Self-assembled multi-drug combination drug-loaded nanoparticles and preparation method and application thereof

Abstract

The application discloses self-assembled multi-drug combination drug-loaded nanoparticles, a preparation method and application thereof, the multi-drug combination drug-loaded nanoparticles are self-assembled by amphiphilic compounds, and at least two kinds of antitumor drugs are encapsulated in the interior of the nanomicelles; and the application further provides application of the multi-drug combination drug-loaded nanoparticles in preparation of a drug for treating lung cancer. The multi-drug combination drug-loaded nanoparticles have excellent water solubility and targeting property, can overcome drug resistance, provide more available means for treatment of lung cancer and drug-resistant lung cancer, and have important practical significance and value for improving the health level of residents in China.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a self-assembled multi-drug co-loaded nanoparticle, its preparation method, and its application. Background Technology

[0002] Currently, lung cancer is the leading cause of cancer-related morbidity and mortality in my country, with non-small cell lung cancer (NSCLC) accounting for approximately 80%-85% of cases. Of these, about 50% are patients with epidermal growth factor receptor (EGFR) mutations. Compared to traditional radiotherapy and chemotherapy, EGFR-TKIs, as small-molecule targeted therapies for lung cancer, have significantly improved the survival rate of patients with intermediate-to-advanced EGFR-mutant lung cancer. Three generations of EGFR-TKIs have been approved for marketing in my country: first-generation gefitinib, erlotinib, and itotinib; second-generation afatinib; and third-generation osimertinib. However, after a period of use (an average of about one year), these targeted drugs still face the serious challenge of drug resistance and exhibit significant toxic side effects. How to prolong the effective duration of these drugs and reduce their side effects is a pressing issue that needs to be addressed.

[0003] Tumors are generally heterogeneous, often containing different subpopulations within the same tumor. This means that single targeted therapies are only effective against the subsets that match sensitive mutations. Given this situation, combination therapies have attracted increasing attention in recent years. One approach is to continue using EGFR-TKIs in combination with chemotherapy after resistance to targeted therapies has developed. Unfortunately, most clinical trial results have been unsatisfactory; the combined treatment is not superior to chemotherapy and may even significantly increase toxic side effects. Therefore, finding methods to further enhance the anticancer effects of targeted and chemotherapeutic drugs while reducing their toxic side effects is of great significance. Summary of the Invention

[0004] The purpose of this invention is to propose a self-assembled multi-drug co-loaded nanoparticle, its preparation method and application, which can improve the solubility of hydrophobic drugs, enhance drug targeting, achieve controllable drug release and reduce drug toxicity, and to a certain extent overcome tumor drug resistance and enhance anti-cancer effects.

[0005] The objective of this invention will be achieved through the following technical solutions:

[0006] This invention provides a self-assembled multi-drug-loaded nanoparticle, which is self-assembled from an amphiphilic compound, with at least two antitumor drugs encapsulated within nanomicelles; the structural formula of the amphiphilic compound is shown in formula a:

[0007]

[0008] Where: R1, R2, R3, R4, R5, R6, R7, R8 are selected from -H and Any one of the following, where n is an integer between 1 and 8.

[0009] Furthermore, the number of -H values ​​in R1, R2, R3, R4, R5, R6, R7, and R8 is 4-8. The number is 0-4.

[0010] Furthermore, the method for preparing the amphiphilic compound includes the following steps:

[0011] (1) The compounds 1 and 2 were dissolved in a THF / H2O mixed solution, and copper sulfate pentahydrate and sodium ascorbate were added. The reaction was carried out at 30℃-70℃ for 2h-6h under nitrogen protection. After the reaction was completed, the solvent was dried, dichloromethane was added and extracted with saturated ammonium chloride solution. The organic phase was dried with MgSO4, filtered, concentrated and then obtained by column chromatography to obtain compound 3.

[0012] (2) Dissolve compound 3 in methanol, slowly add excess ethylenediamine, react at 20℃-40℃ for 2-4 days, remove the solvent, purify the crude product by dialysis, and freeze dry to obtain compound 4.

