Vesicle-loaded lupilate nanoparticles, methods of making and using the same

Abstract

This invention relates to vesicle-loaded lupeol derivative nanoparticles, their preparation method, and applications. The invention utilizes probiotic-constructed artificial membrane vesicles loaded with the chemical drug lupeol quaternary ammonium salt derivative, effectively addressing the shortcomings of lupeol quaternary ammonium salt derivatives in terms of targeting and solubility in colorectal cancer. The carrier material is derived from natural intestinal probiotics, possessing a certain degree of biosafety; simultaneously, the carrier material itself also exhibits intestinal function regulation and immune-inducing effects, and can synergistically enhance the anti-colorectal cancer efficacy of lupeol quaternary ammonium salt derivatives, achieving an immunotherapy effect.

Description

Technical Field

[0001] This invention relates to the field of drug synthesis technology, and in particular to a vesicle-loaded lupeol derivative nanoparticle, its preparation method, and its application. Background Technology

[0002] Colorectal cancer is one of the most common malignant tumors of the digestive tract worldwide, ranking third in incidence and second in mortality among common malignant tumors, seriously threatening human life and health. Currently, clinical treatment for colorectal cancer primarily involves surgery, supplemented by chemotherapy. However, traditional chemotherapy drugs have limitations such as drug resistance, nonspecificity, and toxic side effects, reducing their clinical effectiveness and necessitating the development of novel anti-colorectal cancer drugs. In recent years, increasing research has focused on developing effective anti-cancer components from traditional Chinese medicine. While the natural product lupeol possesses anti-colorectal cancer activity, its lipid solubility limits its clinical application.

[0003] Patent 202010654852.8 discloses that the molecular structure of lupeol was modified to obtain a lupeol quaternary ammonium salt derivative (LD-1), which has a stronger anti-colorectal cancer effect than lupeol and low toxicity to normal cell lines.

[0004] However, LD-1 drugs face similar challenges to traditional chemotherapy drugs, such as insufficient targeting ability, requiring larger doses and leading to severe toxic side effects; poor stability, easily metabolized in vivo; and even poor water solubility due to their physical properties, resulting in complex administration methods. These problems have, to some extent, restricted the research progress on their anti-tumor function and hindered their translation into clinical applications. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide vesicle-loaded lupeol derivative nanoparticles, their preparation method and application, effectively addressing the shortcomings of lupeol quaternary ammonium salt derivatives in terms of targeting and solubility in colorectal cancer.

[0006] First aspect:

[0007] A nanoparticle loaded with lupeol derivative in vesicles, the nanoparticles being composed of probiotic vesicles encapsulating lupeol quaternary ammonium salt derivative (LD-1), the surface of the probiotic vesicles being modified with phospholipid-disulfide bond-polyethylene glycol-folic acid (DSPE-SS-PEG-FA). The structural formula of the lupinol quaternary ammonium salt derivative is as follows: ; The structural formula of the phospholipid-disulfide bond-polyethylene glycol-folic acid is:

[0008] Among them, the molecular weight of polyethylene glycol is ≥2000.

[0009] This invention utilizes probiotic-constructed artificial membrane vesicles loaded with the chemical drug lupeol quaternary ammonium salt derivative, effectively addressing the shortcomings of lupeol quaternary ammonium salt derivative in terms of targeting and solubility in colorectal cancer. The carrier material is derived from natural intestinal probiotics, possessing a certain degree of biocompatibility; simultaneously, the carrier material itself also exhibits intestinal function regulation and immune induction effects, and can synergistically enhance the anti-colorectal cancer efficacy of lupeol quaternary ammonium salt derivative, achieving the effect of immunotherapy.

[0010] Specifically, in the structure of phospholipid-disulfide bond-polyethylene glycol-folic acid, FA (folic acid) enables active targeting and enhances accumulation at the tumor site; PEG (polyethylene glycol) prolongs circulation time and reduces immune clearance; and the DSPE (phospholipid) structure facilitates assembly and stability.

[0011] The vesicle-loaded lupeol derivative nanoparticles of this invention treat colorectal cancer through a synergistic mechanism of targeted delivery and immune regulation. They can be formulated into any dosage form, such as tablets, capsules, solutions, suspensions, ointments, lyophilized powders, pellets, films, liposomes, etc. Specifically, they can be formulated into health foods that aid in the treatment of colorectal cancer; they can also be used as food additives, added in liquid or solid powder form to beverages, cheese, bread, flour, and other foods.

[0012] As a preferred embodiment, the probiotic vesicles include Lactobacillus plantarum vesicles.

[0013] The second aspect: A method for preparing vesicle-loaded lupeol derivative nanoparticles as described in the first aspect comprises the following steps: S1: Centrifuge the probiotic culture medium and collect the precipitated bacterial cells; S2: The precipitated bacterial cells are resuspended in lysozyme solution, shaken to mix, centrifuged, and the precipitated protoplasts are collected. S3: The precipitated protoplasts are resuspended in phosphate buffer solution, then the membrane is broken by sonication, the precipitate is removed by centrifugation, and the supernatant containing probiotic membrane fragments is retained.

[0014] S4: The supernatant containing probiotic membrane fragments is centrifuged for the first time to remove organelles and other impurities, and the supernatant is retained. The membrane probiotic fragments are separated by a second centrifugation to obtain a precipitate containing probiotic membrane fragments. S5: The precipitate containing probiotic membrane fragments is resuspended in phosphate buffer solution, and then the lupeol quaternary ammonium salt derivative and the phospholipid-disulfide bond-polyethylene glycol-folic acid (DSPE-SS-PEG-FA) are added, and the mixture is sonicated to form nanospheres. S6: The nanospheres are extruded using a thin film extrusion method to prepare vesicle-loaded lupeol derivative nanoparticles.

