Preparation method of rhizoma smilacis glabrae exosome and application of rhizoma smilacis glabrae exosome in bladder cancer

Abstract

The invention belongs to the technical field of biological medicines, and discloses a preparation method of a rhizoma smilacis glabrae exosome and application of the rhizoma smilacis glabrae exosome in bladder cancer. The preparation method comprises the following steps: cleaning, soaking and slicing a rhizoma smilacis glabrae medicinal material, adding a phosphate buffer solution, juicing in a juicer, and filtering to obtain crude juice; sequentially carrying out low-speed, medium-speed and ultra-high-speed centrifugal treatment through a differential ultra-speed centrifugal method to obtain a rhizoma smilacis glabrae precipitate; and resuspending by using a phosphate buffer solution, carrying out ultra-high-speed centrifugation and filtering by using a filter membrane to obtain the rhizoma smilacis glabrae exosome. System experiments prove that the exosome-like nano-particles extracted from rhizoma smilacis glabrae can be effectively taken by bladder cancer cells, can significantly inhibit the growth of bladder cancer in vivo and in vitro by inhibiting proliferation and invasion of cancer cells, inducing oxidative stress, apoptosis, cell cycle arrest and other mechanisms, and has no obvious toxic or side effect on normal tissues; good biological safety and clinical application prospects are realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing Smilax glabra exosomes and their application in bladder cancer. Background Technology

[0002] Bladder cancer (BCa) is a common malignant tumor of the urinary system, and its treatment still faces challenges such as high recurrence rates, chemotherapy resistance, and toxic side effects. Currently, commonly used clinical regimens combining surgery with chemotherapy or immunotherapy have limited efficacy and significantly impact patients' quality of life. Therefore, the development of novel, highly effective, and low-toxicity anti-bladder cancer drugs is of great significance.

[0003] In recent years, plant-derived exosome-like nanoparticles have been considered promising drug delivery systems. However, existing research has largely focused on a few plants, such as ginseng and pumpkin, whose antitumor activities and mechanisms are source-specific. Smilax glabra Roxb (SGR) is a traditional Chinese medicine, but its conventional extracts may have their composition and activity stability affected by factors such as heating during processing, thus impacting efficacy. Furthermore, research on its active ingredients and antitumor mechanisms, particularly in the form of SGR-derived exosome-like nanoparticles (SGR-ELNs), remains lacking. Summary of the Invention

[0004] To address the shortcomings of the aforementioned technologies, this invention provides a method for preparing exosomes from Smilax glabra and their application in bladder cancer. This invention successfully isolates exosome-like nanoparticles from fresh Smilax glabra and systematically demonstrates their significant and safe inhibitory effect on bladder cancer in vitro and in vivo, providing a solution for bladder cancer treatment and offering a drug or health product that effectively improves bladder cancer.

[0005] The technical solution of this invention: A method for preparing Smilax glabra exosomes includes the following steps: (1) Take Smilax glabra medicinal material, wash it, soak it in deionized water for 22-26 hours, take it out and slice it, add 1000-3000 mL of phosphate buffer solution with pH 5-9, put it into a juicer and juice it to form a paste, filter it with gauze filter, collect the filtrate, which is Smilax glabra crude juice, for use. (2) After pre-cooling the crude juice of Smilax glabra obtained in step (1) to 3-5℃, pour it into a centrifuge and centrifuge at low speed of 1000-5000×g for 10-40 min, at medium speed of 7000-13000×g for 40-80 min and at ultra-high speed of 80000-130000×g for 50-90 min. Centrifugation process to obtain Smilax glabra exosome precipitate for later use. (3) After resuspending the precipitate of Smilax glabra obtained in step (2) by adding phosphate buffer, centrifuge at 80,000-130,000×g for 50-90 min to obtain the resuspension of Smilax glabra exudate for later use. (4) Filter the resuspended Smilax glabra in step (3) through a 0.1-0.3 μm filter membrane to obtain 1-10 mL of Smilax glabra exosomes with a particle size of 155-170 nm and a protein concentration of 1-3 mg / mL.

[0006] In step (1) above, take the Smilax glabra medicinal material, wash it, soak it in deionized water for 23-25 ​​hours, take it out and slice it, add 1500-2500mL of phosphate buffer solution with pH 7-8, put it into a juicer and juice it to form a paste, filter it with a gauze filter, collect the filtrate, which is the crude juice of Smilax glabra, for later use.

[0007] Specifically, in step (1) above, the Smilax glabra medicinal material is washed, soaked in deionized water for 24 hours, sliced, and then 2000 mL of phosphate buffer solution with pH 7.4 is added. The mixture is put into a juicer to extract juice and form a paste. The paste is filtered through a gauze filter and the filtrate is collected as Smilax glabra crude juice for later use.

[0008] In step (2) above, the crude juice of Smilax glabra obtained in step (1) is pre-cooled to 3.5-4.5℃ and then poured into a centrifuge for sequential centrifugation at low speed of 2000-4000×g for 20-35min, medium speed of 9000-11000×g for 50-70min, and ultra-high speed of 90000-110000×g for 60-80min. The centrifugation process yields Smilax glabra exosome precipitate for later use.

[0009] Specifically, in step (2) above, the crude juice of Smilax glabra obtained in step (1) is pre-cooled to 4°C and then poured into a centrifuge for sequential centrifugation at 3000×g low speed for 30 min, 10000×g medium speed for 60 min and 100000×g ultra-high speed for 70 min. The centrifugation process yields Smilax glabra exosome precipitate for later use.

[0010] In step (3) above, after adding phosphate buffer to the precipitate of Smilax glabra obtained in step (2) for resuspending, centrifuge at 90000-110000×g for 60-80 min to obtain Smilax glabra resuspension for later use.

[0011] Specifically, in step (3) above, after adding phosphate buffer to the precipitate of Smilax glabra obtained in step (2) for resuspending, centrifuge at 100000×g for 70 min to obtain Smilax glabra resuspension for later use.

