A tumor cell and mitochondria dual-level targeting tripterygium wilfordii liposome and its preparation and application

Abstract

The present invention discloses a tripterygium wilfordii nanoliposome, which is composed of a liposome core and an outer layer of wrapping material, wherein the liposome core contains a lipid material, TPP-TPGS and tripterygium wilfordii, and the wrapping material is hyaluronic acid, wherein the hyaluronic acid can actively target the overexpressed CD44 receptor on the surface of tumor cells, thereby increasing the accumulation of the drug carried in the tumor site, and the TPP-TPGS can further deliver the drug to the mitochondria, thereby increasing the active oxygen content of tumor cells, reducing the mitochondrial membrane potential, destroying the mitochondrial function, and further causing apoptosis. Therefore, the liposome is a tumor cell and mitochondrial dual-level targeting liposome, which is beneficial to reducing the toxicity of tripterygium wilfordii and improving the efficacy of the drug against intrahepatic cholangiocarcinoma.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and relates to a preparation method and application of a tumor cell and mitochondria dual-stage targeted tripterygium wilfordii nanoliposome. Background Art

[0002] Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor of biliary epithelial cells that originates from the secondary and higher intrahepatic bile duct branches. Its incidence is second only to hepatocellular carcinoma, accounting for about 10% to 15% of primary liver malignancies, and is gradually increasing. ICC has a complex etiology, is highly invasive, and has a poor prognosis. Currently, radical surgery is still the main means to cure intrahepatic cholangiocarcinoma, but most patients with intrahepatic cholangiocarcinoma lose the opportunity for surgery due to their hidden onset and advanced stage at the time of initial diagnosis. Pathological molecular studies have shown that the complexity and significant heterogeneity of the pathogenesis of ICC hinder effective clinical treatment. Whether patients with non-surgical treatment can accept effective anticancer drugs is still a huge challenge to the current scientific research status of ICC. Therefore, the development of safe and efficient new targeted drugs and treatment strategies is of great significance to controlling the occurrence and development of ICC.

[0003] Celastrol (Cela) is a triterpenoid quinine methylate extracted from the traditional Chinese medicine Tripterygium wilfordii. As a natural biologically active compound, it has anti-inflammatory, metabolic pathway-affecting, autophagy-inducing or apoptosis-inducing, and anti-tumor functions. Studies have shown that Celastrol can inhibit the occurrence of intrahepatic cholangiocarcinoma by regulating the reprogramming of sugar metabolism. Specifically, Celastrol can exert anti-tumor effects by increasing the level of reactive oxygen species (ROS) in tumor cells, affecting mitochondrial function, and inhibiting the migration, invasion, and proliferation of tumor cells.

[0004] However, Celastrol has low solubility, poor permeability, low bioavailability, and significant liver, kidney, heart, and reproductive toxicity. It also has low organ and tissue selectivity and requires high doses to achieve effective concentrations at the lesion site. In short, Celastrol's low solubility and potential off-target toxicity limit its clinical application.

[0005] Nanoliposomes can improve the solubility of poorly soluble drugs and promote drug absorption. In addition, liposomes have a sustained release function that can protect drugs from being degraded by enzymes before reaching the lesion site. Further, passive enhanced permeability and retention (EPR effect) can be relied on to increase the accumulation of drugs at the target site and reduce systemic toxicity. At present, a large number of studies have proved its importance in the research of modern drug preparations. Triphenylphosphine-D-α-tocopherol polyethylene glycol 1000 succinate (TPP-TPGS) is a cationic polymer that can interact with negatively charged hyaluronic acid (HA) or mitochondrial membranes through electrostatic effects. Liposomes modified with TPP-TPGS have mitochondrial targeting effects, and the outer layer can be coated with HA. HA can specifically bind to the CD44 receptor overexpressed on the surface of tumor cells. Therefore, after modifying the outer layer of drug-loaded nanoparticles with HA, drugs can be delivered through receptor-mediated targeting, improving the uptake efficiency of tumor cells to drug-loaded particles and increasing anti-tumor efficacy.

[0006] Based on the above background, the applicant designed a nanoliposome delivery system of tripterygium wilfordii with dual-level targeting of tumor cells and mitochondria, and demonstrated that the drug delivery system has better efficacy against ICC. Summary of the invention

[0007] The purpose of the present invention is to provide a tripterygium wilfordii nanoliposome with dual-level targeting of tumor cells and mitochondria, and a preparation method and application thereof, in order to improve the efficacy of the anticancer drug tripterygium wilfordii in treating intrahepatic bile duct carcinoma.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] A tripterygium wilfordii nanoliposome, which consists of a liposome inner core and an outer layer wrapping material, wherein the liposome inner core contains a lipid material, TPP-TPGS and tripterygium wilfordii, wherein the lipid material consists of phospholipids and cholesterol, and the wrapping material is hyaluronic acid, which can actively target CD44 receptors overexpressed on the surface of tumor cells to increase the accumulation of the drug carried in the tumor site, and the TPP-TPGS can further deliver the drug to mitochondria to increase the active oxygen content of tumor cells, reduce mitochondrial membrane potential, destroy mitochondrial function, and further induce cell apoptosis. The liposome is a tumor cell and mitochondria dual-level targeting liposome.