[0015] As a preferred embodiment, in step S1, when the probiotic culture medium is centrifuged, the centrifugal force is 1000-2000×g, the temperature is 4℃, and the time is 20-40min.

[0016] As a preferred embodiment, in step S2, the lysozyme solution includes at least one of animal lysozyme, microbial lysozyme, and egg white lysozyme; the concentration of the lysozyme solution is 1-10 mg / mL; after adding the lysozyme solution, it is treated at 35-40℃ for 12-48 h; during centrifugation, the centrifugal force is 3000-10000×g, and the time is 5-20 min.

[0017] As a preferred embodiment, in step S3, the membrane is broken by ultrasonication in an ice bath at 0-4℃, with an ultrasonic frequency of 20-50kHz and an ultrasonic time of 5-30min; during centrifugation, the centrifugal force is 2000-4000×g, the temperature is 4℃, and the time is 10-30min.

[0018] As a preferred embodiment, in step S4, during the first centrifugation, the centrifugal force is 15000-20000×g, the temperature is 4℃, and the time is 30-60min; during the second centrifugation, the centrifugal force is 80000-100000×g, the temperature is 4℃, and the time is 30-60min.

[0019] As a preferred embodiment, in step S5, the ultrasound is performed in an ice bath at 0-4℃, with an ultrasound frequency of 40-60kHz and an ultrasound time of 30-60min; the protein concentration in the precipitate containing probiotic membrane fragments is 100-200μg / ml; the mass ratio of the precipitate containing probiotic membrane fragments, the lupeol quaternary ammonium salt derivative, and the phospholipid-disulfide bond-polyethylene glycol-folic acid is 50-400:1:25-200, and the precipitate containing probiotic membrane fragments is based on protein mass.

[0020] If the protein concentration in the precipitate containing probiotic membrane fragments is too high, it may cause the membrane fragments to self-aggregate, affecting the particle size of the nanoparticles prepared subsequently. If the protein concentration is too low, it may not be able to effectively encapsulate the lupeol quaternary ammonium salt derivative, which will affect the drug loading efficiency of the nanoparticles. If the phospholipid-disulfide bond-polyethylene glycol-folic acid content is too low, there will be too few folic acid groups grafted onto the surface of the nanoparticles to play a targeting role, which may lead to insufficient ability of the nanoparticles to target tumors.

[0021] As a preferred embodiment, in step S6, the film used in the film extrusion method includes a polycarbonate film with a pore size of 100-200 nm, and the film is extruded by repeated extrusion in an ice bath at 0-4°C for 10-20 times.

[0022] Third aspect: The application of the vesicle-loaded lupeol derivative nanoparticles described in the first aspect to pharmaceutical compositions, health product compositions, or food.

[0023] Specifically, in pharmaceutical compositions, drug-acceptable carriers or excipients can be added according to pharmaceutical and formulation requirements. Dosage forms include tablets, capsules, injections, injectable solutions, oral solutions, solutions, suspensions, ointments, lyophilized powders, pellets, films, liposomes, etc.

[0024] The health product composition can be in dosage forms such as tablets, injections, capsules, injectable solutions, or oral solutions.

[0025] In food, it can be used as a functional additive, incorporated into everyday foods such as beverages, cheese, bread, and flour in liquid or solid powder form. Attached Figure Description

[0026] Figure 1 This is a flowchart of the preparation process of LD-1@MV-FA nanoparticles.

[0027] Figure 2 This is a particle size distribution diagram of LD-1@MV-FA nanoparticles.

[0028] Figure 3 This is a transmission electron microscope (TEM) image of LD-1@MV-FA nanoparticles.

[0029] Figure 4 This is a graph showing the release of LD-1 from LD-1@MV-FA nanoparticles in simulated normal physiological environments and simulated tumor microenvironments.

[0030] Figure 5 This is a fluorescence image of HCT-116 cells uptake LD-1@MV-FA nanoparticles. Figure 6 This is a graph showing the cytotoxicity of LD-1@MV-FA nanoparticles against RAW264.7 (A), HCoEpic (B), and HCT-116 (C).

[0031] Figure 7 This is a graph showing the effect of LD-1@MV-FA nanoparticles on apoptosis in HCT-116 cells.

[0032] Figure 8 This is a diagram showing the effect of LD-1@MV-FA nanoparticles on the migration of HCT-116 cells. Figure 9 This is a diagram showing the effect of LD-1@MV-FA nanoparticles on the morphology of RAW264.7 cells.

[0033] Figure 10This is a graph showing the effect of LD-1@MV-FA nanoparticles on NO production in RAW264.7 cells.

[0034] Figure 11 This is a diagram illustrating the therapeutic effect of LD-1@MV-FA nanoparticles on a zebrafish colorectal cancer model. Figure 12 This is a graph evaluating the in vivo tumor targeting effect of LD-1@MV-FA nanoparticles on a mouse model of colorectal cancer.

[0035] Figure 13 This is a graph showing the changes in tumor volume in a mouse model of colorectal cancer after treatment with LD-1@MV-FA nanoparticles.

[0036] Figure 14 This is a graph showing the changes in tumor size and weight in a mouse model of colorectal cancer after treatment with LD-1@MV-FA nanoparticles. Detailed Implementation

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0040] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0041] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0042] A nanoparticle loaded with lupeol derivative in vesicles, the nanoparticles being composed of probiotic vesicles encapsulating a lupeol quaternary ammonium salt derivative (LD-1), the surface of the probiotic vesicles being modified with phospholipid-disulfide bond-polyethylene glycol-folic acid (DSPE-SS-PEG-FA).

