A ginsenoside Rg3-based colon-targeting lipid nanoparticle, a calcium alginate microsphere thereof, and application thereof in treating ulcerative colitis

Abstract

The application is a colon-targeting lipid nanoparticle based on ginsenoside Rg3, a calcium alginate microsphere thereof and application thereof in treating ulcerative colitis, and belongs to the technical field of new excipients and new dosage forms of pharmaceutical preparations, and particularly relates to an oral colon-targeting delivery system for treating ulcerative colitis. The system replaces cholesterol in traditional lipid nanoparticles with ginsenoside Rg3, constructs lipid nanoparticles capable of efficiently loading TNF-alpha siRNA, and effectively improves the targeting property and lysosome escape ability of the lipid nanoparticles on inflammatory macrophages. In order to further realize colon-specific delivery, the above-mentioned lipid nanoparticles are loaded in a pH-responsive calcium alginate microsphere, so that the drug is kept stable in the upper gastrointestinal tract and is released in a responsive manner in the colon. Pharmacodynamic studies show that the drug delivery system can significantly relieve the symptoms of colitis, effectively silence the expression of TNF-alpha, repair the intestinal barrier, and regulate the intestinal microbiota. The application provides a safe and efficient new strategy for solving the problems of poor gastrointestinal stability and low targeting efficiency of oral delivery of nucleic acid drugs.

Description

Technical Field

[0001] This invention belongs to the technical field of new pharmaceutical excipients and dosage forms, specifically relating to a novel micro / nano formulation for colon-targeted delivery of siRNA. This invention achieves highly efficient targeting of macrophages at inflammatory sites and lysosomal escape by constructing siRNA lipid nanoparticles using ginsenoside Rg3 instead of traditional cholesterol; and utilizes pH-responsive calcium alginate microspheres for encapsulation, achieving colon-specific delivery and effective release of siTNF-α to ulcerative colitis lesions. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease with complex etiologies, characterized by persistent inflammation of the colonic mucosa, clinically manifested as diarrhea, abdominal pain, and rectal bleeding. Its core pathogenesis lies in the excessive activation of immune cells, particularly the massive secretion of inflammatory factors such as tumor necrosis factor-α (TNF-α) by macrophages, which is a key factor driving the occurrence and development of inflammation in UC. Therefore, silencing TNF-α is considered an effective strategy for the precise treatment of UC.

[0003] Small interfering RNA (siRNA) can specifically silence pathogenic genes at the mRNA level through RNA interference (RNAi) mechanisms. For example, silencing TNF-α siRNA (TNF-α siRNA) can theoretically effectively inhibit inflammatory responses, providing a new strategy for the precision treatment of ulcerative colitis (UC). However, siRNA drugs themselves have disadvantages such as strong hydrophilicity, negative charge, and easy degradation by nucleases, making it difficult to penetrate cell membranes to exert their effects. In addition, oral administration, as the most ideal route for treating local intestinal diseases, faces challenges from multiple physiological barriers, including gastric acid, digestive enzymes, complex mucus layers, and intestinal emptying.

[0004] Lipid nanoparticles (LNPs) are among the most mature nucleic acid drug delivery carriers, effectively protecting siRNA and promoting its cellular uptake. Traditional LNPs typically contain a large amount of cholesterol (20%-50%) to maintain the stability and fluidity of the lipid bilayer. However, the presence of cholesterol can trigger adverse reactions such as complement activation-related pseudohypersensitivity (CARPA); more importantly, the Niemann-Pick C1 (NPC1) protein on the lysosomal membrane recognizes and binds to cholesterol, leading to the large-scale extracellular expulsion of cholesterol-containing LNPs, significantly reducing the lysosomal escape efficiency of siRNA and resulting in poor gene silencing effects. Simultaneously, traditional LNPs lack active targeting to macrophages at inflamed sites and are ill-equipped to withstand the complex gastrointestinal environment. To address the problems of low lysosomal escape efficiency, poor targeting, and physiological barriers encountered in oral delivery of nucleic acid drugs, the inventors propose a solution: a novel oral siRNA delivery system that overcomes the gastrointestinal barrier, possesses active targeting capability to inflamed macrophages, achieves efficient lysosomal escape, and exhibits higher biocompatibility, thereby advancing nucleic acid drug therapy for ulcerative colitis. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of traditional LNPs in existing technologies, such as adverse reactions due to the use of cholesterol, low lysosomal escape efficiency, lack of targeting, and difficulties in oral delivery. It also addresses the gastrointestinal barrier problem encountered in the oral delivery of siRNA drugs. This invention provides a novel lipid nanoparticle that replaces cholesterol with ginsenoside Rg3. This nanoparticle significantly improves targeting to inflammatory macrophages and lysosomal escape efficiency. Furthermore, this invention provides a pH-responsive calcium alginate (CA) microsphere encapsulating this lipid nanoparticle, enabling oral colon-targeted delivery of siRNA, and ultimately providing a safe and efficient UC treatment strategy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a colon-targeting lipid nanoparticle (LNP) based on ginsenoside Rg3, comprising ionizable cationic lipids, neutral phospholipids, ginsenoside Rg3, PEG-modified lipids, and siRNA.

