Cabozantinib and osimertinib co-loaded targeting liposome and application thereof in overcoming EGFR-TKI drug resistance

Abstract

The invention particularly relates to a cabozantinib and osimertinib co-loaded targeting liposome and application thereof in overcoming EGFR-TKI drug resistance, and belongs to the technical field of biological medicine. The co-drug-loaded targeting liposome disclosed by the invention is coated with osimertinib and cabozantinib at the same time, and a GE11 peptide targeting ligand is connected to a membrane material of the co-drug-loaded targeting liposome; wherein the mass ratio of osimertinib to cabozantinib is 1: (0.5-2), preferably 1: 1; the membrane material comprises hydrogenated soybean phosphatidylcholine, cholesterol and DSPE-PEG2000, and the mass ratio of the hydrogenated soybean phosphatidylcholine to the cholesterol to the DSPE-PEG2000 is (2.5-3.5): 1: 1. According to the co-drug-loading targeting liposome disclosed by the invention, a drug can be synergistically delivered to a tumor site through active targeting mediated by GE11 peptide, an EGFR main pathway and key drug-resistant bypasses such as MET are efficiently inhibited, and the co-drug-loading targeting liposome shows a remarkable synergistic interaction effect in the aspect of reversing acquired drug resistance of osimertinib.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a targeted liposome co-loaded with cabozantinib and osimertinib and application thereof in overcoming EGFR-TKI drug resistance. BACKGROUND

[0002] Lung cancer is one of the malignant tumors with the highest morbidity and mortality worldwide, and non-small cell lung cancer (NSCLC) accounts for about 80% of all lung cancer cases. Due to atypical early symptoms, most patients are diagnosed at an advanced stage, losing the opportunity for radical surgery. For the EGFR mutant population accounting for about 50% of Chinese NSCLC patients, EGFR-TKI has become the first-line standard treatment. Especially the third-generation EGFR-TKI osimertinib, which irreversibly inhibits EGFR sensitive mutations and T790M drug-resistant mutations, has shown excellent efficacy in the key phase III FLAURA study, with a median progression-free survival (PFS) of 18.9 months and a median overall survival (OS) of 38.6 months, significantly better than the previous generation of drugs, establishing its gold standard position in the treatment of advanced EGFR mutant NSCLC.

[0003] However, the clinical application of EGFR-TKI has always been faced with a serious challenge-acquired drug resistance. Most patients will experience disease progression after about 1 year of osimertinib treatment. The drug resistance mechanisms are complex and diverse, mainly including secondary mutations of the EGFR gene (such as C797S), activation of bypass signaling pathways (such as MET gene amplification and AXL overexpression), and histological type transformation. Once drug resistance occurs, it means treatment failure and disease recurrence, and patients have limited subsequent treatment options and poor prognosis.

[0004] To effectively overcome acquired drug resistance, combination therapy has become an inevitable trend in current research. By combining osimertinib with other drugs with different mechanisms of action, it is expected to achieve multi-pathway synergistic inhibition and overcome or delay drug resistance. However, "who to combine with" and "how to combine" to maximize efficacy and overcome drug resistance have become the core problems faced by those skilled in the art, and are the focus and breakthrough of current research. SUMMARY

[0005] To address the lack of efficient combination therapy options for acquired resistance to osimertinib in existing technologies, as well as the technical challenges of low efficiency in drug combination selection and synergistic delivery, this invention provides a co-loaded targeted liposome based on the synergistic effect of cabozantinib and osimertinib, its preparation method, and its application. This method constructs a synergistic drug delivery system by co-encapsulating cabozantinib and osimertinib in a specific ratio within GE11 peptide-modified liposomes. This system simultaneously inhibits the main EGFR pathway and key resistance pathways such as MET / VEGFR2 / AXL, achieving precise co-delivery and synergistic enhancement of the two drugs in tumor tissues, providing a novel solution for overcoming acquired resistance to EGFR-TKIs.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention discloses a co-loaded drug-targeting liposome, which simultaneously encapsulates osimertinib and cabozantinib, and has a GE11 peptide targeting ligand attached to its membrane material.

[0008] Cabozantinib is an orally bioavailable small-molecule multi-target tyrosine kinase inhibitor. Its unique characteristic lies in its highly efficient and simultaneous inhibition of multiple key signaling pathways associated with tumor growth, angiogenesis, and drug resistance development, including MET, VEGFR1 / 2 / 3, AXL, RET, and KIT. Its potent inhibition of MET and AXL is particularly relevant to overcoming acquired resistance to EGFR-TKIs, as activation of these pathways is a crucial bypass mechanism for tumor cells to evade targeted therapy. Therefore, cabozantinib is considered a highly promising candidate for combination therapy with osimertinib to reverse drug resistance.

