Acid-responsive drug delivery nanoparticles adaptive to pancreatic cancer and preparation method of acid-responsive drug delivery nanoparticles

Abstract

The invention belongs to the technical field of nano drug loading, and particularly relates to an acid-responsive drug delivery nanoparticle adaptive to pancreatic cancer and a preparation method of the acid-responsive drug delivery nanoparticle. According to the polyphenol polymer disclosed by the invention, a biodegradable polyethylene glycol (PEG) polyphenol polymer is used as a skeleton, and then a hydrophobic drug improved gemcitabine and leflunomide are entrapped by using a metal ion chelation effect to form nanoparticles. The nanoparticles can respond to a special environment in tumor cells to release improved gemcitabine and leflunomide, can induce cell ferroptosis by regulating and controlling pancreatic tumor cell mitochondrial metabolism, can induce mitochondrial exocytosis to realize deep drug accumulation, and can realize deep drug accumulation through multi-cell immune-metabolic network regulation and control in tumor focus tissues. The immunosuppression microenvironment is relieved, and pancreatic tumors are inhibited.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano drug delivery, and in particular relates to an acid-responsive drug delivery nanoparticle adapted for pancreatic cancer and a preparation method thereof. Background Art

[0002] Pancreatic tumors are considered one of the most dangerous and challenging tumor models in clinical practice, with a five-year survival rate of only 9%. The main reason for its high malignancy is that pancreatic tumor cells exhibit metabolic reprogramming, that is, changes in multiple metabolic pathways occur within tumor cells to meet the bioenergy and synthesis needs required for their 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 dense matrix and high expression characteristics of immunosuppressive cells in the pancreatic cancer microenvironment. Starting with regulating tumor cell metabolism and improving the entire microenvironment by intervening in single cells is a potential pancreatic cancer treatment strategy.

[0003] Mitochondria are the hub of cell metabolism, involved in the biosynthesis of a variety of amino acids, lipids and nucleotides, and bear the important functions of providing basic energy and maintaining signal transduction. In addition, mitochondria play a key role in a variety of cell death processes, including ferroptosis, apoptosis, autophagy, etc., and their functions are essential for maintaining cell activity. For tumor cells, mitochondria are key organelles that coordinate their metabolic reprogramming. They maintain tumor cell activity by integrating a variety of cell signals, enabling them to adapt and grow in the dynamic and complex tumor microenvironment. Therefore, targeting tumor cell mitochondria is a potential therapeutic approach. It is worth noting that when cells are stimulated by external stimuli, they release damaged mitochondria by forming migratory bodies to achieve mitochondrial quality control. This process is called mitochondrial exocytosis. Previous studies have induced mitochondrial exocytosis to achieve intercellular transport of nano-drug delivery systems in the tumor microenvironment, promote drug penetration deep into the tumor site, and exert sustained anti-tumor effects. It is suggested 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, mitochondrial metabolism of other cells in the tumor microenvironment also affects 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 effect of anti-tumor therapy. Therefore, in tumor therapy 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] As a first-line chemotherapy drug for pancreatic cancer, gemcitabine is mainly used as a nucleoside analog to inhibit the biosynthesis of tumor cells and inhibit their activity. However, as the treatment process progresses, pancreatic tumor cells often develop drug resistance. Studies have shown that the pyrimidine biosynthesis pathway plays a key role in the gemcitabine resistance process. DHODH is a key enzyme in pyrimidine biosynthesis and can play two roles in the cell. On the one hand, it can partially reverse the resistance of tumor cells to gemcitabine by regulating pyrimidine metabolism. On the other hand, DHODH can resist the effect of ferroptosis inducers by reducing ubiquinone. In this application, levofloxacin is used as a DHODH inhibitor. This isoxazole immunosuppressant with antiproliferative activity has shown good potential in multiple 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. In addition, due to the phenomenon of metabolic reprogramming, pancreatic tumors show the characteristics of dense matrix, which further limits the ability of drugs to reach deep tumor tissues. Therefore, the unique size characteristics and functional diversity of nano drug delivery systems can help overcome the dense matrix barrier of pancreatic cancer, prolong the circulation time of therapeutic drugs, and achieve efficient pancreatic cancer treatment through targeted drug delivery. Although many studies have explored the application of various nano drug delivery systems in the treatment of pancreatic tumors, no substantial progress has been made in the clinic, mainly due to problems such as complex preparation processes and biosafety of preparations. Therefore, it is urgent to develop a new drug delivery system that can not only deliver gemcitabine to deep tumor lesions but also combine other strategies to improve gemcitabine resistance. Summary of the invention

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

[0006] Therefore, the present application proposes a one-pot method for preparing polyphenol-iron chelated nanoparticles for co-delivery of modified gemcitabine and leflunomide. The nanoparticle preparation process is simple and has good repeatability. Active targeting of the nanoparticles is achieved by modifying the surface of the nanoparticles with a polypeptide that can target EGFR highly expressed in tumor cells. Nanoparticles can interfere with mitochondrial metabolism and promote deep delivery of nanomedicines through mitochondrial exocytosis, thereby synergistically enhancing the effect and inducing tumor cell ferroptosis and mitochondrial autophagy. By regulating tumor cell metabolism, this method is expected to improve the immunosuppressive microenvironment of pancreatic cancer and enhance the overall therapeutic effect of small molecule chemotherapy drugs.

[0007] The purpose of the present invention is to provide a formulation design strategy based on the pathological characteristics of pancreatic cancer and the current status of clinical technology, which can achieve targeted tumor lesions, responsive drug release, enhance drug penetration by inducing cell mitochondrial exocytosis, achieve regulation of tumor cell metabolism, and enhance the therapeutic efficacy of tumors. Specifically, the present invention provides an acid-responsive drug delivery nanoparticle adapted for pancreatic cancer and a preparation method. Due to the biosafety and versatility of polyphenol materials, the present invention uses polyphenol materials to chelate iron ions to deliver drugs. This polymer can chelate iron ions to form nanoparticles, and encapsulate modified gemcitabine and leflunomide through the cavity. This nanoparticle can encapsulate small molecule drugs, and the particle size is uniform and the morphology is round. It has the characteristics of responding to the acid response of tumor cells, releasing iron ions and drugs, and has excellent tumor accumulation characteristics, and the nanoparticles can induce mitochondrial exocytosis, promote deep penetration of nanoparticles, regulate metabolism-induced ferroptosis reactions, alleviate gemcitabine resistance and achieve tumor immunosuppression.

