Tripdiolide nano-liposome as well as preparation and application thereof

Abstract

The invention discloses a tripdiolide nano-liposome, a preparation method thereof and application of the tripdiolide nano-liposome in preparation of drugs for treating esophageal squamous carcinoma. The preparation method comprises the following steps: firstly, mixing lecithin, cholesterol, polyethylene glycol-phospholipid and tripdiolide to prepare a lipid membrane, then adding an ethanol solution of tripdiolide and a PBS (Phosphate Buffer Solution) to prepare primary emulsion, and then carrying out high-pressure homogenization to obtain the nano-liposome. According to the invention, the water solubility and in-vivo circulation stability of tripdiolide can be enhanced, the drug delivery efficiency can be improved, the enrichment of drugs at tumor sites can be realized, and the damage to normal tissues can be reduced.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the construction of a nanomedicine delivery system and its application in the field of anti-tumor therapy, and particularly to the preparation method of triptolide nanoliposomes and their application in the preparation of drugs for the treatment of esophageal squamous cell carcinoma. Background Technology

[0002] The antitumor potential of triptolide: Tripterygium wilfordii is the active ingredient of plants in the Tripterygium genus. It has anti-inflammatory, immunosuppressive, and antitumor effects, but its poor water solubility, short circulating half-life, and high cytotoxicity to normal cells limit its clinical application. Currently, this problem is often addressed by delivery using traditional solvents (such as DMSO) or liposomes. However, conventional solvent delivery requires high-concentration co-solvents due to the poor water solubility of triptolide (solubility <1 μg / mL), which can lead to significant systemic toxicity and uncontrollable drug release, easily causing dose peaks and troughs. Liposome delivery, on the other hand, has uneven particle size distribution (>500 nm), poor stability (aggregation occurs after 15 days at room temperature), insufficient targeting, and limited killing efficiency against tumor cells. Summary of the Invention

[0003] To address the problems of poor water solubility of triptolide, bioavailability of traditional formulations (<10%), the need for repeated administration, and high toxicity risks, this invention provides a novel method for preparing triptolide nanoliposomes. This method can enhance the water solubility and in vivo circulation stability of triptolide, improve drug delivery efficiency, and achieve drug accumulation at tumor sites while reducing damage to normal tissues.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: A triptolide nanoliposome with a particle size of 100-150 nm; its preparation method includes the following steps: 1) Preparation of lipid membrane: lecithin (PC), cholesterol (Chol), polyethylene glycol-phospholipid (PEG-DSPE) and triptolide were mixed and a chloroform-methanol mixture was added. The mixture was ultrasonically dissolved under ice-water bath conditions, and then the solvent was removed by rotary evaporation to obtain a uniform and transparent lipid membrane. 2) Preparation of colostrum: Add ethanol solution of triptolide and PBS solution to the obtained lipid membrane, emulsify by rotation under normal pressure and then treat with pulsed ultrasound to obtain a semi-transparent colostrum. 3) Preparation of nanoliposomes: The prepared colostrum was filtered through a microporous membrane and then homogenized under high pressure. The resulting homogenate was then subjected to centrifugation and ultrafiltration, and the resulting precipitate was the triptolide nanoliposomes.

[0005] Furthermore, the molar ratio of lecithin, cholesterol and polyethylene glycol-phospholipid used in step 1) is 3:1:0.15.

[0006] Furthermore, the mass ratio of triptolide to total phospholipids (the total amount of lecithin and polyethylene glycol-phospholipids) used in step 1) is 1:50.

[0007] Further, in the chloroform-methanol mixture described in step 1), the volume ratio of chloroform to methanol is 2:1.

[0008] Furthermore, the ultrasound power in step 1) is 200W and the duration is 10 min.

[0009] Furthermore, in step 1), the rotary evaporation temperature is 37°C, the rotation speed is 120 r / min, the vacuum degree is -0.08 MPa, and the time is approximately 20 min.

[0010] Furthermore, the lipid membrane obtained in step 1) needs to be vacuum dried at -0.1 MPa and 25°C for 2 h to completely remove solvent residue.

[0011] Further, the concentration of the triptolide ethanol solution in step 2) is 10 mg / mL.

[0012] Furthermore, the PBS solution described in step 2) has a pH of 7.4 and is preheated to 37°C.

