Nuciferine-based digestive tract tumor targeting nano preparation and preparation method thereof

Abstract

The invention relates to the technical field of medicine, and discloses a nuciferine-based nano preparation for targeting digestive tract tumors and a preparation method thereof.The nuciferine-based nano preparation for targeting digestive tract tumors comprises nuciferine, 1, 2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine, cholesterol, cholesterol hemisuccinate, 1, 2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine, cholesterol hemisuccinate, 1, 2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine, cholesterol hemisuccinate, 1 the lipid vesicle is composed of 1, 2-dioleoyl-sn-glycerol-3-phosphatidyl ethanolamine and distearoyl phosphatidyl ethanolamine-polyethylene glycol (2000)-cyclo (essence-glycerol-radix asparagi-styrene-acrylic-lysine) polypeptide, and the lipid vesicle is composed of 2, 2-dioleoyl-sn-glycerol-3-phosphatidyl ethanolamine and distearoyl phosphatidyl ethanolamine-polyethylene glycol (2000)- The preparation method of the nuciferine-based digestive tract tumor targeting nano preparation comprises the steps of film dispersion, hydration, high-pressure extrusion and purification. The active targeting effect of the cyclo (essence-glycerol-asparagi-styrene-acrylic-lysine) polypeptide ligand is utilized. Synergistic release of drugs in a tumor microenvironment is achieved, the targeting efficiency and local drug concentration of nuciferine are improved, and the anti-tumor effect is enhanced.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a nano-formulation based on lotus leaf alkaloids that targets gastrointestinal tumors and its preparation method. Background Technology

[0002] Gastrointestinal tumors are among the most common and deadliest malignant tumors worldwide. Lotus leaf alkaloid, an apophene alkaloid extracted from lotus plants, has been shown in recent years to have broad-spectrum anti-tumor activity, especially in inhibiting the proliferation of gastrointestinal tumor cells (such as pancreatic cancer and colorectal cancer) and inducing their apoptosis.

[0003] However, the physicochemical properties and pharmacokinetic defects of lotus leaf alkaloids themselves severely restrict their clinical application. Lotus leaf alkaloids have poor water solubility, poor stability in body fluids, and are easily metabolized and eliminated by the body, resulting in an extremely short half-life and very low bioavailability after oral or intravenous injection. This makes it difficult for the free drug to reach and maintain an effective therapeutic concentration at the tumor site, thus limiting its efficacy.

[0004] To overcome these obstacles, existing technologies attempt to encapsulate lotus leaf alkaloids using nanocarriers. While this strategy can improve drug stability and prolong its circulation time in vivo to some extent, most traditional nanocarriers lack tumor specificity. Their distribution in vivo mainly relies on passive targeting, and they cannot actively recognize and bind to tumor cells, resulting in limited drug accumulation efficiency at the tumor site. Most carriers are still cleared by the mononuclear phagocyte system or misdistributed in healthy tissues, making it difficult to achieve precise treatment.

[0005] Furthermore, controlling the efficient drug release of nanocarriers after reaching the tumor site is another major challenge facing current technologies. Many highly stable nanocarriers exhibit slow drug release upon reaching tumor tissue, preventing timely release of drugs from the carrier to exert cytotoxic effects. Although studies have developed single tumor microenvironment-responsive release systems, these systems are often not sensitive enough to environmental changes or release drugs insufficiently, failing to fully adapt to the complex characteristics of the tumor microenvironment, which features both low pH and high enzyme activity. This results in lower-than-expected drug release efficiency, affecting the final therapeutic effect. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a nano-formulation based on lotus leaf alkaloids that targets gastrointestinal tumors and its preparation method. This invention solves the problems of low bioavailability of lotus leaf alkaloids, lack of active tumor targeting by traditional nano-delivery carriers, and insufficient drug release in response to the tumor microenvironment in existing technologies.

[0007] To achieve the above objectives, this invention provides a nano-formulation based on lotus leaf alkaloids that targets gastrointestinal tumors and its preparation method.

[0008] In a first aspect, the present invention provides a nano-formulation based on lotus leaf alkaloids that targets gastrointestinal tumors, employing the following technical solution: The lotus leaf alkaloid-based nano-formulation targeting gastrointestinal tumors is a liposome formulation comprising: Lotus leaf alkaloid; Vesicle structures formed by lipid excipients, wherein the lipid excipients comprise: 1,2-Dipalmitoyl-sn-glycerol-3-phosphatidylcholine; cholesterol; Cholesterol hemisuccinate; 1,2-Dioleoyl-sn-glycerol-3-phosphatidylethanolamine; Distearate phosphatidylethanolamine-polyethylene glycol (2000)-cyclo(arginine-glycine-aspartic-phenylpropanoid-lysine) polypeptide; The molar ratio of the 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine, cholesterol, cholesterol hemisuccinate, 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine and distearate phosphatidylethanolamine-polyethylene glycol (2000)-cyclo(arginine-glycolic acid-aspartic acid-phenylpropanoid-lysine) polypeptide is (45-55):(15-25):(10-20):(10-20):(1-3); Furthermore, the total mass ratio of the lotus leaf alkaloid to the lipid excipient is 1:(10-20).

[0009] By adopting the above technical solution, the nano-formulation provided by the present invention utilizes the chemical properties and proportions of specific lipid components to construct a delivery function that integrates active targeting and pH / enzyme dual response synergy.

[0010] First, this invention utilizes distearylphosphatidylethanolamine-polyethylene glycol (2000)-cyclic (arginine-aspartic-phenylpropanoid-lysine) polypeptide as a targeting ligand. The cyclic (arginine-aspartic-phenylpropanoid-lysine) polypeptide moiety in this targeting ligand can specifically recognize and bind to αvβ3 integrin, which is highly expressed on the surface of gastrointestinal tumor cells (such as pancreatic cancer cells). Through receptor-mediated endocytosis, the nanoparticles are actively enriched into tumor cells, improving targeting efficiency.