[0010] The mass ratio of the phospholipid to TPP-TPGS is 5-15:1, preferably 6-12:1, and optimally 10:1.

[0011] Wherein, the mass ratio of the phospholipid to cholesterol is 10-20:1, preferably 12-15:1, and optimally 14:1.

[0012] Wherein, the mass ratio of the phospholipid to tripterygium wilfordii is 10-30:1, preferably 15-20:1, and optimally 15:1.

[0013] Wherein, the mass ratio of hyaluronic acid to TPP-TPGS is 1-5:1, preferably 2-4:1, and optimally 2:1.

[0014] According to a specific embodiment of the present invention, the optimal raw material ratio of the nanoliposome is: the mass ratio of the phospholipid to TPP-TPGS is 10:1; the mass ratio of the phospholipid to cholesterol is 14:1; the mass ratio of the phospholipid to tripterygium wilfordii is 15:1; the mass ratio of the hyaluronic acid to TPP-TPGS is 2:1. This preferred embodiment has the advantages of high drug encapsulation rate, large drug loading, uniform particle size distribution, etc.

[0015] The present invention further provides a method for preparing the tripterygium wilfordii nanoliposome: firstly, a conventional thin film dispersion method is used to prepare a liposome inner core containing lipid material, TPP-TPGS and tripterygium wilfordii, and then an ultrasonic method is used to wrap hyaluronic acid in the outer layer of the liposome inner core.

[0016] The present invention also provides the use of the tripterygium wilfordii nanoliposome in the preparation of a drug for treating intrahepatic cholangiocarcinoma. The nanoliposome dual-stage targets tumor cells and mitochondria, promotes apoptosis of cholangiocarcinoma cells, and exerts a better therapeutic effect on intrahepatic cholangiocarcinoma.

[0017] The beneficial effects of the present invention are:

[0018] The present invention uses hyaluronic acid to encapsulate liposomes, which can, on the one hand, cover up the cationic charge of the inner core liposomes and improve the in vivo safety of the liposomes; on the other hand, hyaluronic acid can actively target the overexpressed CD44 receptors on the surface of tumor cells, increase the accumulation of the drug carried in the tumor site, and reduce the accumulation in other organs, greatly reducing the systemic toxicity of tripterygium wilfordii, especially the toxicity to the heart and kidney. Further, TPP-TPGS can further deliver tripterygium wilfordii to mitochondria, and significantly increase the content of active oxygen in tumor cells, reduce mitochondrial membrane potential, and destroy mitochondrial function, thereby accelerating tumor cell apoptosis. At the same time, TPGS contains a long hydrophilic chain of polyethylene glycol, so it has good surface activity and properties and water solubility, can significantly increase the absorption of poorly soluble drugs, and improve the stability and sustained release effect of liposomes. It has been proved that the tumor cell and mitochondrial dual-stage targeted tripterygium wilfordii nanoliposomes prepared by the present invention have better anti-intrahepatic cholangiocarcinoma effects.

[0019] The present invention uses a thin film dispersion method combined with an ultrasonic method to prepare a dual-stage targeted liposome, and screens the process parameter conditions according to the physicochemical properties of the loaded drug tripterygium wilfordii. The prepared liposome has the advantages of high encapsulation rate, large drug loading capacity, uniform particle size distribution, etc., and has good drug sustained-release effect and quality stability, and high biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the particle size and potential change diagram of polyester ratio screening of HCTL.

[0021] Figure 2 It is the UV absorption spectrum of HCTL, Cela API and carrier.

[0022] Figure 3 These are scanning electron micrographs of HCTL and two comparative drugs, CL and CTL.

[0023] Figure 4 It is the in vitro release curve of HCTL and two comparative drugs CL, CTL and Cela raw material.

[0024] Figure 5 These are the results of hemolytic experiments of HCTL and two comparative drugs, CL and CTL.

[0025] Figure 6 These are the low-temperature storage stability and serum stability results of HCTL and two comparative drugs, CL and CTL.

[0026] Figure 7 This is the HUCCT1 cell uptake diagram of HCTL and two comparative drugs CL, CTL and Cela raw material.

[0027] Figure 8 It is the mitochondrial co-localization map, fluorescence co-localization line scan map and Pearson coefficient map of HUCCT1 of HCTL and two comparative drugs CL and CTL.

