Irinotecan and afatinib maleate co-loaded liposome preparation as well as preparation method and application thereof

Abstract

The invention discloses an irinotecan and afatinib maleate co-loaded liposome preparation as well as a preparation method and application thereof, irinotecan and afatinib maleate are taken as effective components of the liposome, and the liposome comprises a liposome carrier, an inner water phase positioned in a liposome membrane and an outer water phase positioned outside the liposome membrane; the irinotecan and the afatinib maleate are encapsulated in the inner water phase; the inner water phase comprises an ammonium salt aqueous solution, and an ammonium salt gradient exists between the inner water phase in the liposome membrane and the outer water phase outside the liposome membrane; the outer water phase is a physiological isotonic solution. According to the present invention, the research results show that the irinotecan and afatinib maleate co-carrying liposome can simultaneously deliver the two drugs with the synergistic ratio into the same tumor cell compared to the irinotecan single drug liposome and the afatinib maleate single drug liposome so as to maximize the synergistic effect of the drugs, and significantly improve the anti-tumor effect;

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a liposome preparation for co-loading irinotecan and afatinib maleate, a preparation method and application thereof. BACKGROUND

[0002] Cancer is a major disease that seriously endangers human life and health. According to the latest statistics of the World Health Organization, colorectal cancer (CRC) accounts for 9.6% of all cancer cases, ranking third in the world.

[0003] Irinotecan (IRI) belongs to a semi-synthetic camptothecin derivative, which can be converted into its active metabolite SN38 under the catalysis of carboxylesterase 2 in vivo. SN38 can covalently bind to the Topo Ⅰ-DNA complex to form a ternary complex, which can collide with the advancing replication fork and cause irreversible replication fork stalling and cell death. Irinotecan is approved for clinical treatment of various cancers. The saturated lactone ring in the structure of irinotecan has pH dependence, which reversibly converts to its carboxylate form under physiological conditions, resulting in reduced antitumor activity. The toxic and side effects of irinotecan injection and its lyophilized powder preparation are large, mainly manifested as neutropenia and delayed diarrhea. Chinese patent CN103948545B discloses a liposome for irinotecan delivery, which can effectively protect the functional groups of irinotecan. The invention also provides a method for preparing a liposome composition.

[0004] EGFR (epidermal growth factor receptor) has been detected in various cancers, and its overexpression or mutation is considered to be an influencing factor for poor prognosis of some cancers, which are associated with more significant clinical progression, including colorectal cancer, pancreatic cancer, lung cancer, breast cancer and head and neck squamous cell carcinoma, etc. In colorectal cancer, the proportion of EGFR overexpression is as high as 80%; the overexpression rate in pancreatic cancer is more than 90%. Overexpressed EGFR activates intracellular multiple pathways through dimerization, leading to cell proliferation, angiogenesis, and tumor invasion and metastasis. Afatinib dimaleate (AFA) is an aminotoluazinamine-substituted quinazoline derivative, which can block the phosphorylation activation of EGFR by covalently binding to the intracellular tyrosine kinase domain of EGFR, thereby blocking the cell signaling pathway, inhibiting tumor cell proliferation, and promoting tumor cell apoptosis. However, the bioavailability of afatinib dimaleate oral tablets is low, which limits the development of clinical application.

[0005] Due to the limited anti-cancer effect of irinotecan monotherapy and the drug resistance of tumor cells, irinotecan is usually used in combination with other drugs in clinical practice, such as FOLFIRI chemotherapy regimen. The overexpression of EGFR in colorectal cancer is associated with poor prognosis of colorectal cancer, and the synergistic administration of irinotecan and EGFR inhibitor afatinib-maleate has the potential to improve the efficacy. However, the combination of drugs may affect the same target cells and cause treatment effect and side effects due to the difference in drug half-life and drug properties.

[0006] Based on the above reasons, it is necessary to co-deliver irinotecan and afatinib-maleate, and to simultaneously deliver the two drugs to the same tumor cells in the best synergistic ratio to maximize the synergistic anti-tumor effect. SUMMARY

[0007] The purpose of the present application is to provide an irinotecan and afatinib-maleate co-loaded liposome preparation and its preparation method and application.

[0008] In view of the technical problems existing in the prior art in the treatment of cancer, especially in the treatment of colorectal cancer, the inventors have found that irinotecan and afatinib-maleate co-loaded liposomes can simultaneously deliver two drugs with a synergistic ratio to the same tumor cells to maximize the synergistic effect of the drugs and significantly improve the anti-tumor effect, compared with irinotecan monotherapy and afatinib-maleate monotherapy.