[0007] Preferably, the ginsenoside Rg3 is used as a membrane stabilizer to replace cholesterol.

[0008] Preferably, the cationic lipid is DOTAP (1,2-dioleoyl-3-trimethylammonium-propane). The neutral phospholipid is DSPC (distearate phosphatidylcholine). The PEG-modified lipid is DMG-PEG2000 (dimyristicoglycerol-polyethylene glycol 2000). The siRNA is a TNF-α silencing siRNA (si@TNF-α).

[0009] Preferably, the lipid nanoparticles are prepared by ethanol injection, wherein the charge ratio (N / P ratio) of the cationic lipid to siRNA is 2:1.

[0010] In a second aspect, the present invention provides a calcium alginate microsphere, which is a calcium alginate microsphere (CA@Rg3@siTNF-α) encapsulating the above-mentioned colon-targeting lipid nanoparticles based on ginsenoside Rg3.

[0011] Preferably, the sodium alginate microspheres comprise the aforementioned colon-targeting lipid nanoparticle (LNP) core and a pH-responsive calcium alginate shell. The calcium alginate microspheres exhibit pH-responsive properties, remaining stable in simulated gastric juice (SGF) at pH 1.2 and simulated small intestinal juice (SIF) at pH 6.8, with a drug release rate of less than 10%. They rapidly disintegrate or swell in simulated colonic juice (SCF) at pH 7.4, quickly releasing the LNP core.

[0012] Preferably, the calcium alginate microspheres and the lipid nanoparticles described above are prepared by an ion crosslinking method: ginsenoside Rg3 lipid nanoparticles are mixed with an aqueous solution of sodium alginate (SA), and then added dropwise to an aqueous solution of calcium chloride (CaCl2) via an injection pump to solidify and form microspheres.

[0013] Preferably, the sodium alginate aqueous solution has a mass-volume concentration of 1-5%, and the calcium chloride aqueous solution has a mass-volume concentration of 1-5%.

[0014] More preferably, the sodium alginate aqueous solution has a mass-volume concentration of 2%, and the calcium chloride aqueous solution has a mass-volume concentration of 2%.

[0015] The unit of mass is gram (g), and the unit of volume is milliliter (mL).

[0016] In a third aspect, the present invention also provides the application of the above-mentioned colon-targeting lipid nanoparticles based on ginsenoside Rg3 and the above-mentioned calcium alginate microspheres in the preparation of drugs for treating intestinal diseases.

[0017] Preferably, the colon-targeting nanoparticles or the calcium alginate microspheres are used for oral administration.

[0018] Alternatively, the drug may be used for ulcerative colitis or Crohn's disease.

[0019] Preferably, the ginsenoside Rg3 in the LNP core serves as a substrate for glucose transporter 1 (GLUT1), enabling active targeted delivery to inflammatory macrophages at the ulcerative colitis lesion site.

[0020] Preferably, the drug exerts its therapeutic effect by improving the lysosomal escape efficiency of the LNP core, regulating the gut microbiota, inhibiting the expression of pro-inflammatory factors, and repairing the intestinal barrier.

[0021] Compared with the prior art, the present invention has the following beneficial effects: Lipid nanoparticles containing ginsenoside Rg3 can target glucose transporter 1 (GLUT1) overexpressed on inflammatory macrophages, significantly improving the cellular uptake efficiency of ginsenoside LNPs. Ginsenoside Rg3 lacks a cholesterol structure and cannot be recognized by the NPC1 protein on the lysosomal membrane, avoiding NPC1-mediated LNP efflux and allowing more ginsenoside LNPs to remain intracellularly. In vitro cell experiments showed that Rg3@siCy5 exhibited superior lysosomal escape performance compared to Chol@siCy5. Replacing cholesterol with ginsenoside Rg3 avoids potential adverse reactions such as CARPA caused by cholesterol. Within the therapeutic concentration range, ginsenoside LNPs showed no significant cytotoxicity. Calcium alginate microspheres containing ginsenoside Rg3 lipid nanoparticles achieved specific drug release in the colon through pH-responsive characteristics. The CA@Rg3@siTNF-α delivery system provided by this invention can not only effectively inhibit inflammation by silencing the TNF-α gene (manifested as a decrease in the levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β, and an increase in the level of anti-inflammatory factor TGF-β1), but also restore intestinal barrier function (increase the expression of tight junction protein ZO-1 and reduce intestinal permeability) and regulate the intestinal flora to normalize (maintain the abundance of Bacteroidetes and Firmicutes and improve flora diversity), thus synergistically treating UC through multiple pathways. Attached Figure Description

[0022] Figure 1 The particle size distribution and transmission electron microscopy (TEM) image of ginsenoside lipid nanoparticles Rg3@siCy5 in Example 1 of this invention are shown.