[0028] Fig. 9 These are the in vivo imaging images of HCTL and two comparative drugs, CL, CTL, and DiR API.

[0029] Fig.10 These are the periodic experimental results of HCTL and two comparative drugs CL, CTL and Cela API.

[0030] Fig.11 These are the apoptosis experiment diagrams of HCTL and two comparative drugs CL, CTL and Cela raw material.

[0031] Fig.12This is a graph showing the in vivo efficacy and body weight changes of HCTL, the comparison drug CTL, and Cela API in the AKT / YAP intrahepatic cholangiocarcinoma model.

[0032] Fig.13 Liver mass graph and liver weight ratio graph of the in vivo efficacy of HCTL, comparison drug CTL and Cela API in the AKT / YAP intrahepatic cholangiocarcinoma model.

[0033] Fig.14 The morphological and pathological changes in the liver of mice treated with HCTL, control drug CTL and Cela raw material in the AKT / YAP intrahepatic cholangiocarcinoma model (liver images, HE staining and CK19 immunohistochemistry).

[0034] Fig.15 This is the efficacy chart of HCTL, comparison drug CTL and Cela API in the AKT / YAP intrahepatic cholangiocarcinoma model in terms of serum liver damage biochemical indicators AST and ALT content.

[0035] Fig.16 HE sections of the heart, kidney, spleen and lung showing the efficacy of HCTL, comparison drug CTL and Cela API in the AKT / YAP intrahepatic cholangiocarcinoma model.

[0036] Fig.17 The graphs of LDH and CK, the serum biochemical indicators of cardiac injury, in the AKT / YAP intrahepatic cholangiocarcinoma model of the efficacy of HCTL, the comparison drug CTL, and Cela raw material.

[0037] Fig.18 These are the cardiac injury serum biochemical indicators CRE and BNU graphs of the efficacy of HCTL, comparison drug CTL and Cela raw material in the AKT / YAP intrahepatic cholangiocarcinoma model. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited to these embodiments, and the embodiments are only used to explain the present invention, but not to limit the scope of the present invention. Based on the following embodiments, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention.

[0039] The TPP-TPGS required by the present invention is homemade in the laboratory. TPP-TPGS is a conjugate of the delocalized lipophilic cation triphenylphosphine (TPP) and tocopherol polyethylene glycol succinate (TPGS). Triphenylphosphine (TPP) is a lipophilic cation with strong lipophilicity. It can be preferentially aggregated in the mitochondria of cells and can be used as a mitochondrial targeting ligand. There is a "tocopherol succinate" part in tocopherol polyethylene glycol succinate (TPGS). Tocopherol succinate can change the function of mitochondrial complex II (CII) by replacing the endogenous ligand ubiquinone of the enzyme. Ubiquinone produces excessive reactive oxygen species (ROS) and induces cell death. Therefore, TPGS and TPP + The conjugate of (TPP-TPGS) can also induce similar mitochondrial directed apoptosis. In addition, TPGS contains long hydrophilic chains of polyethylene glycol, so it has good surface activity, properties and water solubility, which can significantly increase the absorption of poorly soluble drugs and improve the stability and sustained release effect of liposomes.

[0040] The remaining reagents are conventional reagents purchased from commercial channels, and the experimental methods not mentioned are conventional experimental methods.

[0041] Example 1 Preparation of celastrol nanoliposomes (HCTL) with dual targeting to tumor cells and mitochondria

[0042] Preparation method: Weigh the prescribed amount of tripterygium wilfordii, soybean lecithin, cholesterol, and TPP-TPGS, dissolve them in chloroform, ultrasonically dissolve, and remove the organic solvent by rotary evaporation to form a uniform thin film of lipid on the wall of the device. Then add ultrapure water to hydrate the film, and place it in a 37°C water bath for 1 hour to fully swell the film. Then ultrasonicate it, filter it through a 0.22μm microporous filter membrane to obtain the liposome core, and finally add HA solution to wrap the liposomes. Ultrasonicate it, and filter it through a 0.22μm microporous filter membrane to obtain HCTL.

[0043] (1) Screening of polyester ratio:

[0044] HCTL liposomes were prepared according to the above method, with a fixed cholesterol-lipid ratio (cholesterol: phospholipid) of 1:10, a drug-lipid ratio (triptertium wilfordii: phospholipid) of 1:30, and a polymer-permeability ratio (hyaluronic acid: TPP-TPGS) of 3:1. The polymer-lipid ratio (TPP-TPGS: phospholipid) was changed to 1:2, 1:4, 1:6, 1:8 and 1:10, and the particle size and Zeta potential were used as indicators to screen the polymer ratio that had a greater impact on the particle size and potential of HCTL liposomes.

