preparation, and there were significant differences compared with the model group (P < 0.01). The tumor inhibition rate of combined administration was slightly higher than that of single administration of the preparation, but there was no statistical difference between the groups, indicating that the addition of disulfide bonds and astragalus polysaccharides did not significantly improve the tumor inhibition rate in nude mice (P > 0.05).

[0072] H&E staining, PCNA detection and TUNEL detection were used to observe tumor tissues to evaluate the therapeutic effect. Figure 7 In the H&E staining diagram, the tumor tissues in the model group were arranged tightly and regularly, with high cell density, deep nuclear staining, and no obvious abnormalities. However, in the treatment group, large areas of necrosis, nuclear condensation, inflammatory infiltration of some cells, and a small amount of red blood cell infiltration and cytoplasmic vacuolation were observed. In the PCNA detection diagram, cells with positive expression were stained red and located in the cell nucleus. In the model group, cells were arranged tightly and proliferated significantly. In each treatment group, cells were arranged loosely, red-stained cells were significantly reduced, and proliferation was inhibited. In the TUNEL detection diagram, cells with positive expression were stained green and located in the cell nucleus. There were very few apoptotic cells in the model group, while the number of apoptotic cells in each treatment group increased significantly. The detection of cell apoptosis by TUNEL method and the detection of proliferation by PCNA method complemented each other. The above results showed that each treatment group played an anti-tumor effect, and its effect was related to inhibiting tumor cell proliferation and promoting tumor cell apoptosis.

[0073] Table 7 Changes in tumor volume after treatment with different drugs (n=5)

[0074] Table 8 Tumor inhibition rate after treatment with different drugs (n=5)

[0075] (6) Safety in animals Depend on Figure 8 It can be seen that the relative weight loss of nude mice in the model group and the positive drug group was relatively large, which may be due to the excessive tumor size in the model group and the hepato-renal toxicity of cisplatin. In contrast, the relative weight of each NCTD preparation did not change significantly, indicating that the NCTD preparation did not have acute toxicity. Fig. 9 As shown, blue represents the cell nucleus and red represents the cytoplasm. The organs of the model group and the NCTD preparation treatment group were normal without obvious lesions, indicating that the NCTD nano preparation did not cause lesions in the organ tissues of nude mice.

[0076] The liver and kidney function indicators (ALT, AST, BUN, CREA) collected from nude mice showed no significant difference in the expression levels of the four factors in each NCTD treatment group compared with the model group (P>0.05), indicating that the nanoparticle preparation had no liver and kidney toxicity in vivo and was biosafe. Fig.10 .

[0077] Summary: In order to investigate Apt-PEG 2000 -ss-NCTD-SLNs combined with APS had an anti-tumor effect on tumor-bearing nude mice. Using normal saline and cisplatin as models and positive controls, the results showed that the cisplatin group had the highest tumor inhibition rate, reaching 72.87±7.59%, while NCTD-SLNs, Apt-PEG 2000 -NCTD-SLNs, Apt-PEG 2000 -ss-NCTD-SLNs and Apt-PEG 2000 The tumor inhibition rates of -ss-NCTD-SLNs+APS were 46.63±9.54, 55.57±9.40, 59.30±8.51, and 61.48±11.03, respectively. The tumor inhibition rate of targeted molecule modification was improved to a certain extent, but the advantages of polysaccharide combination and disulfide bond modification were not reflected. Compared with the model group, the tumor inhibition rates of all groups were significantly different ((P<0.01)). Tumor tissue staining also confirmed that each treatment group had a good effect in inhibiting tumor growth. PCNA detection and TUNEL detection showed that the inhibition of tumor growth may be related to the inhibition of tumor cell proliferation and the promotion of tumor cell apoptosis. Although anticancer drugs such as cisplatin show certain tumor-suppressing effects, they are also accompanied by weight loss and death. However, the NCTD preparation group did not show obvious weight loss. In addition, the results of tissue staining showed that the NCTD preparation had no obvious tissue toxicity, and there was no significant difference in the liver and kidney factors in the plasma of each group compared with the model group (P>0.05). These experimental results show that the nanopreparation has no obvious toxic side effects and has a certain degree of biosafety.