[0013] (3) Dissolve compound 5 in DMF, add EDC / NHS, activate for 0.5h-2h, add compound 4, react at 25℃-60℃ for 1-3 days, remove solvent, purify crude product by dialysis, and freeze dry to obtain target compound 6;

[0014] The reaction equation for the above method is shown in equation i:

[0015]

[0016] Where: R1, R2, R3, R4, R5, R6, R7, R8 are selected from -H and Any one of the following, where n is an integer between 1 and 8.

[0017] Furthermore, in step (1), the volume ratio of THF / H2O is 3-5:1, the molar ratio of compound 1 to compound 2 is 1-1.2:1, the molar ratio of compound 2 to copper sulfate pentahydrate is 5-10:1, and the molar ratio of compound 2 to sodium ascorbate is 2.5-5:1.

[0018] Furthermore, in step (2), the molar ratio of ethylenediamine to compound 3 is 30-100:1.

[0019] Furthermore, in step (3), the molar ratio of EDC to NHS is 1-5:1, the molar ratio of NHS to compound 5 is 2-10:1, and the molar ratio of compound 5 to compound 4 is 1-4:1.

[0020] Furthermore, the specific steps include the following:

[0021] (1) Compound 1 and Compound 2 were dissolved in a mixed solution of THF / H2O with a volume ratio of 4:1. Copper sulfate pentahydrate and sodium ascorbate were added and reacted at 60°C for 5 h under nitrogen protection. After the reaction was completed, the solvent was dried, dichloromethane was added and extracted with saturated ammonium chloride solution. The organic phase was dried with MgSO4, filtered, concentrated and then obtained by column chromatography to obtain Compound 3.

[0022] (2) The compound 3 was dissolved in methanol, and excess ethylenediamine was slowly added. The mixture was reacted at 30°C for 2 days. The solvent was removed, and the crude product was purified by dialysis and lyophilized to obtain the target compound 4.

[0023] (3) Compound 5 was dissolved in DMF, EDC / NHS was added, and after activation for 0.5 h, compound 4 was added. The reaction was carried out at 30 °C for 3 days. The solvent was removed, and the crude product was purified by dialysis and lyophilized to obtain target compound 6.

[0024] Furthermore, the antitumor drug is doxorubicin and a targeted drug, wherein the targeted drug is any one or more of gefitinib, erlotinib, and osimertinib.

[0025] Furthermore, the method for preparing self-assembled multi-drug loaded nanoparticles includes the preparation of doxorubicin loaded nanoparticles and the preparation of targeted drug loaded nanoparticles.

[0026] The preparation of the doxorubicin-loaded nanoparticles specifically includes the following steps: dissolving doxorubicin and the amphiphilic compound in methanol, adding excess triethylamine, then removing the solvent by rotary evaporation to form a thin film, then adding deionized water for dispersion, hydrating at 60°C for 0.5 h, filtering through a filter membrane, and purifying by dialysis to obtain the doxorubicin-loaded nanoparticles;

[0027] The preparation of the targeted drug-loaded nanoparticles specifically includes the following steps: dissolving the targeted drug and the amphiphilic compound in methanol, slowly adding the mixture to deionized water under ultrasonic assistance, stirring at room temperature for 16 hours, removing methanol by rotary evaporation, and filtering through a filter membrane to obtain the targeted drug-loaded nanoparticles.

[0028] Another aspect of the present invention provides the application of multi-drug-loaded nanoparticles in the preparation of drugs for treating lung cancer.

[0029] The outstanding effects of this invention are:

[0030] This invention selects folic acid-functionalized amphiphilic dendritic molecules as carriers for the combined use of targeted drugs and chemotherapy drugs. These amphiphilic dendritic molecules can spontaneously assemble into structures similar to micelles and vesicles of dendritic macromolecules. By carrying different types of targeted drugs and chemotherapy drugs and adjusting the proportion and content of different drugs, tumor drug resistance can be overcome and anti-cancer effects can be enhanced. The IC50 of the optimal combination drug (erlotinib nanoparticles and doxorubicin nanoparticles) is 1251 times lower than that of erlotinib single targeted drug.

[0031] The multi-drug loaded nanoparticles of this invention have good water solubility, avoiding the use of organic solvents, improving the solubility of hydrophobic drugs, and enhancing the targeting of drugs.

