pH-responsive hollow mesoporous polydopamine loaded salidomide nanofomulation, preparation method and application thereof

Abstract

The present application relates to the field of traditional Chinese medicine small molecule nano-preparation, and particularly relates to a pH-responsive hollow mesoporous polydopamine loaded rhodiola glycoside nano-preparation, a preparation method and application thereof. The nano-preparation is mainly composed of a hollow mesoporous polydopamine carrier, rhodiola glycoside loaded on the hollow mesoporous polydopamine carrier, and a pH-responsive enteric material coated on the outer layer of the hollow mesoporous polydopamine loaded with rhodiola glycoside. The nano-preparation produces a coupling synergistic effect through the synergistic innovation of structural design, targeted delivery and pharmacodynamic enhancement, breaks through the technical bottleneck of traditional nano-carriers in drug loading efficiency, targeting accuracy and safety, and realizes efficient and safe treatment of ulcerative colitis and various animal bacterial diarrhea.

Description

Technical Field

[0001] This invention relates to the field of small molecule nano-preparations of traditional Chinese medicine, specifically to pH-responsive hollow mesoporous polydopamine-loaded rhodioloside nano-preparations, their preparation methods, and applications. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, relapsing, idiopathic autoimmune disease characterized by epithelial barrier dysfunction and intestinal ulcers. Clinical manifestations include diarrhea, hematochezia, abdominal pain, and weight loss, which seriously affect the quality of life of patients. Currently, the clinical treatment of UC relies on aminosalicylate, corticosteroids, immunomodulators, and biological agents, but there are obvious limitations: (1) Traditional oral preparations are not absorbed regularly in the gastrointestinal tract and are easily destroyed by gastric acid, resulting in insufficient local drug concentration in the colon; (2) Long-term use of hormones or immunosuppressants can easily cause systemic adverse reactions; (3) 30-40% of patients do not respond to treatment or lose response. In addition, in the field of livestock and poultry farming, the incidence of Escherichia coli diarrhea in chicks and goats is high and the harm is serious, often leading to growth retardation, dehydration, death, and significant economic losses. Clinical treatment relies on antibiotics such as ciprofloxacin, but it is easy to cause drug resistance, drug residues, liver and kidney damage, and intestinal flora disorder, which does not meet the requirements of green farming and food safety. Therefore, it is of great significance to develop a delivery system that can efficiently target the disease area and reduce systemic toxic side effects.

[0003] In recent years, nano-oral delivery systems have provided new directions for intestinal targeted therapy. Polydopamine (PDA) has good biocompatibility, is biodegradable and easy to functionalize, but existing PDA carriers still have defects: (1) poor surface drug loading stability, which makes them prone to burst release in the gastrointestinal tract; (2) lack of pH-responsive design, which makes them unable to be stable in gastric acid (pH 1.2-2.0) and release at specific points in the colon (pH 7.0-7.5); (3) no reports have been made of colon-targeted systems that combine a high drug loading core with a pH-responsive coating.

[0004] Rhodioloside (Sal) possesses anti-inflammatory, antioxidant, immunomodulatory, intestinal barrier protective, and antibacterial activities, and can inhibit the NF-κB pathway and downregulate pro-inflammatory factors such as TNF-α and IL-6. However, its oral bioavailability is low (only 8-12%), its metabolism is rapid, and its local concentration is insufficient, limiting its clinical application. Furthermore, there are no reports of integrated nano-formulations that combine the dual indications of "human ulcerative colitis and animal coliform diarrhea," high drug loading, pH responsiveness, intestinal targeting, anti-inflammatory and antibacterial effects, and protection of liver and kidney function. Therefore, in response to the current lack of traditional Chinese medicine nano-formulations that combine antibacterial and anti-inflammatory properties, intestinal targeting, liver and kidney protection, low residue, and antibiotic alternatives, this invention provides a pH-responsive hollow mesoporous polydopamine-loaded rhodioloside nano-formulation, its preparation method, and its applications. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a pH-responsive hollow mesoporous polydopamine-loaded rhodioloside nanoparticle formulation, its preparation method, and its application. The aim is to achieve high drug loading through hollow mesoporous polydopamine, and to achieve gastric acid protection and targeted release into the colon / intestinal tract through pH-responsive enteric coating, significantly improving the bioavailability of rhodioloside. This formulation can treat human ulcerative colitis, and can also effectively and safely treat Escherichia coli-induced diarrhea in chicks and goats, reducing antibiotic use and lowering the risk of drug resistance and residues.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: Firstly, a pH-responsive hollow mesoporous polydopamine-loaded rhodioloside nanoformulation is provided, wherein the nanoformulation is mainly composed of a hollow mesoporous polydopamine carrier, rhodioloside loaded on the hollow mesoporous polydopamine carrier, and a pH-responsive enteric material coated on the outer layer of the hollow mesoporous polydopamine loaded with rhodioloside. The mass ratio of the hollow mesoporous polydopamine carrier to the rhodioloside is 1:1 to 1:3, preferably 1:2; The total mass ratio of the hollow mesoporous polydopamine carrier and the rhodioloside to the pH-responsive enteric material is 1:0.5 to 1:2.

[0007] The polydopamine is self-assembled and does not contain any inorganic template residue; the rhodioloside is loaded onto the hollow mesoporous polydopamine carrier through π-π stacking and hydrogen bonding.

[0008] Based on the above technical solution, the present invention can be further improved as follows.

[0009] Furthermore, the drug loading of the nano-formulation is 40-50%, and the encapsulation efficiency is 75-85%. The cumulative release rate of the nano-formulation in simulated gastric fluid at pH 1.2 is ≤10% after 2 hours, and the cumulative release rate in simulated intestinal fluid at pH 7.4 is ≥80% after 24 hours.

[0010] Furthermore, the hydrated particle size of the nano-formulation is 250~350 nm, and the Zeta potential is -20 mV to -10 mV.

[0011] Furthermore, the hollow mesoporous polydopamine carrier is prepared by a soft template method, and the particle size of the hollow mesoporous polydopamine carrier is 200~400 nm, with a specific surface area of ​​50~200 m². 2 / g, pore volume 0.2~0.8 cm³ 3 / g; The pH-responsive enteric material includes at least one of Eudragit L100, Eudragit S100, Eudragit L30D-55, and Eudragit L100-55.

[0012] In addition, the rhodioloside is a β-configuration rhodioloside.

[0013] Secondly, the preparation method of pH-responsive hollow mesoporous polydopamine-supported rhodioloside nanoparticles includes the following steps: (1) Preparation of hollow mesoporous polydopamine carrier: Hollow mesoporous polydopamine carrier was prepared by soft template method; (2) Loading of rhodioloside: The hollow mesoporous polydopamine nanocarrier and rhodioloside were dispersed in ultrapure water, mixed evenly, and the free rhodioloside was removed to obtain hollow mesoporous polydopamine loaded with rhodioloside. (3) pH-responsive coating: The hollow mesoporous polydopamine loaded with rhodioloside is mixed evenly with the pH-responsive enteric material, and the precipitate is collected to obtain a pH-responsive hollow mesoporous polydopamine loaded with rhodioloside nanoformulation.

