Oral formulations of therapeutic gut microbiota exosomes encapsulating lupeol and methods of making and using the same

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to an oral preparation of lipoxygenase packaged with lipoxygenase by healthy donor treatment type intestinal flora exosome and a preparation method and application thereof. The oral preparation of lipoxygenase packaged with lipoxygenase by healthy donor treatment type intestinal flora exosome comprises 0.18-0.78 wt% of lipoxygenase, 0.24-0.48 wt% of fucoidan, 1.0-2.0 wt% of tetrahydropyrimidine and healthy donor treatment type intestinal flora exosome supplementing to 100% in terms of weight percentage. The present application adopts an ultrasonic method to prepare the lipoxygenase packaged with lipoxygenase by healthy donor treatment type intestinal flora exosome, uses the treatment type intestinal flora exosome as a natural drug carrier, successfully solves the technical problem of low oral bioavailability of lipoxygenase, and provides a new effective means for enhancing immunity and preventing and treating tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to oral formulations of therapeutic gut microbiota exosomes encapsulating lupeol, their preparation methods, and applications. Background Technology

[0002] Lupeol is a triterpenoid compound. Studies have shown that lupeol has antioxidant, anti-inflammatory, skin-healing-promoting, and tumor cell-inhibiting effects. It can inhibit the growth of breast cancer, prostate cancer, and mouse melanoma. It can be used as an adjuvant for clinically approved drugs to enhance efficacy and reduce drug resistance.

[0003] However, lupeol is almost insoluble in water and has low bioavailability, which has long troubled researchers in terms of administration and limited its clinical efficacy. Lupeol is poorly soluble in gastrointestinal fluids, limiting its absorption and hindering oral absorption through the intestinal epithelium. Current research shows that the oral bioavailability of lupeol is less than 1%. Its high lipid solubility also allows it to accumulate in lipid-rich organs such as adipose tissue, liver, and central nervous system, leading to both safety limitations and long-term therapeutic side effects. Chinese invention patent application CN121102510A discloses an injectable formulation prepared from a hyaluronic acid-modified lupeol zeolite imidazole framework material, but no oral formulation of lupeol has yet overcome the bioavailability problem in the prior art.

[0004] Nanoscale lipid vesicles released by gut microbiota, also known as exosomes, play a vital biomedical role in intraspecific, interspecific, and cross-species communication between cells and bacteria. Therapeutic gut microbiota exosomes from healthy individuals, as an independent secretory system, can potentially target invading microorganisms, maintain population balance, prevent external colonization or pathogen overgrowth, and maintain intestinal immune homeostasis. Overcoming the localization and inefficiency of microbiota transplantation as adjuvant therapy for tumors, these can be termed therapeutic gut microbiota exosomes. These gut-derived microbiota exosomes possess unique biocompatibility and low immunogenicity, enabling them to cross biological barriers such as the intestinal epithelial barrier and the blood-brain barrier, providing a new pathway for drug delivery.

[0005] Existing technologies include drug delivery methods using exosomes. For example, Chinese invention patent CN202511382256.8 discloses the application of CAR exosome-loaded drugs in tumor treatment, and Chinese invention patent CN121102166A19.0 discloses the application of exosome-delivered miR-155-5p inhibitors targeting synovial inflammatory macrophages for the treatment of osteoarthritis. However, these existing technologies are mostly for injections, and the exosomes are all cell-derived. Research on developing oral formulations using therapeutic gut microbiota exosomes for drug delivery has not been reported. Different encapsulation procedures, such as co-incubation at room temperature, electroporation, surfactant treatment, extrusion, freeze-thaw cycles, and sonication, can encapsulate small molecule drugs into exosomes. However, different drug properties require different encapsulation procedures: co-incubation and freeze-thaw cycles are relatively simple and widely applicable, but have low encapsulation efficiency; electroporation and extrusion are suitable for small-scale preparation; surfactant treatment is more suitable for hydrophobic drugs, but it is difficult to remove residues, affecting efficacy. Studies have shown that modifying the exosome membrane structure through ultrasound treatment can make it more effective as a drug carrier and for preserving its contents. Based on the above research, this invention provides a highly efficient method for preparing therapeutic gut microbiota exosomes encapsulating lupeol and its oral formulation, and verifies its application effect in tumor prevention and treatment. Summary of the Invention

[0006] The purpose of this invention is to improve the bioavailability of lupeol in order to achieve highly effective immune enhancement and improve its role in preventing and treating intestinal and distal intestinal tumors.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of the present invention provides an oral formulation of therapeutic gut microbiota exosomes encapsulating lupeol, the oral formulation comprising therapeutic gut microbiota exosomes derived from a healthy donor, lupeol, fucoidan, and tetrahydropyrimidine.

