An oral nano-medicine antigen delivery system, its construction method and application

Abstract

This invention relates to an oral nanomedicine antigen delivery system and its construction method and application, belonging to the field of oral nanomaterials technology; the construction method includes the following steps: (1) preparation of OM nanoparticle suspension; (2) preparation of cationic liposome suspension; (3) preparation of cationic liposome suspension loaded with OM / BF; (4) construction of oral nanomedicine antigen delivery system. This invention breaks through multiple barriers in the gastrointestinal tract, improves drug bioavailability, and achieves precise immune tracking through the OVA tumor antigen model, simulating in vivo anti-tumor CTL response, thereby exerting anti-tumor effects synergistically at the cellular, tissue, and immune levels, effectively solving the problems of poor water solubility and low delivery efficiency of the active ingredient bufotoxin in traditional Chinese medicine. At the same time, through macrophage-mediated immune regulation and precise drug release, it improves targeting and treatment efficiency, opening up a new avenue for tumor immunotherapy with oral nanomedicine.

Description

Technical Field

[0001] This invention relates to an oral nano-medicine antigen delivery system, its construction method and application, belonging to the field of oral nanomaterials technology. Background Technology

[0002] Nanocarriers and delivery systems are rapidly developing in the field of cancer therapy. Through nanoscale carriers, they not only improve the delivery efficiency of anti-tumor drugs but also specifically address multiple challenges in cancer treatment, promoting the synergistic application of various treatment modalities and bringing new hope for cancer cure. Therefore, the research and development of anti-tumor nanomedicine carriers and formulations has become a cutting-edge interdisciplinary field involving pharmacy, nanomaterials science, and biomedicine, with continuous innovative achievements emerging.

[0003] Nanocarriers, with their nanoscale size, high specific surface area, and tunable physicochemical properties, can significantly improve the in vivo processes and therapeutic effects of drugs. For example, loading drugs into or onto the surface of nanocarriers can enhance their stability in the physiological environment, prolong systemic circulation time, and enable on-demand, controllable release, thereby improving the therapeutic index of the drug. The selection of materials for nanocarriers is diverse, including natural polymers such as phospholipids, chitosan, and dextran, as well as synthetic polymers such as polylactic-co-glycolic acid copolymer (PLGA), and inorganic materials such as metal nanoparticles and mesoporous silica, providing ample space for functional design.

[0004] Despite significant advancements in nanotechnology and the clinical application of some nano-formulations, its full potential in medicine remains to be explored. Current optimization efforts focus on the following aspects: First, utilizing hydrophilic nanocarriers to encapsulate hydrophobic drugs, improving their solubility and chemical stability; second, employing nanoencapsulation or biodegradable polymer conjugation to protect drugs from enzymatic degradation and rapid clearance; third, developing functionalized nano-formulations by surface-modifying targeting ligands to enhance their selective recognition and penetration of tumor cells or the matrix, thereby optimizing drug biodistribution and targeting efficiency; fourth, designing stimulus-responsive nanocarriers to precisely release drugs under specific signals in the tumor microenvironment (such as pH, enzymes, redox conditions) or exogenous stimuli (such as light, magnetism, and ultrasound); and fifth, constructing multifunctional integrated nanosystems to achieve combined goals such as multi-drug therapy, multiple targeting, and integrated diagnosis and treatment, thereby enhancing synergistic anti-tumor efficacy and personalized treatment levels.

[0005] Currently, mainstream vaccines (such as those administered subcutaneously or intramuscularly) primarily stimulate systemic humoral immunity, resulting in limited cellular immune responses, making them ineffective against diseases that require strong cellular immunity (such as tumors).

[0006] Traditional oral nanodelivery systems suffer from insufficient stability when subjected to degradation by gastric acid and digestive enzymes; simultaneously, they struggle to efficiently cross the intestinal epithelial barrier, resulting in low drug bioavailability. Most nanodelivery systems lack the ability to target drugs from the intestine to distant lesions, failing to achieve active accumulation and penetration of drugs at the lesion site, thus limiting their targeting efficiency.

[0007] Existing strategies have limitations in stimulating a comprehensive anti-tumor immune response, making it difficult to simultaneously and effectively activate both innate and adaptive immunity and regulate the tumor immune microenvironment.