[0045] The results are shown in Figure 1A. As the polyester-lipid ratio decreases, the content of cationic polymer TPP-TPGS in the liposomes decreases, and the negatively charged HA adsorbed by electrostatic interaction also decreases accordingly, accompanied by a significant decrease in the liposome particle size. When the polyester-lipid ratio is 1:6, 1:8, and 1:10, the changes in the particle size and Zeta potential of the liposomes tend to a plateau, which is speculated to be the ratio range in which HA just fully covers the liposome surface ( Figure 1 AB). In order to obtain negatively charged liposomes with smaller particle size and for cost considerations, the optimal polyester-lipid ratio was selected as 1:10.

[0046] (2) Screening of the ratio of penetration to polymerization, the ratio of cholesteryl lipids and the ratio of drug to lipids:

[0047] The polyester-lipid ratio was fixed at 1:10, and the factors that had a greater impact on the encapsulation efficiency and drug loading of HCTL liposomes were selected as the factors to be investigated. For comprehensive screening index, orthogonal test table L9(3 4 ) were used to conduct experiments and optimize the formulation process. The orthogonal factor selection, orthogonal test results, and variance analysis results are shown in Tables 1, 2, and 3.

[0048] Table 1 Orthogonal test factor level table

[0049]

[0050] Table 2 Orthogonal test results

[0051]

[0052]

[0053] Table 3 Analysis of variance table

[0054] factor Sum of Squared Deviations Degrees of Freedom F Ratio F critical value Significance A 5.402 2 216.080 19.000 * B 0.444 2 17.760 19.000 C 33.610 2 1344.400 19.000 * error 0.03 2

[0055] The results showed that the results of intuitive analysis and variance analysis indicated that the polyester-lipid ratio and drug-lipid ratio had a significant effect on the encapsulation efficiency and drug loading capacity of liposomes (P<0.05), and the degree of influence of each factor was C>A>B. Therefore, the optimal prescription of HCTL was A1B3C1, that is, the mass ratio of the polyester-lipid ratio was 2:1, the mass ratio of the cholesterol-lipid ratio was 1:14, and the mass ratio of the drug-lipid ratio was 1:15.

[0056] (3) Parallel verification test of optimal prescription

[0057] Three groups of parallel validation tests were conducted on the optimal prescription of HCTL obtained from the orthogonal experiment. The test results are shown in Table 4.

[0058] Table 4 Parallel validation test results

[0059]

[0060] The results showed that when the mass ratio of soybean phospholipids (SPC): cholesterol (Chol): Cela: TPP-TPGS: HA was 15: 1.1: 1.0: 1.5: 3.0, the comprehensive screening index was the highest value (20.92), that is, the successfully loaded Cela accounted for the highest proportion of the total mass of liposomes. The verification results showed that the encapsulation efficiency of the three batches of HCTL samples was 97.25%, 98.57%, and 95.85%, with an RSD of 1.36%; the drug loading was 4.50%, 4.57%, and 4.44%, with an RSD of 0.07%; the particle sizes were 170.2, 171.4, and 171.1 nm, respectively, with an RSD of 0.4%; the potentials were -25.2mv, -25.4mv, and -25.3mv, respectively, with an RSD of 0.4%. The test had good reproducibility.

[0061] According to the above experiments, the preparation process of HCTL was finally determined as follows: 2.0 mg of tripterygium wilfordii, 30.0 mg of soybean lecithin, 2.1 mg of cholesterol, and 3.0 mg of TPP-TPGS were weighed and dissolved in chloroform, ultrasonically dissolved, and the organic solvent was removed by rotary evaporation to form a uniform thin film of lipid on the wall of the device, and then 10 mL of ultrapure water was added to hydrate the film, and the film was fully swollen in a 37°C water bath for 1 hour, followed by ultrasonication for 6 minutes, and then filtered through a 0.22 μm microporous membrane for 4 times to obtain the liposome core, and finally hyaluronic acid solution (containing 6.0 mg of hyaluronic acid) was added to encapsulate the liposome, ultrasonicated for 10 minutes, and filtered through a 0.22 μm microporous membrane for 8 times to obtain HCTL.

[0062] Figure 2 The ultraviolet absorption spectrum showed that HCTL liposomes were successfully loaded with tripterygium wilfordii, and all excipients had no interfering absorption at the maximum absorption wavelength of 425 nm for tripterygium wilfordii, and would not affect the determination of tripterygium wilfordii content.

[0063] Example 2 Pharmaceutical Testing of HCTL

[0064] Comparative drug 1 - Preparation of common tripterygium wilfordii liposomes (CL): The method is the same as Example 1, except that TPP-TPGS and HA are not used.