[0014] Further, the preparation of the hollow mesoporous polydopamine carrier in step (1) includes the following specific steps: polyether F127 and trimethylbenzene TMB are dissolved in a mixture of anhydrous ethanol and ultrapure water, and the mixture is ultrasonically treated to form a milky white liquid. Tris buffer and dopamine hydrochloride are then added, mixed evenly, and the precipitate is collected. Finally, the mixture is washed with a mixture of ethanol and acetone in a volume ratio of 1:1 to 3:1 and then freeze-dried to obtain the hollow mesoporous polydopamine carrier.

[0015] Furthermore, the mass ratio of the polyether F127 to the trimethylbenzene TMB is 2:1 to 5:1, preferably 4:1; The volume ratio of the polyether F127 to the mixture system only needs to meet the requirements; the volume ratio of anhydrous ethanol to ultrapure water in the mixture system only needs to meet the requirements; the volume ratio of the polyether F127 to the Tris buffer only needs to meet the requirements; and the ratio of the amount of Tris buffer to the amount of dopamine hydrochloride only needs to meet the requirements. Specifically, the preparation of the hollow mesoporous polydopamine carrier in step (1) includes the following: ① Add 25 mL of deionized water and 25 mL of anhydrous ethanol to a beaker. First, weigh 0.3 g of F127 and dissolve it completely in the above mixture. Then, accurately measure 500 μL of TMB and add it to the above liquid. Stir at 500 rpm for 2 hours at room temperature. ② After stirring for 2 hours, the above mixture was ultrasonically broken down to form a milky white liquid. Then, 90 mg Tris and 15 mg dopamine hydrochloride were added, and the mixture was thoroughly mixed. The mixture was then stirred at 500 rpm for 24 hours. ③ After 24 hours, collect the solution from the beaker into a centrifuge tube, centrifuge at 12000 rpm for 20 minutes at 4℃, collect the black precipitate, wash the collected precipitate several times with acetone and anhydrous ethanol, and freeze-dry it into powder using a vacuum freeze dryer for later use.

[0016] The ultrasonic treatment was performed using an ultrasonic cell disruptor with a power of 400 W, operating for 5 seconds and then intermittently for 5 seconds, for a total time of 10 minutes. The washing process uses a mixture of ethanol and acetone with a volume ratio of 1:1 to 3:1.

[0017] Thirdly, the application of the pH-responsive hollow mesoporous polydopamine-loaded rhodioloside nanoparticles in the preparation of drugs for treating ulcerative colitis.

[0018] Fourthly, the application of the pH-responsive hollow mesoporous polydopamine-loaded rhodioloside nanoparticles in the preparation of drugs for treating Escherichia coli-induced diarrhea in livestock and poultry.

[0019] Furthermore, the livestock and poultry include at least one of chickens, goats, sheep, deer, pigs, and ducks.

[0020] This invention successfully prepared a pH-responsive hollow mesoporous polydopamine-loaded rhodioloside nanoparticle formulation (L100@H / SaL). Through synergistic innovation in structural design, targeted delivery, and drug efficacy enhancement, this formulation generates a coupled synergistic effect, breaking through the technical bottlenecks of traditional nanocarriers in terms of drug loading efficiency, targeting accuracy, and safety, and achieving efficient and safe treatment of ulcerative colitis and various bacterial diarrheas in animals.

[0021] The pH-responsive hollow mesoporous polydopamine-loaded rhodioloside nanoparticles prepared in this invention possess a unique multi-linked synergistic controlled-release mechanism: the hollow mesoporous structure is prepared using a soft template method without inorganic residues, which can simultaneously form hydrogen bonds and π-π stacking forces with rhodioloside; the enteric coating forms a dense, compressible membrane in an acidic environment, which dissociates after entering the neutral intestinal tract, while the inflamed site is enriched by the EPR effect; this multi-linked controlled-release and targeted enrichment mechanism is not an inherent property of existing materials, but rather an unpredictable synergistic effect.

[0022] The beneficial effects of this invention are: (1) Dual design: This invention successfully constructed a high-drug-loaded, colon-targeted, long-acting sustained-release rhodioloside nanodelivery system through a dual design of "hollow mesoporous structure and pH-responsive coating". This effectively overcomes the technical difficulties of oral drugs being degraded by gastric acid, poor colon targeting, and low bioavailability of rhodioloside in the prior art. At the same time, this preparation has shown significantly better efficacy and safety than existing antibiotics in the treatment of ulcerative colitis and Escherichia coli diarrhea in goats and chicks. It has the potential to replace antibiotics and has broad prospects for industrial application.

[0023] (2) Hollow mesoporous structure achieves high drug loading performance: This invention uses hollow mesoporous polydopamine as a drug carrier, which has a higher specific surface area and pore volume compared with traditional solid or ordinary mesoporous structures. Its hollow structure provides sufficient loading space for rhodioloside, while the mesoporous shell facilitates the controlled release of the drug. Experimental results show that the drug loading of the nano-formulation of this invention reaches 45.27% and the encapsulation efficiency is 82.71%, which is significantly better than traditional polydopamine nanocarriers (usually with a drug loading of <20%), effectively solving the technical pain points of poor water solubility and low drug loading efficiency of rhodioloside.

[0024] (3) pH-responsive coating for colon-targeted delivery: This invention uses a pH-responsive enteric coating material (e.g., Eudragit L100) to endow the nano-formulation with good gastric acid protection and colon-targeted release characteristics. In vitro release experiments show that the cumulative release rate of this formulation in simulated gastric juice (pH 1.2) is <10% after 2 hours, which can effectively avoid premature drug degradation in gastric juice; and the cumulative release rate in simulated intestinal juice (pH 7.4) is >85% after 24 hours, achieving high concentration accumulation of the drug in the colon. Compared with nano-formulations without pH-responsive coating, the colon-targeting efficiency of the formulation of this invention is significantly improved, effectively reducing the risk of systemic drug exposure and related adverse reactions.

[0025] (4) Broad-spectrum efficacy and outstanding comprehensive advantages: The L100@H / SaL nano-formulation prepared in this invention has shown excellent effects in the treatment of ulcerative colitis and various bacterial diarrheas in livestock and poultry. ① Significantly improves the treatment effect of ulcerative colitis: Animal experimental results show that L100@H / SaL nano-formulation can significantly improve the clinical symptoms of ulcerative colitis in mice: Relieves systemic symptoms: Significantly inhibits weight loss ( P <0.01 vs model group), rectal bleeding symptoms basically disappeared after day 5, and the Disease Activity Index (DAI) score decreased significantly ( P <0.001 vs model group); Improved colonic pathology: Significantly restored colon length ( P<0.001 vs model group), reduce colonic tissue pathological damage, and restore histological indicators such as intestinal gland depth and mucosal layer thickness; Repairing the intestinal barrier: Significantly upregulated the expression of tight junction proteins ZO-1 and Occludin (P<0.01 vs model group), repairing intestinal barrier function damage caused by ulcerative colitis.