[0011] Secondly, the vesicle structure of this invention is ingeniously designed, possessing dual pH and enzyme responsiveness. Its drug release mechanism is described in detail below: pH Response Mechanism: Cholesterol hemisuccinate, an excipient in the formulation, is a key pH-sensitive lipid. In a normal physiological environment (pH 7.4), its terminal carboxyl group dissociates, giving the liposome surface a negative charge, which maintains the stability of the formulation in the bloodstream through electrostatic repulsion. When the nano-formulation accumulates in the weakly acidic tumor microenvironment (pH 6.5), the carboxyl group of cholesterol hemisuccinate undergoes protonation, becoming neutral, leading to a reversal of the liposome surface charge. This charge change promotes the electrostatic adsorption and fusion of the formulation with the negatively charged tumor cell membrane. Furthermore, the protonation of cholesterol hemisuccinate disrupts the dense arrangement of the lipid bilayer and induces a transition of 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine from a lamellar phase to a hexagonal II phase, resulting in vesicle membrane destabilization, increased membrane permeability, and triggering the first phase of drug release.

[0012] Enzyme response mechanism: 1,2-Dipalmitoyl-sn-glycerol-3-phosphatidylcholine in the excipients is a major structural component of the liposome membrane and also a specific hydrolytic substrate for secretory phospholipase A2, which is highly expressed in the tumor microenvironment. When the formulation is exposed to a high concentration of phospholipase A2, phospholipase A2 specifically hydrolyzes the sn-2 ester bond of 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine, degrading it into lysophosphatidylcholine and free fatty acids. These two degradation products have strong membrane-dissolving effects, leading to irreversible disintegration of the vesicle structure and achieving large-scale drug release in the second stage.

[0013] Synergistic Enhancement Mechanism: The key to this invention lies in the synergistic effect of the pH response mechanism and the enzyme response mechanism. In the tumor microenvironment, the initial pH response (protonation of cholesterol hemisuccinate and phase transition of 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine) not only triggers preliminary drug release, but more importantly, it leads to a loosening and disordering of the lipid membrane arrangement, thereby increasing the exposure of the cleavage sites on the 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine molecule. This allows phospholipase A2 to more easily access and efficiently hydrolyze its substrate. Therefore, the pH response accelerates the rate and extent of the enzyme response, achieving a good synergistic drug release effect.

[0014] In summary, this invention achieves drug enrichment at the tumor site through an active targeting mechanism, and then achieves precise and efficient release of lotus leaf alkaloids at the target site through the synergistic effect of pH response and enzyme response, thereby improving the local concentration and anti-tumor activity of the drug and effectively reducing its toxic side effects on normal tissues.

[0015] In a preferred embodiment, the molar ratio of the 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine, cholesterol, cholesterol hemisuccinate, 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine and distearate phosphatidylethanolamine-polyethylene glycol (2000)-cyclo(arginine-glycine-aspartic-phenylpropanoid-lysine) polypeptide is (48-52):(18-22):(13-17):(13-17):(1.5-2.5), and the total mass ratio of the lotus leaf alkaloid to the lipid excipient is 1:(13-17).

[0016] By adopting the above technical solution, the ratio range is the optimized range of synergistic effect of each component, which can enable nano-formulations to obtain better encapsulation rate, stability and response release efficiency.

[0017] In a preferred embodiment, the distearylphosphatidylethanolamine-polyethylene glycol (2000)-cyclo(arginine-aspartic-phenylpropanoid-lysine) polypeptide is prepared by activating the terminal carboxyl group of the distearylphosphatidylethanolamine-polyethylene glycol (2000)-carboxyl group with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and then coupling it with the cyclo(arginine-aspartic-phenylpropanoid-lysine) polypeptide in the presence of triethylamine.

[0018] In a preferred embodiment, the lotus leaf alkaloid-based nano-formulation targeting gastrointestinal tumors exhibits a negative potential in a buffer solution with a pH of 7.2-7.6 and a positive potential in a buffer solution with a pH of 6.2-6.8 due to the protonation of cholesterol hemisuccinate.

[0019] By adopting the above technical solution, the pH range in which the nano-formulation undergoes potential reversal is clearly defined. This characteristic is an important characterization of the pH response mechanism and enhances the electrostatic adsorption of the nano-formulation to the cell membrane in the tumor microenvironment.

[0020] In a preferred embodiment, the lotus leaf alkaloid is encapsulated within vesicles formed from the lipid excipient.

[0021] By adopting the above technical solution, the stability of lotus leaf alkaloids during systemic circulation can be ensured, and premature leakage before reaching the tumor site can be avoided.

[0022] Secondly, the present invention provides a method for preparing a nano-formulation targeting gastrointestinal tumors based on lotus leaf alkaloids, using the following technical solution: The preparation method of a nano-formulation targeting gastrointestinal tumors based on lotus leaf alkaloids includes the following steps: S1. Lotus leaf alkaloid, 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine, cholesterol, cholesterol hemisuccinate, 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine and distearate phosphatidylethanolamine-polyethylene glycol (2000)-cyclo(arginine-glycine-aspartic-phenylpropanoid-lysine) polypeptide are dissolved in an organic solvent to form a mixed solution; S2. The mixed solution is subjected to rotary evaporation to remove the organic solvent and form a drug lipid film on the inner wall of the container; S3. Add an aqueous buffer solution to the container to hydrate the drug lipid film and obtain a crude liposome suspension. S4. The crude liposome suspension is extruded through a high-pressure extrusion device to obtain a liposome solution with uniform particle size. S5. The liposome solution is purified to remove unencapsulated free lotus leaf alkaloids, thereby obtaining the lotus leaf alkaloid-based nano-formulation targeting gastrointestinal tumors.

[0023] By employing the above technical solution, the present invention provides a method for preparing a nano-formulation targeting gastrointestinal tumors based on lotus leaf alkaloids, namely, thin-film dispersion and high-pressure extrusion. This method is a mature and highly controllable liposome preparation process that can stably prepare nano-formulations that meet pharmaceutical requirements. First, by co-dissolving the drug and all lipid excipients in an organic solvent (step S1), and then forming a uniform drug-lipid mixture film during the subsequent evaporation process (step S2), the molecular-level dispersion of the drug within the lipid backbone is ensured, laying the foundation for the subsequent formation of liposomes with high encapsulation efficiency. Through a high-pressure extrusion device (step S4), the multilayered coarse liposomes formed after hydration are repeatedly passed through a filter membrane with a specific pore size, effectively reducing the vesicle particle size and homogenizing them, resulting in nanoparticles with a narrow size distribution, suitable for intravenous injection and effective tumor targeting in vivo. The final purification step (step S5) effectively removes unencapsulated free drug, ensuring the purity of the formulation and reducing the immediate systemic toxicity caused by free drug.

