Dual-drug synergistic delivery nanomaterials, methods of making and using the same

Abstract

This invention discloses a dual-drug synergistic delivery nanomaterial, its preparation method, and its application, belonging to the field of biomedical technology. The dual-drug synergistic delivery nanomaterial of this invention is a polyethylene glycol-polyamino acid block copolymer (PEG). m -b-(PAA) x -co-PBB y Using methoxylated polyethylene glycolamine as an initiator, and through reasonable regulation based on... L By adjusting the polymerization ratio of N-carboxylic acid anhydride monomers of γ-glutamic acid-γ-benzyl ester and methionine, a ROS-responsive polymer material was synthesized, reducing the preparation cycle in an acid-catalyzed oil-water system from 3 days to 3 hours. This nanomaterial can simultaneously load lenvatinib and gefitinib, effectively solving the problem of single-drug crystallization precipitation through a "dual-drug cluster" mechanism, and achieving synergistic delivery of the two drugs. It shows broad application prospects in the field of liver cancer treatment drug preparation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a dual-drug synergistic delivery nanomaterial, its preparation method, and its application. Background Technology

[0002] Lenvatinib, an oral multi-target tyrosine kinase inhibitor, potently inhibits key targets such as vascular endothelial growth factor receptors (VEGFR1-3), fibroblast growth factor receptors (FGFR1-4), and platelet-derived growth factor receptor (PDGFRα). Furthermore, lenvatinib demonstrated non-inferiority to sorafenib and a higher objective response rate (ORR: 24.1% vs 9.2%) in the REFLECT phase III clinical trial. Therefore, it has become a first-line standard treatment for advanced hepatocellular carcinoma (HCC) (Kudo, et al., Lancet, 2018, 391, 1163-1173). However, the clinical application of lenvatinib is subject to two core limitations:

[0003] First, the drug's physicochemical defects lead to low bioavailability and significant side effects. Lenvatinib belongs to the BCS class II drugs, has extremely poor water solubility (approximately 0.001 mg / mL), and exhibits high hydrophobicity and easy crystallization. This results in its easy aggregation or adsorption by biomolecules such as proteins in the body environment, rapid metabolism after oral administration, and an absolute bioavailability of only 18.8%-29.8%. To maintain an effective drug concentration in the blood, clinical practice tends to use high doses (12 mg / day for patients weighing ≥ 60 kg, 8 mg / day for patients weighing < 60 kg) and frequent administration, which may induce dose-dependent adverse reactions such as hypertension, proteinuria, and diarrhea.

[0004] Second, there are limitations in the efficacy of monotherapy and the issue of drug resistance. Although lenvatinib significantly improves the objective response rate (ORR) compared to sorafenib, the efficacy rate of monotherapy is still less than 25%, and most patients inevitably develop primary or acquired resistance. A groundbreaking study published in Nature by Jin et al. revealed a possible resistance mechanism: while lenvatinib inhibits targets such as FGFR, it also feedback-activates the epidermal growth factor receptor (EGFR) and its downstream PAK2-ERK5 signaling axis in hepatocellular carcinoma cells, triggering bypass escape (Jin, et al., Nature, 2021, 595, 730-734). This study further confirmed through CRISPR-Cas9 synthetic lethal screening that the combination of EGFR phosphorylation inhibition and lenvatinib has a significant synergistic lethal effect on hepatocellular carcinoma, laying a theoretical foundation for combination therapy strategies.

[0005] Based on the above mechanism, the combination of lenvatinib and EGFR inhibitors has rapidly moved from basic research to clinical trials. A prospective single-arm exploratory clinical trial conducted by Shi et al. showed that in lenvatinib-resistant advanced HCC patients (n=12), the combination with gefitinib demonstrated good safety and significant efficacy: 4 patients achieved confirmed partial remission, 4 had stable disease, and the objective response rate reached 33.3% (Shi, et al., Signal Transduct Target Ther, 2024, 9, 359). However, the core limitation of existing studies lies in the fact that the dosing regimen still uses high-dose, high-frequency free drug forms (e.g., in preclinical studies, the dose of lenvatinib and gefitinib dual therapy exceeded 80 mg / kg), failing to address the inherent low bioavailability of lenvatinib and the toxic side effects caused by non-target organ distribution, severely limiting its clinical translation potential. As Jin et al. pointed out, innovation in drug delivery systems is urgently needed to effectively translate the synergistic mechanism into clinical practice.

[0006] To address the issues of low solubility and poor bioavailability of poorly soluble drugs, nanomedicine delivery systems are an important strategy for improving efficacy and reducing toxic side effects. An ideal nanocarrier should effectively encapsulate hydrophobic drugs, prolong circulation time, and achieve passive tumor targeting through enhanced permeation-retention (EPR) effects, thereby increasing drug concentration at the lesion site and reducing systemic toxicity. For example, Hao et al. utilized the high expression of reactive oxygen species (ROS) in activated hepatic stellate cells (HSCs) in liver fibrosis lesions to design ROS-responsive polymethionine-polylysine copolymer micelles for targeted delivery of resveratrol, achieving on-demand drug release at the lesion site and significantly improving liver fibrosis (Hao, et al., ACS Nano, 2022, 16, 2073-2088). In addition, Miyata et al. constructed a polymer micelle with biocompatibility, endosome escape and DNA condensation functions by designing a polyethylene glycol-polyasparagine derivative-polylysine (PEG-PAsp(DET)-PLys) triblock copolymer, which enabled systematic gene delivery to pancreatic tumor tissues. This demonstrated a strategy of achieving multifunctional vector integration through ingenious molecular design (Miyata, et al., Pharm Res, 2008, 25, 2924-2936).

[0007] However, existing research still faces significant technical bottlenecks when applying nanotechnology to the delivery of lenvatinib and its combination therapies: on the one hand, the high hydrophobicity and easy crystallization properties of lenvatinib make it prone to precipitation due to supersaturation when prepared using traditional nanoprecipitation and emulsification methods, resulting in low encapsulation efficiency, insufficient drug loading, and poor batch reproducibility; on the other hand, even if the local single drug concentration in the tumor is increased through nanocarriers, the drug resistance problem caused by EGFR feedback activation still exists, ultimately limiting the anti-tumor effect. Summary of the Invention

[0008] Purpose of the invention: The first purpose of this invention is to provide a dual-drug synergistic delivery nanomaterial; the second purpose of this invention is to provide a preparation method and application of the dual-drug synergistic delivery nanomaterial; the nanomaterial of this invention achieves efficient co-loading and synergistic anti-tumor effects of lenvatinib and gefitinib.

[0009] Technical solution: The dual-drug synergistic delivery nanomaterial of the present invention is a polyethylene glycol-polyamino acid block copolymer, and the block copolymer has the structure shown in formula (I):

[0010] PEG m -b-(PAA x -co-PBB y (I),

[0011] Among them, PEG m This indicates polyethylene glycol segments with a number-average molecular weight of 2000 Da, 3000 Da, 3400 Da, 5000 Da, 6000 Da, 8000 Da, or 10000 Da, where m is the degree of polymerization of polyethylene glycol, m = 45, 68, 77, 113, 136, 182, or 227; PAA x This represents a poly(amino acid A) segment with a degree of polymerization of x, wherein the amino acid A is selected from... L -Glutamic acid-γ-benzyl ester, L -Phenylalanine, L -Tyrosine or L - Tryptophan or any derivative of the above amino acids; PBB y This represents a poly(amino acid B) segment with a degree of polymerization of y, wherein the amino acid B is selected from... L - Methionine or any of its amino acid derivatives; co indicates random copolymerization; b indicates that the PEG segment and the [PAA-co-PBB] segment are block-linked; x takes a value of 10-100, y takes a value of 50-100, and x:y is (1-5):(1-20), the total molecular weight of the copolymer is 10-30 kDa, and the dispersion is... It ranges from 1.05 to 1.21.

[0012] Furthermore, PEGm This indicates polyethylene glycol segments with a number-average molecular weight of 3400 Da, 5000 Da, or 6000 Da, where m is the degree of polymerization of polyethylene glycol, m = 68, 77, or 113; PAA x This represents a poly(amino acid A) segment with a degree of polymerization of x; wherein amino acid A is... L -Glutamic acid-γ-benzyl ester or a derivative thereof, wherein amino acid B is... L -Methionine; the x value is 10-50, the y value is 50-90, and the x:y ratio is (1-3):(1-10); the total molecular weight of the copolymer is 15-23 kDa, and the dispersity is... It is 1.05-1.06.

[0013] More preferably, the number-average molecular weight of the polyethylene glycol segments is 5000 Da, corresponding to a degree of polymerization m = 113; the amino acid A is... L -Glutamic acid-γ-benzyl ester, amino acid B is L -Methionine; where x is 20, y is 80, and x:y is 20:80; the total molecular weight of the copolymer is 19-20 kDa, and the dispersity is... It is 1.05.

[0014] The preparation method of the dual-drug synergistic delivery nanomaterial of the present invention includes the following steps:

[0015] (1) The amino acid, triphosgene, and epoxide are reacted in the first organic solvent to obtain a product containing... N Crude product of -carboxylate intracyclic anhydride (NCA);

[0016] (2) Take the product obtained in step (1) containing N The crude product of α-carboxycyclic intracyclic anhydride (NCA) was directly dissolved in a second organic solvent, and trimethylacetic acid, pH buffer and initiator were added to form an oil-water biphase system for interfacial ring-opening polymerization; the dual-drug synergistic delivery nanomaterial was obtained after purification.

[0017] Further, the epoxide in step (1) is selected from any one of epichlorohydrin and epichlorohydrin; the first organic solvent is selected from any one of tetrahydrofuran, dioxane, diethyl ether or methyl tert-butyl ether; the second organic solvent in step (2) is selected from any one of chloroform, dichloromethane or 1,2-dichloroethane; the initiator is a methoxylated polyethylene glycolamine with a number average molecular weight of 2000 Da, 3000 Da, 3400 Da, 5000 Da, 6000 Da, 8000 Da or 10000 Da; the buffer solution is a buffer solution with a pH of 2-3.

[0018] Preferably, the epoxide in step (1) is propylene oxide; the first organic solvent is tetrahydrofuran; the second organic solvent in step (2) is chloroform; the buffer solution is a buffer solution with a pH of 3; and the methoxy polyethylene glycol amine is PEG. 113 -NH2.

[0019] Further, in step (2), the molar ratio of trimethylacetic acid to initiator is (10-60):(1-3); the volume ratio of the second organic solvent to pH buffer is (0.5-5):(0.5-3); and the total reaction time for steps (1) and (2) is 3-5 h.