[0043] The structural formula of lupeol quaternary ammonium salt derivative (LD-1) is as follows:

[0044] The structural formula of phospholipid-disulfide bond-polyethylene glycol-folic acid (DSPE-SS-PEG-FA) is as follows:

[0045] Among them, the molecular weight of polyethylene glycol is ≥2000, and the probiotic vesicles include Lactobacillus plantarum vesicles.

[0046] A method for preparing vesicle-loaded lupeol derivative nanoparticles includes the following steps: S1: Centrifuge the probiotic culture medium at a centrifugation force of 1000-2000×g, a temperature of 4℃, and a time of 20-40min, and collect the precipitated bacterial cells.

[0047] S2: Resuspend the precipitated bacterial cells in lysozyme solution, shake well, centrifuge at 3000-10000×g for 5-20 min, and collect the precipitated protoplasts. The lysozyme solution includes at least one of animal lysozyme, microbial lysozyme, and egg white lysozyme, with a concentration of 1-10 mg / mL.

[0048] S3: Resuspend the precipitated protoplasts in phosphate-buffered saline (PBS), then sonicate them in an ice bath at 0-4°C for 5-30 min at a frequency of 20-50 kHz. Remove the precipitate by centrifugation at 2000-4000 × g at 4°C for 10-30 min, and retain the supernatant containing probiotic membrane fragments.

[0049] S4: Centrifuge the supernatant containing probiotic membrane fragments at a centrifugation force of 15000-20000×g and a temperature of 4℃ for 30-60 min to remove organelles and other impurities. Keep the supernatant and then centrifuge at a centrifugation force of 80000-100000×g and a temperature of 4℃ for 30-60 min to separate the membrane probiotic fragments and obtain a precipitate containing probiotic membrane fragments.

[0050] S5: The precipitate containing probiotic membrane fragments was resuspended in phosphate-buffered saline (PBS), and then lupeol quaternary ammonium salt derivative and phospholipid-disulfide bond-polyethylene glycol-folic acid (DSPE-SS-PEG-FA) were added. The mixture was then sonicated in an ice bath at 0-4℃ for 30-60 kHz for 30-60 min to prepare nanospheres.

[0051] The protein concentration in the precipitate containing probiotic membrane fragments is 100-200 μg / ml; the mass ratio of the precipitate containing probiotic membrane fragments, lupeol quaternary ammonium salt derivatives, and phospholipid-disulfide bond-polyethylene glycol-folic acid is 50-400:1:25-200, and the precipitate containing probiotic membrane fragments is based on protein mass.

[0052] S6: The nanospheres are placed in an ice bath at 0-4℃ and extruded into a film by repeated extrusion 10-20 times using a thin film extrusion method. After extrusion, the mixture is filtered through a 0.22μm filter membrane for sterilization to prepare vesicle-loaded lupeol derivative nanoparticles. The film used includes a polycarbonate membrane with a pore size of 100-200nm.

[0053] The prepared vesicle-loaded lupeol derivative nanoparticles can be used in pharmaceutical compositions, health product compositions, or food.

[0054] Specifically, in pharmaceutical compositions, drug-acceptable carriers or excipients can be added according to pharmaceutical and formulation requirements. Dosage forms include tablets, capsules, injections, injectable solutions, oral solutions, solutions, suspensions, ointments, lyophilized powders, pellets, films, liposomes, etc.

[0055] The health product composition can be in dosage forms such as tablets, injections, capsules, injectable solutions, or oral solutions.

[0056] In food, it can be used as a functional additive, incorporated into everyday foods such as beverages, cheese, bread, and flour in liquid or solid powder form.

[0057] Example 1 of the present invention is a preferred embodiment, and Examples 4-7 use the sample prepared in Example 1 for testing.

[0058] Example 1 A method for preparing vesicle-loaded lupeol derivative nanoparticles (LD-1@MV-FA), such as... Figure 1 As shown, it includes the following steps: S1 was prepared using MRS broth medium at a ratio of 5.4g of medium per 100ml of distilled water. After preparation, it was subjected to high-temperature autoclaving.

[0059] A volume ratio of 100:3 (MRS broth medium: Lactobacillus plantarum cryopreservation solution) was used to inoculate the frozen Lactobacillus plantarum culture into the culture medium, and the medium was placed in a shaker and incubated at 37°C for 48 hours. When the Lactobacillus plantarum reached a suitable state (i.e., the optical density measured at 600 nm under UV light reached 1.0-1.5, or it entered the stationary phase, with a colony count ≥2.7 × 10^9 cfu / mL), the Lactobacillus plantarum culture was collected. The obtained Lactobacillus plantarum culture was centrifuged at 1500 × g at 4°C for 20 min, and the resulting precipitate of bacterial cells was collected.

[0060] S2, Subsequently, the collected precipitated bacterial cells were resuspended in 1 ml of egg white lysozyme solution with a concentration of 10 mg / ml and treated in a shaker at 37°C for 24 h. After 24 h, the protoplasts obtained by lysozyme treatment were centrifuged at 3500×g and 4°C for 10 min, and the precipitated protoplasts were collected.

[0061] S3, the collected protoplasts were resuspended in 1 ml of PBS and placed in a cell disruptor. Cell disruption was performed by sonication at 24 kHz and 4°C for 30 min on ice. After sonication, the precipitate was removed by centrifugation at 3500 × g and 4°C for 10 min, and the supernatant containing Lactobacillus plantarum membrane fragments was retained.