[0008] In a preferred embodiment, the oral formulation comprises, by weight percentage, 0.18-0.78 wt% lupeol, 0.24-0.48 wt% fucoidan, 1.0-2.0 wt% tetrahydropyrimidine, and therapeutic gut microbiota exosomes to make up the balance to 100%.

[0009] In a preferred embodiment, the therapeutic gut microbiota exosomes include therapeutic gut microbiota exosomes from healthy child donors and / or therapeutic gut microbiota exosomes from healthy adult donors. The therapeutic gut microbiota exosomes are derived from healthy donors, and more than 95% are exosomes of gut bacteria.

[0010] The preparation methods for therapeutic intestinal flora exosomes from adult donors and pediatric donors used in this invention are both based on Examples I-II of Chinese Invention Patent Application "A Therapeutic Intestinal Flora Exosome and Its Preparation Method" (202610179640.6). The specific preparation methods are as follows: Methods for preparing therapeutic gut microbiota exosomes from adult donors: Freshly collected fecal samples from healthy adult donors (approximately 200g / sample) were filtered through an 80-mesh filter bag to remove food residue. The fecal samples were mixed with 20 mmol / L PBS buffer (pH=7.4) at a mass-to-volume ratio of 1:3 and vortexed for 30 seconds to ensure thorough mixing. The mixture was then centrifuged at 10000×g for 10 minutes at 4°C to allow bacterial cells to settle. The supernatant was collected and washed twice, collecting all supernatant. This supernatant was then centrifuged again at 18000×g for 30 minutes at 4°C to remove cell debris, and the second supernatant was collected. The second supernatant was aliquoted and leveled, then ultracentrifuged at 180000×g for 60 minutes at 4°C. The supernatant was discarded, yielding a crude exosome precipitate. The precipitate was resuspended in approximately 1L PBS buffer and washed by ultracentrifugation at 150000×g for 120 minutes at 4°C.

[0011] The crude exosome extract was resuspended in PBS buffer and sonicated at 40 Hz for 30 minutes to ensure thorough mixing and suspension; then the volume was adjusted to 1 L.

[0012] The resuspended suspension was sequentially passed through hollow fiber columns with pore sizes of 0.65µm, 0.45µm, and 0.22µm for tangential flow filtration, and the filtrate outside the membrane was recovered.

[0013] Add an equal volume of 1 wt% Triton X-114 solution to the filtrate and stir in an ice bath for 60 minutes. Then transfer to a 45°C water bath and allow to separate into layers. Finally, centrifuge at 15000×g for 15 minutes and collect the supernatant. Repeat this step twice.

[0014] Add 100 mL / L of polymyxin B agarose magnetic beads to the supernatant; after thorough stirring to adsorb endotoxins, adsorb the magnetic beads through a magnetic separator, collect the effluent, and detect its endotoxin content.

[0015] β-propiolactone was added to the effluent to a final concentration of 0.1 wt%, and after thorough mixing, the virus was inactivated at 37°C for 3 hours. Subsequently, 0.1 mol / L disodium bicarbonate solution was added to adjust the pH to 7.4 to neutralize and hydrolyze the β-propiolactone.

[0016] The neutralized solution was dialyzed for 3 hours at 0.1 MPa using a 100 kDa tangential flow filtration device to thoroughly remove residual Triton X-114, small protein molecules, and other impurities. The concentrate in the dialysis bag was recovered, and the sample was treated with RNase A at a final concentration of 10 ng / μL at 37°C for 15 minutes to remove free RNA; then, it was ablated with proteinase K at a final concentration of 500 ng / μL at 37°C for 30 minutes to degrade free large protein particles, thus obtaining therapeutic gut microbiota exosomes.

[0017] Methods for preparing therapeutic gut microbiota exosomes from pediatric donors: Freshly collected fecal samples (approximately 100g) from healthy children were filtered through an 80-mesh filter to remove food residue. The fecal sample was mixed with 20 mmol / L PBS buffer (pH=7.4) at a mass-to-volume ratio of 1:5 and vortexed for 30 seconds to ensure thorough mixing. The mixture was then centrifuged at 4°C and 10000×g for 10 minutes to allow bacterial cells to settle. The supernatant was collected and washed three times, collecting all supernatant. This supernatant was then centrifuged again at 4°C and 18000×g for 30 minutes to remove cell debris, and the second supernatant was collected. The second supernatant was aliquoted, leveled, and ultracentrifuged at 4°C and 180000×g for 90 minutes. The supernatant was discarded, yielding a crude exosome precipitate. The precipitate was resuspended in approximately 1L of PBS buffer and washed by ultracentrifugation at 4°C and 150000×g for 120 minutes.