[0020] Preferably, the molar ratio of trimethylacetic acid to initiator in step (2) is 30:1; the volume ratio of the second organic solvent to pH buffer is 1:1; and the total reaction time for steps (1) and (2) is 3 h.

[0021] Furthermore, the dual-drug co-loaded nanomedicine in the dual-drug synergistic delivery nanomaterial of the present invention is lenvatinib and gefitinib.

[0022] The present invention discloses a method for preparing a dual-drug co-loaded nanomedicine, comprising the following steps:

[0023] (1) Lenvatinib, gefitinib and dual-drug synergistic delivery nanomaterials were dissolved in an organic solvent to form a mixed solution;

[0024] (2) The mixed solution obtained in step (1) is added dropwise to water and stirred. After dialysis purification, nanomedicine is formed.

[0025] Further, in step (1), the mass ratio of the dual-drug synergistic delivery nanomaterial (polyethylene glycol-polyamino acid block copolymer), lenvatinib, and gefitinib is (15-25):(1-5):(0.5-10); the organic solvent is selected from... N , N -Dimethylformamide, dimethyl sulfoxide, N , N -Dimethylacetamide, N Any of the following: -methylpyrrolidone.

[0026] Preferably, in step (1), the mass ratio of the dual-drug synergistic delivery nanomaterial, lenvatinib, and gefitinib is 20:1:4.2 (lenvatinib:gefitinib = 1:4, mol / mol); the organic solvent is... N , N -Dimethylformamide.

[0027] Furthermore, the stirring speed in step (2) is 300-800 rpm, and the dripping time is 5-10 min.

[0028] Preferably, the stirring speed in step (2) is 400 rpm and the dropping time is 30 min.

[0029] The application of the dual-drug synergistic delivery nanomaterial or the dual-drug co-loaded nanomedicine described in this invention in the preparation of drugs for treating liver cancer.

[0030] This nanomaterial achieves efficient encapsulation of lenvatinib (a multi-target tyrosine kinase inhibitor), a highly hydrophobic and easily crystallizing drug. Simultaneously, gefitinib (an EGFR phosphorylation inhibitor) is introduced for co-assembly, constructing a dual-drug synergistic delivery system. The dual-drug combination (lenvatinib and gefitinib) used in this invention exhibits synergy, with therapeutic effects far exceeding those of single-drug therapy. Furthermore, the two drugs also demonstrate synergy at the loading mechanism level; within a certain proportion, they form relatively stable "dual-drug molecular clusters" in water. These clusters increase the interaction between drug molecules, significantly inhibiting the crystallization tendency and aggregation behavior of individual drug molecules, and promoting the stable formation of the nanomedicine. Finally, the designed responsive carrier has a simple synthetic route, readily available raw materials, and excellent biocompatibility. After screening for the optimal drug combination and material ratio, efficient dual-drug loading can be achieved. The resulting nanomedicine not only significantly improves the therapeutic effect of hepatocellular carcinoma but also reduces the toxic side effects of the original drugs.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0032] (1) The dual-drug synergistic delivery nanomaterials of the present invention are constructed by molecular design to form a functional polymer carrier with a hydrophilic-hydrophobic block structure; by utilizing its synergistic assembly behavior with drug molecules, the highly hydrophobic and easily crystallized lenvatinib and gefitinib (multi-target tyrosine kinase inhibitors) are efficiently loaded, and the resulting drug-loaded nanoparticles have the characteristics of uniform particle size, high stability, and extended in vivo circulation time.

[0033] (2) The present invention efficiently prepared polyethylene glycol-polyamino acid materials by acid-catalyzed oil-water two-phase polymerization, which shortened the synthesis cycle from the traditional 3 days (including monomer preparation, purification and polymerization steps) to 3 hours. Moreover, the polymerization process is controllable and the material composition is clear, so that the nanodelivery system has good batch-to-batch consistency.

[0034] (3) The application of the dual-drug synergistic delivery nanomaterials described in this invention in the preparation of drugs for treating liver cancer. This invention reveals for the first time the phenomenon of "drug molecule clusters" formed by lenvatinib and gefitinib in a specific ratio, solving the problem of rapid precipitation caused by crystallization when single-drug loading is used. The synergistic relationship between the two drugs in inhibiting the proliferation of liver cancer cells was verified using the Synergy Finder 3.0 program. At the same time, after comprehensively considering the drug dosage and the effect of inhibiting the proliferation of liver cancer cells, the optimal combination ratio of lenvatinib and gefitinib was determined to be 1:4 (molar ratio). In addition, the two drugs in N , N The dual-drug clusters formed in the dimethylformamide / water system can be effectively encapsulated by the hydrophobic regions of the carrier, significantly improving the stability and drug loading efficiency of the nanoparticles after dual-drug co-loading.

[0035] (4) The ROS-responsive nanodelivery system constructed in this invention exhibits excellent drug controlled release capability in the tumor microenvironment: methionine, as a ROS-responsive unit, achieves a hydrophobic-hydrophilic transition in tumor tissues with high ROS levels, triggering micelle disintegration to release the drug. Cell experiments show that PEG... 113 -b-(PBLG 20 -co-PMET 80 The ROS-responsive nanomedicine formed after loading the two drugs significantly inhibited Huh-7 and Hep3B liver cancer cells. Animal experiments further confirmed that, compared with non-responsive nanomedicines, this nanomedicine had a more significant anti-tumor effect at the same dose, while effectively reducing the metabolic damage to the liver and kidneys caused by free lenvatinib and gefitinib. Attached Figure Description

[0036] Figure 1 Preparation of PEG, a dual-drug synergistic delivery nanomaterial, for Example 1 113 -b-(PAA x -co-PBB y Synthesis route diagram of (material PEM);

[0037] Figure 2 PEG, a dual-drug synergistic delivery nanomaterial prepared in Example 1 113 -b-(PAA x -co-PBB y GPC (gel permeation chromatography) chromatogram of ) Figure 2 (a) in the figure shows the GPC diagrams for different monomer materials; Figure 2 (b) in the figure shows the GPC diagrams for materials with different monomer ratios;

[0038] Figure 3 It is PEG in Example 1 113 -b-(PBLG 20 -co-PMET80 The hydrogen NMR spectrum of the material;

[0039] Figure 4 Preparation of PEG in Comparative Example 2 113 -b-(PBLG 20 -co-PnLeu 80 Synthesis route diagram for material PEnL;

[0040] Figure 5 These are the GPC spectra of different dual-drug synergistic delivery nanomaterials in Example 1. Figure 5 In Comparative Example 1, (a) shows the PEG obtained from the conventional ring-opening polymerization of NCA. 113 -b-(PBLG 20 -co-PMET 80 GPC plot; Figure 5 (b) in the example is PEG from Comparative Example 2. 113 -b-(PBLG 20 -co-PnLeu 80 GPC spectra of the material;

[0041] Figure 6 It is the PEG of Comparative Example 2 in Example 1. 113 -b-(PBLG 20 -co-PnLeu 80 The hydrogen NMR spectrum of the material;

[0042] Figure 7 This refers to the synergy between lenvatinib and gefitinib in Synergy Finder 3.0 in Example 2 (score > 10 indicates a strong synergistic relationship).

[0043] Figure 8 This is a drug dose-cell survival curve of Huh-7 liver cancer cells with different ratios of lenvatinib and gefitinib drug combinations in Example 3.

[0044] Figure 9 This is a schematic diagram of the preparation method of dual-drug co-loaded nanomedicine by nanoprecipitation method in Example 4;

[0045] Figure 10 The differences in drug structure and nanoparticle stability before and after loading are shown in Example 4 and Comparative Example 2 for lenvatinib and gefitinib.

[0046] Figure 11 The difference in nanoparticle stability when the nanomaterials for dual-drug synergistic delivery are loaded with different proportions of lenvatinib and gefitinib in Example 5;

[0047] Figure 12This is the result of the stability study of the single / dual drug complex formed by lenvatinib and gefitinib in Example 5. Figure 12 (a) in the figure is the particle size distribution of a single drug dispersed in water; Figure 12 (b) in the figure is the particle size distribution diagram of the single drug dispersed in water for 24 h; Figure 12 (c) in the figure is the particle size distribution diagram of the two drugs dispersed in water; Figure 12 (d) in the figure is the particle size distribution diagram after the two drugs are dispersed in water for 24 h;

[0048] Figure 13 This is a responsive characterization of methionine under incubation with different concentrations of hydrogen peroxide in Example 6. Figure 13 (a) in the diagram is a schematic diagram and equation for the oxidation of polymethionine; Figure 13 (b) shows the NMR spectra before and after oxidation;

[0049] Figure 14 This is Example 7, which describes the release of lenvatinib and gefitinib from the dual-drug synergistic delivery nanomaterial under different concentrations of hydrogen peroxide. Figure 14 (a) in the figure represents the release curve of lenvatinib; Figure 14 (b) in the figure is the gefitinib release curve;

[0050] Figure 15 This is the UV absorption spectrum of the delivery system loaded with different proportions of lenvatinib and gefitinib in Experiment Example 5;

[0051] Figure 16 This is the hemolysis experiment of free drug, carrier solution, and nanodelivery system in Example 10;

[0052] Figure 17 Example 11 shows the drug dose-cell survival curves of Huh-7 liver cancer cells after a series of dual-drug synergistic delivery nanomaterials loaded with lenvatinib and gefitinib.

[0053] Figure 18 Example 12 is a study on the uptake of a series of dual-drug synergistic delivery nanomaterials by liver cancer cells;

[0054] Figure 19 Example 13 is a study on the material distribution of hepatocellular carcinoma cells after taking up nanomaterials delivered by a dual-drug synergistic delivery system.

[0055] Figure 20 Example 14 shows the drug dose-cell survival curves of Huh-7 liver cancer cells after dual-drug synergistic delivery of nanomaterials loaded with different proportions of lenvatinib and gefitinib.

[0056] Figure 21 This is an in vivo antitumor activity evaluation of each drug group against mouse hepatocellular carcinoma in Example 15; Figure 21(a) in the diagram is a schematic diagram of the mouse treatment experiment process; Figure 21 (b) shows the tumor images of mice in each group after treatment; Figure 21 (c) represents the tumor weight of mice in each group after treatment; Figure 21 (d) in the figure represents the tumor volume change curves of mice in each group during the treatment process; Figure 21 (e) in the figure represents the curve of mouse body weight change in each group during the treatment process;

[0057] Figure 22 Example 16 describes a study of liver and kidney damage in Huh-7 tumor mice after treatment with a free drug, a non-ROS-responsive delivery system, and a ROS-responsive delivery system. The study included the detection of liver markers, ALT (alanine aminotransferase) and AST (aspartate aminotransferase); and kidney markers, CRE (creatinine) and BUN (blood urea nitrogen).