[0012] In step (4) above, the resuspension of Smilax glabra from step (3) is filtered through a 0.22 μm filter membrane to obtain 5 mL of Smilax glabra exosomes with a protein size of 163.7 nm and a protein content of 1.5 mg / mL.

[0013] The aforementioned application of Smilax glabra exosomes in the preparation of products for the prevention or treatment of bladder cancer.

[0014] The aforementioned products for bladder cancer are either pharmaceuticals or health supplements.

[0015] Beneficial effects of this invention: 1. This invention provides a method for extracting exosomes from Smilax glabra. By successfully isolating and characterizing SGR-ELNs from fresh Smilax glabra, the median particle size of which is 163.7 nm was determined, and the complex pharmacodynamic material basis was elucidated, providing a new entity for the development of nanomedicines derived from traditional Chinese medicine.

[0016] 2. In vitro and in vivo experiments on the SGR-ELNs of the present invention have fully demonstrated that SGR-ELNs can effectively inhibit bladder cancer by inhibiting proliferation and invasion, inducing oxidative stress, apoptosis and cell cycle arrest, with clear effects and significant therapeutic efficacy.

[0017] 3. Through the targeting verification of the SGR-ELNs of the present invention in bladder cancer cell lines, the experiment showed that SGR-ELNs can be effectively taken up by bladder cancer cells, especially showing high targeting in the UMUC-3 cell line, suggesting that it has a natural targeting advantage.

[0018] 4. Based on the safety of the SGR-ELNs of the present invention in in vivo experiments, SGR-ELNs showed low cytotoxicity to normal cells and no damage to the liver, kidney function and major organs of model animals at effective therapeutic doses, demonstrating good drug safety.

[0019] 5. The SGR-ELNs of this invention are extracted from natural plants, possess biodegradability, good biocompatibility, and low immunogenicity, which are superior to some synthetic nanomaterials and have great potential for clinical application. Attached Figure Description

[0020] Figure 1 Schematic diagram of SGR-ELNs separation process; Figure 2 Extract images of physical objects from different stages; Figure 3Physicochemical characterization of S. sarcodactylis exosome-like nanoparticles (A: NTA analysis of the size and concentration of SGR-ELNs (X-axis: diameter / nm, Y-axis: particle concentration / mL); B: zeta potential of SGR-ELNs; C: TEM images of SGR-ELNs at different scales (1000nm, 300nm, 100nm)). Figure 4 Metabolomics analysis of SGR-ELNs based on UPLC-MS / MS (A: Total ion chromatogram of SGR-ELNs in positive ion mode; B: Total ion chromatogram of SGR-ELNs in negative ion mode). Figure 5 Analysis of the pharmacodynamic material basis of SGR-ELNs (A: Classification ratio of all components of SGR-ELNs; B: Classification ratio of the top 100 components); Figure 6 Typical diagram of SGR-ELNs being taken up by cells; Figure 7 Comparison of uptake efficiency of SGR-ELNs by three bladder cancer cell lines (A: Quantitative and differential analysis of uptake efficiency of fluorescently labeled SGR-ELNs by different bladder cancer cell lines (UMUC-3, T24 and 5637) at 12 hours; B: Quantitative and differential analysis of uptake efficiency of fluorescently labeled SGR-ELNs at 24 hours). Figure 8 Inhibitory effects of SGR-ELNs on cell viability of different cell lines (A: UMUC-3 cells co-cultured with SGR-ELNs, cell viability was measured using CCK8 at 24h and 48h; B: T24 cells co-cultured with SGR-ELNs, cell viability was measured using CCK8 at 24h and 48h; C: 5637 cells co-cultured with SGR-ELNs, cell viability was measured using CCK8 at 24h and 48h; D: SV-HUC-1 cells co-cultured with SGR-ELNs, cell viability was measured using CCK8 at 24h and 48h). Figure 9 Inhibitory effects of SGR-ELNs on the proliferation of UMUC-3 and T24 cells (A: UMUC-3 cell colony formation assay and quantitative analysis; B: T24 cell colony formation assay and quantitative analysis; D: UMUC-3 cell PCNA protein blot analysis and quantitative analysis; G: T24 cell PCNA protein blot analysis and quantitative analysis; H: T24 cell PCNA protein blot analysis and quantitative analysis). Figure 10 The inhibitory effect of SGR-ELNs on the invasive ability of UMUC-3 and T24 cells (A: Measurement and quantitative analysis of UMUC-3 cell invasive ability; B: Measurement and quantitative analysis of T24 cell invasive ability; D: Measurement and quantitative analysis of T24 cell invasive ability). Figure 11 SGR-ELNs induce oxidative stress in UMUC-3 and T24 cells (A: Oxidative stress changes ROS levels in UMUC-3 cells and its quantitative analysis; B: Oxidative stress changes ROS levels in T24 cells and its quantitative analysis; D) Figure 12 SGR-ELNs induced significant damage to UMUC-3 and T24 cells (A: SGR-ELNs induced UMUC-3 cell contraction and aggregation (scale bar: 100 μm); B: SGR-ELNs induced T24 cell contraction and aggregation (scale bar: 100 μm)). Figure 13 SGR-ELNs induce apoptosis in UMUC-3 and T24 cells (A: Flow cytometry analysis of apoptosis rate and quantitative analysis of SGR-ELNs-treated UMUC-3 cells; B: Apoptosis rate and quantitative analysis of T24 cells; D: Western blot analysis and quantitative analysis of cleaved-caspase 3 protein in UMUC-3 cells after SGR-ELNs treatment; G: Western blot analysis and quantitative analysis of cleaved-caspase 3 protein in T24 cells; H: Western blot analysis and quantitative analysis of cleaved-caspase 3 protein in T24 cells). Figure 14 SGR-ELNs induce G2 phase cell cycle arrest in UMUC-3 and T24 cells (A: Flow cytometry analysis of cell cycle distribution and quantitative analysis of SGR-ELNs-treated UMUC-3 cells; B: Flow cytometry analysis of cell cycle distribution and quantitative analysis of SGR-ELNs-treated T24 cells; D: Western blot analysis and quantitative analysis of CDK1, Cyclin B1, and p21 proteins in UMUC-3 cells after SGR-ELNs treatment (FH); I: Western blot analysis and quantitative analysis of CDK1, Cyclin B1, and p21 proteins in T24 cells (JL)). Figure 15 SGR-ELNs inhibited tumor growth in bladder cancer xenografts. (A: Body weight changes in different groups of animal models (phosphate-buffered saline PBS: control group, SGR-ELNs-L: 5 mg / kg, SGR-ELNs-H: 10 mg / kg); B: Images of tumor-bearing mice in different groups; C: Images of tumor tissues in different groups in vitro; D: Analysis of tumor weight differences in different groups of mice; E: Analysis of tumor volume differences in different groups of mice; F: Immunohistochemical (Ki-67) staining sections of mouse tumor tissue (scale bar: 50 μm); G: H&E staining of mouse tumor tissue (scale bar: 50 μm)). Figure 16Safety assessment results of SGR-ELNs (A: ALT detection results in different groups of mice; B: AST detection results in different groups of mice; C: CREA detection results in different groups of mice; D: H&E staining results of heart, liver, spleen, lung and kidney tissues of mice in each group (scale bar: 50um)). Detailed Implementation