[0065] Comparative drug 2 - Preparation of mitochondrial targeted tripterygium wilfordii liposomes (CTL): The method is the same as Example 1, except that HA is not used.

[0066] (1) Particle size distribution and Zeta potential

[0067] The prepared liposomes were appropriately diluted and the particle size and Zeta potential of the liposomes were measured using a Malvern particle size analyzer. The results were as follows:

[0068] Size(nm) PDI Zeta potionial (mv) CL 126.9±1.3 0.266±0.010 -19.2±0.3 CTL 92.6±0.3 0.207±0.002 15.2±0.1 HCTL 172.3±0.6 0.280±0.005 -29.6±0.1

[0069] The results showed that compared with the particle size of the tripterygium wilfordii liposome CL without the addition of targeting materials, the particle size of the CTL group with the addition of TPP-TPGS was reduced. This is because it contains both the lipophilic group of vitamin E and the hydrophilic long chain of polyethylene glycol, so it has better surfactant properties and water solubility, which is more conducive to the formation of liposomes. The HCTL group electrostatically adsorbed a layer of negatively charged hyaluronic acid polysaccharide skeleton on the outer layer of the CTL group structure, and its particle size increased, and the potential reversal was achieved, which is conducive to improving the biocompatibility of liposome nanobody.

[0070] (2) Morphological characterization

[0071] The prepared liposomes were stained with 5% phosphotungstic acid and their morphology was characterized by transmission electron microscopy. Figure 3 shown.

[0072] The results showed that HCTL, CL and CTL liposomes were all spherical nanoparticles with narrow size dispersion and regular edges in transmission electron microscopy. Compared with cationic liposomes CTL, the HCTL liposomes covered with HA on the surface showed a concentric ring shape in the cross section of the hyaluronic acid shell, and the structure was clearly visible, indicating that hyaluronic acid was successfully adsorbed to the CTL surface by electrostatics, and the obtained HCTL liposomes were multilamellar liposomes.

[0073] (3) In vitro drug release

[0074] The in vitro release of Cela API, HCTL, CL and CTL liposomes in phosphate buffer solution at pH 7.4 was studied by dialysis bag diffusion method. 1 mL of Cela API, HCTL, CL and CTL liposome solution was transferred to a dialysis bag and then immersed in 20 mL of PBS (pH 7.4) containing 1% (weight percentage) Tween 80 at 37°C. 2 mL of PBS release medium was removed at predetermined time intervals and replaced with 2 mL of fresh medium. The drug concentration was determined by ultraviolet spectrophotometry (UV) and the cumulative drug release was calculated.

[0075] The drug release of each group was as follows Figure 4 As shown in the figure, free tripterygium wilfordii was released rapidly, with a release rate of 28.2% in the first 2 hours and complete release within 18 hours. At 72 hours, the cumulative release rates of CL, CTL, and HCTL were 64.0%, 60.6%, and 49.4%, respectively. This indicates that the HA coating on the surface of HCTL can block the drug release channel and achieve a better sustained release effect of the drug.

[0076] (4) Safety evaluation

[0077] Hemolysis test was used to evaluate the safety of intravenous injection of CL, CTL and HCTL liposomes. Liposome suspensions of different concentrations were mixed with 10% red blood cell suspension and incubated at 37°C for 2 h. The OD value at 540 nm was measured using an xMark spectrophotometer (Bio-Rad, USA), and the hemolysis rate was calculated and safety was evaluated.

[0078] The experimental results showed that when the blood compatibility of the three liposomes was evaluated for safety, CL and HCTL had lower hemolysis rates, indicating good biocompatibility ( Figure 5 ).

[0079] (5) Stability test

[0080] The stability of HCTL, CL and CTL liposomes was investigated. The particle size of the liposomes stored in a 4°C refrigerator was continuously measured to compare the low-temperature stability of the three groups of liposomes. After the drug-containing liposome suspension was incubated with culture medium containing 10% fetal bovine serum at different time points, the particle size distribution of the liposomes was measured to investigate the serum stability.

[0081] like Figure 6 The particle size of HCTL stored in a 4°C refrigerator as shown in A increased by only 2.8% in half a month, which was better than 3.7% and 9.9% of CL and CTL, respectively. Similarly, the particle size polydispersity index (PDI) of HCTL showed smaller fluctuations than that of the other two groups of liposomes during co-incubation with 10% fetal bovine serum culture medium ( Figure 6 B). In conclusion, HCTL exhibited better low-temperature storage stability and serum stability.