[0018] The crude exosome extract was resuspended in PBS buffer, sonicated at 40 Hz for 30 minutes to ensure thorough mixing, and then brought to a final volume of 1 L.

[0019] The resuspended suspension was sequentially passed through hollow fiber columns with pore sizes of 0.65µm, 0.45µm, and 0.22µm for tangential flow filtration, and the filtrate outside the membrane was recovered.

[0020] Add a 2wt% Triton X-114 solution (Triton X-114 solution to filtrate volume ratio of 1:3) to the filtrate and stir in an ice bath for 60 minutes. Then transfer to a 45°C water bath and allow to separate into layers. Finally, centrifuge at 15000×g for 15 minutes and collect the supernatant. Repeat this step twice.

[0021] Add 50 mL / L of polymyxin B agarose magnetic beads to the supernatant; after thorough stirring to adsorb endotoxins, adsorb the magnetic beads through a magnetic separator, collect the effluent, and detect its endotoxin content.

[0022] β-propiolactone was added to the suspension to a final concentration of 0.1 wt%, and after thorough mixing, the virus was inactivated at 37°C for 2 hours. Then, 0.1 mol / L disodium bicarbonate solution was added to adjust the pH to 7.4 to neutralize and hydrolyze the β-propiolactone.

[0023] The neutralized solution was dialyzed using a 100 kDa tangential flow filtration device for 1 hour to thoroughly remove residual Triton X-114, small protein molecules, and other impurities. The concentrate was recovered from the permeabilized membrane and treated with RNase A at a final concentration of 10 ng / μL at 37°C for 15 minutes to remove free RNA. Then, it was ablated with proteinase K at a final concentration of 500 ng / μL at 37°C for 30 minutes to degrade free large protein particles, yielding concentrated therapeutic intestinal flora exosomes.

[0024] A second aspect of this invention provides a method for preparing an oral formulation of therapeutic gut microbiota exosomes encapsulating lupeol, comprising the following steps: S1. Add lupin alcohol powder to a therapeutic gut microbiota exosome PBS solution to obtain a suspension; S2. The suspension is subjected to ultrasonic treatment, and after ultrasonic treatment, the suspension is placed in a 37°C water bath to recover for 1-2 hours; S3. Centrifuge to remove unencapsulated lupeol particles; centrifuge again to collect therapeutic gut microbiota exosomes encapsulated with lupeol; S4. The collected therapeutic gut microbiota exocrine secretions containing lupeol were resuspended in PBS, and fucoidan and tetrahydropyrimidine were added to obtain the oral formulation.

[0025] In a preferred embodiment, the final concentration of lupeol in S1 is 1 mg / mL to 2 mg / mL.

[0026] As a preferred embodiment, the ultrasonic treatment in S2 specifically includes: in an ultrasonic breaker with a power of 300w-500w, a 2mm probe, an amplitude of 20%-40%, ultrasonic treatment for 0.5-1.5min, followed by an ice bath for 2min, and repeating 5-7 cycles.

[0027] In a preferred embodiment, S3 specifically includes: centrifuging at 2000-4000 rpm for 10-15 min to remove unencapsulated lupeol particles; and centrifuging at 120000g-150000g for 90-120 min to collect therapeutic gut microbiota exosomes encapsulated with lupeol.

[0028] The concentration of the therapeutic gut microbiota exosomes was 10. 10 -10 12 / mL A third aspect of the present invention provides the application of an oral formulation of therapeutic gut microbiota exosomes encapsulating lupeol.

[0029] In one preferred embodiment, the oral formulation is used for the prevention or treatment of colorectal cancer, breast cancer, cervical cancer, prostate cancer, and / or liver cancer.

[0030] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. Compared to cell-derived exosomes, which require energy- and nutrient-intensive processes such as cell culture, the therapeutic gut microbiota exosomes of this invention are derived from feces, i.e., human waste products. This represents a highly efficient utilization of biotechnological resources and significantly reduces production costs. Furthermore, the healthy donor therapeutic gut microbiota exosomes possess unique biocompatibility and low immunogenicity, enabling them to cross multiple biological barriers, reduce the clearance rate of internal drugs, and effectively deliver lupeol to target tissues in the intestine and distal intestinal regions, greatly improving drug bioavailability and therapeutic efficacy.

[0031] 2. The oral formulation developed in this invention can improve the bioavailability of lupeol while maintaining the balance of the intestinal microecology and exert the natural immunomodulatory effect of therapeutic intestinal flora exosomes.