[0078] Example 4 (Study on the preparation of Apt-PEG2000-ss-NCTD-SLNs) (1) Determination of encapsulation efficiency by dialysis method 1) Establishment of dialysis balance Accurately pipette 50 ml of water as the dialysis medium, draw 5 ml of NCTD-SLNs into the dialysis bag, and dialyze at 25 °C and 300 r / min magnetic stirring. Take 10 ml of the dialysis medium and concentrate it to 2 ml at 30 min, 45 min, 60 min, 75 min, 90 min, 120 min, 150 min, and 180 min of dialysis, and inject it through the membrane. The results are shown in the figure below. Fig.11 It can be seen that as the dialysis time increases, the free norcantharidin in the bag is continuously released to the outside of the bag. From 45min to 75min, the dialysis shows a balanced trend. As time continues to increase, the NCTD content outside the bag increases suddenly, indicating that the norcantharidin in the solid lipid nanoparticles in the bag is dialyzed out. This may be because norcantharidin has weak water solubility and the drug is easily dissolved from the lipid in a medium with a large amount of water. Since the trend of equilibrium is shown from 45min to 75min, it is the dialysis equilibrium section, so 45min is selected as the dialysis time point in the future.

[0079] 2) Determination of encapsulation efficiency Accurately pipette 1.0 mL of NCTD-SLNs test sample, add methanol to make it 5 mL to break the emulsion, sonicate, let it stand overnight, sonicate again, make it constant and inject it through the membrane to get the total drug amount; accurately pipette 5.0 mL of NCTD-SLNs into the dialysis bag, dialyze for 45 minutes, take 10 mL of dialysis medium and spin dry, add methanol to make it 2 mL, inject it through the membrane to get the free amount. The encapsulation efficiency (EE) is calculated as follows:

[0080] 3) Determination of drug loading Accurately pipette 2.0 mL of NCTD-SLNs into a weighing bottle that has been dried to a constant weight, freeze-dry for 48 h, weigh, calculate the net weight of the lyophilized powder of NCTD-SLNs, and inject the lyophilized powder; the calculation formula for drug loading (Drμg Loading, DL) is as follows:

[0081] 4) Single factor investigation of NCTD-SLNs prescription and preparation process The encapsulation efficiency and drug loading of NCTD-SLNs were determined according to the above method. Another amount of NCTD-SLNs was taken, filtered through a 0.22μm water-based microporous filter membrane, placed in a sample pool, and the average particle size, PDI and Zeta potential of NCTD-SLNs were determined using a laser particle size analyzer. Single factor investigation was performed using average particle size, PDI, Zeta potential, encapsulation efficiency and drug loading as evaluation indicators.

[0082] (2) Selection of preparation method The preliminary experiments screened the thin film dispersion method, ethanol injection method and emulsification ultrasonic dispersion method. The NCTD-SLNs prepared by the thin film dispersion method had incomplete or granular film formation, and the film was not easy to be washed off; while the sample prepared by the ethanol injection method had poor stability. Therefore, considering the actual experimental conditions and the feasibility of the preparation method, the emulsification ultrasonic dispersion method commonly used in the laboratory was selected for preparation. This method is simple to operate and can reduce the use of toxic reagents such as chloroform. The specific experimental steps of this method are as follows: accurately weigh the prescribed amount of monostearate glyceryl, soybean lecithin and norcantharidin, dissolve them in 5 mL of methanol to form an organic phase, take 15 mL of a certain concentration of poloxamer aqueous solution to form an aqueous phase, heat the two phases to 65 ℃ at the same time, and slowly inject the aqueous phase into the organic phase under 1000 r / min magnetic stirring in a 65 ℃ water bath. Stir at constant temperature for 1h to evaporate the methanol, place it in an ultrasonic cell crusher while hot, cool and solidify at room temperature, and pass through a 0.22μm microporous filter membrane to obtain NCTD-SLNs.