[0013] Further, in step 2), 100 μL of triptolide ethanol solution and 2 mL of PBS are added to each gram of lipid membrane.

[0014] Furthermore, in step 2), the ambient pressure rotary emulsification temperature is 37°C, the rotation speed is 80 r / min, and the processing time is 40 min.

[0015] Furthermore, in step 2), the pulsed ultrasound consists of 2 seconds of ultrasound followed by a 1-second pause, with a power of 200W and a total processing time of 10 minutes.

[0016] Furthermore, the microporous filter membrane used in step 3) has a pore size of 0.8 μm to remove large particle agglomerates.

[0017] Further, the pressure of the high-pressure homogenization in step 3) is 100 MPa, and the cycle is repeated 5 times for 5 minutes each time.

[0018] Furthermore, in step 3), the centrifugal ultrafiltration speed is 13000 r / min and the time is 20 min.

[0019] The triptolide nanoliposomes can be used to prepare antitumor drugs.

[0020] Furthermore, the triptolide nanoliposomes can induce pyroptosis in esophageal squamous cell carcinoma cells by regulating the c-myc / BAX-caspase-3 / GSDME and ROS / inflammasome-caspase-1 / GSDMD pathways, and therefore can be used to prepare drugs for the treatment of esophageal squamous cell carcinoma.

[0021] The significant advantages of this invention are: 1) Improve drug delivery efficiency: This invention uses nanoliposomes for encapsulation, with an encapsulation rate of >85%, which can increase the water solubility of triptolide to over 10 μg / mL and enhance its in vivo circulation stability.

[0022] 2) Reduced toxicity: This invention uses nanoliposome encapsulation to achieve drug enrichment at the tumor site, reducing exposure to normal tissues and damage to normal tissues.

[0023] 3) Optimize formulation stability: By controlling the nanoparticle size (100-200 nm) and storing at low temperature (4℃), the shelf life of liposomes can be extended, allowing them to remain translucent even after 30 days of storage at 4℃. After rewarming, the particle size can be restored to the nanoscale, making them suitable for clinical cold chain transportation.

[0024] 4) Multiple anti-tumor effects: The nanoliposomes prepared in this invention can simultaneously inhibit cell viability, invasiveness and 3D aggregation ability, thus blocking tumor progression in all aspects. Attached Figure Description

[0025] Figure 1 Figures (a, b) show the inhibition of esophageal squamous cell carcinoma KYSE-150 and TE-1 cells by different treatment times with the triptolide nanoliposomes prepared according to the examples, and the effect on the viability of normal cells (BEAS-2B, HPCs) (c).

[0026] Figure 2 The diagram shows the invasive ability of triptolide nanoliposomes prepared for this example against esophageal squamous cell carcinoma KYSE-150 and TE-1 cells.

[0027] Figure 3 The diagram shows the inhibition of 3D clustering of esophageal squamous cell carcinoma TE-1 cells by triptolide nanoliposomes prepared for the example.

[0028] Figure 4 Microscopic images of pyroptosis in esophageal squamous cell carcinoma KYSE-150 and TE-1 cells after treatment with triptolide nanoliposomes prepared in the examples.

[0029] Figure 5 Western blot images of pyroptosis-related proteins in esophageal squamous cell carcinoma KYSE-150 and TE-1 cells after treatment with triptolide nanoliposomes prepared in the examples.

[0030] Figure 6 Figure showing the secretion of IL-1β by KYSE-150 and TE-1 cytokines in esophageal squamous cell carcinoma after treatment with triptolide nanoliposomes by ELISA. Detailed Implementation