[0009] The purpose of the present application can be achieved by the following technical solutions:

[0010] In a first aspect, the present application claims an irinotecan and afatinib-maleate co-loaded liposome, which uses irinotecan and afatinib-maleate as effective components and includes a liposome carrier, an inner water phase in the liposome membrane and an outer water phase outside the liposome membrane.

[0011] The irinotecan and afatinib-maleate are encapsulated in the inner water phase.

[0012] The inner water phase contains an aqueous solution of ammonium salt, and there is an ammonium salt gradient between the inner water phase in the liposome membrane and the outer water phase outside the membrane; the outer water phase is a physiological isotonic solution.

[0013] The irinotecan is irinotecan base or irinotecan hydrochloride.

[0014] Further, the molar ratio of irinotecan to afatinib-maleate is 1:2 to 6:1 (preferably 1:2 to 4:1).

[0015] Furthermore, the liposome membrane is made of neutral phospholipids and cholesterol, wherein the molar ratio of cholesterol to neutral phospholipids is 1:1.2 to 1.8, and the molar ratio of irinotecan to neutral phospholipids is 1:5 to 10.

[0016] Furthermore, the neutral phospholipid is selected from one or more of natural phospholipids and synthetic phospholipids; the natural phospholipid is one or more of soybean phospholipids and lecithin; the synthetic phospholipid is one or more of hydrogenated soybean lecithin (HSPC), distearylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), disqualylphosphatidylcholine (DEPC), and 1,2-dioleoylphosphatidylcholine (DOPC).

[0017] Furthermore, the liposome membrane material also contains polyethylene glycol-modified phospholipid DSPE-PEG2000, wherein the weight ratio of polyethylene glycol-modified phospholipid DSPE-PEG2000 to neutral phospholipid is 0.05~0.1:1.

[0018] Furthermore, the ammonium salt is selected from one or more of ammonium sulfate, ammonium phosphate, ammonium chloride, ammonium sucrose octasulfate, ammonium methanesulfonate, and ammonium hydroxyethylsulfonate, and the concentration of ammonium ions in the internal aqueous phase is 200 mM to 600 mM.

[0019] Furthermore, the physiological isotonic solution is selected from 0.9% (w / v) sodium chloride aqueous solution, 5% (w / v) glucose aqueous solution or 10% (w / v) sucrose aqueous solution, and the pH of the external aqueous phase is 5.0 to 8.0.

[0020] Secondly, this invention claims protection for a method for preparing the above-mentioned irinotecan and afatinib maleate co-loaded liposomes, the method comprising the following steps:

[0021] (1) Preparation of blank liposomes: Dissolve the material of the liposome membrane in an organic solvent, form blank liposomes by thin film dispersion or solvent injection, and homogenize to the required particle size;

[0022] In a specific embodiment of the present invention, blank liposomes are prepared by the method described in A or B below:

[0023] A. According to the formula, select phospholipids and cholesterol, dissolve them in anhydrous ethanol or anhydrous ethanol-tert-butanol mixed solvent, mix with ammonium salt aqueous solution, remove the solvent to obtain blank crude liposomes, and use a high-pressure homogenizer and / or extrusion equipment to prepare blank liposomes to the required particle size.

[0024] B. According to the formula, select phospholipids and cholesterol, dissolve them in chloroform or chloroform-methanol mixed solvent, and rotary evaporate to form a lipid film. Add ammonium salt aqueous solution for hydration to obtain blank crude liposomes. Use a high-pressure homogenizer and / or extrusion equipment to prepare blank liposomes to the required particle size.

[0025] (2) Generation of ion gradient between water phase inside and outside liposome membrane: Replace the water phase outside the blank liposome membrane to generate an ion gradient between the water phase inside and outside the liposome membrane;

[0026] (3) Preparation of drug-containing liposomes: Irinotecan and afatinib maleate mixed solution were added to a blank liposome dispersion with an ion gradient, heated and stirred and incubated to obtain drug-containing liposomes;

[0027] (4) Removal of free drug and concentration of sample: Add buffer medium to liposome dispersion, remove unencapsulated drug using tangential flow device, and concentrate liposome dispersion;

[0028] (5) Volume adjustment, sterilization and dispensing: Adjust the concentration of liposome drug, adjust the volume, filter and sterilize, dispense to obtain liposome injection solution; or add lyophilization protectant to liposome drug, adjust the concentration of liposome drug, adjust the volume, filter and sterilize, dispense, freeze dry to obtain lyophilized powder injection.