[0008] Based on the characteristics of pancreatic cancer microenvironment, the present invention utilizes the characteristics of polyphenol nanoparticles chelating metal ions to form a cavity to carry drugs, which has the purpose of responding to the acidic intracellular environment of tumor cells to achieve deep penetration and release of drugs. The structure of the polymer is designed as follows: a biodegradable polyethylene glycol (PEG) polyphenol polymer is used as a skeleton, and then the hydrophobic drug modified gemcitabine and leflunomide are encapsulated to form nanoparticles by metal ion chelation. By combining the GE11 peptide that targets tumor tissues that highly express epidermal growth factor receptors with the material, the targeting ability of nanoparticles to pancreatic tumor tissues is improved. The nanoparticles can exist stably in the systemic circulation and target tumor tissues under the action of the targeting functional molecule GE11 peptide. The nanoparticles are promoted to penetrate deep tumor tissues by inducing tumor cell mitochondrial exocytosis. After being taken into tumor cells, the polyphenol iron chelated nanoparticles disintegrate, regulate the mitochondrial metabolism of tumor cells, and achieve regulation of the entire microenvironment through the metabolism-immune network of the tumor microenvironment.

[0009] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions: The present invention provides an acid-responsive drug delivery nanoparticle adapted for pancreatic cancer, which is obtained by encapsulating a DHODH inhibitor and a chemotherapeutic drug for pancreatic cancer by chelating iron ions with an amphiphilic polyphenol polymer. The polymer comprises: an amphiphilic polymer 1 modified with polyphenols, and an amphiphilic polymer 2 modified with polyphenols and having a targeting polypeptide modified at one end.

[0010] Preferably, a macromolecular chain of the polymer comprises a polyethylene glycol hydrophilic segment and a polylysine-polyphenol block in sequence from one end to the other end. Preferably, the chemotherapy drug for pancreatic cancer is modified gemcitabine.

[0011] Preferably, the targeting polypeptide in the polymer 2 is GE11.

[0012] Preferably, the DHODH inhibitor is leflunomide.

[0013] More preferably, the drug loading of leflunomide is 7.31±0.32%, and the drug loading of improved gemcitabine is 3.01±1.26%.

[0014] More preferably, the structural formula of polymer 1 is as shown in formula I; the structural formula of polymer 2 is as shown in formula II;

[0015] Formula I

[0016] Formula II More preferably, the mass ratio of polymer one to polymer two is 60:40.

[0017] 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.

[0018] In the present invention, the nuclear magnetic resonance hydrogen spectrum test characterizes formula I and obtains that 11 PCAs are connected to the polymer.

[0019] In the present invention, the GE11 peptide has a simplified formula as shown in Formula III, and this polypeptide segment is a polypeptide segment that can specifically bind to the epidermal growth factor receptor.

[0020]

[0021] Formula III In the present invention, the polymer 1 is a polyphenol block CH3O-PEG-poly-Lysine-PCA (Formula I), and its synthesis method is as follows: (1) generating Lys(z)-NCA (Formula IV) through a condensation reaction; (2) generating CH3O-PEG-poly-Lysine(z) (Formula V) and Mal-PEG-poly-Lysine(z) (Formula VI) through a ring-opening polymerization reaction; (3) then connecting the polypeptide by the reaction of maleimide and thiol to obtain GE11-PEG-poly-Lysine(z) (Formula VII); (4) reacting trifluoroacetic acid with a 33% hydrobromic acid solution dissolved in acetic acid to obtain CH3O-PEG-poly-Lysine (Formula VIII) and GE11-PEG-poly-Lysine (Formula IX); (5) then reacting CH3O-PEG-poly-Lysine or GE11-PEG- Poly-Lysine and 3,4-dihydroxybenzaldehyde (PCA) are reacted to obtain CH3O-PEG-poly-Lysine-PCA (Formula X) and GE11-PEG-poly-Lysine-PCA (Formula XI); (6) Gemcitabine is reacted with octadecanoyl chloride by acylation reaction to obtain modified gemcitabine (Formula XII). CH3O-PEG-poly-Lysine-PCA and GE11-PEG-poly-Lysine-PCA are formed into a mixed polymer in a mass ratio of 60:40, chelated iron ions, and entrapped leflunomide and modified gemcitabine to form nanoparticles.

[0022] The present invention also provides a method for preparing the above-mentioned acid-responsive drug delivery nanoparticles adapted for pancreatic cancer, which specifically comprises the following steps: (1) N6-benzyloxycarbonyl-L-lysine is reacted 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:

[0023] 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:

[0024] Formula V Or b: Mal-PEG-NH2 and Lys(z)-NCA are subjected to a ring-opening polymerization reaction to obtain Mal-PEG-poly-Lysine(z); the reaction process is shown in Formula VI:

[0025] Formula VI (3) Mal-PEG-poly-Lysine (z) is subjected to a click reaction with the GE11 polypeptide to obtain GE11-PEG-poly-Lysine (z); the reaction process is shown in Formula VII:

[0026] Formula VII (4) a: CH3O-PEG-poly-Lysine (z) reacts with trifluoroacetic acid and 33% hydrobromic acid solution dissolved in acetic acid to obtain CH3O-PEG-poly-Lysine; the reaction process is shown in Formula VIII:

[0027] Formula VIII Or, b: GE11-PEG-poly-Lysine (z) reacts with trifluoroacetic acid and 33% hydrobromic acid solution dissolved in acetic acid to obtain GE11-PEG-poly-Lysine; the reaction process is shown in Formula IX:

[0028] Formula IX (5) a: CH3O-PEG-poly-Lysine is subjected to a Schiff base synthesis reaction with 3,4-dihydroxybenzaldehyde (PCA) and triethylamine to obtain CH3O-PEG-poly-Lysine-PCA; the reaction process is shown in Formula X:

[0029] Formula Ⅹ Or, b: GE11-PEG-poly-Lysine is subjected to a Schiff base synthesis reaction with 3,4-dihydroxybenzaldehyde and triethylamine to obtain GE11-PEG-poly-Lysine-PCA; the reaction process is shown in Formula XI:

[0030] Formula XI (6) Gemcitabine is subjected to Friedel-Crafts acylation reaction with octadecanoyl chloride to obtain improved gemcitabine: the reaction process is shown in Formula XII,

[0031] Formula Ⅻ (7) The acid-responsive drug delivery nanoparticles adapted for pancreatic cancer are prepared by a one-pot method, and the specific steps are as follows: polymer 1, polymer 2, modified gemcitabine and leflunomide are dissolved in a solvent, and an aqueous solution of ferric chloride is added dropwise under high-speed stirring. After stirring for 8 hours, the acid-responsive drug delivery nanoparticles adapted for pancreatic cancer are obtained by dialysis.