[0031] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto. Example

[0032] A triptolide nanoliposome, the preparation method of which includes the following steps: 1) Mix lecithin (PC), cholesterol (Chol), and polyethylene glycol-phospholipid (PEG-DSPE) in a molar ratio of 3:1:0.15, and add triptolide at a mass ratio of 1:50 to total phospholipids (total amount of lecithin and PEG-DSPE). Then add a mixture of chloroform and methanol (2:1, v / v), and sonicate for 10 min (ultrasonic power of 200W) under ice-water bath conditions to dissolve it. Then place it in a 37℃ constant temperature water bath and remove the solvent by rotary evaporation at a vacuum of -0.08MPa and a rotation speed of 120r / min until a uniform and transparent lipid film is formed on the bottle wall. Then dry it in a vacuum drying oven at -0.1 MPa and 25℃ for 2 h. 2) Accurately weigh 1 mg of triptolide and add it to 100 μL of anhydrous ethanol. Vortex for 30 s to obtain an ethanol solution of 10 mg / mL triptolide. Preheat the PBS solution (containing 10 mM Tris-HCl and 0.15 M NaCl) at pH 7.4 to 37°C. Then add 100 μL of the ethanol solution of triptolide and 2 mL of PBS solution to 1 g of dried lipid membrane. Emulsify by rotation at 37°C and normal pressure for 40 min (rotation speed 80 r / min) to form a cloudy proemulsion. Then treat with pulsed sonication for 10 min in a 2 s, 1 s pause mode (ultrasonic power 200 W) to obtain a translucent proemulsion. 3) The prepared colostrum was filtered through a polycarbonate membrane with a pore size of 0.8 μm and then homogenized five times at 100 MPa using an ATS-1000 high-pressure homogenizer for 5 minutes each time. After each cycle, samples were taken and the particle size was detected by dynamic light scattering (DLS). 4) Transfer the obtained homogenate to an ultrafiltration tube and centrifuge at 4℃ and 13000r / min for 20min to obtain triptolide nanoliposomes.

[0033] 1. The physical properties of the obtained triptolide nanoliposomes were tested, and the results are as follows: Particle size: 120nm (Dynamic Light Scattering, DLS), polydispersity index (PDI) <0.2, surface potential -20-30mV.

[0034] Table 1. Stability study of Tripterygium wilfordii nanoliposomes

[0035] The stability test results show that: 1) Compared with room temperature, liposomes placed at 4°C showed slower particle size growth, indicating that low-temperature storage is beneficial for long-term preservation.

[0036] 2) Temperature responsiveness: After being stored at 4℃ for 30 days, the liposomes are “milky white and translucent”, but they can be restored to transparency after being heated at 40℃ for 5 minutes, which proves that they have temperature responsiveness (this property is beneficial for them to function in a specific temperature environment in the body, or to be quickly reconstituted by heating when used).

[0037] The results above show that the prepared triptolide nanoliposomes have good physical properties (small particle size, uniform distribution, suitable surface potential), excellent dispersibility, storage stability and temperature response performance.

[0038] 2. In vitro antitumor activity of the obtained triptolide nanoliposomes: Cell viability inhibition was tested using the CCK8 assay. Specifically, human esophageal squamous cell carcinoma cells KYSE-150 and TE-1, human lung epithelial cells BEAS-2B, and hematopoietic progenitor cells (HPCs) in logarithmic growth phase were collected and their cell concentrations were adjusted to 5 × 10⁶ cells / mL using RPMI-1640 medium containing 10% FBS. 3 Cell suspension was obtained by adding 100 μL of cell suspension per well. The cells were divided into experimental, positive, negative, and blank control groups in 96-well plates, with three replicates per group. Except for the blank control group, 100 μL of cell suspension was added to each well. The plates were then incubated at 37°C with 5% CO2 for 24 hours until cell adhesion. The old culture medium was removed from the 96-well plates. Culture medium containing different concentrations of triptolide nanoliposomes was added to the experimental group wells, culture medium containing different concentrations of free triptolide was added to the positive control group wells, and blank culture medium without the drug was added to the blank and negative control group wells (100 μL / well). Incubation continued for 48 hours. At 24 and 48 hours of incubation, 50 μL of old culture medium was removed from each well, and 10 μL of CCK8 reagent + 90 μL of fresh culture medium was added. After incubation in the dark for 2 hours, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader, and cell viability was calculated using the following formula. Simultaneously, based on the 48-hour cell viability data, the IC50 of the nanoliposomes against KYSE-150 and TE-1 cells was calculated using GraphPad Prism software.50 Value (half-maximal inhibitory concentration).

[0039] Cell viability (%) = [(OD value of experimental group - OD value of blank control) / (OD value of negative control - OD value of blank control)] × 100%.

