Liposome, its preparation method and application in preparing drugs for treating PARP inhibitor-resistant breast cancer

Abstract

The present invention provides a liposome, a preparation method thereof, and an application thereof in the preparation of a drug for treating PARP inhibitor-resistant breast cancer, relating to the field of biotechnology. A preparation method of a liposome provided by the present invention includes: injecting an ethanol solution dissolved with soybean phospholipid and cholesterol into an aqueous histidine solution for hydration under stirring conditions, volatilizing the ethanol component after the hydration ends, and then preparing the liposome after ultrasonic treatment. This preparation method is simple and convenient, and the encapsulation efficiency of the prepared liposome loaded with histidine is high. Through research by the inventor, it is found that histidine metabolic reprogramming occurs during the process of PARPi resistance in breast cancer cells, enabling the maintenance of DNA synthesis and repair. After treating PARPi-resistant breast cancer cells with the liposome of the present invention, the histidine content in breast cancer cells can be increased, making PARPi-resistant cells sensitive to PARPi, thereby realizing the treatment of PARPi-resistant breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a liposome and a preparation method thereof, and an application thereof in preparing a drug for treating PARP inhibitor-resistant breast cancer. Background Art

[0002] Approximately 5% of breast cancer patients harbor mutations or deletions in the BRCA tumor suppressor genes, which repair DNA double-strand breaks (DSBs) by homologous recombination (HR). Therefore, in a clinical trial, a synthetic lethal interaction between BRCA deficiency and poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) treatment was established and led to promising improved responses. Briefly, PARP is a nuclear protein involved in multiple cellular processes, including chromatin remodeling and DNA damage repair. Upon DNA damage, PARP is rapidly recruited to the break site, where it participates in the synthesis of protein-conjugated polymers of ADP-ribose, a process known as PARylation. Treatment of BRCA-deficient breast cancer with poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) has shown promising clinical efficacy. In PARPi-treated BRCA-deficient cells, DSBs are not repaired, ultimately triggering apoptosis. Despite the promising rate of improvement, durable clinical response rates in patients with advanced disease are limited, primarily due to acquired resistance. Resistance mechanisms can be divided into the following categories: upregulation of drug efflux pumps, restoration of BRCA function, and restoration of fork stability. However, current progress in overcoming PARPi resistance does not address all cases of PARPi resistance, and there is currently no targeted therapy. Therefore, understanding the underlying mechanisms and identification of targets remains urgently needed.

[0003] Metabolic reprogramming is a hallmark of cancer cells. The key features of the cancer metabolome are altered glucose metabolism, increased fatty acid consumption, and altered dependence on amino acids. The 20 standard proteinogenic amino acids contribute to a range of important processes for cell proliferation, including the biosynthesis of proteins, nucleotides, lipids, glutathione, glucosamine, and polyamines, and the replenishment of carbon in the tricarboxylic acid (TCA) cycle (anaplerosis). Cancer cells are characterized by upregulated glutamine hydrolysis and increased dependence on serine and methionine. Glutamine is a non-essential amino acid that promotes cancer cell proliferation as an important nitrogen donor for amino acid and nucleotide synthesis. The high rate of glutamine hydrolysis supports rapid proliferation by promoting the supply of precursors for biosynthetic pathways while providing cells with aKG, oxaloacetate, or pyruvate under conditions of glutamine starvation. However, the understanding of amino acid metabolic reprogramming is far from satisfactory. Targeting the lethal interaction between amino acids and cancer cells is a promising therapeutic strategy. However, there are currently few reports on metabolic reprogramming of PARPi resistance.

[0004] In addition, liposomes are a good drug carrier that can achieve targeted drug delivery. However, there are currently many methods for synthesizing liposomes, and there are problems such as low encapsulation efficiency and poor stability.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The first object of the present invention is to provide a method for preparing liposomes. The liposomes prepared by the method have a high encapsulation rate and can be used for the treatment of PARP inhibitor-resistant breast cancer to solve the above-mentioned problem.

[0007] The second object of the present invention is to provide a liposome.

[0008] The third object of the present invention is to provide the use of the above liposome in the preparation of a drug for treating PARP inhibitor-resistant breast cancer.

