A chonglou-si-mu-gi-xi-tai-bin-lipid-liposome, a preparation method and application

Abstract

This invention belongs to the field of biomedical technology, and particularly relates to a gemcitabine liposome, its preparation method, and its application. The gemcitabine liposome comprises the following components in parts by weight: 5-15 parts phospholipid, 1-2 parts ferruginin A, and 1 part gemcitabine; the gemcitabine liposome does not contain cholesterol. In the application of this gemcitabine liposome in the preparation of drugs for treating pancreatic cancer, it can significantly reduce the proportion of immunosuppressive cells and increase the proportion of cytotoxic cells. The gemcitabine liposome provided by this invention exhibits good stability, anti-multidrug resistance, synergistic effect, and drug synergistic effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a gemcitabine liposome, its preparation method, and its application. Background Technology

[0002] Pancreatic cancer, a malignant tumor that seriously threatens human health, has a similar incidence and mortality rate, with an extremely poor prognosis; its 5-year survival rate is less than 10%. Currently, surgical resection remains the best treatment option for achieving long-term survival. However, more than 80% of patients are diagnosed with advanced-stage pancreatic cancer at the time of initial diagnosis, including local progression or distant metastasis, leading to a low surgical resection rate. Furthermore, the poor efficacy and related side effects of gemcitabine-based chemotherapy regimens have resulted in no significant improvement in the overall 5-year survival rate for pancreatic cancer patients. Effective treatment of pancreatic cancer remains a challenge, and the development of new treatment options is hoped for.

[0003] Gemcitabine (GEM) is a novel pyrimidine deoxynucleoside analogue that exerts its antitumor effect through antimetabolism. It primarily acts on the DNA synthesis phase, effectively preventing cell progression from the G1 phase to the S phase. It is a well-tolerated and highly effective chemotherapeutic drug, but it is rapidly metabolized in the blood by deaminases into inactive compounds and excreted through the kidneys. Its short half-life, poor selectivity, susceptibility to drug resistance, and adverse reactions in the bone marrow and gastrointestinal tract limit its widespread use.

[0004] Liposomes are spherical vesicles formed by the self-assembly of phospholipid molecules in water. They possess advantages such as good biocompatibility, ease of modification, improved drug distribution in vivo, and ability to overcome tissue and cellular uptake barriers. Furthermore, they exhibit high drug loading efficiency for drugs with varying solubility, allowing for the simultaneous encapsulation and delivery of multiple drugs. However, conventional liposome formulations of anticancer drugs have several drawbacks. For example, intravenous administration lacks targeting, resulting in low tumor tissue uptake and adverse reactions such as hand-foot syndrome. Furthermore, batch-to-batch variations, improving the encapsulation efficiency of chemotherapy drugs, and addressing organic solvent residues are all significant concerns. While conventional cholesterol liposomes can improve the physical stability of liposomes, their structure, being a polycyclic monounsaturated alcohol, contains functional groups prone to structural derivatization, such as double bonds and hydroxyl groups. During processing and storage, they are susceptible to the effects of external conditions such as oxygen, light, and metals, leading to cholesterol oxidation products. These cholesterol oxidation products are diverse and possess certain cytotoxic, mutagenic, and potentially carcinogenic properties, significantly promoting atherosclerosis and thus affecting the stability and safety of liposome formulations, ultimately harming human health. Furthermore, excessive cholesterol intake carries the risk of hyperlipidemia and tumors. Additionally, most current research modifies liposomes with targeting ligand molecules or immunomodulators to directly target tumor cells and exert synergistic anti-tumor effects. However, these modifications involve various synthetic steps, are costly, and complex. Therefore, finding a liposome that is easy to prepare yet simultaneously possesses tumor-targeting properties is particularly important.