[0026] ② Highly effective treatment for E. coli-induced diarrhea in goats: In a goat E. coli-induced diarrhea model, the L100@H / SaL nanoformulation showed significantly better therapeutic effects than ofloxacin. The therapeutic efficacy indicators were significantly improved: the cure rate reached 89.0±4.2%, significantly higher than that of the antibiotic group; the recurrence rate (14.3±3.1%) and mortality rate (10.0±4.5%) were both significantly reduced, and the average recovery time (4.2±0.8 days) was significantly shortened. P <0.05 or P <0.01); The antibacterial effect is long-lasting and highly effective: the decrease in fecal E. coli count after treatment is significantly better than that in the antibiotic group. P <0.01), the value on day 14 of the experiment was much lower than that of the antibiotic group, indicating a more lasting antibacterial effect; Overall improvement in physical condition: white blood cell count, absolute neutrophil count and percentage returned to normal range, red blood cell count and hemoglobin significantly increased, and hematocrit returned from elevated to normal level, indicating that it can effectively control bacterial infection, improve the body's nutrition and oxygen-carrying function, and correct dehydration symptoms. Significant safety advantages: During treatment, liver and kidney function indicators such as ALT, AST, Cr, and BUN remained at normal levels, while those in the antibiotic group showed significant increases (P<0.05), indicating that the formulation of this invention has higher drug safety while ensuring efficacy.

[0027] ③ Highly effective treatment for E. coli-induced diarrhea in chicks: In clinical trials of E. coli-induced diarrhea in chicks, the L100@H / SaL nanoformulation showed significantly better therapeutic effects than ciprofloxacin. The efficacy indicators were significantly improved: the cure rate reached 90.0±6.2%, the diarrhea rate decreased to 6.7±4.1%, and the mortality rate decreased to 0. All efficacy indicators were significantly better than those of the antibiotic control group (P<0.01). Long-lasting and highly effective antibacterial effect: On the 5th day of treatment, the number of fecal E. coli decreased to 3.5 × 10⁻⁶. 3 ±0.5×10 3 The CFU / g level was close to that of the blank control group after the experiment, while the antibiotic group maintained a higher level, and its ability to control bacteria was significantly better than that of ciprofloxacin (P<0.01). Overall improvement in physical condition: white blood cell count and neutrophil count returned to normal range, red blood cell count, hemoglobin and hematocrit significantly improved, indicating that infection can be effectively controlled, nutritional status can be improved and dehydration can be corrected; Significant safety advantages: During treatment, liver and kidney function indicators such as ALT, AST, Cr, and BUN remained at normal levels, while the ciprofloxacin group showed a significant increase, confirming that the preparation of this invention has a good protective effect on liver and kidney function during treatment.

[0028] It is worth noting that the pathological mechanisms of ulcerative colitis (autoimmune inflammation) and bacterial enteritis (pathogenic microbial infection) are fundamentally different. The nano-formulation described in this invention has demonstrated excellent efficacy against both of these intestinal diseases with different mechanisms, and its efficacy surpasses that of standard treatments in their respective fields. Its broad-spectrum therapeutic activity reflects a non-obvious technical effect (see Results and Discussion section for details). ④ Excellent biocompatibility: Cytotoxicity experiments showed that the nano-formulation of this invention maintained a cell viability of over 85% in both RAW264.7 and Caco-2 cells at a concentration of 250 μg / mL, meeting the safety requirements for biomedical materials. In vivo imaging results showed that the formulation could remain in the colonic region for more than 24 hours, significantly prolonging its in vivo circulation time. Compared with traditional Western medicines and ordinary nano-formulations, this invention exhibits superior safety due to the excellent biocompatibility of the polydopamine carrier and the low systemic exposure characteristics resulting from targeted delivery; furthermore, the nano-formulation of this invention has synergistic antioxidant effects. Attached Figure Description

[0029] Figure 1 The diagram shows the preparation process and characterization of nanoparticles; where A is a schematic diagram of the nanoparticle preparation process, B is an EDS elemental mapping analysis diagram, C is a transmission electron microscope image, and D and E are specific surface area and pore size distribution diagrams. Figure 2 The image shows the characterization of the nanoparticles; where A is the hydrated particle size, B is the Zeta potential, and C is the polydispersity index. Figure 3 The graph shows the results of the drug loading and in vitro drug release performance evaluation; where A is the standard curve of rhodioloside, B is the drug loading rate and encapsulation rate of different drug-to-material ratios, and C is the in vitro drug release curve. Figure 4 The figures show the results of cell compatibility and clearance assays; where A represents the cell compatibility of RAW264.7, B represents the cell compatibility of Caco-2, C represents the in vitro DPPH clearance assay, and D represents the in vitro ABTS clearance assay. Figure 5The image shows the sustained release and distribution of the nano-formulation in mice; where A represents in vivo imaging and quantitative analysis of mice, B represents in vivo imaging and quantitative analysis of the entire intestinal segment, and C represents in vivo imaging and quantitative analysis of the colon. Figure 6 The images show the therapeutic effects on mice with ulcerative colitis. A represents fecal bleeding during treatment in the mouse ulcerative colitis model; B represents weight changes during treatment; C represents changes in the disease activity index (DAI) during treatment; D represents a photograph of the colon length after treatment; E represents a statistical graph of the colon length after treatment; F represents a photograph of the spleen after treatment; and G represents a statistical graph of the spleen index after treatment. Figure 7 The images show the results of colonic histopathology and intestinal barrier function evaluation; where A is a colonic H&E stained section after treatment in mice, B is a colonic ab~pas stained section after treatment in mice, C is the depth of intestinal glands, mucosal layer thickness, muscle layer thickness and intestinal wall thickness after treatment in mice, D is a section stained with ZO-1 immunofluorescence antibody, E is a section stained with Occludin immunofluorescence antibody, F is a semi-quantitative analysis of ZO-1, and G is a semi-quantitative analysis of Occludin. Detailed Implementation

[0030] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0031] Examples 1 to 6: Preparation and characterization of pH-responsive hollow mesoporous polydopamine-loaded rhodioloside nanoformulations.

[0032] 1. Preparation of pH-responsive hollow mesoporous polydopamine-supported rhodioloside nanoparticles ( Figure 1 (A)

[0033] (1) Preparation of hollow mesoporous polydopamine (HMPDA) carrier: Weigh 0.3 g of poloxamer F127 and 0.5 mL of trimethylbenzene (TMB), dissolve them in a mixture of 25 mL of anhydrous ethanol and 25 mL of ultrapure water, and stir at room temperature for 2 h at a stirring speed of 300–500 rpm. Treat the mixture using an ultrasonic cell disruptor (400 W, 5 s operation / 5 s interval, total 10 min) to form a milky white liquid. Add 90 mg of Tris buffer to the mixture, then add 15 mg of dopamine hydrochloride, and continue stirring at room temperature for 24 h at a stirring speed of 300–500 rpm. After the reaction is complete, centrifuge at 12000 rpm for 15 min to collect the precipitate. Wash the precipitate three times with a 1:1 (v / v) mixture of ethanol and acetone. Freeze-dry the collected precipitate at -55 °C for 12 h. Store the freeze-dried product for later use to obtain the hollow mesoporous polydopamine nanocarrier, labeled HMPDA.

[0034] (2) Loading of rhodioloside: The hollow mesoporous polydopamine nanocarrier prepared in step (1) was dispersed in ultrapure water at a mass ratio of 1:2 with rhodioloside and stirred at room temperature for 24 h. After centrifugation at 12000 rpm for 15 min, the free drug was removed and the precipitate was collected to obtain hollow mesoporous polydopamine loaded with rhodioloside (labeled as H@SaL), which was stored for later use.