[0008] Although current oral nanodelivery systems based on macrophage "hitchhiking" strategies have shown significant advantages in improving targeting, enhancing immunomodulation, and improving drug bioavailability, how to systematically integrate the multi-level delivery process of "gut-systemic circulation-cell-subcellular" within the same nanoparticle remains a bottleneck that current technologies have not yet overcome. Summary of the Invention

[0009] The purpose of this invention is to provide an oral nano-medicine antigen delivery system, its construction method, and its application to solve the technical problems existing in the prior art as described above.

[0010] The technical solution provided by this invention is as follows:

[0011] One objective of this invention is to provide a method for constructing an oral nanomedicine antigen delivery system, comprising the following steps:

[0012] (1) Preparation of OM nanoparticle suspension: Ovalbumin OVA was dissolved in PBS to prepare OVA solution. An equal volume of KMnO4 aqueous solution was slowly added under stirring. The reaction was carried out at room temperature. The precipitate was collected by centrifugation and washed with deionized water to obtain OM nanoparticles. The OM nanoparticles were resuspended in PBS to obtain OM nanoparticle suspension.

[0013] (2) Preparation of cationic liposome suspension: Weigh phosphatidylcholine, cholesterol and DOTAP ((2,3-dioleoxypropyl)trimethylammonium chloride), dissolve in anhydrous chloroform, rotary evaporate in a water bath to form a lipid film, and then dry, hydrate and sonicate to form cationic liposome suspension;

[0014] (3) Preparation of cationic liposome suspension loaded with OM / BF: Add ethanol solution of BF (bufotoxin) and OM nanoparticle suspension from step (1) to the cationic liposome suspension in step (2), and incubate in a water bath under nitrogen protection to obtain cationic liposome suspension loaded with OM / BF.

[0015] (4) Construction of oral nano-medicine antigen delivery system: Yeast β-glucan (YGB) was dissolved in PBS to prepare yeast β-glucan solution. The solution was added dropwise to the cationic liposome suspension loaded with OM / BF in step (3) under stirring. The reaction was carried out under stirring at room temperature and centrifuged to obtain yeast β-glucan modified nanoparticles. The yeast β-glucan modified nanoparticles were resuspended in PBS to obtain the oral nano-medicine antigen delivery system.

[0016] Further, in step (1), the mass ratio of OVA to KMnO4 is 5:2; the reaction time at room temperature is 3-6 h; the centrifugation speed is 120000-150000 ×g and the centrifugation time is 20-40 min.

[0017] Furthermore, the concentration of the OM nanoparticle suspension was 1-1.5 mg / mL; the concentration of the cationic liposome suspension was 2-2.5 mg / mL; and the concentration of the cationic liposome suspension loaded with OM / BF was 2.5-3 mg / mL.

[0018] Further, in step (2), the molar ratio of phosphatidylcholine, cholesterol, and DOTAP is 5:3:2; the temperature of the water bath is 45-60 ℃; the drying temperature is 37-45 ℃, and the drying time is 30-60 min; the hydration is performed using 10 mL of citrate buffer solution with pH 6.5-6.8; and the power of the ultrasonic treatment is 200-300 W.

[0019] Further, in step (3), the mass ratio of the cationic liposome suspension, the ethanol solution of BF and the OM nanoparticle suspension is 20:1:4, and the concentration of the ethanol solution of BF is 1-1.5 mg / mL; the temperature of the water bath shaking incubation is 50-65 ℃, and the time is 2-4 h.

[0020] Further, in step (4), the mass ratio of yeast β-glucan to cationic liposome suspension loaded with OM / BF is 5:1; the stirring reaction time at room temperature is 6-8 h; the centrifugation speed is 120000-150000 ×g and the time is 45-60 min.

[0021] The second objective of this invention is to provide an oral nano-medicine antigen delivery system constructed using the method described above.

[0022] The third objective of this invention is to provide the application of the oral nano-medicine antigen delivery system described above in oral nano-drug delivery.