[0021] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention.

[0022] Example 1: Preparation of Smilax glabra exosomes Wash the Smilax glabra herb, soak it in deionized water for 22 hours, take it out and slice it, add 1000mL of phosphate buffer solution with pH 5, put it in a juicer and juice it to form a paste, filter it with a gauze filter, collect the filtrate, which is the crude juice of Smilax glabra, for later use. After pre-cooling the crude juice of Smilax glabra obtained in step (1) to 3°C, pour it into a centrifuge and centrifuge it sequentially at a low speed of 1000×g for 10 min, a medium speed of 7000×g for 40 min, and an ultra-high speed of 80000×g for 50 min. Centrifugation process yields Smilax glabra exosome precipitate for later use. After resuspending the precipitate of Smilax glabra obtained in step (2) by adding phosphate buffer, centrifuge at 80000×g for 50 min to obtain Smilax glabra resuspension for later use. The resuspension of Smilax glabra in step (3) was filtered through a 0.1 μm filter membrane to obtain 1 mL of Smilax glabra exosomes with a protein size of 155 nm and a protein content of 1 mg / mL.

[0023] Example 2: Preparation of Smilax glabra exosomes Wash the Smilax glabra herb, soak it in deionized water for 23 hours, take it out and slice it, add 1500mL of phosphate buffer solution with pH 6, put it in a juicer and juice it to form a paste, filter it with a gauze filter, collect the filtrate, which is the crude juice of Smilax glabra, for later use. After pre-cooling the crude juice of Smilax glabra obtained in step (1) to 3.5℃, pour it into a centrifuge and centrifuge at 2000×g for 20 min, 8000×g for 50 min and 90000×g for 60 min in sequence. Centrifugation process to obtain Smilax glabra exosome precipitate for later use. After resuspending the precipitate of Smilax glabra obtained in step (2) by adding phosphate buffer, centrifuge at 90000×g for 60 min to obtain Smilax glabra resuspension for later use. The resuspension of Smilax glabra in step (3) was filtered through a 0.2 μm filter membrane to obtain 3 mL of Smilax glabra exosomes with a protein content of 1.5 mg / mL and a particle size of 160 nm.

[0024] Example 3: Preparation of Smilax glabra exosomes Wash the Smilax glabra herb, soak it in deionized water for 24 hours, take it out and slice it, add 2000mL of phosphate buffer solution with pH 7, put it in a juicer and juice it to form a paste, filter it with a gauze filter, collect the filtrate, which is the crude juice of Smilax glabra, for later use. After pre-cooling the crude juice of Smilax glabra obtained in step (1) to 4°C, it was poured into a centrifuge and centrifuged at 3000×g for 25 min, 9000×g for 60 min, and 100000×g for 70 min in sequence. The centrifugation process yielded Smilax glabra exosome precipitate for later use. After resuspending the precipitate of Smilax glabra obtained in step (2) by adding phosphate buffer, centrifuge at 100,000×g for 70 min to obtain Smilax glabra resuspension for later use. The resuspension of Smilax glabra in step (3) was filtered through a 0.22 μm filter membrane to obtain 5 mL of Smilax glabra exosomes with a protein size of 162 nm and a protein concentration of 2 mg / mL.

[0025] Example 4: Preparation of Smilax glabra exosomes Wash the Smilax glabra herb, soak it in deionized water for 25 hours, take it out and slice it, add 2500mL of phosphate buffer solution with pH 8, put it in a juicer and juice it to form a paste, filter it with a gauze filter, collect the filtrate, which is the crude juice of Smilax glabra, for later use. After pre-cooling the crude juice of Smilax glabra obtained in step (1) to 4.5℃, pour it into a centrifuge and centrifuge at 4000×g for 30 min, 10000×g for 70 min and 110000×g for 80 min in sequence. Centrifugation process to obtain Smilax glabra exosome precipitate for later use. After resuspending the precipitate of Smilax glabra obtained in step (2) by adding phosphate buffer, centrifuge at 110000×g for 80 min to obtain Smilax glabra resuspension for later use. The resuspension of Smilax glabra in step (3) was filtered through a 0.3 μm filter membrane to obtain 8 mL of Smilax glabra exosomes with a protein size of 165 nm and a protein content of 2.5 mg / mL.