[0058] Figure 23 Example 15 is a schematic diagram of the preparation of a responsive delivery system for the treatment of hepatocellular carcinoma. Detailed Implementation

[0059] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0060] To further illustrate the preparation and application of the dual-drug synergistic delivery nanomaterials of the present invention and to achieve the intended purpose of the invention, the present invention will be further described in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0062] Acetonitrile (HPLC grade) was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd., catalog number A3862; water (HPLC grade) was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number B610001-0006; methoxy polyethylene glycol amine (PEG) 113 -NH2) was purchased from Xiamen Sainobang Biotechnology Co., Ltd., product number 06020100206; methoxy polyethylene glycol amine (PEG) 45 -NH2) was purchased from Xiamen Sainobang Biotechnology Co., Ltd., product number 06020100202; methoxy polyethylene glycol amine (PEG) 227 -NH2) was purchased from Xiamen Sainuobang Biotechnology Co., Ltd., product number 06020100209; L - Methionine was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number M101130-25 g;L -Glutamic acid-γ-benzyl ester was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number G110915-25g; propylene oxide was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., catalog number P816084-100ml; L - Leucine was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number L814752-25g; L -Phenylanine was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number L816180-25g; L -Tyrosine was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number L818844-25g; L Tryptophan was purchased from Shanghai Maclean's Biochemical Technology Co., Ltd., catalog number L818799-25g; triphosgene was purchased from Shanghai Maclean's Biochemical Technology Co., Ltd., catalog number T818744-100g; creatinine (CRE) test kit was purchased from Nanjing Jiancheng Bioengineering Institute, catalog number C011-2-1; blood urea nitrogen (BUN) test kit was purchased from Nanjing Jiancheng Bioengineering Institute, catalog number C013-2-1; aspartate aminotransferase (AST) test kit was purchased from Nanjing Jiancheng Bioengineering Institute. The following products were purchased: alanine aminotransferase (ALT) test kit (C009-2-1) from Nanjing Jiancheng Bioengineering Institute; N-hydroxysuccinimide ester (Cy5) (NHS) from Shanghai Maclean Biochemical Technology Co., Ltd. (C849970-25mg); and 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyltetrazolium bromide thiazolyl blue (MTT) from Dalian Meilun Biotechnology Co., Ltd. (MB4698-1).

[0063] Male BALB / c-Nude nude mice, SPF grade, 5-6 weeks old, weighing 18-20 g, were purchased from Changzhou Cavens Laboratory Animal Co., Ltd., and housed in a specific pathogen-free (SPF) grade animal laboratory. All animal experiments were conducted in accordance with the guidelines of the Laboratory Animal Management and Ethics Committee of Soochow University.

[0064] Human liver cancer cells (Huh-7 and Hep3B) were donated by Professor Yin Lichen's research group at Soochow University. Huh-7 cells were cultured in DMEM medium containing 10% FBS, and Hep3B cells were cultured in MEM medium containing 10% FBS.

[0065] The buffer solution used in this invention can refer to the "Easily prepared wide range buffer series" reported by Carmody, J. Chem. Educ. 1961, 38, 559-560; different pH ranges can be designed and prepared according to actual application requirements. Depending on the required pH value, common glycine buffer, citrate buffer, etc., can also achieve the same effect.

[0066] For ease of explanation, the specific preparation method of the buffer solution of this invention is as follows: Citric acid (231.2 mg, 1.1 mM) and boric acid (272.1 mg, 4.4 mM) are weighed and dissolved in 22 mL of water, and the resulting solution is designated as solution A; sodium phosphate (114.1 mg, 0.3 mM) is weighed and dissolved in 3 mL of water, and the resulting solution is designated as solution B. Solution A and solution B are then mixed at a volume ratio of 22:3 to obtain a buffer solution with a pH of 3.

[0067] Example 1: PEG nanomaterial for dual-drug synergistic delivery 113 -b-(PAA 20 -co-PBB 80 Synthesis of

[0068] To synthesize PEG 113 -b-(PBLG 20 -co-PMET 80 For example, at room temperature, weigh... L -Glutamic acid-γ-benzyl ester (15.0 mg, 0.07 mmol) and L Methionine (41.8 mg, 0.28 mmol) was mixed and placed in a reaction flask. 3 mL of tetrahydrofuran (THF) was added to uniformly disperse the amino acids. Subsequently, propylene oxide (196.6 mg, 3.4 mmol) and triphosgene (50.2 mg, 0.17 mmol) were added sequentially. The suspended solids were observed to gradually dissolve and clarify. The reaction was carried out at room temperature for approximately 3 h until the solution was completely clear, at which point stirring was stopped. The reaction solvent and some impurities were removed by vacuum evaporation to obtain a yellow, oily crude NCA mixture. The solvent and propylene oxide were removed under vacuum until dry. The crude NCA product was dissolved in chloroform (1 mL), and chloroform, hydrogen chloride, and triphosgene were precipitated in n-hexane (14 mL) to remove impurities such as chloroform, hydrogen chloride, and triphosgene. The solvent was removed again under vacuum to determine the mass of the crude NCA product (32.3 mg, 0.27 mmol). The corresponding amount of initiator PEG was weighed. 113-NH2 (13.5 mg, 0.0027 mmol), trimethylacetic acid (8.27 mg, 0.081 mmol). The crude NCA product was redissolved in chloroform (1.35 mL), and trimethylacetic acid was added with stirring to disperse. Then, a pH 3 buffer (1.35 mL) was added, and the mixture was stirred rapidly for approximately 30 s. PEG was immediately added. 113 - Stirring with NH2. Reaction kinetics were monitored using infrared spectroscopy; the reaction was considered complete when monomer conversion > 99% (reaction time 0.5 h). The polymer was initially purified by precipitation with n-hexane, and then... N , N Dimethylformamide was used to dissolve the polymer and disperse it in water to form a polymer solution. The polymer solution was then dialyzed using a dialysis bag with a molecular weight cutoff of 3500 Da, with the water changed every 4 hours for a total of 3-5 times. After dialysis, the solution was freeze-dried to obtain the PEG material. 113 -b-(PBLG 20 -co-PMET 80 The specific synthetic route is as follows: Figure 1 As shown.

[0069] Using PEG as a synthetic material 113 -b-(PBLG 20 -co-PMET 80 Using the same preparation method, different dual-drug synergistic delivery nanomaterials PEG were prepared. 113 -b-(PAA 20 -co-PBB 80 The difference is: L -Glutamic acid-γ-benzyl ester was replaced with phenylalanine (0.07 mmol, 11.6 mg), respectively. L -Tyrosine (0.07 mmol, 12.7 mg), L -Tryptophan (0.07 mmol, 14.3 mg) was used to obtain the material PEG. 113 -b-(PPHE 20 -co-PMET 80 PEG 113 -b-(PTYR 20 -co-PMET 80 PEG 113 -b-(PTRP 20 -co-PMET 80 ).

[0070] Using PEG as a synthetic material 113 -b-(PBLG 20 -co-PMET 80Using the same preparation method, other dual-drug synergistic delivery nanomaterials PEG with different proportions were prepared. 113 -b-(PBLG x -co-PMET y The difference is: PEG 113 -b-(PBLG 50 -co-PMET 50 Weigh out the following during preparation: L -Glutamic acid-γ-benzyl ester (15 mg, 0.068 mmol), methionine (10.28 mg, 0.068 mmol); PEG 113 -b-(PBLG 30 -co-PMET 70 Weigh out the following during preparation: L -Glutamic acid-γ-benzyl ester (9.93 mg, 0.045 mmol), methionine (15.88 mg, 0.105 mmol); PEG 113 -b-(PBLG 10 -co-PMET 90 Weigh out the following during preparation: L -Glutamic acid-γ-benzyl ester (7.50 mg, 0.034 mmol), methionine (46.27 mg, 0.306 mmol); PEG 113 -b-PMET 100 During preparation, only methionine (15.12 mg, 0.1 mmol) should be weighed.

[0071] This invention prepares other dual-drug synergistic delivery nanomaterials PEG in different proportions. x -b-(PBLG 20 -co-PMET 80 ), using PEG as a synthetic material 113 -b-(PBLG 20 -co-PMET 80 The same preparation method, the difference being: PEG 113 -NH2 initiator replaced with PEG 45 -NH2 (5.4 mg, 0.0027 mmol) and PEG 227 -NH2 (27 mg, 0.0027 mmol) yielded the material PEG. 45 -b-(PBLG 20 -co-PMET 80 ) and PEG 227 -b-(PBLG 20 -co-PMET 80 ).

[0072] To trace the delivery of nanomaterials in cell experiments, after copolymerization, the copolymer was precipitated and dissolved in an aqueous solution of NaHCO3 (0.2 M), the pH of the solution was adjusted to 8, and then Cy5 NHS ester (50 μL, 1.6 μmol) was added and stirred overnight. The reaction was then lyophilized by dialyzing to obtain fluorescently labeled PEM-Cy5 nanomaterials.

[0073] Experimental Example 1

[0074] Molecular weight and compositional characterization of the dual-drug synergistic delivery nanomaterial. PEG prepared in Example 1. 113 -b-(PBLG 20 -co-PMET 80 Taking the characterization method of ) as an example, the number-average molecular weight (M) of the copolyamino acids was determined by gel permeation chromatography (GPC). n ) and molecular weight distribution (ε). Take 5 mg of PEG. 113 -b-(PBLG 20 -co-PMET 80 The material was dissolved in chromatographic grade DMF containing LiBr (0.1 M) (5 mg / mL), and then filtered through a PTFE membrane (0.22 μm) to remove undissolved impurities before GPC analysis. Simultaneously, the actual proportions of the copolyamino acids in different components were calculated by nuclear magnetic resonance spectroscopy (NMR). 5 mg of PEG was used... 113 -b-(PBLG 20 -co-PMET 80 The material was dissolved in 500 μL of deuterated trifluoroacetic acid (10 mg / mL) and characterized by NMR. Other dual-drug delivery nanomaterials with different proportions were characterized using the same method. GPC spectra of the dual-drug synergistic delivery nanomaterials: [Image caption: PEG-coated nanomaterials]. 113 -b-(PBLG 20 -co-PMET 80 Taking the spectrum of PEG as an example, the data obtained from the GPC test above is imported into Origin software to obtain PEG. 113 -b-(PBLG 20 -co-PMET 80 Spectra. The spectra of other dual-drug synergistic delivery nanomaterials with different proportions were obtained using the same testing method.