[0009] Preferably, the mass ratio of osimertinib to cabozantinib in the targeted liposomes is 1:(0.5-2), more preferably 1:1. This ratio is the optimal ratio screened based on numerous in vitro synergistic experiments, and can produce a significant synergistic antitumor effect.

[0010] Preferably, the GE11 peptide is coupled to the target liposome membrane via DSPE-PEG2000.

[0011] Among them, GE11 peptide is a short targeting peptide with high affinity for EGFR and extremely low immunogenicity. Compared with traditional targeting ligands such as antibodies or full-length EGF protein, its advantages are: small molecular weight, less likely to induce immune clearance and better tumor penetration; simple structure, convenient chemical synthesis, easy to stably conjugate with carrier materials (such as DSPE-PEG2000), and high batch-to-batch consistency.

[0012] Preferably, the membrane material of the targeted liposomes comprises hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, and DSPE-PEG2000, with a mass ratio of (2.5-3.5):1:1, and most preferably 3:1:1. This specific membrane material composition is optimized to simultaneously achieve efficient encapsulation of osimertinib and cabozantinib, two drugs with different properties, while maintaining the long-term stability of the liposome structure.

[0013] More importantly, the co-loaded drug-targeting liposomes prepared using the above-mentioned technical solution of the present invention have an encapsulation rate of not less than 95% for both osimertinib and cabozantinib, indicating that the preparation process can maximize the encapsulation of drugs inside the liposomes, reduce drug loss, and facilitate the synergistic delivery of drugs.

[0014] Secondly, the present invention also provides a pharmaceutical formulation comprising the above-mentioned co-loaded drug-targeting liposomes and a pharmaceutically acceptable carrier, such as a filler, wetting agent, binder, disintegrant or lubricant, which can be prepared into a dosage form suitable for injection administration.

[0015] Thirdly, the present invention also discloses the use of the above-mentioned co-loaded drug-targeting liposomes or pharmaceutical formulations in the preparation of a medicament for treating non-small cell lung cancer resistant to osimertinib.

[0016] Preferably, the non-small cell lung cancer resistant to osimertinib is non-small cell lung cancer with EGFR T790M mutation and / or c-MET amplification.

[0017] The targeted liposomes of this invention can accumulate in tumor tissue through the active targeting effect of GE11 peptide, and synergize with osimertinib by inhibiting key drug resistance pathways such as c-MET using cabozantinib, thereby effectively reversing drug resistance and inhibiting tumor growth.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) This invention is the first to co-load osimertinib and cabozantinib onto the same targeting liposome and, in a specific combination (mass ratio 1:1), produce an unexpected synergistic effect in reversing osimertinib resistance, with a significantly better effect than the combination of osimertinib and anlotinib in the prior art.

[0020] (2) The present invention uses GE11 peptide instead of EGF protein as the target, providing a targeting solution with smaller molecular weight, lower immunogenicity and easier industrial production.

[0021] (3) The present invention achieves an encapsulation rate of more than 95% for both drugs by using a specific liposome membrane material composition, thus ensuring the efficiency and consistency of drug delivery. Attached Figure Description

[0022] Figure 1 This study validated the successful construction of osimertinib (Osi) resistant cell lines and demonstrated drug sensitivity testing results. Figure 1 The AC values ​​represent the effects of osimertinib on cell viability in three cell lines (NCI-H1975, PC-9, and HCC827), respectively. These values ​​were determined using the CCK-8 assay and fitted with the inhibition rate. Figure 1 The DF values ​​were obtained by measuring the effect of osimertinib on the cell viability of three corresponding drug-resistant cell lines (NCI-H1975 OR, PC-9 OR, and HCC827 OR) using the CCK-8 assay and fitting the inhibition rate (number of experiments n=4).

[0023] Figure 2 In the middle section, A is a representative image of cell colony formation after drug treatment of the drug-resistant cell line NCI-H1975 OR, and B is a statistical analysis of the representative images of colony formation.

[0024] Figure 3 A shows transmission electron microscopy images of four types of targeted liposomes; B shows the Zata potential results of the four types of targeted liposomes; and C shows the particle diameter of the four types of liposomes measured by DLS.

[0025] Figure 4 In Figure A, the curve showing the effect of conventional combination therapy on the cell activity of the drug-resistant cell line NCI-H1975 OR is shown. In Figure B, the curve showing the effect of the dual-loaded nanoliposomes of the present invention on the cell activity of the drug-resistant cell line NCI-H1975 OR is shown.