[0026] Example 5: Preparation of Smilax glabra exosomes Wash the Smilax glabra herb, soak it in deionized water for 26 hours, take it out and slice it, add 3000mL of phosphate buffer solution with pH 9, put it in a juicer and juice it to form a paste, filter it with a gauze filter, collect the filtrate, which is the crude juice of Smilax glabra, for later use. After pre-cooling the crude juice of Smilax glabra obtained in step (1) to 5°C, pour it into a centrifuge and centrifuge it sequentially at a low speed of 5000×g for 40 min, a medium speed of 13000×g for 80 min, and an ultra-high speed of 130000×g for 90 min. Centrifugation process yields Smilax glabra exosome precipitate for later use. After resuspending the precipitate of Smilax glabra obtained in step (2) by adding phosphate buffer, centrifuge at 130000×g for 90 min to obtain Smilax glabra resuspension for later use. The resuspension of Smilax glabra in step (3) was filtered through a 0.22 μm filter membrane to obtain 10 mL of Smilax glabra exosomes with a protein size of 170 nm and a protein content of 3 mg / mL.

[0027] Example 6: Preparation of Smilax glabra exosomes Wash the Smilax glabra herb, soak it in deionized water for 24 hours, take it out and slice it, add 2000mL of phosphate buffer solution with pH 7.4, put it in a juicer and juice it to form a paste, filter it with a gauze filter, collect the filtrate, which is the crude juice of Smilax glabra, for later use. After pre-cooling the crude juice of Smilax glabra obtained in step (1) to 4°C, pour it into a centrifuge and centrifuge it sequentially at a low speed of 3000×g for 30 min, a medium speed of 10000×g for 60 min, and an ultra-high speed of 100000×g for 70 min. Centrifugation process yields Smilax glabra exosome precipitate for later use. After resuspending the precipitate of Smilax glabra obtained in step (2) by adding phosphate buffer, centrifuge at 100,000×g for 70 min to obtain Smilax glabra resuspension for later use. (4) Filter the Smilax glabra resuspension from step (3) through a 0.22 μm filter membrane to obtain 5 mL of Smilax glabra exosomes with a protein size of 163.7 nm and a protein concentration of 1.5 mg / mL.

[0028] To obtain the solution of this invention and verify its technical effects, the inventors conducted extensive experimental research, some of which are recorded below: 1. Methodology and efficacy evaluation of the preparation of Smilax glabra exosomes 1.1 Chemical reagents and antibodies Fresh Smilax glabra rhizome was purchased from Guangxi Zhuang Autonomous Region, China, and identified as fresh Smilax glabra rhizome by Associate Professor Tian Minyi of Guizhou University. The super-enhanced cell counting kit (CCK8 kit, catalog number MA0225-2) used in the experiment was purchased from Meilun Biotechnology Co., Ltd.; other kits and reagents (including RIPA lysis buffer (catalog number P0013B), protease and phosphatase inhibitor mixture (catalog number P1045), SDS-PAGE protein loading buffer (catalog number P0015), Annexin V-FITC / PI apoptosis detection kit (catalog number C1062), ROS detection kit (catalog number S0033), 1,1'-bis(octadecyl-3,3,3',3'-tetramethylindole carbocyanine perchlorate (Dil) fluorescent probe (catalog number C1036)) were all purchased from Beyotime Biotechnology Co., Ltd.; primary antibodies included PCNA (catalog number 10205-2-AP) and cleaved-caspase 3. 3. The following substances were purchased from Proteintech Biotechnology Co., Ltd.: cyclin-dependent kinase 1 (CDK1, catalog number 19532-1-AP), cyclin B1 (Cyclin B1, catalog number 55004-1-AP), PI3K (catalog number 67071-1-Ig), phosphorylated PI3K (p-PI3K, catalog number 28842-1-AP), AKT (catalog number 60203-2-Ig), phosphorylated AKT (p-AKT, catalog number 66444-1-Ig), Ki67 (catalog number 27309-1), beta-actin (catalog number 20536-1-AP), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, catalog number 60004-1-Ig); phosphate-buffered saline (PBS, catalog number C0221B) was purchased from Beyotime Biotechnology Co., Ltd.

[0029] 1.2 Preparation method of SGR-ELNs A method for preparing Smilax glabra exosomes is described below. Figure 1 , Figure 2 Its characteristics include the following steps: Wash the Smilax glabra herb, soak it in deionized water for 24 hours, take it out and slice it, add 2000mL of phosphate buffer solution with pH 7.4, put it in a juicer and juice it to form a paste, filter it with a gauze filter, collect the filtrate, which is the crude juice of Smilax glabra, for later use. After pre-cooling the crude juice of Smilax glabra obtained in step (1) to 4°C, pour it into a centrifuge and centrifuge it sequentially at a low speed of 3000×g for 30 min, a medium speed of 10000×g for 60 min, and an ultra-high speed of 100000×g for 70 min. Centrifugation process yields Smilax glabra exosome precipitate for later use. After resuspending the precipitate of Smilax glabra obtained in step (2) by adding phosphate buffer, centrifuge at 100,000×g for 70 min to obtain Smilax glabra resuspension for later use. The resuspension of Smilax glabra in step (3) was filtered through a 0.22 μm filter membrane to obtain 5 mL of Smilax glabra exosomes with a particle size of 163.7 nm and a protein concentration of 1.5 mg / mL, which were then obtained as SGR-ELNs.

[0030] 1.3 Identification of SGR-ELNs Guangzhou Nuowo Biotechnology Co., Ltd. was commissioned to determine the morphology, particle size, and zeta potential of SGR-ELNs using TEM and NTA. The particle size and zeta potential were detected using the Particle Metrix ZetaView system. After the samples were washed with pure water and diluted to an appropriate concentration, real-time images of the particles were acquired using ZetaView software, and the results were analyzed and recorded. The ultrastructure of SGR-ELNs was observed using a transmission electron microscope (FEI Tecnai G2 Spirit T12). 5-10 μL of sample was dropped onto a copper grid, and after adsorption for 10 min, excess liquid at the edge was absorbed with filter paper. 5-10 μL of phosphotungstic acid was added, and after standing for 3 min, excess liquid was absorbed again. The samples were air-dried at room temperature before being tested.