[0075] Table 1 shows the prepared PEG nanomaterials with different proportions for dual-drug synergistic delivery. 113 -b-(PBLG x -co-PMET y The molecular weight and structural composition characterization data of ).

[0076]

[0077] Note: a E refers to nanomaterials used for dual-drug synergistic delivery. L -Glutamic acid-γ-benzyl ester, M refers to methionine in the dual-drug synergistic delivery nanomaterial; F refers to phenylalanine in the dual-drug synergistic delivery nanomaterial; Y refers to phenylalanine in the dual-drug synergistic delivery nanomaterial. L -Tyrosine, W refers to the dual-drug synergistic delivery nanomaterials L -Tryptophan, L-finger dual-drug synergistic delivery nanomaterials L - Leucine.

[0078] Combining Table 1 and Figure 2 GPC spectrum analysis of (a) shows that when the amino acid is L When one of the following is present: γ-glutamic acid benzyl ester, phenylalanine, tyrosine, and tryptophan, it is composed of... L The copolymer involving γ-glutamic acid benzyl ester exhibited the narrowest dispersion (1.05) and its molecular weight distribution conformed to the expected range (theoretical molecular weight 19.9 kDa, actual molecular weight 19.0 kDa). Other copolymers, however, showed large dispersion and shoulder peaks in their GPC spectra, indicating non-uniform molecular weight distribution and unsuitability as candidate components for delivery materials. Therefore, in subsequent experiments, [the copolymer was selected]. L -Glutamic acid-γ-benzyl ester and methionine are the building blocks of nanomaterials.

[0079] Simultaneously, based on the dual-drug synergistic delivery nanomaterial PEG in Table 1... 113 -b-(PBLG x -co-PMET y GPC and NMR characterization data of the polymer system were analyzed, and the comprehensive analysis showed that the polymerization system exhibited excellent controllability and reproducibility. In terms of molecular weight, the actual molecular weight (18.5-22.5 kDa) of all samples deviated from the theoretical value by less than 5%, with E... 50 -M 50 and E 10 -M 90 The groups are perfectly matched, PEG 113 -b-(PBLG x -co-PMET y Overall dispersion ( (The value is between 1.05 and 1.06.) See also: Figure 2 GPC spectral analysis in (b) shows the presence of PEG nanomaterials with different proportions for dual-drug synergistic delivery. 113 -b-(PBLG x -co-PMET yAll samples showed narrow dispersion, with no shoulder peaks or other extraneous peaks. This indicates that the molecular weight distribution of materials in different proportions is uniform, with minimal batch-to-batch variation. Regarding material composition, the actual composition measured by NMR (E:M = 47:53 to 10:90) deviates from the theoretical feed ratio by no more than 5%. Figure 3 The integral result is used to calculate PEG. 113 -b-(PBLG x -co-PMET y The actual copolymer molar ratio corresponding to this was 22:78, which is highly consistent with the initial feed ratio (i.e., 20:80), confirming the controllability of the oil-water polymerization system. This method not only significantly shortens the material preparation cycle but also eliminates the preparation step of amino acid NCA monomers, greatly simplifying the synthesis process of copolymerized amino acid materials.

[0080] Experimental Example 2

[0081] Segment selection for dual-drug synergistic delivery nanomaterials with different polyethylene glycol (PEG) segments. The PEG prepared in Example 1... x -b-(PBLG 20 -co-PMET 80 The solubility and drug loading capacity were evaluated. Specifically, the material (5 mg) and drug (1 mg) were dissolved in DMF (200 μL), and then added dropwise to stirred water to complete the loading. Experiments showed that when the PEG chain length was 45, the hydrophilic segment was too short, resulting in low solubility of the copolymer in water. It easily precipitated during nanoprecipitation and could not form a stable micelle solution. The fundamental reason is that its hydrophilic segment is too short to effectively shield the hydrophobic core and maintain colloidal stability. While the copolymer had better solubility when the PEG chain length was 227, its self-assembly ability decreased significantly. During drug loading, the drug precipitated in large quantities before it could be encapsulated by the hydrophobic segment of the carrier, indicating that the excessively long hydrophilic segment made it difficult for the hydrophobic segment to effectively aggregate, hindering the orderly formation of drug-loaded micelles. Although intermediate chain lengths (68, 77, 113, 136 and 182) can all form micelles, the PEG chain length of 113 has the best overall performance. Therefore, methoxy polyethylene glycol amine with a chain length of 113 was selected for subsequent experiments.

[0082] Comparative Example 1: Preparation of PEG by conventional NCA ring-opening polymerization 113 -b-(PBLG 20 -co-PMET 80 )

[0083] This comparative example is similar to Example 1, which synthesizes the dual-drug synergistic delivery nanomaterial PEG. 113 -b-(PBLG 20 -co-PMET 80The steps are the same, except that in Example 1, the two amino acids are mixed and directly reacted to obtain a crude mixed NCA product, which can be carried out without purification before subsequent reactions; while in Comparative Example 1, crude BLG-NCA and MET-NCA products need to be prepared separately, purified and then copolymerized.

[0084] The specific steps are as follows: After obtaining the crude BLG-NCA product according to Example 1, the crude product needs to be dissolved in THF (20 mL), and 4 times the volume of n-hexane is slowly added along the flask wall for recrystallization (overnight). The recrystallization step is repeated three times, and the crystals are then vacuum dried for 6 h to obtain white needle-like BLG-NCA crystals. After obtaining the crude MET-NCA product according to Example 1, it needs to be purified by column chromatography using petroleum ether / ethyl acetate = 5:1 (v / v) (2-3 times). The product is dissolved in 10 mL of dichloromethane, and the solvent is removed in a rotary evaporator to obtain colorless oily MET-NCA. The purified BLG-NCA (14.2 mg, 0.054 mmol) and MET-NCA (37.8 mg, 0.22 mmol) are weighed separately and dissolved in 0.7 mL of a mixed solvent (DMF / dichloromethane, v / v = 1:1) to obtain an NCA mixed solution. Then, PEG is weighed... 113 -NH2 (0.0027 mmol, 13.5 mg) was dissolved in 100 μL of a mixed solvent and added to the NCA solution. The mixture was stirred rapidly, and the reaction was allowed to proceed for approximately 24 h. Reaction kinetics were monitored using infrared spectroscopy, and the reaction was considered complete when the monomer conversion was >99%. The polymer was initially purified by precipitation with n-hexane, and then further purified using... N , N Dimethylformamide was used to dissolve and disperse the polymer in water to form a polymer solution. The polymer solution was then dialyzed using a dialysis bag with a molecular weight cutoff of 3500 Da, with the water changed every 4 hours for a total of 3-5 times. After dialysis, the solution was freeze-dried to obtain the PEG material. 113 -b-(PBLG 20 -co-PMET 80 ).

[0085] For PEG synthesized by traditional methods 113 -b-(PBLG 20 -co-PMET 80 Materials, characterization methods, and PEG prepared by oil-water polymerization in Example 1 113 -b-(PBLG 20 -co-PMET 80 The materials are consistent. See also Figure 5(a) GPC spectral analysis shows that the material exhibits not only significant dispersion but also a multi-peak distribution. This is primarily due to excessive impurities in MET-NCA, which may be initiated by H2O during column chromatography purification. Furthermore, some impurities may be difficult to remove completely, hindering subsequent polymerization and causing polymer contamination from side reactions, resulting in a cluttered spectral pattern. Simultaneously, GPC results indicate that the actual molecular weight (34.6 kDa) is significantly greater than the theoretical molecular weight (19.0 kDa). This is again mainly due to the difficulty in removing impurities from MET-NCA, which deactivates some initiators during polymerization, leading to a much higher actual molecular weight. Therefore, for special monomers like MET-NCA, the oil-water polymerization method employed in this invention offers unique advantages.

[0086] By comparing the materials prepared in Example 1 (oil-water polymerization) with those prepared in Comparative Example 1 (conventional method), it was found that Example 1, by directly polymerizing the crude NCA product without purification using an oil-water polymerization method, shortened the polymerization time from three days to three hours. Furthermore, the materials obtained in Example 1 exhibited controllable polymerization, a narrow molecular weight distribution, and a well-defined composition and structure. Subsequent experiments all used the method described in Example 1 to prepare dual-drug delivery nanomaterials to ensure the rigor and reliability of the experimental results.

[0087] Comparative Example 2: Non-responsive dual-drug delivery nanomaterial PEG 113 -b-(PBLG 20 -co-PnLeu 80 Synthesis of

[0088] This comparative example is similar to the synthesis of the dual-drug delivery nanomaterial PEG in Example 1. 113 -b-(PBLG 20 -co-PMET 80 The steps are the same, except that methionine is replaced with ortholeucine (36.7 mg, 0.28 mmol). The rest of the steps are identical. The specific synthetic route is as follows: Figure 4 As shown.

[0089] Synthetic PEG 113 -b-(PBLG 20 -co-PnLeu 80 The materials and characterization methods are the same as described above. See [link / reference]. Figure 5 Analysis of the GPC test results in (b) shows that the actual molecular weight of the material is 17.5 kDa, which is close to the theoretical molecular weight of 18.4 kDa, and the dispersion is 1.05, indicating a narrow molecular weight distribution. Furthermore, the integral area obtained from the 1H NMR spectrum of the material can be used to calculate... L The actual molar ratio of -glutamic acid-γ-benzyl ester residues to oroleucine residues is 1:3.66 (e.g. Figure 6The corresponding copolymer molar ratio is 22:78, which is close to the preset ratio, ensuring the accurate synthesis of the experimental materials.

[0090] PEG 113 -b-(PBLG 20 -co-PnLeu 80 Different from PEG 113 -b-(PBLG 20 -co-PMET 80 Its characteristic is that the material does not contain ROS-responsive sulfur atoms, and the rest is related to PEG. 113 -b-(PBLG 20 -co-PMET 80 The results are completely consistent. Therefore, this material was used as a non-responsive ROS carrier, and the nanodelivery system formed after loading the two drugs had no ROS responsiveness, thus serving as a control group compared with the ROS responsive delivery system.