[0024] In a preferred embodiment, in step S1, the organic solvent is a mixture of chloroform and methanol, wherein the volume ratio of chloroform to methanol is 2:1 to 3:1.

[0025] By adopting the above technical solution, the mixed solvent system has excellent solubility for lotus leaf alkaloids and various lipid excipients with large polarity differences. It can ensure that a clear and uniform single-phase solution is formed in step S1, avoid uneven film formation due to incomplete dissolution, and thus ensure batch-to-batch quality stability of the final product.

[0026] In a preferred embodiment, the temperature of rotary evaporation in step S2 is 45-55°C, and the temperature of hydration in step S3 is 45-55°C.

[0027] By adopting the above technical solution, the operating temperature is controlled above the phase transition temperature (approximately 41°C) of the main lipid component 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine. This allows the lipid molecular chains to be in a more fluid liquid crystal state, which is beneficial for forming a uniform film with orderly arrangement and no phase separation during evaporation. Furthermore, it promotes the orderly self-assembly of lipid molecules into complete vesicle structures during hydration, thereby improving the encapsulation efficiency of the drug.

[0028] In a preferred embodiment, the extrusion in step S4 includes sequentially passing the material through polycarbonate filter membranes with pore sizes of 150-250 nm and 80-120 nm, with each pore size being extruded repeatedly 10-20 times.

[0029] By employing the above-mentioned technical solution and using extrusion with a two-stage decreasing pore size, the liposome particle size can be reduced gently and gradually, avoiding vesicle rupture and drug leakage caused by a single, intense mechanical shearing force. This parameter setting can stably obtain single-compartment liposomes with an average particle size of around 100 nm. This size range is beneficial for enrichment at the tumor site through enhanced permeability and long retention effects, while effectively preventing excessively rapid clearance by the mononuclear phagocyte system.

[0030] In a preferred embodiment, the purification in step S5 is performed by dialysis for 18-30 hours.

[0031] By adopting the above technical solution, dialysis is a gentle purification method that uses molecular size differences to separate small-molecule free drugs from colloidal solutions of nano-formulations. The entire process has little impact on the integrity of the liposome vesicle structure, which can minimize drug leakage during purification and ensure the quality of the final product.

[0032] This invention provides a nano-formulation based on lotus leaf alkaloids targeting gastrointestinal tumors and its preparation method. It possesses the following beneficial effects: 1. This invention modifies the surface of nano-formulations with distearate phosphatidylethanolamine-polyethylene glycol (2000)-cyclic (arginine-aspartic-phenylpropanoid-lysine) polypeptide. Utilizing the specific affinity of its cyclic (arginine-aspartic-phenylpropanoid-lysine) polypeptide ligand for αvβ3 integrin highly expressed on the surface of gastrointestinal tumor cells, the nano-formulations achieve active targeting of tumor cells, thereby improving the accumulation and cellular uptake efficiency of lotus leaf alkaloids at the tumor site and enhancing the precision of treatment.

[0033] 2. This invention utilizes the pH-dependent charge-reversal properties of cholesterol hemisuccinate and the enzymatic response of 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine to phospholipase A2, which is highly expressed in the tumor microenvironment, to construct a pH / enzyme dual-response synergistic release mechanism. In the tumor microenvironment, the membrane destabilization caused by the pH response accelerates the subsequent enzymatic disintegration of the lipid membrane by phospholipase A2, ensuring the efficient and programmed release of lotus leaf alkaloids at the target site and increasing the local drug concentration.

[0034] 3. This invention encapsulates lotus leaf alkaloids within specific lipid vesicles and prepares them using thin-film dispersion and high-pressure extrusion methods. This effectively protects the chemical stability of lotus leaf alkaloids in systemic circulation, preventing premature degradation or leakage before reaching the lesion. Simultaneously, the polyethylene glycol segments on the surface of the nano-formulation provide long-cycle characteristics, collectively overcoming the shortcomings of low bioavailability and short in vivo half-life of free lotus leaf alkaloids, thus providing the necessary pharmacokinetic basis for exerting its antitumor efficacy. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the preparation examples, comparative examples, and test examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Preparation Example 1: Synthesis and Characterization of the Targeting Ligand Disteaylphosphatidylethanolamine-Polyethylene Glycol (2000)-Cyclic (Argin-Glycer-Aspartic-Phenylacetyl) Peptide This preparation example describes the process of covalently linking a cyclo(arginine-aspartic-phenylpropanoid-lysine) polypeptide to the carboxyl terminus of distearate phosphatidylethanolamine-polyethylene glycol (2000) via N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide coupling chemistry.

[0037] 28.5 mg of distearylphosphatidylethanolamine-polyethylene glycol (2000)-carboxyl group was dissolved in 2 mL of anhydrous dimethyl sulfoxide and placed in a dry reaction flask. Under nitrogen protection and magnetic stirring, 5.7 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 3.5 mg of N-hydroxysuccinimide were added to the solution. The reaction mixture was stirred at room temperature (25 °C) in the dark for 4 hours to completely activate the terminal carboxyl group of distearylphosphatidylethanolamine-polyethylene glycol (2000)-carboxyl group to the N-hydroxysuccinimide active ester.

[0038] In another dry reaction flask, 7.4 mg of the cyclic (arginine-glycine-aspartic-phenylpropanoid-lysine) peptide was dissolved in 0.5 mL of anhydrous dimethyl sulfoxide, followed by the addition of 4.2 μL of triethylamine. This peptide solution was then slowly added dropwise to the activated N-hydroxysuccinimide active ester solution. The reaction system was continued under nitrogen protection and stirred at room temperature (25 °C) in the dark for 24 hours.

[0039] After the reaction was completed, the reaction mixture was transferred to a dialysis bag, and dialysis was performed at 4°C using 2000 mL of deionized water as the dialysate for 48 hours. During this period, the dialysate was changed every 4-6 hours to thoroughly remove unreacted cyclic (arginine-aspartic-phenylpropanoid-lysine) peptides, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, N-hydroxysuccinimide byproducts, and other small molecule impurities.