[0023] The technical solution provided by this invention has the following advantages compared with the prior art:

[0024] 1. This invention utilizes the natural acid-resistant and enzymatic-resistant properties of yeast β-glucan to protect the drug-loaded nanosystem. Through its ability to specifically target the Dectin-1 receptor on intestinal M cells, it actively mediates the nanosystem's crossing of the intestinal mucosal barrier and entry into the gut-associated lymphoid tissue, solving the challenges of stability and absorption efficiency in oral delivery. Through a multi-level delivery pathway—M cell transport → macrophage / DC recognition and uptake → macrophage homing to the lesion site—active targeting and deep penetration from intestinal absorption to lymphatic circulation and then to tumor tissue are achieved, significantly improving the resolution and efficiency of targeted drug delivery.

[0025] 2. This invention overcomes the limitations of existing technologies that rely solely on single immune activation through a synergistic mechanism. It utilizes yeast β-glucan to polarize macrophages into an anti-tumor M1 phenotype, thereby activating innate immunity. OM nanoparticles can efficiently deliver OVA antigens to lymph nodes, and manganese ions are used to improve the tumor immune microenvironment, activating antigen-specific CD8. + T-cell immunity enhances adaptive immunity; simultaneously, it combines with bufotoxin to induce tumor cell apoptosis, producing a synergistic effect with immunotherapy, aiming to stimulate a powerful innate and adaptive anti-tumor immune response.

[0026] 3. This invention, based on a macrophage "hitchhiking" strategy, constructs an oral nanomedicine delivery system. This system uses yeast β-glucan as a targeting "compass," achieving efficient drug delivery in gut-associated lymphoid tissue through its specific recognition of the Dectin-1 receptor on the macrophage surface. Utilizing the natural homing ability of macrophages, oral nanoliposomes loaded with bufotoxin are precisely delivered to the tumor microenvironment, achieving multi-level targeted delivery from the gut to the lymphatic system to the tumor. This system not only overcomes multiple barriers in the gastrointestinal tract, improving drug bioavailability, but also achieves precise immune tracking through an OVA tumor antigen model, simulating in vivo anti-tumor CTL responses, thereby synergistically exerting anti-tumor effects at the cellular, tissue, and immune levels. It effectively solves the problems of poor water solubility and low delivery efficiency of the active ingredient bufotoxin, while simultaneously improving targeting and therapeutic efficiency through macrophage-mediated immune regulation and precise drug release, opening a new avenue for tumor immunotherapy based on oral nanomedicine.

[0027] 4. This invention utilizes a precisely designed cationic liposome structure modified with yeast β-glucan, synergistically combining the antigen delivery and immune activation functions of OM nanoparticles with the lipophilic properties of bufotoxin and the proton buffering capacity of PEI, to construct a YGB-C-Lip-OM / BF composite nanosystem with hierarchical recognition and responsive release capabilities. This system not only integrates multiple modules of polysaccharides, lipids, and inorganic mineralization components in its composition, but also functionally overcomes the intestinal epithelial barrier, achieves specific recognition by M cells / macrophages, guides targeted lymphatic migration, and ultimately induces macrophage M1 polarization and antigen-specific immune responses in the tumor microenvironment. This allows for precise drug delivery and synergistic therapy at the systemic, cellular, and immune microenvironment levels. This strategy, with its controllable components, clear pathways, and coherent mechanisms, constructs a multi-level targeted therapeutic pathway that cannot be replicated by other single-function nanosystems. Attached Figure Description

[0028] Figure 1 This is a comparison chart of particle size in Examples 1-4 of the present invention;

[0029] Figure 2 These are potential comparison diagrams for embodiments 1-4 of the present invention;

[0030] Figure 3 This is a transmission electron microscope image of Embodiment 1 of the present invention;

[0031] Figure 4 This is a hydration particle size distribution diagram (three replicates) for Example 4 of the present invention.

[0032] Figure 5 This is a potential distribution diagram (repeated three times) of Embodiment 4 of the present invention;

[0033] Figure 6 This is a comparison chart showing the M-cell transport efficiency and specific binding ability to the macrophage Dectin-1 receptor of the delivery systems in Comparative Example 2 and Example 4 of the present invention.

[0034] Figure 7 This is a comparison chart of the encapsulation efficiency of the delivery systems of Comparative Example 3, Comparative Example 4 and Example 4 of the present invention in a simulated intestinal fluid.

[0035] Figure 8 This is a comparison chart of the encapsulation efficiency of the delivery systems of Comparative Example 3, Comparative Example 4 and Example 4 of the present invention in simulated gastric fluid.