[0005] Fritillaria cirrhosaside A is one of the main active ingredients extracted from Fritillaria cirrhosa. It is a pentacyclic triterpenoid saponin composed of hydrophilic glycosidic chains and hydrophobic glycosidic aglycones, exhibiting a steroidal structure similar to cholesterol, but with several advantages. Currently, no "co-loaded liposomes using Fritillaria cirrhosaside A instead of cholesterol as a bilayer membrane" prepared by active drug delivery methods have been found. This experiment, using Fritillaria cirrhosaside A as a lipid membrane regulator to replace cholesterol and encapsulate it in liposomes, yields novel functional liposomes with pharmacological activity, which shows greater application potential. Furthermore, most saponins currently suffer from poor solubility, low bioavailability, and hemolytic effects. Using Fritillaria cirrhosaside A in liposome membrane materials can not only reduce toxicity and enhance efficacy, but also, when combined with appropriate formulations, encapsulate gemcitabine, an anticancer drug, in liposomes for synergistic effects.

[0006] Therefore, selecting the optimal drug combination and developing the best preparation process to produce a novel gemcitabine liposome with better efficacy, lower toxicity, and quality and other indicators that meet drug requirements, in order to comply with drug application requirements, requires extensive research and technological breakthroughs. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for preparing and applying a novel liposome containing gemcitabine and fritillaria thunbergii. The fritillaria thunbergii liposomes of this invention are stable, have small particle size, high drug encapsulation efficiency, good in vivo compatibility, better efficacy, and lower toxicity. In particular, it provides an optimal ratio of gemcitabine and fritillaria thunbergii for synergistic anticancer action, and further provides a novel synergistic anticancer liposome targeting pancreatic cancer.

[0008] The technical solution is as follows:

[0009] The first objective of this invention is to provide a gemcitabine liposome containing 5-15 parts soybean lecithin (SL), 1-2 parts tubuside A (Tub I), and 1 part gemcitabine (GEM); wherein the gemcitabine liposome does not contain cholesterol.

[0010] In one embodiment, in the gemcitabine liposome, the gemcitabine A forms a phospholipid membrane with the phospholipid.

[0011] In one embodiment, the phospholipid membrane preferably further includes soybean lecithin.

[0012] In one embodiment, preferably, the inner side of the phospholipid membrane is an inner aqueous phase, the outer side of the phospholipid membrane is an outer aqueous phase, and the gemcitabine is encapsulated in the inner aqueous phase.

[0013] In one embodiment, the HPLC purity of the fritillary glycoside A is ≥99%.

[0014] In one embodiment, the gemcitabine liposome does not contain cholesterol.

[0015] The second objective of this invention is to provide a method for preparing fritillary glycoside A and gemcitabine liposomes, comprising the following steps: Precisely weighed amounts of soybean lecithin and gemcitabine are dissolved in chloroform; fritillary glycoside A is dissolved in methanol, and the solutions are mixed and evaporated at 60°C for 20 minutes using a rotary evaporator to form a fixed membrane. This process encapsulates fritillary glycoside A in the lipids. The resulting membrane is hydrated in phosphate-buffered saline (PBS) at 60°C for 1 hour to obtain a concentrated suspension of liposomes. The suspension is then sonicated for 30 minutes to form liposomes, followed by 2 seconds of sonication and then standing for 3 seconds. The liposomes are filtered through a 0.22 μm filter and stored at 4°C.

[0016] The reagents and raw materials used in this invention are all commercially available.

[0017] The third objective of this invention is to provide a blank liposome for preparing gemcitabine liposomes, wherein the blank liposome is a blank liposome with gemcitabine liposome as the membrane material, the blank liposome has a membrane composed of a phospholipid membrane and gemcitabine liposome, and the blank liposome does not contain cholesterol.

[0018] The fourth objective of this invention is to provide a pharmaceutical formulation containing gemcitabine liposomes, which is made from gemcitabine liposomes and other pharmaceutically acceptable excipients.