[0032] 3. This invention employs an ultrasonic method to prepare an oral formulation of lupeol encapsulated in exosomes. Utilizing therapeutic gut microbiota exosomes as a natural drug carrier, it successfully solves the technical challenge of low oral bioavailability of lupeol, providing a new and effective approach for tumor prevention and treatment. Drug delivery via therapeutic gut microbiota exosomes from healthy donors may achieve multi-target systemic therapeutic effects, synergistically enhancing the various anti-cancer effects of lupeol. Significant effects have been demonstrated in in vitro inhibition experiments against colorectal cancer, breast cancer, prostate cancer cells, and liver cancer cells.

[0033] 4. The present invention employs an ultrasonic method for preparing exosome-encapsulated lupeol with an encapsulation rate of 74%-78%, which is 12 to 19 times higher than the 4%-6% encapsulation rate of the cyclic freeze-thaw method. Compared with the electroporation method, it is suitable for mass production. The preparation method is simple and easy to implement, and is easy to scale up, which enhances the practicality and market competitiveness of the present invention. Attached Figure Description

[0034] Figure 1 This is a graph showing the particle size distribution of the therapeutic gut microbiota exosome sample before lupeol loading in Example I of nanoflow cytometry detection. Figure 2 This is a graph showing the particle size distribution of the therapeutic gut microbiota exosome sample after lupeol was loaded into the sample in Example I of nanoflow cytometry. Figure 3 This is a graph showing the particle size change of an oral formulation sample stored at -4°C for 90 days in Example I of Dynamic Light Scattering Detection. Figure 4 This is a graph showing the concentration of exosome particles in an oral formulation sample before freezing at -80°C, as described in Example I of nanoflow cytometry detection. Figure 5 This is a graph showing the exosome particle concentration of an oral formulation sample after being frozen at -80°C for 90 days using nanoflow cytometry in Example I. Figure 6 For the example, a significant increase in the half-hemolytic index (HC50) in the drug administration hemolysis test indicates enhanced humoral immunity: (A) HC50 at different administration times; (B) Significant differences in HC50 at different administration times ("T1", 0d; "T2", 5d; "T3", 12d; "T4", 0d; "T50", 0d; "T50", 0d; "T6", 0d; "T7", 0d; "T8", 0d; "T9", 0d; "T1", 0d; "T2", 0d; "T3 ... "P<0.05; " "P<0.01; " (P<0.001). Figure 7 For the example sample, a significant increase in the phagocytic index in the clearance test of mice at different drug administration times indicates enhanced cellular immunity: (“ "P<0.05; " "P<0.01;" (P<0.001). Figure 8 The following images show the enhanced immune response of spleen tissue before and after administration of cyclophosphamide to the splenic subcellular populations CD4 and CD8 using single-staining fluorescence (20×): (A) Comparison of CD4 and CD8 fluorescence intensities before administration (0d) of the sample in Example I after cyclophosphamide immunodeficiency membrane formation; (B) Comparison of CD4 and CD8 fluorescence intensities 5d after administration of the sample in Example I; (C) Comparison of CD4 and CD8 fluorescence intensities 12d after administration of the sample in Example I. Figure 9 This is a graph showing the average CD4 / CD8 fluorescence intensity ratio of spleen cells at different drug administration times in the example samples; Figure 10 The IC50 values ​​of the sample for different tumor cells are as follows: "HCT 116", colorectal cancer cells; "MDA-MB-231", triple-negative breast cancer cells; "HeLa", cervical cancer cells; "PC3", chemotherapy-resistant prostate cancer cells; "Hep3B", liver cancer cells. Detailed Implementation

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

[0036] Example I A method for preparing an oral formulation of lupeol loaded onto pediatric donor therapeutic gut microbiota exosomes includes the following steps: S1. Prepare a suspension by adding lupeol (Maclean L875611, 95%, CAS No.: 545-47-1) to a final concentration of 1 mg / mL to freshly prepared pediatric donor therapeutic intestinal flora exosome PBS solution; S2. Place the suspension in an ultrasonic disruptor with a power of 300W, a 2mm probe, an amplitude of 40%, sonicate for 0.5min, then in an ice bath for 2min, repeating 7 cycles; after ultrasonic treatment, place the suspension in a 37℃ water bath for 1h to recover. S3. Centrifuge at 2000 rpm for 15 min to remove unencapsulated lupeol particles; centrifuge at 150000 g for 90 min to collect pediatric donor therapeutic gut microbiota exosomes encapsulated with lupeol; S4. The collected exoglobulin from pediatric donors containing lupeol was resuspended in 1 / 40 of the original volume of PBS. Fucoidan (Yuanye S11142, BR85%; CAS No.: 9072-19-9) at a weight percentage of 0.24 wt% of the resuspension was added as a stabilizer, and tetrahydropyrimidine (Shenzhen Zhongke Xinyang Biotechnology Co., Ltd.; 98%; CAS No.: 96702-03-3) was added as a cryoprotectant to obtain the oral formulation.