[0091] Example 2: Synergistic effect of lenvatinib and gefitinib combination on Huh-7 and Hep3B cells

[0092] Synergistic effect analysis of lenvatinib (hereinafter referred to as Len) and gefitinib (hereinafter referred to as Gef) on Huh-7 and Hep3B liver cancer cells. Taking the synergistic analysis of lenvatinib and gefitinib combination on Huh-7 cells as an example, Huh-7 cells (5 × 10⁻⁶ cells) were prepared the night before. 3Cells were evenly seeded in 96-well plates and allowed to reach 80% confluence before use in experiments. Lenvatinib (2.5 mg, 0.0059 mmol) and gefitinib (10.5 mg, 0.023 mmol) were weighed and dissolved in DMSO (150 μL). The solutions were then diluted three times sequentially with DMEM (FD) cell culture medium containing 10% fetal bovine serum, each time a 10-fold dilution, to obtain a mixed solution of 40 μM lenvatinib and 160 μM gefitinib. The pre-set concentrations of lenvatinib in the mixed drug solution were 0, 1.25, 2.5, 5, 10, and 20 μM; the concentrations of gefitinib in the mixed drug solution were 0, 5, 10, 20, 40, and 80 μM. Taking a mixed solution of 20 μM lenvatinib and 80 μM gefitinib as an example, 200 μL of 40 μM lenvatinib and 160 μM gefitinib solutions need to be mixed. Other drug solutions of different concentrations are obtained using the same algorithm. Then, 100 μL of each dual-drug solution is added to a 96-well plate (n = 3), with one well reserved for a control group containing the same volume of PBS. The plates are incubated for 48 h. After incubation, 100 μL of LTT / FD (1:4, v / v) solution is added and incubated for another 4 h. The supernatant is carefully aspirated, and 200 μL of DMSO is added to dissolve the formazan crystals. After complete dissolution, the absorbance at 570 nm is measured using a microplate reader. Using the control group as a 100% reference, the cell viability of other groups is calculated, and the average value of each group is taken. The data is imported into Synergy Finder 3.0 to obtain the synergistic relationship between the two drugs.

[0093] The synergistic test of the combination of lenvatinib and gefitinib on Hep3B cells was consistent with the test of the combination of lenvatinib and gefitinib on Huh-7 cells in Example 2 above, except that Huh-7 cells were replaced with Hep3B cells and the culture medium was changed to MEM medium.

[0094] See Figure 7 In the ZIP model analysis using Synergy Finder 3.0, a score less than 0 was considered antagonistic; a score of 0 was considered additive; and a score greater than 10 was considered synergistic. Experiments demonstrated that the combination of lenvatinib and gefitinib exhibited significant synergistic effects in both Huh-7 and Hep3B cells, proving the feasibility of dual-drug therapy. Further observation revealed significant differences in the degree of synergy among different drug ratios, indicating the existence of an optimal drug ratio that can significantly enhance the inhibition of liver cancer cell proliferation. This can be used to further optimize the dosing regimen for a more efficient and precise liver cancer treatment strategy.

[0095] Example 3: Test on the inhibitory effect of different proportions of free drug on liver cancer cells

[0096] Inhibitory effects of different ratios of free drugs on liver cancer cells. Taking the Len:Gef (1:4, mol / mol) drug combination treatment of liver cancer cells as an example, Huh-7 liver cancer cells (5 × 10⁻⁶) were treated one night in advance. 3 Cells were evenly seeded in 96-well plates until 80% confluence was achieved. Lenvatinib (0.25 mg, 0.00059 mmol) and gefitinib (1.05 mg, 0.0023 mmol) were weighed and dissolved in DMSO (15 μL). The solutions were then diluted three times sequentially with FD (diluted 10-fold each time) to obtain a mixed solution of 40 μM lenvatinib and 160 μM gefitinib. This solution was then diluted halved eight times to obtain nine gradient concentration solutions. Each solution was then added to a 96-well plate (100 μL) using a multipipe (n = 6). One well was reserved for a control group containing the same volume of PBS. The plates were incubated for 48 h. After incubation, 100 μL of MTT / FD (1:4, v / v) mixed solution was added and incubated for 4 h. The supernatant was carefully aspirated and DMSO was added to dissolve the formazan crystals. After complete dissolution, the absorbance at 570 nm was measured using an ELISA reader. The inhibitory effect of the drug on Huh-7 cells was analyzed based on the results.

[0097] The inhibitory effects of other free drug combinations in different proportions on liver cancer cells were tested using the same method as in Example 3 above, except that lenvatinib and gefitinib were weighed according to the following Len:Gef molar ratios of 1:0.5, 1:1, 1:2 and 1:6 for testing.

[0098] See Figure 8 Cell survival data from different groups were imported into Prism software to obtain inhibition curves of Huh-7 cells for different drug combinations. Using a 50% cell survival rate as the evaluation criterion, with a fixed lenvatinib ratio, the concentration of lenvatinib required to kill 50% of tumor cells decreased as the proportion of gefitinib increased, until the molar ratio reached 1:4 and 1:6. At this point, the inhibitory effects of the two drug combinations on liver cancer cells were similar, and further increasing the drug ratio no longer significantly increased the inhibitory effect on cells. Because the 1:4 drug combination requires less drug and has fewer toxic side effects while maintaining the inhibitory effect on liver cancer cells compared to the 1:6 ratio, the optimal molar ratio of lenvatinib to gefitinib was initially determined to be 1:4, and this ratio was used in subsequent experiments.

[0099] Example 4: Preparation method of dual-drug co-loaded nanomedicine

[0100] PEG prepared in Example 1 113 -b-(PBLG 20-co-PMET 80 Taking Len:Gef (1:4, mol / mol) loading as an example, weigh out the PEG prepared in Example 1. 113 -b-(PBLG 20 -co-PMET 80 The material was used as a dual-drug loading carrier (5 mg), lenvatinib (0.25 mg, 0.00059 mmol), and gefitinib (1.05 mg, 0.0023 mmol), all of which were completely dissolved in DMF solvent (200 μL). This solution was then added dropwise to stirred water (4 mL) for 30 min. The dispersed solution was then transferred to a dialysis bag with a molecular weight cutoff of 3500 Da for dialysis, with water changed every 4 hours for a total of 3-5 times. After removing free drug and DMF, the dual-drug co-loaded nanomedicine solution was obtained. For specific preparation methods, please refer to [link to preparation method]. Figure 9 .

[0101] The preparation method for dual-drug co-loaded nanomedicines using nanomaterials with different monomer ratios for dual-drug synergistic delivery is the same as in Example 4 above, except that the dual-drug synergistic delivery nanomaterial PEG is used. 113 -b-(PBLG 20 -co-PMET 80 Replace with the following material: PEG 113 -b-(PBLG 50 -co-PMET 50 PEG 113 -b-(PBLG 30 -co-PMET 70 PEG 113 -b-(PBLG 10 -co-PMET 90 PEG 113 -b-PMET 100 .

[0102] The solvent selection method in the preparation of dual-drug co-loaded nanomedicines is the same as in Example 4, except that the organic solvent DMF is replaced with dimethyl sulfoxide. N , N -Dimethylacetamide, N One of the methylpyrrolidones.

[0103] Comparative Example 3: Preparation method of single-drug loaded nanomedicine

[0104] Weigh 5 mg of the PEG prepared in Example 1 113 -b-(PBLG 20 -co-PMET 80The material was used as a carrier, and 1.25 mg of lenvatinib was dissolved in DMF (200 μL). The solution was then added dropwise to stirred water (4 mL) for 30 min. The dispersed solution was transferred to a dialysis bag (3500 Da) to remove the free drug and DMF, thus obtaining the single-drug nanoparticle.

[0105] Weigh out 5 mg of PEG 113 -b-(PBLG 20 -co-PMET 80 1.25 mg of gefitinib was completely dissolved in DMF (200 μL), and then added dropwise to stirred water (4 mL) for 30 min. The dispersed solution was transferred to a dialysis bag (3500 Da) to remove the free drug and DMF, thus obtaining the single-drug nanoparticle.

[0106] See Figure 10 Highly efficient loading of two drugs can be achieved using nanoprecipitation. However, in Comparative Example 3, when two single drugs were loaded separately, a large amount of drugs precipitated during the nanoprecipitation stage, resulting in a low loading rate. Therefore, the two drugs may interact when dispersed in water, inhibiting the crystallization and aggregation of drug molecules and providing conditions for loading the hydrophobic region of the material. Comparative experiments further demonstrate that this invention innovatively achieves dual drug loading through a simple nanoprecipitation method.

[0107] PEG nanomaterials for dual-drug synergistic delivery with different monomer ratios 113 -b-(PBLG x -co-PMET y Preparation of dual-drug co-loaded nanomedicines with particle size and PEG 113 -b-(PBLG 20 -co-PMET 80 The prepared dual-drug co-loaded nanomedicines are close.

[0108] Experimental Example 3: PEG Nanomaterial for Dual Drug Synergistic Delivery 113 -b-(PBLG 20 -co-PMET 80 Difference in particle size between single-drug and dual-drug loaded nanoparticles

[0109] Different nanomedicines, namely PEG, were obtained using the methods described in "Example 4" and "Comparative Example 3". 113 -b-(PBLG 20 -co-PMET 80Two single drugs and a 1:4 ratio of two drugs were loaded onto the carrier. The aqueous solution of the carrier was prepared in the same way, except that no drug was added during the nanoprecipitation process. The DMF solution of the carrier was directly added to the water. After the purification steps were the same to obtain the nanodrug solution, the concentration of the obtained solution was uniformly 1 mg / mL, and 1 mL was taken for DLS particle size test.

[0110] See Figure 10 The PEG prepared in Example 1 can be obtained from DLS data. 113 -b-(PBLG 20 -co-PMET 80 The material used as a drug carrier forms micelles with a size of 90-100 nm. After loading lenvatinib or gefitinib monotherapy, a large amount of micron-sized drug precipitates appeared, so monotherapy was almost impossible to load. However, after loading lenvatinib and gefitinib dual therapy (Example 4), the obtained drug-loaded nanoparticles were more uniform in size and there were no micron-sized precipitates.

[0111] Therefore, the combined use of the two drugs not only has a synergistic effect in inhibiting the proliferation of liver cancer cells, but also innovatively solves the problem of the difficulty in loading lenvatinib or gefitinib monotherapy via nanoprecipitation. Furthermore, the results also indicate that there is some interaction between the two drugs during the loading of the dual-drug delivery nanomaterial prepared in Example 1, which promotes the smooth progress of the nanoprecipitation process.

[0112] Example 5: PEG nanomaterial for dual-drug synergistic delivery 113 -b-(PBLG 20 -co-PMET 80 The particle size distribution of drug-loaded nanoparticles after loading different proportions of dual drugs.