[0032] Preferably, step (1): N6-benzyloxycarbonyl-L-lysine and triphosgene are placed in a two-necked flask, evacuated, and the air is isolated by argon gas. Anhydrous tetrahydrofuran is added to the flask to dissolve the mixture, and the mixture is stirred and reacted at 50° C. to obtain a white block solid Lys(z)-NCA.

[0033] Further preferably, the molar ratio of N6-benzyloxycarbonyl-L-lysine to triphosgene in step (1) is 2.35:1.

[0034] Further preferably, in step (1), N6-benzyloxycarbonyl-L-lysine and triphosgene are added to anhydrous tetrahydrofuran, and the reaction efficiency is higher after the sample is completely dissolved.

[0035] Further preferably, the reaction time of step (1) is 3 hours; after the reaction is completed and the system returns to room temperature, the reaction solution is added dropwise to anhydrous n-hexane at -20°C to precipitate a solid, which is then filtered to obtain a solid product.

[0036] Preferably, in step (2) a, CH3O-PEG-NH2 and Lys(z)-NCA are placed in a two-necked flask, evacuated, and air is isolated by argon gas, and anhydrous dimethyl sulfoxide is added to the flask to dissolve, and then stirred at 50°C for reaction, and dialyzed and freeze-dried after 24 hours to obtain the product CH3O-PEG-poly-Lysine(z); Further preferably, the molar ratio of CH3O-PEG-NH2 to Lys(z)-NCA in step (2)a is 1:16.

[0037] Further preferably, in step (2) a, CH3O-PEG-NH2 and Lys(z)-NCA are placed in a two-necked flask, evacuated under argon protection and repeated three times until the air is completely evacuated, and then anhydrous dimethyl sulfoxide is added; during dialysis, a dialysis bag (MWCO: 3500) is used to dialyze the loaded reactants in pure water, the water is changed every 12 hours, the dialysis is performed for 24 hours, and the product is obtained after freeze-drying.

[0038] Preferably, in step (2) b, Mal-PEG-NH2 and Lys(z)-NCA are placed in a two-necked flask, evacuated, and the air is isolated by argon gas. Anhydrous dimethyl sulfoxide is added to the flask to dissolve, and the mixture is stirred at 50° C. to react. After 24 hours, the mixture is dialyzed and freeze-dried to obtain the product Mal-PEG-poly-Lysine(z).

[0039] Further preferably, the molar ratio of Mal-PEG-NH2 to Lys(z)-NCA in step (2)b is 1:16.

[0040] Further preferably, in step (2) b, Mal-PEG-NH2 and Lys(z)-NCA are placed in a two-necked flask, evacuated, and isolated from the air by argon gas, and then anhydrous dimethyl sulfoxide is added to the flask to dissolve; during dialysis, a dialysis bag (MWCO: 3500) is used to dialyze the loaded reactants in pure water, the water is changed every 12 hours, the dialysis is performed for 24 hours, and the product is obtained after freeze-drying. Preferably, the molar ratio of Mal-PEG-poly-Lysine (z) to GE11 polypeptide in step (3) is 1:1 Further preferably, the solvent used in step (3) is a mixed solvent of PBS at pH 7.4 and anhydrous DMSO; after the reaction is completed, a dialysis bag (MWCO: 3500) is used to load the reactants and dialyze them in deionized water for 24 h, with the water being changed every 12 h, and the product is obtained after freeze-drying.

[0041] Preferably, in step (4) a, CH3O-PEG-poly-Lysine (z) is placed in a single-necked flask, trifluoroacetic acid and a 33% hydrobromic acid solution dissolved in acetic acid are added for reaction, the reaction is stirred at room temperature, and after 3 hours, the product CH3O-PEG-poly-Lysine is obtained by dialyzing and freeze-drying.

[0042] Preferably, the volume ratio of trifluoroacetic acid to 33% hydrobromic acid solution dissolved in acetic acid in step (4) a is 95:5.

[0043] Further preferably, the reaction in step (4) a is strictly controlled at room temperature for 3 h. 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 h, with the water being changed every 8 h. The product is obtained after freeze-drying.

[0044] Preferably, in step (4) b, GE11-PEG-poly-Lysine (z) is placed in a single-necked flask, trifluoroacetic acid and a 33% hydrobromic acid solution dissolved in acetic acid are added to react, the reaction is stirred at room temperature, and after 3 hours, the product GE11-PEG-poly-Lysine is obtained by dialyzing and freeze-drying; Further preferably, the volume ratio of trifluoroacetic acid to 33% hydrobromic acid solution dissolved in acetic acid in step (4) b is 95:5; Further preferably, 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, with the water being changed every 12 hours. The product is obtained after freeze-drying.

[0045] Preferably, CH3O-PEG-poly-Lysine and triethylamine in step (5) a are placed in a two-necked flask at a molar ratio of 1:1, evacuated, 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 dialysis bag with a MWCO of 7000 and dialyzed in deionized water for 24 h, with the water being changed every 8 h. The product is obtained after freeze-drying.