[0062] S4. Centrifuge the supernatant containing *Lactobacillus plantarum* membrane fragments at 20,000 × g and 4 °C for 30 min to remove organelles and other impurities, and retain the supernatant. Continue centrifuging at 100,000 × g and 4 °C for 60 min to obtain a precipitate containing *Lactobacillus plantarum* membrane fragments.

[0063] S5, mix 200 μg / ml of *Lactobacillus plantarum* membrane fragments with 6 μl of 0.1 mM LD-1, and simultaneously add 10 μl of 10 mg / ml DSPE-SS-PEG-FA. Sonicate the mixture at 40 kHz in an ice bath at 4°C for 30 min to prepare nanospheres.

[0064] S6. The nanospheres were extruded 20 times using a manual liposome extruder equipped with a 100nm polycarbonate film. After extrusion, the mixture was filtered through a 0.22μm filter membrane for sterilization, and finally, vesicle-loaded lupeol derivative nanoparticles (LD-1@MV-FA) were prepared.

[0065] Example 2 A method for preparing vesicle-loaded lupeol derivative nanoparticles (LD-1@MV-FA) includes the following steps: S1 was prepared using MRS broth medium at a ratio of 5.4g of medium per 100ml of distilled water. After preparation, it was subjected to high-temperature autoclaving.

[0066] A volume ratio of 100:3 (MRS broth medium: Lactobacillus plantarum cryopreservation solution) was used to inoculate the frozen Lactobacillus plantarum culture into the culture medium, and the medium was placed in a shaker and incubated at 37°C for 48 hours. When the Lactobacillus plantarum reached a suitable state (i.e., the optical density measured at 600 nm under UV light reached 1.0-1.5, or it entered the stationary phase, with a colony count ≥2.7×10^9 cfu / mL), the Lactobacillus plantarum culture was collected. The obtained Lactobacillus plantarum culture was centrifuged at 2000×g at 4°C for 20 min, and the resulting precipitate of bacterial cells was collected.

[0067] S2, Subsequently, the collected precipitated bacterial cells were resuspended in 1 ml of egg white lysozyme solution with a concentration of 10 mg / ml and treated in a shaker at 37°C for 24 h. After 24 h, the protoplasts obtained by lysozyme treatment were centrifuged at 10000×g at 4°C for 5 min, and the precipitated protoplasts were collected.

[0068] S3, the collected protoplasts were resuspended in 1 ml of PBS and placed in a cell disruptor. Cell disruption was performed by sonication at 24 kHz and 4°C for 30 min on ice. After sonication, the precipitate was removed by centrifugation at 3500 × g and 4°C for 10 min, and the supernatant containing Lactobacillus plantarum membrane fragments was retained.

[0069] S4. Centrifuge the supernatant containing *Lactobacillus plantarum* membrane fragments at 15000×g, 4℃ for 60 min to remove organelles and other impurities, and retain the supernatant. Continue centrifuging at 100000×g, 4℃ for 30 min to obtain a precipitate containing *Lactobacillus plantarum* membrane fragments.

[0070] S5, mix 200 μg / ml of *Lactobacillus plantarum* membrane fragments with 6 μl of 0.2 mM LD-1, and simultaneously add 10 μl of 10 mg / ml DSPE-SS-PEG-FA. Sonicate the mixture at 40 kHz in an ice bath at 4 °C for 30 min to prepare nanospheres.

[0071] S6. The nanospheres were extruded 20 times using a manual liposome extruder equipped with a 100nm polycarbonate film. After extrusion, the mixture was filtered through a 0.22μm filter membrane for sterilization, and finally, vesicle-loaded lupeol derivative nanoparticles (LD-1@MV-FA) were prepared.

[0072] Example 3 A method for preparing vesicle-loaded lupeol derivative nanoparticles (LD-1@MV-FA) includes the following steps: S1 was prepared using MRS broth medium at a ratio of 5.4g of medium per 100ml of distilled water. After preparation, it was subjected to high-temperature autoclaving.

[0073] A volume ratio of 100:3 (MRS broth medium: Lactobacillus plantarum cryopreservation solution) was used to inoculate the frozen Lactobacillus plantarum culture into the culture medium, and the medium was placed in a shaker and incubated at 37°C for 48 hours. When the Lactobacillus plantarum reached a suitable state (i.e., the optical density measured at 600 nm under UV light reached 1.0-1.5, or it entered the stationary phase, with a colony count ≥2.7 × 10^9 cfu / mL), the Lactobacillus plantarum culture was collected. The obtained Lactobacillus plantarum culture was centrifuged at 1000 × g at 4°C for 40 min, and the resulting precipitate of bacterial cells was collected.

[0074] S2, Subsequently, the collected precipitated bacterial cells were resuspended in 1 ml of egg white lysozyme solution with a concentration of 10 mg / ml and treated in a shaker at 37°C for 24 h. After 24 h, the protoplasts obtained by lysozyme treatment were centrifuged at 6000×g at 4°C for 8 min, and the precipitated protoplasts were collected.

[0075] S3, the collected protoplasts were resuspended in 1 ml of PBS and placed in a cell disruptor. Cell disruption was performed by sonication at 24 kHz and 4°C for 30 min on ice. After sonication, the precipitate was removed by centrifugation at 4000 × g and 4°C for 10 min, and the supernatant containing Lactobacillus plantarum membrane fragments was retained.

[0076] S4. Centrifuge the supernatant containing *Lactobacillus plantarum* membrane fragments at 18000×g, 4℃ for 30 min to remove organelles and other impurities, and retain the supernatant. Continue centrifuging at 80000×g, 4℃ for 45 min to obtain a precipitate containing *Lactobacillus plantarum* membrane fragments.