[0023] Figure 2 This is a gel electrophoresis image of ginsenoside lipid nanoparticles Rg3@siCy5 in Example 2 of the present invention under different N / P conditions.

[0024] Figure 3 This is a diagram showing the in vitro release of Rg3@siCy5 in lipase and PBS solution in Example 2 of the present invention.

[0025] Figure 4This is a cellular uptake diagram of conventional Chol@siCy5 and Rg3@siCy5 in RAW264.7 cells in Example 3 of the present invention; ns: P ≥ 0.05. P<0.05 P<0.01 (all two-tailed t-tests).

[0026] Figure 5 This is a cell uptake diagram of Chol@siCy5 and Rg3@siCy5 in a normal DMEM culture medium environment and a high glucose environment in Example 3 of the present invention; ns: P ≥ 0.05. P<0.05 P < 0.0001 (all two-tailed t-tests).

[0027] Figure 6 This is a Pearson colocation coefficient plot of Chol@siCy5 and Rg3@siCy5 in Embodiment 3 of the present invention; ns: P ≥ 0.05. P<0.05 P<0.01 P<0.001 (all two-tailed t-tests).

[0028] Figure 7 Pearson colocation coefficients of Chol@siCy5 and Rg3@siCy5 in pure PBS solution and PBS solution containing ezetimibe in Example 3 of this invention; ns: P ≥ 0.05 P<0.05 P<0.01 (all two-tailed t-tests).

[0029] Figure 8 The morphology characterization of CA@Rg3@siCy5 in Embodiment 4 of the present invention is shown in CLSM bright field image and scanning electron microscope image.

[0030] Figure 9 The particle size distribution and potential diagram of CA@Rg3@siCy5 provided in Embodiment 4 of the present invention.

[0031] Figure 10 The in vitro release curve of CA@Rg3@siCy5 in simulated gastrointestinal fluid (SGF, SIF, SCF) provided in Embodiment 4 of the present invention.

[0032] Figure 11Figure A shows the tissue distribution of CA@Rg3@siCy5 provided in Example 5 of this invention in C57BL / 6 colitis model mice. Figure A is a fluorescence imaging map of the distribution of each group of preparations in live mice, and Figure B is a quantitative fluorescence map; ns: P ≥ 0.05. P<0.05 P<0.01 P < 0.0001 (all two-tailed t-tests).

[0033] Figure 12 Figure A shows the tissue distribution of CA@Rg3@siCy5 provided in Example 5 of this invention in C57BL / 6 colitis model mice. Figure A is a fluorescence imaging map of the distribution of each group of preparations in isolated tissues, and Figure B is a quantitative fluorescence map; ns: P ≥ 0.05. P<0.05 P<0.01 P<0.001 P < 0.0001 (all two-tailed t-tests).

[0034] Figure 13 This image shows the pharmacodynamic experiment of CA@Rg3@siCy5 in C57BL / 6 colitis model mice provided in Example 6 of this invention. Wherein, A: weight changes in mice of different treatment groups; B: spleen changes in mice of different treatment groups; C: colon length in mice of different treatment groups; D: DAI score in mice of different treatment groups; E: colon images in mice of different treatment groups; F: spleen images in mice of different treatment groups; ns: P ≥ 0.05. P<0.05 P<0.01 P<0.001 P < 0.0001 (all two-tailed t-tests).

[0035] Figure 14The levels of serum colitis-related factors in the therapeutic effect of CA@Rg3@siCy5 on C57BL / 6 colitis model mice provided in Example 6 of this invention are shown in the figure. Wherein, A: transforming growth factor-β1 level in mouse serum; B: tumor necrosis factor-α level in mouse serum; C: interleukin-1-β level in mouse serum; D: interleukin-6 level in mouse serum; E: myeloperoxidase level in mouse serum; ns: P ≥ 0.05. P<0.05 P<0.01 P<0.001 P < 0.0001 (all two-tailed t-tests).

