An acid-responsive drug delivery nanoparticle for pancreatic cancer and a preparation method thereof

Abstract

The application belongs to the technical field of nano drug loading, and particularly relates to an acid response drug delivery nanoparticle suitable for pancreatic cancer and a preparation method. The polyphenol polymer is a polymer with a biodegradable polyethylene glycol (PEG) polyphenol as a skeleton, and then a metal ion chelation effect is used to load a hydrophobic drug, improved gemcitabine and leflunomide, to form a nanoparticle. The nanoparticle can release the improved gemcitabine and leflunomide in response to a special environment in tumor cells, can regulate mitochondrial metabolism of pancreatic tumor cells, induce cell ferroptosis, and at the same time, the nanoparticle can induce mitochondrial exocytosis to realize deep drug accumulation, regulate an immune-metabolic network of multiple cells in a tumor lesion tissue, relieve an immunosuppressive microenvironment, and inhibit pancreatic tumors.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine delivery technology, specifically relating to an acid-responsive drug delivery nanoparticle adapted for pancreatic cancer and its preparation method. Background Technology

[0002] Pancreatic cancer is considered one of the most dangerous and challenging tumor models in clinical practice, with a five-year survival rate of only 9%. Its high malignancy is primarily due to metabolic reprogramming of pancreatic tumor cells, meaning that multiple metabolic pathways within tumor cells undergo changes to meet their bioenergy and synthetic demands for excessive growth. This metabolic abnormality not only affects the tumor cells themselves but also gradually extends to the entire tumor microenvironment, ultimately leading to the formation of a dense matrix and the high expression of immunosuppressive cells in the pancreatic cancer microenvironment. Regulating tumor cell metabolism and improving the entire microenvironment through single-cell intervention is a promising therapeutic strategy for pancreatic cancer.

[0003] Mitochondria are the hub of cellular metabolism, participating in the biosynthesis of various amino acids, lipids, and nucleotides, and playing a vital role in providing basic energy and maintaining signal transduction. Furthermore, mitochondria play a crucial role in various cell death processes, including ferroptosis, apoptosis, and autophagy, and their function is essential for maintaining cellular viability. For tumor cells, mitochondria are key organelles coordinating their metabolic reprogramming, maintaining tumor cell viability by integrating multiple cellular signals, enabling them to adapt and grow in the dynamic and complex tumor microenvironment. Therefore, targeting tumor cell mitochondria is a promising therapeutic approach. Notably, when cells are stimulated, they release damaged mitochondria by forming migration bodies, achieving mitochondrial quality control—a process known as mitochondrial exocytosis. Previous studies have used induced mitochondrial exocytosis to facilitate the intercellular transport of nanomedicine delivery systems within the tumor microenvironment, promoting drug penetration deep into tumor sites and exerting sustained anti-tumor effects. This suggests that this phenomenon can be used to promote drug penetration into tissues, and mitochondrial exocytosis provides a potential method for penetrating dense tumor parenchyma. In addition, the mitochondrial metabolism of other cells in the tumor microenvironment also influences tumor progression. For example, interfering with the mitochondrial metabolism of CD8+ T cells can promote the transformation of effector T cells into memory T cells, thereby synergistically enhancing the efficacy of anti-tumor therapies. Therefore, in tumor therapies targeting mitochondrial metabolism, selecting targets that specifically interfere with the mitochondrial metabolism of tumor cells is crucial for killing tumor cells and activating anti-tumor immune responses.

[0004] Gemcitabine, a first-line chemotherapy drug for pancreatic cancer, primarily inhibits its activity by incorporating into the biosynthesis of tumor cells as a nucleoside analog. However, as treatment progresses, pancreatic tumor cells often develop resistance, and studies have shown that the pyrimidine biosynthesis pathway plays a crucial role in gemcitabine resistance. DHODH, a key enzyme in pyrimidine biosynthesis, plays two roles intracellularly: firstly, it can partially reverse tumor cell resistance to gemcitabine by regulating pyrimidine metabolism; secondly, DHODH can counteract the effects of ferroptosis inducers by reducing ubiquinone. In this application, leflunomide is used as a DHODH inhibitor. This is an isoxazole immunosuppressant with antiproliferative activity, which has shown promising potential in several anti-tumor clinical studies. However, the application of small molecule drugs in chemotherapy is limited by their physicochemical properties, resulting in insufficient solubility, stability, and targeting in vivo. Furthermore, due to metabolic reprogramming, pancreatic tumors exhibit a dense matrix, which further limits the ability of drugs to reach deep tumor tissues. Therefore, leveraging the unique size characteristics and functional diversity of nanomedicine delivery systems can help overcome the dense matrix barrier of pancreatic cancer, prolong the circulation time of therapeutic drugs, and achieve highly efficient pancreatic cancer treatment through targeted drug delivery. Although many studies have explored the application of various nanomedicine delivery systems in the treatment of pancreatic tumors, substantial progress has not been made in clinical practice, mainly due to issues such as complex manufacturing processes and biosafety of formulations. Therefore, there is an urgent need to develop a new drug delivery system that can not only deliver gemcitabine to deep tumor lesions but also combine it with other strategies to improve gemcitabine resistance. Summary of the Invention

[0005] Polyphenols are found in a variety of natural plants and have good biocompatibility. They can chelate metal ions to form nanoparticles by forming coordination bonds. At the same time, this binding is pH dependent and can dechelate in response to the acidic environment inside tumor cells. Therefore, they are superior carriers for delivering therapeutic drugs and achieving corresponding release from tumors.

[0006] Therefore, this application proposes a one-pot method for preparing polyphenol-iron chelate nanoparticles for the co-delivery of modified gemcitabine and leflunomide. The nanoparticle preparation process is simple and reproducible. Active targeting of the nanoparticles is achieved by modifying the nanoparticle surface with peptides that target EGFR, which is highly expressed in tumor cells. The nanoparticles can interfere with mitochondrial metabolism and promote deep delivery of nanomedicines through mitochondrial exocytosis, thereby synergistically inducing ferroptosis and mitophagy in tumor cells. By regulating tumor cell metabolism, this method holds promise for improving the immunosuppressive microenvironment of pancreatic cancer and enhancing the overall therapeutic effect of small molecule chemotherapeutic drugs.

[0007] The purpose of this invention is to provide a formulation design strategy based on the pathological characteristics and current clinical technology of pancreatic cancer, to achieve targeted tumor lesion release, enhanced drug penetration by inducing mitochondrial exocytosis, and regulation of tumor cell metabolism to enhance the therapeutic efficacy of tumor treatment. Specifically, this invention provides acid-responsive drug delivery nanoparticles adapted for pancreatic cancer and their preparation method. Due to the biocompatibility and multifunctionality of polyphenolic materials, this invention utilizes polyphenolic materials to chelate iron ions for drug delivery. This polymer can chelate iron ions to form nanoparticles, which are then encapsulated with modified gemcitabine and leflunomide through cavities. These nanoparticles can encapsulate small molecule drugs and have uniform particle size and spherical morphology. They exhibit the characteristic of responding to the acid response of tumor cells, releasing iron ions and drugs, and possessing superior tumor accumulation properties. Furthermore, these nanoparticles can induce mitochondrial exocytosis, promote deep penetration of nanoparticles, regulate metabolism to induce ferroptosis, alleviate gemcitabine resistance, and achieve tumor immunosuppression.