[0029] The liposomes of the present invention can be prepared using conventional liposome preparation methods. Those skilled in the art can select various methods to prepare them according to the liposome formulation provided by the present invention. For the liposome formulation selected by the present invention, the preferred preparation method is active drug delivery technology.

[0030] Thirdly, the present invention seeks to protect the use of the above-mentioned irinotecan and afatinib maleate co-loaded liposomes in the preparation of therapeutic drugs for colorectal cancer and pancreatic cancer.

[0031] The beneficial effects of this invention are:

[0032] This invention discovers that the combined use of irinotecan and afatinib maleate in tumor treatment can exert a synergistic effect. By encapsulating irinotecan and afatinib maleate in an optimal synergistic ratio in liposomes, the differences in the half-life and distribution of these two drugs in vivo are overcome, allowing them to be delivered simultaneously to the same tumor cells in the optimal synergistic ratio, thereby maximizing their synergistic anti-tumor effect. In vitro and in vivo experiments have demonstrated that the prepared co-loaded liposomes significantly improve the anti-tumor efficacy compared to both irinotecan and afatinib maleate monotherapy and combination therapy. Specifically, it has the following technical advantages:

[0033] (1) This invention employs active drug delivery technology to prepare co-loaded liposomes for co-delivering irinotecan and afatinib maleate. The encapsulation efficiencies of the two drugs are similar and both are greater than 90%. The prepared co-loaded liposomes have a particle size of approximately 100–120 nm and a uniform particle size distribution. They exhibit good storage stability at 4°C. In vitro release experiments show that irinotecan and afatinib maleate exhibit significant sustained-release behavior, and their release is synchronous. The liposome preparation process of this invention is simple, controllable, and reproducible, making it easy for industrial production.

[0034] (2) This invention demonstrates through in vitro cytotoxicity test, apoptosis test and in vivo pharmacodynamic experiment in tumor-bearing nude mice that irinotecan and afatinib maleate co-loaded liposomes can significantly enhance the anti-tumor effect compared with single drug and two-drug combination administration.

[0035] (3) This invention studies the mechanism of the synergistic effect of afatinib maleate on the anti-tumor activity of irinotecan, and proves that afatinib maleate enhances the anti-tumor proliferation effect of irinotecan by reducing the protein expression level of Rad51. Attached Figure Description

[0036] Figure 1 Characterization and preliminary stability investigation of co-loaded liposomes. In the figures, a) shows the appearance and TEM images of the co-loaded liposomes; b) shows the particle size, particle size distribution, and zeta potential of the co-loaded liposomes; and c) shows the preliminary stability investigation results of the co-loaded liposomes.

[0037] Figure 2 The results show the in vitro release of irinotecan and afatinib maleate from co-loaded liposomes in PBS (pH 7.4) at 37°C.

[0038] Figure 3 This study investigates the synergistic antitumor effect and mechanism of irinotecan and afatinib maleate. Figure a shows the Fa-CI combination index curves of irinotecan and afatinib maleate at different molar ratios; figure b shows the survival rate of HT-29 cells after 48 hours of incubation with different formulations; and figure c shows the apoptosis of cells after 48 hours of incubation with different formulations.

[0039] Figure 4 This study investigates the mechanism by which afatinib maleate enhances the antitumor effect of irinotecan. Image a shows the Western blotting results of Rad51 protein in HT-29 cells after incubation with irinotecan and afatinib maleate; image b shows the quantitative statistical graph of Rad51 protein in HT-29 cells after incubation with irinotecan and afatinib maleate.

[0040] Figure 5 This study evaluates the in vivo antitumor efficacy of co-loaded liposomes. Figure a shows a statistical graph of mouse tumor weight; figure b shows a statistical graph of tumor inhibition rates for different formulations.

[0041] Figure 6 Images of Ki67 immunofluorescence staining in tumor tissues with different formulations.