[0035] (3) pH-responsive coating: The hollow mesoporous polydopamine loaded with rhodioloside prepared in step (2) was mixed with Eudragit L100 at a mass ratio of 3:1, stirred at room temperature for 12 h, and the precipitate was collected by centrifugation to obtain pH-responsive hollow mesoporous polydopamine loaded with rhodioloside nanoparticles (labeled as L100@H / SaL).

[0036] Examples 2-5 follow the preparation method of Example 1, adjusting the amounts of rhodioloside, HMPDA, and Eudragit L100 according to the mass ratios in Table 1, with the remaining operations being the same.

[0037] Table 1 2. Characterization of nano-formulation structure.

[0038] The hydration particle size, polydispersity index (PDI), and zeta potential of the prepared L100@H / SaL particles were determined using dynamic light scattering (DLS). The results showed that... Figure 2 The hydrated particle size (A, B, and C) was 311.8 ± 5.96 nm, the Zeta potential was -15.5 ± 0.57 mV, and the PDI was 0.32 ± 0.018, indicating that the nano-formulation has good uniformity and stability.

[0039] The morphology of the nano-formulation was observed using transmission electron microscopy (TEM), and the results showed that ( Figure 1 The nanoparticles (containing C) are spherical, exhibit good dispersibility, and have a hollow central structure. Elemental analysis was performed using energy-dispersive X-ray spectroscopy (EDS). Figure 1 (B) The results showed that C, N, and O elements were uniformly distributed in the nano-formulation; Specific surface area and pore size distribution were determined using nitrogen adsorption-desorption experiments. Figure 1 (D, E), the results show that the hollow mesoporous polydopamine carrier has a typical mesoporous structure.

[0040] 3. Evaluation of drug loading capacity and in vitro drug release performance.

[0041] (1) Establishment of the standard curve for rhodioloside: Rhodioloside standard solutions with concentrations of 0.125, 0.25, 0.5, 1.0, and 2.0 mg / mL were precisely prepared, and the absorbance at 275 nm was measured. A standard curve was plotted with concentration on the x-axis and absorbance on the y-axis. Figure 3 (A). The resulting regression equation is Y = 0.5234X + 0.0123 (R²). 2 =0.9992), with a linear range of 0.125~2.0 mg / mL.

[0042] (2) Determination of encapsulation efficiency and drug loading rate: Five drug-to-particle ratios (rhodioloside:HMPDA:Eudragit L100, mass ratio) were set at 1:2:1, 1:1.5:1, 1:1:1, 1.5:1:1.5, and 2:1:2, and each nano-formulation was prepared according to the method in Example 1. The supernatant was collected by centrifugation, and the absorbance at 275 nm was measured. The free drug concentration was calculated based on the standard curve, and the encapsulation efficiency (EE) and drug loading (LC) were calculated using the following formulas: Encapsulation rate (%) = (Drug feed amount - Free drug amount) / Drug feed amount × 100%; Drug loading rate (%) = (Drug input amount - Free drug amount) / Total mass of nano-formulation × 100%; The results are as follows Figure 3 As shown in Figure B, the encapsulation efficiency is the highest at 82.71% when the drug-to-material ratio is 1:2:1, and the drug loading is 45.27%.

[0043] (3) In vitro drug release behavior: The L100@H / SaL nanoparticles prepared in Example 1 were placed in a dialysis bag (molecular weight cutoff 3500 Da). 50 mL of simulated gastric fluid (pH 1.2, containing 0.2% Tween 80) and simulated intestinal fluid (pH 7.4, containing 0.2% Tween 80) were added, respectively. The bag was then placed in a 37°C water bath shaker (100 rpm) for in vitro release experiments. At preset time points (0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h), 1 mL of release medium was collected, and an equal volume of fresh medium was added. The absorbance at 275 nm was measured using ultraviolet spectrophotometry, and the cumulative release rate was calculated.

[0044] The results are as follows Figure 3 As shown in Figure C, the cumulative release rate of L100@H / SaL in simulated gastric fluid (pH 1.2) was 8.3±1.2% after 2 hours, indicating that the formulation can effectively avoid premature drug release in gastric fluid; in simulated intestinal fluid (pH 7.4), the cumulative release rate reached 86.5±3.1% after 24 hours and 94.2±2.5% after 48 hours, showing good pH-responsive sustained-release characteristics.

[0045] 4. Evaluation of cell compatibility and antioxidant activity.

[0046] (1) Cell culture: Mouse mononuclear macrophages RAW264.7 and human colorectal adenocarcinoma cells Caco-2 (both purchased from Suzhou Xinsaimei Biotechnology Co., Ltd.) were cultured in DMEM high glucose medium and MEM medium (both purchased from Wuhan Boster Biological Engineering Co., Ltd.) containing 10% fetal bovine serum, respectively, and were routinely cultured in an incubator at 37°C and 5% CO2.

[0047] (2) Cytotoxicity test: RAW264.7 and Caco-2 cells were respectively planted at a density of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 μg / mL in 96-well plates and cultured for 24 h. Then, different concentrations (0 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, and 250 μg / mL, based on the total mass of the nanoparticles) of free rhodioloside (Sal), hollow mesoporous polydopamine (HMPDA), rhodioloside-loaded hollow mesoporous polydopamine (H / SaL), and pH-responsive hollow mesoporous polydopamine-loaded rhodioloside (L100@H / SaL) solutions were added, with 5 replicates per group. After another 24 h of culture, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated in the dark for 4 h. The absorbance was measured at 450 nm using a microplate reader to calculate cell viability.

[0048] The results are as follows Figure 4As shown in Figures A and B, at concentrations below 250 μg / mL, the cell viability of all formulation groups was higher than 85%, indicating that the nano-formulations prepared in this invention have good biocompatibility.

[0049] (3) Antioxidant activity experiment: The antioxidant activity of the nano-formulation was evaluated using ABTS and DPpH free radical scavenging assays. ABTS and DPpH working solutions were prepared separately, and different concentrations of Sal, HMPDA, H / SaL, and L100@H / SaL solutions were added. After the reaction was carried out in the dark, the absorbance was measured, and the free radical scavenging rate was calculated.

[0050] The results are as follows Figure 4 As shown in C and D, the L100@H / SaL group exhibited concentration-dependent free radical scavenging activity, which was significantly superior to the free Sal group at the same concentration. P The value <0.05 indicates that the nanocarrier and rhodioloside have a synergistic antioxidant effect.

[0051] 5. Sustained release and distribution of nano-formulations in mice.

[0052] (1) Fluorescent labeling: The L100@H / SaL nanoformulation was fluorescently labeled using Cy5.5 NHS ester. Cy5.5 was dissolved in DMSO and added to the L100@H / SaL solution at a mass ratio of Cy5.5:nanoformulation of 1:100. The mixture was stirred in the dark for 4 h, and the free Cy5.5 was removed by centrifugation. The Cy5.5-labeled L100@H / SaL was collected for later use.