[0026] Figure 5 Figure A shows the effect of different treatment groups on the cell cycle distribution of NCI-H1975 OR cells; Figure B shows the statistical analysis results of the data in Figure A; Figure C shows the effect of different treatment groups on apoptosis of NCI-H1975 OR cells; and Figure D shows the statistical analysis results of the data in Figure C. PE represents phycoerythrin fluorescent labeling, FITC represents fluorescein isothiocyanate fluorescent labeling, and PerCP represents phycoerythrin-polyacrylamide complex fluorescent labeling.

[0027] Figure 6 The effects of four targeted liposome drugs on the migration ability of NCI-H1975 OR cells; among them, Figure 6 A and B are representative images (magnification: 200×) of the cell scratch experiment under different treatment groups, respectively, and their quantitative statistical analysis (number of experiments repeated n=3).

[0028] Figure 7 The effects of four targeted liposome drugs on the migration and invasion abilities of NCI-H1975 OR cells were investigated; among them, Figure 7Images A and B represent representative images (magnification: 200×) from a cell migration experiment and their quantitative statistical analysis, respectively. Figure 7 The CD images are representative images (magnification: 200×) of the cell invasion experiment and their quantitative statistical analysis (experimental repetition number n=3).

[0029] Figure 8 Western blot was used to detect the effects of four targeted liposomal drugs on PI3K / AKT and RAS / RAF / MEK pathway proteins;

[0030] Figure 9 In Figure A, the enrichment results of four targeted liposome drugs in BEAS-2B cells are shown, and in Figure B, the enrichment results of four targeted liposome drugs in NCI-H1975 OR cells are shown.

[0031] Figure 10 Figure A shows xenograft tumors in nude mice after treatment with four targeted liposome drugs; Figure B shows the growth curve of tumor volume in nude mice after treatment with four targeted liposome drugs; Figure C shows the mass statistics of tumors in nude mice after 14 days of treatment with four targeted liposome drugs; and Figure D shows the change in weight of nude mice with the treatment time of four targeted liposome drugs.

[0032] Figure 11 The effects of the targeted liposomes and comparative liposome drugs of this invention at different concentrations on the viability of NCI-H1975 OR cells were investigated.

[0033] Figure descriptions: The IC50 value mentioned in the figure represents the half-maximal inhibitory concentration, which is the concentration required to inhibit 50% of cell activity after drug treatment, in μmol / L; In the figure, "*" indicates P < 0.05, i.e., the result is statistically significant; "**" indicates P < 0.01, i.e., the result is highly statistically significant; "***" indicates P < 0.001, i.e., the result is extremely statistically significant; "****" indicates P < 0.0001, i.e., the result is extremely statistically significant; "ns" indicates that the result is not statistically significant. Detailed Implementation

[0034] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0035] The present invention will be further illustrated below with reference to the accompanying drawings and examples. It should be noted that the following examples are only for illustrating the present invention and are not intended to limit the present invention. Any non-essential improvements and adjustments made on the basis of the present invention shall fall within the scope of protection claimed by the present invention.

[0036] The materials used in the embodiments of this invention are sourced from the following sources:

[0037] Cell lines: human lung adenocarcinoma cell line NCI-H1975, human lung adenocarcinoma cell line PC-9, and human non-small cell lung cancer cell line HCC827 were purchased from the National Collection of Authenticated Cell Cultures, Chinese Academy of Sciences; human bronchial normal epithelial cell line BEAS-2B was purchased from the National Collection of Authenticated Cell Cultures, Chinese Academy of Sciences.

[0038] Materials: Female BALB / c nude mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.; cabozantinib and osimertinib were purchased from Selleck, Inc., USA.

[0039] Reagents: Anti-PI3K, p-PI3K, MEK, and p-MEK antibodies were purchased from Cell Signaling Technology, USA; anti-AKT, ERK, and p-ERK antibodies were purchased from Proteintech, Wuhan Sanying Biotechnology Co., Ltd.; K-RAS and RAF antibodies were purchased from Santa Cruz Biotechnology, USA; Hoechst 33342 dye was purchased from Beyotime Biotechnology Co., Ltd., Shanghai; GE11 peptide, FITC (fluorescent label), soybean phosphatidylcholine (HSPC), and cholesterol were purchased from MedChemexpress Biotechnology, USA.

[0040] The IC50 value mentioned in the embodiments and comparative examples of this invention represents the half-maximal inhibitory concentration, that is, the concentration required to inhibit 50% of cell activity after drug treatment, in μmol / L.

[0041] Example 1: In vitro inhibitory effect of cabozantinib and osimertinib combination therapy on osimertinib-resistant cell lines

[0042] 1.1 Construction of drug-resistant cell lines

[0043] By long-term exposure of osimertinib-sensitive cell lines (NCI-H1975, PC-9, HCC827) to 1 μM osimertinib, corresponding osimertinib-acquired resistance cell lines (NCI-H1975 OR, PC-9 OR, HCC827 OR) were induced and established.