[0036] Figure 9 Particle size and potential diagram of the YGB-C-Lip-OM / BF delivery system of the present invention as a function of time;

[0037] Figure 10This diagram illustrates the expression of the macrophage M1 marker cytokine IL-6 by the delivery system YGB-C-Lip-OM / BF of the present invention.

[0038] Figure 11 This diagram illustrates the expression of the macrophage M1 marker cytokine TNF-α by the delivery system YGB-C-Lip-OM / BF of the present invention.

[0039] Figure 12 A graph showing the change in transmembrane resistance of cells was constructed for the M-cell model of this invention;

[0040] Figure 13 This is a diagram illustrating the effect of yeast-β-glucan in crossing the M cell and Caco2 barrier according to the present invention.

[0041] Figure 14 This is a comparison chart showing the ability of yeast-β-glucan to cross the Caco2 barrier according to the present invention;

[0042] Figure 15 This is a comparison diagram of the ability of yeast-β-glucan to cross the M cell barrier according to the present invention;

[0043] Figure 16 The bar chart shows the safety evaluation of the delivery system YGB-C-Lip-OM / BF of the present invention against RAW 264.7.

[0044] Figure 17 A bar chart showing the safety evaluation of the delivery system YGB-C-Lip-OM / BF of the present invention against FHC;

[0045] Figure 18 A bar chart showing the in vitro antitumor efficacy evaluation of the delivery system YGB-C-Lip-OM / BF of the present invention over time;

[0046] Figure 19 This is a bar chart showing the in vitro antitumor effect of the delivery system YGB-C-Lip-OM / BF of the present invention as a function of concentration. Detailed Implementation

[0047] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0048] Example 1: Preparation of OM nanoparticle suspension

[0049] Ovalbumin (OVA) was dissolved in phosphate-buffered saline (PBS, pH 7.4) to prepare a 1 mg / mL solution. An equal volume of 0.4 mg / mL KMnO4 aqueous solution was slowly added under continuous stirring, and the reaction was carried out at room temperature for 3 h. After the reaction, the precipitate was collected by centrifugation (120000 × g, 20 min), washed three times with deionized water to remove unreacted ions, and OM nanoparticles were obtained. The OM nanoparticles were then resuspended in 10 mL of pH 7.4 PBS to obtain a 1 mg / mL OM nanoparticle suspension, which was stored at 4 °C for later use.

[0050] The particle size and potential comparison diagrams of the substances prepared in Example 1 are shown below. Figure 1 and Figure 2 As shown, the hydrated particle size of the OM nanoparticle suspension was determined to be 38±1.5 nm, the polydispersity index (PDI) was 0.16-0.2, and the zeta potential was -10±3 mV by dynamic light scattering (DLS). Figure 3 The image shows a transmission electron microscope (TEM) image of the obtained OM nanoparticles. As can be seen from the image, the OM nanoparticles exhibit a regular structure, uniform particle size distribution, and good dispersion, with no obvious aggregation or structural collapse observed. Their particle size is basically consistent with the DLS measurement results. Combined with the low PDI and negative Zeta potential, this indicates that the nanosystem was successfully constructed and possesses good structural stability.

[0051] Example 2: Preparation of cationic liposome (C-Lip) suspension

[0052] Phosphatidylcholine (PC), cholesterol (Chol), and the cationic lipid material DOTAP ((2,3-dioleoxypropyl)trimethylammonium chloride) were weighed in a molar ratio of 5:3:2 and dissolved in 10 mL of anhydrous chloroform. A uniform lipid film was then formed by rotary evaporation in a 45 °C water bath, followed by overnight drying in a 37 °C vacuum oven to completely remove the organic solvent. The film was then hydrated with 10 mL of pH 6.5 citrate buffer and sonicated at 45 °C (300 W, 2 s on, 3 s off) until a uniform cationic liposome (C-Lip) suspension of 2 mg / mL was formed. Finally, the suspension was aseptically filtered through a 0.22 μm microporous membrane to obtain cationic liposomes with a particle size of 110 ± 10 nm and a Zeta potential of +35 ± 2 mV. The particle size and potential comparison diagrams of the substances prepared in Example 2 are shown below. Figure 1 and Figure 2 As shown.