[0053] (2) In vivo imaging: Healthy ICR mice (purchased from Chengdu Dashuo Experimental Animal Co., Ltd.) were randomly divided into three groups (n=3), and administered Cy5.5-labeled L100@H / SaL, H / SaL, and SaL by gavage, respectively. Fluorescence images were acquired using a small animal in vivo imaging system (excitation wavelength 640 nm, emission wavelength 700 nm) at 2 h, 4 h, 8 h, 16 h, and 24 h after administration to observe the distribution of the nano-formulation in vivo.

[0054] The results are as follows Figure 5 As shown in Figures A to C, the free Cy5.5 group showed strong systemic distribution 2 h after administration, and the fluorescence signal weakened significantly after 8 h; while the L100@H / SaL group showed fluorescence signal in the colon region 4 h after administration, and the fluorescence intensity in the colon region reached its peak at 8-16 h, and a significant fluorescence signal could still be detected at 24 h, indicating that the nano-formulation of the present invention has good colon targeting and in vivo sustained-release ability.

[0055] Comparative Example 1: Solid polydopamine loaded with rhodioloside (without hollow structure).

[0056] Solid polydopamine nanoparticles were prepared using conventional methods. By controlling the types of template agents, regulators, and polymerization conditions, MPDA with different structures and loaded with an equal amount of rhodioloside could be obtained. The results showed that the solid structure had low pore volume and a drug loading of only 18.35%; moreover, the drug adsorption was weak, with a release rate of up to 37.62% in gastric juice after 2 hours, failing to achieve gastric acid protection and exhibiting poor colonic accumulation capacity.

[0057] Comparative Example 2: Preparation of HMPDA using silica hard template (with inorganic residues).

[0058] Hollow mesoporous polydopamine was prepared using a traditional SiO2 hard template method. First, SiO2 nanospheres were prepared. Then, in an ammonia solution, dopamine underwent oxidative self-polymerization, forming a PDA shell on the SiO2 surface, resulting in a SiO2@PDA core-shell structure. The SiO2 template was then removed by etching with hydrofluoric acid (HF) or sodium hydroxide (NaOH) solution, leaving trace amounts of inorganic silicon impurities after etching. Results showed that the biocompatibility of this carrier was reduced, with a Caco-2 cell survival rate of only 72.3%. Furthermore, the residual impurities interfered with drug binding, resulting in a drug loading of only 29.61%, significantly lower than the template-free soft template process of this invention.

[0059] Comparative Example 3: Uncoated naked nanoparticles (H / SaL).

[0060] Uncoated drug-loaded nanoparticles were prepared by removing the Eudragit L100 coating. Results showed that the uncoated nanoparticles rapidly disintegrated in acidic gastric juice, with a drug release rate as high as 41.25% after 2 hours. Severe gastric acid degradation prevented intestinal targeting, and the cure rate for colitis in animals was only 53.2%.

[0061] Comparative Example 4: Pure Uteqi physically encapsulated rhodioloside (without nanocarrier).

[0062] Rhodioloside powder was directly encapsulated using Eudragit L100 without the addition of HMPDA carrier. Results showed that the drug loading in this system was only 12.74%, with no adsorption or fixation effect; in vivo diffusion was uneven, accumulation at inflammatory sites was low, and the antibacterial and anti-inflammatory effects were significantly weaker than those of this invention. It is evident that hollow mesoporous polydopamine alone cannot achieve gastric acid stability; enteric coating alone cannot achieve high drug loading; and rhodioloside alone cannot achieve intestinal accumulation. The combination of these three components produces a synergistic effect, achieving technical effects that cannot be achieved by individual components or combinations of two components, and is not a conventional material superposition.

[0063] Experimental Example 1: Evaluation of the therapeutic effect of nano-formulation on mice with ulcerative colitis.

[0064] 1. Establishment of an ulcerative colitis model and drug administration regimen: Male ICR mice aged 6–8 weeks were randomly divided into 5 groups (n=6): normal control group, model group, free Sal group (50 mg / kg), H / SaL group (50 mg / kg as Sal), and L100@H / SaL group (50 mg / kg as Sal). Except for the normal control group, the mice in the other groups were given free access to 3% (w / v) sodium dextran sulfate (DSS) solution for 7 consecutive days to induce an ulcerative colitis model. Simultaneously with model establishment, the treatment groups were administered the corresponding drugs by gavage daily, while the normal control group and model group received an equal volume of physiological saline for 7 consecutive days.

[0065] 2. Efficacy evaluation indicators: (1) Weight changes, rectal bleeding and disease activity index (DAI) score Mouse weight, fecal characteristics, and blood in stool were recorded daily. The DAI score was calculated using the following criteria: weight loss (0: none; 1: 1-5%; 2: 5-10%; 3: 10-15%; 4: >15%), fecal characteristics (0: normal; 2: loose; 4: watery stool), and blood in stool (0: normal; 2: positive for occult blood; 4: visible blood in stool). The DAI score was the sum of the three scores divided by 3.

[0066] The results are as follows Figure 6 As shown in Figures A, B, and C, the model group mice exhibited significant weight loss, hematochezia, and elevated DAI scores after DSS induction; the free Sal group showed some improvement, but the effect was limited; the H@SaL group showed better improvement than the free Sal group; and the L100@H / SaL group significantly inhibited weight loss. P <0.01 vs model group), rectal bleeding symptoms basically disappeared after day 5, and the DAI score decreased significantly ( P <0.001 vs model group), the efficacy was superior to the H / SaL group ( P <0.05).

[0067] (2) Colon length and spleen index: After treatment, the mice were euthanized, the colon was dissected, and its length was measured. The spleen was harvested, weighed, and the spleen index was calculated. The formula for calculating the spleen index is as follows: Spleen index = spleen weight / body weight × 100%.

[0068] The results are as follows Figure 6 As shown in D, E, F, and G, the colon length in the model group was significantly shortened ( P <0.001 vs normal group), spleen index significantly increased ( P <0.001 vs normal group); the colon length in the L100@H / SaL group was significantly increased compared to the model group (P <0.001), spleen index significantly decreased ( P <0.01), and the therapeutic effect was superior to that of the free Sal group and the H / SaL group.

[0069] (3) Histopathological evaluation of colon tissue: Mouse colon tissue was collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (AB-PAS), respectively. Histopathological changes were observed under an optical microscope, and the depth of intestinal glands, thickness of mucosal layer, thickness of muscle layer and thickness of intestinal wall were measured.

[0070] The results are as follows Figure 7 As shown in Figures A, B, and C, the colonic mucosal structure in the model group was severely damaged, with extensive inflammatory cell infiltration, and significantly reduced intestinal gland depth, mucosal layer thickness, muscular layer thickness, and intestinal wall thickness. P <0.001 vs normal group); the pathological damage to colon tissue in the L100@H / SaL group was significantly reduced, and all thickness indicators were significantly restored ( P <0.01 vs model group), which is better than the free Sal group and H@SaL group.

[0071] (4) Evaluation of intestinal barrier function: Immunofluorescence staining was used to detect the expression of tight junction proteins ZO-1 and Occludin in colon tissue. After antigen retrieval and blocking, colon tissue sections were incubated overnight with primary antibodies against ZO-1 and Occludin, respectively. The next day, they were incubated with secondary fluorescent antibodies, and the cell nuclei were counterstained with DAPI. The fluorescence intensity was observed and semi-quantitatively analyzed under a fluorescence microscope.