[0009] The fourth object of the present invention is to provide the use of the above liposome combined with PARP inhibitor in the preparation of a drug for treating PARP inhibitor-resistant breast cancer.

[0010] The fifth object of the present invention is to provide a drug for treating PARP inhibitor-resistant breast cancer.

[0011] In order to achieve the above objectives, the following technical solutions are adopted:

[0012] In a first aspect, the present invention provides a method for preparing liposomes, comprising: injecting an ethanol solution containing soybean lecithin and cholesterol into a histidine aqueous solution under stirring conditions for hydration, volatilizing the ethanol component after the hydration, and then preparing liposomes after ultrasonic treatment;

[0013] The concentration of the histidine aqueous solution is 0.1-0.15 mg / mL;

[0014] In the ethanol solution, the concentration of soybean lecithin is 8-12 mg / mL, and the concentration of cholesterol is 2-3 mg / mL;

[0015] The volume ratio of the histidine aqueous solution to the ethanol solution is 4:1-1:1;

[0016] The hydration temperature is 55-65°C.

[0017] As a further technical solution, the concentration of the histidine aqueous solution is 0.125 mg / mL;

[0018] And / or, in the ethanol solution, the concentration of soybean lecithin is 10 mg / mL, and the concentration of cholesterol is 2.5 mg / mL;

[0019] And / or, the volume ratio of the histidine aqueous solution to the ethanol solution is 2:1.

[0020] As a further technical solution, during the hydration process, a solubilizing agent is added;

[0021] And / or, the hydration time is 0.5-2h;

[0022] And / or, the hydration temperature is 60°C.

[0023] As a further technical solution, the stirring speed is 400-600r / min;

[0024] And / or, the power of the ultrasound is 150-250W, the ultrasound mode is intermittent ultrasound, working for 1-3s, stopping for 1-3s, and the ultrasound time is 5-15min.

[0025] In a second aspect, the present invention provides a liposome prepared by the above-mentioned preparation method.

[0026] In a third aspect, the present invention provides the use of the above liposome in the preparation of a drug for treating PARP inhibitor-resistant breast cancer.

[0027] As a further technical solution, the PARP inhibitors include Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib and Pamiparib.

[0028] In a fourth aspect, the present invention provides the use of the above-mentioned liposome combined with a PARP inhibitor in the preparation of a drug for treating PARP inhibitor-resistant breast cancer.

[0029] As a further technical solution, the PARP inhibitors include olaparib, rucaparib, niraparib, talazoparib, fluzoparib and pamiparib.

[0030] In a fifth aspect, the present invention provides a drug for treating PARP inhibitor-resistant breast cancer, comprising the above-mentioned liposome and a PARP inhibitor.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The method for preparing the liposome provided by the invention is simple and convenient, and the prepared liposome loaded with histidine has a high encapsulation rate.

[0033] The inventors have found that histidine metabolism reprogramming occurs during the PARPi resistance process in breast cancer cells, allowing DNA synthesis and repair to be maintained. After the PARPi-resistant breast cancer cells are treated with the liposomes provided by the present invention, the histidine content in the breast cancer cells can be increased, making the PARPi-resistant cells sensitive to PARPi again, thereby achieving the treatment of PARPi-resistant breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 :The experimental results of histidine metabolic reprogramming conferring PARPi resistance to cancer cells;

[0036] Figure 2 :The experimental results show that liposomal histidine inhibits DNA damage repair but does not affect HR and fork stability;

[0037] Figure 3 The particle size and potential analysis of the liposomes in Comparative Example 1;

[0038] Figure 4 This is the particle size and potential analysis of the liposomes in Example 1. DETAILED DESCRIPTION

[0039] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0040] In a first aspect, the present invention provides a method for preparing liposomes, comprising: injecting an ethanol solution containing soybean lecithin and cholesterol into a histidine aqueous solution under stirring conditions for hydration, volatilizing the ethanol component after the hydration, and then preparing liposomes after ultrasonic treatment;

[0041] The concentration of the histidine aqueous solution may be, for example, but not limited to, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL or 0.15 mg / mL;

[0042] In the ethanol solution, the concentration of soybean lecithin can be, for example, but not limited to 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL or 12 mg / mL, and the concentration of cholesterol can be, for example, but not limited to 2 mg / mL, 2.5 mg / mL or 3 mg / mL;

[0043] The volume ratio of the histidine aqueous solution to the ethanol solution may be, for example, but not limited to, 4:1, 3:1, 2:1 or 1:1;

[0044] The hydration temperature may be, for example, but not limited to, 55°C, 57°C, 59°C, 61°C, 63°C or 65°C.