[0083] (3) Selection of phospholipid types The main phospholipids used in solid lipid nanoparticles include egg yolk lecithin, soybean lecithin, and hydrogenated soybean lecithin. Other factors were kept constant, and solid lipid nanoparticles of three different phospholipids were prepared by emulsification ultrasonic dispersion method. The appearance was observed, and the particle size, PDI, Zeta potential, encapsulation efficiency and drug loading were measured. The results are shown in Table 9. During the experimental operation, the sample prepared by hydrogenated soybean lecithin was relatively turbid, while the encapsulation efficiency of soybean lecithin was higher than that of egg yolk lecithin. The results of other indicators were similar and had no obvious effect on the formulation, so soybean lecithin was selected for subsequent experimental optimization.

[0084] Table 9 Phospholipid types

[0085] (4) Dosage According to the literature review, the different properties of drugs and lipids will affect the stability of drugs in lipids. When preparing NCTD-SLNs, the dosage will have a great impact on its encapsulation rate. Fixed other factors, the dosage of NCTD was set to 4mg, 6mg, 8mg and 10mg for investigation, and the appearance was observed. Its particle size, PDI, Zeta potential, encapsulation rate and drug loading were used as indicators for evaluation. The results are shown in Table 10. The increase in dosage will increase the encapsulation first and then decrease, indicating that when the lipid is a certain amount, as the drug increases, part of the drug is encapsulated, and after reaching saturation, the drug is no longer encapsulated, so that the free drug increases and the encapsulation rate decreases. The encapsulation rate of 6mg of drug is high, and with the increase of dosage, the encapsulation rate decreases significantly, so the dosage of norcantharidin in subsequent experiments is 6mg.

[0086] Table 10 Dosage investigation

[0087] (5) Temperature investigation Since lipid materials are solid at room temperature, the preparation temperature should be controlled to be higher than the melting point of lipids so that the drug and lipids are mixed and melted. At the same time, the emulsification temperature will affect the emulsification ability of the emulsifier, and too high a temperature will destroy the emulsification effect.

[45] Therefore, other factors were fixed in this experiment. On the basis of the previous preliminary experiment, the emulsification temperature was set at 55℃, 60℃, 65℃ and 70℃ for investigation. The appearance was observed and the particle size, PDI, Zeta potential, encapsulation efficiency and drug loading were used as indicators for evaluation. The results are shown in Table 11. At 65℃, the encapsulation efficiency reached the maximum value of 76.12%, and the encapsulation efficiency decreased significantly as the temperature continued to increase. Therefore, it can be concluded that temperature is the largest factor affecting the encapsulation efficiency, so the temperature was selected for subsequent process optimization investigation.

[0088] Table 11 Temperature investigation

[0089] (6) Ultrasonic time investigation The ultrasonic process is to redisperse the particles in the system by adding external energy. The longer the ultrasonic time, the smaller the nanoparticle size. However, if the ultrasonic time is too long, the temperature of the solution system will rise, the nanoparticles will aggregate, adhesion will occur, and the drug will leak easily, resulting in instability of the system. And too long ultrasonic time may cause damage to the instrument and metal contamination to the sample. In this experiment, other factors are fixed and the ultrasonic time is set to 3min, 5min, 10min and 15min for investigation. The appearance is observed and evaluated by its particle size, PDI, Zeta potential, encapsulation rate and drug loading. The results are shown in Table 12. If the ultrasonic time is too short, such as 3min, the particle redispersion effect is not good, the particle size and dispersion index are too large, and the encapsulation rate is also small. It may be that the nanoparticles are mostly in an adhesion state, which makes them unstable and the drug is easy to leak out; as the ultrasonic time is prolonged, the encapsulation rate gradually increases, and the maximum value appears at 10min of ultrasonic, and the particle size dispersion is relatively uniform; the encapsulation rate appears at a minimum value at 15min, which may be due to damage to the nanoparticles caused by too long time. From the above results, it can be seen that the ultrasonic time is a factor that has a greater impact on the formulation, so the ultrasonic time is selected to continue the process optimization investigation.