[0008] This invention, based on the characteristics of the pancreatic cancer microenvironment, utilizes the chelating properties of polyphenol nanoparticles with metal ions to form a cavity for drug delivery. This allows for deep penetration and drug release in response to the acidic intracellular environment of tumor cells. The polymer structure is designed as follows: a biodegradable polyethylene glycol (PEG) polyphenol polymer serves as the backbone, followed by the encapsulation of hydrophobic modified gemcitabine and leflunomide into nanoparticles using metal ion chelation. By combining the GE11 peptide, which highly expresses the epidermal growth factor receptor in tumor tissue, with the material, the targeting ability of the nanoparticles to pancreatic tumor tissue is enhanced. The nanoparticles remain stable in systemic circulation and target tumor tissue under the influence of the GE11 peptide. Inducing mitochondrial exocytosis in tumor cells promotes the penetration of nanoparticles into deep tumor tissue. Upon uptake into tumor cells, the polyphenol-iron chelated nanoparticles disintegrate, regulating mitochondrial metabolism in tumor cells. This regulation of the entire tumor microenvironment is achieved through the metabolic-immune network of the tumor microenvironment.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides an acid-responsive drug delivery nanoparticle adapted for pancreatic cancer, which is obtained by encapsulating iron ions with an amphiphilic polyphenol polymer and a DHODH inhibitor and a chemotherapeutic drug targeting pancreatic cancer. The polymer includes: an amphiphilic polymer one modified with polyphenols, and an amphiphilic polymer two modified with polyphenols and a targeting peptide at one end.

[0011] Preferably, the polymer macromolecular chain comprises, from one end to the other, a polyethylene glycol hydrophilic segment and a polylysine-polyphenol block.

[0012] Preferably, the chemotherapy drug for pancreatic cancer is a modified gemcitabine.

[0013] Preferably, the targeting peptide in polymer II is GE11.

[0014] Preferably, the DHODH inhibitor is leflunomide.

[0015] More preferably, the leflunomide loading is 7.31±0.32%, and the modified gemcitabine loading is 3.01±1.26%.

[0016] More preferably, the polymer one has the structural formula shown in Formula I; the polymer two has the structural formula shown in Formula II;

[0017]

[0018] Formula I

[0019]

[0020] Formula II

[0021] More preferably, the mass ratio of polymer one to polymer two is 60:40.

[0022] Preferably, the acid-responsive drug delivery nanoparticles adapted for pancreatic cancer have an average particle size of 150 nm and a zeta potential of 1.26 mV.

[0023] In this invention, the polymer was characterized by 11 PCA atoms by nuclear magnetic resonance hydrogen spectroscopy.

[0024] In this invention, the GE11 peptide, as shown in Formula III, is a polypeptide segment that can specifically bind to the epidermal growth factor receptor.

[0025]

[0026] Formula III

[0027] In this invention, the polymer is a polyphenol block CH3O-PEG-poly-Lysine-PCA (Formula I), and its synthesis method is as follows: (1) Lys(z)-NCA (Formula IV) is generated by condensation reaction; (2) CH3O-PEG-poly-Lysine(z) (Formula V) and Mal-PEG-poly-Lysine(z) (Formula VI) are generated by ring-opening polymerization reaction; (3) The polypeptide is then linked by the reaction of maleimide and thiol group to obtain GE11-PEG-poly-Lysine(z) (Formula VII); (4) CH3O-PEG-poly-Lysine (Formula VIII) and GE11-PEG-poly-Lysine (Formula IX) are obtained by reacting trifluoroacetic acid with a 33% hydrobromic acid solution dissolved in acetic acid; (5) CH3O-PEG-poly-Lysine or GE11-PEG- Poly-Lysine was reacted with 3,4-dihydroxybenzaldehyde (PCA) to obtain CH3O-PEG-poly-Lysine-PCA (Formula X) and GE11-PEG-poly-Lysine-PCA (Formula XI); (6) Gemcitabine was reacted with octadecyl chloride by acylation to obtain modified gemcitabine (Formula XII). CH3O-PEG-poly-Lysine-PCA and GE11-PEG-poly-Lysine-PCA were mixed polymerized in a mass ratio of 60:40, chelated iron ions, and encapsulated leflunomide and modified gemcitabine to form nanoparticles.

[0028] The present invention also provides a method for preparing the above-mentioned acid-responsive drug delivery nanoparticles adapted for pancreatic cancer, specifically comprising the following steps:

[0029] (1) N6-benzyloxycarbonyl-L-lysine reacts with triphosgene according to the Fuchs-farthing method to obtain N6-benzyloxycarbonyl-L-lysine-N-carboxylic anhydride (Lys(z)-NCA); the reaction process is shown in Formula IV:

[0030]

[0031] Formula IV

[0032] (2) a. CH3O-PEG-NH2 and Lys(z)-NCA undergo a ring-opening polymerization reaction to obtain CH3O-PEG-poly-Lysine(z); the reaction process is shown in Formula V:

[0033]

[0034] Formula V

[0035] Or b: Mal-PEG-poly-Lysine(z) is obtained by ring-opening polymerization of Mal-PEG-NH2 and Lys(z)-NCA; the reaction process is shown in Formula VI:

[0036]

[0037] Formula VI

[0038] (3) Mal-PEG-poly-Lysine(z) and GE11 polypeptide undergo a Click reaction to obtain GE11-PEG-poly-Lysine(z); the reaction process is shown in Formula VII:

[0039]

[0040] Formula VII

[0041] (4) a: CH3O-PEG-poly-Lysine(z) reacts with trifluoroacetic acid dissolved in a 33% hydrobromic acid solution of acetic acid to obtain CH3O-PEG-poly-Lysine; the reaction process is shown in formula VIII:

[0042]

[0043] Formula VIII

[0044] Alternatively, b: GE11-PEG-poly-Lysine(z) reacts with trifluoroacetic acid dissolved in a 33% hydrobromic acid solution of acetic acid to obtain GE11-PEG-poly-Lysine; the reaction process is shown in Formula IX:

[0045]

[0046] Formula IX

[0047] (5) a: CH3O-PEG-poly-Lysine reacts with 3,4-dihydroxybenzaldehyde (PCA) and triethylamine in a Schiff base synthesis reaction to obtain CH3O-PEG-poly-Lysine-PCA; the reaction process is shown in Formula X:

[0048]

[0049] Formula X

[0050] Alternatively, b: GE11-PEG-poly-Lysine reacts with 3,4-dihydroxybenzaldehyde and triethylamine in a Schiff base synthesis reaction to obtain GE11-PEG-poly-Lysine-PCA; the reaction process is shown in Formula XI:

[0051]

[0052] Formula XI

[0053] (6) Gemcitabine undergoes a Friedel-Crafts acylation reaction with octadecyl chloride to obtain a modified gemcitabine: the reaction process is shown in Formula XII.