[0082] Example 3 Investigation of the targeting of HCTL and evaluation of its efficacy on ICC

[0083] (1) Cell uptake experiment

[0084] Coumarin 6 (C6) with green fluorescence was loaded into liposomes (named C6@CL, C6@CTL and C6@HCTL, respectively) according to the same preparation method to replace tripterine to characterize the drug uptake in cells. First, cholangiocarcinoma cell HUCCT 1 was cultured in a 6-well plate for 24 hours. The original culture medium was replaced with a new culture medium containing C6@CL, C6@CTL and C6@HCTL. The concentration of coumarin 6 in each well was 500 ng / mL. After 4 hours of culture, the cell uptake in each group was observed using an inverted fluorescence microscope. In addition, to confirm that the cell uptake of HCTL is related to CD44 receptor-mediated endocytosis, 3 mg of excess hyaluronic acid was added to the wells, and then HCTL was added. After 6 hours, the cells were treated as described above.

[0085] like Figure 7As shown in the figure, at 4h after administration, the fluorescence intensity of CTL was significantly enhanced compared with that of ordinary liposome C6@CL, which may be due to the mutual attraction between the positively charged TPP part and the negatively charged tumor cell membrane. It is worth noting that HCTL modified with HA has the highest uptake, and after the cells are pre-saturated with hyaluronic acid in advance, they are treated with the same amount of HCTL, resulting in a significant decrease in the green fluorescence intensity in the cells. This shows that the HA shell of HCTL can specifically bind to the CD44 receptor overexpressed on the surface of tumor cells, thereby significantly promoting the cell uptake of HCTL. At the same time, free HA can competitively inhibit this binding.

[0086] (2) Mitochondrial targeting assay

[0087] Similarly, liposomes loaded with coumarin 6 were used instead of celastrol liposomes to investigate the mitochondrial targeting of HCTL. HUCCT 1 cells in the logarithmic growth phase were inoculated in six-well plates. After culturing for 24 hours, the cells were cultured with the same concentration of drug-containing culture medium. After 4 hours, the drug-containing culture medium was removed, and the cell mitochondria were stained with Mitotracker-red (50nM) at 37°C for 30 minutes. Then, after fixation with 4% paraformaldehyde, the cells were stained with the fluorescent dye Hoechst33342 (10μg / mL) at room temperature in the dark for 10 minutes. After washing with PBS, the mitochondrial co-localization was observed with a laser confocal microscope (Nikon C2; Japan). The confocal images were analyzed using Image J software and line scanning was performed. The data were imported into GraphPad Prism 9.0 to obtain the corresponding line scanning diagram of the fluorescence spatial position.

[0088] like Figure 8 As shown in (a)-(c), C6@CL has no targeting property. After entering the cell, it is scattered in the cytoplasm and has a low overlap with mitochondria. However, in the C6@CTL and C6@HCTL groups, the red and green fluorescence mostly overlap, and yellow fluorescence is displayed after superposition, indicating that they both have better mitochondrial targeting ability. Similarly, the line scans corresponding to the combined images of each group also show that the position and intensity of the red and green fluorescence signals of the C6@CTL and C6@HCTL groups are more synchronized. In addition, quantitative analysis was performed by calculating the Pearson's coefficient. The results showed that the P values ​​of C6@CTL and C6@HCTL were 2.3 times and 2.8 times higher than those of C6@CL, respectively ( Figure 8 (d)). The above results indicate that the TPP group has the ability to target mitochondria.

[0089] (3) In vivo tumor targeting assay

[0090] To further verify the tumor targeting ability of HA envelope, HUCCT1 cells (1×10 7 The xenograft model was prepared by subcutaneously injecting 100 μL of RPMI 1640 (100 μL) of 4-week-old female BALB / c nude mice. 3 At the same time, each nude mouse was injected with different near-infrared fluorescent dye (DiR) preparations (free DiR, DiR@CL, DiR@CTL and DiR@HCTL) through the tail vein, and the DiR injection dose for each mouse was 0.2 mg / kg. The mice were anesthetized with isoflurane at the specified time points and quickly photographed at EM530 / 635nm fluorescence intensity using a small animal in vivo imaging system. 24 hours after the injection, the mice were anesthetized with isoflurane and killed. Tumors and major organs (heart, liver, spleen, lung, kidney) were then collected and photographed.

[0091] like Fig. 9 As shown in the figure, HCTL can actively target tumors with the help of the HA shell. DiR fluorescence enrichment can be observed at the tumor site 1 hour after administration. The active targeting transport speed is significantly higher than that of the other two nanoliposomes that rely solely on the EPR effect for passive transport. The free drug group had the worst targeting effect, and there was no obvious tumor accumulation trend within 24 hours of administration. In addition, strong fluorescence can still be observed at the tumor site in the HCTL group 24 hours after administration, which is consistent with the in vitro results, further demonstrating that HCTL has stable tumor targeting ability.