[0037] Example II A method for preparing an oral formulation of lupeol loaded onto pediatric donor therapeutic gut microbiota exosomes includes the following steps: S1. Prepare a suspension by adding lupeol (Maclean L875611, 95%, CAS No.: 545-47-1) to a final concentration of 2 mg / mL to freshly prepared adult donor therapeutic intestinal flora exosome PBS solution; S2. Place the suspension in an ultrasonic disruptor with a power of 500W, a 2mm probe, an amplitude of 20%, sonicate for 1.5 minutes, then in an ice bath for 2 minutes, repeating 5 cycles; after ultrasonic treatment, place the suspension in a 37℃ water bath for 2 hours to recover. S3. Centrifuge at 4000 rpm for 10 min to remove unencapsulated lupeol particles; centrifuge at 120000 g for 120 min to collect adult donor therapeutic gut microbiota exosomes encapsulated with lupeol; S4. The collected exocrine secretions of therapeutic gut microbiota from pediatric donors, loaded with lupeol, were resuspended in 1 / 20 of the original volume of PBS. Fucoidan (Yuanye S11142, BR85%; CAS No.: 9072-19-9) at a weight percentage of 0.48 wt% of the resuspended solution was added as a stabilizer, and tetrahydropyrimidine (Shenzhen Zhongke Xinyang Biotechnology Co., Ltd.; 98%; CAS No.: 96702-03-3) was added as a cryoprotectant to obtain the oral formulation.

[0038] Performance testing The oral formulations of Examples I-II were subjected to the following tests: (a) Load factor Detection method: The concentration (S) of lupeol in the supernatant after ultracentrifugation of pediatric donor therapeutic intestinal flora exosomes and adult donor therapeutic intestinal flora exosomes in S3 was analyzed by high performance liquid chromatography (HPLC). The determination of terpenoids was performed in accordance with the standard GB / T5009.268 Determination of phytosterols in food and EP2.2.29 High performance liquid chromatography. The total concentration T (mg / mL) was calculated by dividing the initial mass (mg) of lupeol added in S1 by 95% and the volume (mL) of the exosome PBS solution. The formula for calculating the load factor is: Load factor % = (TS) ÷ T × 100% Results of Example I: At a concentration of 1 mg / mL (2.34 mM), when exosomes were prepared with 0.1% lupeol (w / v), a large amount of lupeol was captured, reaching 740.2 ± 8.3 μg / mL. 1 The encapsulation rate of lupeol in Example I was approximately 74%.

[0039] Results of Example II: At a concentration of 2 mg / mL (4.68 mM), the encapsulation capacity of lupeol was significantly increased when a concentration of 0.2% was used, reaching a total encapsulated lupeol amount of 1559.1 ± 9.5 μg / mL. 1 The loading rate of lupeol in Example 2 was approximately 78%.

[0040] Combining the two concentrations in Examples I and II, no significant difference was observed in the lupeol loading rate.

[0041] (ii) Stability After lupeol was loaded into exosomes, the average size of the nanofluidic vesicles changed, with the volume increasing by approximately 17%–21%. Figure 1 and Figure 2contrast); The oral formulation prepared in Example I was stored at 4°C for 90 days. The results showed no significant statistical differences in the average hydrodynamic diameter and exosome particle number at 30 and 60 days (p<0.01). After 60 days, changes occurred in Example I, with a decrease in the number of vesicles, leaving 83%. After 90 days, both the number and size of exosome particles in Example I changed significantly. The increase in these values ​​was associated with increased volume, possibly accompanied by phenomena such as self-aggregation, coagulation, flocculation, and precipitation. Dynamic optical dispersion detection showed extremely non-uniform particle size with multiple peaks. Figure 3 ).

[0042] After being stored at -80°C for 90 days, oral formulations showed a slight decrease in exosome count only in the initial period, i.e., after thawing the day after freezing, with the number of exosome particles being 87%–92% of that before freezing; however, no significant changes were observed after 30, 60, and 90 days of freezing. Figure 4-5 ).

[0043] (III) Immunization Enhancement Test Select 4-6 week old C57BL infants, half male and half female.

[0044] Immunodeficiency model: Cyclophosphamide 80mg / kg was administered via intraperitoneal injection for 3 consecutive days.

[0045] Therapeutic gut microbiota exosome administration: 0.9 mL once daily by gavage, at a concentration of 10. 10 / mL; Sampling method: Blood was collected after modeling and on days 0, 5, and 12 after drug administration.

