Preparation method and application of broad-spectrum anti-tumor nano-liposome

Abstract

The invention provides a preparation method and application of broad-spectrum anti-tumor nano-liposome, and the preparation method comprises the following steps: dissolving 15mg of ginsenoside Rg3 and 50mg of PL-100M in 10ml of absolute ethyl alcohol together, then transferring into an eggplant-shaped bottle, and carrying out reduced pressure rotary evaporation until a solvent is volatilized and a uniform film is visible on the bottle wall; the preparation method comprises the following steps: dissolving 5mg of cysteine and 0.5 g of glucose in 10ml of pure water, adding an aqueous solution into an eggplant-shaped bottle, and carrying out ultrasonic hydration until a film completely falls off to obtain a lipidosome crude suspension; and carrying out ultrasonic treatment on the suspension for 30 times under 270W by using a probe ultrasonic instrument to form the Rg3-Lp / Cysteine liposome. The novel cell death type, namely disulfur death, is targeted to tumor metabolism, the target spot is GLUT1 which is generally and highly expressed on the surfaces of tumor cells, and the anti-tumor effect of the nano-liposome has broad spectrum and has no influence on normal tissues.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a preparation method and application of a broad-spectrum anti-tumor nanoliposome. Background Art

[0002] Conventional therapies such as radiotherapy, chemotherapy and targeted therapy have been widely used in clinical practice and are common treatments for tumors, but they still have limitations. Radiotherapy and chemotherapy have poor targeting, are prone to damage normal tissues, and are prone to drug resistance. Targeted therapy is highly targeted and has few side effects, but its scope of application is limited, the efficacy varies from individual to individual, and the cost of treatment is high. Targeted metabolism is a new means of selectively acting on tumor cells. Since tumor cells usually undergo changes in their metabolic profiles, the metabolic-related phenotypes and genes that are differentially expressed between tumor cells and normal cells can be used as key targets for tumor metabolic therapy. However, the therapeutic effect of metabolic targeted drugs is limited by their off-target toxicity to immune cells and high-dose-mediated side effects. Therefore, the present invention proposes a preparation method and application of a broad-spectrum anti-tumor nanoliposome. Summary of the invention

[0003] In view of the technical problems mentioned in the background technology, the present invention provides a preparation method and application of a broad-spectrum anti-tumor nanoliposome.

[0004] The technical solution adopted by the present invention to solve its technical problem is:

[0005] A method for preparing a broad-spectrum anti-tumor nanoliposome comprises the following steps:

[0006] Dissolve 15 mg of ginsenoside Rg3 and 50 mg of PL-100M in 10 ml of anhydrous ethanol, then transfer to an eggplant-shaped bottle and perform rotary evaporation under reduced pressure until the solvent is evaporated and a uniform film can be seen on the bottle wall;

[0007] Dissolve 5 mg of cysteine ​​and 0.5 g of glucose in 10 ml of pure water, add the aqueous solution into an eggplant-shaped bottle, and ultrasonically hydrate until the film completely falls off to obtain a crude liposome suspension;

[0008] The suspension was sonicated 30 times at 270 W (working time 5 s, rest time 5 s, protection temperature 25° C.) using a probe sonicator to form Rg3-Lp / Cysteine ​​liposomes.

[0009] Another object of the present invention is to provide a method for preparing broad-spectrum anti-tumor nanoliposomes and use the Rg3-Lp / Cysteine ​​liposomes prepared in anti-tumor applications.

[0010] As a further technical solution of the present invention, the application includes:

[0011] In vivo efficacy evaluation: A 4T1 tumor-bearing mouse model was established and used. 2×10 6 4T1 cells were suspended in RPMI-1640 medium and inoculated subcutaneously on the right back of BALB / c female mice. The tumor size was measured with a digital caliper and the tumor volume was calculated. When the in situ tumor volume increased to 50-100 mm 3 The mice were randomly divided into 3 groups (n=5): Control, Rg3+Cysteine ​​and Rg3-Lp / Cysteine; each mouse was given drug (Rg3: 1500 μg / mL, 200 μL) via tail vein, once every 2 days, for a total of 3 injections. The tumor volume and body weight of the mice were recorded every other day during the treatment. When the tumor volume was greater than 2000 mm 3 Or when the mice died, the mice were considered dead. After 17 days, the experimental animals were killed and the tumor volume inhibition rate TIR was calculated.

