Application of drug-loaded lipid nanocarriers in the preparation of drugs for the treatment of drug-resistant lung cancer

Abstract

The present application provides the use of drug-loaded lipid nanocarriers in the preparation of drugs for treating drug-resistant lung cancer, wherein the drug-loaded lipid nanocarriers are made of raw materials including lecithin, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol-amino, osimertinib, and anlotinib hydrochloride, and are modified with EGF. The drug-loaded lipid nanocarriers can be significantly enriched in tumor tissues, effectively inhibit the proliferation of non-small cell lung cancer cells, and effectively reduce the acquired drug resistance of non-small cell lung cancer to EGFR-TKI. In vivo experiments in mice have shown that the nanoliposome drug delivery system has a better tumor inhibition effect than the combination of simple osimertinib and anlotinib.

Description

Technical Field

[0001] The present application belongs to the field of anti-tumor drugs. Specifically, the present application provides the use of drug-loaded lipid nanocarriers in the preparation of drugs for treating drug-resistant lung cancer. Background Art

[0002] Lung cancer is one of the most common types of cancer, of which non-small cell lung cancer accounts for a large number. The treatment of early lung cancer is mainly based on surgery, but due to the lack of obvious early symptoms, many patients have missed the best time for surgery when they are diagnosed, and lung cancer also has a high recurrence and metastasis rate after surgery. For patients who have missed the surgical indications, most clinical treatment plans are based on radiotherapy and chemotherapy. However, the effect of radiotherapy and chemotherapy has reached a bottleneck period and no further treatment breakthroughs can be achieved.

[0003] Targeted therapy is a new breakthrough in the treatment of lung cancer. In recent years, with the development of precision medicine, more and more lung cancer driver genes have been discovered. Among them, EGFR mutation is the most common driver gene mutation in patients with non-small cell lung cancer (NSCLC) in my country, present in about half of the patients. Fortunately, the emergence of EGFR-TKI has brought hope of long-term survival to patients with advanced NSCLC with EGFR mutations. Among them, osimertinib has established a new standard for the first-line treatment of advanced NSCLC with EGFR mutations with its excellent efficacy and safety, and has been unanimously recommended as a first-line preferred option by domestic and international guidelines.

[0004] However, the use of osimertinib for treatment faces a serious problem: the vast majority of lung cancer patients will develop varying degrees of drug resistance about one year after treatment, leading to disease recurrence and treatment failure, which is called acquired drug resistance.

[0005] Therefore, how to study the mechanism of EGFR-TKI resistance and reverse resistance and apply the multi-target tyrosine kinase inhibitor anlotinib to the preparation of non-small cell lung cancer drugs is an urgent problem that technicians in this field need to solve. Summary of the invention

[0006] On the one hand, the present application provides the use of a drug-loaded lipid nanocarrier in the preparation of a drug for treating drug-resistant lung cancer, wherein the drug-loaded lipid nanocarrier is made of raw materials including 100 to 400 parts by weight of lecithin, 50-100 parts by weight of cholesterol and 20 to 60 parts by weight of distearoyl phosphatidylethanolamine-polyethylene glycol-amino, 50 to 100 parts by weight of osimertinib, and 5 to 20 parts by weight of anlotinib hydrochloride, and is modified with EGF; the drug-resistant lung cancer is osimertinib-resistant lung cancer.

[0007] Furthermore, the lung cancer is non-small cell lung cancer.

[0008] Furthermore, the weight ratio of osimertinib to anlotinib hydrochloride in the drug-loaded lipid nanocarrier is 8:1.

[0009] Furthermore, the total concentration of osimertinib and anlotinib hydrochloride in the drug is 1-5 mg / mL.

[0010] Furthermore, the drug loading amount of osimertinib in the drug-loaded lipid nanocarrier is 7-8% w / w, and the drug loading amount of anlotinib hydrochloride is 0.9-1% w / w.

[0011] Furthermore, the drug-loaded lipid nanocarrier is prepared by a method comprising the following steps:

[0012] (1) Phosphatidylcholine, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol-amino and osimertinib are fully dissolved in anhydrous ethanol, injected into an ionic aqueous solution, stirred, and the resulting solution is passed through a polycarbonate membrane to obtain osimertinib-loaded nanoliposomes;

[0013] (2) replacing the solution system of osimertinib liposomes obtained in step (1) with 5% glucose, adding anlotinib hydrochloride, and then replacing the solution system with PBS solution to prepare nanoliposomes co-loaded with osimertinib / anlotinib drugs;

[0014] (3) Take EGF, add EDC and NHS to activate the carboxyl group of EGF, and incubate it with the nanoliposomes co-loaded with osimertinib / anlotinib drugs obtained in step (2).