[0090] Table 12 Ultrasound time investigation

[0091] (7) Glyceryl monostearate (GMS) input Glyceryl monostearate is a nonionic surfactant. It has both hydrophilic and lipophilic groups, and has multiple functions such as wetting and emulsification. It is soluble in methanol, ethanol, chloroform, acetone and other solutions, and is often used in the research of solid lipid nanoparticle molding process. The total amount of lipid has a great influence on the molding of the preparation. Increasing the amount of lipid can provide more dissolution space for the drug and increase the drug encapsulation rate, but it will increase the particle size, make the sample turbid, and make it difficult to mold, resulting in reduced stability. Therefore, exploring the amount of lipid input is the key to the molding of solid lipid nanoparticles. In this experiment, other factors were fixed unchanged, and the amount of glyceryl monostearate was set to 25mg, 50mg, 75mg and 100mg for investigation, and the appearance was observed, and its particle size, PDI, Zeta potential, encapsulation rate and drug loading were used as indicators for evaluation. The results are shown in Table 13. With the increase of GMS input, the encapsulation rate first increased and then decreased. When the input amount was 50 mg, the sample was turbid, and the particle size and polydispersity index were also large, while the encapsulation rate of the input amount of 100 mg was extremely low, indicating that both too high and too low GMS inputs would affect the molding of the preparation, indicating that the lipid input is the key factor affecting the molding, so the GMS input was selected to continue the process optimization investigation.

[0092] Table 13 Investigation on the input amount of glyceryl monostearate (GMS)

[0093] (8) Investigation of total stabilizer concentration (poloxamer F68 and soybean lecithin) Literature reports that SLNs prepared by the use of poloxamer and soybean lecithin often have smaller particle size and higher storage stability. They are the most commonly used surfactants, which can promote the formation of preparations. In addition, injection-grade soybean lecithin and poloxamer can meet the requirements of intravenous administration. Combined with preliminary experimental studies, this paper selects poloxamer F68 as an emulsifier. The ratio of the two groups is fixed (poloxamer F68: soybean lecithin is 2:1), and the total stabilizer content is set to 75mg, 150mg, 225mg and 300mg for investigation. The appearance is observed, and its particle size, PDI, Zeta potential, encapsulation rate and drug loading are used as indicators for evaluation. The results are shown in Table 14. Taking the maximum encapsulation rate as the principle and the principle of economy, combined with other indicators, there is no significant effect on the preparation formation. The encapsulation rate reaches the maximum at 150mg, and the encapsulation rate between the groups is not significantly different, so the subsequent experiment is carried out with a total stabilizer content of 150mg.

[0094] Table 14 Total stabilizer concentration investigation

[0095] (9) Investigation of the ratio of poloxamer F68 to soybean lecithin The other factors were fixed and the ratio of poloxamer F68 to soybean lecithin was changed. The ratio of poloxamer F68 to soybean lecithin was set to 4:1, 2:1, 1:1, and 1:2 for investigation. The appearance was observed, and the particle size, PDI, Zeta potential, encapsulation efficiency, and drug loading were measured. The results are shown in Table 15. The encapsulation efficiency was taken as the principle of taking the maximum value, and other indicators were combined to have no significant effect on the formulation. The encapsulation efficiency was the largest at a ratio of 2:1, but the encapsulation efficiency differences between the groups were not significant, so the subsequent experiments were carried out at a ratio of 2:1.

[0096] Table 15 Investigation of the ratio of poloxamer (F-68) to egg lecithin

[0097] (10) Box-Behnken experimental design of NCTD-SLNs formulation and preparation process 10.1 Box-Behnken Experimental Design Based on the results of the above single factor investigation, it was found that hydration temperature (A), GMS input (B), and ultrasonic time (C) had a greater impact on the encapsulation rate of the prescription. Therefore, they were selected as investigation factors to further optimize the prescription. Three levels (-1, 0, +1) were designed, and the encapsulation rate was used as an indicator for evaluation.