[0113] The preparation method for dual-drug co-loaded nanomedicines with different drug combinations is the same as in Example 4 above, except that the Len:Gef (1:4, mol / mol) drug combination is replaced with the following molar ratios: 1:0.5, 1:1, 1:2, and 1:6 to obtain dual-drug co-loaded nanomedicines with different ratios. The concentration of the resulting solutions is uniformly set to 1 mg / mL, and 1 mL is taken for DLS particle size analysis.

[0114] See Figure 11DLS data showed that when the molar ratio of lenvatinib to gefitinib was 1:1, 1:2, and 1:4, the nanomedicine exhibited a unimodal distribution, uniform particle size, and good loading effect; however, at ratios of 1:0.5 and 1:6, a multimodal distribution appeared, indicating that the nanosystem was not homogeneous. These results indicate that the two drugs can only form a stable complex within a specific ratio range (1:1 to 1:4), thus constructing a stable drug-loaded nanosystem. More importantly, during the construction of the drug-loaded system, it was found that the carrier could not effectively load either lenvatinib or gefitinib alone, but could simultaneously load both drugs at a specific ratio and exhibit excellent stability. This phenomenon suggests that there may be a specific intrinsic mechanism for synergistic loading of the two drugs.

[0115] Experimental Example 4: Particle size changes after different proportions of free mono / dual drugs are dispersed in water

[0116] Weigh out 5 mg of lenvatinib and gefitinib respectively and dissolve them in DMF to prepare a 1 mg / mL mixed drug solution. Then, take 50 μL of each solution and mix them in an EP (Eppendorf) tube, and slowly add the mixture dropwise to water (1 mL) for DLS testing.

[0117] Weigh 5 mg of lenvatinib and dissolve it in DMF to prepare a 0.5 mg / mL drug solution. Then, slowly add 50 μL of the solution to 1 mL of water for DLS testing.

[0118] Weigh 5 mg of gefitinib and dissolve it in DMF to prepare a 0.5 mg / mL drug solution. Then, slowly add 50 μL of the solution dropwise to 1 mL of water for DLS testing.

[0119] from Figure 12 As shown in the single-drug particle size distribution spectra (a) and (b) in the figure, the particle sizes of both free lenvatinib and gefitinib changed significantly after being dispersed in water, and a large amount of precipitate gradually formed during the experiment. In contrast, from... Figure 12 As shown in the particle size distribution spectra (c) and (d) of the dual-drug mixture, the particle size distribution of the dual drugs ranges from 100 to 1000 nm, and the solution remains clear after the initial dispersion. After 24 hours, the particle size of the free dual drugs mostly stabilizes in the range of 300 to 500 nm. This demonstrates the formation of dual-drug molecular clusters, and that these clusters possess a certain degree of stability, which is beneficial for subsequent drug loading. This is the key to the unique loading advantage of this invention.

[0120] Example 6: Evaluation of the responsiveness of the methionine unit

[0121] The PEG prepared in Example 1 113 -b-PMET 100(5 mg) of material was dispersed in water (4 mL) using a nanoprecipitation method. Subsequently, different amounts of hydrogen peroxide (0 mM, 1 mM, and 10 mM) were added according to the final concentration of hydrogen peroxide (H2O2), and the mixture was heated at 37°C. o The reaction was stirred overnight at C, followed by dialysis using a dialysis bag with a molecular weight cutoff of 3500 Da. The water was changed every 4 hours for a total of 3-5 times. After dialysis, the mixture was freeze-dried, dissolved in D2O, and subjected to NMR analysis.

[0122] See Figure 13 Analysis (a) shows that after methionine is oxidized by peroxide, the thioether group is converted into a sulfoxide group. See also... Figure 13 Analysis (b) shows that the methyl hydrogen atom in the methionine residue underwent a significant chemical shift (from 2.13 ppm to 2.75 ppm) after incubation with different concentrations of hydrogen peroxide. This indicates that even under low concentrations of hydrogen peroxide (1 mM), the material can achieve a hydrophobic-to-hydrophilic transition, thereby causing micelle disintegration and releasing the drug.

[0123] Example 7: Drug release of Len:Gef@PEM under different conditions

[0124] Taking a hydrogen peroxide concentration of 1 mM as an example, after obtaining the nanomedicine according to the method in "Example 4", 5 mL of the nanomedicine solution was added to a dialysis bag with a molecular weight cutoff of 3500 Da, while maintaining the hydrogen peroxide concentration in the dialysis bag at 1 mM. The two ends were tied tightly with thread to prevent drug leakage. The dialysis bag was carefully placed in 25 mL of a 1 mM DMF / H2O (1:9, v / v) mixed solvent and placed at 37°C. o Gently shake the solution in a shaker at temperature C. Take 1 mL of the solution at different time points, and immediately replenish with 1 mM DMF / H2O solvent. Perform HPLC analysis on the taken solutions and calculate the cumulative drug release rate at a series of time points to obtain the drug release under the condition of 1 mM hydrogen peroxide concentration.

[0125] Drug release tests under other different conditions were all performed using the same method.

[0126] See Figure 14 The release curve of lenvatinib in (a) and Figure 14Analysis of the gefitinib release curve (b) shows that, under simulated normal physiological conditions (close to 0 mM H2O2), the cumulative release rate of the two drugs in the dual-drug co-loaded nanomedicine prepared in Example 4 of this invention was approximately 20% within 48 hours, indicating minimal drug leakage in normal tissues and thus reducing its toxic side effects. However, in a release medium containing 10 mM H2O2 (simulating ROS levels in the tumor microenvironment), the cumulative drug release rate reached over 70% within 48 hours, exhibiting typical ROS-responsive release characteristics. In summary, this nanomedicine can achieve responsive drug release under high ROS conditions simulating the hepatocellular carcinoma microenvironment, and is expected to effectively reduce toxic side effects on normal tissues.

[0127] Example 8: Drug loading effect of different dual-drug synergistic delivery nanomaterials loaded with dual drugs

[0128] PEG prepared in Example 1 113 -b-(PBLG 20 -co-PMET 80 Taking a Len:Gef (1:4, mol / mol) material loading as an example, the nanodrug solution obtained by the last method in "Example 4" was mixed with methanol (1:4, v / v). 200 μL of the solution was added to 800 μL of methanol, allowing the micelles to disintegrate in the organic solvent and release the loaded drug for UV testing. Data in the 500–200 nm band were obtained. After subtracting the baseline and the material's own absorbance, the concentrations of lenvatinib and gefitinib were calculated, thus obtaining the actual drug loading and efficiency.

[0129] The concentration UV tests for the remaining drugs with different loading ratios were all performed using the same method.

[0130] The drug loading efficiency and drug loading capacity of the dual-drug synergistic delivery nanomaterials prepared in Example 1, loaded with lenvatinib and gefitinib according to the method in Example 4, are shown in Table 2:

[0131] Table 2. Results of drug loading efficiency and drug loading capacity characterization tests for lenvatinib and gefitinib.

[0132]

[0133] As shown in Table 2, the drug loading efficiency and drug loading data reveal that, with... L The decrease in γ-glutamic acid benzyl ester (E) content and the increase in methionine (M) content led to a decline in the drug loading efficiency of both drugs. This is because... L The benzene ring structure of γ-glutamic acid benzyl ester exhibits both hydrophobic and π-π stacking interactions with the drug, and a reduction in the benzene ring of the carrier is detrimental to drug loading. Specifically, for lenvatinib, when L-Glutamic acid-γ-benzyl ester has a polymerization degree from 50 (E) 50 -M 50 ) decreased to 20 (E 20 -M 80 When the drug loading efficiency decreased from 66.8% to 62.4%, and the drug loading amount decreased from 3.2% to 2.8%, the decrease was relatively small; however, for gefitinib, the drug loading efficiency decreased from 73.1% to 64.5% and the drug loading amount decreased from 11.9% to 10.7% with the same proportional change, the decrease was more significant. L The degree of polymerization of γ-glutamic acid benzyl ester further decreased to 10 (E). 10 -M 90 ) or completely remove (M 100 When lenvatinib was used, the drug loading efficiency of both drugs decreased to approximately 52.7-58.7%, and the drug loading reached its lowest level (lenvatinib 2.2%, gefitinib 9.0-9.1%), indicating that... L The content of γ-glutamic acid benzyl ester is positively correlated with the drug loading effect.

[0134] It is worth noting that when L The degree of polymerization of -glutamic acid-γ-benzyl ester is 20 (i.e., E). 20 -M 80 At the same time, the drug loading efficiency remained at a high level: lenvatinib had a drug loading efficiency of 62.4% and a drug loading amount of 2.8%; gefitinib had a drug loading efficiency of 64.5% and a drug loading amount of 10.7%. Considering the requirement of methionine as a response unit, L The degree of polymerization of γ-glutamic acid benzyl ester is not necessarily better the higher it is; a balance needs to be struck between drug loading capacity and responsiveness. (Compared to E...) 50 -M 50 In comparison, E 20 -M 80 The drug loading efficiency of lenvatinib decreased by only about 6.6%, gefitinib by about 11.8%, while the degree of polymerization of methionine increased by 60, which will significantly improve the material's oxidative responsiveness. Therefore, E 20 -M 80 It is an optimal ratio that balances drug loading performance and responsiveness.

[0135] Example 9: Drug loading effect of different proportions of drug combinations in dual-drug synergistic delivery nanomaterials

[0136] This embodiment is the same as Example 8, except that it uses the PEG prepared in Example 1. 113 -b-(PBLG 20 -co-PMET 80 The materials were loaded with Len:Gef in the following different molar ratios (mol / mol): 1:0.5, 1:1, 1:2, 1:4 and 1:6.

[0137] Table 3 shows the characterization results of drug loading efficiency and drug loading amount of nanomaterials loaded with different proportions of lenvatinib and gefitinib for dual-drug synergistic delivery:

[0138] Table 3. Characterization of drug loading efficiency and drug loading amount of lenvatinib and gefitinib with different loading ratios.

[0139]

[0140] Table 3 shows the drug loading efficiency and amount of lenvatinib and gefitinib loaded at different ratios. Lenvatinib's drug loading efficiency initially increased and then decreased with increasing gefitinib ratio, reaching its highest value (64.8%) at a 1:4 ratio. Gefitinib's drug loading efficiency showed a similar trend (55.2%→65.9%→49.1%), and the drug loading amount also conformed to this result. The combined drug loading effect of the two drugs initially increased and then decreased with increasing ratio. From a 1:0.5 ratio to 1:2, the combined drug loading reached 13.9%, then gradually decreased. At a 1:4 ratio, the combined drug loading still reached 13.5%, providing sufficient drug loading for subsequent cell and animal experiments.