[0040] The solution in the dialysis bag was collected and freeze-dried to obtain a white, fluffy solid product, namely, distearylphosphatidylethanolamine-polyethylene glycol (2000)-cyclic (arginine-glycine-aspartic-phenylpropanoid-lysine) polypeptide, with the yield calculated by weighing. The product was sealed and dried at -20°C for later use. The structure of the product was confirmed by proton nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, indicating that the cyclic (arginine-glycine-aspartic-phenylpropanoid-lysine) polypeptide was successfully coupled to the end of the distearylphosphatidylethanolamine-polyethylene glycol (2000)-carboxyl group.

[0041] Examples 1-3: Example 1: This embodiment provides a nano-formulation for targeting gastrointestinal tumors based on lotus leaf alkaloids. The preparation method of the nano-formulation for targeting gastrointestinal tumors based on lotus leaf alkaloids is based on thin film dispersion and high-pressure extrusion, and specifically includes the following steps: Accurately weigh 2.92 mg of lotus leaf alkaloid, 33.03 mg of 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine, 9.68 mg of cholesterol, 4.87 mg of cholesterol hemisuccinate, 7.44 mg of 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine, and 3.43 mg of distearylphosphatidylethanolamine-polyethylene glycol (2000)-cyclo(arginine-aspartic-phenylpropanoid-lysine) polypeptide prepared in the preparation example. The molar ratio of each lipid component is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine: cholesterol: cholesterol hemisuccinate: 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine: distearate-phosphatidylethanolamine-polyethylene glycol (2000)-cyclo(arginine-aspartic-phenylpropanoid-lysine) polypeptide = 45:25:10:10:1, and the mass ratio of lotus leaf alkaloid to total lipids is 1:20.

[0042] Place all components together in a round-bottom flask, add 10 ml of a chloroform and methanol mixture (volume ratio 3:1) to dissolve them completely, forming a clear organic phase solution.

[0043] The round-bottom flask was connected to a rotary evaporator and rotary evaporated for 30 minutes under a vacuum of -0.08 MPa in a water bath at 50°C, forming a uniform drug lipid film on the inner wall of the flask.

[0044] Add 5 ml of phosphate buffered saline with pH 7.4 to the flask and continue to hydrate by rotation at 50°C for 60 min to obtain crude liposome suspension.

[0045] The crude liposome suspension was passed through a high-pressure extrusion device and then through polycarbonate filter membranes with pore sizes of 200 nm and 100 nm in sequence. Each pore size was repeatedly extruded 15 times to obtain a liposome solution with uniform particle size.

[0046] The liposome solution was transferred to a dialysis bag and dialyzed in phosphate-buffered saline at 4°C for 24 hours to remove unencapsulated free lotus leaf alkaloids, thus obtaining the lotus leaf alkaloid-based nano-formulation targeting gastrointestinal tumors of this embodiment.

[0047] Example 2: This embodiment provides a nano-formulation for targeting gastrointestinal tumors based on lotus leaf alkaloids. The preparation method of the nano-formulation for targeting gastrointestinal tumors based on lotus leaf alkaloids is based on thin film dispersion and high-pressure extrusion, and specifically includes the following steps: Accurately weigh 4.65 mg of lotus leaf alkaloid, 36.70 mg of 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine, 7.73 mg of cholesterol, 7.30 mg of cholesterol hemisuccinate, 11.16 mg of 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine, and 6.86 mg of the compound prepared in the preparation example. The molar ratio of each lipid component is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine: cholesterol: cholesterol hemisuccinate: 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine: distearate-phosphatidylethanolamine-polyethylene glycol (2000)-cyclic (arginine-aspartic-phenylpropanoid-lysine) polypeptide = 50:20:15:15:2, and the mass ratio of lotus leaf alkaloid to total lipids is 1:15.

[0048] Place all components together in a round-bottom flask, add 10 ml of a chloroform and methanol mixture (volume ratio 2:1) to dissolve them completely, forming a clear organic phase solution.

[0049] The round-bottom flask was connected to a rotary evaporator and rotary evaporated for 30 minutes under a vacuum of -0.08 MPa in a water bath at 50°C, forming a uniform drug lipid film on the inner wall of the flask.

[0050] Add 5 ml of phosphate buffered saline with pH 7.4 to the flask and continue to hydrate by rotation at 50°C for 60 min to obtain crude liposome suspension.

[0051] The crude liposome suspension was passed through a high-pressure extrusion device and then through polycarbonate filter membranes with pore sizes of 200 nm and 100 nm in sequence. Each pore size was repeatedly extruded 15 times to obtain a liposome solution with uniform particle size.

[0052] The liposome solution was transferred to a dialysis bag and dialyzed in phosphate-buffered saline at 4°C for 24 hours to remove unencapsulated free lotus leaf alkaloids, thus obtaining the lotus leaf alkaloid-based nano-formulation targeting gastrointestinal tumors of this embodiment.

[0053] Example 3: This embodiment provides a nano-formulation for targeting gastrointestinal tumors based on lotus leaf alkaloids. The preparation method of the nano-formulation for targeting gastrointestinal tumors based on lotus leaf alkaloids is based on thin film dispersion and high-pressure extrusion, and specifically includes the following steps: Accurately weigh 8.07 mg of lotus leaf alkaloid, 40.37 mg of 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine, 5.80 mg of cholesterol, 9.73 mg of cholesterol hemisuccinate, 14.88 mg of 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine, and 10.30 mg of the [specific ingredient] prepared in the preparation example. The molar ratio of each lipid component is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine: cholesterol: cholesterol hemisuccinate: 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine: distearate-phosphatidylethanolamine-polyethylene glycol (2000)-cyclic (arginine-aspartic-phenylpropanoid-lysine) polypeptide = 55:15:20:20:3, and the mass ratio of lotus leaf alkaloid to total lipids is 1:10.

[0054] Place all components together in a round-bottom flask, add 10 ml of a chloroform and methanol mixture (volume ratio 2:1) to dissolve them completely, forming a clear organic phase solution.