[0046] 1. The mouse hemolysin test showed that it enhances humoral immunity. 1.1 Microplate Reader Method: Sample wells and blank control wells were set up. For sample wells: serum was diluted with SA buffer (generally 200-500 times); 50 μL of diluted serum was added to each well. For blank control wells: 50 μL of SA buffer was added to each well, followed by 25 μL of 10% (v / v) SRBC and 50 μL of complement (diluted 1:8 with SA solution). The mixture was incubated at 37℃ for 30 min, and the reaction was stopped by placing it on ice. The mixture was then centrifuged horizontally at 1500 rpm for 10 min. 50 μL of the supernatant from each sample and blank control well was added to another 96-well plate, along with 150 μL of DuPont's reagent. Simultaneously, half of the wells were set up for hemolysis; 12.5 μL of 10% (v / v) SRBC was added, followed by DuPont's reagent to a final volume of 200 μL. The mixture was thoroughly mixed with a shaker, and after standing for 10 min, the optical density of each well was measured at 540 nm using an automated microplate reader.

[0047] The amount of hemolysin is determined by the half-maximal hemolysin value (HC). 50 The result, calculated according to Equation 1, is shown in Table 1.

[0048] Formula 1HC 50 =

[0049] Table 1. Detection indicators and average values ​​of mouse hemolysin test in Examples I and II.

[0050]

[0051] 1.2 Data Processing and Result Analysis: After immunizing animals with SRBCs, hemolysin (SRBC antibody) appears in the serum. With the participation of complement, hemolysin, when incubated with SRBCs, can cause a hemolytic reaction, releasing hemoglobin. The level of hemolysin in the animal serum, i.e., the antibody level, indicates differences in humoral immune function. Animals were grouped according to different drug administration and detection times: T1 (0 days), T2 (5 days), and T3 (12 days), and comparisons were made between the two groups. Figure 6 The results showed that the mean half-hemolytic index (HC) of the T2 group (5 days after administration) was significantly higher than that of the T1 group (0 days before administration after cyclophosphamide immunodeficiency modeling) in each embodiment. 50 The difference was highly significant (P < 0.01); the difference was also highly significant compared to the T3 group after 12 days of administration (P < 0.001), indicating a positive result for this test. Furthermore, compared to the T2 group after 5 days of administration (T3 group vs. T2 group), the mean half-maximal hemolytic index (HC) was significantly lower after 12 days of administration. 50 The difference was also significant (P<0.05), indicating that the antibody levels in the serum of immunodeficient mice continued to increase with increasing drug dosage, and humoral immunity continued to be enhanced.

[0052] 2. Mouse clearance test showed enhanced cellular immunity. 2.1 Dilute the Indian ink stock solution 3-4 times with physiological saline. Inject the diluted Indian ink (10 mL / kg) into the tail vein of mice, starting immediately after injection. 2 and 10 minutes after injection, collect 20 μL of blood from the internal canthal venous plexus and immediately add it to 2 mL of 0.1% Na₂CO₃ solution. Measure the optical density (OD) at 600 nm using a 721 spectrophotometer, with the Na₂CO₃ solution as a blank control. Alternatively, an ELISA reader can be used to measure the OD at 600 nm.

[0053] Mice were euthanized, and their livers and spleens were harvested. Blood was blotted from the surface of the organs with filter paper, and each organ was weighed. The phagocytic index was used to represent the carbon clearance capacity of the mice. Within a certain range, the rate of carbon particle clearance in the body showed an exponential relationship with blood carbon concentration. A linear relationship was found between the logarithm of blood carbon concentration on the ordinate and time on the abscissa. The slope of this linear relationship (…) K Equation 2), i.e., the carbon clearance index, can represent the phagocytic rate. The weight of the animal's liver and spleen affects the phagocytic rate, which is generally expressed by the corrected phagocytic index a (Equation 3). The experimental results are statistically shown in Table 1. Formula 2: K =(lgOD1-lgOD2) / (t2-t1) Equation 3: a =

[0054] Table 2 shows the detection indicators and average values ​​of the mouse clearance test in Examples I and II.