[0019] The fifth objective of this invention is to provide an application of gemcitabine liposomes in the preparation of drugs for treating pancreatic cancer.

[0020] In one embodiment, the liposome of gemcitabine methyl terbin can significantly reduce the proportion of immunosuppressive cells and increase the proportion of cytotoxic cells.

[0021] Combining all the above technical solutions, the advantages and positive effects of this invention are as follows: the gemcitabine liposomes provided by this invention have good stability, anti-multidrug resistance, synergistic effect, and drug synergistic effect. Taking the gemcitabine liposomes of the embodiment as an example, replacing cholesterol in the liposomes with gemcitabine A not only reduces the excessive intake of cholesterol by the human body, especially lesion cells, but also maintains the stability of the liposomes and enhances their medicinal value. Its efficacy is significantly better than that of gemcitabine monomers and ordinary gemcitabine liposomes. This demonstrates that gemcitabine A plays a better role as a "drug, excipient, and membrane material" in the gemcitabine liposomes of the embodiment.

[0022] Specifically, this is reflected in:

[0023] The efficacy of the medicine has been significantly improved.

[0024] 1. In particular, the Tub I-GEM-Lps group showed the best efficacy, with a significantly enhanced tumor inhibition rate compared to the C-GEM-Lps group, the GEM+Tub I group, and the GEM group. This indicates that the novel liposomes are significantly more effective than gemcitabine alone and also significantly more effective than conventional gemcitabine liposomes. This demonstrates that the membrane structure of tusiviridine A has a significant advantage in drug release and synergistic effects of the novel liposomes.

[0025] 2. Significantly reduced toxic side effects.

[0026] The liposomes prepared according to the formulation of this invention did not result in mouse mortality in the Tub I-GEM-Lps group. The CCK-8 assay further verified that the novel liposomes group inhibited cell viability more strongly than other groups.

[0027] 3. The liposomes of gemcitabine methyl terbin of the present invention have a particle size D90 ≤ 150 nm and an encapsulation efficiency ≥ 80%.

[0028] In addition, the inventive step evidence for the claims of this invention is also reflected in the following important aspects:

[0029] (1) The technical solution of the present invention greatly improves the prognosis of cancer patients after transformation, and has significant commercial value and socio-economic effects.

[0030] (2) The technical solution of the present invention solves the technical problem that people have long desired to improve the targeting of drug-loaded liposomes and simultaneously have conditional tumor immune function. Attached Figure Description

[0031] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this disclosure and, together with the description, serve to explain the principles of this disclosure;

[0032] Figure 1 This is a graph showing the cytotoxicity of free GEM and different types of GEM-loaded liposomes on Panc02 cells, where: TubI is tebufenozide A; GEM is gemcitabine; LPs are liposomes; and C is cholesterol.

[0033] Figure 2 This is a graph showing the effects of free GEM and different types of GEM-loaded liposomes on apoptosis in Panc02 cells; where: Figure 2 A is a flow cytometry diagram showing apoptosis; Figure 2 B is a statistical graph of apoptosis; *P<0.05, **P<0.01 and

[0034] ***P<0.001, (n=3; mean±SD);

[0035] Figure 3 This is a graph showing the cellular uptake of novel coumarin-6 liposomes and conventional liposomes by Panc02 cells; in which: Figure 3 A is the uptake rate of liposomes by tumor cells detected by fluorescence microscopy; Figure 3 B represents the uptake rate of liposomes by tumor cells as detected by flow cytometry; *P<0.05, **P<0.01 and ***P<0.001, (n=3; mean±SD);

[0036] Figure 4 This is a graph showing the in vivo anticancer activity of gemcitabine (GEM)-loaded liposomes containing fritillaria cirrhosa; where: Figure 4 A shows photographs of each group of tumors; Figure 4 B represents the tumor weight statistics for each group of tumors; (n = 4; mean ± standard deviation); Figure 4C is the tumor volume of each group of tumors (n=5; mean ± SD) *P<0.05, **P<0.01, ***P<0.001;