[0040] Figure 1 The graph shows the inhibition of esophageal squamous cell carcinoma KYSE-150 and TE-1 cells at different treatment times in the experimental group and the positive control group, as well as the effect on the viability of human lung epithelial cells BEAS-2B and hematopoietic progenitor cells (HPCs). Figure 1 The results showed that triptolide nanoliposomes had an IC50 effect on esophageal squamous cell carcinoma cells KYSE-150 and TE-1. 50 The values ​​were 1.8 ng / mL (48 h) and 2.1 ng / mL (48 h), respectively, which were significantly lower than those of the free drug (IC50 > 10 ng / mL), and its toxicity to normal cells (BEAS-2B, HPCs) was lower than that of the free drug.

[0041] The Transwell assay was used to test the inhibition of invasion. Specifically, Matrigel was diluted 1:8 with serum-free medium on ice, and 50 μL of the diluted Matrigel was added to the upper chamber of a Transwell chamber. The chamber was then incubated at 37°C for 4 hours to allow the Matrigel to solidify and form an "artificial basement membrane." Subsequently, logarithmic-phase human esophageal squamous cell carcinoma cells KYSE-150 and TE-1 were harvested and their concentrations adjusted to 2 × 10⁻⁶ cells with serum-free medium. 4 Cell suspension was obtained by adding 200 μL of cell suspension per cell. 200 μL of cell suspension was added to the upper chamber of the transwell chamber. Simultaneously, serum-free culture medium containing different concentrations of triptolide nanoliposomes was added to the experimental groups, while an equal volume of blank serum-free culture medium was added to the control group. 600 μL of culture medium containing 20% ​​FBS was added to the lower chamber, and the chamber was incubated at 37°C with 5% CO2 for 24 h to allow cells to penetrate the Matrigel and chamber membrane. The transwell chamber was then removed, the liquid in the upper chamber was aspirated, and the cells were gently washed twice with PBS. Invading cells on the lower surface of the chamber were fixed with 4% paraformaldehyde (room temperature, 30 min). The fixative was then aspirated, the cells were washed twice with PBS, and stained with 0.1% crystal violet (room temperature, 20 min). The chamber was then gently rinsed with distilled water to remove unbound stain, and uninvaded cells in the upper chamber were wiped away with a cotton swab. The cells were observed under an inverted microscope, and the number of invading cells was counted in five randomly selected fields from each group, and the average value was calculated. Statistical analysis was performed using SPSS software to compare the differences between the experimental group and the control group (P < 0.01 was considered statistically significant). The results are as follows: Figure 2 As shown.

[0042] Figure 2The results showed that when the concentration of triptolide nanoliposomes was ≥1.25 ng / mL, the number of invasive esophageal squamous cell carcinoma KYSE-150 and TE-1 cells was reduced by >50% (P<0.01).

[0043] 3D clustering suppression results are as follows Figure 3 As shown. Figure 3 The results showed that in GravityPLUS TM In the 3D culture system, 2.5 ng / mL of nanoliposomes can reduce the volume of TE-1 cell clusters by 60% (P < 0.01).

[0044] 3. Pyroptosis Induction: Sterile cell slides were placed into 6-well plates, washed once with PBS, and sterilized by UV irradiation for 30 min. Then, KYSE-150 and TE-1 cells in logarithmic growth phase were taken and their concentration adjusted to 1×10⁻⁶. 5 2 mL of culture medium was added to each well of a 6-well plate (covering the cell slide). The plates were incubated at 37°C and 5% CO2 for 24 hours to allow cell adhesion. The old culture medium was removed. The experimental group received 2 mL of culture medium containing 2.5 ng / mL triptolide nanoliposomes (2 mL / well), while the control group received an equal volume of drug-free culture medium. Incubation continued for 48 hours, after which the culture medium was removed. The cell slides were gently washed three times with PBS (5 min each time). Cells were then fixed with 4% paraformaldehyde (room temperature, 15 min), washed three times with PBS, and then 0.1% Triton X-100 (diluted with PBS) was added. The plates were incubated at room temperature for 10 min to allow cell membrane permeability. After washing three times with PBS, the cells were observed under a microscope. Five fields of view were randomly selected from each group for photographing, and cell morphology was recorded. The results are shown below. Figure 4 As shown.

[0045] Figure 4 The results showed that the cells treated in the experimental group exhibited cell membrane rupture and cytoplasmic swelling, consistent with pyroptosis.