[0036] Figure 15 This invention relates to the effect of CA@Rg3@siCy5 provided in Example 6 on intestinal permeability in C57BL / 6 colitis model mice. Wherein, A: FITC fluorescence intensity detected in intestinal tissue of mice in different treatment groups; B: fluorescence intensity of ZO-1 protein detected in intestinal tissue of mice in different treatment groups after staining; ns: P ≥ 0.05. P<0.05 P<0.01 P<0.001 P < 0.0001 (all two-tailed t-tests).

[0037] Figure 16 This is a HE slice of the colon of a C57BL / 6 colitis model mouse provided in Example 6 of the present invention, using CA@Rg3@siCy5.

[0038] Figure 17 In the efficacy experiment of CA@Rg3@siCy5 against C57BL / 6 colitis provided in Example 7 of this invention, the liver and kidney function indicators of mice in different treatment groups were analyzed. Wherein A: white blood cell level in mice in different treatment groups; B: alanine aminotransferase (ALT) level in the serum of mice in different treatment groups; C: aspartate aminotransferase (AST) level in the serum of mice in different treatment groups; D: blood urea nitrogen (BUN) level in the serum of mice in different treatment groups; E: creatinine (CRE) level in the serum of mice in different treatment groups; ns: P ≥ 0.05. P<0.05 P<0.01 P<0.001 (all two-tailed t-tests).

[0039] Figure 18 This invention relates to the effect of CA@Rg3@siCy5 provided in Example 7 on the α-diversity of the gut microbiota in C57BL / 6 colitis model mice. Where A: Observed species; B: Chao 1 index; C: Shannon index; D: Simpson index; ns: P ≥ 0.05. P<0.05 P<0.01 P<0.001 (all two-tailed t-tests).

[0040] Figure 19 Principal component analysis (PCA) diagram of the intestinal flora of C57BL / 6 colitis model mice provided in Example 7 of this invention using CA@Rg3@siCy5.

[0041] Figure 20 The effect of CA@Rg3@siCy5 provided in Example 7 of this invention on the species composition of the intestinal flora in C57BL / 6 colitis model mice. Detailed Implementation

[0042] The following specific examples further illustrate the above-mentioned content of the present invention. These embodiments are only used to illustrate the present invention and do not constitute a limitation on the scope of the present invention.

[0043] Example 1: Preparation and characterization of ginsenoside Rg3 lipid nanoparticles (Rg3@siTNF-α) Preparation: An ethanol injection method was used. DOTAP, DSPC, ginsenoside Rg3, and DMG-PEG2000 were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 (organic phase). TNF-α siRNA was dissolved in DEPC-treated water (aqueous phase). Under vortexing conditions, the organic phase was rapidly injected into the aqueous phase (volume ratio 1:3), and vortexing was continued for 30 minutes. Afterward, it was incubated at 37°C for 20 minutes, and finally, the ethanol was removed by centrifugation (4000 rpm, 5 min) using a 100 kDa ultrafiltration centrifuge tube, yielding Rg3@siTNF-α. Rg3@siCy5 loaded with Cy5-labeled siRNA was prepared using the same method for tracking, as well as a control formulation Chol@siCy5 using cholesterol instead of ginsenoside Rg3.

[0044] Characterization: As shown in Table 1, Malvern particle size analyzer measurements revealed that the particle size of Rg3@siCy5 was 155.0 ± 4.158 nm, the PDI was 0.229 ± 0.028, the Zeta potential was +23.4 ± 0.794 mV, and the encapsulation efficiency was as high as 94.17%. Transmission electron microscopy (TEM) Figure 1 B) It shows that it has a regular spherical shape.

[0045] Table 1: Particle size, potential and encapsulation efficiency of Rg3@siCy5 Example 2: Screening for the optimal N / P ratio of Rg3@siTNF-α and in vitro release assay Prepare a 2% agarose gel by dissolving 2.0 g of agarose in 50 × TAE (2 mL) electrophoresis buffer and heating in a microwave oven until melted. After slight cooling, add 10 μL of GoldView dye to the agarose gel solution, mix well, and then cast the gel. Mix 10 μL of Rg3@siTNF-α with 1 μL of 10 × Loading Buffer, load the sample, connect the positive and negative electrodes, and run at 110 V for 30 min. Observe the siRNA bands using a spectrophotometer. Figure 2 As shown, the optimal encapsulation ratio can be achieved when N / P is 2:1.

[0046] An in vitro lipase-simulated environment was used to investigate the in vitro release behavior of LNPs, compared with a PBS (pH 7.4) environment. 10 μL of RNase was added to 1 mL of Rg3@siTNF-α and incubated at 37 ℃ on a shaker (150 rpm). Samples were taken at predetermined time points (0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h), and 5 times the volume of methanol solution was added for demulsification. Figure 3The release amount and rate of Rg3@siTNF-α under LPS conditions were much higher than those under pH 7.4 PBS conditions, indicating that lipase helps release LNPs, improves drug release efficiency, and has better safety.