[0037] Figure 5 These are representative hematoxylin and eosin stained sections of the heart, liver, lungs, and kidneys (scale bar = 100 μm);

[0038] Figure 6 This is a diagram of the immune microenvironment; in which: Figure 6 A is a flow cytometry analysis of the relative abundance and histogram of CD4+ and CD8+ cells in tumor tissues treated with different groups. Figure 6 B is a flow cytometry and histogram analysis of the relative abundance of CD11b+Ly6G+ MDSCs in tumors treated with different groups. *P<0.05

[0039] **P<0.01 and***P<0.001, (n=3; mean±SD). Detailed Implementation

[0040] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The present invention is further illustrated below by way of examples, but is not limited thereto to the scope of the described embodiments. Experimental methods in the following examples, unless otherwise specified, were performed according to conventional methods and conditions, or as selected according to the trade instructions.

[0041] Experimental drugs: Fritillaria cirrhosaside A (≥98% by mass, Shanghai Yuanye Biotechnology Co., Ltd.); Cholesterol (>98% by mass, Shanghai Yuanye Biotechnology Co., Ltd.); Soybean lecithin (PC-95, >97% by mass, Aivit Shanghai Pharmaceutical Technology Co., Ltd., batch number SY-SO-220602); Methanol (chromatographic grade, ≥99.9% by mass, Shanghai Aladdin Biochemical Technology Co., Ltd.); Chloroform (≥99% by mass, Tianjin Bohua Chemical Reagent Co., Ltd.); Hoechst 33342 (Beyotime Biotechnology Co., Ltd.); Fetal bovine serum (FBS, VivaCell); Culture medium (DMEM, Solarbio Biotechnology Co., Ltd.); Coumarin-6 (≥98% by mass, aladdin).

[0042] Experimental instruments: AB135-S type 0.0001 g balance (Mettler-Toledo Ltd.); Shimadzu LC-20AT type high performance liquid chromatograph (Shimadzu Corporation, Japan); N-1300 rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, RE-2000A); KQ-250DE type CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Zetasizer Nano ZS90 type laser particle size analyzer (Malvin Ltd., UK); Leica inverted fluorescence microscope (Leica Microsystems Ltd., Germany); Spark 20M microplate reader (Tecan Trading AG, Switzerland).

[0043] Experimental cells and animals:

[0044] Mouse pancreatic cancer cells were purchased from the American College of Cell Bank (ATCC).

[0045] Strain: C57; Sex: Female; Weight: 18g±2g; Age: 6-8 weeks. Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Housed in the animal facility of Nankai Hospital. Ambient temperature: 22±2℃; Relative humidity: 50±5%. Feed, water, and bedding were aseptically treated and replenished and replaced regularly. All animal experimental protocols were approved by the Laboratory Animal Ethics Committee of Nankai Hospital, and all animal experiments were conducted in accordance with the relevant guidelines for the husbandry and use of laboratory animals at Nankai Hospital.

[0046] Example 1: Effect of the ratio of phospholipid to fritillary glycoside A on the particle size and PDI of novel fritillary glycoside A liposomes

[0047]

[0048] Results Analysis: Using the above-mentioned thin-film hydration method, qualified liposomes co-loaded with fritillary glycoside A and gemcitabine can be prepared. Specifically, when the amount of Tub I is fixed at 3, the particle size gradually increases as the amount of SL is gradually increased. When SL is 10 parts, the particle size is 159±0.33, and the PDI is 0.27±0.018, which is the smallest and most stable among all groups. When SL is fixed at 10 parts, and SL:Tub I = 10:2, the particle size is 112±0.21, and the PDI is 0.26±0.006. This ratio results in the smallest particle size and a more stable liposome solution.