[0054]

[0055] Formula XII

[0056] (7) The acid-responsive drug delivery nanoparticles adapted to pancreatic cancer were prepared by a one-pot method. The specific steps are as follows: Polymer I, Polymer II, modified gemcitabine and leflunomide were dissolved in a solvent, and ferric chloride aqueous solution was added dropwise under high speed stirring. After stirring for 8 hours, the acid-responsive drug delivery nanoparticles adapted to pancreatic cancer were obtained by dialysis.

[0057] Preferably, in step (1): N6-benzyloxycarbonyl-L-lysine and triphosgene are placed in a two-necked flask, vacuumed, and argon isolating the air. Anhydrous tetrahydrofuran is added to the flask to dissolve the mixture, and the mixture is stirred at 50 °C to obtain a white blocky solid Lys(z)-NCA.

[0058] More preferably, the molar ratio of N6-benzyloxycarbonyl-L-lysine to triphosgene in step (1) is 2.35:1.

[0059] In a further preferred embodiment, in step (1), N6-benzyloxycarbonyl-L-lysine and triphosgene are added to anhydrous tetrahydrofuran, and the reaction efficiency is high after the sample is completely dissolved.

[0060] More preferably, the reaction time in step (1) is 3 hours; after the reaction is complete, the system is allowed to return to room temperature, and the reaction liquid is added dropwise to anhydrous n-hexane at -20 °C to precipitate a solid precipitate, which is then filtered to obtain a solid product.

[0061] Preferably, in step (2)a, CH3O-PEG-NH2 and Lys(z)-NCA are placed in a two-necked flask, vacuumed, and argon isolating the air. Anhydrous dimethyl sulfoxide is added to the flask to dissolve the flask, and the mixture is stirred at 50 °C to carry out the reaction. After 24 h, the product CH3O-PEG-poly-Lysine(z) is obtained by dialyzing and freeze-drying.

[0062] More preferably, the molar ratio of CH3O-PEG-NH2 and Lys(z)-NCA in step (2)a is 1:16.

[0063] In a further preferred embodiment, in step (2)a, CH3O-PEG-NH2 and Lys(z)-NCA are placed in a two-necked flask, and the evacuation is repeated three times under argon protection until the air is completely purged. Then anhydrous dimethyl sulfoxide is added. During dialysis, a (MWCO: 3500) dialysis bag is used to dialyze the loaded reactants in pure water, and the water is changed every 12 h. After dialysis for 24 h, the product is obtained after freeze-drying.

[0064] Preferably, in step (2)b, Mal-PEG-NH2 and Lys(z)-NCA are placed in a two-necked flask, vacuumed, and argon isolating the air. Anhydrous dimethyl sulfoxide is added to the flask to dissolve the flask, and the mixture is stirred at 50 °C to carry out the reaction. After 24 h, the product Mal-PEG-poly-Lysine(z) is obtained by dialyzing and freeze-drying.

[0065] More preferably, the molar ratio of Mal-PEG-NH2 to Lys(z)-NCA in step (2)b is 1:16.

[0066] In a further preferred embodiment, in step (2)b, Mal-PEG-NH2 and Lys(z)-NCA are placed in a two-necked flask, evacuated, and isolated from air by argon. Anhydrous dimethyl sulfoxide is then added to the flask to dissolve them. During dialysis, a (MWCO: 3500) dialysis bag is used to dialyze the loaded reactants in pure water. The water is changed every 12 hours, and the dialysis is repeated for 24 hours. The product is then obtained after freeze-drying.

[0067] Preferably, in step (3), the molar ratio of Mal-PEG-poly-Lysine(z) to GE11 peptide is 1:1.

[0068] More preferably, the solvent used in step (3) is a mixture of PBS with pH 7.4 and anhydrous DMSO; after the reaction is completed, the reactants are loaded into a (MWCO: 3500) dialysis bag and dialyzed with deionized water for 24 h, with the water changed every 12 h, and the product is obtained after freeze drying.

[0069] Preferably, in step (4) a, CH3O-PEG-poly-Lysine(z) is placed in a single-necked flask, and trifluoroacetic acid and 33% hydrobromic acid solution dissolved in acetic acid are added to react. The reaction is carried out by stirring at room temperature, and after 3 hours, the product CH3O-PEG-poly-Lysine is obtained by dialyzing and freeze-drying.

[0070] Preferably, the volume ratio of trifluoroacetic acid to 33% hydrobromic acid solution dissolved in acetic acid in step (4)a is 95:5.

[0071] In a further preferred embodiment, step (4)a involves strictly controlling the reaction at room temperature for 3 hours. After the reaction is completed, the reactants are loaded into a dialysis bag with a MWCO of 3500 and dialyzed in deionized water for 24 hours. The water is changed every 8 hours. The product is obtained after freeze-drying.

[0072] Preferably, in step (4)b, GE11-PEG-poly-Lysine(z) is placed in a single-necked flask, and trifluoroacetic acid and 33% hydrobromic acid solution dissolved in acetic acid are added to react. The reaction is carried out by stirring at room temperature. After 3 hours, the product GE11-PEG-poly-Lysine is obtained by dialyzing and freeze-drying.

[0073] More preferably, in step (4)b, the volume ratio of trifluoroacetic acid to the 33% hydrobromic acid solution dissolved in acetic acid is 95:5;

[0074] In a further preferred embodiment, the reaction in step (4)b is strictly controlled at room temperature for 3 hours. After the reaction is completed, the reactants are loaded into a dialysis bag with a MWCO of 3500 and dialyzed in deionized water for 24 hours. The water is changed every 12 hours. The product is obtained after freeze drying.

[0075] Preferably, in step (5)a, CH3O-PEG-poly-Lysine and triethylamine are placed in a two-necked flask at a molar ratio of 1:1, vacuumed, and stirred at room temperature for 30 min under argon protection. Then, PCA dissolved in anhydrous dimethyl sulfoxide is added to the flask at a molar ratio of 1:16, and the reaction is carried out for 18 h. After the reaction is completed, the reactants are loaded into a 7000 MWCO dialysis bag and dialyzed with deionized water for 24 h, with the water changed every 8 h. The product is obtained after freeze drying.

[0076] Preferably, in step (5)b, GE11-PEG-poly-Lysine and triethylamine are placed in a two-necked flask at a molar ratio of 1:1, vacuumed, and stirred at room temperature for 30 min under argon protection. Then, PCA dissolved in anhydrous dimethyl sulfoxide is added to the flask at a molar ratio of 1:16, and the reaction is carried out for 18 h. After the reaction is completed, the reactants are loaded with a (MWCO: 7000) dialysis bag and dialyzed with deionized water for 24 h, with the water changed every 8 h. The product is obtained after freeze drying.

[0077] Preferably, in step (6), gemcitabine and triethylamine are placed in a two-necked flask, evacuated, and isolated from air by argon. Anhydrous N,N-dimethylformamide is added to the flask to dissolve the mixture, and the temperature is lowered to 0°C. Then, sclerosyl chloride is added to the system and placed in an oil bath at 35°C. The mixture is stirred to carry out the reaction. After 24 hours, the reaction is monitored by thin-layer chromatography. Modified gemcitabine is obtained by column chromatography purification.