[0047] Example 3: In vitro cell experiments The mouse macrophage RAW264.7 was used as a model.

[0048] Cellular uptake differences: at 6 × 10 5 RAW264.7 cell suspension at a density of 10 cells / mL was evenly seeded into 12-well plates and cultured overnight in a cell culture incubator to allow for cell adhesion. After adhesion, the original culture medium was discarded, and Chol@siCy5 and Rg3@siCy5 (Cy5 siRNA concentration of 100 nM) diluted with serum-free medium was added. The plates were then incubated again in a cell culture incubator, and the plates were removed at set time points (0.5 h, 2 h, and 4 h), and the drug-containing culture medium was discarded. Preheated PBS (pH 7.4) was added to stop uptake. The cells were washed three times with cold PBS (pH 7.4), digested with trypsin, centrifuged (1000 rpm, 3 min), and redispersed with PBS (pH 7.4). The cell suspension was collected after filtration and analyzed by flow cytometry. Flow cytometry results ( Figure 4 This indicates that the cellular uptake efficiency of Rg3@siCy5 is significantly higher than that of Chol@siCy5, and its uptake can be competitively inhibited by a high glucose environment. Figure 5 This confirms the GLUT1-mediated targeted uptake mechanism.

[0049] Lysosomal escape: The lysosomal escape ability of LNPs was investigated using CLSM. Lysosomes were labeled with LysoTracker Green and cell nuclei were labeled with Hoechst. Chol@siCy5 and Rg3@siCy5 were located using Cy5 markers. RAW264.7 cells were divided into groups of 6 × 10⁻⁶ cells. 5The cells were evenly seeded at a density of 100 cells / mL into confocal culture dishes and transferred to a cell culture incubator for overnight culture to allow them to adhere and grow. The original culture medium was discarded, and Chol@siCy5 and Rg3@siCy5 diluted with serum-free medium were added to each well to achieve a siRNA Cy5 concentration of 100 nM per well. The dishes were then incubated again in a cell culture incubator. At set time points (4 h, 6 h), the confocal dishes were removed, the drug-containing culture medium was discarded, and PBS (pH 7.4) was added to stop uptake. The dishes were washed three times with cold PBS (pH 7.4), and incubated with LysoTracker Green for 30 min. The LysoTracker Green was discarded, and the dishes were washed three times with cold PBS (pH 7.4). Hoechst was added and incubated for 15 min. The Hoechst was discarded, and the dishes were washed three times with PBS (pH 7.4). The Pearson colocalization coefficient was calculated based on CLSM colocalization. Figure 6 The colocalization coefficient of the Rg3@siCy5 group was significantly lower than that of the Chol@siCy5 group, indicating that it had a stronger lysosomal escape ability. Figure 7 After using the NPC1 inhibitor ezetimibe, the escape ability of Chol@siCy5 was improved, while Rg3@siCy5 showed no significant change, confirming that its escape advantage stemmed from avoiding NPC1 recognition.

[0050] Example 4: Preparation and characterization of drug-loaded calcium alginate microspheres (CA@Rg3@siTNF-α) Preparation: Prepare a 2% (w / v) sodium alginate (SA) aqueous solution. Mix the Rg3@siTNF-α prepared in Example 1 with an equal volume of 2% SA solution, vortex until homogeneous, to obtain a 1% SA LNPs mixture. Add this mixture dropwise to a 2% (w / v) CaCl2 aqueous solution using a syringe pump (flow rate 1800 μL / min). + Crosslinking with sodium alginate to form gel microspheres. Washing three times with deionized water to remove excess ions yields CA@Rg3@siTNF-α.

[0051] Characterization and formulation optimization: CA@Rg3@siTNF-α was shown to be spherical under laser confocal microscopy (CLSM) and scanning electron microscopy (SEM). Figure 8 ), uniform particle size distribution, and decreased potential ( Figure 9 The release behavior of microspheres prepared with different SA concentrations (1%, 2%, and 3%) in simulated gastric juice (SGF), simulated intestinal juice (SIF), and simulated colonic juice (SCF) was compared. Figure 10The optimal concentration of SA was determined to be 2%, which releases very little (<10%) in SGF / SIF, but rapidly releases LNPs in SCF, demonstrating excellent colon-targeted drug release characteristics.

[0052] Example 5: In vivo tissue distribution study The DSS-induced C57BL / 6 female colitis mouse model was used.

[0053] Establishment of colitis model in mice: Mice were fed purified water containing 3% DSS (w / v), ensuring they received a fresh 3% DSS solution daily and had access to it continuously. After 6 days of continuous feeding, DSS solution feeding was discontinued, and the mice were replaced with DSS-free water. The mice's activity, weight changes, fecal characteristics (e.g., mucus, bloody stool), and fecal occult blood were recorded.