[0012] As a further technical solution of the present invention, the application includes:

[0013] In vitro cytotoxicity assay: CCK-8 colorimetric assay was used to detect the cytotoxicity of Rg3-Lp / Cysteine ​​to breast cancer cell 4T1, melanoma cell B16, lung cancer cell LLC and colon cancer cell CT26. The cells were plated at 1×10 4 (100 μL) cells were seeded in a 96-well plate and cultured overnight. After cell adhesion, C-Lp, Rg3, Cysteine, Rg3-Lp / Cysteine ​​or Rg3+Cysteine ​​(free drug) was diluted with culture medium; 100 μL (Rg3: 15 μg / mL) was added to each well, and culture was continued for 36 hours. The culture medium was removed and 100 μL of 10% CCK-8 culture medium was added and incubated for another 2 hours. The OD value of the cells at 450 nm was recorded with an enzyme marker. In addition, mouse mammary epithelial cells HC11 were used to investigate the toxicity of Rg3-Lp / Cysteine ​​to normal mouse cells.

[0014] As a further technical solution of the present invention, the application includes:

[0015] Fluorescence staining of actin: Tumor cells 4T1, CT26 and normal cells HC11 were used as model cells. The model cells were cultured at 1.5×10 5The cells were inoculated at a density of 10 cells / mL in a 20 mm glass-bottomed culture dish and incubated overnight in an incubator. After cell adhesion, the original culture medium was discarded and the cells were treated with 2 mL of PBS, Rg3+Cysteine, and Rg3-Lp / Cysteine ​​culture medium (Rg3: 15 μg / mL), respectively. After incubation in a 37°C incubator for 4 h, the culture medium was removed and the cells were washed twice with PBS (pH 7.4), fixed with 4% paraformaldehyde on ice for 15 min, the fixed cells were permeabilized with a membrane permeabilization solution for 5 min at room temperature, and then washed twice with PBS. The cells were incubated in phalloidin at room temperature in the dark for 20 min, actin was stained, and finally the cells were sealed with DAPI and stained at the same time, and observed using a laser confocal microscope.

[0016] As a further technical solution of the present invention, the application includes:

[0017] NADP(H) determination: Tumor cells 4T1, CT26 and normal cells HC11 were used as model cells, and intracellular NADPH and NADP(H) total (NADPH+NADP + ) using NADP + / NADPH detection kit (WST-8), cells were counted at 4×10 5 (2ml) cells were inoculated into 6-well plates. After the cells were confluent, the culture medium was aspirated and 200μl of NADP was added to each well. + / NADPH extract and gently blow to promote cell lysis, centrifuge the lysate for 10 min (12000g, 4°C), pipette 50μl supernatant into a 96-well plate, add 100μl G6PDH working solution to each well, incubate at 37°C in the dark for 10 min, add 10μl colorimetric solution to each well, mix well, incubate at 37°C in the dark for 30 min, measure the absorbance at 450nm, and measure NADP(H) total ;

[0018] After centrifugation, the lysate was heated in a 60°C water bath for 30 min and the NADPH content was determined according to the above method. total Subtract NADPH to calculate NADP + concentration.

[0019] The beneficial effects of the present invention are:

[0020] The present invention provides a preparation method and application of a broad-spectrum anti-tumor nanoliposome based on a disulfide death strategy. Disulfide death is different from other existing forms of cell death and is a new form of regulated cell death (RCD), which reveals a new mechanism for organisms to resist the malignant progression of tumors and provides new perspectives and targets for tumor research and treatment. Disulfide death is mainly based on the inhibition of NADPH production and the subsequent accumulation of disulfide cystine to induce the shrinkage of actin. With glucose starvation as a prerequisite, pharmacological blockade of GLUT1 to inhibit glucose uptake will induce tumor cell death. When Rg3 is used as a liposome membrane stabilizer to insert into a bilayer phospholipid, the exposed sugar group can bind to GLUT1 on the surface of tumor cells, inhibiting the uptake of glucose by tumor cells through GLUT1, inhibiting the accumulation of NADPH in tumor cells, and promoting disulfide stress. The present invention uses the GLUT1 targeted binding of Rg3 for the first time to study its anti-tumor effect based on glucose metabolism interference, which is a new perspective for Rg3 anti-tumor research.