[0046] Preferably, the GE11-PEG-poly-Lysine and triethylamine in step (5) b are placed in a two-necked flask at a molar ratio of 1:1, evacuated, 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, a dialysis bag (MWCO: 7000) is used to load the reactants and dialyze them in deionized water for 24 h, with the water being changed every 8 h. The product is obtained after freeze-drying. Preferably, in step (6), gemcitabine and triethylamine are placed in a two-necked flask, evacuated, and the air is isolated by argon gas, and anhydrous N,N-dimethylformamide is added to the flask to dissolve, and then the temperature is lowered to 0°C, and then hard acyl chloride is added to the system and placed in an oil bath at 35°C, stirred for reaction, and the reaction is monitored by thin layer chromatography after 24 hours; and the improved gemcitabine is purified by column chromatography; Further preferably, the molar ratio of gemcitabine, triethylamine and octadecanoyl chloride in step (6) is 1:20:11. First, gemcitabine and triethylamine are added to anhydrous DMF and stirred at room temperature for 5 minutes, and then cooled to 0°C; octadecanoyl chloride is dissolved in 2 mL of anhydrous N,N-dimethylformamide, slowly added dropwise within 15 minutes, and stirred at room temperature for 24 hours. Then, it is washed three times with saturated brine, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and after removing ethyl acetate by rotary evaporation, a white waxy solid is obtained after purification under the condition of a dichloromethane: methanol volume ratio of 20:1.

[0047] The present invention also provides the use of the acid-responsive drug delivery nanoparticles adapted to pancreatic cancer in the preparation of drugs for treating pancreatic cancer.

[0048] Compared with the prior art, the present invention has the following advantages: (1) The nano drug delivery system described in the present invention has high biosafety.

[0049] (2) The present invention can release drugs in response to the pancreatic tumor microenvironment, inducing multimodal death of tumor cells. Iron ions and leflunomide synergistically induce ferroptosis, and leflunomide and gemcitabine synergistically induce autophagy of tumor cells. The combination of ferroptosis and autophagy can effectively kill tumor cells.

[0050] (3) The method of the present invention uses modified gemcitabine to improve the drug resistance of small molecule chemotherapy drugs.

[0051] (4) The present invention provides a nano drug delivery system that can penetrate the external matrix of pancreatic cancer and achieve deep drug accumulation.

[0052] (5) The nano drug delivery system described in the present invention realizes global regulation from a single cell perspective and improves the immunosuppressive microenvironment of pancreatic cancer tumors through metabolic-immune crosstalk. It interferes with cell mitochondrial metabolism, induces mitochondrial exocytosis of tumor cells, inhibits T cell exhaustion, and promotes the transformation of effector T cells into memory T cells by affecting intercellular crosstalk. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is the nuclear magnetic resonance spectrum of CH3O-PEG-poly-Lysine-PCA in Example 1 of the present invention.

[0054] Figure 2 This is the nuclear magnetic resonance spectrum of GE11-PEG-poly-Lysine-PCA in Example 1 of the present invention.

[0055] Figure 3 The graphs are the particle size and zeta potential of the nanoparticles in Example 1 of the present invention and the graph of the stable particle size change over 7 days; wherein, Graph A is the particle size of the nanoparticles, Graph B is the potential, and Graph C is the graph of the stable particle size change over 7 days.

[0056] Figure 4 Figure 1 is a flow cytometry result diagram of the nanoparticles with different target head modification degrees being taken up by tumor cells in the present invention; wherein, Figure A is a flow cytometry result diagram, and Figure B is a quantification of Figure A.

[0057] Figure 5 Figure 1 is the targeting effect of the nanoparticles of the present invention in KPC Luci orthotopic pancreatic cancer-bearing mice; Figure A is the distribution result of the nanoparticles in the tumor-bearing mice, and Figure B is the quantitative result of the main organs.

[0058] Figure 6 These are the results of the anti-tumor therapeutic effect investigation of nanoparticles in mice bearing KPC Luci orthotopic pancreatic cancer and the weight changes of mice after treatment; Figure A shows the tumor growth signal in the mouse, and Figure B shows the weight changes of the mouse.

[0059] Figure 7These are the results of investigating the changes in metabolites in tumor tissues of nanoparticles in mice bearing KPC orthotopic pancreatic cancer; Figure A shows the results of GSH in tumor tissues after treatment; Figure B shows the results of NADP+ / NADPH in tumor tissues after treatment; Figure C shows the results of LPO in tumor tissues after treatment; and Figure D shows the results of ROS in tumor tissues after treatment.

[0060] Figure 8 Figure 5 is the flow cytometry result of nanoparticles in KPC orthotopic pancreatic cancer mice; Figure A is the quantitative result of memory T cells; Figure B is the quantitative result of mature DC in lymph nodes; Figure C is the quantitative result of CTL cells in tumor tissue; Figure D is the quantitative result of Th1 cells in tumor tissue; Figure E is the quantitative result of Treg cells in tumor tissue; Figure F is the quantitative result of MDSC cells in tumor tissue; Figure G is the quantitative result of PD1+ CD8+ T cells in tumor tissue; Figure H is the quantitative result of LAG3+ CD8+ T cells in tumor tissue; Figure I is the quantitative result of TIM3+ CD8+ T cells in tumor tissue; Figure J is the quantitative result of exhausted T cells in tumor tissue; Figure K is the ratio of early exhausted T cells and terminal exhausted T cells in tumor tissue. DETAILED DESCRIPTION

[0061] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0062] Example 1 An acid-responsive drug delivery nanoparticle adapted for pancreatic cancer and a preparation method thereof, wherein the drug delivery nanoparticle synthesis method comprises the following steps: (1) 5 g, 17.85 mmol N6-benzyloxycarbonyl-L-lysine and 2.25 g, 7.6 mmol triphosgene were added under vacuum, 50 mL of anhydrous tetrahydrofuran was added, argon was filled in an oil bath and heated to 50 °C with stirring for reaction. After 3 h, the solvent was concentrated by rotary evaporation, and the reaction solution was then added dropwise to 300 mL of anhydrous n-hexane precooled at -20 °C to precipitate a solid precipitate, which was filtered to obtain Lys(z)-NCA.