[0015] Furthermore, in steps (1) and (2) of the method, 100 to 400 parts by weight of lecithin, 50 to 100 parts by weight of cholesterol, 20 to 60 parts by weight of distearoyl phosphatidylethanolamine-polyethylene glycol-amino, 50 to 100 parts by weight of osimertinib, 1000 to 4000 parts by weight of ethanol solution, and 5 to 20 parts by weight of anlotinib hydrochloride are used; preferably 200 to 300 parts of lecithin, 60 to 80 parts of cholesterol, 40 to 50 parts of distearoyl phosphatidylethanolamine-polyethylene glycol-amino, 60 to 80 parts of osimertinib, 2000 to 3000 parts of ethanol solution, and 10 to 15 parts of anlotinib hydrochloride.

[0016] Furthermore, the weight ratio of EGF to nanoliposome used in step (3) is 1:50-500; preferably 1:100-200.

[0017] Furthermore, the medicine is in the form of an injection.

[0018] Furthermore, the medicine also contains pharmaceutically acceptable excipients.

[0019] Depending on the dosage form, those skilled in the art can routinely select excipients for the drug of the present application based on knowledge in the pharmaceutical field, including but not limited to fillers, adhesives, lubricants, surfactants, coating agents, capsule shells, taste masking agents, flavoring agents, solvents, co-solvents, suspending agents, pH adjusters, antioxidants, etc.

[0020] The present invention provides an EGF-modified nanoliposome drug delivery system loaded with osimertinib and anlotinib, or an application of the EGF-modified nanoliposome drug delivery system loaded with osimertinib and anlotinib obtained by the above preparation method in the preparation of anti-non-small cell lung cancer drugs.

[0021] The drug-carrying system provided by the present invention uses liposomes as carriers, and after being modified with EGF, osimertinib and anlotinib are loaded. The liposome nanomedicine provided by the present invention has a high degree of biocompatibility and biodegradability because the structure of the liposome is extremely similar to that of the cell membrane. Therefore, the liposome can protect the drug from being degraded by enzymes before reaching the lesion site. At the same time, the drug is "hidden" inside the liposome in the form of physical encapsulation, which can improve the stability of the drug, reduce the toxicity of the drug, increase the dosage, and exert a better therapeutic effect. In addition, the liposome shows that after being modified with EGF, the effective retention time of the liposome in the lesion site can be extended, thereby achieving efficient targeted drug delivery.

[0022] Anlotinib is a small molecule, multi-target tyrosine kinase receptor inhibitor that can effectively inhibit kinases such as VEGFR 1-3, PDGFR α / β, FGFR 1-4, c-Kit, etc., and has the effects of anti-tumor angiogenesis and inhibiting tumor growth. Anlotinib can promote the distribution of targeted drugs in tissues. The present invention combines anlotinib and osimertinib to inhibit the activity of drug-resistant cancer cells; after being packaged in nanoliposomes modified with EGF, the drug can be significantly enriched in lung cancer cells and tissue lesions; it can more effectively inhibit the proliferation of cancer cells and induce their apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1The figure shows the changes of cell activity of various cells under the action of osimertinib and / or anlotinib; Part A shows the cell activity curves of H1975 and H1975OR cells under different concentrations of osimertinib; Part B shows the cell activity curves of PC9 and PC9OR cells under different concentrations of osimertinib; Part C shows the cell activity curves of H1975, H1975OR, PC9, and PC9OR cells after applying different concentrations of anlotinib; Part D shows the cell activity curves of H1975OR cells after applying different molar ratios of osimertinib Part A shows the cell activity curves of PC9OR cells after application of osimertinib and anlotinib, as well as the CI (combination index) curve when the molar ratio of osimertinib: anlotinib is 8:1; Part E shows the cell activity curves of PC9OR cells after application of different molar ratios of osimertinib and anlotinib, as well as the CI (combination index) curve when the molar ratio of osimertinib: anlotinib is 1:2; Part F shows the cell colony formation images of H1975OR and PC9OR when osimertinib is used alone, anlotinib is used alone, and anlotinib + osimertinib is used in combination.