[0032] The multi-drug combined drug-loaded nanoparticles of the present invention can enhance drug targeting, achieve controllable drug release and reduce drug toxicity and side effects, and overcome tumor drug resistance and enhance anti-cancer effects to a certain extent through the combination therapy of targeted drugs and chemotherapy drugs.

[0033] The multi-drug-loaded nanoparticles proposed in this invention have excellent water solubility and targeting properties, and can overcome drug resistance, providing more available means for the treatment of lung cancer and drug-resistant lung cancer, which has important practical significance and value for improving the health level of Chinese residents.

[0034] The following detailed description of specific embodiments of the present invention will make the technical solution of the present invention easier to understand and master. Attached Figure Description

[0035] Figure 1 The image shows the hydration diameter size and distribution (DLS) of the self-assembled multi-drug-loaded nanoparticles prepared in Example 1 of this invention.

[0036] Figure 2 This is a TEM image of the self-assembled multi-drug co-loaded nanoparticles prepared in Example 1 of the present invention;

[0037] Figure 3 This is a hemolytic test diagram of the amphiphilic compound prepared in Example 1 of the present invention;

[0038] Figure 4 This is a graph showing the cytotoxicity results of the self-assembled multi-drug-loaded nanoparticles prepared in Example 1 of this invention. Detailed Implementation

[0039] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0040] All reagents used in this invention are commercially available. All experimental methods used in this invention are conventional methods and techniques in the art.

[0041] Example 1

[0042] The method for preparing self-assembled multi-drug co-loaded nanoparticles in this embodiment includes the following steps:

[0043] (1) Compound 1-1 (1-azidooctadecane) (163 mg, 0.55 mmol) and compound 2 (714 mg, 0.5 mmol) were dissolved in a mixed solution of THF / H2O (v:v) = 4 / 1. Copper sulfate pentahydrate (12 mg, 0.05 mmol) and sodium ascorbate (19.8 mg, 0.1 mmol) were added, and the mixture was reacted at 60 °C for 5 h under nitrogen protection. After the reaction was completed, the solvent was dried, dichloromethane was added, and the mixture was extracted three times with saturated ammonium chloride solution. The organic phase was dried with MgSO4, filtered, concentrated, and then subjected to column chromatography to obtain 707 mg of compound 3-1, with a yield of 82%.

[0044] (2) Compound 3-1 (517 mg, 0.3 mmol) was dissolved in methanol, and ethylenediamine (902 mg, 15 mmol) was slowly added. The mixture was stirred at 30 °C for 2 days. After removing the solvent, the crude product was purified by dialysis through a dialysis membrane with MWCO = 1000. Then, it was freeze-dried to obtain 544 mg of the target compound 4-1, with a yield of 93%.

[0045] (3) Compound 5 (folic acid, 132 mg, 0.3 mmol) was dissolved in DMF, and EDC (9.0 mmol, 1725 mg) and NHS (3.0 mmol, 345 mg) were added. After activation by stirring at room temperature for 0.5 h, compound 4-1 (195 mg, 0.1 mmol) was added, and the reaction was carried out at 30 °C for 3 days. The solvent was removed, and the crude product was purified by dialysis through a dialysis membrane with MWCO = 1000. The product was then lyophilized to obtain amphiphilic compound 6-1. Each amphiphilic molecule was linked to The average number is 2.3.

[0046] The reaction equation for the above method is shown below:

[0047]

[0048] (4) 3.0 mg of compound 6-1 was dissolved in 2.0 mL of methanol, and 2.0 mg of doxorubicin and 1.0 μL of triethylamine were added. The solvent was removed by rotary evaporation, forming a thin film at the bottom of the flask. Then, 2.0 mL of deionized water was added, and the mixture was stirred and hydrated at 60 °C for 0.5 h. After filtration through a 0.22 μm filter membrane, the mixture was purified by dialysis through a 1000 MWCO membrane to obtain doxorubicin-loaded nanoparticles. The encapsulation efficiency was 81%, the drug loading was 36%, and the doxorubicin concentration was 805 mg / L, as determined by an enzyme-linked immunosorbent assay (ELISA) reader.

[0049] μL / mL;

[0050] (5) 3.0 mg of compound 6-1 and 1.0 mg of gefitinib were dissolved in 1.0 mL of methanol. Under ultrasonic assistance, the mixture was slowly added to 3.0 mL of deionized water. The mixture was stirred at room temperature for 16 h to evaporate the methanol. The remaining methanol was then removed by rotary evaporation. After 0.22...