[0021] In 2000, Rg3 was developed and approved as Shenyi Capsule in China as a clinical first-line anticancer drug. A large number of clinical trials have shown that Shenyi Capsule significantly improves the therapeutic effect of chemotherapy and reduces the side effects of chemotherapy, indicating the safety of Rg3. Compared with highly selective GLUT1 inhibitors, off-target toxicity and high-dose mediated side effects to immune cells and normal cells are avoided. Cysteine ​​is an essential amino acid for the body and an intermediate product of amino acid metabolism. It is required for the normal activities of various cells and is non-toxic. It is only oxidized to cystine in an oxidative environment caused by glucose starvation, resulting in the accumulation of disulfide, generating disulfide stress, and causing cell death. Therefore, due to the safety of Rg3 and cysteine ​​and its simple formulation design, the present invention does not have the side effects of anti-tumor therapies such as radiotherapy and chemotherapy.

[0022] The present invention utilizes a new type of cell death - disulfide death, which targets tumor metabolism. The target is GLUT1 which is commonly and highly expressed on the surface of tumor cells. The anti-tumor effect of nanoliposomes has a broad spectrum and has no effect on normal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a characterization diagram of Rg3-Lp / Cysteine.

[0024] Figure 2 The selective tumor cell cytotoxicity graph of Rg3-Lp / Cysteine.

[0025] Figure 3 This is a wrinkled image of actin.

[0026] Figure 4 This is the determination diagram of NADP(H).

[0027] Figure 5 This is a diagram of the selective targeting of Rg3-Lp to tumor cells.

[0028] Figure 6 This is a diagram for in vivo drug efficacy evaluation.

[0029] Figure 7 This is a safety evaluation diagram. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The present invention provides a method for preparing a broad-spectrum anti-tumor nanoliposome, comprising the following steps:

[0032] Dissolve 15 mg of ginsenoside Rg3 and 50 mg of PL-100M in 10 ml of anhydrous ethanol, then transfer to an eggplant-shaped bottle and perform rotary evaporation under reduced pressure until the solvent is evaporated and a uniform film can be seen on the bottle wall;

[0033] Dissolve 5 mg of cysteine ​​and 0.5 g of glucose in 10 ml of pure water, add the aqueous solution into an eggplant-shaped bottle, and ultrasonically hydrate until the film completely falls off to obtain a crude liposome suspension;

[0034] The suspension was sonicated 30 times at 270 W (working time 5 s, rest time 5 s, protection temperature 25° C.) using a probe sonicator to form Rg3-Lp / Cysteine ​​liposomes.

[0035] The particle size of Rg3-Lp / Cysteine ​​was investigated by laser particle size analyzer, and the morphology of Rg3-Lp / Cysteine ​​was observed by transmission electron microscopy (TEM).

[0036] See Figure 1 The particle size of Rg3-Lp / Cysteine ​​is (110.37±0.87) nm, the PDI is 0.094±0.011, and the liposomes are evenly dispersed and are round-like spheres with smooth surfaces.

[0037] The fluorescent dye-loaded liposomes were prepared by thin film dispersion method. 15 mg of ginsenoside Rg3, 2 mg of FITC, and 50 mg of PL-100M were dissolved in 10 ml of anhydrous ethanol to prepare FITC-Rg3-Lp liposomes.

[0038] Another object of the present invention is to provide a method for preparing broad-spectrum anti-tumor nanoliposomes and use the Rg3-Lp / Cysteine ​​liposomes prepared in anti-tumor applications.