[0072] The results are as follows Figure 7 As shown in D, E, F, and G, the fluorescence intensity of ZO-1 and Occludin in the model group was significantly reduced ( P <0.001 vs normal group), indicating impaired intestinal barrier function; the fluorescence intensity of ZO-1 and Occludin in the L100@H / SaL group was significantly restored ( P <0.01 vs model group), showing a significant difference compared to the free Sal group and H / SaL group ( P <0.05), indicating that the nano-formulation of the present invention can effectively repair intestinal barrier damage caused by ulcerative colitis.

[0073] Experimental Example 2: Clinical efficacy trial of L100@H / SaL against goat Escherichia coli-induced diarrhea.

[0074] This study aims to verify the efficacy and safety of pH-responsive hollow mesoporous polydopamine-loaded rhodioloside nanoparticles (L100@H / SaL) in treating Escherichia coli-induced diarrhea in goats, and to provide experimental evidence for its veterinary clinical application.

[0075] 1. Test Procedure: Sixty goats weighing 10–15 kg were selected from a large-scale sheep farm that had naturally developed the disease and were diagnosed with E. coli-induced diarrhea by clinical symptom observation and PCR testing. Twenty healthy goats from the same farm were also selected as a blank control. The diarrhea cases were randomly divided into three groups of 20 goats each, as follows: Blank control group: healthy goats, without medication and with routine feeding and management; Antibiotic group: goats with diarrhea, administered commercially available florfenicol preparation orally at the clinically recommended dose once daily for 5 consecutive days; Nanoparticle preparation group: goats with diarrhea, administered the nanoparticle preparation prepared in Example 1 of this invention once daily for 5 consecutive days.

[0076] Each group was kept in a separate area of ​​the same sheep farm, with consistent feed, water, environment, and feeding management conditions. No other antibiotics, antidiarrheal drugs, or additives that could affect the experimental results were used during the experiment.

[0077] During the experiment, the mental state, appetite, body temperature, fecal characteristics, and frequency of diarrhea in goats were observed and recorded daily. The time to improvement, recovery, cure rate, improvement rate, relapse rate, and mortality rate were also recorded. Blood samples were collected from the jugular vein before the experiment, on day 5 of treatment, and on day 14 of the experiment to measure complete blood count and serum biochemical indicators, including ALT, AST, BUN, Cr, and electrolyte levels. Fecal samples were collected to determine the number of Escherichia coli colonies, and pathogenic Escherichia coli were detected using PCR.

[0078] Criteria for determining efficacy: Cure is defined as formed stool, normal mental state and appetite, stable body temperature, and no recurrence for 3 consecutive days; Improvement is defined as reduced frequency of diarrhea, improved stool consistency, and improved mental state and appetite; Ineffective is defined as no improvement or worsening of symptoms, or even death.

[0079] 2. Evaluation of therapeutic effect: (1) Treatment efficacy for diarrhea The treatment efficacy for goat diarrhea is shown in Table 2. The blank control group had no diarrhea symptoms and a mortality rate of 0. The antibiotic group had a cure rate of 71.1±5.7%, a relapse rate of 14.3±3.1%, a mortality rate of 11.0±4.6%, and an average recovery time of 4.2±0.8 days. The L100@H / SaL group had a cure rate of 89.0±4.2%, a relapse rate of 4.8±2.2%, a mortality rate of 2.7±2.3%, and an average recovery time of 2.8±0.6 days. All efficacy indicators were significantly better than those of the antibiotic group (P<0.05 or P<0.01), with a higher cure rate, lower relapse rate and mortality rate, and shorter recovery and improvement times.

[0080] Table 2. Statistical table of efficacy for treating goat diarrhea in each group. Note: Intergroup comparisons were analyzed using SPSS software; "—" indicates no relevant data; experimental data are expressed as mean ± standard deviation (x ± s). *Comparison with the antibiotic group P< 0.05, ** P< 0.01* P< 0.05 indicates a significant difference.

[0081] (2) Results of Escherichia coli count in goat feces: The statistical results of the number of Escherichia coli in the feces of goats in each group are shown in Table 3: The number of Escherichia coli in the feces of the blank control group remained at a low level throughout the process (approximately 1.2 × 10⁻⁶). 3 CFU / g). Before the experiment, there was no significant difference in E. coli count between the antibiotic group and the L100@H / SaL group (approximately 8.7 × 10⁻⁶ CFU / g). 6 The CFU / g levels in the L100@H / SaL group were significantly higher than those in the control group. On day 5 of treatment, the CFU / g level in the L100@H / SaL group decreased to 8.5 × 10⁻⁶. 4 ±1.0×10 4 The CFU / g level was significantly better than that of the antibiotic group (3.2 × 10⁻⁶). 5 ±0.8×10 5 CFU / g (P<0.01); On day 14 of the experiment, the CFU / g level in the L100@H / SaL group further decreased to 1.7×10⁻⁶. 4 ±0.4×10 4 CFU / g was significantly lower than the 5.7 × 10⁻⁶ CFU / g in the antibiotic group. 4 ±1.2×10 4 CFU / g. This demonstrates that the L100@H / SaL group showed significantly better inhibitory effects on goat fecal Escherichia coli than conventional Western medicine.

[0082] Table 3. Statistics of Escherichia coli count in goat feces of each group (CFU / g) (3) Results of routine blood tests: The results of routine blood tests are shown in Table 4. The L100@H / SaL nanoparticle formulation showed significant efficacy in treating diarrhea-related bacterial infections. On day 5 of treatment, the white blood cell count in the L100@H / SaL group was (9.5±1.5×10⁻⁶). 9 / L), absolute neutrophil count (4.4±1.2×10 9 The levels of both the neutrophil count ( / L) and neutrophil percentage (41.3±4.4%) were significantly lower than before the experiment, approaching the normal range of the blank control group (8.3±1.3×10⁻⁶). 9 / L, 3.5±1.1×109 The recovery rate was 44.3 ± 5.3% (L), and the degree of recovery was better than that of the antibiotic group (12.4 ± 1.7 × 10⁹ / L). 9 / L, 7.3±1.5×10 9 The levels of [unclear text - likely a typo, should be 57.5 ± 4.3%] indicate that it can effectively control bacterial infection. Meanwhile, the red blood cell count was [unclear text - likely a typo, should be 11.7 ± 2.2 × 10⁹ / L]. 12 The levels of hemoglobin (98.6±8.6 g / L) and hematocrit (0.39±0.05 L / L) significantly increased, indicating improved nutritional status and oxygen-carrying capacity. Hematocrit (0.39±0.05 L / L) returned to normal levels, reflecting effective correction of dehydration symptoms. All six core indicators were close to normal and showed highly significant differences compared to the antibiotic group (P<0.01), confirming that the L100@H / SaL nanoparticle formulation has a definite comprehensive therapeutic effect in controlling infection, improving the body's condition, and correcting dehydration, and is significantly superior to the antibiotic group.