[0055] The round-bottom flask was connected to a rotary evaporator and rotary evaporated for 30 minutes under a vacuum of -0.08 MPa in a water bath at 50°C, forming a uniform drug lipid film on the inner wall of the flask.

[0056] Add 5 ml of phosphate buffered saline with pH 7.4 to the flask and continue to hydrate by rotation at 50°C for 60 min to obtain crude liposome suspension.

[0057] The crude liposome suspension was passed through a high-pressure extrusion device and then through polycarbonate filter membranes with pore sizes of 200 nm and 100 nm in sequence. Each pore size was repeatedly extruded 15 times to obtain a liposome solution with uniform particle size.

[0058] The liposome solution was transferred to a dialysis bag and dialyzed in phosphate-buffered saline at 4°C for 24 hours to remove unencapsulated free lotus leaf alkaloids, thus obtaining the lotus leaf alkaloid-based nano-formulation targeting gastrointestinal tumors of this embodiment.

[0059] Comparative Examples 1-5: Comparative Example 1: Compared to Example 2, the difference is that the distearylphosphatidylethanolamine-polyethylene glycol (2000)-cyclo(arginine-aspartic-phenylpropionic-lysine) polypeptide prepared in the preparation example is not added, but is replaced with an equimolar amount of distearylphosphatidylethanolamine-polyethylene glycol (2000)-methoxy. The remaining components and preparation steps are the same.

[0060] Comparative Example 2: Compared to Example 2, the difference is that the pH-responsive components cholesterol hemisuccinate and 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine are not added; instead, the molar amounts of cholesterol hemisuccinate and 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine are replaced with an equimolar amount of cholesterol. All other components and preparation steps are the same.

[0061] Comparative Example 3: The difference from Example 2 is that the enzyme-responsive component 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine is replaced with an equimolar amount of distearyl-sn-glycerol-3-phosphatidylcholine. All other components and preparation steps are the same.

[0062] Comparative Example 4: Compared to Example 2, the difference lies in the following: cholesterol hemisuccinate and 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine are not added, and the molar amounts of cholesterol hemisuccinate and 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine are replaced with an equimolar amount of cholesterol; simultaneously, 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine is replaced with an equimolar amount of distearate-sn-glycerol-3-phosphatidylcholine. All other components and preparation steps are the same.

[0063] Comparative Example 5: This comparative example uses a free lotus leaf alkali solution.

[0064] Preparation method: Accurately weigh lotus leaf alkaloids and dissolve them in a phosphate buffer solution with a pH of 7.4 containing 5% dimethyl sulfoxide, so that the concentration is consistent with the final drug concentration of lotus leaf alkaloids in Example 2.

[0065] Test Examples 1-4: Test Example 1: This test case aims to evaluate the basic physicochemical characteristics of the lotus leaf alkaloid-based nanoformulations targeting gastrointestinal tumors prepared in Examples 1-3, including their particle size, polydispersity index, zeta potential, drug encapsulation efficiency, and drug loading under different pH conditions.

[0066] Particle size, polydispersity index and zeta potential determination: The nano-formulation suspensions prepared in Examples 1, 2 and 3 were diluted to a total lipid concentration of 1 mg / mL using phosphate-buffered saline (10 mmol / L) at pH 7.4 and phosphate-buffered saline (10 mmol / L) at pH 6.5, respectively. The diluted sample was placed in a dynamic light scattering instrument and equilibrated at 25°C. The hydration particle size and polydispersity index of the sample were determined using dynamic light scattering technology. Subsequently, the zeta potential in the corresponding pH buffer was determined using laser Doppler velocimetry with the same sample and instrument.

[0067] Encapsulation efficiency and drug loading determination: The content of nuciferine was determined by high performance liquid chromatography (HPLC). The chromatographic conditions were set as follows: C18 reversed-phase column (4.6 x 250 mm, 5...). The mobile phase was methanol:water (containing 0.1% triethylamine, pH adjusted to 3.0) = 70:30; the flow rate was 1.0 mL / min; the column temperature was 30℃; and the detection wavelength was 272 nm. A standard curve was established by measuring a series of lotus leaf alkaloid standard solutions of known concentrations.

[0068] All phosphate-buffered saline solutions used in the dialysis process during the preparation of Examples 1-3 were collected, combined, and precisely diluted to volume. Samples were taken, and the concentration of lotus leaf alkaloid in the dialysate was determined by high-performance liquid chromatography (HPLC). Based on this, the total amount of unencapsulated free drug was calculated. .

[0069] According to the initial feed amount of lotus leaf alkaloid in Examples 1-3 and total lipid input The encapsulation efficiency and drug loading are calculated using the following formulas: Encapsulated drug amount : ; Encapsulation efficiency (EE): ; Drug loading capacity LC: .

[0070] in, This represents the initial total amount of lotus leaf alkaloids added. To determine the total amount of unencapsulated free drug obtained; This represents the initial amount of total lipids fed.

[0071] Experimental data: The physicochemical characterization data of Examples 1-3 are summarized in Table 1.

[0072] Table 1: Physicochemical property characterization data of Examples 1-3 Note: The "-" in the encapsulation efficiency and drug loading columns in Table 1 above is because encapsulation efficiency and drug loading are not pH responsiveness test items, so they do not need to be listed again in the pH 6.5 responsiveness test row, and are therefore left blank.

[0073] The data from Test Example 1 confirms the feasibility and stability of the method for preparing the lotus leaf alkaloid-based nano-formulation targeting gastrointestinal tumors provided by this invention. Examples 1, 2, and 3 all successfully prepared lotus leaf alkaloid-based nano-formulations targeting gastrointestinal tumors with uniform particle size (average particle size 108-117 nm) and good dispersibility (polydispersity index < 0.2). All examples achieved efficient encapsulation of lotus leaf alkaloids (encapsulation rate > 88%) and a preset drug loading gradient.

[0074] Key data show that the surface charge of the lotus leaf alkaloid-based nano-formulation targeting gastrointestinal tumors is pH-dependent. In a simulated normal body fluid environment (pH 7.4), all embodiments carried a negative charge (-18.4 mV to -21.3 mV). This negative charge helps reduce non-specific adsorption to serum proteins and maintain the stability of the nano-formulation in blood circulation.