[0031] 1.4 Metabolomics Analysis of SGR-ELNs The components of SGR-ELNs were analyzed by UPLC-MS / MS at Wuhan Maiwei Metabolic Biotechnology Co., Ltd.: 500 μL of 70% methanol extract containing internal standard was added to the sample and vortexed for 2 min; then the sample was subjected to liquid nitrogen quick-freezing for 5 min, thawing on ice for 5 min, and vortexing for 2 min, and this cycle was repeated 3 times; the sample was centrifuged at 12000 r / min at 4°C for 10 min, the supernatant was collected and concentrated to dryness; the dried residue was reconstituted with 100 μL of 70% methanol aqueous solution without internal standard, vortexed for 3 min and then sonicated in an ice bath for 10 min; the sample was centrifuged at 12000 r / min at 4°C for 3 min, and the supernatant was collected for detection. Chromatographic conditions: An Agilent SB-C18 column (1.8 μm, 2.1 mm × 100 mm) was used; mobile phase A was ultrapure water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid; gradient elution program: at 0.00 min, the concentration of mobile phase B was 5%, linearly increased to 95% within 9.00 min and maintained for 1 min, decreased to 5% from 10.00 to 11.10 min, and then equilibrated to 14 min; flow rate was 0.35 mL / min, column temperature was 40°C, and injection volume was 5 μL. Mass spectrometry conditions: Electrospray ionization (ESI) source temperature 500°C; ion spray voltage 5500 V in positive ion mode and -4500 V in negative ion mode; gas flow rates for GSI, GSII, and CUR were 50, 60, and 25 psi, respectively; collision-induced dissociation parameter was set to high; triple quadrupole (QQQ) scanning was performed using multiple reaction monitoring (MRM) mode, with collision gas set to medium; declustering voltage and collision energy were optimized, and specific MRM ion pairs were monitored according to the elution cycle. Based on a self-built MWDB database, metabolites were qualitatively analyzed using secondary mass spectrometry information, and quantitative analysis was performed using MRM mode.

[0032] 1.5 Results We successfully isolated SGR-ELNs from fresh Smilax glabra and characterized their physicochemical properties. NTA results are shown below. Figure 3 The median particle size of SGR-ELNs was 163.7 nm, which meets the particle size requirements of exosomes. Furthermore, its zeta potential was -47.91 mV, consistent with the negative potential characteristics of exosomes, indicating good physical stability. TEM images showed that SGR-ELNs exhibited a typical cup-shaped morphology, consistent with the morphological characteristics of exosomes. UPLC-MS / MS analysis clarified the biological and pharmacological characteristics of SGR-ELNs: Total ion current chromatograms (TIC) of SGR-ELNs in both positive and negative ion modes showed their complete compositional profile (see [link to UPLC-MS / MS analysis]). Figure 4A total of 527 components were identified, classified into 9 categories, mainly including lipids, flavonoids, phenolic acids, terpenes, and alkaloids. Notably, the top 100 compounds by relative abundance accounted for 88.1% of the identified compounds, divided into 8 categories. These main compounds also belong to lipids, phenolic acids, flavonoids, and terpenes. (See [link to flavonoid classification] for more details.) Figure 5 And Table 1.

[0033] Table 1 Top 100 Material Compositions 2. Validation of the targeting ability of SGR-ELNs in bladder cancer cell lines 2.1 Method SGR-ELNs were labeled with Dil (a membrane labeling dye). 1 μL of Dil dye (1 mg / mL) was added to 100 μL of SGR-ELN suspension, and the mixture was incubated at 37°C in the dark for 30 min. Unbound dye was removed by centrifugation at 100,000 × g for 70 min at 4°C, and the precipitate was resuspended in PBS. UMUC-3, T24, and 5637 cells (1 × 10⁻⁶) were then used as the control group. 5 Cells were seeded at 37°C for 12 h and 24 h after adding SGR-ELNs (product number WG801001, Service Bio) in a confocal culture dish. After incubation, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, and stained with DAPI for 5 min. Cell uptake of SGR-ELNs was observed using a laser confocal microscope (Leica, Germany), and the uptake level was quantified using ImageJ software.

[0034] 2.2 Experimental Objectives and Results To clarify the targeting ability of SGR-ELNs on different bladder cancer cell lines in vitro, the uptake efficiency of SGR-ELNs by three bladder cancer cell lines at different time points was compared through co-culture experiments. Figure 6 The results showed that the uptake of SGR-ELNs by cells gradually increased with prolonged co-incubation time; compared with 12 h of incubation, the intracellular red-labeled SGR-ELN signal was enhanced after 24 h of incubation. Furthermore, UMUC-3 cells showed higher uptake of SGR-ELNs at both 12 h and 24 h than T24 and 5637 cells, with 5637 cells exhibiting the weakest uptake capacity. These results indicate that there are significant differences in the uptake efficiency of SGR-ELNs among different cell lines, with UMUC-3 cells showing the strongest uptake capacity. Figure 7 .

[0035] 3. Verify the anti-bladder cancer effect of SGR-ELNs We first evaluated the effects of SGR-ELNs on different tumor cell phenotypes in in vitro experiments. The cell lines (UMUC-3, T24, 5637, SV-HUC-1) and their dedicated complete culture media used in this experiment were purchased from Pronosei Biotechnology Co., Ltd. Cells were cultured at 37°C and 5% CO2 and identified by short tandem repeat (STR) typing. The following are the methodological and results regarding the effects of SGR-ELNs on cell viability, clonal capacity, invasiveness, oxidative stress, apoptosis, and cell cycle: 3.1 Cell viability assay: 3.1.1 Method The effect of SGR-ELNs on the viability of UMUC-3, T24, 5637, and SV-HUC-1 cells was detected using the CCK8 assay kit: cells were loaded at a concentration of 1×10⁻⁶ cells / mL. 5 Cells were seeded at a density of 10 cells / mL in 96-well plates and treated with SGR-ELNs at concentrations of 0, 8, 16, 32, 64, 128, and 256 μg / mL for 24 h or 48 h. 10 μL of CCK8 solution was added to each well, and the plates were incubated at 37°C for 2 h. The absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA). Cell viability was analyzed using GraphPad Prism 10.1.2 software.