[0083] Table 4. Statistical table of core blood routine indicators of goats in each group (4) Serum biochemical index test results: The results of serum biochemical index monitoring are shown in Table 5. The serum biochemical indexes of the blank control group were all within the normal range (ALT 38.5±5.4 U / L, AST 71.0±8.5 U / L, Cr 82.0±6.5 μmol / L, BUN 6.1±1.2 mmol / L). On day 5 of the experiment, the levels of ALT (47.7±6.2 U / L), AST (112.0±10.8 U / L), Cr (129.0±8.4 μmol / L), and BUN (8.8±1.5 mmol / L) in the antibiotic group were significantly elevated. In the L100@H / SaL group, all indicators (ALT 41.2±5.5 U / L, AST 76.0±9.2 U / L, Cr 88.0±8.3 μmol / L, BUN 7.1±1.2 mmol / L) were significantly lower than those in the antibiotic group (P<0.05), and were close to the normal levels of the blank control group. Therefore, the L100@H / SaL group showed significantly better protective effects against liver and kidney damage in goats than the antibiotic group, and demonstrated higher safety.

[0084] Table 5. Statistical table of serum biochemical indicators of goats in each group. Experimental Example 3: Clinical efficacy trial of L100@H / SaL against Escherichia coli-induced diarrhea in chicks.

[0085] This study aims to verify the efficacy and safety of pH-responsive hollow mesoporous polydopamine-loaded rhodioloside nanoparticles (L100@H / SaL) in treating Escherichia coli-induced diarrhea in chicks, providing experimental evidence for its veterinary clinical application.

[0086] 1. Test Procedure: Animal grouping and administration: Ninety 14-day-old yellow-feathered broiler chickens that had spontaneously developed the disease and were confirmed to be infected with *E. coli* by bacterial isolation and identification were randomly divided into three groups of 30 chickens each. There were no significant differences in body weight or disease severity among the groups (P>0.05). The control group consisted of 30 healthy yellow-feathered broiler chickens that were neither infected nor treated; the drug control group consisted of infected chicks that received ciprofloxacin orally at the standard clinical dose once daily for 5 days; and the nano-preparation group consisted of infected chicks that received L100@H / SaL nano-preparation orally once daily for 5 days. The experiment lasted 10 days, with uniform feeding and management and consistent environmental conditions throughout.

[0087] Sampling and observation indicators: The mental state, diarrhea severity, feed intake, and mortality of chicks were observed daily. Ten chicks were randomly selected from each group before the experiment, on day 5 of treatment, and after the experiment. Wing vein blood was collected (for complete blood count and serum biochemical indicators), and fresh feces were collected (for E. coli count). Observation indicators included: clinical symptoms (diarrhea rate, mortality rate, cure rate), complete blood count indicators (WBC, absolute neutrophil count), serum biochemical indicators (ALT, AST, Cr, BUN), and fecal E. coli count.

[0088] 2. Evaluation of treatment effectiveness: (1) Comprehensive evaluation of therapeutic effect: Table 6 shows that during the experiment, the 30 healthy chicks in the blank control group showed no diarrhea or mortality. In the western medicine control group, after treatment, the cure rate of the 30 infected chicks was 64.5±8.3%, with 30.0±8.2% still exhibiting diarrhea symptoms, and the mortality rate was 6.8±5.2%. The L100@H / SaL group showed significantly better treatment results than the western medicine control group (P<0.01), with a cure rate of 91.0±6.2%, a diarrhea rate reduced to 6.8±5.3%, and a mortality rate of 0.0±0.0%. All efficacy indicators were close to those of the blank control group. In conclusion, the L100@H / SaL nano-formulation can significantly improve the treatment effect of E. coli-induced diarrhea in chicks, reduce the diarrhea rate and mortality rate, and its efficacy is superior to conventional ciprofloxacin treatment.

[0089] Table 6. Statistical table of core data on the efficacy of treatment for diarrhea in chicks of each group. (2) Effect of L100@H / SaL on the number of Escherichia coli in the feces of infected chicks in each group: Table 7 shows that there was no significant difference in the number of *E. coli* in the feces of infected chicks among the groups before the experiment (P>0.05), while the ciprofloxacin group and the L100@H / SaL group were significantly higher than the blank control group. On day 5 of treatment and at the end of the experiment, the number of *E. coli* in the feces of infected chicks decreased in both groups, but the decrease in the L100@H / SaL group was significantly greater than that in the western medicine control group (P<0.01), dropping to 3.5 × 10⁻⁶ on day 5 of treatment. 3 ±0.4×10 3 CFU / g decreased to 1.4 × 10⁻⁶ after the experiment. 3 ±0.5×10³ CFU / g, close to the level of the blank control group (approximately 1.2×10³ CFU / g). 3 (CFU / g), while the control group treated with Western medicine had a level of 4.9 × 10 on day 5. 5 ±0.6×10 5 The CFU / g level remained at 5.6 × 10⁻⁶ after the experiment. 4 ±1.1×10 4 The L100@H / SaL nanoformulation showed a higher level of CFU / g. In summary, the L100@H / SaL nanoformulation exhibited a more significant and longer-lasting effect in inhibiting the proliferation of Escherichia coli in chick feces, and its antibacterial ability was superior to that of conventional ciprofloxacin.

[0090] Table 7. Count of E. coli in feces of chicks in each group (CFU / g) (3) Effects of L100@H / SaL on routine blood parameters: The effects of L100@H / SaL on blood routine indicators are shown in Table 8. In the blank control group, all indicators were within the normal range (total white blood cell count 7.6 ± 1.2 × 10⁻⁶). 9 / L, absolute neutrophil count 3.6±0.8×10 9 / L, neutrophil percentage 46.3±4.6%, red blood cell count 3.8±0.7×10 12 / L, hemoglobin 106.3±8.5 g / L, hematocrit 0.34±0.02 L / L. On day 5 of treatment in the ciprofloxacin group, the total white blood cell count was (11.7±1.8×10) 9 / L) and neutrophils (6.8±1.4×10 9 The blood count (57.9 ± 4.5%) was still significantly elevated, indicating that the bacterial infection was not effectively controlled; the red blood cell count (3.1 ± 0.6 × 10⁹ / L, 57.9 ± 4.5%) was also significantly elevated, suggesting that the bacterial infection was not effectively controlled; 12 The levels of hemoglobin (87.3±7.1 g / L) and hematocrit (0.24±0.07 L / L) were significantly lower in the nanoparticle group, reflecting nutritional depletion, decreased oxygen-carrying capacity, and uncorrected dehydration caused by diarrhea. In contrast, the levels of all indicators in the nanoparticle group on day 5 of treatment were significantly lower (total white blood cell count 8.7±1.4×10⁻⁶). 9 / L, absolute neutrophil count 4.2±0.8×10 9 / L, neutrophil percentage 46.4±4.1%, red blood cell count 3.6±0.2×10 12 The levels of hemoglobin (103.5±7.7 g / L) and hematocrit (0.31±0.05 L / L) were significantly better than those in the ciprofloxacin group (P<0.01), and were close to the normal levels of the blank control group. In conclusion, the L100@H / SaL nanoparticle formulation effectively controlled the inflammatory response induced by E. coli infection in chicks, significantly improved the body's infection, nutritional, and dehydration status, and its overall efficacy was superior to conventional ciprofloxacin treatment.