[0049] Example 2: Effect of drug-to-phospholipid ratio on encapsulation efficiency and drug loading

[0050]

[0051] Results Analysis: The above-described thin-film hydration method can prepare qualified liposomes co-loaded with fritillary glycoside A and gemcitabine, and these liposome membranes are cholesterol-free. When SL:Tub I:GEM = 10:2:1, the encapsulation efficiency of Tub I and gemcitabine is good. As the amount of gemcitabine and GEM decreases, the encapsulation efficiency of gemcitabine and GEM decreases sharply. As the amount of GEM increases, the encapsulation efficiency also decreases. Therefore, the optimal ratio is SL:Tub I:GEM = 10:2:1.

[0052] Example 3: In vitro detection of the effect of novel fritillary glycoside A liposome on tumor cell proliferation

[0053] Panc02 cells were selected for culture.

[0054] Panc02 cell culture and passage: Cell resuscitation was performed according to the Panc02 cell instructions. DMEM full culture (containing 10% FBS and 1% penicillin-dextrose antibody) was prepared, and cells were transferred to the DMEM full culture and cultured in an incubator. When the cells almost completely filled the entire microscopic field of view, the culture medium was discarded, and the cells were treated with 0.05% trypsin (25cm²). 2 Use 0.5 mL, 75 cm³ culture flasks. 2 Digest cells in a culture flask using 1.5 mL of DMEM for 6 minutes until completely digested. Then, add an appropriate amount of DMEM to stop the digestion. Transfer all cell solution to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add an appropriate amount of DMEM, resuspend the cells by pipetting, passage, and then incubate in a constant temperature incubator.

[0055] CCK-8 test

[0056] Panc02 cells in the logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 5 100 μL of cells / well was seeded into a 96-well plate. Six wells were reserved with only 100 μL of culture medium (without cells) as apoptosis wells. The plates were then incubated at 37°C and 5% CO2 for 24 h.

[0057] Experimental groups: Free GME solution (GME), free TubI / GME mixed solution (TubI / GME), GME-loaded ordinary cholesterol liposomes (C-GME-Lp), and GME-loaded novel terbufotoxin A liposomes (TubI-GEM-Lp) were prepared using complete culture medium. Gemcitabine was diluted at concentration gradients from 0.2 μg / mL to 12 μg / mL, with the amount of terbufotoxin A corresponding to the prescribed dosage. The old culture medium was removed, and 100 μL of the pre-prepared free drug solution and drug-loaded liposome solution (n=6) were added according to the concentration gradient. Six wells were reserved for control (culture medium only). After addition, the 96-well plates were gently tapped to mix the drugs and incubated at 37°C in a 5% CO2 cell culture incubator for 24 h. Then, the drug solution was aspirated, and 10 μL of LCK-8 solution was added to each well. The cells were then incubated at 37°C with 5% CO2 for 30 min, followed by incubation on a microplate shaker with low-speed shaking for 20 min. The absorbance at 570 nm was then measured using a microplate reader, and the survival rate of each drug on Panc02 cells was calculated. The calculation formula is as follows:

[0058]

[0059] Results analysis: such as Figure 1 As shown, free GEM, free TubI / GME, GME-loaded ordinary cholesterol liposomes (C-GME-Lp), and GME-loaded novel liposomes containing terbinafine (TubI-GEM-Lp) all inhibited the proliferation of mouse pancreatic cancer cells Panco2 in a concentration-dependent manner, indicating that gemcitabine itself has a good anti-proliferative effect on pancreatic cancer cells Panco2. Although we used terbinafine (TubI) instead of cholesterol as the membrane material for the liposomes, it also has anti-cancer activity. Therefore, we designed the same concentration of free drug group according to the formulation amount of novel terbinafine liposomes. Compared with GME, free TubI / GME mixed solution (TubI / GME), GME-loaded ordinary cholesterol liposomes (C-GME-Lp), and GME-loaded novel terbinafine liposomes (TubI-GEM-Lp) all showed significant anti-proliferative effects. I-GEM-Lp exhibits stronger cell-killing ability, and the novel liposomes show more significant tumor-inhibiting effects, thus significantly improving drug efficacy. These results suggest that when terbinafine is used as the membrane material of liposomes, it can produce a synergistic effect with gemcitabine, and is superior to the free state of the two substances used alone.