[0054] In vivo imaging: Chol@siCy5, Rg3@siCy5, CA@Chol@siCy5, and CA@Rg3@siCy5 were administered orally, respectively. Observation using a small animal in vivo imaging system showed ( Figure 11 The fluorescence signal of the LNPs group without microspheres was significantly reduced after 12 hours, while the two groups with CA microspheres were still able to detect strong fluorescence signals in the abdomen (colon region) after 24 hours.

[0055] Distribution of tissues in vitro: Mice were sacrificed at different time points after drug administration (2 h, 6 h, and 48 h after drug administration), and the heart, liver, spleen, lung, kidney, stomach, small intestine, and large intestine were removed. After the surface blood was absorbed with filter paper, in vitro imaging observation was performed. Figure 12 The results showed that the fluorescence intensity of CA@Rg3@siCy5 and CA@Chol@siCy5 in the colon was much higher than that in other groups, and their distribution in other organs (heart, liver, spleen, lung, kidney) was extremely low, demonstrating the excellent colon-targeting and protective effects of calcium alginate microspheres.

[0056] Example 6: In vivo pharmacodynamic evaluation Prophylactic administration: Administration began on day 1 of DSS modeling. Mice in the 5-ASA group received 5-ASA (200 mg / kg / day) orally via gavage daily. The Rg3@siTNF-α treatment group received Rg3@siTNF-α lipid nanoparticles (TNF-α siRNA dose of 200 μg / kg) orally via gavage daily. The CA@Rg3@siTNF-α group received drug-loaded calcium alginate microspheres (TNF-α siRNA dose of 200 μg / kg) orally via gavage daily. The CA@Chol@siTNF-α group received drug-loaded calcium alginate microspheres (TNF-α siRNA dose of 200 μg / kg) orally via gavage daily. The CA@Rg3@siScr group received drug-loaded calcium alginate microspheres (Scramble siRNA dose of 200 μg / kg) orally via gavage daily. The drug was administered at a dose of μg / kg until day 7. Both the normal control group and the model group were given the same volume of physiological saline. During the experiment, the mice were weighed daily, and their living conditions and fecal characteristics were observed. On day 7 of modeling, the mice were fed purified water without DSS, and drug administration was stopped. On day 8, the mice's eyes were removed for blood collection, and the C57BL / 6 mice were then euthanized by cervical dislocation. The abdominal cavity was quickly opened, and the intestinal segment from the terminal cecum to the anus was separated. The length of the colon was measured, and the volume and weight of the spleen were measured and weighed to help determine the degree of relief of colitis. The colon was washed with sterile physiological saline to remove feces, secretions, and blood. A 2 cm section of colon tissue from the most inflammatory distal colon was fixed in 4% paraformaldehyde, stained with H&E, and then subjected to histopathological observation. Blood was collected from the eyeballs of mice and placed in a negative pressure coagulation collection tube. The blood was centrifuged at 4000 rpm for 10 min. The mouse serum was then collected and the levels of colitis-related inflammatory factors (IL-1β, TNF-α, IL-6, TGF-β1) and myeloperoxidase (MPO) activity in the mouse serum were measured using an ELICA kit.

[0057] During the experiment, the control group mice had shiny black fur, were active and responsive, and had normal fecal color and consistency. The model group mice exhibited bloody stools and occult blood, lethargy, and rough fur. Figure 13 As shown, during the experiment, the body weight of the control group mice remained relatively stable with a slight upward trend. Mice given the modeling agent experienced a continuous decrease in body weight over the first four days. Compared to the model group, the CA@Rg3@siTNF-α treatment group most effectively alleviated the weight loss in mice. Figure 13 A), improves colon shortening ( Figure 13 C) and splenomegaly ( Figure 13 B), in addition, such as Figure 13According to the DAI scores of mice plotted by D, CA@Rg3@siTNF-α can significantly reduce the disease activity index of mice and improve the disease status of mice better than the 5-ASA group; no significant improvement was observed in the Rg3@siTNF-α treatment group, and the condition of mice was almost the same as that of the model group.

[0058] Anti-inflammatory and antioxidant effects: Serum from each group of mice was collected, and the levels of inflammatory factors in the colitis were detected by ELISA to examine the degree of inflammation remission. Figure 14 The CA@Rg3@siTNF-α group significantly upregulated the expression level of the anti-inflammatory cytokine TGF-β1 in the serum of colitis mice, and downregulated the expression levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β. While the other treatment groups also alleviated the expression of inflammatory factors to varying degrees, these effects were significantly different from those observed in the CA@Rg3@siTNF-α group. Furthermore, the CA@Rg3@siTNF-α group significantly inhibited myeloperoxidase activity.