[0046] Western blot experiments were conducted to verify the molecular mechanism. The specific procedure involved taking KYSE-150 and TE-1 cells after 48 hours of incubation, removing the culture medium, washing twice with PBS, and adding 150 μL of IRA lysis buffer (containing 1% protease inhibitor) to each well. Lysis was performed on ice for 30 minutes, during which cells were scraped off using a cell scraper. The lysed mixture was then centrifuged at 12000 rpm for 15 minutes at 4°C, and the supernatant (containing total protein) was transferred to a new EP tube. Standards and working solutions were prepared according to the BCA kit instructions. Standards and samples (1:10 dilution) were added to 96-well plates and incubated at 37°C for 30 minutes. OD values ​​were then measured at 562 nm using a microplate reader. The protein concentration in the samples was calculated based on the standard curve, and all sample concentrations were adjusted to the same level (e.g., 2 μg / μL). 20 μg of protein was taken from each sample and added to 5× loading buffer (1:4 volume ratio), and the sample was boiled at 100°C for 5 minutes for denaturation. A 10% separating gel and a 5% stacking gel were prepared according to the kit instructions. After sample loading, perform electrophoresis at 80V for 30 min in the stacking gel stage and at 120V for 60 min in the separating gel stage, until the bromophenol blue indicator reaches the bottom of the gel. Cut a PVDF membrane of the same size as the gel, activate it by soaking it in methanol for 1 min, and then assemble it into a transfer sandwich with filter paper and gel in the following order: negative electrode → filter paper → gel → PVDF membrane → filter paper → positive electrode. Transfer the membrane at a constant current of 200mA for 90 min under ice bath conditions (GSDMD / GSDME molecular weight approximately 50-60kDa). After transfer, place the PVDF membrane in blocking buffer containing 5% skim milk powder (diluted with TBST) and block it on a shaker at room temperature for 1 h. Remove the blocking buffer, add primary antibodies diluted with TBST (anti-GSDMD: 1:1000; anti-GSDME: 1:1000; anti-GAPDH: 1:5000), and incubate overnight on a shaker at 4°C. The following day, the PVDF membrane was washed three times with TBST (10 min each time), and HRP-labeled secondary antibody (1:5000 diluted in TBST) was added. The membrane was incubated on a shaker at room temperature for 1 h. After washing three times with TBST (10 min each time), ECL chemiluminescence reagent was added, and protein band images were acquired using a chemiluminescence imaging system. Results are as follows: Figure 5 As shown.

[0047] Figure 5 The results showed increased cleavage of GSDMD and GSDME proteins, suggesting the formation of pyroptosis channels.

[0048] To detect IL-1β levels in culture supernatant using ELISA, the specific procedure is as follows: Collect KYSE-150 and TE-1 cell culture supernatants after 48 hours of drug treatment and drug-free treatment (as experimental and control groups, respectively). Centrifuge at 3000 rpm for 10 minutes at 4°C to remove cell debris. Aliquot the supernatant and store at -80°C for later use. Take out the standards, antibody working solution, and chromogenic solution from the ELISA kit and equilibrate at room temperature for 30 minutes. Dilute the standards according to the instructions (concentration gradient: 0, 15.625, 31.25, 62.5, 125, 250, 500, 1000 pg / mL) for later use. Add 100 μL of standards or samples (3 replicates per group) to each 96-well microplate, and add 100 μL of PBS to the blank wells. Seal the plate and incubate at 37°C for 2 hours. Discard the liquid in the wells, wash the plate 5 times with washing buffer (30 seconds each time), and pat dry any remaining liquid. Add 100 μL of IL-1β antibody working solution to each well, seal the plate, and incubate at 37°C for 1 h. Repeat the washing step, adding 100 μL of chromogenic solution (equal volumes of substrate A and B) to each well, and incubate at 37°C in the dark for 15 min. Add 50 μL of stop solution (sulfuric acid solution) to each well, gently shake to stop the reaction (the color changes from blue to yellow). Within 15 min, measure the OD value of each well at 450 nm using a microplate reader. Plot a standard curve based on the OD values ​​of the standards, substitute the sample OD values ​​to calculate the actual concentration of IL-1β (pg / mL), and statistically analyze the differences between the experimental group and the control group (P < 0.001 is considered highly significant). The results are shown in [Figure number missing]. Figure 6 .

[0049] like Figure 6 As shown, the IL-1β content in the culture supernatant was significantly increased (P < 0.001), confirming the activation of the inflammasome.