[0060] Example 4: In vitro detection of the effect of novel fritillary glycoside A liposome on tumor cell apoptosis

[0061] Cell flow cytometry

[0062] Panc02 cells were planted at a density of 1 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells per well in 12-well plates. Experimental groups were as described above, and experiments were conducted according to the Annexin V-FITC apoptosis detection kit instructions. Apoptotic cells were measured using an EXFLOW flow cytometer.

[0063] Results analysis: Flow cytometry results for apoptosis detection in free GEM, free Tubi / GME mixed solution, and various drug-loaded GEM liposome groups are as follows: Figure 2 The results showed that, compared with the untreated PBS group, free GEM significantly promoted late apoptosis. While the physical mixture solution and conventional liposomes showed similar effects in promoting late apoptosis, the novel liposomes significantly promoted late apoptosis more than both the conventional liposome group and the physical mixture group. This indicates that Tub I-Lps, as a membrane material, is more effective than conventional cholesterol liposomes in promoting pancreatic cancer cell apoptosis. Tub I-GEM-Lps showed approximately 1.7 times more late apoptosis than C-GME-Lps. These results suggest that the novel liposomes loaded with gemcitabine can better exert a synergistic effect, promoting apoptosis in mouse pancreatic cancer cells (Panc02), especially late apoptosis (early apoptosis is reversible, late apoptosis is irreversible).

[0064] Example 5: Detection of tumor cells' ability to take up novel fritillary glycoside A liposomes

[0065] Cellular uptake

[0066] Preparation of liposomes carrying coumarin-6

[0067] Preparation method of coumarin-6 solution: Weigh an appropriate amount of coumarin-6 solid powder in the dark, dissolve it in an appropriate amount of anhydrous ethanol and chloroform (1:1) to prepare a coumarin-6 solution with a final concentration of 2 mg / ml.

[0068] Membrane material formulations for novel liposomes loaded with coumarin-6 and ordinary cholesterol liposomes. Weigh out the prescribed amounts of membrane material, add 10 μL of the above-prepared coumarin-6 solution, and dissolve in appropriate amounts of anhydrous ethanol and chloroform (1:1, mL / mL). The remaining preparation methods are the same as above.

[0069] Qualitative assessment of cell uptake: Panc02 cells were cultured at a density of 1 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of 1000 cells per well in 6-well plates and incubated at 37°C with 5% CO2 for 24 h. A novel coumarin-loaded terbinafine liposome and a regular liposome were added to each well at a final concentration of 500 ng / mL. The cells were incubated for 4 h, then Hoechst 33342 was added, and the cells were stained in the dark for 30 min. The culture medium was discarded, and the cells were washed three times with pre-cooled PBS. Qualitative observation was then performed under a confocal microscope.

[0070] Quantitative assessment of cellular uptake: The fluorescence intensity of coumarin-6 in cells was measured by flow cytometry.

[0071] Results analysis: Images obtained under a confocal fluorescence microscope showed that Tub I-Lp exhibited significantly enhanced fluorescence intensity in Panc02 cells compared to C-Lp, indicating that cells took up more Tub I-Lp. Flow cytometry results further confirmed that the cellular uptake rate of Tub I-Lp was approximately 3 times higher than that of C-Lp.