[0077] S5, mix 200 μg / ml of *Lactobacillus plantarum* membrane fragments with 6 μl of 0.4 mM LD-1, and simultaneously add 10 μl of 10 mg / ml DSPE-SS-PEG-FA. Sonicate the mixture at 40 kHz in an ice bath at 4 °C for 30 min to prepare nanospheres.

[0078] S6. The nanospheres were extruded 20 times using a manual liposome extruder equipped with a 100nm polycarbonate film. After extrusion, the mixture was filtered through a 0.22μm filter membrane for sterilization, and finally, vesicle-loaded lupeol derivative nanoparticles (LD-1@MV-FA) were prepared.

[0079] Example 4 Characterization and performance analysis of LD-1@MV-FA nanoparticles (1) The particle size of the LD-1@MV-FA nanoparticles prepared in Example 1 was measured using a laser particle size analyzer, and the results are as follows: Figure 2 As shown. The LD-1@MV-FA nanoparticles prepared in Example 1 have an average particle size of 165.3 nm, which is within the suitable particle size range for nano-drug loading (generally referring to 1-1000 nm, of which 20-200 nm particle size range is more common). Its larger volume provides more space for subsequent drug loading.

[0080] (2) The morphology of the LD-1@MV-FA nanoparticles prepared in Example 1 was examined using transmission electron microscopy. The results are as follows: Figure 3 As shown. The LD-1@MV-FA nanoparticles prepared in Example 1 are spherical and have a significant phospholipid bilayer structure. Furthermore, the vesicle size in the electron microscopy image is consistent with the dynamic light scattering results, indicating that LD-1@MV-FA was successfully obtained.

[0081] (3) In vitro drug controlled release experiment To investigate the release behavior of LD-1 in LD-1@MV-FA nanoparticles, we examined whether the disulfide bonds on the modifier could be broken due to the difference in the content of reduced glutathione (GSH) between the tumor microenvironment and the normal environment, thereby accelerating drug release.

[0082] The experiment began with LD-1@MV-FA, prepared in Example 1, placed in a dialysis bag with a molecular weight of 500 Da. This bag was then aliquoted into experimental groups with different GSH concentrations (2 mM in normal tissue and 8 mM in the tumor microenvironment). The dialysis bag and buffer solution were placed in a 37°C constant-temperature shaker and horizontally oscillated at 120 rpm to simulate the dynamic environment of the human body. During the experiment, 50 μl samples were precisely taken from the release medium at predetermined intervals. An equal volume of PBS buffer was immediately added after each sampling to maintain a constant total volume and ensure the stability of the experimental conditions. The 50 μl samples were transferred to 96-well plates, and their absorbance at 248 nm was measured. By comparing the absorbance with a standard curve, the absorbance values ​​were converted into corresponding drug concentrations, and the total drug release was calculated.

[0083] Drug controlled release effect such as Figure 4 As shown, under simulated normal physiological conditions (GSH concentration of 2 mM), the cumulative release of LD-1 remained at a low level. This phenomenon indicates that LD-1@MV-FA nanoparticles have good stability during blood circulation, effectively preventing premature drug release before reaching the target site, thus ensuring complete drug delivery to the site of action. However, under simulated tumor microenvironment conditions (GSH concentration of 8 mM), the release behavior of LD-1 changed significantly, with a significant increase in cumulative release.

[0084] Example 5 In vitro cell evaluation experiment of LD-1@MV-FA nanoparticles (1) Cellular uptake of LD-1@MV-FA nanoparticles LD-1@MV-FA was labeled using a lipophilic fluorescent cell membrane far-infrared fluorescence staining kit (DiD) under light-protected conditions. First, a six-well plate was prepared, with an appropriate amount of complete culture medium added to each well, followed by a glass slide. HCT-116 colorectal cancer cells were seeded into the six-well plate at a density of 50% per well and incubated for 24 hours. When the cell density reached approximately 90% under a microscope, the drug delivery experiment could proceed. Fresh culture medium was prepared, with 150 μl of the prepared DiD-labeled LD-1@MV-FA added to every 850 ml of complete culture medium. After thorough mixing, the medium was added to the six-well plate via medium exchange. The cells were incubated for another 24 hours, then the supernatant was removed, and the cells were washed 2-3 times with PBS buffer. Cells were fixed with paraformaldehyde for approximately 15 minutes, then the paraformaldehyde was removed, and the cells were washed 2-3 times with PBS buffer. The glass slides were removed, mounted with mounting medium, and finally observed under an inverted fluorescence microscope.

[0085] The results are as follows Figure 5 As shown, LD-1@MV was labeled with fluorescein isothiocyanate (FITC) to obtain LD-1@MV-FITC. In the LD-1@MV-FITC group, the green fluorescent channel and the FITC green channel showed significant overlap. Furthermore, comparison with cell localization in bright field confirmed that the prepared LD-1@MV-FA nanoparticles could be taken up by HCT-116 cells.

[0086] (2) Cytotoxicity assay of LD-1@MV-FA nanoparticles The effects of LD-1@MV-FA nanoparticles on the proliferation activity of HCT-116 colorectal cancer cells, HCOepic colonic epithelial cells, and RAW264.7 macrophages were investigated using a commercially available CCK-8 cell proliferation assay kit.

[0087] The results are as follows Figure 6 As shown, when the protein concentration of LD-1@MV-FA solution was greater than 100 μg / ml, the survival rate of HCT-116 cells decreased to 2.45%. At this point, the LD-1 content increased, and its ability to inhibit cell proliferation was significantly enhanced. This indicates that the prepared LD-1@MV-FA possesses significant anti-proliferative capacity at the cellular level. Conversely, at an administration concentration of 100 μg / ml, the minimum survival rates of HCoEpic and RAW264.7 cells were both greater than 85%. This indicates that within the effective range, LD-1@MV-FA has minimal effect on macrophages and normal colorectal epithelial cells.