[0045] The method for preparing the liposome provided by the invention is simple and convenient, and the prepared liposome loaded with histidine has a high encapsulation rate.

[0046] In some optional embodiments, the concentration of the histidine aqueous solution is 0.125 mg / mL;

[0047] In some optional embodiments, in the ethanol solution, the concentration of soybean lecithin is 10 mg / mL, and the concentration of cholesterol is 2.5 mg / mL;

[0048] In some optional embodiments, the volume ratio of the histidine aqueous solution to the ethanol solution is 2:1.

[0049] By further optimizing and adjusting the ratio of each component, the prepared liposome has a high encapsulation rate for histidine and good stability.

[0050] In some optional embodiments, a solubilizing agent is added during the hydration process;

[0051] The solubilizing agent includes but is not limited to polysorbates or polyoxyethylene fatty acid esters;

[0052] The polysorbates include Tween 80;

[0053] In some optional embodiments, during the hydration process, Tween 80 is added at a final concentration of 0.1%-0.3%;

[0054] In some optional embodiments, the hydration time may be, for example, but not limited to, 0.5 h, 1 h, 1.5 h or 2 h.

[0055] In some optional embodiments, the hydration temperature is 60°C.

[0056] In some optional embodiments, the stirring speed may be, for example, but not limited to, 400 r / min, 450 r / min, 500 r / min, 550 r / min or 600 r / min;

[0057] In some optional embodiments, the power of the ultrasound is 150-250W, the ultrasound mode is intermittent ultrasound, working for 1-3s, stopping for 1-3s, and the ultrasound time is 5-15min.

[0058] Through further optimization and adjustment of the preparation parameters of the present invention, the prepared liposome has a high encapsulation rate for histidine and good stability.

[0059] In a second aspect, the present invention provides a liposome prepared by the above-mentioned preparation method.

[0060] The liposome provided by the invention has good stability and high histidine content.

[0061] In a third aspect, the present invention provides the use of the above liposome in the preparation of a drug for treating PARP inhibitor-resistant breast cancer.

[0062] The inventors have found that histidine metabolic reprogramming occurs during the PARPi resistance process in breast cancer cells, allowing DNA synthesis and repair to be maintained. After the PARPi-resistant breast cancer cells are treated with the liposomes provided by the present invention, the histidine content in the breast cancer cells can be increased, making the PARPi-resistant cells sensitive to PARPi again, thereby achieving the treatment of PARPi-resistant breast cancer.

[0063] In some optional embodiments, the PARP inhibitor includes but is not limited to olaparib, rucaparib, niraparib, talazoparib, fluzoparib and pamiparib.

[0064] In a fourth aspect, the present invention provides the use of the above-mentioned liposome combined with a PARP inhibitor in the preparation of a drug for treating PARP inhibitor-resistant breast cancer.

[0065] The inventors have found that the liposomes provided by the present invention can increase the histidine content in breast cancer cells, thereby making PARPi-resistant cells sensitive to PARPi again, thereby improving the effect of PARP inhibitors on PARPi-resistant breast cancer.

[0066] In some optional embodiments, the PARP inhibitor includes but is not limited to olaparib, rucaparib, niraparib, talazoparib, fluzoparib and pamiparib.

[0067] In a fifth aspect, the present invention provides a drug for treating PARP inhibitor-resistant breast cancer, comprising the above-mentioned liposome and a PARP inhibitor.

[0068] The drug could be used to treat ARPi-resistant breast cancer.

[0069] In some optional embodiments, the PARP inhibitor includes but is not limited to olaparib, rucaparib, niraparib, talazoparib, fluzoparib and pamiparib.