[0059] Intestinal barrier repair: FITC-Dextran refers to a fluorescein isothiocyanate-modified dextran. As a fluorescent probe, FITC-Dextran is widely used to assess in vitro and in vivo cell permeability, colitis permeability, and other studies, depending on the molecular weight of the dextran. Figure 15 As shown in Figure A, the green fluorescence intensity detected in the intestines of control group mice was only about 1 µg / mL, demonstrating the integrity of the normal mouse intestines. However, the fluorescence signal detected in the model group was close to 4 µg / mL, indicating that the intestinal barrier in DSS-induced colitis mice was completely disrupted, leading to increased permeability and elevated serum FITC-Dextran concentration. Compared with other treatment groups, the CA@Rg3@siTNF-α treatment group significantly reduced the level of FITC-Dextran in mouse serum, greatly alleviating the increased intestinal permeability caused by colitis, indicating that CA@Rg3@siTNF-α can effectively inhibit the disruption of the intestinal tight junction barrier. Furthermore, tight junction protein ZO-1 is a core component for maintaining intestinal barrier integrity; its downregulation significantly affects intestinal permeability, increasing the likelihood of toxins entering the bloodstream via the intestines, thereby triggering systemic infection. Figure 15 B. In the control group, the red fluorescence signal of ZO-1 in the colonic tissue was significantly stronger than that in the model group. Both the 5-ASA group and the CA@Chol@siTNF-α group increased ZO-1 expression to some extent. Compared with other drug-treated groups, oral administration of CA@Rg3@siTNF-α can increase the expression level of ZO-1 in the colonic tissue of mice with high DSS-induced colitis and improve the intestinal barrier damage in colitis mice.

[0060] like Figure 16As shown, compared with the normal group of mice, the model control group showed the disappearance of crypts and goblet cells. Pathological sections of the 5-ASA, Rg3@siTNF-α, CA@Chol@siTNF-α, CA@Rg3@siScr, and CA@Chol@siScr control groups showed varying degrees of crypt loss, goblet cell loss, and inflammatory cell infiltration. Sections of the CA@Rg3@siTNF-α treatment group showed crypts and goblet cells, but no obvious inflammatory cell infiltration.

[0061] Example 7: Safety and Gut Microbiota Analysis Starting from day 7 of the DSS-induced colitis model, mice were administered 5-ASA (200 mg / kg / day) orally via gavage daily. The Rg3@siTNF-α treatment group received Rg3@siTNF-α lipid nanoparticles (TNF-α siRNA dose of 200 μg / kg) orally via gavage daily. The CA@Rg3@siTNF-α group received drug-loaded calcium alginate microspheres (TNF-α siRNA dose of 200 μg / kg) orally via gavage daily. The CA@Chol@siTNF-α group received drug-loaded calcium alginate microspheres (TNF-α siRNA dose of 200 μg / kg) orally via gavage daily. The CA@Rg3@siScr group received drug-loaded calcium alginate microspheres (Scramble siRNA dose of 200 μg / kg) orally via gavage daily. The CA@Chol@siScr group received drug-loaded calcium alginate microspheres (Scramble siRNA dose of 200 μg / kg) orally via gavage daily. The mice were administered the drug (μg / kg) for 6 consecutive days. The control and model groups received no drug treatment. On day 7 of modeling, the mice were fed DSS-free purified water and the drug was administered for 6 consecutive days. After this period, the mice's eyeballs were removed and blood was collected. C57BL / 6 female mice were then euthanized by cervical dislocation, and biological samples from the spleen and colon were collected. Blood from the mouse eyeballs was placed in a negative pressure coagulation collection tube and centrifuged at 4000 rpm for 10 min. A separate sample was placed in a heparin blood collection tube for Seville sampling to measure blood routine and assess liver and kidney safety. The colon and rectum of the mice were removed using a sterile scalpel. The contents of the desired intestinal segments were cut and separated, collected in sterile EP tubes, and stored at -80°C. The samples were then sent to Shanghai Ling'en Biotechnology Co., Ltd. on dry ice for analysis via 16S rRNA sequencing. α-diversity analysis, composition analysis, core common species analysis, and LEfSe analysis were performed.

[0062] like Figure 17As shown in Figure A, the white blood cell levels in the model group and the Rg3@siTNF-α group mice were significantly elevated and exceeded the normal range. However, CA@Rg3@siTNF-α reduced the white blood cell levels in mice to the normal range, and the reduction was more significant compared to 5-ASA and CA@Chol@siTNF-α. Therefore, CA@Rg3@siTNF-α can effectively inhibit the inflammatory state of acute colitis.