[0024] Figure 2 Parameter diagrams and images of co-drug-loaded liposomes and EGF-modified liposomes; Part A shows the average particle size, polydispersity coefficient, and potential data of liposomes; Part B shows projection electron micrographs of co-drug-loaded liposomes and EGF-modified liposomes; Part C shows the drug release curves of osimertinib and anlotinib in nanomedicines.

[0025] Figure 3 The effect of combined use of anlotinib + osimertinib and nano-drug on H1975OR cells; Part A is the cell activity curve obtained by combining anlotinib + osimertinib and nano-drug on H1975OR cells for 5 days; Part B is the cell colony formation image of H1975OR after combined use of anlotinib + osimertinib and nano-drug; Part C is the flow cytometry detection of the apoptotic cell ratio of H1975OR after combined use of anlotinib + osimertinib and nano-drug; Part D is the immunoblotting experiment to detect the expression of apoptosis-related proteins in H1975OR cells after combined use of anlotinib + osimertinib and nano-drug;

[0026] Figure 4 The localization of liposome drugs in cells and organisms; Part A uses a confocal microscope to observe the cellular localization of liposome drugs carrying FITC fluorescence labels in human normal bronchial epithelial B2B cells and drug-resistant cells; Part B uses a small animal imager to observe the tissue localization of liposome drugs carrying Cy5.5 fluorescence labels in a nude mouse subcutaneous transplant tumor model.

[0027] Figure 5The effect of the combined use of anlotinib + osimertinib and nanodrug on tumor volume; Part A is a schematic diagram of the tumor morphology in H1975OR nude mice in vivo after the combined use of anlotinib + osimertinib and nanodrug, and Part B is a schematic diagram of the tumor volume in H1975OR nude mice in vivo after the combined use of anlotinib + osimertinib and nanodrug. DETAILED DESCRIPTION

[0028] The following examples are provided to facilitate a better understanding of the present invention, but are not limited thereto. These examples are only used for illustrative purposes and in no way limit the scope of protection of the present invention.

[0029] Example 1 Effect of combined use of anlotinib and osimertinib on the drug sensitivity of osimertinib-acquired resistant H1975OR cells and PC9OR cells

[0030] Preparation of osimertinib-acquired resistance cell lines:

[0031] Osimertinib-sensitive cell lines PC9 cells and H1975 cells were exposed to osimertinib for a long time to establish osimertinib-resistant cell lines PC9OR and H1975OR. During the culture process, 1 μM / L osimertinib was continuously added to the DMEM medium to maintain its drug resistance. PC9 and H1975 cells were purchased from the cell bank of the Chinese Academy of Sciences, and the above cells were preserved by Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital.

[0032] Four types of cells were cultured:

[0033] PC9, PC9OR, H1975 and H1975OR cells were cultured with DMEM medium containing 10% FBS, respectively. The cells were placed in a 10 cm cell culture dish in an incubator at 37°C and 5% CO2 saturated humidity until the cells were in the logarithmic growth phase. Cells were passaged with 0.25% trypsin-EDTA digestion solution. At the same time, anlotinib and osimertinib were dissolved in DMSO solution.

[0034] Cells in the logarithmic growth phase were taken and 7.5×10 3Cells were plated on a 96-well plate, and the 96-well plate was placed in a cell incubator for overnight culture. After the cells adhered to the wall, the cells were treated with anlotinib and osimertinib, respectively. The concentrations of the drugs in each group were increased in sequence according to the concentration gradient of 0μmol / L, 0.125μmol / L, 0.25μmol / L, 0.5μmol / L, 1μmol / L, 2μmol / L, 4μmol / L, 8μmol / L, 16μmol / L, and 32μmol / L, respectively. There were 4 replicate wells in each group, and the culture medium in each well was 100 μL. After 48 h of treatment, the 96-well plate was taken out, 10 μL of CCK-8 solution was added to each well, and the cells were cultured in the dark at 37°C for 1 h. The absorbance of each well at OD450nm was measured with an enzyme marker. The average value of each replicate well was taken, and the drug inhibition rate was calculated by the formula: (absorbance value of the untreated group - absorbance value of the experimental group) / absorbance value of the untreated group × 100%. Finally, the IC50 concentration and cell activity curve of anlotinib and osimertinib were obtained. Each group of experiments was repeated 3 times. Figure 1 As shown in Part A of the Figure 2, the IC50 of osimertinib for H1975 is 0.19 μmol / L, while the IC50 of H1975OR is 8.13 μmol / L. Figure 1 As shown in Part B, the IC50 of osimertinib for PC9 is 0.86μmol / L, while the IC50 of the corresponding resistant strain PC9OR is 7.39μmol / L. The above shows that the osimertinib-resistant cell line we constructed has much higher resistance to osimertinib than the parental sensitive cells (P<0.01); Figure 1 As shown in Part C, the IC50 of anlotinib for H1975, H1975OR, PC9, and PC9OR were 2.05 μmol / L, 1.26 μmol / L, 3.97 μmol / L, and 7.32 μmol / L, respectively.