[0070] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0071] The reagents and instruments used in the following examples include:

[0072] Reagents:

[0073] name Production batch number Soybean lecithin Avituo (Shanghai) Pharmaceutical Technology Co., Ltd. SY-SO-220401 cholesterol Shanghai MacLean Biochemical Technology Co., Ltd. Anhydrous ethanol Tianjin Zhiyuan Chemical Reagent Co., Ltd. 20230501 Twain 80 Shanghai MacLean Biochemical Technology Co., Ltd. C14134834 Ninhydrin Tianjin Damao Chemical Reagent Factory Reduced ninhydrin dihydrate Aladdin #E2124179

[0074] Experimental instruments:

[0075]

[0076]

[0077] Example 1

[0078] A liposome, the preparation method is as follows:

[0079] Accurately measure 2.5 mg of histidine and dissolve it in 20 ml of pure water as the aqueous phase. Weigh 100 mg of soybean lecithin and 25 mg of cholesterol and dissolve them in 10 ml of ethanol as the oil phase. Preheat the oil phase to 60°C, and use a syringe to uniformly inject 10 ml of the oil phase into the 20 ml of the aqueous phase. Hydrate for 1 hour under 60°C magnetic stirring (500 r / min) and add 0.2% Tween 80. After hydration, evaporate the ethanol under reduced pressure. Then, ultrasonicate with a probe (200 W, work for 2 seconds, stop for 2 seconds) for 10 minutes to obtain histidine-liposomes.

[0080] Example 2

[0081] A liposome, the preparation method is as follows:

[0082] Accurately measure 2 mg of histidine and dissolve it in 20 ml of pure water as the aqueous phase. Weigh 80 mg of soybean lecithin and 30 mg of cholesterol and dissolve them in 10 ml of ethanol as the oil phase. Preheat the oil phase to 55°C, and use a syringe to uniformly inject 50 ml of the oil phase into the 20 ml of the aqueous phase. Hydrate for 1 hour under 55°C magnetic stirring (500 r / min) and add 0.1% Tween 80. After hydration, evaporate the ethanol under reduced pressure. Then subject to probe ultrasound (150 W, work 3 seconds, stop 1 second) for 15 minutes to obtain histidine-liposomes.

[0083] Example 3

[0084] A liposome, the preparation method is as follows:

[0085] Accurately measure 3 mg of histidine and dissolve it in 20 ml of pure water as the aqueous phase. Weigh 120 mg of soybean lecithin and 20 mg of cholesterol and dissolve them in 10 ml of ethanol as the oil phase. Preheat the oil phase to 65°C, and use a syringe to uniformly inject 10 ml of the oil phase into the 10 ml of the aqueous phase. Hydrate for 1 hour under 65°C magnetic stirring (500 r / min) and add 0.3% Tween 80. After hydration, evaporate the ethanol under reduced pressure. Then subject to probe ultrasound (250 W, work 1 s, stop 3 s) for 5 min to obtain histidine-liposomes.

[0086] Comparative Example 1

[0087] A liposome, which differs from that of Example 1 in that it does not contain histidine.

[0088] Example 4

[0089] The encapsulation efficiency, particle size and potential of the liposomes provided in Example 1 were measured. The experimental process and results are as follows:

[0090] Determination of encapsulation rate (encapsulation rate is an important indicator for evaluating liposomes. It refers to the percentage of drug content encapsulated in the lipid bilayer to the total dosage, which can reflect the degree of drug encapsulation in liposomes):

[0091] Take 2 mg of histidine and dilute it to 10 ml of water. Use the ninhydrin method to develop the color and then measure the ultraviolet absorption.

[0092] The encapsulation efficiency of the liposomes was determined by ultrafiltration. 400ul of liposomes were placed in an ultrafiltration tube, centrifuged and the clear solution (free histidine was filtered out) was removed, and the ultraviolet absorption was determined after color development using the ninhydrin method.

[0093] Take 500ul of liposomes into a volumetric flask, dilute to 10ml with methanol, and break the emulsion by ultrasonication for 10min. Use the ninhydrin method to develop the color and measure the ultraviolet absorption.

[0094] Test results: The method measured the liposome encapsulation efficiency to be 83.3%, and the drug loading to be 0.225 mg / ml. In addition, the liposomes provided in Example 2-3 also achieved good encapsulation of histidine after testing.