[0042] Figure 7 This is for monitoring changes in mouse body weight during drug administration. In the figure, a is the mouse body weight change curve; b is the mouse body weight statistical graph. Detailed Implementation

[0043] The present invention will be further illustrated below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0044] Example 1

[0045] prescription

[0046]

[0047] The specific steps are as follows:

[0048] (1) Accurately weigh the prescribed amounts of cholesterol, HSPC, and DSPE-PEG2000, and dissolve them in an appropriate amount of anhydrous ethanol to obtain a lipid solution; mix the above lipid solution with 100 mL of ammonium sulfate solution (ammonium ion concentration of 300 mM), and remove the ethanol under reduced pressure to obtain crude blank liposomes. Homogenize three times using a high-pressure homogenizer at 1000 bar, and then granulate them sequentially through polycarbonate membranes of 400 nm, 200 nm, and 100 nm to obtain blank liposomes with uniform particle size;

[0049] (2) Use a tangential flow ultrafiltration device to dialyze blank liposomes, and continuously replenish water for injection in between to create an ion gradient between the aqueous phase inside the liposome and the aqueous phase outside.

[0050] (3) Prepare a mixed solution of irinotecan hydrochloride and afatinib maleate with water for injection, add it to the blank liposomes above according to the prescription ratio, heat and stir at 60°C, incubate for 60 min, use ion gradient to drive the drug to penetrate the lipid membrane and exist stably in the inner aqueous phase, and cool in an ice water bath after incubation.

[0051] (4) Tangential flow dialysis was used to remove unencapsulated drugs, and the sample was concentrated to about 50 mL. 0.45 g of sodium chloride was added to adjust the osmotic pressure.

[0052] (5) Adjust the drug concentration, make up the volume, filter and sterilize with a 0.22μm filter membrane, fill with nitrogen and seal in a vial to obtain irinotecan hydrochloride and afatinib maleate co-loaded liposomes (Co-IRI / AFA Lip).

[0053] Comparative Example 1: Preparation of Irinotecan Hydrochloride Liposomes

[0054] The main steps are the same as in Example 1. The difference is that in step (3) of Comparative Example 1, irinotecan hydrochloride solution is added when incubating with blank liposomes. The other steps are the same, and irinotecan hydrochloride liposomes (IRI Lip) are obtained.

[0055] Comparative Example 2: Preparation of afatinib maleate liposomes

[0056] The main steps are the same as in Example 1. The difference is that in step (3) of Comparative Example 1, afatinib maleate solution is added when incubating with blank liposomes. The other steps are the same, and afatinib maleate liposomes (AFA Lip) are obtained.

[0057] Example 2 Characterization of co-loaded liposomes

[0058] The co-loaded liposome samples prepared in Example 1 were visually observed. The samples and 2% (w / v) phosphotungstic acid were added dropwise onto a copper grid for negative staining, and the microstructure of the co-loaded liposomes was observed using a transmission electron microscope. Using ultrapure water as the dispersion medium, the particle size, particle size distribution, and zeta potential of the co-loaded liposomes were determined using a laser scattering particle size analyzer.

[0059] The encapsulation efficiency of the two drugs was determined using dextran gel column chromatography. The co-loaded liposome sample prepared in Example 1 was added to the chromatography column, eluted with PBS (pH 6.5) buffer, and the eluent was collected. The liposomes were then disrupted with methanol to release the encapsulated drug, and the sample was filtered and injected to determine the encapsulated drug content. The encapsulation efficiency was calculated as follows:

[0060]

[0061] The co-loaded liposomes prepared in Example 1 were stored at 4°C, and samples were taken every week for observation. The particle size and particle size distribution were measured by a laser scattering particle size analyzer to examine their storage stability at 4°C.

[0062] from Figure 1 As can be seen from 'a', the co-loaded liposomes appear as a milky white suspension, which shows a pale blue opalescence after dilution. Transmission electron microscopy reveals that the microstructure of the co-loaded liposomes is spherical with a uniform size distribution.

[0063] from Figure 1 As can be seen from b, the particle size of the co-loaded liposomes is 120.35 nm, the PDI is 0.197, and the Zeta potential is -10.0 mV;

[0064] from Figure 1 As can be seen from 'c', the co-loaded liposomes exhibit good stability under storage conditions at 4℃.

[0065] The particle size, zeta potential, and encapsulation efficiency of the prepared co-loaded liposomes are shown in Table 1.

[0066] Table 1. Particle size, zeta potential, and encapsulation efficiency of co-loaded liposomes

[0067]

[0068] Three batches of co-loaded liposomes were prepared, and the encapsulation efficiency of irinotecan hydrochloride and afatinib maleate reached over 94%, with the assay method exhibiting good reproducibility.

[0069] Example 3 In vitro drug release assay

[0070] The in vitro release behavior of the co-loaded liposomes prepared in Example 1 was investigated using dialysis as the release method.