[0088] (3) Detection of the effect of LD-1@MV-FA nanoparticles on HCT-116 cell apoptosis The effect of LD-1@MV-FA on apoptosis in HCT-116 cells was detected using a commercial flow cytometry apoptosis detection kit. After the samples were tested, the data were collected and the cell DNA content was analyzed using FlowJo software.

[0089] The results are as follows Figure 7 As shown, Figure 7 A shows the apoptosis status of HCT-116 cells after 24 h of treatment with the control group, LD-1, simple bacterial vesicles (MV), and LD-1@MV, as detected by flow cytometry. The premature apoptosis rate in the control group (7A) was 6.15%, while the premature apoptosis rates in the LD-1 group (7B) and LD-1@MV-FA group were 13.45% and 16.47%, respectively. This indicates that nano-LD-1@MV-FA has a similar pro-apoptotic effect to the drug. Significance analysis (…) Figure 7 B), LD-1 and LD-1@MV-FA showed significant pro-apoptotic effects compared to the control group, while MV did not show obvious pro-apoptotic effects.

[0090] (4) Detection of the effect of LD-1@MV-FA nanoparticles on HCT-116 cell migration like Figure 8 As shown, Figure 8 A demonstrates the effect of LD-1@MV-FA nanoparticles on HCT-116 cell migration using the cell scratch assay. Figure 8 B and Figure 8 C shows the scratch area analysis results at 12h and 24h. After 12h of treatment, both the LD-1 group and the LD-1@MV-FA group showed significant cell migration inhibition compared to the control group, while the inhibition effect of the MV group was relatively weak. After 24h of treatment, only the LD-1@MV-FA group showed significant cell migration inhibition. This indicates that LD-1@MV-FA not only possesses the cell migration inhibition ability of LD-1 drug, but also has a more sustained migration inhibition effect compared to the LD-1 group alone. This phenomenon may be attributed to the sustained-release properties of LD-1@MV-FA.

[0091] (5) Observation of RAW264.7 cell morphology RAW264.7 mouse macrophages were cultured to a confluence density of 60% and then seeded into six-well plates. Once the cell density in the six-well plates reached 60% again, the appropriate concentrations of the culture medium were prepared using complete culture medium according to the control group, LD-1 group, MV group, and LD-1@MV-FA group, and the drugs were administered via medium exchange. After culturing the cells in an incubator for 24 hours, the supernatant was removed, PBS was added, and the morphological changes of the RAW264.7 mouse macrophages were observed under a light microscope.

[0092] The results are as follows Figure 9 As shown, Figure 9 Figures A, B, C, and D show the morphological changes of RAW264.7 cells after treatment with the BLANK, LD, MV, and LD-1@MV-FA groups for 48 hours, respectively. Observation of the morphology of RAW264.7 cells revealed significant dendritic morphology in both the MV and LD-1@MV-FA groups, indicating that the prepared MV and LD-1@MV-FA can promote the polarization of RAW264.7 cells.

[0093] (6) Detection of nitric oxide concentration The effect of LD-1@MV-FA nanoparticles on nitric oxide production in RAW264.7 cells was investigated using a commercially available nitric oxide assay kit.

[0094] The results are as follows Figure 10 As shown, 24 hours after drug administration, the average NO concentration was 2.2 μM in the control group, 3.4 μM in the LD-1 group, 14.7 μM in the MV group, and 17.0 μM in the LD-1@MV-FA group. Compared with the control group and the LD-1 group, the MV group and the nanoparticle group significantly promoted NO production. Since NO is a marker of macrophage polarization to the M1 type, this indicates that MV and LD-1@MV-FA can promote macrophage polarization to the M1 type to some extent.

[0095] Example 6 Therapeutic effects of LD-1@MV-FA nanoparticles on a zebrafish colorectal carcinoma model Normally developing 48 hpf zebrafish embryos were selected and placed on agarose gel. Approximately 30 nL of HCT-116 tumor cells labeled with an orange cell membrane fluorescent probe (DiI) were injected into the zebrafish yolk sac using a microinjection apparatus. The embryos were then incubated at 28.5°C for 24 h. After incubation, zebrafish embryos with similar tumor size and no metastasis were selected under a fluorescence microscope and photographed, thus constructing a zebrafish colorectal cancer model.

[0096] Ten zebrafish with colorectal cancer were placed in 48-well plates. Four groups were set up: a blank control group (PBS), an LD-1 group (500 μM), an MV group (200 μg / mL), and an LD-1-MV group (200 μg / mL). At 72 hpf and 120 hpf, the above solutions were injected into the yolk sacs of the zebrafish using a microinjector to observe their effect on the number of yolk sac vesicles. At 144 hpf, the zebrafish were observed and photographed under a fluorescence microscope to observe the proliferation of tumor cells. The fluorescence area of ​​the tumor cells was analyzed using ImageJ, and the number of macrophages within the tumor was counted.

[0097] The results are as follows Figure 11 As shown, Figure 11 A presents fluorescence microscopic images of tumor cells and macrophages in zebrafish, and plots them based on their fluorescence area and cell number. Figure 11 B and Figure 11 C. Figure 11 B shows that at 144 hpf, the fluorescence area of ​​tumor cells (red fluorescence) in zebrafish in the MV group was 27870.90±1926.92 pixels, which was not significantly different from that in the blank control group (30231.10±2648.89 pixels) (p>0.05). In addition, the fluorescence areas of tumor cells in zebrafish in the LD-1 group and the LD-1@MV-FA group were 21166.80±1608.33 pixels and 18579.20±1571.82 pixels, respectively, which were significantly different from those in the blank control group (30231.10±2648.89 pixels) (p<0.05).