[0095] Determination of particle size and potential:

[0096] The particle size was measured by a particle size analyzer (such as Figure 3 and Figure 4 As shown), the particle size of the blank liposome (Comparative Example 1) was 51.4 nm, and the PDI was 0.164. The particle size of the drug-loaded liposome (Example 1) was 67.5 nm, and the PDI was 0.211.

[0097] The microscopic morphology of liposomes is generally spherical. Lipid suspensions can be observed by transmission electron microscopy or freeze-dried powders can be observed by scanning electron microscopy. Qualified liposomes need to have regular morphology and uniform dispersion. The particle size and distribution of liposomes determine the part that interacts with cells in the body, as well as absorption and distribution. The morphology and particle size of liposomes can affect the encapsulation rate and stability. Liposomes are evenly distributed, highly stable, slowly released, and have high oral bioavailability. Liposomes with the smallest particle size are preferred, and small particle size liposomes have more advantages.

[0098] Example 5

[0099] To clarify the amino acid metabolic remodeling that occurs during PARPi resistance in breast cancer, PARPi-resistant cell lines (PDXR7 and PDXR8) were first constructed and the amino acid content in different cells was analyzed using metabolome sequencing. The results showed that the contents of 19 amino acids in PARPi-resistant cells were significantly increased, but only the content of histidine was significantly decreased. Subsequently, another PARPi-resistant strain was constructed and the content of histidine and its metabolic intermediates were detected in samples of PARPi-resistant patients. Since metabolic remodeling is mainly achieved by changes in the content or activity of enzymes that catalyze each metabolic step, transcriptome sequencing and KEGG analysis were also performed. The results showed that the histidine metabolic pathway was significantly changed. Metabolome sequencing and transcriptome sequencing were used in combination to clarify and analyze the amino acid metabolic remodeling that occurs during PARPi resistance in breast cancer. The results show that histidine metabolic remodeling plays an important role in the PARPi resistance of breast cancer.

[0100] In addition, considering that PARPi-resistant cells exhibit impaired histidine uptake ability, when cells are incubated with a medium containing a high concentration of histidine, the intracellular histidine level is limited. Therefore, liposomes (Liposome His, Lipo His) containing histidine (His), i.e., the liposomes provided in Example 1, and empty liposomes (Liposome Ctrl, LipoCtrl), i.e., the liposomes provided in Comparative Example 1, were synthesized.

[0101] PDXR7 and PDXR8 cells were treated with His liposomes and Olaparib, and the intracellular histidine level was measured. Liposome His treatment significantly increased the intracellular histidine level ( Figure 1 The specific experimental steps are as follows:

[0102] First, PDXR7 and PDXR8 cells treated with His liposomes and empty liposomes were collected, and necessary processing steps such as extraction and purification were performed to obtain the histidine samples to be tested. Then a series of histidine standard solutions with known concentrations were prepared for the construction of a standard curve. A high-resolution mass spectrometer was used to set the instrument parameters according to the experimental requirements, including scanning mode, fragmentation voltage, etc. The histidine sample solution to be tested and the standard solution were injected into the mass spectrometer through the injection system. The mass spectrometer was started for sample analysis, the mass spectrum was recorded, and the relative content of histidine was determined based on the mass spectrum of the standard. The obtained data were processed using mass spectrometry software, including peak area calculation, curve fitting, etc. The relative content of histidine to be tested was calculated based on the standard curve and the peak area of ​​the sample to be tested. The experimental conditions, mass spectrum, data processing results, and relative content calculation results were recorded. The results showed that the histidine level in cells treated with His liposomes, whether PDXR7 or PDXR8, was higher than that in the blank control group.