[0039] The applications include:

[0040] In vitro cytotoxicity assay: CCK-8 colorimetric assay was used to detect the cytotoxicity of Rg3-Lp / Cysteine ​​to 4T1, B16, LLC and CT26 tumor cells. 4 (100 μL) cells were inoculated in a 96-well plate and cultured overnight. After cell adhesion, C-Lp, Rg3, Cysteine, Rg3-Lp / Cysteine ​​or Rg3+Cysteine ​​(free drug) was diluted with culture medium; 100 μL (Rg3: 15 μg / mL) was added to each well, and culture was continued for 36 hours. The culture medium was removed and 100 μL of 10% CCK-8 culture medium was added for further incubation for 2 hours. The OD value of the cells at 450 nm was recorded with an enzyme marker. In addition, mouse mammary epithelial cells HC11 were used to investigate the toxicity of Rg3-Lp / Cysteine ​​to normal mouse cells.

[0041] See Figure 2 , the viability of tumor cells after treatment with Rg3+Cysteine ​​(free drug) and Rg3-Lp / Cysteine ​​was less than 50%. Compared with Rg3+Cysteine, Rg3-Lp / Cysteine ​​was more toxic to 4T1, B16, and LLC, and the cell viability was less than 5%. After treatment with Rg3-Lp / Cysteine, the viability of CT26 cells was 32.19%, which was significantly different from the control group. This shows that Rg3 and Cysteine ​​have significant tumor cell toxicity in synergy, and the liposome nano-drug delivery system can promote drug uptake and further enhance the killing effect of drugs on tumor cells. There was no significant difference in the cell viability of mammary epithelial cells HC11 after drug treatment, proving that the preparation is non-toxic to normal cells.

[0042] The synergistic treatment of Rg3 and Cysteine ​​has a broad spectrum of tumor cell toxicity and has no obvious effect on normal cells. This is because tumor cells generally highly express GLUT1, and Rg3-Lp / Cysteine ​​has a stronger affinity for tumor cells and selectively inhibits the glucose uptake of tumor cells. It is a safe and effective treatment strategy.

[0043] Fluorescence staining of actin: Tumor cells 4T1, CT26 and normal cells HC11 were used as model cells. The model cells were cultured at 1.5×10 5The cells were inoculated at a density of 10 cells / mL in a 20 mm glass-bottomed culture dish and incubated overnight in an incubator. After cell adhesion, the original culture medium was discarded and the cells were treated with 2 mL of PBS, Rg3+Cysteine, and Rg3-Lp / Cysteine ​​culture medium (Rg3: 15 μg / mL), respectively. After incubation in a 37°C incubator for 4 h, the culture medium was removed and the cells were washed twice with PBS (pH 7.4), fixed with 4% paraformaldehyde on ice for 15 min, the fixed cells were permeabilized with a membrane permeabilization solution for 5 min at room temperature, and then washed twice with PBS. The cells were incubated in phalloidin at room temperature in the dark for 20 min, actin was stained, and finally the cells were sealed with DAPI and stained at the same time, and observed using a laser confocal microscope.

[0044] See Figure 3 Compared with the control group, the morphology of 4T1 actin microfilaments was destroyed, and CT26 actin was obviously wrinkled, indicating that Rg3+Cysteine ​​(free drug) and Rg3-Lp / Cysteine ​​treatment caused the disorder of tumor cell cytoskeleton actin. Compared with the control group, HC11 actin did not change significantly. Both free administration and preparation administration will destroy the cytoskeleton actin structure of tumor cells, but have no effect on HC11, which is consistent with the cytotoxicity results. It shows that the main reason for the synergistic induction of tumor cell death by Rg3 and Cysteine ​​is the destruction of the cytoskeleton, and the cells undergo disulfide death.

[0045] NADP(H) determination: Tumor cells 4T1, CT26 and normal cells HC11 were used as model cells, and intracellular NADPH and NADP(H) total (NADPH+NADP + ) using NADP + / NADPH detection kit (WST-8), cells were counted at 4×10 5 (2ml) cells were inoculated into 6-well plates. After the cells were confluent, the culture medium was aspirated and 200μl of NADP was added to each well. + / NADPH extract and gently blow to promote cell lysis, centrifuge the lysate for 10 min (12000g, 4°C), pipette 50 μl of supernatant into a 96-well plate, add 100 μl of G6PDH working solution to each well, incubate at 37°C in the dark for 10 min, add 10 μl of color development solution to each well, mix well, incubate at 37°C in the dark for 30 min, measure the absorbance at 450 nm, and measure NADP (H) total ;

[0046] After centrifugation, the lysate was heated in a 60°C water bath for 30 min and the NADPH content was determined according to the above method. total Subtract NADPH to calculate NADP+ concentration.