[0075] When the environment was switched to acidic conditions simulating the tumor microenvironment (pH 6.5), the Zeta potential of all formulations reversed, exhibiting a positive charge (+6.8 mV to +9.2 mV). This potential reversal is attributed to the pH-sensitive component cholesterol hemisuccinate in the formulation. At pH 7.4, the terminal carboxyl group of cholesterol hemisuccinate dissociates and becomes negatively charged; at pH 6.5 (close to or below its acidity coefficient), the terminal carboxyl group of cholesterol hemisuccinate is protonated, causing the negative charge on the surface of the nanoformulation to be shielded or neutralized, thus converting it to a positive charge overall.

[0076] This acid-triggered charge reversal characteristic is one of the key mechanisms by which the invention achieves tumor microenvironment responsiveness, validating the effectiveness of the pH-responsive gating design. This positive charge enhances the electrostatic adsorption between the formulation and the negatively charged tumor cell membrane, thereby promoting cellular uptake.

[0077] Test Example 2: This test case aims to simulate the drug release behavior of the drug carrier in a normal physiological environment (pH 7.4) and a tumor microenvironment (pH 6.5, containing a high concentration of phospholipase A2) to verify the pH and enzyme dual-response drug release mechanism designed in this invention.

[0078] Experimental steps: The lyophilized powders from Example 2, Comparative Example 1, Comparative Example 3, Comparative Example 4, and Comparative Example 5 were resuspended, and 5 mL of each powder was placed in a dialysis bag. Immerse the dialysis bags in conical flasks containing 50 mL of release medium. Four different release media were used in the experiment: Group A: Phosphate buffer solution with a pH of 7.4.

[0079] Group B: Phosphate buffer solution with a pH of 6.5.

[0080] Group C: Phosphate buffer at pH 7.4 containing 10 U / mL phospholipase A2.

[0081] Group D: Phosphate buffer at pH 6.5 containing 10 U / mL phospholipase A2.

[0082] Place all conical flasks in a constant temperature shaking water bath at 37°C (100 rpm). At preset time points (e.g., 0.5, 1, 2, 4, 8, 12, 24 hours), remove 1 mL of sample from the release medium and immediately replenish with 1 mL of fresh release medium of the corresponding amount. The concentration of lotus leaf alkaloid in the sample was determined by high performance liquid chromatography, and the cumulative release rate of the drug was calculated.

[0083] Experimental data: The cumulative drug release rate (%) of each formulation in different release media over 24 hours is summarized in Table 2.

[0084] Table 2: Cumulative drug release rate of each formulation under different conditions In vitro release data systematically validated the pH / enzyme dual response mechanism of the nanoformulation of the present invention.

[0085] Stability verification: All formulations exhibited low drug release rates (<15%) in medium A, which simulates a normal physiological environment, indicating that the carrier has good stability and can effectively prevent premature drug leakage in the bloodstream. The non-responsive comparative example 4 showed extremely low release rates under all conditions, demonstrating its stable carrier structure.

[0086] pH response verification: In medium B with only acidic stimulation, both Example 2 and Comparative Example 3 (pH response only) showed drug burst release (approximately 51%), while Comparative Example 5 (phospholipase A2 response only) showed no significant change, thus demonstrating the effectiveness of the pH-responsive component in the nano-formulation.

[0087] Enzyme response verification: In medium C, which is stimulated only by phospholipase A2, both Example 2 and Comparative Example 5 (phospholipase A2 response only) showed drug release (approximately 59%), while Comparative Example 3 (pH response only) showed no significant change, thus demonstrating the effectiveness of the enzyme-responsive component in the nanoformulation.

[0088] Synergistic effect verification: Most importantly, in medium D, which simultaneously simulates the acidic and high-enzyme environment of tumors, the cumulative release rate of Example 2 reached 89.3%, far exceeding any single response mechanism (54.8% of Comparative Example 3 and 61.3% of Comparative Example 5) and the non-response control group (14.1% of Comparative Example 4). This indicates that the acidic environment not only triggers drug release itself but also loosens the liposome structure, thereby increasing the efficiency of phospholipase A2 in approaching and hydrolyzing its substrate, ultimately producing a good synergistic drug release effect.

[0089] In summary, through direct comparison with Comparative Examples 3 and 5, this test case fully demonstrates the independent effectiveness and synergistic effect of the pH and enzyme dual response mechanism designed in this invention, providing the technical basis for achieving precise and efficient drug release from tumor sites.

[0090] Test Example 3: This test case aims to evaluate, through quantitative analysis, the cellular uptake efficiency of the nanoformulation of the present invention mediated by both targeting ligands and charge reversal in a simulated tumor acidic microenvironment.

[0091] Experimental steps: The human gastric cancer cell line MGC-803 (highly expressing integrin αvβ3) was selected as the model cell. In the experiment, coumarin-6 was used as a fluorescent probe, and fluorescently labeled nano-formulations loaded with coumarin-6 were prepared using the same preparation method as in Example 2 and Comparative Example 1. MGC-803 cells were seeded in 6-well plates at a density of 1×10⁵ cells per well and placed in RPMI-1640 medium containing 10% fetal bovine serum. The plates were then incubated at 37°C in a 5% CO₂ incubator for 24 hours. After cultivation, the original culture medium was discarded. The nanoformations of Example 2 and Comparative Example 1 containing coumarin-6 were diluted with serum-free culture media at pH 7.4 and pH 6.5, respectively, to achieve a final concentration of 1... Add the diluted formulation solution to the corresponding 6-well plate, with 3 replicates per group; Cells and nanoparticles were co-incubated at 37°C for 4 hours. After incubation, the cells were washed three times with pre-cooled phosphate-buffered saline to remove non-specifically adsorbed nanoparticles from the cell surface. Cells were then detached by digestion with 0.25% trypsin-EDTA, collected by centrifugation, and treated with 400... L-phosphate buffered saline resuspension; The average fluorescence intensity of cells in each group was detected and analyzed using flow cytometry.

[0092] Experimental data: Table 3 summarizes the efficiency data of uptake by MGC-803 cells in Example 2 and Comparative Example 1 under different pH conditions.