[0103] Next, in order to reveal the biological importance of histidine in PARPi resistance. Two systems were established as described below. PARPi-resistant PDXR cells (PDXR7 and PDXR8) were treated with liposomal His and Olaparib to interfere with histidine metabolic reprogramming, and a control group was set up. PARPi-sensitive cells (PDXS1, PDXS2) were treated with conditioned medium containing low concentrations of histidine (0.05 times His) and Olaparib and conditioned medium containing high concentrations of histidine (1 times His) and Olaparib to simulate metabolic reprogramming. The results showed that increased histidine levels significantly inhibited the proliferation, self-renewal and survival of PARPi-resistant PDXS cells, which was manifested as decreased cell viability, decreased tumor sphere formation ability, and increased apoptosis rate ( Figure 1 B, D, F, H in which, Figure 1 The vertical axes of D and E in the figure represent the logarithmic fraction of no response. Let's take an example to illustrate the meaning of the horizontal and vertical axes: the inventor inoculated 10 cells on a plate, and after two weeks of culture, only 9 tumor spheres were observed. That is to say, only one cell was not observed as a tumor sphere, the proportion of no response was 1 / 10, and the logarithmic proportion of no response was -1. Therefore, the point (10, -1) was obtained in the figure. The horizontal axis represents the number of inoculated cells. ). As expected, histidine deficiency inhibited PARPi-induced cell apoptosis and promoted cell proliferation and sphere formation ( Figure 1 The specific experimental steps are as follows:

[0104] First, the CCK8 experiment was performed. PDX cells were cultured using standard culture techniques to ensure that the cells were in a good growth state and were distributed into culture dishes. Experimental and control groups were set up. PARPi-resistant PDXR cells treated with His liposomes and Olaparib were the experimental group, and those treated with empty liposomes and Olaparib were the control group; PARPi-sensitive PDXS cells treated with low-concentration histidine (0.05 times His) and Olaparib conditioned medium were the experimental group, and those treated with normal concentration histidine (1 times His) and Olaparib were the control group. The differently treated cells were added to the cell culture medium in the culture dish and treated according to the predetermined concentration and treatment time. After the treatment, CCK-8 reagent was added to each culture dish according to the kit instructions or the experimental optimization method. The culture dish was continued to be incubated to allow the cells to fully react with the CCK-8 reagent and allow it to be reduced to produce a detectable product. Then, the absorbance in each culture dish was measured using an ELISA reader or a photometer, and the optical density values ​​of the experimental group and the control group were measured at an appropriate wavelength. The absorbance values ​​of each experimental group and the control group were recorded. Subtract the absorbance value of the control group from the absorbance value of each experimental group to obtain the relative absorbance value. Then, according to the characteristics of the CCK-8 reagent and the experimental design, the relative absorbance value is converted into a relative cell proliferation rate or cytotoxicity. The relative cell proliferation rate of each experimental group and the treatment time are plotted into a curve graph, and the morphology and change trend of the curve are analyzed. The experimental results are statistically analyzed, such as analysis of variance (ANOVA) or t-test, to evaluate the significant differences in cell proliferation rate under different treatment conditions. The results are shown in Figure 1 As shown in Figure B, increased histidine levels can significantly inhibit the proliferation of PARPi-resistant PDXR cells, which is manifested by decreased cell viability. Figure 1 As shown in C, histidine deficiency promoted PARPi-induced cell proliferation.

[0105] Next, a limiting dilution experiment is performed. The treated PDX cells are cultured in an appropriate culture medium until the logarithmic growth phase. The cells are counted using a hemocytometer or an automated cell counter. The tumor cell line is serially diluted to obtain cell suspensions of different dilutions. Each dilution of the cell suspension is dispensed into individual culture dishes or wells of a culture plate according to a predetermined number of cells. The cell suspension is cultured under non-adhesive conditions, such as in a culture medium containing an appropriate concentration of agar or polyvinyl alcohol. Appropriate culture conditions, including temperature, humidity, and CO, are maintained. 2concentration, etc. to promote the formation of tumor spheres. Regularly observe and record the formation of tumor spheres, including the size, shape and number of the spheres. After the culture is completed, perform relevant experimental operations, such as staining, imaging or other analysis. Calculate the ratio or percentage of tumor sphere formation at different dilutions to determine the dilution limit of tumor sphere formation. Count the number of tumor spheres formed at each dilution to evaluate the effect of different dilutions on tumor sphere formation. Analyze the morphological characteristics of tumor spheres, such as size, shape and surface structure, to evaluate their biological characteristics. Repeat the experiment to verify the repeatability and stability of the results. Perform statistical analysis on the experimental data, including mean, standard deviation and significance test, to evaluate the differences between different treatment groups. The results are shown in Figure 1 As shown in Figure D, increased histidine levels can significantly inhibit the self-renewal of PARPi-resistant PDXR cells, which is manifested by a decrease in the ability to form tumor spheres. Figure 1 As shown in E, histidine depletion promoted PARPi-induced tumor sphere formation.