[0091] Table 8. Statistical Table of Core Blood Routine Indicators in Each Group of Chicks (4) Effects of L100@H / SaL on liver and kidney function in experimental chicks: As shown in Table 9, in the blank control group, the serum ALT (36.7±5.4 U / L), AST (73.2±7.8 U / L), Cr (81.3±7.8 μmol / L), and BUN (6.1±1.0 mmol / L) levels of chicks were all within the normal range. On day 5 of treatment in the ciprofloxacin group, all four indicators (ALT 47.5±6.6 U / L, AST 108.3±10.5 U / L, Cr 123.2±8.7 μmol / L, and BUN 8.9±1.3 mmol / L) were significantly elevated, indicating liver and kidney damage. On day 5 of treatment, the L100@H / SaL group showed significantly lower levels of four indicators (ALT 39.1±5.1 U / L, AST 73.8±9.1 U / L, Cr 82.5±8.2 μmol / L, BUN 6.9±1.6 mmol / L) compared to the ciprofloxacin group (P<0.05), and were close to the normal levels of the blank control group. In conclusion, the L100@H / SaL nanoparticle formulation effectively protects liver and kidney function in chicks while treating E. coli-induced diarrhea, demonstrating significantly better safety than conventional ciprofloxacin.

[0092] Table 9. Statistical table of serum biochemical indicators of chicks in each group. In summary, the pH-responsive hollow mesoporous polydopamine-loaded rhodioloside nanoformulation (L100@H / SaL) provided by this invention effectively solves the technical challenges of traditional drugs, such as low drug loading efficiency, poor targeting, insufficient safety, and narrow therapeutic spectrum, through structural innovation, targeted modification, and efficacy optimization. This formulation significantly improves the bioavailability and therapeutic effect of rhodioloside, demonstrating significant advantages in the treatment of ulcerative colitis and various bacterial diarrheas in animals, and possesses important patent protection value and broad industrialization potential.

[0093] The nanoparticles prepared in this invention have a hydrated particle size of 311.8 ± 5.96 nm, a drug loading of 45.27%, and an encapsulation efficiency of 82.71%. In vitro release experiments showed that the release rate of this formulation was less than 10% in simulated gastric fluid (pH 1.2) after 2 hours, and the cumulative release rate was over 85% in simulated intestinal fluid (pH 7.4) after 24 hours. Animal experiments demonstrated that this formulation significantly improved weight loss, hematochezia, colon shortening, and intestinal barrier function impairment in mice with ulcerative colitis; it also effectively treated E. coli-induced diarrhea in chicks and goats, significantly improving the cure rate, reducing diarrhea and mortality rates, effectively inhibiting the proliferation of E. coli in feces, improving inflammatory states and liver and kidney function, and exhibiting superior safety compared to conventional antibiotics. This invention utilizes a hollow mesoporous structure to achieve high drug loading and combines pH-responsive coating to achieve intestinal targeted delivery, which can effectively improve the bioavailability and therapeutic effect of rhodioloside, and can be used to prevent or treat ulcerative colitis, Escherichia coli diarrhea in chicks and Escherichia coli diarrhea in goats.

[0094] The nanoparticles prepared in this invention have a hydrated particle size of 311.8 ± 5.96 nm, a drug loading of 45.27%, and an encapsulation efficiency of 82.71%. In vitro release experiments showed that the release rate of this formulation was less than 10% in simulated gastric juice (pH 1.2) after 2 hours, and the cumulative release rate was over 85% in simulated intestinal juice (pH 7.4) after 24 hours. Animal experiments showed that this formulation significantly improved weight loss, rectal bleeding, colonic shortening, and intestinal barrier function impairment in mice with ulcerative colitis. Although embodiments of the present invention have been shown and described above, it is understood that these embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A pH-responsive hollow mesoporous polydopamine-supported rhodioloside nanoparticle formulation, characterized in that, The nano-formulation mainly consists of a hollow mesoporous polydopamine carrier, rhodioloside loaded on the hollow mesoporous polydopamine carrier, and a pH-responsive enteric-coated material coating the outer layer of the hollow mesoporous polydopamine carrier loaded with rhodioloside. The mass ratio of the hollow mesoporous polydopamine carrier to the rhodioloside is 1:1 to 1:

3. The total mass ratio of the hollow mesoporous polydopamine carrier and the rhodioloside to the pH-responsive enteric material is 1:0.5 to 1:

2.

2. The pH-responsive hollow mesoporous polydopamine-supported rhodioloside nanoformulation according to claim 1, characterized in that, The drug loading of the nano-formulation is 40-50%, and the encapsulation rate is 75-85%.

3. The pH-responsive hollow mesoporous polydopamine-supported rhodioloside nanoformulation according to claim 1, characterized in that, The hydrated particle size of the nano-formulation is 250~350 nm, and the zeta potential is -20 mV to -10 mV.

4. The pH-responsive hollow mesoporous polydopamine-supported rhodioloside nanoformulation according to any one of claims 1 to 3, characterized in that, The hollow mesoporous polydopamine carrier was prepared by a soft template method. The particle size of the hollow mesoporous polydopamine carrier was 200-400 nm, and the specific surface area was 50-200 m². 2 / g, pore volume 0.2~0.8 cm³ 3 / g; The pH-responsive enteric material includes at least one of Eudragit L100, Eudragit S100, Eudragit L30D-55, and Eudragit L100-55.

5. A method for preparing pH-responsive hollow mesoporous polydopamine-supported rhodioloside nanoparticles according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Preparation of hollow mesoporous polydopamine carrier: Hollow mesoporous polydopamine carrier was prepared by soft template method; (2) Loading of rhodioloside: The hollow mesoporous polydopamine nanocarrier and rhodioloside were dispersed in ultrapure water, mixed evenly, and the free rhodioloside was removed to obtain hollow mesoporous polydopamine loaded with rhodioloside. (3) pH-responsive coating: The hollow mesoporous polydopamine loaded with rhodioloside is mixed evenly with the pH-responsive enteric material, and the precipitate is collected to obtain a pH-responsive hollow mesoporous polydopamine loaded with rhodioloside nanoformulation.

6. The method for preparing the pH-responsive hollow mesoporous polydopamine-supported rhodioloside nanoparticles according to claim 5, characterized in that, Step (1) Preparation of hollow mesoporous polydopamine carrier includes the following specific steps: dissolve polyether F127 and trimethylbenzene TMB in a mixture of anhydrous ethanol and ultrapure water, sonicate to form a milky white liquid, add Tris buffer and dopamine hydrochloride, mix evenly, collect the precipitate, and finally wash and freeze dry to obtain hollow mesoporous polydopamine carrier.

7. The method for preparing the pH-responsive hollow mesoporous polydopamine-supported rhodioloside nanoparticles according to claim 6, characterized in that, The mass ratio of the polyether F127 to the trimethylbenzene TMB is 2:1 to 5:

1.

8. The use of the pH-responsive hollow mesoporous polydopamine-loaded rhodioloside nanoformulation according to any one of claims 1 to 4 in the preparation of a drug for treating ulcerative colitis.

9. The use of the pH-responsive hollow mesoporous polydopamine-loaded rhodioloside nanoformulation according to any one of claims 1 to 4 in the preparation of a drug for treating Escherichia coli-induced diarrhea in livestock and poultry.

10. The application according to claim 9, characterized in that, The livestock and poultry include at least one of chickens, goats, sheep, deer, pigs, and ducks.

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