[0051] After filtration through a μm filter membrane, gefitinib-loaded nanoparticles were obtained. Microplate reader analysis revealed an encapsulation efficiency of 56%, a drug loading of 16%, and a gefitinib concentration of 278 μL / mL.

[0052] (6) 3.0 mg of compound 6-1 and 1.0 mg of erlotinib were dissolved in 1.0 mL of methanol. Under ultrasonic assistance, the mixture was slowly added to 3.0 mL of deionized water. The mixture was stirred at room temperature for 16 h to evaporate the methanol. The remaining methanol was then removed by rotary evaporation. After 0.22...

[0053] After filtration through a μm filter membrane, erlotinib-loaded nanoparticles were obtained. Microplate reader analysis revealed an encapsulation efficiency of 64%, a drug loading of 18%, and an erlotinib concentration of 312 μL / mL.

[0054] (7) 3.0 mg of compound 6-1 and 1.0 mg of osimertinib were dissolved in 1.0 mL of methanol. Under ultrasonic assistance, the mixture was slowly added to 3.0 mL of deionized water. The mixture was stirred at room temperature for 16 h to evaporate the methanol. The remaining methanol was then removed by rotary evaporation. After 0.22...

[0055] After filtration through a μm filter membrane, osimertinib-loaded nanoparticles were obtained. Encapsulation efficiency was 49%, drug loading was 14%, and osimertinib concentration was 236 μg / mL, as determined by an enzyme-linked immunosorbent assay (ELISA).

[0056] Example 2

[0057] The method for preparing self-assembled multi-drug co-loaded nanoparticles in this embodiment includes the following steps:

[0058] (1) Compound 1-2 (1-azidoeicosane) (178 mg, 0.55 mmol) and compound 2 (714 mg, 0.5 mmol) were dissolved in a mixed solution of THF / H2O (v:v) = 4 / 1. Copper sulfate pentahydrate (12 mg, 0.05 mmol) and sodium ascorbate (19.8 mg, 0.1 mmol) were added, and the mixture was reacted at 60 °C for 5 h under nitrogen protection. After the reaction was completed, the solvent was dried, dichloromethane was added, and the mixture was extracted three times with saturated ammonium chloride solution. The organic phase was dried with MgSO4, filtered, concentrated, and then subjected to column chromatography to obtain 675 mg of compound 3-2, with a yield of 77%.

[0059] (2) Compound 3-2 (526 mg, 0.3 mmol) was dissolved in methanol, and ethylenediamine (902 mg, 15 mmol) was slowly added. The mixture was stirred at 30 °C for 3 days. After removing the solvent, the crude product was purified by dialysis through a dialysis membrane with MWCO = 1000. Then, it was freeze-dried to obtain 563 mg of the target compound 4-2, with a yield of 95%.

[0060] (3) Compound 5 (folic acid, 132 mg, 0.3 mmol) was dissolved in DMF, and EDC (9.0 mmol, 1725 mg) and NHS (3.0 mmol, 345 mg) were added. After activation by stirring at room temperature for 0.5 h, compound 4-2 (198 mg, 0.1 mmol) was added, and the reaction was carried out at 30 °C for 3 days. The solvent was removed, and the crude product was purified by dialysis through a dialysis membrane with MWCO = 1000. The product was then lyophilized to obtain the amphiphilic compound 6-2. Each amphiphilic molecule was linked to The average quantity is 2.5;

[0061] The reaction equation for the above method is shown below:

[0062]

[0063] (4) 3.0 mg of compound 6-2 was dissolved in 2.0 mL of methanol, 2.0 mg of doxorubicin and 1.0 μL of triethylamine were added, and the solvent was removed by rotary evaporation to form a thin film at the bottom of the flask. Then, 2.0 mL of deionized water was added, and the mixture was stirred and hydrated at 60 °C for 0.5 h. After filtration through a 0.22 μm filter membrane, the mixture was purified by dialysis through a dialysis membrane with an MWCO of 1000 to obtain doxorubicin-loaded nanoparticles. The encapsulation efficiency was 75%, the drug loading was 30%, and the doxorubicin concentration was 758 μg / mL, as determined by an enzyme-linked immunosorbent assay (ELISA).