[0044] 1.2 Culture of Six Cell Types

[0045] NCI-H1975, PC-9, HCC827, NCI-H1975 OR, PC-9 OR, and HCC827 OR cells were cultured in DMEM medium containing 10% FBS. The cells were placed in 10 cm cell culture dishes and incubated at 37 ℃ with 5% CO2 saturated humidity until the cells were in the logarithmic growth phase. Then, the cells were passaged using 0.25% trypsin-EDTA digestion solution.

[0046] Osimertinib (Osi) and cabozantinib (Cabo) were dissolved in DMSO solution.

[0047] 1.3 Successful verification of the construction of drug-resistant cell lines

[0048] Cells in the logarithmic growth phase were collected and stored at a density of 7.5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μL in 96-well plates and cultured overnight until cell adhesion. Cells were treated with osimertinib at concentrations of 0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μmol / L, with four replicates per group and 100 μL of culture medium per well. After 48 hours of treatment, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37 °C in the dark for 1 hour. The absorbance at 450 nm was measured using a microplate reader. The average value of the replicates was taken, and the inhibition rate was calculated using the formula (absorbance of untreated group - absorbance of experimental group) / absorbance of untreated group × 100%. An IC50 curve was then fitted to the inhibition rate. Each experiment was independently repeated four times.

[0049] The results are as follows Figure 1 As shown, the IC50 values ​​of the parental cell lines NCI-H1975, PC-9, and HCC827 against osimertinib, as determined by the CCK-8 assay, were 1.430 μM, 1.964 μM, and 1.139 μM, respectively. Figure 1 (A–C); while the IC50 values ​​for the corresponding drug-resistant cell lines NCI-H1975OR, PC-9 OR, and HCC827 OR increased to 7.449 μM, 3.947 μM, and 11.47 μM, respectively. Figure 1 The D–F cell lines showed a significant decrease in sensitivity to osimertinib (P < 0.01), indicating that the drug-resistant cell model was successfully constructed.

[0050] 1.4 Effects of conventional combination therapy of cabozantinib and osimertinib on osimertinib-resistant cell lines

[0051] To evaluate the effect of cabozantinib on the sensitivity of osimertinib-resistant cell lines, the cells were divided into an osimertinib monotherapy group and a conventional combination therapy group. The conventional combination therapy ratio was determined based on the IC50 values ​​of the two drugs, as shown in the figure below. Figure 4The results in Table A and Table 6 show that, taking NCI-H1975 OR cells as an example (IC50 osimertinib = 7.449 μmol / L, IC50 cabozantinib = 11.13 μmol / L), the IC50 ratio was approximately 1:1. Different ratio groups were set up, and each group was administered drugs according to a concentration gradient to ensure that a fixed ratio was maintained at each concentration point. The cell inhibition rate was detected by the CCK-8 assay, and the combination index (CI) was calculated using CompuSyn software based on the Chou-Talalay method to evaluate the synergistic effect (CI < 1 indicates synergy, CI = 1 indicates additive effect, CI > 1 indicates antagonism).

[0052] Table 1. CI values ​​for different effective doses of osimertinib (Osi) and cabozantinib (Cabo) in the NCI-H1975 OR cell line resistant to drugs.

[0053]

[0054] Table 2. CI values ​​for different effective doses of osimertinib (Osi) and cabozantinib (Cabo) in the drug-resistant cell line PC-9 OR.

[0055]

[0056] Table 3. CI values ​​for different effective doses of osimertinib (Osi) and cabozantinib (Cabo) in the drug-resistant cell line HCC827 OR.

[0057]

[0058] Abbreviations: Osi: osimertinib; Cabo: cabozantinib; CI: combination index; ED: effective dose; ED50: half-maximal effective dose; ED75: 75% effective dose; ED90: 90% effective dose; ED95: 95% effective dose.

[0059] Table 4. Inhibition rate of osimertinib (Osi) at different concentrations in drug-resistant cell lines.

[0060]

[0061] The results are shown in Tables 1-4. In the three drug-resistant cell lines NCI-H1975 OR, PC-9 OR and HCC827 OR, the conventional combination therapy group showed better cell inhibition effect than osimertinib monotherapy. The 1:1 combination ratio showed a more significant synergistic effect at most effective dose levels.

[0062] 1.5 Colony Formation Experiment

[0063] In drug-resistant cell lines NCI-H1975 OR, PC-9 OR, and HCC827 OR, control, Osi monotherapy, Cabo monotherapy, and Osi+Cabo combination therapy groups were established. Cells were treated with different drug concentrations for 7 days. Colony formation assay results are shown below. Figure 2 As shown, the combination of the two drugs suggests a synergistic antiproliferative effect.