[0078] Further preferably, in step (6), the molar ratio of gemcitabine, triethylamine, and octadecyl chloride is 1:20:11. First, gemcitabine and triethylamine are added to anhydrous DMF and stirred at room temperature for 5 min, then cooled to 0°C. Octadecyl chloride is dissolved in 2 mL of anhydrous N,N-dimethylformamide and slowly added dropwise over 15 min, with stirring at room temperature for 24 h. Then, it is washed three times with saturated brine, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and the ethyl acetate is removed by rotary evaporation. After purification under a dichloromethane:methanol volume ratio of 20:1, a white waxy solid is obtained.

[0079] The present invention also provides the application of the above-mentioned acid-responsive drug delivery nanoparticles adapted to pancreatic cancer in the preparation of drugs for treating pancreatic cancer.

[0080] Compared with the prior art, the present invention has the following advantages:

[0081] (1) The nano-drug delivery system described in this invention has high biosafety.

[0082] (2) The present invention can release drugs in response to the pancreatic tumor microenvironment, induce multimodal tumor cell death, iron ions and leflunomide synergistically induce ferroptosis, leflunomide and gemcitabine synergistically induce tumor cell autophagy, and combine ferroptosis and autophagy to efficiently kill tumor cells.

[0083] (3) The method of the present invention uses modified gemcitabine to improve resistance to small molecule chemotherapy drugs.

[0084] (4) The present invention provides a nano-drug delivery system that can penetrate the external matrix of pancreatic cancer and achieve deep drug accumulation.

[0085] (5) The nano-drug delivery system described in this invention achieves global regulation starting from a single cell, and improves the immunosuppressive microenvironment of pancreatic cancer tumors through metabolic-immune crosstalk. It interferes with mitochondrial metabolism, induces mitochondrial exocytosis in tumor cells, and inhibits T cell exhaustion by affecting intercellular crosstalk, thereby promoting the transformation of effector T cells into memory T cells. Attached Figure Description

[0086] Figure 1 This is the nuclear magnetic resonance spectrum of CH3O-PEG-poly-Lysine-PCA in Example 1 of the present invention.

[0087] Figure 2 This is the nuclear magnetic resonance spectrum of GE11-PEG-poly-Lysine-PCA in Example 1 of the present invention.

[0088] Figure 3The figures show the particle size and zeta potential of the nanoparticles in Example 1 of this invention, as well as the stable particle size change over 7 days; wherein, Figure A shows the particle size of the nanoparticles, Figure B shows the potential, and Figure C shows the stable particle size change over 7 days.

[0089] Figure 4 The images show flow cytometry results of nanoparticles with different target modification degrees being taken up by tumor cells in this invention; where A is the flow cytometry result and B is the quantification of A.

[0090] Figure 5 Figure A shows the targeting effect of the nanoparticles of this invention on mice bearing KPC Luci orthotopic pancreatic cancer; Figure B shows the distribution of the nanoparticles in the tumor-bearing mice and the quantitative results of the major organs.

[0091] Figure 6 The results show the antitumor therapeutic effect of nanoparticles on KPC Luci-bearing orthotopic pancreatic cancer mice and the changes in mouse body weight after treatment; Figure A shows the tumor growth signal in mice, and Figure B shows the changes in mouse body weight.

[0092] Figure 7 Figure 1 shows the results of the study on the changes in metabolites of tumor tissue in mice with KPC-bearing orthotopic pancreatic cancer. Figure 2 shows the results of GSH in tumor tissue after treatment. Figure 3 shows the results of NADP+ / NADPH in tumor tissue after treatment. Figure 4 shows the results of LPO in tumor tissue after treatment. Figure 5 shows the results of ROS in tumor tissue after treatment.

[0093] Figure 8 Figure 1 shows the flow cytometry results of nanoparticles in mice with KPC-bearing orthotopic pancreatic cancer; Figure A shows the quantitative results of memory T cells; Figure B shows the quantitative results of mature DCs in lymph nodes; Figure C shows the quantitative results of CTL cells in tumor tissue; Figure D shows the quantitative results of Th1 cells in tumor tissue; Figure E shows the quantitative results of Treg cells in tumor tissue; Figure F shows the quantitative results of MDSC cells in tumor tissue; Figure G shows the quantitative results of PD1+CD8+ T cells in tumor tissue; Figure H shows the quantitative results of LAG3+CD8+ T cells in tumor tissue; Figure I shows the quantitative results of TIM3+CD8+ T cells in tumor tissue; Figure J shows the quantitative results of exhausted T cells in tumor tissue; Figure K shows the ratio of early-stage exhausted T cells to terminal-stage exhausted T cells in tumor tissue. Detailed Implementation

[0094] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0095] Example 1

[0096] An acid-responsive drug delivery nanoparticle adapted for pancreatic cancer and its preparation method, wherein the drug delivery nanoparticle synthesis method includes the following steps:

[0097] (1) 5 g, 17.85 mmol N6-benzyloxycarbonyl-L-lysine and 2.25 g, 7.6 mmol triphosgene were added under vacuum, and 50 mL of anhydrous tetrahydrofuran was added. The mixture was protected with argon gas, heated in an oil bath to 50 °C and stirred. After 3 h, the solvent was concentrated by rotary evaporation. Then, the reaction solution was added dropwise to 300 mL of anhydrous n-hexane pre-cooled at -20 °C to precipitate a solid precipitate. The precipitate was obtained by filtration. Lys(z)-NCA was obtained.

[0098] (2) a. 500 mg, 0.1 mmol CH3O-PEG-NH2 and 398 mg, 1.6 mmol Lys(z)-NCA were evacuated under argon protection and repeated 3 times until the air was completely purged. 20 mL of anhydrous dimethyl sulfoxide was added, and the mixture was heated to 50 °C in an oil bath and stirred to carry out the reaction. The mixture was stirred at 50 °C for 24 h. After that, the reaction mixture was loaded with a (MWCO: 3500) dialysis bag and dialyzed with pure water. The water was changed every 12 h. After freeze drying, the product CH3O-PEG-poly-Lysine(z) was obtained.

[0099] b: 500 mg, 0.1 mmol Mal-PEG-NH2 and 398 mg, 1.6 mmol Lys(z)-NCA were placed in a two-necked flask, evacuated, and then 20 mL of anhydrous dimethyl sulfoxide was added to the flask under argon protection. The mixture was heated in an oil bath to 50 °C with stirring. After 24 h, the reactants were dialyzed against pure water using a (MWCO: 3500) dialysis bag, with the water changed every 12 h. After freeze-drying, the product Mal-PEG-poly-Lysine(z) was obtained.

[0100] (3) 100 mg, 0.012 mmol Mal-PEG-poly-Lysine(z) and 20 mg, 0.012 mmol GE11 polypeptide were dissolved in a 1:1 mixture of PBS (pH 7.4) and anhydrous DMSO and reacted at room temperature overnight. After the reaction was completed, the reactants were loaded into a dialysis bag (MWCO: 3500) and dialyzed with deionized water for 24 h. The water was changed every 12 h. After freeze drying, the product GE11-PEG-poly-Lysine(z) was obtained.