[0053] Example 3: Preparation of cationic liposome suspension loaded with OM / BF (C-Lip-OM / BF)

[0054] The cationic liposome suspension prepared in Example 2 was mixed with an ethanol solution of bufotalin (BF) (1 mg / mL) and an OM nanoparticle suspension prepared in Example 1 (the mass ratio of cationic liposome suspension, ethanol solution of BF, and OM nanoparticle suspension was 20:1:4). Under nitrogen protection, the mixture was incubated in a 50 °C water bath with shaking for 2 h to efficiently encapsulate the drug and antigen, resulting in a 2.5 mg / mL cationic liposome (C-Lip-OM / BF) suspension with a particle size of 140±10 nm and a Zeta potential of +25±3 mV. The particle size and potential comparison diagrams of the substances prepared in Example 3 are shown below. Figure 1 and Figure 2 As shown in the figure. The cationic liposome suspension loaded with OM / BF was then dialyzed for 24 h to remove unencapsulated free drug and OM nanoparticles. The final formulation showed a BF encapsulation efficiency of 88.5 ± 2.3% as determined by high-performance liquid chromatography (HPLC).

[0055] Example 4: Construction of an oral nano-medicine antigen delivery system (YGB-C-Lip-OM / BF)

[0056] 25 mg of yeast β-glucan (YGB) was dissolved in 50 mL of PBS at pH 7.4 to obtain a yeast β-glucan solution. This solution was then added dropwise to 5 mg of the C-Lip-OM / BF suspension prepared in Example 3 under gentle stirring. The reaction was continued at room temperature for 6 h with stirring, achieving stable modification of the YGB on the surface of cationic liposomes through electrostatic adsorption. After the reaction, the yeast β-glucan-modified nanoparticles were collected by ultracentrifugation (120,000 × g, 45 min) and resuspended in 20 mL of pH 7.4 PBS to obtain a 3 mg / mL yeast β-glucan-modified nanoparticle suspension, which is the oral nanomedicine antigen delivery system (YGB-C-Lip-OM / BF). The particle size and potential comparison diagrams of the substances prepared in Example 4 are shown below. Figure 1 and Figure 2 As shown, the final YGB-C-Lip-OM / BF hydrated particle size was 145±8 nm, the PDI was 0.18, and the Zeta potential dropped to +5±1 mV after DLS characterization, indicating that YGB was successfully coated. Figure 4 The hydration particle size distribution diagram of YGB-C-Lip-OM / BF is shown. Figure 5The potential distribution of YGB-C-Lip-OM / BF is shown in the diagram. The hydrated particle size distribution shows a single, concentrated peak, indicating excellent monodispersity and a highly uniform particle size distribution. This uniform nanoscale size is beneficial to the physical stability of the oral delivery system in the gastrointestinal environment and subsequent efficient cellular uptake. The potential distribution shows a sharp single peak in the Zeta potential distribution of YGB-C-Lip-OM / BF. The original cationic liposomes (C-Lip-OM / BF) have a high positive charge. However, after introducing YGB through electrostatic adsorption, the Zeta potential of the system significantly decreases to a near-neutral micro-positive level due to the coating and charge shielding effect of YGB. This characteristic change in surface charge directly proves that YGB has been successfully and stably modified onto the surface of the cationic liposomes. The particle size and potential characterization results together confirm that we have successfully prepared a YGB-coated oral nanoantigen delivery system (YGB-C-Lip-OM / BF) using an electrostatic adsorption strategy. The system is not only of moderate size and uniformly distributed, but the change in surface charge also confirms the effective encapsulation of YGB, laying a good physicochemical foundation for its use as an oral nanovaccine for antigen delivery.

[0057] Comparative Example 1

[0058] Referring to Example 4, the optimal encapsulation ratio of yeast β-glucan was verified by changing the mass ratio of C-Lip-OM / BF to yeast β-glucan and measuring the Zeta potential of YGB-C-Lip-OM / BF. As the yeast β-glucan ratio increased (from 1:1 to 1:5), the Zeta potential of the delivery system YGB-C-Lip-OM / BF gradually decreased, as shown in Table 1. This indicates that yeast β-glucan was successfully encapsulated on the liposome surface and its cationic properties were effectively neutralized, providing a surface chemical basis for subsequent targeted recognition and intracellular delivery based on the Dectin-1 receptor.