[0035] In order to further observe the sensitivity of drug-resistant cells to osimertinib after the addition of anlotinib, the cells were divided into two groups: osimertinib group (using concentration gradient: 0μmol / L, 0.25μmol / L, 0.5μmol / L, 1μmol / L, 2μmol / L, 4μmol / L, 8μmol / L, 16μmol / L, 32μmol / L) and combination group. The two groups were administered according to the method in Example 1, where the combination ratio was determined according to the IC50 of the two drugs. Taking PC9OR as an example, IC50 (osimertinib) = 7.39μmol / L, IC50 (anlotinib) = 7.32μmol / L, and the IC50 ratio of the two drugs in this cell line was about 1:1, so three subgroups with a combination ratio of 1:1, 2:1, and 1:2 were set based on this as the baseline. Taking the 2:1 ratio combination group as an example, the concentration gradient of osimertinib in this group is: 0μmol / L, 0.25μmol / L, 0.5μmol / L, 1μmol / L, 2μmol / L, 4μmol / L, 8μmol / L, 16μmol / L, 32μmol / L, and the concentration gradient of anlotinib is: 0μmol / L, 0.125μmol / L, 0.25μmol / L, 0.5μmol / L, 1μmol / L, 2μmol / L, 4μmol / L, 8μmol / L, 16μmol / L. In this way, a fixed combination ratio is ensured under each concentration gradient, and the same is true for the other combination groups. CCK8 experiments were performed on each treatment group to obtain the inhibition rate of the drug under different concentration gradients of each treatment group. Next, we use CompuSyn software based on the classic Chou-Talalay method to calculate the drug combination index (CI) and evaluate the synergistic effect of the drugs. CompuSyn is a software commonly used for drug combination effect analysis. It calculates the combination index by analyzing the drug dose-effect curve. The "drug dose" here refers to the concentration gradients we set, the "effect" is the drug inhibition rate corresponding to each concentration gradient, and the combination index is the output result of the software, which can reflect the synergistic effect when the drugs are used in combination. A CI value less than 1 indicates a synergistic effect, equal to 1 indicates an additive effect, and greater than 1 indicates an antagonistic effect. Figure 1 As shown in parts D and E of the figure, the combination treatment groups of the two drug-resistant cells have significantly better tumor cell survival inhibition than single-agent osimertinib, among which the H1975OR 8:1 combination group and the PC9OR1:2 combination group showed better synergistic effects.

[0036] Colony formation assay was used to examine the effects of different drug treatments on tumor cell proliferation:

[0037] In the colony formation experiment, the cell lines we used were H1975OR and PC9OR, which were parallel to each other. The drug treatment groups were: NC negative control group (containing an equal volume of DMSO to dissolve the drug), osimertinib group (1μmol / L), anlotinib group ((1μmol / L)), and combination group (1μmol / L osimertinib + 1μmol / L anlotinib). The cells were treated with different drug concentrations according to the above-mentioned drug concentrations for 7 days. The results of the colony formation experiment are shown in the figure. Figure 1 As shown in part F, the number of colonies formed in the combination drug group was significantly less than that in the NC group and the single drug group.

[0038] Example 2 Construction of nanoliposomes loaded with osimertinib and anlotinib.

[0039] Preparation of osimertinib-loaded liposomes:

[0040] An appropriate amount of lecithin, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG2000-NH2) and a certain amount of osimertinib were fully dissolved in anhydrous ethanol and injected into 5-10 volumes of deionized water solution (50-60°C) at a rate of 1 mL / min. After magnetic stirring for 30 minutes, the resulting solution was sequentially extruded through polycarbonate membranes with pore sizes of 400 nm, 200 nm, 100 nm, and 50 nm 10 times each to prepare osimertinib-loaded nanoliposomes with uniform particle size.