[0098] Table 16 Box-Behnken factor level design

[0099] Table 17 Box-Behnken factor level design

[0100] Table 18 Box-Behnken ANOVA

[0101] The data were analyzed using Design-Expert 12 software, and binomial equation fitting and variance analysis were performed on the relationship between the encapsulation efficiency and each investigated factor, resulting in the regression equation: EE%=80.93-3.62A-3.41B+2.62C+3AB+6.65AC+0.0312BC-9.54A 2 -7.86B 2 -5.77C 2 (R 2 =0.9566). The results of variance analysis are shown in Table 14. The results show that the P value of the encapsulation rate model is less than 0.001, and the P value of the dependent variable model lack of fit term is greater than 0.05, indicating that the model fits well and the error is small; the regression equation correction determination coefficient (R 2 adj) was 0.9008, indicating that the model can explain 90.08% of the variation in the response value; the coefficient of variation (CV%) of the model was 4.20%, indicating that the model had high accuracy; the precision value was the ratio of the effective signal to the noise, which was 12.4921>4, indicating that the model was reasonable and that the model could be used for design analysis. In addition, hydration temperature, GMS input, and ultrasonic time all had significant effects on the encapsulation rate (P<0.05), and there was an interaction between hydration temperature and ultrasonic time (P value of AC<0.05), and there was no interaction between the other two items (P values ​​of AB and BC>0.05); the relationship between the factors was not a simple first-order relationship, but a second-order relationship (A 2 , B 2 , C 2 The P value is <0.05). The 3D effect diagram and contour diagram of the response surface of the interaction factors with the encapsulation rate as the evaluation index are shown in Fig.12 .

[0102] 10.2 NCTD-SLNs verification experiment The mathematical model constructed above was comprehensively analyzed using Design-Expert 12 software. Under the premise of ensuring the highest encapsulation rate and fit, the optimal values ​​of each factor were finally determined: hydration temperature 64°C, GMS addition amount 69 mg, and ultrasonic time 11 min. The validation experiment was repeated 3 times for the optimized formulation. The experimental value of the encapsulation rate was consistent with the predicted value, indicating that the prediction results of the Box-Behnken experimental design were accurate. The results are shown in Table 19.

[0103] Table 19 Analysis of NCTD-SLNs verification results

[0104] (11) Long-circulating chain addition amount (mal-PEG2000-DSPE) Based on the preliminary experiments, different masses of mal-PEG were inserted into the optimized prescription. 2000 -DSPE (3mg, 6mg, 9mg, 12mg and 15mg), observed the appearance, measured the particle size, PDI, Zeta potential, encapsulation efficiency and drug loading. The results are shown in Table 20. With the addition of the guiding molecule, the particle size of the nanoparticles became smaller and the absolute value of the Zeta potential became larger and larger. This may be because the PEG modification changed the internal density of the nanoparticles and made their spatial structure more complex. There was no significant change in the encapsulation efficiency among the groups. Considering that the PDI of the 9mg and 12mg additions was less than 0.3, suggesting that they may have better storage stability, and the potential was about -6, according to the principle of economy, the addition amount of the long circulation chain was set at 9mg.

[0105] Table 20 Investigation of the amount of mal-PEG2000-DSPE added

[0106] (12) Investigation of the binding capacity of APS613-1 aptamer 12.1 APS613-1 adapter connection method According to the literature, based on the high expression of GPC3 protein in liver tumor cells, GPC3 protein was selected as the target, and nucleic acid aptamer technology was introduced. The aptamer APS613-1 with high specificity and strong affinity for GPC3 protein was screened by SELEX technology. Its 3' was modified with thiol and incubated with maleimide on mal-PEG2000-NCTD-SLNs in a nitrogen environment at 4°C in the dark for 24 hours (Apt: mal-PEG2000-NCTD-SLNs ratio was 1:50, 1:100, 1:150, 1:200). Agarose gel electrophoresis was used to qualitatively examine whether the aptamer was connected to the solid lipid nanoparticles.

[0107] 12.2 Principle of Agarose Gel Electrophoresis Agarose gel electrophoresis is an electrophoresis method that uses agarose as a medium. Agarose gel has a network structure, and the molecules of substances will encounter resistance when passing through. The larger the molecules, the greater the resistance, and it is difficult for them to move in the pores of the gel. At the same time, different nucleic acids will carry different charges according to different pH values, and the forces they are subjected to in the electric field are different, so different nucleic acids can be separated. Therefore, this method has both charge effect and molecular sieve effect. After consulting the literature, it is known that large molecular weight solid lipid nanoparticles will be retained in the spotting hole, while free aptamers will move with the current to form bright strips.