[0050] Mechanism model: Tripterygium acetate nanoliposomes induce pyroptosis through a dual mechanism: inhibiting c-myc, activating the BAX-caspase-3 pathway to cleave GSDME, and simultaneously inducing ROS generation to activate the NLRP3 inflammasome-caspase-1-GSDMD pathway.

[0051] The above demonstrates that the prepared triptolide nanoliposomes can simultaneously inhibit cell viability, invasiveness, and 3D aggregation ability, thus comprehensively blocking tumor progression.

[0052] Comparative Example 1 A triptolide nanoliposome, the preparation method of which includes the following steps: 1) Preparation of lipid membrane: Lecithin (PC), cholesterol (Chol), and polyethylene glycol-phospholipid (PEG-DSPE) were mixed in a molar ratio of 3:1:0.15, and triptolide was added at a mass ratio of 1:50 to total phospholipids (total amount of lecithin and PEG-DSPE). Then, a mixture of chloroform and methanol (2:1, v / v) was added. The mixture was sonicated for 10 min (ultrasonic power of 200W) under ice-water bath conditions to dissolve the lecithin membrane. The mixture was then placed in a 37℃ constant temperature water bath and the solvent was removed by rotary evaporation at a vacuum of -0.08MPa and a rotation speed of 120r / min until a uniform and transparent lipid membrane was formed on the bottle wall. The membrane was then dried in a vacuum drying oven at -0.1 MPa and 25℃ for 2 h. 2) Preparation of colostrum: Accurately weigh 1 mg of triptolide and add it to 100 μL of anhydrous ethanol. Vortex for 30 s to obtain an ethanol solution of 10 mg / mL triptolide. Preheat PBS solution (containing 10 mM Tris-HCl and 0.15 M NaCl) at pH 7.4 to 37°C. Then add 100 μL of the ethanol solution of triptolide and 2 mL of PBS solution to 1 g of dried lipid membrane. Emulsify by rotation at 37°C and normal pressure for 40 min (rotation speed 80 r / min) to form a cloudy colostrum. Then treat with pulsed sonication for 10 min in a 2 s, 1 s pause mode (ultrasonic power 200 W) to obtain a translucent colostrum. 3) The prepared colostrum was filtered through a polycarbonate membrane with a pore size of 0.8 μm, and then homogenized at 100 MPa for 5 min using an ATS-1000 high-pressure homogenizer. The particle size was then measured by dynamic light scattering (DLS). 4) Transfer the obtained homogenate to an ultrafiltration tube and centrifuge at 4℃ and 13000r / min for 20min to obtain triptolide nanoliposomes.

[0053] Comparative Example 2 A triptolide nanoliposome, the preparation method of which includes the following steps: 1) Preparation of lipid membrane: Lecithin (PC), cholesterol (Chol), and polyethylene glycol-phospholipid (PEG-DSPE) were mixed in a molar ratio of 3:1:0.15, and triptolide was added at a mass ratio of 1:50 to total phospholipids (total amount of lecithin and PEG-DSPE). Then, a mixture of chloroform and methanol (2:1, v / v) was added. The mixture was sonicated for 10 min (ultrasonic power of 200W) under ice-water bath conditions to dissolve the lecithin membrane. The mixture was then placed in a 37℃ constant temperature water bath and the solvent was removed by rotary evaporation at a vacuum of -0.08MPa and a rotation speed of 120r / min until a uniform and transparent lipid membrane was formed on the bottle wall. The membrane was then dried in a vacuum drying oven at -0.1 MPa and 25℃ for 2 h. 2) Preparation of colostrum: Accurately weigh 1 mg of triptolide and add it to 100 μL of anhydrous ethanol. Vortex for 30 s to obtain an ethanol solution of 10 mg / mL triptolide. Preheat PBS solution (containing 10 mM Tris-HCl and 0.15 M NaCl) at pH 7.4 to 37°C. Then add 100 μL of the ethanol solution of triptolide and 2 mL of PBS solution to 1 g of dried lipid membrane. Emulsify by rotation at 37°C and normal pressure for 40 min (rotation speed 80 r / min) to form a cloudy colostrum. Then treat with pulsed sonication for 10 min in a 2 s, 1 s pause mode (ultrasonic power 200 W) to obtain a translucent colostrum. 3) The prepared colostrum was filtered through a polycarbonate membrane with a pore size of 0.8 μm and then homogenized five times at 50 MPa using an ATS-1000 high-pressure homogenizer for 5 min each time. After each cycle, samples were taken and the particle size was detected by dynamic light scattering (DLS). 4) Transfer the obtained homogenate to an ultrafiltration tube and centrifuge at 4℃ and 13000r / min for 20min to obtain triptolide nanoliposomes.