[0041] Preparation of co-loaded osimertinib / anlotinib liposomes:

[0042] The osimertinib liposome solution prepared above was replaced with 5% glucose using a Millipore ultrafiltration system (100 kD). After the solution system was heated to 50-60°C, a certain amount of anlotinib hydrochloride was added, and then the solution system was replaced with PBS solution (PH=7.4) using a Millipore ultrafiltration system to obtain nanoliposomes co-loaded with osimertinib / anlotinib drugs, which were stored at 4 degrees for future use.

[0043] Preparation of EGF-modified drug-loaded liposomes:

[0044] Take 1 mg of human recombinant epidermal growth factor (EGF), add 0.1 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 0.1 mg of N-hydroxysuccinimide (NHS) as catalyst to activate the carboxyl group in EGF; and incubate with the above-prepared co-drug-loaded nanoliposomes (50-500 mg, preferably 100-200 mg) at 20°C overnight; obtain EGF-modified co-drug-loaded liposome solution. Remove free drugs and EGF through Sephadex G25 gel column to obtain EGF-modified co-drug-loaded nanoliposomes, and then ultrafiltration and concentration to a liposome solution with a certain drug concentration, and store in a refrigerator at 4°C for later use.

[0045] The materials and drug portions in the above preparation process are: 100-400 parts of lecithin, 50-100 parts of cholesterol and 20-60 parts of distearoyl phosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG2000-NH2), 50-100 parts of osimertinib; 1000-4000 parts of ethanol solution; 5-20 parts of anlotinib hydrochloride; preferably 200-300 parts of lecithin, 60-80 parts of cholesterol and 40-50 parts of distearoyl phosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG2000-NH2), 60-80 parts of osimertinib; 2000-3000 parts of ethanol solution; 10-15 parts of anlotinib hydrochloride.

[0046] The mass ratio of the two drugs, osimertinib (molecular weight 499.61) and anlotinib hydrochloride (molecular weight 480.36), is 8:1.

[0047] The EGF-modified liposomes co-loaded with osimertinib / anlotinib prepared above, wherein: the concentration in step 3 is to a certain drug concentration, preferably the total concentration of the two drugs is 1-5 mg / mL.

[0048] Determination of particle size potential:

[0049] The prepared drug liposomes were diluted with pure water at a ratio of 1:100, and the particle size and surface Zeta potential of the sample in pure water were measured by Malvern Laser Particle Sizer 3000. The results are as follows: Figure 2 Part A of

[0050] Transmission electron microscopy observation of the morphology of PEGylated drug liposomes:

[0051] The copper mesh was dipped into a small amount of the co-loaded liposome suspension, negatively stained with 2% phosphotungstic acid, and observed under a transmission electron microscope after drying (Japan JEOL 2010). Figure 2 In part B, it can be seen that the drug liposomes are regular spheres.

[0052] The encapsulation efficiency and drug loading of osimertinib and anlotinib in the co-loaded particles were detected by high performance liquid chromatography (HPLC):

[0053] Establish OSI standard curve: accurately weigh 2 mg osimertinib standard (actually high-purity original drug) into a volumetric flask; add acetonitrile to fully dissolve and make a 100 μg / mL stock solution, and gradually dilute with acetonitrile to make solutions with concentrations of 40, 20, 10, 5, and 2.5 μg / ml. After filtering, take 20 μL of the above solutions respectively, inject into high-performance liquid chromatography, and measure the absorption peak area of ​​the sample at a wavelength of 340 nm. The specific chromatographic conditions were: Agilent LC1200; Diamonsil C18 chromatographic column (150 × 4.6 mm, 5 μm), column temperature of 25°C, mobile phase of pure water-acetonitrile, gradient elution, gradient elution program: 0-0.5 min, acetonitrile 10%; 0.5-3.0 min, acetonitrile 10%-95%; 3.0-5.0 min, acetonitrile maintained at 95%; 5.0-5.1min, acetonitrile 95%-10%; 5.1-10 min, acetonitrile 10%; flow rate of 1 ml / min; detection wavelength of 340 nm, injection volume of 20 μL. The linear analysis of the absorption peak area y of osimertinib concentration x was performed to obtain the relevant standard curve I, specifically y =141.13x - 19.323 (R² = 0.9991; n= 5), and the linear concentration range was 2.5-40 μg / ml. The encapsulation efficiency and drug loading of osimertinib in the nanoparticles were also determined: 5 mg of freeze-dried targeted co-loaded drug nanoparticles were accurately weighed, 5 mL of anhydrous methanol / acetonitrile was added to dissolve the freeze-dried sample, the treated sample was injected into HPLC, the absorption peak area at a wavelength of 340 nm was determined, and the encapsulation efficiency and drug loading of osimertinib in the nanoparticles were calculated using the standard curve, where encapsulation efficiency I = amount of osimertinib encapsulated in co-loaded particles / total amount of osimertinib drug input × 100% = 95.3%; drug loading I = amount of osimertinib encapsulated in co-loaded particles / total mass of targeted co-loaded particles × 100% = 7.45%.