[0036] 3.1.2 Objectives and Results To verify the anticancer effect of SGR-ELNs, their inhibitory effect on the activity of various bladder cancer cell lines (UMUC-3, T24, 5637) and the normal ureteral epithelial cell line SV-Huc-1 was first evaluated. Figure 8 The results showed that SGR-ELNs inhibited the viability of the three bladder cancer cell lines in a concentration- and time-dependent manner. UMUC-3 and T24 cells were more sensitive to SGR-ELNs, with significantly greater inhibition of viability compared to 5637 cells. Furthermore, SGR-ELNs had almost no significant effect on the viability of SV-Huc-1 cells, indicating that SGR-ELNs have a selective killing effect on cancer cells. This is consistent with the results of cell tracing experiments: UMUC-3 cells, which took up the most SGR-ELNs, showed the most significant decrease in viability. Therefore, in subsequent experiments, UMUC-3 and T24 cells, which are more sensitive, were selected for further validation.

[0037] 3.2 Colony Formation Experiment 3.2.1 Method UMUC-3 and T24 cells were seeded at a density of 800 cells per well in 6-well plates. After adhesion, the cells were treated with SGR-ELNs at concentrations of 0, 8, and 16 μg / mL, respectively, and cultured for approximately 10 days. The culture medium was then discarded, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 30 min, and stained with 0.1% crystal violet for 15 min. The number of colonies was counted and analyzed using ImageJ software.

[0038] 3.3 Cell invasion assay 3.3.1 Method The matrix gel (Corning product) was diluted 1:9 with serum-free medium. 100 μL of the diluted matrix gel was added to the upper chamber of a Transwell chamber (8 μm pore size, Corning product) and incubated at 37°C for 30 min to solidify. UMUC-3 and T24 cells were resuspended in serum-free medium to a density of 1 × 10⁻⁶ cells / mL. 5 Cells were added at a concentration of 10% fetal bovine serum to the upper chamber. 200 μL of cell suspension containing 0, 8, or 16 μg / mL SGR-ELNs was added to the upper chamber, and 600 μL of culture medium containing 10% fetal bovine serum was added to the lower chamber. After incubation at 37°C and 5% CO2 for 24 h, uninvaded cells in the upper chamber were wiped off. Cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 15 min. The number of invading cells was counted and analyzed using ImageJ software.

[0039] 3.3.2 Western Blot Experimental Protocol Proteins were extracted using RIPA lysis buffer (Beyotime Biotechnology, catalog number P0013) and protein concentrations were determined using a BCA protein quantification kit (Beyotime Biotechnology, catalog number P0012). 20 μg of protein was loaded onto a 4%–20% FuturePAGE™ pre-prepared gel (ACE Biotechnology, catalog number X12420), and after electrophoresis, transferred to a Millipore polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with QuickBlock™ blocking buffer (Beyotime Biotechnology, catalog number P0252) and incubated overnight at 4°C with primary antibodies (PCNA and β-actin, both diluted 1:1000). After incubation with the corresponding secondary antibodies, the protein expression levels were detected using ECL chemiluminescence reagent (Millipore, USA). ImageJ software was used to evaluate and analyze the protein expression levels.

[0040] 3.3.3 Results Tumor cells possess strong proliferative and invasive capabilities, which are often the cause of disease progression and poor prognosis in cancer patients. This study evaluated the effects of SGR-ELNs on UMUC-3 and T24 cells using colony formation assays, Western blotting (WB) assays, and Transwell assays. Compared with the control group, SGR-ELNs significantly reduced colony numbers, indicating that they inhibited the proliferation of these two cell types in a dose-dependent manner. Proliferating cell nuclear antigen (PCNA) is a key protein involved in DNA replication and is closely related to the proliferation level of tumor cells; SGR-ELNs significantly downregulated PCNA expression levels in UMUC-3 and T24 cells, confirming its significant inhibitory effect on bladder cancer cell proliferation. Figure 9 Furthermore, compared with the control group, SGR-ELNs significantly weakened the invasive ability of bladder cancer cells. Figure 10 In summary, SGR-ELNs can significantly inhibit the proliferation and invasiveness of bladder cancer cells.

[0041] 3.4 Detection of intracellular oxidative stress levels ROS levels were quantified using a specific ROS detection kit (Beyotime Biotechnology, catalog number S0033): UMUC-3 and T24 cells (1×10⁻⁶) were tested. 5 Cells were treated with 0, 8, or 16 μg / mL SGR-ELNs for 24 h, then 10 μM DCFH-DA probe was added and incubated at 37°C for 30 min. After washing the cells three times with PBS, the fluorescence intensity was observed using a fluorescence microscope, and the data were analyzed using ImageJ software.

[0042] 3.5 Apoptosis Analysis 3.5.1 Method Apoptosis was assessed using the Annexin V-FITC / PI kit (Beyotime Biotechnology, catalog number C1062): UMUC-3 and T24 cells (1×10⁻⁶) were used. 5 Cells (cells / mL) were treated with 0, 8, and 16 μg / mL SGR-ELNs for 24 h, and then collected. Staining was performed according to the kit instructions. Detection was performed using a flow cytometer (BD Biosciences, FACS Celesta model), and apoptosis rate was calculated using FlowJo software (version 10.8.1). Protein extraction was performed as described above. Cells were incubated overnight at 4°C with primary antibodies (cleaved-caspase 3 and GAPDH, both diluted 1:1000). After incubation with the corresponding secondary antibodies, detection was performed using ECL chemiluminescence reagent (Millipore, USA). Protein expression levels were assessed and analyzed using ImageJ software.