[0072] Example 6: Effects and Toxicity Evaluation of Novel Fritillariae Toxicin A Liposome on the Growth of Pancreatic In Situ Carcinoma in Mice

[0073] Animal experiments

[0074] The cells were kept in C57 for 12 hours with alternating light and dark conditions, and were fed and watered regularly each day. Simultaneously, cells in the logarithmic growth phase were harvested, digested with trypsin, and centrifuged at 1200 rpm for 5 minutes to collect the cell pellet. The supernatant was discarded, and the cell pellet was resuspended in PBS to adjust the cell density to 2.5 × 10⁻⁶ cells / day. 7 / ml, take a cell suspension of this density and inoculate it in situ into the mouse pancreas, with a cell number of 5×10⁶ cells. 6 Each mouse was inoculated with 0.2 ml of solution. After all mice were inoculated, their condition was observed daily to assess whether the tumor model had been successfully established in vivo.

[0075] Experimental grouping and administration method

[0076] The mice that successfully underwent in vivo modeling were randomly divided into 5 groups: the model group, the free gemcitabine group, the free gemcitabine monomer group, and the free fritillary glycoside A monomer mixed solution group. There were 6 mice in each group of ordinary gemcitabine liposomes and novel fritillary glycoside A liposomes. Intraperitoneal administration began on the third day after successful in situ tumor implantation.

[0077] The dosage is as follows:

[0078] Model group: 0.2 ml of PBS (negative control) was administered intraperitoneally once every other day;

[0079] Free gemcitabine group (5 mg / kg): 0.2 ml was administered intraperitoneally once every other day;

[0080] Mixture of free gemcitabine monomer and free fritillary glycoside A monomer: 0.2 ml was administered intraperitoneally once every other day;

[0081] Regular gemcitabine liposomes (5 mg / kg): 0.2 ml, administered intraperitoneally once every other day.

[0082] Novel Fritillaria cirrhosa glycoside A liposome (5mg / kg): 0.2ml, administered intraperitoneally once every other day.

[0083] Tumor sampling and testing methods

[0084] Panc02 cells were inoculated into the pancreas of C57 mice in situ, and changes at the inoculation site were observed daily. After 3 days, intraperitoneal administration was performed daily. After 15 days of intraperitoneal administration, the mice were euthanized by cervical dislocation, and tumor tissue was dissected from the pancreas. The corresponding tumor volume and weight were calculated. The formula for calculating tumor volume (TV) is: V = 1 / 2 × a × b², where a represents the longest diameter of the tumor, and b is the maximum transverse diameter perpendicular to the longest diameter.

[0085] Safety evaluation---HE staining

[0086] After the pharmacodynamic experiments were completed, the heart, liver, lungs, and kidneys of C57 mice were removed, and these organs were prepared into paraffin-embedded sections and stained with hematoxylin and eosin (HE). These HE-stained sections were then observed and images were acquired under an inverted fluorescence microscope.

[0087] Results analysis: Measurements of tumor weight and volume showed that the novel liposomes exhibited significantly better in vivo anticancer effects than the conventional liposome group and the combination therapy of gemcitabine and fritillaria cirrhosa extract. Pathological results indicated that the novel fritillaria cirrhosa extract liposome delivery system has high safety and holds promise for clinical application.

[0088] Example 7: Effects of Novel Fritillariaein A Liposome on the Tumor Microenvironment of Pancreatic Orthotopic Carcinoma in Mice

[0089] Isolation of immune cells from spleen tissue:

[0090] Mouse spleens were collected and placed on a 40 μm cell sieve in a cell culture dish. A small amount of PBS was added, and the spleen was gently ground on ice using the end of a 1 mL syringe plunger to separate and filter the spleen cells. The collected cell filtrate was centrifuged at 1500 rpm at 4°C for 5 min, the supernatant was discarded, 2 mL of erythrocyte lysis buffer was added, and the mixture was gently mixed and incubated for 10 min. After incubation, 5 times the volume of PBS was added to terminate lysis, and the mixture was centrifuged at 1500 rpm at 4°C for 5 min. The supernatant was discarded, 1 mL of PBS was added to resuspend the cells, and the mixture was filtered through a 200-mesh sieve to obtain a suspension of single spleen cells.