[0141] However, when the drug loading ratio is 1:0.5 and 1:6, the drug loading effect is significantly worse than the intermediate ratio. Especially at a drug loading ratio of 1:6, the ratio of lenvatinib to gefitinib deviates too much from the preset ratio. When the loading ratio is 1:4, the dual-drug loading ratio is close to the preset ratio, which is better than the 1:6 design. A possible explanation for this phenomenon is that the dual drugs can form stable "molecular clusters" within a certain ratio combination, but when the proportion of one drug is too high, the excess drug cannot stably form "molecular clusters," thus drug crystallization is detrimental to loading. This result is consistent with the drug loading mechanism explored in this invention. Based on the above analysis, subsequent experiments will use a loading ratio of 1:4 for testing.

[0142] Experimental Example 5: Ultraviolet spectra of nanomaterials loaded with different proportions of drugs for dual-drug synergistic delivery

[0143] The 500~200 nm band data obtained from Example 9 were imported into Origin software to obtain a series of spectra.

[0144] See Figure 15 The gray dashed line represents the UV absorption of the material itself. There is no signal at the gefitinib absorption peak at 330 nm, but there is partial absorption at the lenvatinib absorption peak at 245 nm. The material's absorbance value should be subtracted when calculating drug concentration. Furthermore, the overall spectrum shows that when the loading ratio is 1:4, both drugs exhibit strong absorption signals, directly demonstrating the successful loading of both drugs.

[0145] Example 10: Hemolysis experiment of free drug, dual-drug synergistic delivery nanomaterials, and dual-drug co-loaded nanomedicines.

[0146] The in vivo biocompatibility of the copolymer amino acids was further evaluated using a blood compatibility test. First, blood was collected from healthy mice (Cavens: BALB / c-Nude nude mice SPF grade, 7-8 weeks old) via orbital sampling in anticoagulated vessels. o Centrifuge at 3000 rpm for 10 min at C20°C to remove the supernatant serum. Dissolve and wash the erythrocyte pellet with PBS, then centrifuge again until the supernatant is colorless. Resuspend the pellet to form a 2% erythrocyte suspension. Mix the carrier aqueous solution, carrier-loaded dual-drug solution (0.1 mg / mL), free dual-drug solution, H2O (positive control, PC), and PBS (negative control, NC) with an equal volume of erythrocyte suspension and incubate at 37°C. o Incubate at C for 4 h. After centrifugation, collect the supernatant and measure its UV absorbance (λ = 570 nm). The hemolysis rate is calculated according to the following formula:

[0147]

[0148] Among them, A S A PC and A NC The absorbance values ​​at λ = 570 nm represent the absorbance of the copolymerized amino acid sample, the positive control, and the negative control, respectively.

[0149] See Figure 16 In the hemolysis experiments of different groups, the hemolysis rate of both the material itself and the drug-loaded nanomedicine Len&GEf@PEM (the dual-drug co-loaded nanomedicine prepared in Example 4 with Len:Gef = 1:4) was less than 1%, demonstrating good biocompatibility. In contrast, the hemolysis rate of the free drug group was close to 50%, confirming the toxic side effects of the free drug. Therefore, the dual-drug co-loaded nanomedicine prepared in Example 4 can effectively avoid the biotoxic interference caused by the free drug, providing a safety guarantee for subsequent animal experiments.

[0150] Example 11: Inhibitory effect of different dual-drug synergistic delivery nanomaterials loaded with dual drugs on liver cancer cells

[0151] PEG prepared in Example 4 113 -b-(PBLG 20 -co-PMET 80 Taking the treatment of liver cancer cells with a dual-drug co-loaded nanomedicine obtained by loading Len:Gef (1:4, mol / mol) onto the material as an example, the liver cancer cells Huh-7 cells (5×10⁶ cells) were treated with the dual-drug co-loaded nanomedicine the night before. 3Cells were evenly seeded in 96-well plates until the cell confluence reached 70-80%. Drug loading was performed using the method described in "Example 4," by mixing the solution with methanol (1:4, v / v) and measuring the drug concentration using a UV spectrophotometer. The solution was diluted with cell culture medium to a mixture of 40 μM lenvatinib and 160 μM gefitinib, and then halved eight times. The resulting series of solutions were added to 96-well plates (100 μL each) using a multi-channel pipette and incubated for 48 h. After incubation, 100 μL of MTT / FD (1:4, v / v) solution was added and incubated for another 4 h. The supernatant was carefully aspirated, and DMSO was added to dissolve the formazan. After complete dissolution, the absorbance at 570 nm was measured using a microplate reader. The inhibitory effect of the nanomedicine on Huh-7 cells was analyzed based on the results.

[0152] The inhibitory effects of other dual-drug synergistic delivery nanomaterials loaded with dual drugs on liver cancer cells were all tested using the same method.

[0153] See Figure 17 Under the premise of the same drug dosage, the inhibitory effects of nanomedicines formed by carriers with different degrees of methionine polymerization on liver cancer cells were compared. The results showed that as the degree of polymerization of the methionine unit increased, the antitumor activity of the nanomedicine gradually increased; however, when PEG... 113 -b-(PBLG x -co-PMET y After the degree of polymerization of methionine in the PEGylated nanoparticles reached 80%, further increasing its proportion resulted in a plateauing of the inhibitory effect. Considering the drug-loading properties of each material and its in vitro antitumor effect after forming nanomedicines, PEG was ultimately selected. 113 -b-(PBLG 20 -co-PMET 80 It serves as the optimal drug carrier for subsequent animal treatment experiments.

[0154] Example 12: Comparison of the uptake capacity of liver cancer cells by different dual-drug synergistic delivery nanomaterials.

[0155] PEG, the material prepared in Example 1 113 -b-(PBLG 20 -co-PMET 80 Taking the uptake experiment of Huh-7 cells as an example, Huh-7 cells were resuspended in fresh DMEM medium and incubated at a ratio of 2 × 10⁶ cells per well. 5 Cells were evenly seeded at a density of 1,000 cells into 12-well plates. The 12-well plates were then placed in a 5% CO2, 37°C environment. o Incubate overnight in a cell culture incubator at C. After ensuring full cell adhesion and acclimatization, replace the culture medium with fresh DMEM (0.01 mg / mL) containing Cy5-labeled material of the present invention, and incubate at 37°C.o Cells were incubated at C for 4 h. After washing three times with cold PBS, cells were collected and their uptake efficiency was analyzed by flow cytometry. The mean fluorescence intensity (MFI) of the cells was calculated using FlowJo software.

[0156] The uptake experiments of other different carrier materials were conducted using the same method.

[0157] See Figure 18 Average fluorescence intensity data show that PEG 113 -b-(PBLG 20 -co-PMET 80 The material exhibited excellent uptake efficiency in both types of liver cancer cells, and the cellular uptake gradually decreased with increasing methionine polymerization degree. 20 -M 80 The group achieved an optimal balance between cellular uptake efficiency and ROS responsiveness—the material retains sufficient aromatic ring structure to ensure efficient cellular uptake while containing adequate methionine for rapid ROS response and drug release. In contrast, E 10 -M 90 and M 100 Although the methionine content exceeds 90%, the low aromatic ring content significantly reduces the cells' ability to take up the nanomedicine formed from it, thus weakening its killing effect on tumor cells. Therefore, PEG... 113 -b-(PBLG 20 -co-PMET 80 The material is the most excellent candidate drug carrier.

[0158] Example 13: Hepatocellular carcinoma cells' response to PEG 113 -b-(PBLG 20 -co-PMET 80 Material uptake analysis

[0159] Taking the uptake behavior analysis of Huh-7 cells as an example, Huh-7 cells were resuspended in fresh DMEM medium and grown at a ratio of 2 × 10⁶ cells per well. 5 Cells were evenly seeded at a density of 1,000 cells into 12-well plates. The 12-well plates were then placed in a 5% CO2, 37°C environment. o Incubate overnight in a cell culture incubator at C. After ensuring full cell adhesion and acclimatization, replace the culture medium with fresh DMEM (0.01 mg / mL) containing Cy5-labeled copolyamino acids, and incubate at 37°C. oCells were incubated at C for 4 h. After washing three times with cold PBS, cells were collected and their uptake efficiency was analyzed by flow cytometry. Cells were incubated with the copolyamino acid material and then stained with Hoechst 33342 (5 μg / mL, nuclear dye) for 15 min and LysoTracker Green (200 nM, endosome / lysosomal dye) for 30 min, respectively. Cells were washed three more times with cold PBS to remove free dye, then added to fresh DMEM medium and subjected to CLSM imaging analysis.

[0160] See Figure 19 Analysis of the CLSM images revealed a high degree of overlap between the red signals of Cy5-labeled copolyamino acids and the green signals of lysosomes, indicating that the material was endocytosed by lysosomes after entering the cell, demonstrating a good uptake effect. Furthermore, the majority of the material was encapsulated within lysosomes, confirming the release of the drug after entering the cell. Specifically, upon entering the lysosome, the high concentration of ROS oxidizes the methionine residues, converting them from hydrophobic to hydrophilic, causing micelle disintegration and promoting drug release into the cytoplasm to kill liver cancer cells.

[0161] Example 14: Inhibitory effect of nanomaterials loaded with different proportions of drugs on liver cancer cells in a dual-drug synergistic delivery system

[0162] Taking the treatment of liver cancer cells with Len:Gef (1:4, mol / mol) as an example, the liver cancer cells Huh-7 cells (5 × 10⁻⁶) were loaded with Len:Gef (1:4, mol / mol) the night before. 3 Cells were evenly seeded in 96-well plates until the cell confluence reached 70-80%. Drug loading was performed using the method described in "Example 4," by mixing the solution with methanol (1:4, v / v) and measuring the drug concentration using a UV spectrophotometer. The solution was diluted with cell culture medium to a mixture of 40 μM lenvatinib and 160 μM gefitinib, and then halved eight times. The resulting series of solutions were added to 96-well plates (100 μL each) using a multi-channel pipette and incubated for 48 h. After incubation, 100 μL of MTT / FD (1:4, v / v) solution was added and incubated for another 4 h. The supernatant was carefully aspirated, and DMSO was added to dissolve the formazan crystals. After complete dissolution, the absorbance at 570 nm was measured using a microplate reader. The inhibitory effect of the nanomedicine on Huh-7 cells was analyzed based on the results.

[0163] The inhibitory effects of other drugs with different loading ratios on liver cancer cells were all tested using the same method.

[0164] See Figure 20Analysis of the inhibition curves shows that the inhibitory trend of the materials loaded with different proportions of drugs on liver cancer cells is basically consistent with the previous pattern of free drugs: the experimental groups loaded with drugs all showed a concentration-dependent inhibitory effect on cells, but the inhibitory effect on cells tended to level off when the loading ratio exceeded 1:4.