[0063] (2) a. Combine 500 mg, 0.1 mmol CH3O-PEG-NH2 and 398 mg, 1.6 mmol Lys(z)-NCA, evacuate under argon protection and repeat 3 times until the air is completely evacuated, add 20 mL of anhydrous dimethyl sulfoxide, heat to 50 °C in an oil bath and stir to react, place at 50 °C and stir to react. After 24 h, use a dialysis bag (MWCO: 3500) to load the reactants and dialyze against pure water, changing the water every 12 h. After freeze-drying, the product CH3O-PEG-poly-Lysine(z) was obtained.

[0064] b: Place 500 mg, 0.1 mmol Mal-PEG- NH2 and 398 mg, 1.6 mmol Lys(z)-NCA in a two-necked flask, evacuate the flask, isolate the air with argon, add 20 mL of anhydrous dimethyl sulfoxide into the flask, fill with argon for protection, heat the oil bath to 50 °C and stir to react, place at 50 °C and stir to react, after 24 hours, use a dialysis bag (MWCO: 3500) to load the reactants and dialyze in pure water, change the water every 12 hours, and freeze-dry to obtain the product Mal-PEG-poly-Lysine(z). (3) 100 mg, 0.012 mmol Mal-PEG-poly-Lysine (z) and 20 mg, 0.012 mmol GE11 peptide were dissolved in a mixed solvent of PBS (pH 7.4) and anhydrous DMSO in a volume ratio of 1:1, and the mixture was allowed to react overnight at room temperature. After the reaction was completed, a dialysis bag (MWCO: 3500) was used to load the reactants and dialyzed against deionized water for 24 h. The water was changed every 12 h, and the product GE11-PEG-poly-Lysine (z) was obtained after freeze-drying.

[0065] (4) a: 200 mg, 0.022 mmol CH3O-PEG-poly-Lysine (z) was reacted with 9.5 mL trifluoroacetic acid and 500 μL 33% hydrobromic acid solution in acetic acid at room temperature for 3 h. After the reaction, the reactants were loaded into a dialysis bag (MWCO: 3500) and dialyzed against deionized water for 24 h. The water was changed every 12 h and the product CH3O-PEG-poly-Lysine was obtained after freeze-drying.

[0066] b: 200 mg, 0.019 mmol GE11-PEG-poly-Lysine (z) was reacted with 9.5 mL trifluoroacetic acid and 500 μL 33% hydrobromic acid solution in acetic acid at room temperature for 3 h. After the reaction was completed, the reactants were loaded into a dialysis bag (MWCO: 3500) and dialyzed in deionized water for 24 h. The water was changed every 12 h, and the product GE11-PEG-poly-Lysine was obtained after freeze-drying.

[0067] (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 and evacuated. The mixture was stirred at room temperature for 30 min under argon protection. Subsequently, 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 allowed to react for 18 h. After the reaction was completed, the reactants were loaded into a dialysis bag (MWCO: 7000) and dialyzed against deionized water for 24 h. The water was changed every 8 h. The product CH3O-PEG-poly-Lysine-PCA was obtained after freeze-drying.

[0068] 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. Subsequently, 48 mg, 0.35 mmol 3, 4-dihydroxybenzaldehyde (PCA) was dissolved in 3 mL anhydrous dimethyl sulfoxide and added to the reaction system for 18 h. After the reaction was completed, the reactants were loaded into a dialysis bag (MWCO: 7000) and dialyzed in deionized water for 24 h. The water was changed every 8 h. The product GE11-PEG-poly-Lysine-PCA was obtained after freeze-drying.

[0069] (6) 30 mg, 0.01 mmol gemcitabine and 20 mg, 0.2 mmol triethylamine were dissolved in anhydrous N, N-dimethylformamide and stirred at room temperature for 5 min. After cooling to 0°C, 33 mg, 0.11 mmol hard acyl chloride was dissolved in 2 mL anhydrous N, N-dimethylformamide and slowly added dropwise to the reaction system within 15 min. The mixture was stirred at room temperature for 24 h. The mixture was then washed three times with saturated brine, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and the ethyl acetate was removed by rotary evaporation. The mixture was purified under the condition of a dichloromethane:methanol volume ratio of 20:1 to obtain a white waxy solid.

[0070] (7) The acid-responsive drug delivery nanoparticles were prepared by a one-pot method, specifically, 6 mg CH3O-PEG-poly-Lysine-PCA (polymer 1), 4 mg GE11-PEG- poly-Lysine-PCA (polymer 2) 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, and stirred at 3000 rpm for 10 min at room temperature. Then, a 1 mg / mL ferric chloride solution was diluted to obtain a solution, and the solution was added dropwise to the above system, and then stirred at 3000 rpm for 12 h at room temperature to obtain nanoparticles (target head modification degree was 40%).

[0071] Comparative Example 1 A kind of acid-responsive drug delivery nanoparticles adapted for pancreatic cancer and a preparation method. The difference between the preparation method and Example 1 is that when 8 mL of water-soluble preparation is added during the nanoparticle preparation stage, it is found that the particle size of the obtained nanoparticles is above 200 nm and the polydispersity coefficient is close to 0.5.

[0072] Comparative Example 2 (corresponding to the target head modification degree of 0) A hypoxia-responsive drug delivery nanoparticle adapted for KRAS mutant pancreatic cancer and a preparation method thereof, which is different from Example 1 in that in step (7), 10 mg of CH3O-PEG-poly-Lysine-PCA (a polymer with a structural formula as shown in Formula I) is dissolved in 10 mL of water, 1 mL of a 1 mg / mL methanol solution of modified gemcitabine, and 1 mL of a 1 mg / mL methanol solution of leflunomide.

[0073] Comparative Example 3 (corresponding to a target head modification degree of 10%) A hypoxia-responsive drug delivery nanoparticle adapted for KRAS mutant pancreatic cancer and a preparation method thereof, which is different from Example 1 in that in step (7), 9 mg of CH3O-PEG-poly-Lysine-PCA (a polymer having a structural formula as shown in Formula I) and 1 mg of GE11-PEG-poly-Lysine-PCA are dissolved in 10 mL of water, 1 mL of a 1 mg / mL methanol solution of modified gemcitabine and 1 mL of a 1 mg / mL methanol solution of leflunomide.