[0047] See Figure 4 For 4T1 and CT26 tumor cells, compared with the Control group, the NADP(H) in the Rg3+Cysteine ​​(free drug) and Rg3-Lp / Cysteine ​​groups total The values ​​of NADP in the Rg3+Cysteine ​​(free drug) and Rg3-Lp / Cysteine ​​groups were significantly decreased compared with the Control group. + / NADPH decreased significantly, while NADPH value had no significant difference, indicating that there may be conversion of cysteine ​​to cystine. Normal cell HC11 NADP(H) total There was no significant difference in the values ​​among the three groups, that is, HC11 had no effect on glucose uptake.

[0048] Cellular uptake of liposomes: Confocal laser scanning microscopy (CLSM) was used to examine the cellular internalization of C-Lp / Cysteine, Rg3-Lp / Cysteine, and free drugs (Rg3+Cysteine) by 4T1, CT26, and HC11 cells. For better observation, we prepared FITC-Rg3-Lp, FITC-C-Lp, and FITC-Rg3 (free drugs). Cells were cultured at 1.5×10 5 The cells were seeded at a density of 10 cells / mL in a 20 mm glass-bottomed culture dish and incubated in an incubator overnight. The original culture medium was discarded, and then 2 mL of FITC-Rg3-Lp, FITC-C-Lp and FITC-Rg3 (free drug) (FITC concentration was 2 μg / mL) were added to the glass-bottomed culture dish. After incubation in a 37°C incubator for 2 and 4 h, the culture medium was removed and the cells were washed twice with PBS (pH 7.4). The cells were fixed with 4% paraformaldehyde on ice for 15 min, and then the cell nuclei were labeled with DAPI, and the images were observed and photographed using a confocal fluorescence microscope.

[0049] See Figure 5For the cell uptake of tumor cells (4T1, CT26), after incubation for 2h in the free drug group (FITC-Rg3), basically no FITC was taken up by cells, and the green fluorescence was negligible. With the extension of incubation time, the amount of cell uptake gradually increased. After incubation for 2h in FITC-C-Lp, only a small amount of FITC was taken up by cells, while the FITC-Rg3-Lp group showed strong green fluorescence, and the cell uptake level was significantly higher than that of the free drug group and the FITC-C-Lp group. This obvious difference indicates that Rg3 liposomes can target tumor cells and help the drugs in liposomes to be taken up specifically and quickly by tumor cells. For the cell uptake of normal cells (HC11), after incubation for 2h and 4h in the free drug group (FITC-Rg3), basically no FITC was taken up by cells. After incubation for 2h in FITC-C-Lp, only a small amount of FITC was taken up by cells, and the cell uptake level of the FITC-Rg3-Lp group was significantly higher than that of the free drug group and the FITC-C-Lp group, which was not much different. The results showed that drugs encapsulated in liposomes were more conducive to cell uptake than drugs in free state. However, due to the low expression of GLUT1 in normal cells, Rg3 liposomes did not show obvious specific uptake in normal cells.

[0050] In vivo efficacy evaluation: A 4T1 tumor-bearing mouse model was established and used. 2×10 6 4T1 cells were suspended in RPMI-1640 medium and inoculated subcutaneously on the right back of BALB / c female mice. The tumor size was measured with a digital caliper and the tumor volume was calculated. When the in situ tumor volume increased to 50-100 mm 3 The mice were randomly divided into 3 groups (n=5): Control, Rg3+Cysteine ​​and Rg3-Lp / Cysteine; each mouse was given drug (Rg3: 1500 μg / mL, 200 μL) via tail vein, once every 2 days, for a total of 3 injections. The tumor volume and body weight of the mice were recorded every other day during the treatment. When the tumor volume was greater than 2000 mm 3 Or when the mice died, the mice were considered dead. After 17 days, the experimental animals were killed and the tumor volume inhibition rate TIR was calculated.