[0059] Table 1 Potential Results

[0060]

[0061] Comparative Example 2

[0062] An oral nano-Chinese medicine antigen delivery system is prepared in a way that differs from that in Examples 1-4 in that the cationic lipid material DOTAP is not introduced for modification during the preparation of the cationic liposome suspension, i.e. it does not have cationic surface properties. The resulting delivery system is named YGB-Lip-OM / BF.

[0063] Comparative Example 2 and the delivery system YGB-C-Lip-OM / BF prepared in this invention were used. Their intestinal epithelial penetration efficiency was evaluated using an M-cell model, and their binding to the macrophage Dectin-1 receptor was observed using confocal microscopy. The results are as follows: Figure 6 As shown. The results show that the delivery system YGB-C-Lip-OM / BF prepared in Example 4 of the present invention, due to its cationic properties, exhibits significantly enhanced M cell transport efficiency and specific binding ability to the macrophage Dectin-1 receptor.

[0064] Comparative Example 3

[0065] An oral nano-medicinal antigen delivery system is prepared in a way that differs from that in Examples 1-4 in that no OM nanoparticles are added during the synthesis process. The delivery system obtained in Comparative Example 3 is named YGB-C-Lip-BF.

[0066] Comparative Example 4

[0067] An oral nano-medicine antigen delivery system is prepared in a way that differs from that in Examples 1-4 in that yeast β-glucan is not added during the synthesis process. The delivery system obtained in Comparative Example 4 is named C-Lip-OM / BF.

[0068] The encapsulation efficiency of Comparative Examples 3 and 4, as well as the YGB-C-Lip-OM / BF system obtained through Examples 1-4, was tested in intestinal simulated fluid (pH=7.6) and gastric simulated fluid (pH=1.2).

[0069] Encapsulation efficiency (EE%) was determined by ultrafiltration centrifugation. The nanoparticle dispersion was centrifuged, and free BF (W) in the filtrate was quantified using high-performance liquid chromatography (HPLC). free ) and the total BF (W) in the system total The encapsulation ratio is calculated using the following formula:

[0070]

[0071] The results are as follows Figure 7 and Figure 8 As shown in the figure. The results show that the encapsulation efficiency of the delivery system prepared in Example 4 of the present invention is significantly higher than that of Comparative Examples 3 and 4. Furthermore, the encapsulation efficiency of the delivery system of the present invention does not change significantly with time, while the encapsulation efficiency of Comparative Examples 3 and 4 decreases significantly. Therefore, the delivery system prepared in this invention can effectively maintain the structural integrity of nanoparticles in complex physiological environments and significantly improve blood circulation stability, demonstrating the key role of yeast β-glucan surface modification in microenvironment regulation and stability assurance.

[0072] Comparative Example 5: OVA / MnOx composite (OM nanoparticles) synthesized by KMnO4 biomineralization.

[0073] Referring to Example 1, the particle size and PDI of the OVA / MnOx complex were optimized by controlling the mass ratio of KMnO4 to OVA. The test results are shown in Table 2. Finally, the 6th group, with small and uniform particle size, was selected, with a KMnO4 to OVA mass ratio of 2:5, a particle size of 37.98 nm, and a PDI of 0.165. Chemical absorption spectroscopy confirmed that the OVA / MnOx complex was encapsulated by liposomes.

[0074] Table 2. Particle size and PDI results of OVA / MnOx composite.

[0075]

[0076] test:

[0077] 1. 72-hour serum stability test of the YGB-C-Lip-OM / BF delivery system

[0078] To evaluate the physiological stability of the delivery system YGB-C-Lip-OM / BF, serum stability testing was performed. Freshly prepared nanoparticle dispersions were homogenized with an equal volume of fetal bovine serum (FBS) in phosphate-buffered saline (PBS, pH 7.4). The mixture was then incubated at 37 °C in a shaker. Samples were removed from the incubation medium at predetermined time points (0, 12, 24, 36, 48, 60, and 72 h). Changes in the hydration size and zeta potential of the nanoparticles were continuously monitored using dynamic light scattering (DLS). The results are shown below. Figure 9 As shown in the figure. The results showed that the YGB-C-Lip-OM / BF delivery system was approximately 118 nm at 0 h and increased only slightly to 131 nm at 72 h, with an absolute change of only about 13 nm (relative change of about 11%). This small fluctuation is well within the physiologically acceptable range for nanocarriers, strongly demonstrating that no serious protein-induced aggregation occurred (if aggregation were true, the particle size would usually increase several times over). The surface charge remained highly stable during the 72 h incubation period, fluctuating only slightly between +3.8 mV and +5.1 mV. The lack of significant changes in particle size and potential confirmed its good colloidal stability, making it suitable for oral delivery, and demonstrating that the nanomaterials prepared in this invention, encapsulated by yeast β-glucan and containing OM particles, have longer gastrointestinal fluid stability and a more significant improvement in the tumor immune microenvironment.