[0074] Comparative Example 4 (corresponding to a target head modification degree of 20%) A hypoxia-responsive drug delivery nanoparticle adapted for KRAS mutant pancreatic cancer and a preparation method thereof, which is different from Example 1 in that in step (7), 8 mg of CH3O-PEG-poly-Lysine-PCA (a polymer having a structural formula as shown in Formula I) and 2 mg of GE11-PEG-poly-Lysine-PCA are dissolved in 10 mL of water, 1 mL of a 1 mg / mL methanol solution of modified gemcitabine and 1 mL of a 1 mg / mL methanol solution of leflunomide.

[0075] Comparative Example 5 (corresponding to a target head modification degree of 30%) A hypoxia-responsive drug delivery nanoparticle adapted for KRAS mutant pancreatic cancer and a preparation method thereof, which is different from Example 1 in that in step (7), 7 mg of CH3O-PEG-poly-Lysine-PCA (a polymer having a structural formula as shown in Formula I) and 3 mg of GE11-PEG-poly-Lysine-PCA are dissolved in 10 mL of water, 1 mL of a 1 mg / mL methanol solution of modified gemcitabine and 1 mL of a 1 mg / mL methanol solution of leflunomide.

[0076] Comparative Example 6 (corresponding to a target head modification degree of 50%) A hypoxia-responsive drug delivery nanoparticle adapted for KRAS mutant pancreatic cancer and a preparation method thereof, which is different from Example 1 in that in step (7), 5 mg of CH3O-PEG-poly-Lysine-PCA (a polymer having a structural formula as shown in Formula I) and 5 mg of GE11-PEG-poly-Lysine-PCA are dissolved in 10 mL of water, 1 mL of a 1 mg / mL methanol solution of modified gemcitabine and 1 mL of a 1 mg / mL methanol solution of leflunomide.

[0077] The following characterization and testing were performed on the drug-loaded nanoparticles: Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of CH3O-PEG-poly-Lysine-PCA in Example 1, indicating that 16 PCAs are connected to one polymer molecular chain.

[0078] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of GE11-PEG-poly-Lysine-PCA in Example 1, indicating that 16 PCAs and 1 GE11 polypeptide are connected to one polymer molecular chain.

[0079] The obtained nanoparticles are in the shape of regular spheres, such as Figure 3 As shown in A, the particle size distribution is uniform, and the average particle size is 150nm; Figure 3 B shows that the zeta potential is 1.26 mV; Figure 3 C shows that the particle size and PDI change little after being placed in PBS with pH 7.4 for 7 days. The HPLC results show that the drug loading of leflunomide is 7.31±0.32%, and the drug loading of modified gemcitabine is 3.01±1.26%.

[0080] Flow cytometry experiments were used to examine the uptake of nanoparticles with different target head modification degrees (target head modification degree refers to the mass ratio of the material containing the target head to the polymer material) by tumor cells. Figure 4 The flow cytometry results of nanoparticles with different modification degrees (Comparative Examples 2-6 and Example 1) being taken up by tumor cells show that when the modification degree of the target head is 40% (Example 1), the tumor cells have the most significant uptake of the nanoparticles. The modification degree of the target head of the nanoparticles in the following experiments is 40%.

[0081] Subsequently, the targeting of nanoparticles in mice bearing KPC orthotopic pancreatic cancer was investigated. First, the hypoxia-responsive nanoparticles were labeled with fluorescent probes. The specific steps were as follows: 6 mg CH3O-PEG-poly-Lysine-PCA (polymer 1), 4 mg GE11-PEG- poly-Lysine-PCA (polymer 2) were weighed and dissolved in 10 mL of water, 1 mL of 1 mg / mL Cy5 probe aqueous solution, and stirred at room temperature for 10 min at 3000 rpm. Then, a 1 mg / mL ferric chloride solution was diluted, added dropwise to the above system, and then stirred at room temperature for 12 h at 3000 rpm to obtain nanoparticles, and Cy5-labeled nanoparticles were obtained.

[0082] The following experimental method is to configure KPC cells into 1 × 10 6 / 100 μL of cell suspension. After the male C57 was fully anesthetized, part of the pancreas of the nude mouse was exposed surgically, and the prepared cell suspension was injected in situ into the pancreas with an insulin needle to establish a PDAC orthotopic tumor model. Different preparations were intravenously administered to pancreatic cancer-bearing mice. At multiple time points within 24 hours, the IVIS Spectrum was used to monitor the in vivo signal distribution of the fluorescent probe encapsulated in the preparation. The mice were dislocated 24 hours after administration. The heart was perfused with physiological saline and paraformaldehyde, and the heart, liver, spleen, lungs, and kidneys of the mice were removed. The distribution of the fluorescent probe signal in the main organs of the mice was further observed under the in vivo optical imaging system. The results are shown in Figure 5As shown, A is the distribution result of nanoparticles in tumor-bearing mice, showing the specific tumor accumulation performance of GGP-Cy5 (indicating the tumor accumulation performance of nanoparticles modified with GE11 peptide polymers), Cy5 indicates nanoparticles, and PP-Cy5 indicates that GE11-PEG-poly-Lysine-PCA is replaced with an equal mass of CH3O-PEG-poly-Lysine-PCA and the nanoparticles are prepared in the same way. B is the quantitative result of ex vivo tissue, indicating that the preparation has a significant targeting effect on tumor tissue in vivo.

[0083] The anti-tumor effect of the nanoparticles in tumor-bearing mice was then investigated (the preparation was injected intravenously into mice bearing orthotopic pancreatic tumors, and the size of the tumor fluorescence signal, as well as the weight changes of the mice and the survival status after administration were monitored). The results are as follows Figure 6 As shown, Figure A is a curve showing the change of tumor signal over time, and Figure B is a curve showing the change of mouse weight over time. Figure 6 G1-G8 represent tumor-bearing mice with different drug treatments. The preparations administered to mice in each group are Group 1: Saline (normal saline), Group 2: Gem 1.5 mg / kg + Lef 3 mg / kg (free drug at the same dose as the preparation), Group 3: Gem 1.5 mg / kg + Nab-Paclitaxel 0.19 mg / kg (clinical free drug at the same dose as the preparation), 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 (non-drug-loaded nanoparticles), Group 7: GPPG (gemcitabine-loaded nanoparticles), Group 8: GPPG / L (final preparation). It shows that the preparation has a good anti-tumor effect in vivo, and does not cause weight loss in mice in vivo, and has good biosafety.