[0091] Flow cytometry detection of immune cell subsets:

[0092] ① Adjust the sample cell concentration to approximately 1×10 using pre-cooled PBS. 6 Aliquots were prepared at 1.5 mL per 1 mL centrifuge tube.

[0093] ② Perform fluorescent labeling and staining according to the following scheme.

[0094] a) CD3 FITC, CD8 APC, CD4 PE (labeling CD4 and CD8 T cells);

[0095] b) CD11b FITC, Ly6G PE (labeling MDSCs cells);

[0096] ③ After staining at 4℃ in the dark for 40 min, wash twice with PBS and detect by flow cytometry.

[0097] Results analysis: As shown in the figure, compared with the model group, the proportion of CD8+ T cell subsets in the novel liposome group was significantly increased, but it could significantly reduce the proportion of MDSCs cells in spleen tissue; indicating that the novel liposome group can significantly reduce the proportion of immunosuppressive cells (MDSCs) and increase the proportion of immune killer cells (CD8+ T), thus exerting an anti-tumor effect.

[0098] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention and within the spirit and principles of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A liposome containing gemcitabine methyl terbin, characterized in that: It comprises the following components in parts by weight: 5-15 parts phospholipids, 1-2 parts fritillary glycoside A, and 1 part gemcitabine; the fritillary glycoside A gemcitabine liposomes do not contain cholesterol, and in the fritillary glycoside A gemcitabine liposomes, fritillary glycoside A and the phospholipids form a phospholipid membrane, the inner side of the phospholipid membrane is an inner aqueous phase, the outer side of the phospholipid membrane is an outer aqueous phase, and gemcitabine is encapsulated in the inner aqueous phase.

2. The liposome of gemcitabine methyl terbin according to claim 1, characterized in that: The optimal ratio of phospholipids, fritillary glycoside A, and gemcitabine is 10:2:

1.

3. The liposome of gemcitabine methyl terbin according to claim 1, characterized in that: The phospholipid membrane also includes soybean lecithin.

4. The liposome of gemcitabine methyl terbin according to claim 1, characterized in that: The HPLC purity of the fritillary glycoside A is ≥99%.

5. A method for preparing gemcitabine liposomes as described in any one of claims 1-4, characterized in that: Includes the following steps; S1: Accurately weigh the prescribed amounts of soybean lecithin and gemcitabine and dissolve them in chloroform; S2: Dissolve fritillary glycoside A in methanol, mix the solutions and evaporate them at 60°C for 20 minutes using a rotary evaporator to form a fixed film; S3: Hydrate the obtained film in phosphate-buffered saline at 60°C for 1 hour to obtain a concentrated suspension of liposomes; S4: Sonicate the suspension for 30 minutes to form liposomes, sonicate for 2 seconds, and then let stand for 3 seconds; S5: Pass through a 0.22 μm filter membrane to obtain Fritillaria cirrhosa glycoside A liposomes and store them in a refrigerator at 4°C.

6. A blank liposome for preparing gemcitabine liposomes as described in any one of claims 1-3, characterized in that: The blank liposomes are blank liposomes with fritillary glycoside A as the membrane material. The blank liposomes have a membrane composed of a phospholipid membrane and fritillary glycoside A. The blank liposomes do not contain cholesterol.

7. A pharmaceutical formulation containing gemcitabine liposomes as described in any one of claims 1-4, characterized in that: It is made from gemcitabine liposomes and other pharmaceutically acceptable excipients.

8. The use of the liposome of gemcitabine methyl terbin as described in any one of claims 1-4 in the preparation of a drug for treating pancreatic cancer.

9. The application according to claim 8, characterized in that: The aforementioned liposome containing gemcitabine can significantly reduce the proportion of immunosuppressive cells and increase the proportion of cytotoxic cells.

Images