[0051] See Figure 6 On day 17, the tumor volumes of both the control group and the free drug group were greater than 1500 mm 3 . Among them, the control group had the largest tumor volume, and the free drug group had a slightly smaller tumor volume, but there was no significant difference between the two groups. Rg3-Lp / Cysteine ​​significantly inhibited tumor growth, and the tumor volume was lower than that of the control group and the free drug group, with a tumor inhibition rate of 36%. This shows that the free drug has almost no anti-tumor efficacy, and the co-drug-loaded liposomes have a stronger anti-tumor effect, suggesting that ginsenoside Rg3 and cysteine ​​have a certain in vivo anti-tumor activity synergistically.

[0052] Safety assessment: In order to investigate the in vivo safety of the drug, normal mice were directly treated with the drug. BALB / c mice were randomly divided into 3 groups (n=5): (1) Control, (2) Rg3-Lp / Cysteine ​​(Low, the same as the therapeutic dose), (3) Rg3-Lp / Cysteine ​​(High, twice the therapeutic dose). Each mouse was injected intraperitoneally with the drug once every 2 days, for a total of 9 injections. The body weight of the mice was recorded every other day during the treatment period. Two days after the last administration, the mice were killed and the five internal organs were taken for H&E staining. The sections were scanned with a microscope slide scanner (PannoramicMIDI) and analyzed and photographed using the accompanying caseviewer software.

[0053] See Figure 7 The HE staining results of the main organs showed that there was no obvious inflammatory response and damage in the heart, liver, spleen, lung, and kidney of each medication group, indicating that Rg3-Lp / Cysteine ​​has good in vivo safety.

[0054] The invention prepares a broad-spectrum anti-tumor nanoliposome based on a disulfide death strategy, which can achieve a broad-spectrum anti-tumor effect and has no side effects on normal tissues.

[0055] Disulfide death is a new type of cell death mediated by disulfide stress. Studies have found that glucose starvation-induced cystine accumulation can lead to cytoskeleton actin disulfide cross-linking and cytoskeleton contraction, peeling off from the plasma membrane, and ultimately leading to cell death. Under normal physiological conditions, cystine is transported into the cell and rapidly reduced to cysteine ​​to offset disulfide stress and maintain cell survival. Cystine and cysteine ​​can be converted into each other in the cell according to the redox status.

[0056] Among them, NADPH provides key reducing power to prevent the formation of disulfide bonds between actin cytoskeletal proteins, thereby inhibiting disulfide stress-induced cell death. NADPH is mainly produced by the pentose phosphate pathway in glucose metabolism. Inhibiting cellular uptake of glucose and then inhibiting subsequent intracellular NADPH production, placing the cell in an oxidative environment, will cause cystine accumulation, causing disulfide stress and disulfide cell death. In existing studies, the induction of glucose starvation is mainly achieved by inhibitors of highly targeted glucose transporter 1 (GLUT1), but since a small amount of GLUT1 is also expressed on normal cells, off-target toxicity may occur. Therefore, we selected the traditional Chinese medicine Rg3, which also has GLUT1 targeting effect, to study its ability to induce disulfide death in tumor cells.

[0057] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0058] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing a broad-spectrum anti-tumor nanoliposome, characterized in that: The following steps are involved: Dissolve 15 mg of ginsenoside Rg3 and 50 mg of PL-100M in 10 ml of anhydrous ethanol, then transfer to an eggplant-shaped bottle and perform rotary evaporation under reduced pressure until the solvent is evaporated and a uniform film can be seen on the bottle wall; Dissolve 5 mg of cysteine ​​and 0.5 g of glucose in 10 ml of pure water, add the aqueous solution into an eggplant-shaped bottle, and ultrasonically hydrate until the film completely falls off to obtain a crude liposome suspension; The suspension was sonicated 30 times at 270 W using a probe sonicator to form Rg3-Lp / Cysteine ​​liposomes.

2. Use of Rg3-Lp / Cysteine ​​liposomes prepared by the method for preparing a broad-spectrum anti-tumor nanoliposome as claimed in claim 1 in anti-tumor.