[0064] The preparation methods for other targeted drug-loaded nanoparticles are basically the same as those in Example 1.

[0065] Example 3

[0066] The method for preparing self-assembled multi-drug co-loaded nanoparticles in this embodiment includes the following steps:

[0067] (1) Compound 1-3 (1-azidohexadecane) (147 mg, 0.55 mmol) and compound 2 (714 mg, 0.5 mmol) were dissolved in a mixed solution of THF / H2O (v:v) = 4 / 1. Copper sulfate pentahydrate (12 mg, 0.05 mmol) and sodium ascorbate (19.8 mg, 0.1 mmol) were added, and the mixture was reacted at 60 °C for 5 h under nitrogen protection. After the reaction was completed, the solvent was dried, dichloromethane was added, and the mixture was extracted three times with saturated ammonium chloride solution. The organic phase was dried with MgSO4, filtered, concentrated, and then subjected to column chromatography to obtain 721 mg of compound 3-3, with a yield of 85%.

[0068] (2) Compound 3-3 (509 mg, 0.3 mmol) was dissolved in methanol, and ethylenediamine (902 mg, 15 mmol) was slowly added. The mixture was stirred at 30 °C for 3 days. After removing the solvent, the crude product was purified by dialysis through a dialysis membrane with MWCO = 1000. Then, it was freeze-dried to obtain 530 mg of the target compound 4-3, with a yield of 92%.

[0069] (3) Compound 5 (folic acid, 132 mg, 0.3 mmol) was dissolved in DMF, and EDC (9.0 mmol, 1725 mg) and NHS (3.0 mmol, 345 mg) were added. After activation by stirring at room temperature for 0.5 h, compound 4-3 (192 mg, 0.1 mmol) was added, and the reaction was carried out at 30 °C for 3 days. The solvent was removed, and the crude product was purified by dialysis through a dialysis membrane with MWCO = 1000. The product was then lyophilized to obtain amphiphilic compound 6-3. Each amphiphilic molecule was linked to The average quantity is 2.2;

[0070] The reaction equation for the above method is shown below:

[0071]

[0072] (4) 3.0 mg of compound 6-2 was dissolved in 2.0 mL of methanol, and 2.0 mg of doxorubicin and 1.0 μL of triethylamine were added. The solvent was removed by rotary evaporation, forming a thin film at the bottom of the flask. Then, 2.0 mL of deionized water was added, and the mixture was stirred and hydrated at 60 °C for 0.5 h. After filtration through a 0.22 μm filter membrane, the mixture was purified by dialysis through a dialysis membrane with an MWCO of 1000 to obtain doxorubicin-loaded nanoparticles. The encapsulation efficiency was 77%, the drug loading was 34%, and the doxorubicin concentration was 616 μg / mL, as determined by an enzyme-linked immunosorbent assay (ELISA).

[0073] The preparation methods for other targeted drug-loaded nanoparticles are basically the same as those in Example 1.

[0074] Experimental Example 1

[0075] Hydration diameter size and distribution of drug-loaded nanoparticles in Example 1

[0076] Different drug-loaded nanoparticle solutions were diluted to 100 μM (calculated based on the concentration of the amphiphilic compound 6-1), filtered through a 0.45 μm pore size filter membrane, and 1-2 mL of the sample was taken. The hydration diameter and particle size distribution of the nanomicelles were determined by DLS. Figure 1 The diagram shows the hydration diameter and distribution of drug-loaded nanoparticles, such as... Figure 1 As shown, the hydration diameter of the four drug-loaded nanoparticles is around 8 nm, exhibiting good uniformity.

[0077] Experiment Example 2

[0078] TEM testing of drug-loaded nanoparticles in Example 1

[0079] Different drug-loaded nanoparticles were diluted to prepare 10 μM nanomicelle solutions. After filtration through a filter membrane with a pore size of 0.22 μm, 10 μL of the sample was dropped onto a carbon-coated copper grid and dried under an infrared lamp until the moisture was completely evaporated. The sample was then stained with 5 μL of 1% phosphotungstic acid solution for 5 min. Excess staining agent was immediately removed with filter paper, and the morphological characteristics of the sample were observed by TEM. Figure 2 TEM images of different drug-loaded nanoparticles, such as Figure 2 As shown, the diameter of the nanoparticles is 8-10 nm, which is consistent with the DLS results.