[0108] 12.3 Agarose gel electrophoresis experiment Weigh 1.4g agarose and dissolve it in 70ml TAE buffer. Heat and boil twice until completely dissolved. Prepare 2% agarose gel solution. Add 7μL Gelred red fluorescent dye solution and shake well. Pour into the glass tank and insert the comb. After the gel cools down for 20 minutes, put it into the electrophoresis pool. Take 5μL of sample and mix with 1μL loading buffer. Load 6μL into each well. Perform electrophoresis at 110V constant voltage for 30 minutes in TAE buffer. Observe and take pictures with gel imaging system. The results are shown in the table. Fig.13 , 14 .

[0109] Depend on Fig.13 It can be seen that the solid lipid nanoparticles without aptamers have almost no fluorescence at the origin, while the solid lipid nanoparticles with aptamers are retained in the wells and emit fluorescence, and there are some free bands, indicating that part of the aptamers have been connected to the solid lipid nanoparticles; Fig.14 It shows that as the relative amount of aptamer added decreases, the brightness of the free aptamer decreases. There is almost no free aptamer at 1:200. Considering the competitive inhibition test of free aptamer on tumor, the concentration with as little free aptamer as possible is selected. At this concentration, the targeting molecules on the surface of solid lipid nanoparticles have reached saturation, which is 1:150. Subsequent experiments determine the amount of aptamer added at this concentration.

[0110] 13 Preliminary study on the physicochemical properties of SLNs modified with various ligands 13.1 Comparison of particle size, PDI, Zeta potential and encapsulation efficiency of Apt-PEG2000-ss-NCTD-SLNs before and after incubation 9 mg of mal-PEG was inserted based on the optimized prescription. 2000 -ss-DSPE, the aptamer was added at a ratio of 1:150 and mixed, and the rest of the process was the same as before, and the particle size, PDI, Zeta potential, and encapsulation efficiency were measured. The results are shown in Table 21. There was no significant change in the indicators before and after incubation, which may be because the aptamer is only a small molecular weight substance relative to the SLNs particles, which is not enough to see the change under macroscopic conditions.

[0111] Table 21 Preliminary characterization results of redox solid lipid nanoparticles

[0112] 13.2 Appearance Visual observation of three types of norcantharidin solid lipid nanoparticles (NCTD-SLNs, Apt-PEG 2000 -NCTD-SLNs, Apt-PEG 2000 -ss-NCTD-SLNs) and took pictures. The results are shown in Fig.15 As can be seen from the figure, the preparation is clear and transparent, with a light blue opalescence, and the sample modified with the targeted molecular chain is even clearer.

[0113] 13.3 Morphological observation Take 1 drop of NCTD-SLNs and Apt-PEG respectively 2000 -ss-NCTD-SLNs suspension was dropped onto a copper grid covered with a support film and allowed to stand and dry at room temperature. The morphology of the nanoparticles was observed under a transmission electron microscopy (TEM). Fig.16 It can be seen that the preparation is spherical, the particle size is relatively uniform, and no serious adhesion phenomenon is observed.

[0114] 13.4 Particle size distribution Take a small amount of solid lipid nanoparticle sample in a cuvette and measure its particle size distribution using a laser particle size analyzer. The particle size distribution diagram is shown in Fig.17 ; The particle size of each NCTD-SLNs is about 100nm.

[0115] 13.5 Zeta potential Take a small amount of solid lipid nanoparticle sample into the sample pool and measure its potential using a laser particle size analyzer. The results are shown in Fig.18 The absolute values ​​of the Zeta potential of each NCTD-SLNs were between 0 and -10.

[0116] Due to Apt-PEG 2000 -ss-NCTD-SLNs is a preparation for injection, so it is necessary to investigate its injection safety. The hemolytic activity of the preparation was evaluated according to the hemolysis and coagulation test method in 1148 of the General Rule 4 of the 2020 Pharmacopoeia.