[0079] 2. Analysis of macrophage M1 polarization capacity

[0080] To evaluate the regulatory effect of YGB-C-Lip-OM / BF on macrophage M1 polarization, quantitative analysis was used to detect changes in the expression of macrophage phenotypic markers. RAW264.7 macrophages were cultured and then treated with PBS (control group NC) at pH 7.4 or YGB-C-Lip-OM / BF for 24 h, respectively. Subsequently, the mRNA and protein expression levels of the M1 macrophage marker cytokines IL-6 and TNF-α were detected by qRT-PCR and ELISA, respectively. Results are shown below. Figure 10 and Figure 11 As shown, compared with the control group NC, the expression of IL-6 and TNF-α in the YGB-C-Lip-OM / BF treatment group was significantly upregulated (P<0.05), suggesting that this nanosystem can effectively induce macrophages to polarize towards the pro-inflammatory and anti-tumor M1 phenotype, thereby helping to activate the anti-tumor immune response in the tumor microenvironment.

[0081] 3. Evaluation of the barrier crossing effect of M cell culture and yeast-β-glucan

[0082] In the M-cell co-culture model, epithelial barrier permeability and the ability of yeast-β-glucan to cross the barrier were mainly assessed using TEER detection combined with fluorescence tracing experiments. Specifically, Caco2 cells were cultured in the upper chamber of a Transwell cell culture until a stable monolayer was formed, and then co-cultured with Raji B cells to induce M-like cell differentiation. During co-culture, TEER values ​​were measured periodically using an epithelial resistance meter. When the TEER decreased by approximately 50% compared to the Caco2 monolayer alone, it indicated successful M-like cell formation and increased barrier permeability. Based on this, fluorescently labeled yeast-β-glucan was added to the upper chamber of the Transwell cell culture. The lower chamber culture medium was collected at set time points (e.g., 0, 2, 4, 6 h), and the fluorescence intensity of the lower chamber was detected using a fluorescence microscope or a fluorescence microplate reader, and compared with the control group (Caco2 monolayer without M-cell induction).

[0083] Specifically, such as Figure 12 As shown, the M-cell model was successfully established. In stark contrast to the persistently elevated TEER values ​​observed in the monoclonal Caco2 cell model, the system co-cultured with Raji B cells exhibited a significant decrease in TEER values ​​after 18 days. Based on the successful establishment of this model, transepithelial transport capacity was subsequently assessed. Figure 13-15Fluorescence assays showed that the transport capacity of yeast-β-glucan-modified nanoparticles (YGB-C-LIP-OM / BF) was extremely low in the standard Caco2 model, but in the M-cell model, the cumulative fluorescence signal in the lower chamber significantly increased over time (2, 4, and 6 h). Furthermore, within the M-cell model, the YGB-modified nanomaterials exhibited significantly higher permeability compared to the unmodified material (C-LIP-OM / BF). Therefore, combining structural validation (reduced TEER) and functional validation (targeted lower chamber fluorescence enhancement), a comprehensive conclusion was drawn regarding the successful establishment of the M-cell model and the specific transepithelial transport capability of the YGB-modified nanocarrier.