[0063] Safety Assessment: The liver and kidneys are the main organs for drug metabolism and excretion, and their functional indicators (such as serum ALT, AST, BUN, etc.) reflect drug-induced liver and kidney damage. For example, elevated ALT and AST levels indicate hepatocellular damage, while abnormal CRE and BUN levels reflect renal dysfunction. Serum samples from mice with colitis were collected and analyzed using ELISA to measure liver and kidney-related indicators in the serum of these mice to assess drug safety.

[0064] The results are as follows Figure 17 B- Figure 17 D. Serum liver function (ALT, AST) and kidney function (BUN, CREA) in mice from both the control and treatment groups were within safe limits and showed no significant differences. This indicates that none of the treatment groups caused significant liver or kidney damage in mice during the experiment, further confirming the in vivo safety of CA@Rg3@siTNF-α of this invention. Furthermore, the progression of colitis can lead to an increase in white blood cell levels in the blood. Gut microbiota analysis: 16S rRNA sequencing was performed on mouse colon contents. α-diversity analysis ( Figure 18 The results showed that CA@Rg3@siTNF-α significantly restored the decline in observed species (Chao1 index) caused by colitis. Principal component analysis (PCA) Figure 19 This indicates that the gut microbiota structure of mice treated with CA@Rg3@siTNF-α was more similar to that of the healthy control group. For example... Figure 20 As shown in the left figure, at the phylum level, CA@Rg3@siTNF-α can effectively regulate the Bacteroidetes phylum ( Bacteroidota ) and Firmicutes ( Firmicutes The proportion of ( ) should be adjusted to bring it closer to normal. For example... Figure 20 As shown in the right figure, at the scientific level, the model group norank_f_Muribaculaceae The bacterial flora decreased significantly, but after treatment with CA@Rg3@siTNF-α and CA@Chol@siTNF-α, the abundance of this flora was restored, significantly regulating the intestinal flora of mice and thus improving ulcerative colitis in mice.

Claims

1. A colon-targeting lipid nanoparticle based on ginsenoside Rg3, characterized in that, The lipid nanoparticles (LNPs) are composed of the following components: a) ionizable cationic lipids; b) neutral phospholipids; c) ginsenoside Rg3; d) polyethylene glycol (PEG) lipids; and e) small interfering RNA (siRNA), wherein the ginsenoside Rg3 is a membrane stabilizer.

2. The colon-targeting lipid nanoparticles according to claim 1, characterized in that, The cationic lipid is DOTAP; the neutral phospholipid is DSPC; and the PEG-modified lipid is DMG-PEG2000.

3. The colon-targeting lipid nanoparticles according to claim 1, characterized in that, The LNP was prepared by ethanol injection.

4. A calcium alginate microsphere, characterized in that, The sodium alginate microspheres comprise a colon-targeting lipid nanoparticle (LNP) core as described in any one of claims 1 to 3 and a pH-responsive calcium alginate shell. The calcium alginate microspheres exhibit pH-responsive properties, remaining stable in simulated gastric juice (SGF) at pH 1.2 and simulated small intestinal juice (SIF) at pH 6.8, with a drug release rate of less than 10%. They rapidly disintegrate or swell in simulated colonic juice (SCF) at pH 7.4, quickly releasing the LNP core.

5. The calcium alginate microspheres according to claim 4, characterized in that, The calcium alginate microspheres are prepared by an ion crosslinking method, specifically by mixing the lipid nanoparticles with an aqueous solution of sodium alginate, and then adding the mixture dropwise into an aqueous solution of calcium chloride to solidify and form microspheres.

6. The calcium alginate microspheres according to claim 5, characterized in that, The sodium alginate aqueous solution has a mass-volume concentration of 1-5%, and the calcium chloride aqueous solution has a mass-volume concentration of 1-5%.

7. The use of the colon-targeting lipid nanoparticles according to any one of claims 1 to 3 or the calcium alginate microspheres according to any one of claims 4 to 6 in the preparation of a medicament for treating intestinal diseases, characterized in that, Preferably, the intestinal disease is ulcerative colitis or Crohn's disease.

8. The application according to claim 7, characterized in that, The ginsenoside Rg3 in the LNP core acts as a substrate for glucose transporter 1 (GLUT1), enabling active targeted delivery to inflammatory macrophages at the lesion site of ulcerative colitis.

9. The application according to claim 7, characterized in that, The colon-targeting nanoparticles or the calcium alginate microspheres are for oral administration.

10. The application according to claim 7, characterized in that, The drug exerts its therapeutic effect by improving the lysosomal escape efficiency of the LNP core, regulating the gut microbiota, inhibiting the expression of pro-inflammatory factors, and repairing the intestinal barrier.

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