[0071] 1 mL of the co-loaded liposomes prepared in Example 1 was accurately measured and encapsulated in a dialysis bag with a molecular weight cutoff of 8–14 kDa. The dialysis bag was completely immersed in a container containing 30 mL of pH 7.4 PBS buffer. The container was sealed and transferred to a 37°C water bath shaker with shaking at 100 times per minute. 1 mL of release medium was collected at 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h, and 1 mL of fresh medium was added immediately after each sampling. The drug content in the sample was detected using high-performance liquid chromatography (HPLC), and the cumulative drug release rate at each time point was calculated. The results are shown below. Figure 2 .

[0072] from Figure 2 It can be seen that the cumulative release rate of irinotecan hydrochloride was only 16.94% and that of afatinib maleate was only 12.97% after 24 hours. The release behavior of both drugs was consistent, indicating that the co-loaded liposomes have a sustained-release effect and that the release rates of the two drugs are comparable. It is speculated that only a small amount of drug leaks from the co-loaded liposomes in the bloodstream, and most of the drug is effectively delivered to the tumor site in a synergistic ratio.

[0073] Example 4

[0074] prescription:

[0075]

[0076] The liposome preparation method was the same as in Example 1, except that hydrochloric acid was added to dissolve irinotecan to a solution pH of 3.5 to obtain an irinotecan solution, which was then co-incubated with afatinib maleate solution into blank liposomes. The encapsulation efficiency and particle size of Co-IRI / AFA Lip are shown in the table below.

[0077] Table 2. Particle size, zeta potential, and encapsulation efficiency of co-loaded liposomes

[0078]

[0079] Example 5

[0080] prescription:

[0081]

[0082] The liposome preparation method was the same as in Example 1, except that blank liposomes were prepared according to the molar ratio of irinotecan hydrochloride to HSPC of 1:4 to 1:12. The encapsulation efficiency and particle size of Co-IRI / AFA Lip are shown in the table below.

[0083] Table 3. Effect of drug-to-liposome ratio on particle size, zeta potential, and encapsulation efficiency of co-loaded liposomes.

[0084]

[0085] The results show that the encapsulation efficiency of irinotecan hydrochloride and afatinib maleate in the drug-to-lipid ratio range of 1:5 to 12 meets the formulation technical requirements (encapsulation efficiency greater than 85%). As the drug-to-lipid ratio decreases, the encapsulation efficiency gradually increases. When the drug-to-lipid ratio reaches 1:12, the encapsulation efficiency of irinotecan and afatinib maleate is basically the same as that of a drug-to-lipid ratio of 1:10. However, the drug-to-lipid ratio is too low, and the formulation cost is too high.

[0086] Example 6

[0087] prescription:

[0088]

[0089] The liposome preparation method was the same as in Example 1, except that blank liposomes were prepared according to a cholesterol to HSPC molar ratio of 1:1 to 1:2. The Co-IRI / AFA Lip encapsulation efficiency and particle size are shown in the table below.

[0090] Table 4. Effects of cholesterol-to-lipid ratio on particle size, zeta potential, and encapsulation efficiency of co-loaded liposomes.

[0091]

[0092] The results showed that the encapsulation efficiency of irinotecan hydrochloride and afatinib maleate within the cholesterol-to-lipid ratio range of 1:1 to 1:1.8 met the formulation technical requirements (encapsulation efficiency greater than 85%). However, when the cholesterol-to-lipid ratio was 1:1, some phospholipid fragments could be observed under a microscope, and the liposome particle size was slightly larger.

[0093] Example 7

[0094] prescription

[0095]

[0096] The liposome preparation method is the same as in Example 1, except that blank liposomes are prepared using different types of neutral phospholipids. The encapsulation efficiency and particle size of Co-IRI / AFA Lip are shown in the table below.

[0097] Table 5. Effects of neutral phospholipid type on particle size, zeta potential, and encapsulation efficiency of co-loaded liposomes.

[0098]

[0099] The results show that the encapsulation efficiency of liposomal irinotecan hydrochloride and afatinib maleate prepared from neutral phospholipids such as HSPC, DSPC, DSPE, DEPC and DOPC meets the formulation technical requirements (encapsulation efficiency greater than 85%).

[0100] Example 8

[0101] prescription

[0102]

[0103] The liposome preparation method is the same as in Example 1. The encapsulation efficiency and particle size of Co-IRI / AFA Lip are shown in the table below.