[0084] Then, the changes in the levels of related metabolites in tumor tissue after administration were investigated. Figure 7 The figure shows 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.

[0085] The proportion and type of immune cells in tumor tissues and lymph nodes after treatment were then examined, and the types and proportions of immune cells were examined by flow cytometry. Memory T cells were marked by 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). DC cells in lymph nodes were marked by 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 marked by 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 marked by 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 marked by 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 by 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 G1-G8 represent tumor-bearing mice with different drug treatments. The preparations administered to mice in each group are Group 1: Saline (normal saline), Group 2: Gem 1.5 mg / kg + Lef 3 mg / kg (free drug at the same dose as the preparation), Group 3: Gem 1.5 mg / kg + Nab-Paclitaxel 0.19 mg / kg (clinical free drug at the same dose as the preparation), 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 (non-drug-loaded nanoparticles), Group 7: GPPG (gemcitabine-loaded nanoparticles), Group 8: GPPG / L (final preparation). It shows that the Group 8 preparation can activate anti-tumor immune response. Figure 8 A represents the proportion of Memory T cells in tumor tissue. Figure 8 B represents the ratio of mature DC cells in lymphoid tissue. Figure 8 C represents the ratio of CTL cells in tumor tissue. Figure 8 D represents the ratio of Th1 cells in tumor tissue. Figure 8 E represents the ratio of Treg cells in tumor tissue. Figure 8 F represents the ratio of MDSC-positive cells in tumor tissue. Figure 8 G represents the ratio of PD1+ cells in tumor tissue. Figure 8 H represents the ratio of LAG3+ positive cells in tumor tissue. Figure 8 I represents the ratio of TIM3+ positive cells in tumor tissues, Figure 8 J represents the proportion of exhausted T cells. Figure 8 K is the ratio of the number of exhausted T cells in the early and terminal stages of tumor tissue.

Claims

1. An acid-responsive drug delivery nanoparticle adapted for pancreatic cancer, characterized in that: The invention is obtained by chelating iron ions with an amphiphilic polyphenol polymer and encapsulating chemotherapeutic drugs and DHODH inhibitors for pancreatic cancer. The polymer comprises: an amphiphilic polymer 1 modified with polyphenols, and an amphiphilic polymer 2 modified with polyphenols and having a targeting polypeptide modified at one end.

2. The acid-responsive drug delivery nanoparticle adapted for pancreatic cancer according to claim 1, characterized in that: The polymer has a macromolecular chain which comprises a polyethylene glycol hydrophilic segment and a polylysine-polyphenol block in sequence from one end to the other end; Preferably, the chemotherapy drug for pancreatic cancer is modified gemcitabine; Preferably, the targeting polypeptide in the polymer 2 is GE11; Preferably, the DHODH inhibitor is leflunomide; More preferably, the drug loading of leflunomide is 7.31±0.32%, and the drug loading of improved gemcitabine is 3.01±1.26%.

3. The acid-responsive drug delivery nanoparticle adapted for pancreatic cancer according to claim 2, characterized in that: The structural formula of the polymer 1 is shown in Formula I; the structural formula of the polymer 2 is shown in Formula II; Formula I Formula II Preferably, the mass ratio of polymer 1 to polymer 2 is 60:40; 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.

4. The method for preparing the acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) N6-benzyloxycarbonyl-L-lysine is reacted 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-NH2 and Lys(z)-NCA are subjected to a ring-opening polymerization reaction to obtain Mal-PEG-poly-Lysine(z); the reaction process is shown in Formula VI: Formula VI (3) Mal-PEG-poly-Lysine (z) is subjected to a click reaction with the 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 and 33% hydrobromic acid solution dissolved in acetic acid to obtain CH3O-PEG-poly-Lysine; the reaction process is shown in Formula VIII: Formula VIII Or, b: GE11-PEG-poly-Lysine (z) reacts with trifluoroacetic acid and 33% hydrobromic acid solution dissolved in acetic acid to obtain GE11-PEG-poly-Lysine; the reaction process is shown in Formula IX: Formula IX (5) a: CH3O-PEG-poly-Lysine is subjected to a Schiff base synthesis reaction with 3,4-dihydroxybenzaldehyde (PCA) and triethylamine to obtain CH3O-PEG-poly-Lysine-PCA; the reaction process is shown in Formula X: Formula X Or, b: GE11-PEG-poly-Lysine is subjected to a Schiff base synthesis reaction with 3,4-dihydroxybenzaldehyde and triethylamine to obtain GE11-PEG-poly-Lysine-PCA; the reaction process is shown in Formula XI: Formula XI (6) Gemcitabine is subjected to Friedel-Crafts acylation reaction with octadecanoyl chloride to obtain improved gemcitabine: the reaction process is shown in Formula XII Formula Ⅻ (7) The acid-responsive drug delivery nanoparticles adapted for pancreatic cancer are prepared by a one-pot method, and the specific steps are as follows: polymer 1, polymer 2, modified gemcitabine and leflunomide are dissolved in a solvent, and an aqueous solution of ferric chloride is added dropwise under high-speed stirring. After stirring for 8 hours, the acid-responsive drug delivery nanoparticles adapted for pancreatic cancer are obtained by dialyzing.

5. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 4, characterized in that: Step (1): Place N6-benzyloxycarbonyl-L-lysine and triphosgene in a two-necked flask, evacuate the flask, isolate the air with argon, then add anhydrous tetrahydrofuran to dissolve, and then stir and react at 50°C to obtain a white block solid Lys(z)-NCA; Preferably, the molar ratio of N6-benzyloxycarbonyl-L-lysine to triphosgene in step (1) is 2.35:1; Preferably, in step (1), N6-benzyloxycarbonyl-L-lysine and triphosgene are added to anhydrous tetrahydrofuran, and the reaction efficiency is higher after the sample is completely dissolved; Preferably, the reaction time of step (1) is 3 hours; after the reaction is completed and the system returns to room temperature, the reaction solution is added dropwise to anhydrous n-hexane at -20°C to precipitate a solid, which is then filtered to obtain a solid product; Preferably, in step (2) a, CH3O-PEG-NH2 and Lys(z)-NCA are placed in a two-necked flask, evacuated, and air is isolated by argon gas, and anhydrous dimethyl sulfoxide is added to the flask to dissolve, and then stirred at 50°C for reaction, and dialyzed and freeze-dried after 24 hours to obtain the product CH3O-PEG-poly-Lysine(z); Further preferably, the molar ratio of CH3O-PEG-NH2 and Lys(z)-NCA in step (2) a is 1:16; Further preferably, in step (2) a, CH3O-PEG-NH2 and Lys(z)-NCA are placed in a two-necked flask, evacuated under argon protection and repeated 3 times until the air is completely evacuated, and then anhydrous dimethyl sulfoxide is added; during dialysis, a dialysis bag (MWCO: 3500) is used to dialyze the loaded reactants in pure water, the water is changed every 12 hours, the dialysis is performed for 24 hours, and the product is obtained after freeze-drying; Preferably, step (2) b: Mal-PEG-NH2 and Lys(z)-NCA are placed in a two-necked flask, evacuated, and isolated from the air by argon gas, and anhydrous dimethyl sulfoxide is added to the flask to dissolve, and then stirred at 50°C for reaction, and dialyzed and freeze-dried after 24 hours to obtain the product Mal-PEG-poly-Lysine(z); Further preferably, the molar ratio of Mal-PEG-NH2 to Lys(z)-NCA in step (2) b is 1:16; Further preferably, in step (2) b, Mal-PEG-NH2 and Lys(z)-NCA are placed in a two-necked flask, evacuated, and isolated from the air by argon gas, and then anhydrous dimethyl sulfoxide is added to the flask to dissolve; during dialysis, the loaded reactants are dialyzed in pure water using a dialysis bag with a MWCO of 3500, the water is changed every 12 hours, the dialysis is performed for 24 hours, and the product is obtained after freeze-drying.

6. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 4, characterized in that: Step (3): dissolving Mal-PEG-poly-Lysine (z) and GE11 polypeptide in a mixed solvent of PBS and DMSO in a volume ratio of 1:1, leaving the mixture to react overnight at room temperature, dialyzing and freeze-drying to obtain the product GE11-PEG-poly-Lysine (z); the molar ratio of Mal-PEG-poly-Lysine (z) to GE11 polypeptide in step (3) is 1:1; Preferably, the solvent used in step (3) is a mixed solvent of PBS with a pH of 7.4 and anhydrous DMSO; 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 h, with the water being changed every 12 h, and the product is obtained after freeze-drying.

7. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 4, characterized in that: Step (4) a: CH3O-PEG-poly-Lysine (z) is placed in a single-necked flask, trifluoroacetic acid and a 33% hydrobromic acid solution dissolved in acetic acid are added to react, the reaction is stirred at room temperature, and after 3 hours, dialyzed and freeze-dried to obtain the product CH3O-PEG-poly-Lysine; Preferably, the volume ratio of trifluoroacetic acid to 33% hydrobromic acid solution dissolved in acetic acid in step (4) a is 19:1; Preferably, step (4) a: the reaction 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 8 hours, and the product is obtained after freeze-drying; Preferably, step (4) b: GE11-PEG-poly-Lysine (z) is placed in a single-necked flask, trifluoroacetic acid and a 33% hydrobromic acid solution dissolved in acetic acid are added to react, the reaction is stirred at room temperature, and after 3 hours, the product GE11-PEG-poly-Lysine is obtained by dialyzing and freeze-drying; Further preferably, the volume ratio of trifluoroacetic acid to 33% hydrobromic acid solution dissolved in acetic acid in step (4) b is 19:1; Further preferably, in step (4) b: the reaction 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 8 hours, and the product is obtained after freeze-drying.

8. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 4, characterized in that: The CH3O-PEG-poly-Lysine and triethylamine in step (5) a are placed in a two-necked flask at a molar ratio of 1:1, and the mixture is evacuated. The mixture is stirred at room temperature for 30 min under argon protection, and then PCA dissolved in anhydrous dimethyl sulfoxide is added to the flask at a molar ratio of 1:

16. The mixture is reacted for 18 h. After the reaction is completed, a dialysis bag (MWCO: 7000) is used to load the reactants and dialyze them in deionized water for 24 h. The water is changed every 8 h, and the product is obtained after freeze-drying; CH3O-PEG-poly-Lysine-PCA; Preferably, step (5) b: GE11-PEG-poly-Lysine and triethylamine are placed in a two-necked flask at a molar ratio of 1:1, evacuated, and stirred at room temperature for 30 minutes 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 hours. After the reaction is completed, a dialysis bag (MWCO: 7000) is used to load the reactants and dialyze them in deionized water for 24 hours, with the water being changed every 8 hours. The product is obtained after freeze-drying; GE11-PEG-poly-Lysine-PCA.

9. The method for preparing acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to claim 4, characterized in that: Step (6): Gemcitabine and triethylamine are placed in a two-necked flask, and the flask is evacuated. After the air is isolated by argon, anhydrous N,N-dimethylformamide is added to the flask to dissolve, and then the temperature is lowered to 0°C. Then, stearoyl chloride is added to the system and placed in an oil bath at 35°C, and the reaction is stirred. After 24 hours, the reaction is monitored by thin layer chromatography; and the improved gemcitabine is purified by column chromatography; Preferably, the molar ratio of gemcitabine, triethylamine and octadecanoyl chloride in step (6) is 1:2:1.

1. First, add anhydrous DMF to gemcitabine and triethylamine, stir at room temperature for 5 minutes, and then cool to 0°C; dissolve stearoyl chloride in 2 mL of anhydrous N,N-dimethylformamide, slowly add dropwise within 15 minutes, stir at room temperature for 24 hours, 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 the condition of a dichloromethane:methanol volume ratio of 20:1 to obtain a white waxy solid.

10. Use of the acid-responsive drug delivery nanoparticles adapted for pancreatic cancer according to any one of claims 1 to 3 in the preparation of a drug for treating pancreatic cancer.

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