[0064] Example 2: Construction of targeted liposomes co-loaded with cabozantinib (Cabo) and osimertinib (Osi)

[0065] To improve drug delivery efficiency, targeted nanoliposomes were constructed. The specific steps are as follows:

[0066] (1) Preparation of blank liposomes: The thin film dispersion-ultrasonic hydration method was used, and the specific steps are as follows:

[0067] Hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, and DSPE-PEG2000 pre-conjugated with GE11 peptide and FITC fluorescent label, respectively, were precisely weighed and mixed at a mass ratio of 3:1:1. The mixture was thoroughly dissolved in dichloromethane organic solvent and allowed to dissolve completely in a 30 °C water bath. Subsequently, a uniform lipid film was formed by rotary evaporation under vacuum in a 60 °C water bath for 30 minutes. 60 mL of deionized water was then added and ultrasonically dispersed (for 30 minutes). The film was then filtered sequentially through 0.45 μm, 0.22 μm (twice), and 0.1 μm (twice) filter membranes, and finally dialyzed to obtain blank liposomes.

[0068] (2) Preparation of drug-loaded liposomes (Osi-nano / Cabo-nano): Based on the blank liposome formulation, 61 mg Osi or 31 mg Cabo were added respectively, and the same method was used for preparation.

[0069] (3) Preparation of dual drug-loaded liposomes (Osi+Cabo-nano): 31 mg of Osi and 31 mg of Cabo were added to the blank liposome formulation and prepared by the same method.

[0070] Table 5 Results of liposome concentration, drug loading, and encapsulation efficiency

[0071]

[0072] The liposomes prepared above were subjected to physicochemical characterization: drug loading and encapsulation efficiency were determined; nanoparticle morphology of the liposomes was observed using transmission electron microscopy; particle size distribution was determined using dynamic light scattering technology; and surface charge characteristics were analyzed using a Zeta potentiometer. The results are as follows: Figure 3 As shown in Table 5.

[0073] from Figure 3 As can be seen from Figure A, the prepared liposome nanoparticles are regular spherical or near-spherical in shape, uniform in size, well-dispersed, and without obvious adhesion or aggregation. Figure 3 The C-cell assay showed that the liposomes had a moderate and uniform hydrodynamic diameter, which suggests that they have good circulation and permeability in vivo. Figure 3 The results from B indicate that the nanoparticles carry a moderate charge on their surface, which helps maintain the long-term stability of the nano-dispersion system through electrostatic repulsion and prevents aggregation and precipitation during storage.

[0074] The drug loading data in Table 5 show that the three drug-loaded liposomes (Osi-nano, Cabo-nano, Osi+Cabo-nano) all achieved encapsulation rates of over 97% for their respective drugs. This reveals that the liposome preparation process used in this invention can efficiently encapsulate drugs inside the liposomes with minimal drug loss during the preparation process.

[0075] Example 3: Validation of the efficacy of targeted liposome drugs

[0076] 3.1 In vitro cytotoxicity assay

[0077] Nano group: Cabozantinib (Cabo) and osimertinib (Osi) are co-encapsulated in the same liposome nanoparticle at a 1:1 mass ratio, as in the dual-drug-loaded liposome prepared in Example 2;

[0078] The conventional combination therapy group (Osi:Cabo=1:1): Cabozantinib (Cabo) and osimertinib (Osi) were simply physically mixed at a concentration ratio of 1:1 and then added to the culture medium for incubation.

[0079] The cytotoxic effects of nanoliposome-based drugs (Nano) and conventional combination therapy (Osi:Cabo = 1:1) on NCI-H1975 OR cells were assessed using the CCK-8 assay. Logarithmic growth phase NCI-H1975 OR cells were collected and cultured at 1 × 10⁶ cells per well. 3Cells were seeded in 96-well plates and cultured overnight to allow cell adhesion. Then, the cells were treated with the corresponding drugs. Both groups were administered the drugs at a fixed ratio. Therefore, the IC50 values ​​were calculated using a concentration gradient of osimertinib, with concentrations of 0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μmol / L. Each group had four replicates, and each well contained 100 μL of culture medium. After three days of treatment, 10 μL of LCK-8 solution was added to each well, and the cells were cultured in the dark for another 1 h. The absorbance at 450 nm was measured using a microplate reader. The cell inhibition rate was calculated as: (Absorbance of untreated group – Absorbance of experimental group) / Absorbance of untreated group × 100%. The experiment was repeated three times to obtain the IC50 values ​​and cell viability curves for both treatment methods. Figure 4 The results in Table B and Table 6 show that the IC50 of the Nano group was 1.394 μmol / L, while the IC50 of the conventional combination drug group was 4.137 μmol / L, indicating that nanoliposome packaging significantly enhanced the toxicity of the drug to tumor cells (P < 0.01).