3. The use of Rg3-Lp / Cysteine ​​liposomes prepared by the method for preparing a broad-spectrum anti-tumor nanoliposome according to claim 2 in anti-tumor, characterized in that: The applications include: In vivo efficacy evaluation: A 4T1 tumor-bearing mouse model was established and used. 2×10 6 4T1 cells were suspended in RPMI-1640 medium and inoculated subcutaneously on the right back of BALB / c female mice. The tumor size was measured with a digital caliper and the tumor volume was calculated. When the in situ tumor volume increased to 50-100 mm 3 The mice were randomly divided into three groups: Control, Rg3+Cysteine ​​and Rg3-Lp / Cysteine. The drug was given to each mouse through the tail vein once every 2 days for a total of 3 injections. The tumor volume and body weight of the mice were recorded every other day during the treatment. When the tumor volume was greater than 2000mm 3 Or the mice were considered dead when they died. After 17 days, the experimental animals were killed and the tumor volume inhibition rate TIR was calculated.

4. The use of Rg3-Lp / Cysteine ​​liposomes prepared by the method for preparing a broad-spectrum anti-tumor nanoliposome according to claim 2 in anti-tumor, characterized in that: The applications include: In vitro cytotoxicity assay: CCK-8 colorimetric assay was used to detect the cytotoxicity of Rg3-Lp / Cysteine ​​to breast cancer cell 4T1, melanoma cell B16, lung cancer cell LLC and colon cancer cell CT26. The cells were plated at 1×10 4 Cells were seeded in a 96-well plate and cultured overnight. After cell adhesion, C-Lp, Rg3, Cysteine, Rg3-Lp / Cysteine ​​or Rg3+Cysteine ​​was diluted with culture medium; 100 μl was added to each well, and culture was continued for 36 hours. The culture medium was removed and 100 μl of 10% CCK-8 culture medium was added for further incubation for 2 hours. The OD value of the cells at 450 nm was recorded with a microplate reader. In addition, mouse mammary epithelial cells HC11 were used to investigate the toxicity of Rg3-Lp / Cysteine ​​to normal mouse cells.

5. The use of Rg3-Lp / Cysteine ​​liposomes prepared by the method for preparing a broad-spectrum anti-tumor nanoliposome according to claim 2 in anti-tumor, characterized in that: The applications include: Fluorescence staining of actin: Tumor cells 4T1, CT26 and normal cells HC11 were used as model cells. The model cells were cultured at 1.5×10 5 The cells were inoculated at a density of 10 cells / mL in a 20 mm glass-bottomed culture dish and incubated overnight in an incubator. After cell adhesion, the original culture medium was discarded and the cells were treated with 2 mL of PBS, Rg3+Cysteine, and Rg3-Lp / Cysteine ​​culture medium, respectively. After incubation in a 37°C incubator for 4 h, the culture medium was removed and the cells were washed twice with PBS, fixed with 4% paraformaldehyde on ice for 15 min, the fixed cells were permeabilized with a membrane permeabilization solution for 5 min at room temperature, and then washed twice with PBS. The cells were incubated in phalloidin at room temperature in the dark for 20 min, actin was stained, and finally the cells were sealed with DAPI and the cell nuclei were stained, and observed using a laser confocal microscope.

6. The use of Rg3-Lp / Cysteine ​​liposomes prepared by the method for preparing a broad-spectrum anti-tumor nanoliposome according to claim 2 in anti-tumor, characterized in that: The applications include: NADP(H) determination: Tumor cells 4T1, CT26 and normal cells HC11 were used as model cells, and intracellular NADPH and NADP(H) total Leveraging NADP + / NADPH detection kit, cells were counted at 4×10 5 The cells were inoculated into 6-well plates. After the cells were fully grown, the culture medium was aspirated and 200 μl of NADP was added to each well. + / NADPH extract and gently blow to promote cell lysis, centrifuge the lysate for 10 minutes, aspirate 50μl supernatant into a 96-well plate, add 100μl G6PDH working solution to each well, incubate at 37℃ in the dark for 10 minutes, add 10μl colorimetric solution to each well, mix well, incubate at 37℃ in the dark for 30 minutes, measure the absorbance at 450nm, and measure NADP(H) total ; After centrifugation, the lysate was heated in a 60°C water bath for 30 min, and the NADPH content was measured. total Subtract NADPH to calculate NADP + concentration.

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