[0098] Therefore, LD-1 and LD-1@MV-FA significantly inhibited the growth of HCT-116 tumor cells in zebrafish. At 144 hpf, the number of macrophages (green fluorescence) in the tumor of zebrafish in the MV group was (7.10±1.32), which was not significantly different from that in the blank control group (5.80±1.02) (p>0.05). Furthermore, from Figure 11 As shown in Figure C, the number of macrophages in the tumor cells of zebrafish in the LD-1 group and the LD-1@MV-FA group were (11.30±1.07) and (13.00±1.09), respectively, which were significantly different from those in the blank control group (5.80±1.02) (p<0.05). Therefore, LD-1 and LD-1@MV-FA can significantly promote the aggregation of macrophages around tumor HCT-116 cells in zebrafish.

[0099] Example 7 Therapeutic effects of LD-1@MV-FA nanoparticles on a mouse model of colorectal cancer (1) A mouse model of colorectal cancer was established using a subcutaneous tumorigenesis method. A control group (PBS), an LD-1 (500 μM) group, an MV (200 μg / mL) group, and an LD-1-MV (200 μg / mL) group were set up. The drugs were injected into the animals via tail vein injection, once every two days, for a total intervention period of approximately 8 days.

[0100] (2) To verify the targeting performance of LD-1@MV-FA, a mouse in vivo imaging experiment was conducted. First, mice with successfully established models were injected with LD-1@MV-Fitc via the tail vein, then anesthetized with isoflurane, and fluorescence imaging was performed using a small animal in vivo imaging system. Fluorescence imaging was conducted at 0h, 2h, 4h, 8h, 12h, and 24h. Twenty-four hours after injection, the mice were euthanized by cervical dislocation, and dissected to remove key organs and tumor tissue for fluorescence imaging.

[0101] The results are as follows Figure 12 As shown. Figure 12 A shows that during tail vein injection, green fluorescence appeared at tumor sites on both sides of the dorsal side of the mouse's forelimb, indicating that the nanoparticles can rapidly target tumor sites through blood circulation and the specific binding of FA to the tumor. Over the next 24 hours, the fluorescence intensity did not significantly decrease under uniform grayscale values, indicating that LD-1@MV-FA can continuously release the drug at the lesion site.

[0102] Figure 12 The superimposed fluorescence images at different time points in B also show that, over time, the nanomedicine gradually penetrates deeper into the tumor. Figure 12 In A, the green fluorescence in the lower body of the mouse increased and converged over time, which is speculated to be due to the accumulation of the fluorescence in the bladder area caused by the mouse's metabolism.

[0103] Figure 12 C shows the results of fluorescence imaging of tumors and vital organs (heart, liver, spleen, lung, and kidney) from mice euthanized 24 hours after drug injection. The tumor sites show significant fluorescence intensity, followed by green fluorescence in major metabolic organs such as the liver and kidneys, while no green fluorescence is observed in other vital organs. This phenomenon indicates that LD-1@MV-FA not only possesses good targeting ability but also significantly reduces its impact on other vital organs, thereby improving drug safety.

[0104] (3) After randomization, before the start of treatment, the tumor volume of each mouse was accurately measured using calipers. Specifically, the maximum length (L) and maximum width (W) of the tumor were measured, and then the volume was calculated using the formula V=(L×W). 2The tumor volume was calculated as () / 2. Every 3 days, the tumor volume data of mice was accurately measured and recorded, and the curve of mouse body weight versus tumor volume change was plotted to evaluate the effect of different drugs on mouse tumors.

[0105] The results are as follows Figure 13 As shown, the dynamic monitoring results of mouse tumor volume during the 28-day modeling and intervention treatment process further verified the above phenomenon. By day 28, the average tumor volume of mice in the control group, LD-1 group, MV group, and LD-1@MV-FA group was 1.79 cm³. 3 0.79cm 3 1.31cm 3 and 0.57cm 3 Statistical significance analysis (p<0.05) showed that the tumor volume in the MV group was slightly lower than that in the control group, indicating that the membrane carrier has a certain bioregulatory effect. However, the LD-1@MV-FA group exhibited the most significant tumor inhibition effect throughout the entire treatment cycle, with a significantly slower tumor volume growth rate and a terminal volume of only about one-third that of the control group, which was significantly better than the LD-1 monotherapy group. This indicates that under the combined effect of the membrane carrier and folic acid targeted modification, the drug accumulation efficiency at the tumor site was significantly improved, thereby enhancing the anti-tumor effect.

[0106] (4) After treatment, to evaluate the inhibitory effect of different drugs on tumors in tumor-bearing mice, tumor tissue samples were collected from Balb / c-nu mice. Mice were euthanized by cervical dislocation, and the tumor tissue was completely removed and washed three times with PBS to remove surface blood and residue. After washing, the tumor weight was measured using an electronic balance and the data was recorded; simultaneously, photographs of the tumor tissue were taken to visually compare the size and appearance differences of tumors in each group.