[0080] Experimental Example 3

[0081] Hemolytic activity test of amphiphilic compound 6-1 in Example 1

[0082] Compound 6-1 was dissolved in physiological saline by vortexing to prepare test samples with concentrations of 2.5, 5, 10, 15, 20, 25, 30, and 40 μg / mL. 1 mL of each concentration was added to a 1.5 mL centrifuge tube. 20 μL of erythrocyte suspension was added to each sample. 20 μL of erythrocyte suspension was added to 1 mL of physiological saline as a negative control, and 20 μL of erythrocyte suspension was added to 1 mL of triple-distilled water as a positive control. The samples were incubated at 37°C for 2 h. Three replicates were set up for both samples and control groups. After incubation, the sample was centrifuged at 3500 rpm for 5 min, and 0.2 mL of the supernatant was transferred to a 96-well plate. The absorbance at 545 nm was measured using a microplate reader. Figure 3 The hemolytic activity test of compound 6-1 showed that the rate of erythrocyte hemolysis gradually increased with increasing concentration. In the concentration range of 2.5–10 μg / mL, the relative erythrocyte hemolysis was <5%, while when the sample concentration exceeded 10 μg / mL, the relative erythrocyte hemolysis was >5%, indicating that high-concentration sample solutions have a certain degree of erythrocyte hemolytic activity.

[0083] Experiment Example 4

[0084] Cytotoxicity of Multidrug-Loaded Nanoparticles in Example 1

[0085] This experiment investigated the anticancer activity of multidrug-loaded nanoparticles against drug-resistant PC9-GR lung cancer cells using the MTT assay. The anticancer drugs were divided into four groups, each with multiple concentrations. Group 1 consisted of erlotinib (concentrations of 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, and 27 μg / mL); Group 2 consisted of erlotinib-loaded nanoparticles (concentrations of 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, and 27 μg / mL); and Group 3 consisted of erlotinib combined with doxorubicin. The first group consisted of two sets of nanoparticles: one containing 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 54 μg / mL, with equal amounts of erlotinib and doxorubicin. The second group consisted of erlotinib-loaded nanoparticles and doxorubicin-loaded nanoparticles (concentrations of 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 54 μg / mL, with equal amounts of erlotinib and doxorubicin nanoparticles). Logarithmic-phase PC9-GR cells were collected, counted, and the cell suspension concentration was adjusted. Cells were then seeded in 96-well plates at 1×10⁶ cells / well. 4 Cells per well were cultured overnight at 37°C with 5% CO2. After grouping and treatment as described above, cells were cultured for 48 hours, and the culture medium was removed. Each well was washed three times with PBS, and 200 μL of medium containing 0.5 mg / mL MTT was added to each well. Cells were incubated at 37°C with 5% CO2 for 4 hours. The supernatant was discarded, and 150 μL of DMSO was added to each well. After gentle shaking for 10 min, the absorbance at 570 nm was measured. Untreated cells were used as a control group. GraphPad Prism 9 was used for plotting and IC50 analysis. 50 calculate.

[0086] like Figure 4 The figure shows the anticancer effect of multi-drug loaded nanoparticles on drug-resistant PC9-GR lung cancer cells. It can be seen that the combined use of erlotinib and doxorubicin loaded nanoparticles (0.02664 μg / mL) has a high IC50. 50 It was the lowest, 1251 times lower than the targeted drug erlotinib (33.33 μg / mL).

[0087] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of a multi-drug-loaded nanoparticle in the preparation of a drug for treating drug-resistant lung cancer, characterized in that: The multi-drug-loaded nanoparticles are self-assembled from amphiphilic compounds. These nanoparticles consist of doxorubicin-loaded nanoparticles and erlotinib-loaded nanoparticles. The doxorubicin-loaded nanoparticles are formed by doxorubicin encapsulating within the nanomicelles of the amphiphilic compounds, and the erlotinib-loaded nanoparticles are formed by erlotinib encapsulating within the nanomicelles of the amphiphilic compounds. The structural formulas of the amphiphilic compounds are shown in Formula 6-1. ， Formula 6-1 Where: R1, R2, R3, R4, R5, R6, R7, R8 are selected from -H and Any one of them, each amphiphilic molecule is connected The average number is 2.