[0080] Table 6 Comparison of half-maximal inhibitory concentrations (IC50) of different administration routes on NCI-H1975 OR cells

[0081]

[0082] 3.2 Cell cycle and apoptosis detection

[0083] NCI-H1975 OR cells in logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 1 / 2 well in 6-well plates, with four treatment groups: blank liposome group (Vehicle), Osi-nano group (1 μmol / L), Cabo-nano group (1 μmol / L), and Osi+Cabo-nano group (osimertinib + cabozantinib, 0.5 μmol / L each). Cells were collected after 48 hours of treatment, and cell cycle and apoptosis staining was performed according to the kit instructions. The results were then analyzed by flow cytometry. Figure 5 As shown.

[0084] Cell cycle results showed ( Figure 5 In both groups A and B, the Osi-nano group arrested tumor cells in the G0 / G1 phase, while the Cabo-nano group had no significant effect on cell cycle distribution. The Osi+Cabo-nano group also induced G0 / G1 phase arrest, and the effect was more significant than that of the Osi-nano group. Apoptosis results are as follows... Figure 5As shown in C–D, both Osi-nano and Cabo-nano can promote tumor cell apoptosis, with the latter showing a lower effect. The Osi+Cabo-nano group significantly increased the apoptosis level, which was significantly higher than that of the single-drug nanogroup, indicating that they have a synergistic enhancing effect in inducing apoptosis.

[0085] 3.3 Cell scratch or migration detection

[0086] Cell scratch assay: NCI-H1975 OR cells in logarithmic growth phase were scratched at a concentration of 5 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of / wells in 6-well plates. Once cells reached confluence exceeding 90%, uniform scratches were made on the cell monolayer in each well using a sterile pipette tip. Cells were gently washed twice with PBS to remove detached cells, and then fresh culture medium containing the following drugs was added: blank liposome group (Vehicle), Osi-nano group (1 μmol / L), Cabo-nano group (1 μmol / L), and Osi+Cabo-nano group (osimertinib and cabozantinib, 0.5 μmol / L each). After culturing for 0, 24, and 48 hours, the scratched areas were observed and photographed under a microscope (magnification: 200×). The scratch area at each time point was measured using image analysis software, and cell migration rate was calculated. Results are as follows: Figure 6 As shown, the Osi-nano group most effectively inhibited the migration ability of NCI-H1975 OR cells, further illustrating the synergistic effect of the technical solution of the present invention in inhibiting the in vitro invasiveness of tumor cells.

[0087] Cell migration and invasion assays: NCI-H1975 OR cells in logarithmic growth phase were resuspended in serum-free medium and the cell density was adjusted. For the migration assay, the cell suspension was added directly to the upper chamber of the Transwell assay; for the invasion assay, the bottom of the upper chamber was pre-coated with matrix gel to simulate the extracellular matrix barrier. Medium containing 10% FBS was added to the lower chamber as a chemical inducer. Four treatment groups were set up: blank liposome group (Vehicle), Osi-nano group (1 μmol / L), Cabo-nano group (1 μmol / L), and Osi+Cabo-nano group (0.5 μmol / L each of osimertinib and cabozantinib). After incubating the cells at 37 ℃ for 48 hours, the chambers were removed, fixed with methanol, stained with crystal violet, and photographed under a microscope (200×) at randomly selected fields of view to count the number of cells that had crossed the membrane. The results are as follows: Figure 7 As shown, compared with the Vehicle group, each single-drug nanogroup (Osi-nano, Cabo-nano) showed a certain degree of migration and invasion inhibition ability, with the Osi+Cabo-nano group showing the most significant inhibition effect.Figure 7 Statistical analysis of B and D showed that the Osi+Cabo-nano group exhibited a synergistic enhancement effect in inhibiting cell migration and invasion, with significantly better results than any single-drug nanogroup.

[0088] 3.4 Detection of signaling pathway proteins

[0089] The technical solution of this invention involves synergistically regulating two intracellular signal transduction pathways that play a core role in tumor development and progression—the PI3K / AKT pathway and the RAS / RAF / MEK / ERK (MAPK) pathway—to achieve a more efficient anti-tumor effect.