[0054] Establish anlotinib standard curve:

[0055] Accurately weigh 2 mg of anlotinib standard (actually high-purity original drug) into a volumetric flask; add deionized water to fully dissolve and make a 100 μg / mL stock solution, dilute it step by step with pure water to make solutions with concentrations of 30, 20, 10, 5, and 2 μg / ml. After filtering, take 20 μL of the above solution and inject it into the high-performance liquid chromatograph to measure the absorption peak area of ​​the sample at a wavelength of 340 nm. The specific chromatographic conditions are: Agilent LC1200; ZOBAX C18 chromatographic column (250 × 4.6 mm, 5 μm), the mobile phase is acetonitrile-ammonium acetate buffer (40:60, adjusted to pH 8.50±0.05 with ammonia water), the flow rate is 1 ml / min; the detection wavelength is 254 nm, the column temperature is 40°C, and the injection volume is 20 μL. The absorption peak area y of anlotinib concentration x was linearly analyzed to obtain the relevant standard curve I, which was specifically y = 358.99x - 406.81 (R² = 0.9985; n= 5), and the linear concentration range was 2-30 μg / ml. The encapsulation efficiency and drug loading of anlotinib in the nanoparticles were also determined: 5 mg of freeze-dried targeted co-loaded drug nanoparticles were accurately weighed, and 5 mL of anhydrous methanol / acetonitrile was added to dissolve the freeze-dried sample. After treatment, the sample was injected into HPLC, and the absorption peak area at a wavelength of 254 nm was determined. The encapsulation efficiency and drug loading of anlotinib in the nanoparticles were calculated using the standard curve, where encapsulation efficiency II = anlotinib drug amount encapsulated in co-loaded particles / total amount of anlotinib drug added × 100% = 97.1%; drug loading II = anlotinib drug amount encapsulated in co-loaded particles / total mass of targeted co-loaded particles × 100% = 0.93%.

[0056] Determination of in vitro drug release profiles in modified co-loaded lipids:

[0057] About 120 mg of freeze-dried EGF-modified co-drug liposomes (inhibitor osimertinib content - 8 mg and anlotinib - 1 mg) were accurately weighed, dissolved in an appropriate amount of PBS, placed in a 5 KD molecular weight dialysis bag, immersed in a beaker containing 200 mL of PBS solution, placed in a 37°C constant temperature water bath oscillator (rate of 100 rpm), and the start time was 0. At the set time points (i.e. 1, 4, 16, 24, 48, 72, and 120 hours), the dialysis bags were taken out and 2 mL of release medium solution was aspirated (blank PBS was added back to the dissolution tank). The above release medium solutions were evaporated to dryness, and then appropriate amounts of acetonitrile or deionized water were added to fully dissolve them. After high-speed centrifugation at 15,000 rpm, 20 uL of the supernatant was taken, filtered, and tested by HPLC (chromatographic conditions as above); the standard curve was used to calculate the in vitro release rate of the drugs osimertinib and anlotinib in the liposomes. Results Figure 2In part C, the release rates of the two drugs were similar, and the cumulative release exceeded 90% at 120 hours.

[0058] Example 3 Verification of the effectiveness of nanoliposome drugs

[0059] Using CCK8 assay to detect the cytotoxicity of nanoliposome drugs (Nano):