[0084] 4. Evaluation of in vitro antitumor efficacy and safety

[0085] The toxicity of the delivery system YGB-C-Lip-OM / BF to tumor cells and normal cells was evaluated using the CCK-8 assay. Results are as follows: Figures 16-19 As shown, the results indicate that this delivery system has a significant inhibitory effect on the proliferation of tumor cells such as B16-OVA, while exhibiting no significant toxicity to normal intestinal epithelial cells, demonstrating its good selective killing ability and biosafety. Specifically, such as Figure 16 and Figure 17 As shown, normal mouse macrophages (RAW 264.7) and normal human intestinal epithelial cells (FHC) maintained extremely high cell viability even at increased concentrations, indicating no significant toxicity to normal tissues and demonstrating in vitro biosafety. In stark contrast, as... Figure 18 and Figure 19 As shown, the drug delivery system exhibited strong concentration-dependent cytotoxicity (IC50) against B16-OVA melanoma cells. 50 The value was 0.8777) and there was a significant time-dependent inhibition of proliferation (the survival rate dropped to below 50% after 48 hours), indicating that it has significant anti-tumor ability.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing an oral nano-traditional Chinese medicine antigen delivery system, characterized in that, Includes the following steps: (1) Preparation of OM nanoparticle suspension: Ovalbumin (OVA) was dissolved in PBS to prepare an OVA solution. KMnO4 aqueous solution was added under stirring. The reaction was carried out at room temperature. The precipitate was collected by centrifugation and washed with deionized water to obtain OM nanoparticles. The OM nanoparticles were resuspended in PBS to obtain an OM nanoparticle suspension. The mass ratio of OVA to KMnO4 was 5:

2. (2) Preparation of cationic liposome suspension: Weigh phosphatidylcholine, cholesterol and DOTAP, dissolve them in anhydrous chloroform, rotary evaporate in a water bath to form a lipid film, and then dry, hydrate and sonicate to form cationic liposome suspension. (3) Preparation of cationic liposome suspension loaded with OM / BF: Add ethanol solution of bufotoxin BF and OM nanoparticle suspension obtained in step (1) to the cationic liposome suspension obtained in step (2), and incubate in a water bath under nitrogen protection to obtain cationic liposome suspension loaded with OM / BF. (4) Construction of oral nano-medicine antigen delivery system: Yeast β-glucan was dissolved in PBS to prepare yeast β-glucan solution, and added dropwise to the cationic liposome suspension loaded with OM / BF obtained in step (3) under stirring. The reaction was carried out by stirring at room temperature and centrifugation was performed to obtain yeast β-glucan modified nanoparticles. The mass ratio of yeast β-glucan to cationic liposome suspension loaded with OM / BF was 5:

1. The yeast β-glucan modified nanoparticles were resuspended in PBS to obtain the oral nano-medicine antigen delivery system.

2. The method according to claim 1, wherein, In step (1), the reaction time at room temperature is 3-6 h; the centrifugation speed is 120000-150000 ×g, and the centrifugation time is 20-40 min.

3. The method of claim 1, wherein the oral nano-traditional Chinese medicine antigen delivery system is constructed by, The concentration of the OM nanoparticle suspension is 1-1.5 mg / mL.

4. The method according to claim 1, wherein, The concentration of the cationic liposome suspension is 2-2.5 mg / mL.

5. The method of claim 1, wherein the oral nano-traditional Chinese medicine antigen delivery system is constructed by, The concentration of the cationic liposome suspension loaded with OM / BF was 2.5-3 mg / mL.

6. The method of claim 1, wherein the oral nano-traditional Chinese medicine antigen delivery system is constructed by, In step (2), the molar ratio of phosphatidylcholine, cholesterol, and DOTAP is 5:3:2; the temperature of the water bath is 45-60 ℃; the drying temperature is 37-45 ℃ and the drying time is 30-60 min; the hydration is performed using 10 mL of citrate buffer solution with pH 6.5-6.8; and the power of the ultrasonic treatment is 200-300 W.

7. The method according to claim 1, wherein, In step (3), the mass ratio of cationic liposome suspension, BF ethanol solution and OM nanoparticle suspension is 20:1:4, and the concentration of BF ethanol solution is 1-1.5 mg / mL; the water bath shaking incubation temperature is 50-65 ℃, and the time is 2-4 h.

8. The method according to claim 1, wherein, In step (4), the stirring reaction time at room temperature is 6-8 h; the centrifugation speed is 120000-150000 ×g and the time is 45-60 min.

9. An oral nano-traditional Chinese medicine antigen delivery system, characterized in that, It is constructed using the method described in any one of claims 1 to 8 for constructing an oral nano-Chinese medicine antigen delivery system.

10. The use of the oral nanomedicine antigen delivery system as described in claim 9 in the preparation of oral nanomedicine delivery formulations.

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