[0090] Regarding the PI3K / AKT signaling pathway, this pathway is a central hub regulating cell growth, metabolism, and survival. Its typical activation mechanism is as follows: Upon receiving a growth signal, the receptor tyrosine kinase (RTK) on the cell membrane recruits and activates phosphatidylinositol 3-kinase (PI3K), which catalyzes the generation of a second messenger. PIP3 then recruits protein kinase B (AKT) to the cell membrane, where it is phosphorylated and activated, forming phosphorylated AKT (p-AKT, the active form). Activated p-AKT phosphorylates a series of downstream target proteins (such as mTOR and GSK-3β), ultimately driving cell proliferation and inhibiting apoptosis. GAPDH is a glyceraldehyde-3-phosphate dehydrogenase that provides energy to the cell. Therefore, p-PI3K and p-AKT are key biomarkers characterizing abnormal activation of this pathway; inhibiting their activity can effectively block pro-survival signals.

[0091] Regarding the RAS / RAF / MEK / ERK signaling pathway, this pathway is another major axis regulating cell proliferation and differentiation. Its cascade activation sequence is as follows: upstream signaling activates the GTPase K-RAS, placing it in its GTP-binding active conformation; active K-RAS then recruits and activates serine / threonine kinases RAF (such as β-RAF); subsequently, RAF phosphorylates and activates the dual-specific kinase MEK, forming phosphorylated MEK (p-MEK); p-MEK specifically phosphorylates and activates its unique substrate, extracellular signal-regulated kinase (ERK), generating phosphorylated ERK (p-ERK). Activated p-ERK translocates to the nucleus, phosphorylating various transcription factors, thereby regulating gene expression related to cell cycle progression. In this pathway, the phosphorylation levels of p-MEK and p-ERK are direct indicators of their signal output intensity. These two pathways exhibit extensive cross-talk and feedback regulatory mechanisms within the cell. For example, inhibiting one pathway may lead to compensatory activation of the other, a significant reason for the limited efficacy and drug resistance of single-target therapies. This invention is based on a deep understanding of this networked signaling mechanism. Through specific drug combinations and delivery systems, it aims to achieve synergistic inhibition of key nodes in two pathways (especially p-AKT and p-MEK / p-ERK), thereby overcoming feedback activation and producing anti-tumor effects superior to single-drug or simple combination therapies. The specific experimental steps are as follows:

[0092] NCI-H1975 OR cells in logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 1 / 2 well in 6-well plates, with four treatment groups: blank liposome group (Vehicle), Osi-nano group (1 μmol / L), Cabo-nano group (1 μmol / L), and Osi+Cabo-nano group (osimertinib + cabozantinib, 0.5 μmol / L each). Cells were collected for protein extraction after 48 hours of treatment. Cells were lysed on ice for 30 minutes using RIPA lysis buffer containing PMSF, and protein concentration was determined using the BCA method. Protein samples were mixed with SDS-PAGE loading buffer, denatured by heating in a metal bath at 100°C, and 30 μg was loaded into each well. Electrophoresis (80 V, 2 h) and transfer to a membrane (100 V, 100 min) were then performed. Cells were blocked with 5% skim milk or BSA for 1 h, and primary antibodies against PI3K, p-PI3K, AKT, ERK, p-ERK, MEK, p-MEK, K-RAS, and RAF were added, and the cells were incubated overnight at 4°C. After washing with TBST, the membrane is incubated with the corresponding secondary antibody, and finally developed and exposed.

[0093] The results are as follows Figure 8As shown, the Osi-nano group had no significant effect on p-PI3K phosphorylation in the PI3K / AKT pathway; the Cabo-nano group had a certain inhibitory effect on p-PI3K and p-AKT phosphorylation in the PI3K / AKT pathway, while the Osi+Cabo-nano group significantly inhibited the activation of the PI3K / AKT pathway. In the RAS / RAF / MEK pathway, the Osi-nano group had no significant effect on p-MEK and p-ERK. The Cabo-nano group, however, had a certain upregulation effect on p-MEK and p-ERK, while the Osi+Cabo-nano group had a strong inhibitory effect on p-MEK, p-ERK, and K-Ras, suggesting that Osi+Cabo-nano treatment significantly inhibited the activation of the RAS / RAF / MEK pathway.

[0094] 3.5 Cell-targeting validation

[0095] Log-phase NCI-H1975 OR cells and the normal human bronchial epithelial cell line BEAS-2B were digested, resuspended, and counted using standard methods, and then divided into 1×10⁶ cells per dish. 4 Cells were evenly seeded at a density of [number] cells per 10 mm glass culture dish. After 24 hours of culture to allow cell attachment, the medium was replaced with FITC-labeled targeted liposome drug and cultured for another 24 hours. The medium was then discarded, and the cells were washed three times with PBS. The culture medium containing Hoechst 33342 dye was added and incubated for 10 minutes to label the cell nuclei. The cells were washed three times again with PBS, replaced with regular culture medium, and immediately observed and photographed using a confocal microscope. Results are as follows: Figure 9 As shown in Figures A and B, the targeted liposome drug showed significant enrichment in NCI-H1975 OR tumor cells.