[0104] Table 6. Particle size, zeta potential, and encapsulation efficiency of co-loaded liposomes

[0105]

[0106] Example 9

[0107] prescription

[0108]

[0109] The liposome preparation method is the same as in Example 1. The encapsulation efficiency and particle size of Co-IRI / AFA Lip are shown in the table below.

[0110] Table 7. Particle size, zeta potential, and encapsulation efficiency of co-loaded liposomes

[0111]

[0112] Example 10: Examination of Synergistic Effect

[0113] The optimal combination ratio of the two drugs was determined through cytotoxicity experiments and the Chou-Talalay method.

[0114] The MTT assay was used to determine the cell viability of HT-29 cells at different concentrations of irinotecan hydrochloride and afatinib maleate. The half-maximal inhibitory concentrations (IC50) of the two drugs were calculated using GraphPad Prism software. 50 ), and based on the obtained IC50 The combination index (CI) of irinotecan hydrochloride and afatinib maleate at different molar concentration ratios was calculated using the following formula:

[0115]

[0116] D1 and D2 are the IC50 values ​​of irinotecan hydrochloride and afatinib maleate, respectively, when used in combination. 50 Values ​​DX1 and DX2 are the IC50 values ​​of irinotecan hydrochloride and afatinib maleate, respectively, during incubation alone. 50 Value. The data was analyzed using CompuSyn software, and a combined exponential curve (Fa-CI) was plotted. The results are as follows: Figure 3 'a' in 'a'.

[0117] CI stands for combination index; a CI < 1 indicates a synergistic effect between the two drugs. Fa represents the inhibition rate, and the CI value within the high-efficiency range of 0.5–0.8 is typically used to assess the effect of the combination therapy. Figure 3 As can be seen from a, irinotecan hydrochloride and afatinib maleate have a better synergistic effect in the molar ratio range of 1:2 to 4:1, and the combination of them in a molar ratio of 4:1 has the best synergistic antitumor effect.

[0118] Example 11 Evaluation of Cytotoxicity and Apoptosis

[0119] Cytotoxicity assay: HT-29 cells in the logarithmic growth phase were seeded at a density of 5000 cells per well and incubated for 24 h. Then, complete culture medium was added to each well, along with blank liposomes (Blank Lip) and complete culture medium containing equal amounts of the drug (IRI concentration 20 μM, AFA concentration 5 μM): Irinotecan hydrochloride solution (IRI Sol), afatinib maleate solution (AFA Sol), a mixture of Irinotecan hydrochloride and afatinib maleate (IRI / AFA Sol), IRI Lip (Control Example 1), AFA Lip (Control Example 2), a mixture of IRI Lip and AFA Lip (IRI Lip / AFA Lip group), and Co-IRI / AFA Lip (Example 1). Each group was in triplicate. Incubation was repeated for 48 h, then each well was replaced with complete culture medium containing 20 μL of MTT solution. After incubation for another 4 h, 150 μL of MTT solution was added to each well. Dissolve the crystals in DMSO with gentle shaking for 10 minutes. Measure the absorbance at 570 nm using a microplate reader. The experimental results were obtained using GraphPad Prism software, such as... Figure 3 As shown in b in the figure.

[0120] Apoptosis assay: HT-29 cells in the logarithmic growth phase were cultured at a density of 1 × 10⁶ cells per well. 6Cells were seeded at the specified density into 6-well plates and incubated for 24 h. Experimental groups were the same as for the cytotoxicity assay, with three replicates per group, and incubation lasted for 48 h. Cells were collected in centrifuge tubes, digested with trypsin, and digestion was terminated by adding complete culture medium. Cells were washed twice with pre-chilled PBS, resuspended in Binding Buffer, and then Annexin V-FITC was added and mixed well. Finally, Propidium Iodide was added, and the cells were incubated at room temperature in the dark for 10 min. Apoptosis was detected using flow cytometry. Data were analyzed using FlowJo software, and the results are as follows: Figure 3 As shown in c in the figure.

[0121] from Figure 3 As can be seen from b, the blank liposomes have good biocompatibility and safety, while Co-IRI / AFALip has significant tumor cell toxicity.

[0122] from Figure 3 As can be seen from c, Co-IRI / AFA Lip can maximize the synergistic pro-apoptotic effect of irinotecan hydrochloride and afatinib maleate.

[0123] Example 12 Research on Synergistic Mechanism

[0124] The synergistic mechanism of irinotecan hydrochloride and afatinib maleate was studied by examining the expression level of the DNA repair protein Rad51 using Western blotting.