[0060] The groups were divided into Nano group and common combination drug group (osi:anlo 8:1). H1975OR cells in logarithmic growth phase were taken and 1×10 3 Cells were plated on a 96-well plate, and the 96-well plate was placed in a cell incubator for overnight culture. After the cells adhered to the wall, the corresponding drugs were used to treat the cells. Since both groups used two drugs in combination and the dosage was in a fixed ratio, the concentration gradient of osimertinib could be used to calculate the corresponding IC50. The concentration gradient was set as: 0μmol / L, 0.125μmol / L, 0.25μmol / L, 0.5μmol / L, 1μmol / L, 2μmol / L, 4μmol / L, 8μmol / L, 16μmol / L, 32μmol / L, increasing in sequence, with 4 replicates in each group, and 100 μL of culture medium in each well. After 5 days of treatment, the 96-well plate was taken out, 10 μL of CCK-8 solution was added to each well, and the culture was continued at 37°C in the dark for 1 hour. The absorbance value of each well at OD450nm was measured with an enzyme marker. The average value of each replicate well was taken, and the drug inhibition rate was calculated by the formula: (absorbance value of the non-drug group - absorbance value of the experimental group) / absorbance value of the non-drug group × 100%. Finally, the IC50 concentration and cell activity curve of the two groups of drug administration were obtained. Each group of experiments was repeated 3 times. Figure 3 As shown in part A, IC50(Nano)=5.099μmol / L, IC50(osi:anlo)=10.95μmol / L, which indicates that the toxicity of the drug to tumor cells was enhanced after nanoliposome packaging (P<0.01).

[0061] Colony formation assay was used to examine the effects of different drug treatments on tumor cell proliferation:

[0062] In the colony formation experiment, the cell lines we used were H1975OR and PC9OR, which were parallel to each other. The drug treatment groups were: NC negative control group (containing an equal volume of DMSO to dissolve the drug), osimertinib group (1μmol / L), anlotinib group ((1μmol / L)), and combination group (1μmol / L osimertinib + 1μmol / L anlotinib). The cells were treated with different drug concentrations according to the above-mentioned drug concentrations for 7 days. The results of the colony formation experiment are shown in the figure. Figure 3As shown in Part B, the number of colonies formed in the combination drug group was significantly less than that in the NC group and the single drug group.

[0063] Cell apoptosis:

[0064] H1975OR cells in the logarithmic growth phase were taken and the cell concentration was 5×10 6 / well, spread on a 6-well plate, and group the cells according to the experimental requirements. It was set into 5 groups for different treatments: NC negative control group (containing an equal volume of DMSO with dissolved drugs), osimertinib group (1μmol / L), anlotinib group (0.125μmol / L), combination group (osimertinib group 1μmol / L + anlotinib group 0.125μmol / L), nanoliposome drug group, and the treatment time was 48h. After treatment, the cells were collected, and a part of the cells were stained with PI according to the specific instructions of the apoptosis kit, and then detected by flow cytometry. The results are as follows Figure 3 As shown in part C, the proportion of apoptotic cells in the nanoliposome drug group was (46.5±3.96)%, which was higher than that in the combined drug group (14.26±1.02)%, and much higher than that in the NC group (6.093±0.59)%, P<0.0001. The other part of the cells was used to extract cell protein, and PMSF (Phenylmethanesulfonyl fluoride) + RIPA (RadioImmunoprecipitation Assay) was used to lyse on ice for 30 minutes, and the protein concentration was measured by BCA method. SDS-PAGE protein buffer was added and mixed and placed in a 100℃ metal bath for heating denaturation. 30μg of denatured protein was loaded, electrophoresed at 80V for 2h, transferred to the membrane at 100V for 100 min, and blocked with 5% skim milk / BSA for 1 h. Primary antibodies for GAPDH, Bcl2, Bax, and cleaved caspase3 were added, incubated overnight at 4℃, washed with TBST the next day, and the corresponding secondary antibodies were added for color development and exposure. The results are as follows Figure 3 As shown in part D, the expression level of Bcl2, an inhibitor of apoptosis, was significantly decreased in the nanoliposome drug group, while the expression level of Bax, a promoter of apoptosis, and cleaved caspase3, an effector of apoptosis, was significantly increased. In addition, the expression level of Bax protein was higher in the combination drug group, which further indicated that nanoliposome drugs were more likely to cause apoptosis of tumor cells.

[0065] In vitro cell-targeted uptake of nanoliposome drugs:

[0066] H1975OR cells and normal human bronchial epithelial cells BEAS-2B cells in the logarithmic growth phase were digested according to the above method, and the cells were resuspended and counted. 4The cells were evenly inoculated on a 10 mm glass-bottomed cell culture dish. After 24 hours of cell attachment, the same concentration (1 μmol / L for osimertinib) of nanoliposome drugs labeled with FITC fluorescence was added to the culture medium. After 24 hours of drug treatment, the original culture medium was discarded, washed 3 times with PBS, and then the culture medium containing Hoechst 33342 dye was added and incubated for 10 minutes. After staining, the PBS was washed 3 times, replaced with ordinary culture medium, and confocal microscopy was performed immediately. Figure 4 As shown in part A, nanoliposome drugs can be significantly enriched in tumor cells.