[0106] Then, conduct a flow cytometry experiment. Collect the treated PDX cells to be tested. Use tools such as a cell counter or microscope to count the cells to determine the number of cells. Select appropriate fluorescent dyes or antibodies to label the cells according to experimental requirements. Place the labeled cell samples into the flow cytometer and set the instrument parameters according to experimental requirements, such as laser wavelength, fluorescence channel, etc. Start the flow cytometer for sample analysis, excite the labeled cells with the laser, detect the fluorescence signal, and record the fluorescence intensity and type of fluorescent dye for each cell. The flow cytometer converts the fluorescence signal into an electrical signal and stores the data in a computer. Use flow cytometer software or other data analysis software to process and analyze the collected data, such as cell counting, fluorescence intensity statistics, etc. Based on the data analysis results, draw charts to display information such as cell labeling and cell subpopulation distribution. The results are as follows: Figure 1 As shown in F and H (left), increased histidine levels can significantly inhibit the survival of PARPi-resistant PDXR cells, which is manifested by increased cell apoptosis rate. Figure 1 As shown in G and H (right), histidine deficiency inhibited PARPi-induced cell apoptosis.

[0107] To investigate the in vivo function of histidine metabolic reprogramming in PARPi resistance, sensitive and resistant cells were subcutaneously transplanted into immunocompromised mice. Mice bearing resistant cancer cells were treated with liposomal Ctrl or liposomal His. Mice bearing sensitive cancer cells were fed a conditioned diet supplemented with low concentrations of histidine (Low Histidine group) or normal concentrations of histidine (Standard group) (the content of histidine in the standard mouse diet was 4.6 g / 100 g feed (for maintenance adult mice) and 3.3 g / 100 g feed (for growth and breeding mice); the content of histidine in the mouse diet of the low histidine group was 0.002 g / 100 g). Mice were treated with olaparib throughout the experiment. PDX cells were cultured and ensured to be in good growth condition. The cultured cells were collected and subjected to necessary treatments, such as trypsin digestion, to obtain a single cell suspension. The cells were counted using tools such as a cell counter or microscope to determine the cell concentration. Inject the counted tumor cell suspension into the subcutaneous tissue of mice at an appropriate concentration. Observe the growth of the animals, measure the tumor volume regularly, and record the growth curve. When the tumor reaches a certain size, terminate the experiment. Euthanize the animals and take out the tumor tissue and adjacent normal tissue for sampling. Perform necessary processing on the sampled tissue samples, such as fixation. Collect data such as tumor growth rate and tumor mass, and analyze the results. Analyze the differences in tumor mass and growth rate between different experimental groups, and evaluate the effects of drugs or treatments on tumor growth. The results are as follows: Figure 1 As shown in Figure 1, mice carrying histidine-mimicking reprogrammed cancer cells showed larger tumor volumes, whereas mice treated with liposomal His had smaller tumor volumes. Figure 1 As shown in J and K, mice carrying cancer cells reprogrammed with mimetic histidine showed faster tumor growth, while mice treated with liposomal His had slower tumor growth. This demonstrates that our synthesized histidine-containing liposomes can be used to treat PAPRi-resistant breast cancer.