[0093] Table 3: Uptake efficiency of Example 2 and Comparative Example 1 in MGC-803 cells The cellular uptake data in this test case validated the independent effectiveness and synergistic effect of the two mechanisms of cyclic (arginine-glycine-aspartic-phenylpropanoid-lysine) peptide targeting and pH charge reversal in this invention.

[0094] First, at pH 7.4, the cellular uptake of Example 2 (targeted formulation) (MFI = 313.2) was more than 2.5 times that of Comparative Example 1 (non-targeted formulation, MFI = 121.4). This result indicates that the cRGD ligand on the surface of the nanoformulation can effectively mediate specific binding to the highly expressed integrin αvβ3 receptor on the surface of MGC-803 cells, thereby enhancing cellular uptake through receptor-mediated endocytosis, confirming the effectiveness of active targeting design.

[0095] Secondly, under acidic conditions (pH 6.5), the uptake of Comparative Example 1 (MFI = 258.9) was also increased compared to that under pH 7.4. This is attributed to the charge reversal driven by the protonation of cholesterol hemisuccinate on the surface of the nanoparticles. The positively charged nanoparticle surface enhanced its electrostatic adsorption with the negatively charged cell membrane, promoting non-specific cell internalization and verifying the amplifying effect of the pH-responsive mechanism on cellular uptake.

[0096] Most importantly, when the active targeting and pH-responsive mechanisms were combined (Example 2, pH 6.5), the cellular uptake efficiency reached its highest level (MFI = 746.5), far exceeding the uptake achieved by any single mechanism. This data indicates that the charge reversal induced by the acidic environment not only increases the affinity between the formulation and the cell membrane but also enhances the probability and efficiency of binding between the cyclic (arginine-glycine-aspartic-phenylpropanoid-lysine) peptide ligand and the receptor by prolonging the residence time of the nanoformulation on the cell surface, ultimately producing a synergistic effect.

[0097] In summary, this test case demonstrates that the nano-formulation can utilize the acidic characteristics of the tumor microenvironment and enhance its active targeting efficiency through charge reversal, thereby achieving efficient internalization of target cancer cells, which forms the basis for its precise drug delivery and therapeutic effect.

[0098] Test Example 4: This test case aims to evaluate the in vitro killing effect of drug-loaded nanoformulations on tumor cells in a simulated tumor acidic microenvironment, in order to verify the synergistic effect of the targeting and pH / enzyme dual responsiveness of the present invention in enhancing antitumor activity.

[0099] Experimental steps: The human gastric cancer cell line MGC-803 (highly expressing integrin αvβ3) was selected as the model cell. MGC-803 cells were seeded into 96-well plates at a density of 5×1035×103 cells per well and cultured at 37°C in a 5% CO2 incubator for 24 hours. The old culture medium was discarded, and the following experimental groups were set up: blank culture medium (control group), free lotus leaf alkaloid, blank nano-formulation (blank carrier of Example 2), Example 2 (liposome formulation loaded with lotus leaf alkaloid), Comparative Example 1 (non-targeted liposome formulation loaded with lotus leaf alkaloid), and Comparative Example 4 (non-responsive liposome formulation loaded with lotus leaf alkaloid). The above preparations were diluted to a series of concentration gradients (based on lotus leaf alkaloid concentration) using RPMI-1640 medium containing 10% fetal bovine serum at pH 7.4 and pH 6.5, respectively. Dilute the formulation solution (100) Add L / hole to the corresponding 96-well plate, with 6 duplicate holes set in each group; The cell plates were placed in culture conditions of pH 7.4 and pH 6.5 (by adjusting the CO2 concentration in the incubator or by using 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer) and incubated at 37°C for 48 hours. After incubation, add 20 mg of [unspecified substance] to each well. L3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazol bromide solution (5 mg / mL), continue incubation for 4 hours; Terminate the culture, carefully aspirate the supernatant, and add 150 ml of the solution to each well. L-dimethyl sulfoxide was shaken at low speed for 10 minutes on an oscillator to fully dissolve the formazan crystals. The absorbance of each well was measured at a wavelength of 490 nm using an ELISA reader. First, calculate the cell viability at each drug concentration using the following formula: Cell viability (%) = (Absorbance value of experimental group - Absorbance value of blank well) / (Absorbance value of control group - Absorbance value of blank well) × 100%; Subsequently, using the cell viability data corresponding to the above series of concentration gradients, nonlinear regression fitting was performed using GraphPad Prism software to calculate and determine the half-maximal inhibitory concentration (IC50) of each formulation. ).

[0100] Experimental data: Table 4 summarizes the half-maximal inhibitory concentration (IC50) data of each formulation on MGC-803 cells after incubation at different pH conditions for 48 hours.

[0101] Table 4: Half-maximal inhibitory concentrations (IC50) of each formulation on MGC-803 cells Note: "-" indicates that the blank culture medium is a blank control group without inhibitors, and its cell viability is defined as 100% and used as a benchmark for calculating the cell viability of other groups. Therefore, the half-maximal inhibitory concentration is not applicable to the blank control group.

[0102] The cytotoxicity data from this test case confirm the tumor microenvironment responsiveness of the nanoformulation of the present invention and its synergistic advantages in mediating antitumor effects.

[0103] The blank nanoformulation did not show significant cytotoxicity under either pH condition (half-maximal inhibitory concentration > 200). This indicates that the carrier material has good biocompatibility.

[0104] In a simulated normal physiological environment (pH 7.4), the half-maximal inhibitory concentration (IC50) values ​​of all nano-formulation groups (Example 2, Comparative Example 1, and Comparative Example 4) were higher than those of free lotus leaf alkaloids, indicating that the drug was stably encapsulated inside the carrier and its activity was effectively shielded, which is beneficial to reducing its toxic side effects in normal tissues.

[0105] When the environment was switched to a simulated tumor microenvironment (pH 6.5), the half-maximal inhibitory concentration (IC50) of free lotus leaf alkaloids showed no significant change. However, in the non-responsive comparative example 4 (IC50 = 38.2%), the IC50 value did not change significantly. The cytotoxicity of Example 2 was only slightly increased, confirming that a stable lipid membrane hindered drug release. In contrast, Example 2, which exhibited pH / enzyme responsiveness (half-maximal inhibitory concentration = 5.8), showed a significantly reduced cytotoxicity. (and Comparative Example 1 (half-maximal inhibitory concentration = 15.3%)) and Comparative Example 2 (half-maximal inhibitory concentration = 15.3%). The cytotoxicity of the nanoformulations was enhanced (the half-maximal inhibitory concentration decreased). This phenomenon is attributed to the acidic environment triggering drug release from the nanoformulations (as shown in Test Example 2), leading to the accumulation of high drug concentrations within the cells.