[0107] The results are as follows Figure 14 As shown, Figure 14 A shows tumor photographs, and the results clearly demonstrate significant differences in tumor growth among the mouse groups. Compared to the control group, both the LD-1@MV-FA group and the LD-1 group exhibited significant tumor inhibition effects, with significantly reduced tumor volume, blurred tumor outlines, and decreased tissue density, showing strong anti-tumor activity. However, mice treated with MV alone still showed some tumor growth, indicating a relatively limited inhibitory effect, suggesting that the carrier itself has a weak anti-tumor effect and mainly functions as a drug delivery agent. Furthermore, further, combined with... Figure 14Analysis of the extracted tumor tissues showed that the average tumor weights of the mice in each group were as follows: control group 1.84g, LD-1 group 0.88g, MV group 1.22g, and LD-1@MV-FA group 0.62g. Compared with the control group, the tumor weights of the LD-1@MV-FA and LD-1 groups were significantly reduced, with the LD-1@MV-FA group showing the most significant tumor-suppressing effect (p<0.01). This result is consistent with the trend of tumor volume changes, further validating the synergistic anti-tumor effect of the LD-1@MV-FA nanosystem in vivo. Based on the above results, it can be inferred that the membrane encapsulation and folic acid modification strategy not only improves the stability and biocompatibility of the drug, but also promotes the specific uptake and retention of the drug by targeting folic acid receptors on the surface of tumor cell membranes, thereby achieving a more efficient tumor-suppressing effect.

[0108] The above embodiments are merely illustrative of several implementations of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the invention patent. For those skilled in the art, any changes, modifications, substitutions, integrations, and parameter alterations to these embodiments without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A type of nanoparticle loaded with lupeol derivative in vesicles, characterized in that, The nanoparticles are composed of probiotic vesicles encapsulating lupeol quaternary ammonium salt derivatives, and the surface of the probiotic vesicles is modified with phospholipid-disulfide bond-polyethylene glycol-folic acid. The structural formula of the lupinol quaternary ammonium salt derivative is as follows: ； The structural formula of the phospholipid-disulfide bond-polyethylene glycol-folic acid is: Among them, the molecular weight of polyethylene glycol is ≥2000.

2. The vesicle-loaded lupeol derivative nanoparticles according to claim 1, characterized in that, The probiotic vesicles include Lactobacillus plantarum vesicles.

3. A method for preparing vesicle-loaded lupeol derivative nanoparticles as described in any one of claims 1 to 2, characterized in that, Includes the following steps: S1: Centrifuge the probiotic culture medium and collect the precipitated bacterial cells; S2: The precipitated bacterial cells are resuspended in lysozyme solution, shaken to mix, centrifuged, and the precipitated protoplasts are collected. S3: The precipitated protoplasts are resuspended in phosphate buffer solution, then the membrane is broken by sonication, the precipitate is removed by centrifugation, and the supernatant containing probiotic membrane fragments is retained. S4: The supernatant containing probiotic membrane fragments is centrifuged for the first time to remove organelles and other impurities, and the supernatant is retained. The membrane probiotic fragments are separated by a second centrifugation to obtain a precipitate containing probiotic membrane fragments. S5: The precipitate containing probiotic membrane fragments is resuspended in phosphate buffer solution, then the lupeol quaternary ammonium salt derivative and the phospholipid-disulfide bond-polyethylene glycol-folic acid are added, and the mixture is sonicated to form nanospheres; S6: The nanospheres are extruded using a thin film extrusion method to prepare vesicle-loaded lupeol derivative nanoparticles.

4. The method for preparing vesicle-loaded lupeol derivative nanoparticles according to claim 3, characterized in that, In step S1, when the probiotic culture medium is centrifuged, the centrifugal force is 1000-200×g, the temperature is 4℃, and the time is 20-40min.

5. The method for preparing vesicle-loaded lupeol derivative nanoparticles according to claim 3, characterized in that, In step S2, the lysozyme solution includes at least one of animal lysozyme, microbial lysozyme, and egg white lysozyme; the concentration of the lysozyme solution is 1-10 mg / mL; after adding the lysozyme solution, it is treated at 35-40℃ for 12-48 h; during centrifugation, the centrifugal force is 3000-10000×g and the time is 5-20 min.

6. The method for preparing vesicle-loaded lupeol derivative nanoparticles according to claim 3, characterized in that, In step S3, the membrane is broken by ultrasonication in an ice bath at 0-4℃, with an ultrasonic frequency of 20-50kHz and an ultrasonic time of 5-30min; during centrifugation, the centrifugal force is 2000-4000×g, the temperature is 4℃, and the time is 10-30min.

7. The method for preparing vesicle-loaded lupeol derivative nanoparticles according to claim 3, characterized in that, In step S4, during the first centrifugation, the centrifugal force is 15000-20000×g, the temperature is 4℃, and the time is 30-60min; during the second centrifugation, the centrifugal force is 80000-100000×g, the temperature is 4℃, and the time is 30-60min.

8. The method for preparing vesicle-loaded lupeol derivative nanoparticles according to claim 3, characterized in that, In step S5, the sample is sonicated in an ice bath at 0-4℃, with an ultrasonic frequency of 40-60kHz and an ultrasonic time of 30-60min; the protein concentration in the precipitate containing probiotic membrane fragments is 100-200μg / ml; the mass ratio of the precipitate containing probiotic membrane fragments, the lupeol quaternary ammonium salt derivative, and the phospholipid-disulfide bond-polyethylene glycol-folic acid is 50-400:1:25-200, and the precipitate containing probiotic membrane fragments is based on protein mass.

9. The method for preparing vesicle-loaded lupeol derivative nanoparticles according to claim 3, characterized in that, In step S6, the film used in the film extrusion method includes a polycarbonate film with a pore size of 100-200 nm, and the film is extruded by repeated extrusion in an ice bath at 0-4°C for 10-20 times.

10. The application of nanoparticles containing vesicle-loaded lupeol derivatives as described in any one of claims 1 to 2, characterized in that, It can be used in pharmaceutical compositions, health product compositions, or food.

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