[0108] Histidine metabolic reprogramming promotes DNA damage repair without affecting homologous recombination and fork stability. Restoration of DNA damage repair capacity is a hallmark of PARPi resistance. First, sensitive / resistant PDX cell lines were treated with Olaparib, and then the expression of DNA damage marker γ-H2A.X in sensitive / resistant PDX cell lines was detected by immunoblotting. As expected, in sensitive PDX cell lines, the level of γ-H2A.X increased under the action of Olaparib; in resistant PDX cell lines, γ-H2A.X showed almost no change after Olaparib treatment (e.g. Figure 2). Next, sensitive and resistant PDX cell lines were treated with His liposomes, blank liposomes, 1x His, and 0.05x His. By immunofluorescence (IF) staining and immunoblotting, we observed that His liposomes induced DNA damage and increased the level of γ-H2A.X. We treated the samples with fluorescently labeled γ-H2A.X antibodies and DAPI. Red light represents γ-H2A.X and blue light represents the nucleus. It is used to detect whether γ-H2A.X is expressed in the nucleus or in the cytoplasm or on the cell membrane. At the same time, the blue light also serves as a control. The intensity of red light represents the expression level of γ-H2A.X protein. If the red light and blue light overlap, it means that the target protein is expressed in the nucleus. In contrast, the conditioned medium containing 0.05x His inhibited DNA damage (as shown in Figure 2A). Figure 2 The decrease in γ-H2A.X levels is associated with the repair of DNA damage. Cells were collected at different time points for immunoblotting analysis to determine the γ-H2A.X levels. The results showed that cells with disrupted histidine degradation pathways had a significantly impaired ability to eliminate γ-H2A.X (e.g. Figure 2 In contrast, in the sensitive PDX cell line treated with low concentrations of histidine, the elimination of γ-H2A.X was superior to that of the control cell line (as shown in D). Figure 2 As shown in E in the figure). Homologous recombination (HR) restoration and fork-dependent DNA repair are the main mechanisms of PARPi resistance. Rad51 is a biomarker of functional HR. The samples were treated with Rad51 antibody and γ-H2A.X antibody for FACS, and Rad51 and γ-H2A.X were detected and statistically processed by fluorescence activated cell sorting (FACS), respectively, and then DNA damage and DNA repair analysis were performed. It is worth noting that compared with the corresponding populations in the control cells, the proportion of Rad51+ cells increased in the cells treated with His liposomes, while the proportion of γ-H2A.X+ cells did not decrease (as shown in Figure 2A). Figure 2 , FH in Figure 3). Since DSBs may occur as a result of unstable replication forks, the effect on replication fork progression was investigated using a DNA fiber assay. Briefly, cells were incubated (30 min) in the corresponding medium (medium containing Olaparib as well as His-liposomes, empty liposomes, 1x His, or 0.05x His), pulsed first with CIdU (15 min) and then with IdU (15 min). Fork velocity (kb / min) was calculated by measuring the total length of CIdU-positive and IdU-positive DNA fibers. Fork stability was determined by the IdU / CIdU ratio. As expected, disrupting the histidine degradation pathway resulted in a decrease in fork velocity without altering fork stability (as shown in Figure 3). Figure 2 , as shown in I and J in FIG.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of liposomes in the preparation of drugs for treating PARP inhibitor-resistant breast cancer; The preparation method of the liposome comprises: Under stirring conditions, an ethanol solution containing soybean lecithin and cholesterol is injected into a histidine aqueous solution for hydration, and after the hydration is completed, the ethanol component is volatilized, and then liposomes are prepared after ultrasonic treatment; The concentration of the histidine aqueous solution is 0.1-0.15 mg / mL; In the ethanol solution, the concentration of soybean lecithin is 8-12 mg / mL, and the concentration of cholesterol is 2-3 mg / mL; The volume ratio of the histidine aqueous solution to the ethanol solution is 4:1-1:1; The hydration temperature is 55-65°C; The PARP inhibitor is olaparib.

2. The use according to claim 1, characterized in that: The concentration of the histidine aqueous solution is 0.125 mg / mL; And / or, in the ethanol solution, the concentration of soybean lecithin is 10 mg / mL, and the concentration of cholesterol is 2.5 mg / mL; And / or, the volume ratio of the histidine aqueous solution to the ethanol solution is 2:

1.

3. The use according to claim 1, characterized in that: During the hydration process, a solubilizer is added; And / or, the hydration time is 0.5-2h; And / or, the hydration temperature is 60°C.

4. The use according to claim 1, characterized in that: The stirring speed is 400-600r / min; And / or, the power of the ultrasound is 150-250W, the ultrasound mode is intermittent ultrasound, working for 1-3s, stopping for 1-3s, and the ultrasound time is 5-15min.

5. Use of the liposomes combined with a PARP inhibitor as claimed in any one of claims 1 to 4 in the preparation of a drug for treating PARP inhibitor-resistant breast cancer; The PARP inhibitor is olaparib.

6. A drug for treating PARP inhibitor-resistant breast cancer, characterized in that: Comprising the liposome described in any one of claims 1 to 4 and a PARP inhibitor; The PARP inhibitor is olaparib.

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