[0092] (4) In vitro pharmacodynamic evaluation

[0093] ① Cycle experiment

[0094] The effect of each group of preparations on the cell cycle was observed by propidium iodide (PI) DNA staining. HUCCT1 cells were inoculated in a small dish and cultured overnight. The same concentration of Tripterygium wilfordii preparation was added to the cells. After 24 hours of culture, the cells were collected and fixed with 70% pre-cooled ethanol. After centrifugation, the cells were washed with PBS and stained in 0.5mL PI / RNase staining solution at room temperature in the dark for 15 minutes. Finally, the cell samples were immediately analyzed by flow cytometry.

[0095] Anti-tumor drugs mainly block tumor cell proliferation in three phases of the cell cycle (i.e., G0 / G1, S, and G2 / M). Fig.10It can be seen that compared with the control group, when CL, CTL and HCTL containing the same concentration of tripterygium wilfordii were used to treat HUCCT 1 cells for 24 hours, the percentage of G0 / G1 phase in the cell cycle increased, and this phenomenon was accompanied by a decrease in the proportion of S and G2 / M phases of the cell cycle. Specifically, compared with the control group (35.7%), the percentage of G0 / G1 phase of cells in the three groups of liposome CL, CTL and HCTL groups increased significantly to 41.8%, 44.4% and 46.6%, respectively, among which HCTL had the most significant effect on the cell cycle. This shows that HCTL can block the cell cycle in the G1 phase, affect DNA replication, and promote cell apoptosis.

[0096] ②Apoptosis experiment

[0097] Annexin V-EGFP apoptosis detection kit was used to detect the cell apoptosis rate of each drug group, and the pro-apoptotic effects of several liposomes were compared. HUCCT 1 cells were inoculated in a six-well plate to allow the cells to fully adhere to the wall. The cells were then placed in a drug-containing culture medium for 24 hours and the cells were collected. Then, the fluorescent dye Annexin V-EGFP was used to stain the early apoptotic cells, and Propidium Iodide (PI) was used to mark the necrotic cells and cells in the late stage of apoptosis. Flow cytometry was then used for analysis.

[0098] like Fig.11 As shown, the total apoptosis rate (sum of early apoptosis rate and late apoptosis rate) of the control group and the four preparations were 7.0%, 13.8%, 31.5%, 35.3% and 39.6%, respectively. Compared with the free tripterygium wilfordii group, the apoptosis rate of the HCTL group increased by 2.9 times. HCTL can significantly increase the apoptosis rate of tripterygium wilfordii on HUCCT1 cells, which is related to the higher uptake and mitochondrial targeting efficiency of HCTL.

[0099] (5) In vivo pharmacodynamic evaluation

[0100] In this experimental part, we used high-pressure tail vein transfection technology to construct a mouse ICC orthotopic tumor model. This method has the advantages of short modeling time, stable modeling, and relatively clear molecular therapeutic targets of the model. Through high-pressure tail vein transfection technology, plasmids carrying activated oncogenes (AKT / Yap) were transferred into mice, so that they were stably expressed in the liver and quickly formed ICC. By administering drugs at the early stage of ICC, the targeting effect and mechanism of HCTL on ICC were investigated. In short, 2mL of physiological saline solution containing three plasmids of AKT, Yap and SB were quickly injected into the tail vein of mice. The mass ratio of the three plasmids was 10:15:1. Subsequently, the successfully transfected mice were randomly divided into four groups (n=8), and the untransfected mice were used as WT controls. All mice were fed a standard diet for 3 weeks. Starting from the fourth week, about 0.2mL of PBS, free tripterygium wilfordii, CTL and HCTL solution were injected into the transfected mice every other day through the tail vein at a dose of 2mg / kg of tripterygium wilfordii. The injections were continued for two weeks, and the weight of the mice was recorded. After the dosing period, the orbital blood of the mice was collected and the mice were killed. The mice were dissected, and the heart, liver, spleen, lungs, kidneys and other organs were separated, photographed and weighed. Some tissues were placed in a centrifuge tube filled with 4% paraformaldehyde, a tissue fixative, for subsequent hematoxylin and eosin staining (H&E) and immunohistochemical analysis. The remaining organ tissues were stored at -80°C for later use. Indicators such as aspartate aminotransferase (ALT) and glutamate aminotransferase (AST), creatinine (CRE), urea nitrogen (BUN), kinase (CK) and lactate dehydrogenase (LDH) in the serum were measured to evaluate the damage of different preparations to major organs such as the liver, kidneys and heart, and to investigate the tumor targeting and toxicity reduction and efficacy enhancement of HCTL.