[0117] 1) Preparation of 2% red blood cell suspension Take blood from a healthy rabbit, stir the plasma with a glass rod to remove the fibrin source to form defibrinated blood, add 10 times the amount of 0.9% sodium chloride solution, shake well, centrifuge at 1000 rpm for 15 min, remove the supernatant, wash the precipitated red blood cells repeatedly with saline solution for 3 times until the supernatant no longer appears red, and add saline to the obtained red blood cells to make a 2% red blood cell suspension.

[0118] 2) Hemolysis test Apt-PEG 2000 -ss-NCTD-SLNs and APS hemolytic activity, 16 10 mL EP tubes were divided into 2 groups, 8 in each group, and numbered. In each group, No. 1-5 were test tubes, and the added sample amounts (NCTD concentration was 0.4 mg / mL, polysaccharide concentration was 2.0 mg / mL) were 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, and 0.5 mL, respectively. No. 6 was the negative control tube, No. 7 was the positive control tube, and No. 8 was the test sample control tube. The specific added amounts are shown in Table 22.

[0119] Table 22 Hemolysis Experiment Protocol

[0120] The EP tube was placed in a 37°C incubator for incubation. After 3 hours, the reaction was terminated by ice bath. The EP tube was centrifuged at low temperature for 10 minutes and then the hemolysis and coagulation reactions were observed. The results were observed with the naked eye and photographed. Fig.19 At the same time, take the supernatant into a cuvette, use an ultraviolet spectrophotometer to measure its absorbance at 576nm, and calculate the hemolysis rate. The results of the hemolysis rate are shown in Tables 2.15 and 2.16. The hemolysis rate is qualified if it is less than 5%. The following is the hemolysis rate calculation formula:

[0121] Among them A 样 is the absorbance value of the sample, A 阴 is the absorbance value of the negative control, A 阳 is the absorbance value of the positive control.

[0122] Depend on Fig.19 It can be observed that the solution in the upper part of the test sample 1-5 tubes is clear and transparent, and almost all the red blood cells sink, which is not significantly different from the negative tube 6, indicating that almost no hemolysis occurs. The hemolysis rate calculated by the ultraviolet spectrophotometer is less than 1%, indicating that there is almost no hemolysis and it can be used for subsequent intravenous administration.

[0123] Table 23 Apt-PEG2000-ss-NCTD-SLNs hemolysis test results

[0124] Table 24 APS hemolysis test results

[0125] The samples prepared by the thin film dispersion method and ethanol injection method in the preliminary experiments were not stable and used highly toxic solvents. The selected emulsified ultrasonic dispersion method is simpler and relatively more stable. The NCTD-SLNs with optimized prescription by Box-Behnken experimental design had an encapsulation efficiency of about 80%, which was consistent with the predicted value and the experimental results were accurate. Mal-PEG of different masses was inserted on the basis of the optimized prescription. 2000 -DSPE (3mg, 6mg, 9mg, 12mg and 15mg), and their particle size, PDI, Zeta potential, encapsulation efficiency and drug loading were measured respectively. With the addition of the guiding molecule, the particle size of the nanoparticles gradually decreased, and the absolute value of the Zeta potential gradually increased, but there was no significant difference in the encapsulation efficiency among the groups. Considering that the PDI of 9mg and 12mg was less than 0.3, suggesting that it may have better storage stability, and the potential was about -6, according to the principle of economy, the amount of guiding molecule added was determined to be 9mg, that is, mal-PEG 2000 -NCTD-SLNs, mal-PEG was prepared by the same method 2000 -ss-NCTD-SLNs.

[0126] mal-PEG 2000 The maleimide group on ss-NCTD-SLNs can react with the thiol group on SH-APS613-1 to form a Michael addition reaction. 2000 -ss-NCTD-SLNs and SH-APS613-1 were incubated in a 4°C nitrogen environment in the dark for 24 hours, and the amount of guiding molecules added was examined by agarose gel electrophoresis. Due to competitive inhibition, a concentration with as little free aptamer as possible should be selected, and at this concentration, the surface targeting molecules of solid lipid nanoparticles have reached saturation. The experimental results showed that the optimal mass ratio of APS613-1 to SLNs was 1:150. Before and after the aptamer connection, the particle size, PDI, Zeta potential, and encapsulation efficiency of SLNs did not change significantly. Further Apt-PEG 2000 The appearance, transmission electron microscopy, and hemolysis of -ss-NCTD-SLNs were evaluated and all met the requirements for liver-targeted preparations of solid lipid nanoparticles and could be used for intravenous injection in subsequent in vivo experiments.