3.

2. The application according to claim 1, characterized in that, The preparation method of the amphiphilic compound includes the following steps: (1) Compound 1-1 and compound 2 were dissolved in a THF / H2O mixed solution, and copper sulfate pentahydrate and sodium ascorbate were added. The reaction was carried out at 30℃-70℃ for 2h-6h under nitrogen protection. After the reaction was completed, the solvent was dried, dichloromethane was added and extracted with saturated ammonium chloride solution. The organic phase was dried with MgSO4, filtered, concentrated and then obtained by column chromatography to obtain compound 3-1. (2) Dissolve the compound 3-1 in methanol, slowly add excess ethylenediamine, react at 20℃-40℃ for 2-4 days, remove the solvent, purify the crude product by dialysis, and freeze dry to obtain compound 4-1; (3) Compound 5 was dissolved in DMF, EDC / NHS was added, and after activation for 0.5-2 hours, compound 4-1 was added. The mixture was reacted at 25-60°C for 1-3 days. The solvent was removed, and the crude product was purified by dialysis and lyophilized to obtain the target compound 6-1. Each amphiphilic molecule was linked to The average quantity is 2.3; The reaction equation for the above method is shown in equation i: ， Formula i Where: R1, R2, R3, R4, R5, R6, R7, R8 are selected from -H and Any one of them.

3. The application according to claim 2, characterized in that: In step (1), the volume ratio of THF / H2O is 3-5:1, the molar ratio of compound 1-1 to compound 2 is 1-1.2:1, the molar ratio of compound 2 to copper sulfate pentahydrate is 5-10:1, and the molar ratio of compound 2 to sodium ascorbate is 2.5-5:

1.

4. The application according to claim 2, characterized in that: In step (2), the molar ratio of ethylenediamine to compound 3-1 is 30-100:

1.

5. The application according to claim 2, characterized in that: In step (3), the molar ratio of EDC to NHS is 1-5:1, the molar ratio of NHS to compound 5 is 2-10:1, and the molar ratio of compound 5 to compound 4-1 is 1-4:

1.

6. The application according to claim 2, characterized in that, Specifically, the steps include the following: (1) The compounds 1-1 and 2 were dissolved in a mixed solution of THF / H2O in a volume ratio of 4:

1. Copper sulfate pentahydrate and sodium ascorbate were added, and the mixture was reacted at 60°C for 5 h under nitrogen protection. After the reaction was completed, the solvent was dried, dichloromethane was added, and the mixture was extracted with saturated ammonium chloride solution. The organic phase was dried with MgSO4, filtered, concentrated, and then subjected to column chromatography to obtain compound 3-1. (2) The compound 3-1 was dissolved in methanol, and excess ethylenediamine was slowly added. The reaction was carried out at 30°C for 2 days. The solvent was removed, and the crude product was purified by dialysis and freeze-dried to obtain the target compound 4-1. (3) Compound 5 was dissolved in DMF, EDC / NHS was added, and after activation for 0.5 h, compound 4-1 was added. The reaction was carried out at 30 °C for 3 days. The solvent was removed, and the crude product was purified by dialysis and lyophilized to obtain target compound 6-1.

7. The application according to claim 1, characterized in that: This includes the preparation of doxorubicin-loaded nanoparticles and erlotinib-loaded nanoparticles; The preparation of the doxorubicin-loaded nanoparticles specifically includes the following steps: dissolving doxorubicin and the amphiphilic compound in methanol, adding excess triethylamine, then removing the solvent by rotary evaporation to form a thin film, then adding deionized water for dispersion, hydrating at 60°C for 0.5 h, filtering through a filter membrane, and purifying by dialysis to obtain the doxorubicin-loaded nanoparticles; The preparation of the erlotinib-loaded nanoparticles specifically includes the following steps: erlotinib and the amphiphilic compound are dissolved in methanol, and the mixture is slowly added to deionized water under ultrasonic assistance. The mixture is stirred at room temperature for 16 hours, and then methanol is removed by rotary evaporation. After filtration through a filter membrane, the erlotinib-loaded nanoparticles are obtained.

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