[0096] 3.6 In vivo tumor inhibition experiment

[0097] The in vivo antitumor effect of targeted liposomal drugs was evaluated using a nude mouse subcutaneous xenograft model. Female BALB / c nude mice, 4–5 weeks old and weighing 18–20 g, were housed in an SPF-grade environment. NCI-H1975 OR cells in the logarithmic growth phase were collected, digested, resuspended, and adjusted to a cell density of approximately 1 × 10⁻⁶ cells / mL. 6 Inoculate 100 µL of the tumor subcutaneously in the right groin of nude mice. Wait until the tumor volume reaches 150-200 mm. 3 At that time, the model was considered successfully established, and the tumor-bearing mice were randomly divided into four groups (n=6): Vehicle group, Osi-nano group, Cabo-nano group, and Osi+Cabo-nano group. Each group received the corresponding drug via tail vein injection for 14 consecutive days. Tumor volume was calculated using the formula (short diameter 2 × long diameter) / 2. The results are as follows...Figure 10 As shown, the tumor volume in the Osi+Cabo-nano group was significantly smaller than that in each single-drug group and the Vehicle group (all P < 0.01), and no significant changes in the body weight of mice in each group were observed during the experiment, indicating that the targeted liposome can effectively inhibit tumor growth in vivo and has good safety.

[0098] Comparative Example

[0099] Comparative document: Chinese patent document, application number 202411162646.X, publication date September 24, 2024, discloses a liposome co-loaded with osimertinib and anlotinib (denoted as Osi+Anlo-nano). To demonstrate the outstanding effect of the present invention, the targeted liposome co-loaded with osimertinib and cabozantinib (Osi+Cabo-nano) prepared in this invention was compared with the liposome in the above-mentioned comparative document under the same experimental conditions to evaluate the cytotoxicity (IC50) of the drug-resistant cell line NCI-H1975 OR.

[0100] Table 7. Statistical analysis of IC50 values ​​of the liposomal drugs of the present invention and comparative examples.

[0101]

[0102] The results are shown in Table 7 and Figure 11 As shown, under the same experimental conditions, the IC50 value of the liposomes of this invention against NCI-H1975 OR cells was significantly lower than that in the prior art. This indicates that, compared with the prior art combination of osimertinib and anlotinib, the co-administration strategy of osimertinib and cabozantinib used in this invention can more effectively inhibit the growth of drug-resistant tumor cells, exhibiting a significantly synergistic and enhanced in vitro antitumor activity, thus verifying the non-obviousness and technological advancement of this invention.

Claims

1. A drug-targeting liposome, characterized in that, The co-loaded drug-targeting liposome encapsulates osimertinib and cabozantinib, and the membrane material of the co-loaded drug-targeting liposome is connected to a GE11 peptide targeting ligand.

2. The drug-loaded targeted liposome according to claim 1, characterized in that, The mass ratio of osimertinib to cabozantinib in the targeted liposomes is 1:(0.5-2).

3. The drug-loaded targeted liposome according to claim 2, characterized in that, The mass ratio of osimertinib to cabozantinib in the targeted liposomes is 1:

1.

4. The drug-loaded targeted liposome according to claim 1, characterized in that, The GE11 peptide is coupled to the target liposome membrane via DSPE-PEG2000.

5. The drug-loaded targeted liposome according to claim 1, characterized in that, The membrane material of the targeted liposomes includes hydrogenated soybean phosphatidylcholine, cholesterol and DSPE-PEG2000, and the mass ratio of hydrogenated soybean phosphatidylcholine, cholesterol and DSPE-PEG2000 is (2.5-3.5):1:

1.

6. The drug-targeting liposome according to claim 5, characterized in that, The mass ratio of hydrogenated soybean phosphatidylcholine, cholesterol, and DSPE-PEG2000 is 3:1:

1.

7. The drug-targeting liposome according to claim 1, characterized in that, The co-loaded drug-targeting liposomes achieved an encapsulation efficiency of ≥95% for osimertinib and cabozantinib.

8. A pharmaceutical preparation, characterized in that, It comprises any one of the drug-targeting liposomes according to claims 1-7 and a pharmaceutically acceptable carrier.

9. The pharmaceutical preparation according to claim 8, characterized in that, Pharmaceutically acceptable carriers include fillers, wetting agents, binders, disintegrants, or lubricants.

10. Use of the co-drug-targeting liposome of any one of claims 1-7 or the pharmaceutical formulation of claim 8 in the preparation of a medicament for treating non-small cell lung cancer resistant to osimertinib.

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