[0127] A person skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of protection of the present application is limited to these examples. In line with the concept of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0128] One or more embodiments of the present application are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the present application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of the present application should be included in the protection scope of the present application.

Claims

1. An anti-liver tumor composition, characterized in that: The invention comprises solid lipid nanoparticles of norcanthaxanthin and astragalus polysaccharide. The concentration of the solid lipid nanoparticles of norcanthaxanthin is 1.0-2.0 μg / mL, the concentration of the astragalus polysaccharide is 300-900 μg / mL, and the concentration ratio of the astragalus polysaccharide to the solid lipid nanoparticles of norcanthaxanthin is in the range of 150-900.

2. The anti-liver tumor composition according to claim 1, characterized in that: The cantharicin solid lipid nanoparticles include cantharicin solid lipid nanoparticles NCTD-SLNs, cantharicin solid lipid nanoparticles Apt-PEG 2000 -NCTD-SLNs and norcanthracen solid lipid nanoparticles Apt-PEG 2000 -ss-NCTD-SLNs at least one of the three; the NCTD-SLNs of the demethylcanthate comprises demethylcanthate and solid lipid nanoparticles; the Apt-PEG solid lipid nanoparticles of the demethylcanthate 2000 -NCTD-SLNs or norcanthracen solid lipid nanoparticles Apt-PEG 2000 -ss-NCTD-SLNs include norcanthracen, solid lipid nanoparticles, long circulating chains and aptamers.

3. The anti-liver tumor composition according to claim 2, characterized in that: The solid lipid nanoparticles are soybean lecithin, and the long circulating chain is Mal-PEG 2000 -ss-DSPE or Mal-PEG 2000 -DSPE.

4. The anti-liver tumor composition according to claim 2, characterized in that: The aptamer is APS613-1.

5. A method for preparing the anti-liver tumor composition according to any one of claims 1 to 4, characterized in that: The preparation method of the norcantharidin solid lipid nanoparticles comprises the following steps: weighing glyceryl monostearate, soybean lecithin and norcantharidin, dissolving them in methanol to form an organic phase, taking a poloxamer aqueous solution to form an aqueous phase, heating the two phases at the same time, slowly injecting the aqueous phase into the organic phase in a water bath, stirring to volatilize methanol, ultrasonicating while hot, cooling and solidifying, and filtering to obtain the norcantharidin solid lipid nanoparticles NCTD-SLNs.

6. The method for preparing the anti-liver tumor composition according to claim 5, characterized in that: The preparation method of the norcanthracen solid lipid nanoparticles further comprises the following steps: adding a long circulating chain Mal-PEG to the norcanthracen solid lipid nanoparticles NCTD-SLNs; 2000 -DSPE or Mal-PEG 2000 -ss-DSPE, forming Mal-PEG 2000 -NCTD-SLNs or Mal-PEG 2000 -ss-NCTD-SLN; the aptamer APS613-1 was combined with the Mal-PEG 2000 -NCTD-SLNs or Mal-PEG 2000 -ss-NCTD-SLN was incubated in a nitrogen environment at 4°C in the dark for 24 h to obtain the cantharicin solid lipid nanoparticles Apt-PEG 2000 -NCTD-SLNs or norcanthracen solid lipid nanoparticles Apt-PEG 2000 -ss-NCTD-SLNs.

7. The method for preparing the anti-liver tumor composition according to claim 6, characterized in that: The mass ratio of the APS613-1 to the demethylcanthacetin solid lipid nanoparticles NCTD-SLNs is 1:

150.

8. The method for preparing the anti-liver tumor composition according to claim 5, characterized in that: The mass ratio of poloxamer to soybean lecithin is 2:

1.

9. The method for preparing the anti-liver tumor composition according to claim 5, characterized in that: The ultrasonic time is 10 min, and the heating temperature is 65°C.

10. Use of the anti-liver tumor composition according to any one of claims 1 to 4 for preparing anti-liver tumor drugs.

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