[0101] (4) a: 200 mg, 0.022 mmol CH3O-PEG-poly-Lysine (z) was mixed with 9.5 mL trifluoroacetic acid and 500 μL of 33% hydrobromic acid solution dissolved in acetic acid and reacted at room temperature for 3 h. After the reaction was completed, the reactants were loaded into a (MWCO: 3500) dialysis bag and dialyzed with deionized water for 24 h. The water was changed every 12 h. After freeze drying, the product CH3O-PEG-poly-Lysine was obtained.

[0102] b: 200 mg, 0.019 mmol GE11-PEG-poly-Lysine(z) was mixed with 9.5 mL trifluoroacetic acid and 500 μL of 33% hydrobromic acid solution dissolved in acetic acid and reacted at room temperature for 3 h. After the reaction was completed, the reactants were loaded into a dialysis bag (MWCO: 3500) and dialyzed against deionized water for 24 h, with the water changed every 12 h. After freeze drying, the product GE11-PEG-poly-Lysine was obtained.

[0103] (5) a: 200 mg, 0.028 mmol CH3O-PEG-poly-Lysine and 2.8 mg, 0.028 mmol triethylamine and 5 mL anhydrous dimethyl sulfoxide were placed in a two-necked flask, vacuumed, and stirred at room temperature for 30 min under argon protection. Then, 62 mg, 0.45 mmol 3,4-dihydroxybenzaldehyde (PCA) was dissolved in 3 mL anhydrous dimethyl sulfoxide and added to the reaction system. The reaction was carried out for 18 h. After the reaction was completed, the reactants were loaded into a (MWCO: 7000) dialysis bag and dialyzed with deionized water for 24 h. The water was changed every 8 h. After freeze drying, the product CH3O-PEG-poly-Lysine-PCA was obtained.

[0104] b: 200 mg, 0.022 mmol GE11-PEG-poly-Lysine, 2.2 mg, 0.022 mmol triethylamine, and 5 mL anhydrous dimethyl sulfoxide were placed in a two-necked flask, evacuated, and stirred at room temperature for 30 min under argon protection. Then, 48 mg, 0.35 mmol 3,4-dihydroxybenzaldehyde (PCA) was dissolved in 3 mL anhydrous dimethyl sulfoxide and added to the reaction system. The reaction was allowed to proceed for 18 h. After the reaction was completed, the reactants were loaded into a (MWCO: 7000) dialysis bag and dialyzed against deionized water for 24 h, with the water changed every 8 h. After freeze-drying, the product GE11-PEG-poly-Lysine-PCA was obtained.

[0105] (6) Dissolve 30 mg, 0.01 mmol gemcitabine, and 20 mg, 0.2 mmol triethylamine in anhydrous N,N-dimethylformamide and stir at room temperature for 5 min. After cooling to 0 °C, dissolve 33 mg, 0.11 mmol stearyl chloride in 2 mL of anhydrous N,N-dimethylformamide and slowly add it dropwise to the reaction system over 15 min. Stir the reaction at room temperature for 24 h. Then wash three times with saturated brine, extract with ethyl acetate, dry with anhydrous sodium sulfate, remove ethyl acetate by rotary evaporation, and purify under a dichloromethane:methanol volume ratio of 20:1 to obtain a white waxy solid.

[0106] (7) The acid-responsive drug delivery nanoparticles were prepared by a one-pot method. The specific steps were as follows: 6 mg of CH3O-PEG-poly-Lysine-PCA (polymer one) and 4 mg of GE11-PEG-poly-Lysine-PCA (polymer two) were weighed and dissolved in 10 mL of water, 1 mL of 1 mg / mL modified gemcitabine methanol solution, and 1 mL of 1 mg / mL leflunomide methanol solution. The mixture was stirred at 3000 rpm at room temperature for 10 min. Then, the solution was diluted to obtain a 1 mg / mL ferric chloride solution, which was added dropwise to the above system. The mixture was then stirred at 3000 rpm at room temperature for 12 h to obtain the nanoparticles (target modification degree of 40%).

[0107] Comparative Example 1

[0108] An acid-responsive drug delivery nanoparticle adapted for pancreatic cancer and its preparation method are disclosed. The preparation method differs from that in Example 1 in that, when 8 mL of water is added to dissolve the preparation in the nanoparticle preparation stage, the obtained nanoparticles have a particle size of more than 200 nm and a polydispersity index close to 0.5.

[0109] Comparative Example 2 (corresponding to a target modification degree of 0)

[0110] A hypoxia-responsive drug delivery nanoparticle adapted for KRAS-mutant pancreatic cancer and its preparation method differ from Example 1 in that, in step (7), CH3O-PEG-poly-Lysine-PCA (polymer with the structural formula shown in Formula I) is 10 mg dissolved in 10 mL of water, 1 mL of 1 mg / mL modified gemcitabine in methanol, and 1 mL of 1 mg / mL leflunomide in methanol.

[0111] Comparative Example 3 (corresponding to a target modification degree of 10%)

[0112] A hypoxia-responsive drug delivery nanoparticle adapted for KRAS-mutant pancreatic cancer and its preparation method differ from Example 1 in that, in step (7), CH3O-PEG-poly-Lysine-PCA (a polymer with the structural formula shown in Formula I) is 9 mg, GE11-PEG-poly-Lysine-PCA is 1 mg, dissolved in 10 mL of water, 1 mL of 1 mg / mL modified gemcitabine methanol solution and 1 mL of 1 mg / mL leflunomide methanol solution.

[0113] Comparative Example 4 (corresponding to a target modification degree of 20%)

[0114] A hypoxia-responsive drug delivery nanoparticle adapted for KRAS-mutant pancreatic cancer and its preparation method differ from Example 1 in that, in step (7), CH3O-PEG-poly-Lysine-PCA (a polymer with the structural formula shown in Formula I) is 8 mg, GE11-PEG-poly-Lysine-PCA is 2 mg, dissolved in 10 mL of water, 1 mL of 1 mg / mL modified gemcitabine methanol solution and 1 mL of 1 mg / mL leflunomide methanol solution.

[0115] Comparative Example 5 (corresponding to a target modification degree of 30%)

[0116] A hypoxia-responsive drug delivery nanoparticle adapted for KRAS-mutant pancreatic cancer and its preparation method differ from Example 1 in that, in step (7), CH3O-PEG-poly-Lysine-PCA (a polymer with the structural formula shown in Formula I) is 7 mg, GE11-PEG-poly-Lysine-PCA is 3 mg, dissolved in 10 mL of water, 1 mL of 1 mg / mL modified gemcitabine methanol solution and 1 mL of 1 mg / mL leflunomide methanol solution.

[0117] Comparative Example 6 (corresponding to a target modification degree of 50%)

[0118] A hypoxia-responsive drug delivery nanoparticle adapted for KRAS-mutant pancreatic cancer and its preparation method differ from Example 1 in that, in step (7), CH3O-PEG-poly-Lysine-PCA (a polymer with the structural formula shown in Formula I) is 5 mg, GE11-PEG-poly-Lysine-PCA 5 mg is dissolved in 10 mL of water, 1 mL of 1 mg / mL modified gemcitabine methanol solution and 1 mL of 1 mg / mL leflunomide methanol solution.