[0125] HT-29 cells in the logarithmic growth phase were cultured at a density of 1 × 10⁻⁶ cells per well. 6 Cells were seeded at the specified density into 6-well plates and incubated for 24 h. Then, complete culture medium, IRI Sol (40 μM), AFA Sol (10 μM), and IRI / AFA Sol (IRI concentration 40 μM, AFA concentration 10 μM) were added to each well, with 3 replicates per group. The cells were incubated in a CO2 incubator for 2 h. Cells were then collected, centrifuged, and the supernatant was discarded. An appropriate amount of RIPA lysis buffer was added, and the cells were lysed on ice for 30 min. After lysis, the supernatant was collected by centrifugation, and the total protein concentration was determined using a BCA kit.

[0126] Protein samples were added to a precast gel and electrophoresed until all samples had escaped the gel. The gel was then transferred to a membrane, blocked, and sequentially incubated with Rad51 antibody and goat anti-rabbit antibody. The gel was then developed and images recorded using a gel imaging system. The images were processed and the grayscale values ​​of the electrophoretic bands were analyzed using ImageLab software. The results are shown below. Figure 4 As shown in a and b in the figure.

[0127] from Figure 4As can be seen from a and b in the figure, afatinib maleate can enhance the anti-proliferative effect on tumor cells by reducing the increase in Rad51 protein expression induced by irinotecan hydrochloride.

[0128] Example 13 Evaluation of in vivo antitumor activity

[0129] Under aseptic conditions, use a syringe needle to draw 100 μL of solution with a density of 1 × 10⁻⁶. 8 HT-29 cell suspension was injected subcutaneously into the axilla of the right forelimb of nude mice. When the tumor volume grew to 150 mm, the tumor was treated. 3 Around 10:00 AM, tumor-bearing nude mice were randomly divided into 5 groups of 5 mice each. The groups were: a 5% (w / v, g / 100 mL) glucose solution control group, an IRI Lip group (Control Example 1), an AFA Lip group (Control Example 2), an IRI Lip / AFA Lip group, and a Co-IRI / AFA Lip group (Example 1). Irinotecan hydrochloride was administered at a dose of 8 mg / kg, and afatinib maleate was administered at a dose equivalent to 2.12 mg / kg.

[0130] The first administration date was recorded as day 0, with administration every three days for a total of three administrations. The weight of the nude mice was measured daily during the administration period, and a weight change curve was plotted. At the end of the administration cycle, the nude mice were euthanized by cervical dislocation, the tumor tissue was completely removed and weighed, and the net weight of the nude mouse was calculated by subtracting the tumor weight from the mouse's body weight.

[0131] The efficacy of co-loaded liposomes in tumor treatment was evaluated by weighing the tumor and using Ki67 immunofluorescence staining of tumor tissue, and the safety of co-loaded liposomes was evaluated by weighing nude mice.

[0132] The tumor inhibition rate (TIR) ​​is calculated using the following formula:

[0133]

[0134] Among them, W1 is the average tumor weight of the 5% glucose solution group, and W2 is the average tumor weight of each treatment group.

[0135] Tumor weight and tumor inhibition rate were obtained through analysis using GraphPad Prism software, and the results are as follows: Figure 5 As shown in a and b in the figure.

[0136] The dissected tumor tissue was fixed in paraformaldehyde for a period of time, dehydrated, dewaxed, hydrated, and then antigen-retrieval was performed on the tissue sections using a retrieval solution. After blocking with BSA, the sections were incubated with Ki67 primary antibody solution, followed by incubation with secondary antibody. Finally, the cell nuclei were stained with DAPI. The fluorescence distribution images of DAPI and Ki67 were observed using a fluorescence microscope. The results are as follows: Figure 6 As shown.

[0137] The nude mouse weight change curve and net weight statistics were also obtained through GraphPad Prism software processing and analysis, and the results are as follows: Figure 7 As shown in a and b in the figure.

[0138] from Figure 5 As can be seen from a and b in the figure, the tumor weight of the co-loaded liposome group was smaller and the tumor inhibition rate was higher, indicating that the co-loaded liposomes had a more significant anti-tumor effect.

[0139] from Figure 6 It can be seen that there were fewer Ki67 positive cells in the liposome-loaded group, indicating that the liposome-loaded group can more significantly inhibit the proliferation of tumor cells.