[0106] Most importantly, the half-maximal inhibitory concentration (IC50) in Example 2 at pH 6.5 was 5.8. The level was lower than that of the untargeted control group 1 (15.3%). This data clearly demonstrates the synergistic effect of the targeting and response mechanisms: the pH 6.5 condition not only triggered rapid drug release, but also induced charge reversal (as shown in Test Example 1) and synergistic ring-cyclic (arginine-glycine-aspartic-phenylpropanoid-lysine) peptide ligand (as shown in Test Example 3) enhanced cellular uptake. Through the combination of efficient endocytosis and simultaneous drug release, Example 2 exhibited significantly higher cell-killing activity than other control groups in the tumor microenvironment, validating the design mechanism of this invention.

Claims

1. A nano-formulation based on lotus leaf alkaloids targeting gastrointestinal tumors, characterized in that, The lotus leaf alkaloid-based nano-formulation targeting gastrointestinal tumors is a liposome formulation comprising: Lotus leaf alkaloid; Vesicle structures formed by lipid excipients, wherein the lipid excipients comprise: 1,2-Dipalmitoyl-sn-glycerol-3-phosphatidylcholine; cholesterol; Cholesterol hemisuccinate; 1,2-Dioleoyl-sn-glycerol-3-phosphatidylethanolamine; Distearate phosphatidylethanolamine-polyethylene glycol (2000)-cyclo(arginine-glycine-aspartic-phenylpropanoid-lysine) polypeptide; The molar ratio of the 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine, cholesterol, cholesterol hemisuccinate, 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine and distearate phosphatidylethanolamine-polyethylene glycol (2000)-cyclo(arginine-glycolic acid-aspartic acid-phenylpropanoid-lysine) polypeptide is (45-55):(15-25):(10-20):(10-20):(1-3); Furthermore, the total mass ratio of the lotus leaf alkaloid to the lipid excipient is 1:(10-20).

2. The nano-formulation for targeting gastrointestinal tumors based on lotus leaf alkaloids according to claim 1, characterized in that, The preferred molar ratio of the 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine, cholesterol, cholesterol hemisuccinate, 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine and distearate phosphatidylethanolamine-polyethylene glycol (2000)-cyclo(arginine-glycolic acid-aspartic acid-phenylpropanoid-lysine) polypeptide is (48-52):(18-22):(13-17):(13-17):(1.5-2.5), and the preferred total mass ratio of the lotus leaf alkaloid to the lipid excipient is 1:(13-17).

3. The nano-formulation for targeting gastrointestinal tumors based on lotus leaf alkaloids according to claim 1, characterized in that, The distearylphosphatidylethanolamine-polyethylene glycol (2000)-cyclo(arginine-aspartic-phenylpropanoid-lysine) polypeptide is prepared by activating the terminal carboxyl group of the distearylphosphatidylethanolamine-polyethylene glycol (2000)-carboxyl group with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinate, and then coupling it with the cyclo(arginine-aspartic-phenylpropanoid-lysine) polypeptide in the presence of triethylamine.

4. The nano-formulation for targeting gastrointestinal tumors based on lotus leaf alkaloids according to claim 1, characterized in that, The aforementioned lotus leaf alkaloid-based nano-formulation targeting gastrointestinal tumors exhibits a negative potential in a buffer solution with a pH of 7.2-7.6, and a positive potential in a buffer solution with a pH of 6.2-6.8 due to the protonation of cholesterol hemisuccinate.

5. A nano-formulation for targeting gastrointestinal tumors based on lotus leaf alkaloids according to claim 1, characterized in that, The lotus leaf alkaloid is encapsulated within vesicles formed from the lipid excipient.

6. A method for preparing a nano-formulation targeting gastrointestinal tumors based on lotus leaf alkaloids, characterized in that, The preparation of a nano-formulation based on lotus leaf alkaloids targeting gastrointestinal tumors as described in any one of claims 1-5 comprises the following steps: S1. Lotus leaf alkaloid, 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine, cholesterol, cholesterol hemisuccinate, 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine and distearate phosphatidylethanolamine-polyethylene glycol (2000)-cyclo(arginine-glycine-aspartic-phenylpropanoid-lysine) polypeptide are dissolved in an organic solvent to form a mixed solution; S2. The mixed solution is subjected to rotary evaporation to remove the organic solvent and form a drug lipid film on the inner wall of the container; S3. Add an aqueous buffer solution to the container to hydrate the drug lipid film and obtain a crude liposome suspension. S4. The crude liposome suspension is extruded through a high-pressure extrusion device to obtain a liposome solution with uniform particle size. S5. The liposome solution is purified to remove unencapsulated free lotus leaf alkaloids, thereby obtaining the lotus leaf alkaloid-based nano-formulation targeting gastrointestinal tumors.

7. The method for preparing a nano-formulation targeting gastrointestinal tumors based on lotus leaf alkaloids according to claim 6, characterized in that, In step S1, the organic solvent is a mixture of chloroform and methanol, and the volume ratio of chloroform to methanol is 2:1 to 3:

1.

8. The method for preparing a nano-formulation targeting gastrointestinal tumors based on lotus leaf alkaloids according to claim 6, characterized in that, In step S2, the temperature for rotary evaporation is 45-55℃, and in step S3, the temperature for hydration is 45-55℃.

9. The method for preparing a nano-formulation targeting gastrointestinal tumors based on lotus leaf alkaloids according to claim 6, characterized in that, In step S4, the extrusion includes sequentially passing the material through polycarbonate filter membranes with pore sizes of 150-250 nm and 80-120 nm, with each pore size being extruded repeatedly 10-20 times.

10. The method for preparing a nano-formulation targeting gastrointestinal tumors based on lotus leaf alkaloids according to claim 6, characterized in that, In step S5, the purification is performed using dialysis, with a dialysis time of 18-30 hours.

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