[0119] The drug-loaded nanoparticles were characterized and tested as follows:

[0120] Figure 1 The 1H NMR spectrum of CH3O-PEG-poly-Lysine-PCA in Example 1 shows that 16 PCA molecules are attached to one polymer chain.

[0121] Figure 2 The 1H NMR spectrum of GE11-PEG-poly-Lysine-PCA in Example 1 shows that 16 PCA molecules and 1 GE11 polypeptide are linked to one polymer molecular chain.

[0122] The resulting nanoparticles are in the form of regular spheres, such as... Figure 3 As shown in Figure A, the particle size distribution is uniform, with an average particle size of 150 nm; Figure 3 From B, we know that the zeta potential is 1.26 mV; from Figure 3 As shown in C, the particle size and PDI changed little after 7 days in PBS at pH 7.4. HPLC results showed that the leflunomide loading was 7.31 ± 0.32%, and the modified gemcitabine loading was 3.01 ± 1.26%.

[0123] The uptake of nanoparticles with different target modification degrees (target modification degree refers to the mass ratio of the target-containing material to the polymer material) by tumor cells was investigated by flow cytometry. Figure 4 Flow cytometry results of tumor cell uptake of nanoparticles with different modification levels (Comparative Examples 2-6 and Example 1) show that tumor cells exhibit the most significant uptake of nanoparticles when the target modification level is 40% (Example 1). The target modification level of the nanoparticles used in the following experiments was 40%.

[0124] The targeting ability of the nanoparticles in mice with KPC-bearing orthotopic pancreatic cancer was then investigated. First, hypoxia-responsive nanoparticles were labeled with a fluorescent probe. The specific steps were as follows: 6 mg of CH3O-PEG-poly-Lysine-PCA (polymer one) and 4 mg of GE11-PEG-poly-Lysine-PCA (polymer two) were dissolved in 10 mL of water, along with 1 mL of a 1 mg / mL Cy5 probe aqueous solution. The mixture was stirred at 3000 rpm at room temperature for 10 min. Then, a 1 mg / mL ferric chloride solution was diluted and added dropwise to the above system. The mixture was then stirred at 3000 rpm at room temperature for 12 h to obtain the Cy5-labeled nanoparticles.

[0125] The subsequent experimental method involved configuring KPC cells at a density of 1 × 10⁶ cells / year. 6 Cell suspensions of 100 μL were prepared. After complete anesthesia of male C57 mice, part of the pancreas was surgically exposed, and the prepared cell suspension was injected in situ into the pancreas using an insulin needle to establish a PDAC in situ tumor model. Different formulations were administered intravenously to pancreatic cancer-bearing mice. The in vivo signal distribution of the fluorescent probes encapsulated in the formulations was monitored at multiple time points within 24 hours using IVIS Spectrum. The mice were cervically dislocated 24 hours after administration. The heart, liver, spleen, lungs, and kidneys of the mice were perfused with physiological saline and paraformaldehyde, and the distribution of the fluorescent probe signals in the major organs was further observed under in vivo optical imaging. Results are as follows: Figure 5 As shown in Figure A, the nanoparticle distribution results in tumor-bearing mice demonstrate the tumor-accumulating properties specific to GGP-Cy5 (indicating the tumor-accumulating properties of nanoparticles modified with GE11 peptide polymers). Cy5 indicates the nanoparticles, and PP-Cy5 represents nanoparticles prepared by replacing GE11-PEG-poly-Lysine-PCA with an equal mass of CH3O-PEG-poly-Lysine-PCA using the same method. Figure B shows the quantitative results in ex vivo tissue, indicating that the formulation has a significant targeting effect on tumor tissue in vivo.

[0126] The antitumor effect of the nanoparticles in tumor-bearing mice was then investigated (mice with orthotopic pancreatic tumors were intravenously injected with the formulation, and the magnitude of tumor fluorescence signal, changes in mouse body weight, and survival after administration were monitored). The results are as follows: Figure 6 As shown, Figure A is the curve of tumor signal change over time, and Figure B is the curve of mouse body weight change over time. Figure 6In the table, G1-G8 represent tumor-bearing mice treated with different formulations. The formulations administered to the mice in each group were as follows: Group 1: Saline (physiological saline), Group 2: Gem 1.5 mg / kg + Lef 3 mg / kg (free drug at the same dose as the formulation), Group 3: Gem 1.5 mg / kg + Nab-Paclitaxel 0.19 mg / kg (clinical free drug at the same dose as the formulation), Group 4: Gem 11 mg / kg + Nab-Paclitaxel 1.38 mg / kg (clinical treatment group), Group 5: PPG / L (non-targeted nanoparticles), Group 6: GPP (unloaded nanoparticles), Group 7: GPPG (gemcitabine-loaded nanoparticles), and Group 8: GPPG / L (final formulation). This indicates that the formulation has good anti-tumor effects in vivo and does not cause weight loss in mice, demonstrating good biocompatibility.

[0127] The changes in the levels of relevant metabolites in tumor tissue after drug administration were then examined. Figure 7 The results show the changes in metabolites in tumor tissue after treatment. Figure A shows the results of GSH after treatment, Figure B shows the results of NADP+ / NADPH after treatment, Figure C shows the results of LPO after treatment, and Figure D shows the results of ROS after treatment.