[0043] 3.5.2 Results To further verify the inhibitory effect of SGR-ELNs on bladder cancer cells, the effects of SGR-ELNs on cellular oxidative stress and apoptosis were assessed using fluorescence microscopy, Western blotting, and flow cytometry. First, compared with the control group, SGR-ELNs significantly increased the levels of reactive oxygen species (ROS) in UMUC-3 and T24 cells (see figure). Figure 11 Secondly, SGR-ELNs induced significant changes in UMUC-3 and T24 cells, including decreased cell density, cell shrinkage, and cell aggregation, suggesting that SGR-ELNs have a potential inhibitory effect on bladder cancer cells. Figure 12 Furthermore, SGR-ELNs significantly increased the apoptosis rate in a concentration-dependent manner and upregulated the expression level of cleaved-caspase 3 in cells (see [link to article]). Figure 13 In summary, SGR-ELNs may inhibit bladder cancer by inducing oxidative stress and promoting apoptosis.

[0044] 3.6 Cell cycle detection 3.6.1 Method UMUC-3 and T24 cells were treated with 0, 8, and 16 μg / mL SGR-ELNs for 24 h, then collected, washed twice with PBS, and fixed overnight with pre-chilled 70% ethanol at 4°C. After centrifugation to remove ethanol, the cells were washed with PBS and then incubated with PI staining solution containing RNase A at 37°C for 30 min in the dark. Cell cycle distribution was detected, and the proportion of cells in each cycle was analyzed using Modifit 5.0 software. Protein extraction was performed as described previously. Cells were incubated overnight at 4°C with primary antibodies (P21, CDK1, Cyclin B1, β-actin, and GAPDH, all diluted 1:1000). After incubation with the corresponding secondary antibodies, the cells were detected using ECL chemiluminescence reagent (Millipore, USA). Protein expression levels were assessed and analyzed using ImageJ software.

[0045] 3.6.2 Results Tumor cells typically possess highly active proliferative capacity, and inducing cell cycle arrest is a common strategy to block cell proliferation. This study evaluated the effects of SGR-ELNs on the cell cycle using flow cytometry and Western blotting. Results are shown in […]. Figure 14 Compared with the control group, SGR-ELNs significantly induced G2 phase cell cycle arrest in UMUC-3 and T24 cells. Furthermore, Western blot results showed that SGR-ELN treatment significantly downregulated the protein expression levels of CDK1 and Cyclin B1 in UMUC-3 and T24 cells, while upregulating the protein expression level of p21; and these changes induced by SGR-ELNs were concentration-dependent. Therefore, SGR-ELNs may inhibit bladder cancer by inducing G2 phase cell cycle arrest in bladder cancer cells.

[0046] 4. Verify the anti-tumor effect of SGR-ELNs in vivo. To verify the anti-bladder cancer effect of SGR-ELNs in vivo, we constructed a nude mouse subcutaneous tumor model using the UMUC-3 cell line and evaluated its in vivo anti-tumor effect.

[0047] 4.1 Methods ① Select 4-week-old male BALB / c nude mice (Beijing Huafukang Biotechnology Co., Ltd.), and inoculate them with 5×10⁻⁶ molluscs in their right axilla. 6 One UMUC-3 cell; tumor formation was confirmed after 7 days (average volume approximately 25 mm). 3 Eighteen mice were randomly divided into three groups (n=6). The mice were administered the drug via gavage every two days for a total of eight doses. The groups were as follows: control group (phosphate-buffered saline), low-concentration SGR-ELNs group (50 mg / kg), and high-concentration SGR-ELNs group (100 mg / kg). Mouse body weight and tumor length / short diameter were recorded every two days during the experiment, calculated using the formula (length × short diameter). 2 The tumor volume was calculated as (1 / 2). This experiment was approved by the Animal Ethics Committee of Guizhou University of Traditional Chinese Medicine (Approval No.: 20250320001). After the experiment, the mice were euthanized, the tumors were collected, weighed, and used for subsequent testing.

[0048] ② HE staining protocol: Tumor tissues from different groups were fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, and cut into 4 μm thick sections. After dewaxing to water, the sections were stained with hematoxylin for 5 min, rinsed with tap water, differentiated with 1% hydrochloric acid-ethanol for 30 s, blued with ammonia for 30 s, and stained with eosin for 3 min. Subsequently, the sections were dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. The tissue morphology was observed under a Nikon microscope.

[0049] ③ Immunohistochemical (IHC) staining Paraffin-embedded tumor tissue was cut into 4 μm thick sections; after dewaxing, antigen retrieval was performed at 95°C for 20 min with 10 mM citrate buffer (pH 6.0); the sections were then incubated at room temperature with 3% H2O2 for 10 min to block endogenous peroxidase, and blocked with 5% bovine serum albumin (BSA) for 30 min; the sections were incubated overnight at 4°C with primary antibody (Ki67, 1:200 dilution), and then incubated with horseradish peroxidase (HRP)-labeled secondary antibody for 30 min; the sections were developed with DAB chromogenic reagent (catalog number PR30010), counterstained with hematoxylin, dehydrated, cleared, and mounted; images were taken using a Nikon microscope.