[0067] In vivo tumor-targeted uptake of nanoliposomal drugs:

[0068] First, a nude mouse subcutaneous tumor model was established. The model used female nude mice aged 4-5 weeks and weighing about 18-20 g, which were raised in an SPF environment. H1975OR cells in the logarithmic growth phase were obtained, digested and resuspended according to the above method, and the cell concentration was adjusted to about 1×10 6 About 100 μL of cells were injected subcutaneously in the right groin of nude mice to form a skin mound. When the tumor grew to 150-200 mm 3 The model was successfully established. The formula for calculating the mouse tumor volume is: tumor volume = (tumor short diameter 2 × tumor long diameter) / 2. The experimental groups were PBS group and nanoliposome drug group. 100 μL of nanoliposome drug labeled with Cy5.5 fluorescent dye was injected into tumor-bearing mice through the tail vein. The control group was injected with the same amount of PBS in the same way. 2 hours after drug injection, the nude mice were placed in an animal in vivo imaging device (IVIS SPECTRUM) for fluorescence imaging. Figure 4 As shown in part B, nanoliposome drugs can be enriched in subcutaneous tumors of mice and have good targeting properties.

[0069] Verification that nanoliposome drugs can significantly inhibit tumor growth in the H1975OR nude mouse model:

[0070] The establishment of the animal model was the same as above. After the tumor was formed, we randomly divided the nude mice with tumors into 5 groups: NC group (drug solvent group), osimertinib group (10 mg / kg / 3d), anlotinib group (1.3 mg / kg / 3d), combined drug group (osimertinib 10 mg / kg / 3d + anlotinib 1.3 mg / kg / 3d), and nanoliposome drug group (osimertinib concentration 10 mg / kg / 3d). Each treatment group contained 5 nude mice. Osimertinib and anlotinib were dissolved in 0.2% sodium carboxymethyl cellulose and administered by gavage; nanoliposome drugs were administered by tail vein injection. The administration time was 28 days. After 28 days of administration, the tumor volumes of the NC group, osimertinib group, anlotinib group, combination group, and nanoliposome group were (1350±361.4), (1267±56.38), (765.2±157.8), (597.9±64.56), and (215.6±128.8), respectively. The tumor volume of the nanoliposome group was significantly smaller than that of the combination group and the single drug group (all P values ​​were <0.01). Figure 5 Part A and B of the

[0071] Finally, it is noted that the above-described embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a drug-loaded lipid nanocarrier in the preparation of a drug for treating drug-resistant lung cancer, characterized in that: The drug-loaded lipid nanocarrier is made of raw materials including 200-300 parts by weight of lecithin, 60-80 parts by weight of cholesterol, 40-50 parts by weight of distearoyl phosphatidylethanolamine-polyethylene glycol-amino, 60-80 parts by weight of osimertinib, and 10-15 parts by weight of anlotinib hydrochloride, and is modified with EGF; the drug-resistant lung cancer is osimertinib-resistant lung cancer; The lung cancer is non-small cell lung cancer; The total concentration of osimertinib and anlotinib hydrochloride in the drug is 1-5 mg / mL; The drug loading amount of osimertinib in the drug-loaded lipid nanocarrier is 7-8% w / w, and the drug loading amount of anlotinib hydrochloride is 0.9-1% w / w; The drug-loaded lipid nanocarrier is prepared by a method comprising the following steps: (1) fully dissolving lecithin, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol-amino and osimertinib in anhydrous ethanol, injecting into an ionic aqueous solution, stirring, and then passing the resulting solution through a polycarbonate membrane to obtain osimertinib-loaded nanoliposomes; (2) replacing the solution system of the osimertinib liposomes obtained in step (1) with 5% glucose, adding anlotinib hydrochloride, and then replacing the solution system with a PBS solution to obtain nanoliposomes co-loaded with osimertinib / anlotinib drugs; (3) taking EGF, adding EDC and NHS to activate the carboxyl group of EGF, and incubating the nanoliposomes co-loaded with osimertinib / anlotinib drugs obtained in step (2); the weight ratio of EGF to the nanoliposomes is 1:100-200.

2. The use according to claim 1, wherein the medicine is in the form of an injection.

3. The use according to claim 2, wherein the medicine further comprises a pharmaceutically acceptable excipient.

Images