[0128] The proportion and type of immune cells in the treated tumor tissue and lymph nodes were then examined, and the types and proportions of immune cells were determined by flow cytometry. Memory T cells were labeled with CD3e (1.25 μL / 100 μL PBS), CD8 (0.5 μL / 100 μL PBS), CD44 (5 μL / 100 μL PBS), and CD62L (0.63 μL / 100 μL PBS). Dendritic cells (DCs) in lymph nodes were labeled with CD11c (1.25 μL / 100 μL PBS), CD80 (1.5 μL / 100 μL PBS), CD86 (0.63 μL / 100 μL PBS), and CD62L (0.63 μL / 100 μL PBS). CTL cells were labeled with CD45 (0.125 μL / 100 μL PBS), CD3 (0.25 μL / 100 μL PBS), and CD8 (0.125 μL / 100 μL PBS). Th1 cells were labeled with CD45 (0.125 μL / 100 μL PBS), CD3 (0.25 μL / 100 μL PBS), and CD4 (1.25 μL / 100 μL PBS). Treg cells were labeled with CD45 (0.125 μL / 100 μL PBS), CD25 (0.24 μL / 100 μL PBS), CD4 (1.25 μL / 100 μL PBS), and Foxp3 (3 μL / 100 μL PBS). MDSCs were labeled with CD11b (0.63 μL / 100 μL PBS), CD45 (0.13 μL / 100 μL PBS), and Gr-1 (0.25 μL / 100 μL PBS). PD-1+ T cells were labeled with CD3e (1.25 μL / 100 μL PBS), CD8 (0.25 μL / 100 μL PBS), and PD-1 (2 μL / 100 μL PBS). LAG3+ T cells were labeled with CD3e (1.25 μL / 100 μL PBS), CD8 (0.25 μL / 100 μL PBS), and LAG3 (5 μL / 100 μL PBS). TIM3+ T cells were labeled with CD3e (1.25 μL / 100 μL PBS), CD8 (0.25 μL / 100 μL PBS), and TIM3 (5 μL / 100 μL PBS).Exhausted T cells were labeled with CD3e (1.25 μL / 100 μL PBS), CD8 (0.25 μL / 100 μL PBS), CD69 (1.25 μL / 100 μL PBS) and Ly108 (2 μL / 100 μL PBS). Figure 8 In the table, G1-G8 represent tumor-bearing mice treated with different formulations. The formulations administered to the mice in each group were as follows: Group 1: Saline (physiological saline); Group 2: Gem 1.5 mg / kg + Lef 3 mg / kg (equal dose of free drug); Group 3: Gem 1.5 mg / kg + Nab-Paclitaxel 0.19 mg / kg (equal dose of clinical free drug); Group 4: Gem 11 mg / kg + Nab-Paclitaxel 1.38 mg / kg (clinical treatment group); Group 5: PPG / L (non-targeted nanoparticles); Group 6: GPP (unloaded nanoparticles); Group 7: GPPG (gemcitabine-loaded nanoparticles); Group 8: GPPG / L (final formulation). This indicates that the formulation in Group 8 can activate the anti-tumor immune response. Figure 8 A represents the proportion of Memory T cells in the tumor tissue. Figure 8 B represents the proportion of mature dendritic cells in lymphoid tissue. Figure 8 C represents the proportion of CTL cells in the tumor tissue. Figure 8 D represents the proportion of Th1 cells in the tumor tissue. Figure 8 E represents the proportion of Treg cells in the tumor tissue. Figure 8 F represents the proportion of MDSC-positive cells in the tumor tissue. Figure 8 G represents the proportion of PD1+ cells in the tumor tissue. Figure 8 H represents the proportion of LAG3+ positive cells in the tumor tissue. Figure 8 I represents the proportion of TIM3+ positive cells in the tumor tissue. Figure 8 J represents the proportion of exhausted T cells. Figure 8 K represents the ratio of exhausted T cells in the early and late stages of tumor tissue.

Claims

1. An acid-responsive drug delivery nanoparticle adapted for pancreatic cancer, characterized in that, The mixture is obtained by chelating iron ions with an amphiphilic polyphenol polymer and encapsulating a chemotherapeutic drug targeting pancreatic cancer and a DHODH inhibitor. The polymer comprises: an amphiphilic polymer one modified with polyphenols, and an amphiphilic polymer two modified with polyphenols and a targeting peptide at one end; the chemotherapeutic drug targeting pancreatic cancer is a modified gemcitabine. The targeting peptide in polymer II is GE11; The DHODH inhibitor is leflunomide; The structural formula of polymer one is shown in Formula I; the structural formula of polymer two is shown in Formula II. Formula I Formula II.

2. The acid-responsive drug delivery nanoparticle adapted for pancreatic cancer according to claim 1, characterized in that, The polymer macromolecular chain comprises, from one end to the other, a polyethylene glycol hydrophilic segment and a polylysine-polyphenol block. The loading of leflunomide was 7.31±0.32%, and the loading of the modified gemcitabine was 3.01±1.26%.

3. The acid-responsive drug delivery nanoparticle adapted for pancreatic cancer according to claim 2, characterized in that, The mass ratio of polymer one to polymer two is 60:

40.

4. The acid-responsive drug delivery nanoparticle adapted for pancreatic cancer according to claim 2, characterized in that, The acid-responsive drug delivery nanoparticles adapted for pancreatic cancer have an average particle size of 150 nm and a zeta potential of 1.26 mV.

5. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to any one of claims 1-4, characterized in that, Includes the following steps: (1) N6-benzyloxycarbonyl-L-lysine reacts with triphosgene according to the Fuchs-farthing method to obtain N6-benzyloxycarbonyl-L-lysine-N-carboxylic anhydride; the reaction process is shown in Formula IV: Formula IV (2) a. CH3O-PEG-NH2 and Lys(z)-NCA undergo a ring-opening polymerization reaction to obtain CH3O-PEG-poly-Lysine(z); the reaction process is shown in Formula V: Formula V Or b: Mal-PEG-poly-Lysine(z) is obtained by ring-opening polymerization of Mal-PEG-NH2 and Lys(z)-NCA; the reaction process is shown in Formula VI: Formula VI (3) Mal-PEG-poly-Lysine(z) undergoes a Click reaction with GE11 polypeptide to obtain GE11-PEG-poly-Lysine(z); the reaction process is shown in Formula VII: Formula VII (4) a: CH3O-PEG-poly-Lysine(z) reacts with trifluoroacetic acid dissolved in a 33% hydrobromic acid solution of acetic acid to obtain CH3O-PEG-poly-Lysine; the reaction process is shown in formula VIII: Formula VIII Alternatively, b: GE11-PEG-poly-Lysine(z) reacts with trifluoroacetic acid dissolved in a 33% hydrobromic acid solution of acetic acid to obtain GE11-PEG-poly-Lysine; the reaction process is shown in Formula IX: Formula IX (5) a: CH3O-PEG-poly-Lysine reacts with 3,4-dihydroxybenzaldehyde (PCA) and triethylamine in a Schiff base synthesis reaction to obtain CH3O-PEG-poly-Lysine-PCA; the reaction process is shown in Formula X: Formula X Alternatively, b: GE11-PEG-poly-Lysine reacts with 3,4-dihydroxybenzaldehyde and triethylamine in a Schiff base synthesis reaction to obtain GE11-PEG-poly-Lysine-PCA; the reaction process is shown in Formula XI: Formula XI (6) Gemcitabine undergoes Friedel-Crafts acylation reaction with octadecyl chloride to obtain modified gemcitabine: the reaction process is shown in Formula XII. Formula XII (7) The acid-responsive drug delivery nanoparticles adapted to pancreatic cancer were prepared by a one-pot method. The specific steps are as follows: polymer I, polymer II, modified gemcitabine and leflunomide were dissolved in a solvent, and ferric chloride aqueous solution was added dropwise under high speed stirring. After stirring for 8 hours, the acid-responsive drug delivery nanoparticles adapted to pancreatic cancer were obtained by dialysis.

6. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 5, characterized in that, Step (1): N6-benzyloxycarbonyl-L-lysine and triphosgene are placed in a two-necked flask, vacuumed, and isolated from air by argon. Then anhydrous tetrahydrofuran is added to dissolve the mixture, and the mixture is stirred at 50 °C to obtain a white blocky solid Lys(z)-NCA.

7. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 5, characterized in that, The molar ratio of N6-benzyloxycarbonyl-L-lysine to triphosgene in step (1) is 2.35:

1.

8. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 5, characterized in that, In step (1), N6-benzyloxycarbonyl-L-lysine and triphosgene are added to anhydrous tetrahydrofuran. The reaction efficiency is high after the sample is completely dissolved.

9. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 5, characterized in that, The reaction time in step (1) is 3 hours. After the reaction is complete, the system is allowed to return to room temperature. The reaction solution is then added dropwise to anhydrous n-hexane at -20 °C to precipitate a solid precipitate. The solid product is then obtained by filtration.

10. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 5, characterized in that, In step (2)a, CH3O-PEG-NH2 and Lys(z)-NCA were placed in a two-necked flask, evacuated, and isolated from air by argon. Anhydrous dimethyl sulfoxide was added to the flask to dissolve the flask, and the mixture was stirred at 50 °C to carry out the reaction. After 24 h, the product CH3O-PEG-poly-Lysine(z) was obtained by dialyzing and freeze-drying.

11. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 10, characterized in that, The molar ratio of CH3O-PEG-NH2 and Lys(z)-NCA in step (2)a is 1:

16.

12. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 10, characterized in that... In step (2)a, CH3O-PEG-NH2 and Lys(z)-NCA were placed in a two-necked flask, and the evacuation was repeated three times under argon protection until the air was completely purged. Then anhydrous dimethyl sulfoxide was added. During dialysis, the reactants were dialyzed in pure water using a (MWCO: 3500) dialysis bag. The water was changed every 12 h, and the dialysis was repeated for 24 h. The product was obtained after freeze-drying.

13. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 5, characterized in that, Step (2)b: Place Mal-PEG-NH2 and Lys(z)-NCA in a two-necked flask, evacuate the air, and then add anhydrous dimethyl sulfoxide to the flask to dissolve it. Stir at 50 °C to carry out the reaction. After 24 h, dialyze and freeze dry to obtain the product Mal-PEG-poly-Lysine(z).

14. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 13, characterized in that, The molar ratio of Mal-PEG-NH2 to Lys(z)-NCA in step (2)b is 1:

16.

15. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 13, characterized in that, In step (2)b, Mal-PEG-NH2 and Lys(z)-NCA were placed in a two-necked flask, evacuated, and isolated from air by argon. Anhydrous dimethyl sulfoxide was then added to the flask to dissolve them. During dialysis, a dialysis bag with a MWCO of 3500 was used to dialyze the loaded reactants in pure water. The water was changed every 12 hours, and the dialysis was performed for 24 hours. The product was obtained after freeze-drying.

16. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 5, characterized in that, Step (3): Dissolve Mal-PEG-poly-Lysine(z) and GE11 peptide in a 1:1 mixture of PBS and DMSO, react at room temperature overnight, and lyophilize by dialyzing to obtain the product GE11-PEG-poly-Lysine(z); the molar ratio of Mal-PEG-poly-Lysine(z) to GE11 peptide in step (3) is 1:

1.

17. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 16, characterized in that, The solvent used in step (3) was a mixture of PBS with pH 7.4 and anhydrous DMSO. After the reaction was completed, the reactants were loaded into a dialysis bag with an MWCO of 3500 and dialyzed with deionized water for 24 h. The water was changed every 12 h. The product was obtained after freeze drying.

18. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 5, characterized in that, Step (4) a: CH3O-PEG-poly-Lysine(z) was placed in a single-necked flask, and trifluoroacetic acid and 33% hydrobromic acid solution dissolved in acetic acid were added to react. The reaction was carried out by stirring at room temperature. After 3 hours, the product CH3O-PEG-poly-Lysine was obtained by dialyzing and freeze-drying.

19. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 18, characterized in that, In step (4)a, the volume ratio of trifluoroacetic acid to 33% hydrobromic acid solution dissolved in acetic acid is 19:

1.

20. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 18, characterized in that, Step (4) a: The reaction was strictly controlled at room temperature for 3 hours. After the reaction was completed, the reactants were loaded into a dialysis bag with an MWCO of 3500 and dialyzed in deionized water for 24 hours. The water was changed every 8 hours. The product was obtained after freeze drying.

21. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 5, characterized in that, Step (4)b: GE11-PEG-poly-Lysine(z) was placed in a single-necked flask, and trifluoroacetic acid and 33% hydrobromic acid solution dissolved in acetic acid were added to react. The reaction was carried out by stirring at room temperature. After 3 hours, the product GE11-PEG-poly-Lysine was obtained by dialyzing and freeze-drying.

22. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 21, characterized in that, In step (4)b, the volume ratio of trifluoroacetic acid to 33% hydrobromic acid solution dissolved in acetic acid is 19:

1.

23. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 21, characterized in that, Step (4)b: The reaction was strictly controlled at room temperature for 3 hours. After the reaction was completed, the reactants were loaded into a dialysis bag with an MWCO of 3500 and dialyzed in deionized water for 24 hours. The water was changed every 8 hours. The product was obtained after freeze drying.

24. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 5, characterized in that, In step (5)a, CH3O-PEG-poly-Lysine and triethylamine were placed in a two-necked flask at a molar ratio of 1:1, and the mixture was evacuated and stirred at room temperature for 30 min under argon protection. Then, PCA dissolved in anhydrous dimethyl sulfoxide was added to the flask at a molar ratio of 1:16, and the reaction was carried out for 18 h. After the reaction was completed, the reactants were dialyzed in a (MWCO: 7000) dialysis bag and dialyzed in deionized water for 24 h, with the water changed every 8 h. After freeze-drying, the product CH3O-PEG-poly-Lysine-PCA was obtained.

25. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 5, characterized in that, Step (5)b: GE11-PEG-poly-Lysine and triethylamine were placed in a two-necked flask at a molar ratio of 1:1, and the mixture was evacuated and stirred at room temperature for 30 min under argon protection. Then, anhydrous dimethyl sulfoxide dissolved PCA was added to the flask at a molar ratio of 1:16, and the reaction was carried out for 18 h. After the reaction was completed, the reactants were dialyzed in a (MWCO: 7000) dialysis bag and dialyzed with deionized water for 24 h, with the water changed every 8 h. After freeze drying, the product GE11-PEG-poly-Lysine-PCA was obtained.

26. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 5, characterized in that, Step (6): Gemcitabine and triethylamine were placed in a two-necked flask, evacuated, and isolated from air by argon. Anhydrous N,N-dimethylformamide was added to the flask to dissolve the mixture. The mixture was then cooled to 0°C. Stearoyl chloride was added to the system and the mixture was placed in an oil bath at 35°C and stirred to carry out the reaction. The reaction was monitored by thin-layer chromatography after 24 hours. Modified gemcitabine was obtained by column chromatography purification.

27. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 26, characterized in that... In step (6), the molar ratio of gemcitabine, triethylamine, and octadecyl chloride is 1:2:1.

1. First, gemcitabine and triethylamine are added to anhydrous DMF and stirred at room temperature for 5 min, and then cooled to 0℃. Stearoyl chloride is dissolved in 2 mL of anhydrous N,N-dimethylformamide and slowly added dropwise over 15 min. The mixture is stirred at room temperature for 24 h, then washed three times with saturated brine, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and the ethyl acetate is removed by rotary evaporation. After purification under the condition of dichloromethane:methanol volume ratio of 20:1, a white waxy solid is obtained.

28. The use of the acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to any one of claims 1-4 in the preparation of a medicament for treating pancreatic cancer.

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