[0054] Table 2 Correlation analysis of Tripterygium wilfordii nanoliposome particle size and biological function

[0055] Table 2 shows that the Tripterygium wilfordii nanoliposomes prepared by high-pressure homogenization in Comparative Example 1 had a particle size of 320 nm, a PDI of 0.42, and a pyroptosis rate of only 21.5%, indicating that large-particle liposomes are easily cleared by RES and cannot effectively induce pyroptosis. The Tripterygium wilfordii nanoliposomes prepared by low-pressure homogenization in Comparative Example 2 had a particle size of 180 nm, a PDI of 0.28, a pyroptosis rate of 38.7%, and an IL-1β release of only 450 pg / mL. In contrast, the Tripterygium wilfordii nanoliposomes prepared in the Example had a particle size of 120 nm, a PDI of 0.18, a pyroptosis rate of 65.2%, and an IL-1β release of 820 pg / mL, demonstrating that they have a good inhibitory effect on esophageal squamous cell carcinoma.

[0056] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for preparing triptolide nanoliposomes, characterized in that, Includes the following steps: 1) Preparation of lipid membrane: lecithin, cholesterol, polyethylene glycol-phospholipid and triptolide were mixed and a chloroform-methanol mixture was added. The mixture was ultrasonically dissolved under ice-water bath conditions, and then the solvent was removed by rotary evaporation to obtain a uniform and transparent lipid membrane. 2) Preparation of colostrum: Add ethanol solution of triptolide and PBS solution to the obtained lipid membrane, emulsify by rotation under normal pressure and then treat with pulsed ultrasound to obtain a semi-transparent colostrum. 3) Preparation of nanoliposomes: The prepared colostrum was filtered through a microporous membrane and then homogenized under high pressure. The resulting homogenate was then subjected to centrifugation and ultrafiltration, and the resulting precipitate was the triptolide nanoliposomes.

2. The method for preparing triptolide nanoliposomes according to claim 1, characterized in that, In step 1), the molar ratio of lecithin, cholesterol, and polyethylene glycol-phospholipids is 3:1:0.15; the mass ratio of triptolide to total phospholipids is 1:

50.

3. The method for preparing triptolide nanoliposomes according to claim 1, characterized in that, In step 1), the volume ratio of chloroform to methanol in the chloroform-methanol mixture is 2:

1.

4. The method for preparing triptolide nanoliposomes according to claim 1, characterized in that, In step 2), 100 μL of triptolide ethanol solution and 2 mL of PBS solution are added to each gram of lipid membrane; wherein the concentration of the triptolide ethanol solution is 10 mg / mL.

5. The method for preparing triptolide nanoliposomes according to claim 1, characterized in that, In step 2), the ambient pressure rotary emulsification temperature is 37℃, the rotation speed is 80r / min, and the processing time is 40 min; the pulsed ultrasound consists of 2 seconds of ultrasound followed by a 1 second pause, the power of the ultrasound used is 200W, and the total processing time is 10 min.

6. The method for preparing triptolide nanoliposomes according to claim 1, characterized in that, The pore size of the microporous filter membrane used in step 3) is 0.8 μm.

7. The method for preparing triptolide nanoliposomes according to claim 1, characterized in that, The high-pressure homogenization in step 3) is performed at a pressure of 100 MPa, with 5 cycles, each lasting 5 minutes.

8. The method for preparing triptolide nanoliposomes according to claim 1, characterized in that, In step 3), the centrifugal ultrafiltration speed is 13000 r / min and the time is 20 min.

9. A triptolide nanoliposome prepared by the method described in claim 1, characterized in that, The resulting nanoliposomes had a particle size of 100-150 nm.

10. The use of triptolide nanoliposomes as described in claim 9 in the preparation of esophageal squamous cell carcinoma treatment drugs.

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