[0101] like Fig.12 As shown in the figure, compared with the normal group (WT), the body weight of the AKT / Yap group (i.e., AKT / Yap transfected mice treated with PBS) increased significantly and rapidly due to the rapid growth of the tumor (P<0.001). However, the body weight of the three treatment groups decreased slightly at the beginning of the treatment and then gradually increased. The slight weight loss may be related to the loss of appetite in mice caused by tripterygium wilfordii as a leptin receptor sensitizer. Compared with the WT group, the weight loss of the three groups was within 10%. Fig.13 The results of liver weight in A showed that the liver weight increased significantly after AKT / Yap transfection, accompanied by tumor malignancy. The liver weight of mice was reduced after administration of the three preparations, and the liver weight was the lowest after HCTL treatment. Fig.13The results of the liver-to-body weight ratio in B show that the weight gain after AKT / Yap modeling is mainly due to the increase in liver weight. This indicator improved after the administration of the three preparations, among which HCTL had the best efficacy. Therefore, in addition to the weight loss effect of the drug, the reason why HCTL caused weight loss was mainly to reduce the weight of the liver by inhibiting tumor growth.

[0102] Fig.14 A is the liver morphology of mice in each group. Compared with the WT group, the liver of mice in the model group was significantly larger, grayish white and dull, with an uneven surface and a granular feel. The interior was covered with tumor agglomerates. The liver morphology of this group was significantly improved after HCTL liposome treatment. Fig.14 In B and C, the liver HE staining histological analysis of each group showed that the tumor had a tubular phenotype and the positive rate of CK19, a specific marker of bile duct cells, was increased, which is similar to the histopathological characteristics of human ICC. The above morphological and pathological changes suggest the occurrence of primary ICC. Moreover, after HCTL liposome treatment, not only the liver mass was reduced to 1 / 2 of that in the model group, but also the tumor volume and number were significantly reduced, the degree of liver fibrosis was reduced, the positive rate of CK19 was reduced, and the serum liver injury markers AST and ALT levels were downregulated to the closest normal values ​​( Fig.15 We further investigated the ability of the dual-stage targeted agent HCTL to reduce the toxicity of tripterygium wilfordii. Fig.16 HE staining sections of the heart, spleen, lung, and kidney of each group. It can be observed from the heart sections that free Cela can cause certain cardiac damage, such as myocardial fiber rupture, the appearance of a large number of disordered gaps, disordered and atrophic myofibril structure, cavitation on myofibers, and the accumulation of inflammatory cells. In addition, some pathological changes can also be seen in the kidney sections of free Cela, specifically moderate and severe atrophy of the volume of some glomeruli, partial necrosis and vacuolar formation of glomerular epithelial cells, and the accumulation of inflammatory cells. Compared with free Cela, due to the active targeting mediated by HA and CD44 receptors, HCTL liposomes can selectively accumulate at the tumor site, thereby reducing the damage of the drug to other organs. In addition, no obvious damage of Celastrol to the lungs and spleen was observed. In addition to the HE staining sections, we also measured a variety of myocardial enzymes in serum, such as creatine kinase (CK) and lactate dehydrogenase (LDH), as well as renal function indicators such as creatinine (CRE) and blood urea nitrogen (BUN). When myocardial cells are damaged, CK and LDH will be released into the blood, causing the levels in the blood to increase; when kidney damage occurs, CRE and BUN will increase because they cannot be excreted from the body. Fig.17 , Fig.18 It can be seen that after treatment with the three preparations, free Cela will lead to an increase in the above four indicators, while HCTL can improve the damage of the drug to the heart and kidney, which is consistent with the results in HE sections.

Claims

1. Use of tripterygium wilfordii nanoliposomes in the preparation of a drug for treating intrahepatic bile duct cancer, characterized in that: The nanoliposome is composed of a liposome inner core and an outer layer of wrapping material, wherein the liposome inner core contains lipid material, TPP-TPGS and tripterygium wilfordii, wherein the lipid material is composed of phospholipid and cholesterol, and the wrapping material is hyaluronic acid, wherein the mass ratio of the phospholipid to TPP-TPGS is 10:1; the mass ratio of the phospholipid to cholesterol is 14:1; the mass ratio of the phospholipid to tripterygium wilfordii is 15:1; and the mass ratio of the hyaluronic acid to TPP-TPGS is 2:

1. The hyaluronic acid can actively target the overexpressed CD44 receptor on the surface of tumor cells, thereby increasing the accumulation of the drug carried in the tumor site. The TPP-TPGS can further deliver the drug to the mitochondria, thereby increasing the active oxygen content of tumor cells, reducing the mitochondrial membrane potential, destroying mitochondrial function, and further causing cell apoptosis. The liposome is a dual-level tumor cell and mitochondrial targeting liposome.

2. The use according to claim 1, characterized in that: First, a conventional thin film dispersion method is used to prepare a liposome inner core containing lipid materials, TPP-TPGS and tripterygium wilfordii, and then an ultrasonic method is used to wrap hyaluronic acid in the outer layer of the liposome inner core.

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