[0140] from Figure 7 As can be seen from a and b in the figure, there was no significant difference in body weight and net weight of mice in each formulation group compared with the control group, indicating that the prepared liposome formulation has good safety.

[0141] The above results demonstrate that Co-IRI / AFA LIP can safely and more effectively inhibit tumor growth in nude mice and exert anti-tumor effects.

[0142] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A liposome co-loaded with irinotecan and afatinib maleate, characterized in that, The liposome uses irinotecan and afatinib maleate as active ingredients and includes a liposome carrier, an inner aqueous phase located inside the liposome membrane, and an outer aqueous phase located outside the liposome membrane. The irinotecan and afatinib maleate are encapsulated in the internal aqueous phase; The inner aqueous phase contains an ammonium salt aqueous solution, and there is an ammonium salt gradient between the inner aqueous phase inside the liposome membrane and the outer aqueous phase outside the membrane; the outer aqueous phase is a physiological isotonic solution.

2. The irinotecan and afatinib maleate co-loaded liposome according to claim 1, characterized in that, The irinotecan mentioned is irinotecan base or irinotecan hydrochloride.

3. The irinotecan and afatinib maleate co-loaded liposome according to claim 1, characterized in that, The molar ratio of irinotecan to afatinib maleate is 1:2 to 6:

1.

4. The irinotecan and afatinib maleate co-loaded liposome according to claim 1, characterized in that, The liposome membrane is made of neutral phospholipids and cholesterol, wherein the molar ratio of cholesterol to neutral phospholipids is 1:1.2 to 1.8, and the molar ratio of irinotecan to neutral phospholipids is 1:5 to 10.

5. The irinotecan and afatinib maleate co-loaded liposome according to claim 4, characterized in that, The neutral phospholipid is selected from one or more of natural phospholipids and synthetic phospholipids; the natural phospholipid is one or more of soybean phospholipids and lecithin; the synthetic phospholipid is one or more of hydrogenated soybean lecithin, distearyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, disqualyl phosphatidylcholine, and 1,2-dioleoyl phosphatidylcholine.

6. The irinotecan and afatinib maleate co-loaded liposome according to claim 4, characterized in that, The liposome membrane material also contains polyethylene glycol-modified phospholipid DSPE-PEG2000, wherein the weight ratio of polyethylene glycol-modified phospholipid DSPE-PEG2000 to neutral phospholipid is 0.05~0.1:

1.

7. The irinotecan and afatinib maleate co-loaded liposome according to claim 1, characterized in that, The ammonium salt is selected from one or more of ammonium sulfate, ammonium phosphate, ammonium chloride, ammonium sucrose octasulfate, ammonium methanesulfonate, and ammonium hydroxyethylsulfonate, and the concentration of ammonium ions in the internal aqueous phase is 200 mM to 600 mM.

8. The irinotecan and afatinib maleate co-loaded liposome according to claim 1, characterized in that, The physiological isotonic solution is selected from 0.9% sodium chloride aqueous solution, 5% glucose aqueous solution or 10% sucrose aqueous solution, and the pH of the external aqueous phase is 5.0 to 8.

0.

9. A method for preparing liposomes co-loaded with irinotecan and afatinib maleate as described in any one of claims 1-8, characterized in that, The preparation method includes the following steps: (1) Preparation of blank liposomes: Dissolve the material of the liposome membrane in an organic solvent, form blank liposomes by thin film dispersion or solvent injection, and homogenize to the required particle size; (2) Generation of ion gradient between water phase inside and outside liposome membrane: Replace the water phase outside the blank liposome membrane to generate an ion gradient between the water phase inside and outside the liposome membrane; (3) Preparation of drug-containing liposomes: Irinotecan and afatinib maleate mixed solution were added to a blank liposome dispersion with an ion gradient, heated and stirred and incubated to obtain drug-containing liposomes; (4) Removal of free drug and concentration of sample: Add buffer medium to liposome dispersion, remove unencapsulated drug using tangential flow device, and concentrate liposome dispersion; (5) Volume adjustment, sterilization and dispensing: Adjust the concentration of liposome drug, adjust the volume, filter and sterilize, dispense to obtain liposome injection solution; or add lyophilization protectant to liposome drug, adjust the concentration of liposome drug, adjust the volume, filter and sterilize, dispense, freeze dry to obtain lyophilized powder injection.

10. Use of irinotecan and afatinib maleate co-loaded liposomes as described in any one of claims 1-9 in the preparation of medicaments for the treatment of colorectal cancer and pancreatic cancer.

Images