[0055]

[0056] 2.2 Data Processing and Result Analysis The carbon clearance assay is a classic method for assessing immune function and phagocytic cell activity in mice by measuring the rate of carbon particle clearance. Analysis of variance (ANOVA) is generally used, but a homogeneity of variance test must be performed first, followed by pairwise comparisons of the means among multiple experimental groups and a control group. In this experiment, groups were divided into groups based on different administration times (0d, 5d, and 12d). The corrected phagocytic index measured 0d before administration of cyclophosphamide in immunodeficiency-induced membrane formation served as the control group. Significance analysis is as follows: Figure 7 The results showed that the corrected mean phagocytic index of the sample in the example administration at 5 days and 12 days was significantly higher than that of the control group, and the statistical results were significantly higher (“ “ "P<0.01) and extremely significant (" A result of "P<0.001" indicates a positive result for this test. The healthy therapeutic gut microbiota exosomes encapsulated with lupeol described in this invention can enhance cellular immunity in a shorter time compared to commercially available immune enhancers (such as thymosin, administered for 15-30 days), significantly improving the immune function of immunodeficient mice.

[0057] 3. T lymphocyte subset (CD4, CD8) assay 3.1 Mouse spleen lymphocyte suspension was prepared using indirect immunofluorescence assay with monoclonal antibody (MCAB), and the cell concentration was adjusted to 1×10⁶ / mL. 6 Cells were treated with 100 μL of fluorescent antibody and 100 μL of 1:5 diluted FIT-labeled anti-mouse IgG antibody. After incubation at 4°C for 30 minutes, the intensity of fluorescently positive CD4 and CD8 T lymphocyte subsets was calculated under a fluorescence microscope. The results are as follows: Figure 8 As shown.

[0058] 3.2 Data processing and results analysis: CD4 cells (helper T cells): dominate immune regulation and help activate other immune cells (such as B cells and CD8 cells); CD8 cells (cytotoxic T cells): directly kill virus-infected cells or tumor cells.

[0059] Image Pro Plus 6.0 software analysis method for immunohistochemical images: When taking photos, try to fill the entire field of view with tissue and ensure consistent background lighting for each image. Using Image Pro Plus 6.0 software, select the same green color as a unified standard for judging positivity in all images. Analyze each image to obtain the fluorescence intensity value (IOD) and corresponding area (AREA) of positive cells. IOD / AREA = Mean Density (mean fluorescence intensity value); and calculate the average fluorescence intensity ratio of CD4 and CD8. Normal CD4 / CD8 ratio range: usually 1.0~2.5 (slight differences may exist between different laboratories). A ratio that is too high or too low may reflect abnormalities in the immune system. A low CD4 / CD8 ratio (e.g., <1.0): suggests decreased immune surveillance function, susceptibility to infection or chronic inflammation; a high CD4 / CD8 ratio (e.g., >2.5): suggests excessive immune activation, which may trigger an autoimmune response. Experimental results are as follows... Figure 9 As shown, the average fluorescence intensity ratio of cyclophosphamide was less than 1.0 on day 0 before administration after immunodeficiency membrane formation, indicating successful immunodeficiency membrane formation. The CD4 / CD8 ratio returned to the normal range (approximately 1.0) on days 5 and 12 after administration, further demonstrating at the spleen tissue level that the two examples of therapeutic gut microbiota exosomes carrying lupeol enhanced the immunity of mice.

[0060] The results of combined hemolysin assay, clearance assay and T lymphocyte subset (CD4, CD8) fluorescence single staining assay showed that the two sample examples of the therapeutic intestinal flora exosome-encapsulated lupeol oral preparation of the present invention significantly enhanced humoral, cellular and tissue immunity.

[0061] (iv) In vitro tumor cell toxicity detection Activated tumor cells: 10g of each of the following were collected: HCT116 colorectal cancer cells, MDA-MB-231 triple-negative breast cancer cells, HeLa cervical cancer cells, PC3 chemotherapy-resistant prostate cancer cells, and Hep3B drug-resistant liver cancer cells. 6 100 μL of T cells and 50 μL of T cells were used, with 3 replicates per group; the cells were co-cultured for 48 hours, i.e., incubated at 5% CO2 and 37°C with low humidity; approximately 1.0 × 10⁶ cells were then collected. 8 Cells were transferred to 24-well cell culture plates and incubated for 24 hours to allow for proper adhesion to the plate bottom. Different volumes (1, 2.5, 5, 10, 15, 20, 30, 40 µL) of the exosome-encapsulated lupeol sample obtained in Examples I-II were added to each well for cytotoxicity testing. The control group was treated with an equal volume of sterile physiological saline (NaCl, 0.9% w / v). After 48 hours, the viable cell concentration was measured according to the CCK8 kit instructions, and the half-maximal inhibitory concentration (IC50) was calculated. The results are as follows: Figure 10 As shown.

[0062] The experimental results are analyzed as follows: (1) Colorectal cancer cells HCT116: The survival rate of cancer cells treated with the sample in the example decreased by 40%-60%; when the concentration of the sample in the example was 20 μM, β-chain protein accumulated in the cell nucleus, the potential for colon cancer cell generation and the expression of Wnt target genes decreased, and apoptosis was induced.