[0165] Therefore, the PEG prepared in Example 1 113 -b-(PBLG 20 -co-PMET 80 The optimal carrier was lenvatinib, and the optimal loading ratio of lenvatinib to gefitinib was 1:4 (mol / mol) for subsequent cell and animal experiments.

[0166] Example 15: Therapeutic effect of liver cancer in mice

[0167] Clinical studies have shown that lenvatinib and gefitinib are rapidly cleared from the bloodstream and randomly distributed in various tissues and organs, resulting in a relatively large dose required for actual treatment and less than ideal efficacy.

[0168] The in vitro experimental data in Example 11 verified the potential antitumor activity of the dual-drug delivery system against hepatocellular carcinoma cells. To further investigate the in vivo antitumor activity of the dual-drug delivery system, a nude mouse subcutaneous hepatocellular carcinoma model was constructed, and the inhibitory effects of PBS (control group), Len:Gef (free drug group), Len:Gef@PEnL (non-responsive drug loading group), and Len:Gef@PEM (responsive drug loading group) on hepatocellular carcinoma were compared and analyzed.

[0169] See Figure 21 In the analysis in (a), Huh-7 cells were subcutaneously inoculated into nude mice lacking a thymus, and the tumor volume grew to 120 mm after approximately four weeks. 3 Mice were divided into groups for treatment. The different experimental groups received lenvatinib (5 mg / kg): gefitinib (21 mg / kg) (1:4, mol / mol) via tail vein injection, administered every two days for the first two weeks and every three days for the following week (for a total of nine injections). The control group received the same volume of PBS. See also Figure 21 In analysis (b) above, tumor tissue was removed and photographed after treatment, resulting in images of tumors in each group of mice. The images show that the Len:Gef@PEnL and Len:Gef@PEM groups exhibited superior tumor growth inhibition compared to the Len:Gef and PBS control groups. In addition, the tumor tissue weight of each experimental group was measured after treatment. See [link to article]. Figure 21Analysis (c) showed that, with the PBS control group as a reference, tumor weight decreased by 38.6%, 67.7%, and 82.5% in the Len:Gef, Len:Gef@PEnL, and Len:Gef@PEM treatment groups, respectively. Tumor volume changes during treatment were also monitored; see [link to relevant documentation]. Figure 21 In the (d) analysis, the tumors in the PBS control group grew very rapidly, with the average tumor volume increasing 8.4 times compared to the start of treatment after day 21. In contrast, the average tumor volume in the Len:Gef, Len:Gef@PEnL, and Len:Gef@PEM treatment groups increased by 4.3, 2.2, and 1.4 times, respectively, with the Len:Gef@PEM treatment group showing the slowest tumor growth rate. Therefore, the combined treatment with these two drugs was quite effective, showing a significant inhibitory effect on tumor growth, but the tumors still maintained a large volume.

[0170] The most significant improvement was observed in the dual-drug delivery system. The Len:Gef@PEnL and Len:Gef@PEM treatment groups showed a substantial increase in tumor growth inhibition, indicating that PEGylated nanoparticles can passively target tumors via the EPR effect, prolonging drug retention time at the tumor site and thus enhancing therapeutic efficacy. Furthermore, the ROS-responsive Len:Gef@PEM group exhibited slightly stronger tumor inhibition than the non-ROS-responsive Len:Gef@PEnL group, suggesting that the responsive carrier releases the drug in response to tumor tissue, achieving targeted therapy. Its response mechanism is as follows: Figure 23 As shown, ROS in liver cancer tissue oxidizes methionine in the carrier, causing a hydrophilic-hydrophobic transition and promoting micelle disintegration to release the drug. Simultaneously, from... Figure 21 As shown in (e), the body weight of mice remained between 21 and 23 g during treatment, with no significant decreasing trend. Only the mice in the free drug group had slightly lower body weight, indicating that the free drug exhibited certain physiological toxicity after being taken up by normal tissues. In conclusion, Len:Gef@PEM nanomedicine demonstrated excellent efficacy and safety in animal experiments, and is of great significance for the treatment of hepatocellular carcinoma.

[0171] Example 16: Damage to the liver and kidneys

[0172] Mice treated in Example 15 were anesthetized with isoflurane and blood was collected. o The blood was left to stand overnight in a refrigerator at room temperature. The blood was centrifuged (1000 rpm, 10 min), and the supernatant serum was collected for testing liver markers, ALT (alanine aminotransferase) and AST (aspartate aminotransferase); and kidney markers, CRE (creatinine) and BUN (blood urea nitrogen). Experiments were performed according to the reagent kit requirements for AST, ALT, CRE, and BUN, and the values ​​for each marker were obtained.

[0173] See Figure 22 Analysis shows that free drugs can cause significant damage to normal tissues, especially metabolically active organs like the liver and kidneys. When these organs are damaged, the concentrations of creatinine, blood urea nitrogen, aspartate aminotransferase (AST), and aminotransferase (GT) in the blood increase. Furthermore, although the blood concentrations of creatinine, urea nitrogen, AST, and GT were within the normal range, the free drug group still caused a certain level of increase in these marker concentrations, indicating some physiological toxicity. This slight toxicity is related to the non-specific distribution of the free drug in the body, leading to a certain degree of increase in various assessment indicators. The nanomedicine constructed in this invention, due to the protection of a responsive carrier (i.e., a dual-drug coordinated delivery nanomaterial), specifically releases the drug at the tumor site while releasing less drug in normal tissues. Therefore, it reduces the toxicity of the free drug while ensuring therapeutic efficacy, which is of great significance for the treatment of hepatocellular carcinoma.

Claims

1. A dual-drug synergistic delivery nanomaterial, characterized in that, The nanomaterial is a polyethylene glycol-polyamino acid block copolymer, and the block copolymer has the structure shown in formula (I): PEG m -b-(CLOSED x -co-PBB y ) (I), Among them, PEG m This indicates polyethylene glycol segments with a number-average molecular weight of 2000 Da, 3000 Da, 3400 Da, 5000 Da, 6000 Da, 8000 Da, or 10000 Da, where m is the degree of polymerization of polyethylene glycol, m = 45, 68, 77, 113, 136, 182, or 227; PAA x This represents a poly(amino acid A) segment with a degree of polymerization of x, wherein the amino acid A is selected from... L -Glutamic acid-γ-benzyl ester, L -Phenylalanine, L -Tyrosine or L - Tryptophan or any derivative of the above amino acids; PBB y This represents a poly(amino acid B) segment with a degree of polymerization of y, wherein the amino acid B is selected from... L - Methionine or any of its amino acid derivatives; co indicates random copolymerization; b indicates that the PEG segment and the [PAA-co-PBB] segment are block-linked; x takes a value of 10-100, y takes a value of 50-100, and x:y is (1-5):(1-20), the total molecular weight of the copolymer is 10-30 kDa, and the dispersion is... It ranges from 1.05 to 1.

21.

2. The dual-drug synergistic delivery nanomaterial according to claim 1, characterized in that, The PEG m The term represents polyethylene glycol segments with a number-average molecular weight of 3400 Da, 5000 Da, or 6000 Da, where m is the degree of polymerization of polyethylene glycol, m = 68, 77, or 113; the PAA... x This represents a poly(amino acid A) segment with a degree of polymerization of x, wherein amino acid A is... L -Glutamic acid-γ-benzyl ester or a derivative thereof, wherein amino acid B is... L -Methionine; the x value is 10-50, the y value is 50-90, and the x:y ratio is (1-3):(1-10); the total molecular weight of the copolymer is 15-23 kDa, and the dispersity is... It is 1.05-1.

06.

3. A method for preparing the dual-drug synergistic delivery nanomaterial according to claim 1, characterized in that, Includes the following steps: (1) The amino acid, triphosgene, and epoxide are reacted in the first organic solvent to obtain a product containing... N - Crude product of carboxyl ring anhydride; (2) Take the product obtained in step (1) containing N The crude product of the carboxyl ring anhydride was directly dissolved in a second organic solvent, and trimethylacetic acid, pH buffer and initiator were added to form an oil-water biphase system for ring-opening polymerization; after purification, dual-drug synergistic delivery nanomaterials were obtained.

4. The method for preparing dual-drug synergistic delivery nanomaterials according to claim 3, characterized in that, The epoxide in step (1) is selected from any one of epichlorohydrin and epichlorohydrin; the first organic solvent is selected from any one of tetrahydrofuran, dioxane, diethyl ether or methyl tert-butyl ether; the second organic solvent in step (2) is selected from any one of chloroform, dichloromethane or 1,2-dichloroethane; the initiator is a methoxylated polyethylene glycolamine with a number average molecular weight of 2000 Da, 3000 Da, 3400 Da, 5000 Da, 6000 Da, 8000 Da or 10000 Da; the buffer solution is a buffer solution with a pH of 2-3.

5. The method for preparing dual-drug synergistic delivery nanomaterials according to claim 3, characterized in that, The molar ratio of trimethylacetic acid to initiator in step (2) is (10-60):(1-3); the volume ratio of the second organic solvent to pH buffer is (0.5-5):(0.5-3); the total reaction time of steps (1) and (2) is 3-5 h.

6. A dual-drug co-loaded nanomedicine based on the dual-drug synergistic delivery nanomaterial of claim 1, characterized in that, The two drugs mentioned are lenvatinib and gefitinib.

7. A method for preparing a dual-drug co-loaded nanomedicine as described in claim 6, characterized in that, Includes the following steps: (1) Lenvatinib, gefitinib and dual-drug synergistic delivery nanomaterials were dissolved in an organic solvent to form a mixed solution; (2) The mixed solution obtained in step (1) is added dropwise to water and stirred. After dialysis purification, nanomedicine is formed.

8. The method for preparing dual-drug synergistic delivery nanomaterials according to claim 7, characterized in that, In step (1), the mass ratio of the dual-drug synergistic delivery nanomaterial, lenvatinib, and gefitinib is (15-25):(1-5):(0.5-10); the organic solvent is selected from... N , N -Dimethylformamide, dimethyl sulfoxide, N , N -Dimethylacetamide, N Any of the following: -methylpyrrolidone.

9. The method for preparing dual-drug synergistic delivery nanomaterials according to claim 7, characterized in that, The stirring speed in step (2) is 300-800 rpm, and the dripping time is 20-40 min.

10. The use of the dual-drug synergistic delivery nanomaterial of claim 1 or the dual-drug co-loaded nanomedicine of claim 6 in the preparation of a drug for treating liver cancer.

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