[0050] 4.2 Results To verify the inhibitory effect of SGR-ELNs on bladder cancer in vivo, a UMUC-3 cell subcutaneous xenograft model in nude mice was established for validation. During the experimental observation period, there were no significant differences in body weight among the groups of mice. Figure 15 SGR-ELNs significantly inhibited the growth of subcutaneous xenografts: compared with the control group, the tumor volume and weight were significantly reduced in both the low-concentration and high-concentration SGR-ELNs groups. Furthermore, SGR-ELNs significantly reduced the expression level of Ki67 in tumor tissue, indicating its inhibitory effect on tumor cell proliferation. HE staining results showed that the tumor cells in the control group were densely packed, with intact cell structure, abundant cytoplasm, and active nuclear division; while the low-concentration SGR-ELNs group (SGR-ELNs-L) and the high-concentration SGR-ELNs group (SGR-ELNs-H) both showed focal necrosis areas, loose tissue arrangement, decreased cell density, and pyknosis.

[0051] 4.3 Evaluation of the application safety of SGR-ELNs To further evaluate the safety of SGR-ELNs, we collected heart, liver, spleen, lung, kidney tissue and serum samples from different groups of mice to assess and analyze the important organs and liver and kidney functions of the mice.

[0052] 4.3.1 Serum Biochemical Indicators Detection Mouse serum samples were centrifuged at 300×g for 10 min, and the supernatant was collected. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (CREA) in the serum were measured according to the instructions.

[0053] 4.3.2 Hematoxylin-eosin (HE) staining Hematoxylin-eosin (HE) staining was performed according to "Method ② HE staining protocol in section 4.1". HE sections of heart, liver, spleen, lung, and kidney tissues were prepared using the above protocol, and structural changes were evaluated under a microscope.

[0054] 4.3.3 Results To evaluate the safety of SGR-ELNs in in vivo experiments, we assessed the vital organs and serum samples from different groups of nude mice. The results showed that SGR-ELNs had no significant effect on ALT, AST, and CREA levels; furthermore, HE staining of major organs (heart, liver, spleen, lung, and kidney) showed no significant differences among the three groups. In conclusion, SGR-ELNs possess high safety. Figure 16 .

Claims

1. A method for preparing exosomes from Smilax glabra, characterized in that: Includes the following steps: (1) Take Smilax glabra medicinal material, wash it, soak it in deionized water for 22-26 hours, take it out and slice it, add 1000-3000 mL of phosphate buffer solution with pH 5-9, put it into a juicer and juice it to form a paste, filter it with gauze filter, collect the filtrate, which is Smilax glabra crude juice, for use. (2) After pre-cooling the crude juice of Smilax glabra obtained in step (1) to 3-5℃, pour it into a centrifuge and centrifuge at low speed of 1000-5000×g for 10-40 min, at medium speed of 7000-13000×g for 40-80 min and at ultra-high speed of 80000-130000×g for 50-90 min. Centrifugation process to obtain Smilax glabra exosome precipitate for later use. (3) After resuspending the precipitate of Smilax glabra exosomes obtained in step (2) by adding phosphate buffer, centrifuge at 80,000-130,000×g for 50-90 min to obtain the resuspension of Smilax glabra exosomes for later use. (4) Filter the suspension of Smilax glabra exosomes from step (3) through a 0.1-0.3 μm filter membrane to obtain 1-10 mL of Smilax glabra exosomes with a protein size of 155-170 nm and a protein content of 1-3 mg / mL.

2. The method for preparing Smilax glabra exosomes according to claim 1, characterized in that, In step (1), the Smilax glabra medicinal material is washed, soaked in deionized water for 23-25 ​​hours, sliced, and then 1500-2500 mL of phosphate buffer solution with pH 7-8 is added. The mixture is put into a juicer to extract juice and form a paste. The paste is filtered through a gauze filter and the filtrate is collected as Smilax glabra crude juice for later use.

3. The method for preparing Smilax glabra exosomes according to claim 2, characterized in that, In step (1), the Smilax glabra medicinal material is washed, soaked in deionized water for 24 hours, sliced, and then 2000 mL of phosphate buffer solution with pH 7.4 is added. The mixture is put into a juicer and juiced to form a paste. The paste is filtered through a gauze filter and the filtrate is collected as Smilax glabra crude juice for later use.

4. The method for preparing Smilax glabra exosomes according to claim 1, characterized in that, In step (2), the crude juice of Smilax glabra obtained in step (1) is pre-cooled to 3.5-4.5℃ and then poured into a centrifuge for sequential centrifugation at low speed of 2000-4000×g for 20-35min, medium speed of 9000-11000×g for 50-70min, and ultra-high speed of 90000-110000×g for 60-80min. The centrifugation process yields Smilax glabra exosome precipitate for later use.

5. The method for preparing Smilax glabra exosomes according to claim 4, characterized in that, In step (2), the crude juice of Smilax glabra obtained in step (1) is pre-cooled to 4°C and then poured into a centrifuge for sequential centrifugation at 3000×g low speed for 30 min, 10000×g medium speed for 60 min and 100000×g ultra-high speed for 70 min. The centrifugation process yields Smilax glabra exosome precipitate for later use.

6. The method for preparing Smilax glabra exosomes according to claim 1, characterized in that, In step (3), after adding phosphate buffer to the precipitate of Smilax glabra obtained in step (2) for resuspending, centrifuge at 90000-110000×g for 60-80 min to obtain Smilax glabra resuspension for later use.

7. The method for preparing Smilax glabra exosomes according to claim 6, characterized in that, In step (3), after adding phosphate buffer to the precipitate of Smilax glabra obtained in step (2) for resuspending, centrifuge at 100000×g for 70 min to obtain Smilax glabra resuspension for later use.

8. The method for preparing Smilax glabra exosomes according to claim 1, characterized in that, In step (4), the resuspension of Smilax glabra from step (3) is filtered through a 0.22 μm filter membrane to obtain 5 mL of Smilax glabra exosomes with a protein size of 163.7 nm and a protein content of 1.5 mg / mL.

9. The use of Smilax glabra exosomes in the preparation of products for the prevention or treatment of bladder cancer according to any one of claims 1-8.

10. The application according to claim 9, characterized in that: The product mentioned is a medicine or health product for bladder cancer.

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