[0063] (2) Triple-negative breast cancer cells MDA-MB-231: Compared with the control group, it inhibited the invasion and adhesion of MDA-MB-231 cells (IC50=26.29μM) and inhibited the growth of MDA-MB-231 cells (IC50=42.38μM).

[0064] (3) Cervical cancer cells HeLa: significantly reduced cell proliferation and survival rate (IC50=43.60μM; p<0.001).

[0065] (4) Prostate cancer chemotherapy-resistant cells PC3: PC3 cells from chemotherapy-resistant prostate cancer patients were obtained, and cell viability was detected at different drug concentrations. IC50≈40μM was calculated. (5) Hep3B liver cancer cells: The growth inhibitory activity of the example group against Hep3B parental cells and drug-resistant cells increased by 72% and 35%, respectively, with an average IC50≈50μM.

[0066] The results of in vitro tumor cell cytotoxicity tests on the sample samples showed that all selected cancer cells exhibited significant cytotoxicity and pro-apoptotic characteristics, and the selected cancer cells showed obvious dose-dependent growth inhibition. This indicates that the oral formulation of therapeutic gut microbiota exosomes encapsulating lupeol described in this invention may not only inhibit the occurrence and development of intestinal tumors such as colorectal cancer and distal intestinal tumors such as cervical cancer, breast cancer, and prostate cancer; but also has a significant inhibitory effect on drug-resistant tumor cells such as PC3 and Hep3B. Therefore, its application prospects in tumor prevention and treatment need to be broadened.

[0067] Based on animal experiments demonstrating immunodeficiency and tumor cell inhibition experiments, the oral formulation of lupeol encapsulated in exosomes of healthy donor gut microbiota described in this invention can be used to rapidly and comprehensively enhance immunity and antagonize the occurrence and development of tumors.

[0068] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A therapeutic intestinal flora exosome-encapsulated lupeol oral formulation, characterized in that, The oral formulation comprises lupeol loaded onto therapeutic gut microbiota exosomes from healthy donors at a content of 0.18% to 0.78 wt%. 0.24–0.48 wt% fucoidan as a stabilizer and 1.0–2.0 wt% tetrahydropyrimidine as a cryoprotectant, with the remainder being a PBS solution containing therapeutic gut microbiota exosomes from healthy donors; The preparation method of oral formulations includes the following steps: (1) A suspension was prepared by adding lupeol powder to a final concentration of 1 mg / mL-2 mg / mL to a PBS solution of therapeutic gut microbiota exosomes derived from healthy donors; (2) Place the exosome lupeol suspension in an ultrasonic disruptor with a power of 300w-500w, a 2mm probe, an amplitude of 20%-40%, sonicate for 0.5-1.5min, then in an ice bath for 2min, and repeat for 5-7 cycles. (3) Place the ultrasound-treated exosomes in a 37°C water bath for 1-2 hours to recover; (4) Centrifuge at 2000-4000 rpm for 10-15 min to remove unencapsulated lupeol powder; (5) Centrifuge at 120,000g-150,000g for 90-120min to collect exosomes containing lupeol; (6) The collected exocrine secretions containing lupeol were suspended in 1 / 40 to 1 / 20 of the original volume of PBS, and then 0.24 to 0.48 wt% fucoidan was added as a stabilizer and 1.0 to 2.0 wt% tetrahydropyrimidine was added as a cryoprotectant. The concentration of exosomes in the therapeutic gut microbiota exosome PBS solution was 10. 10 -10 12 / mL, including therapeutic gut microbiota exosomes from healthy child donors and / or therapeutic gut microbiota exosomes from healthy adult donors, with more than 95% being gut bacterial exosomes.

2. The application of the therapeutic intestinal flora exosome-encapsulated lupeol oral formulation according to claim 1, characterized in that, Application in the preparation of drugs for the prevention and treatment of colorectal cancer.

3. The application of the therapeutic intestinal flora exosome-encapsulated lupeol oral formulation according to claim 1, characterized in that, Application in the preparation of drugs for the prevention and treatment of breast cancer.

4. The application of the therapeutic intestinal flora exosome-encapsulated lupeol oral formulation according to claim 1, characterized in that, Application in the preparation of drugs for the prevention and treatment of cervical cancer.

5. The application of the therapeutic intestinal flora exosome-encapsulated lupeol oral formulation according to claim 1, characterized in that, Application in the preparation of drugs for the prevention and treatment of prostate cancer.

6. The application of the therapeutic intestinal flora exosome-encapsulated lupeol oral formulation according to claim 1, characterized in that, Application in the preparation of drugs for the prevention and treatment of liver cancer.

Images