Ginsenoside-loaded dual-network cross-linked microspheres as well as preparation method and application thereof

Abstract

The invention discloses a ginsenoside-loaded dual-network cross-linked microsphere as well as a preparation method and application thereof, and belongs to the technical field of oral drug delivery and biomedical microsphere materials. The microspheres are composed of a first network formed by polysaccharide-multivalent cation crosslinking and a second network containing dynamic disulfide bonds formed by lipoic acid under illumination, and an interpenetrating double-network structure is formed. The preparation method comprises the following steps: mixing polysaccharide, lipoic acid, a photoinitiator and ginsenoside to form a precursor solution, carrying out multivalent cation crosslinking to form a sphere, and then carrying out light treatment to obtain the bi-crosslinked microspheres. The method is mild in process and simple and convenient to operate. The microspheres can reduce early release of drugs in the stomach environment, improve the adhesion and retention ability of the drugs in intestinal inflammation parts, show responsive release characteristics in inflammation-related redox abnormity environments, and are suitable for oral delivery of inflammatory bowel diseases and other intestinal inflammation-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of oral drug delivery and biomedical microsphere materials technology, specifically relating to a dual-network cross-linked microsphere loaded with ginsenosides, its preparation method, and its applications. The microspheres consist of a first network formed by polysaccharide-polyvalent cationic cross-linking and a dynamic disulfide network formed by lipoic acid (LA) under light irradiation, forming an interpenetrating double network (IPN) structure. The microspheres can be loaded with ginsenosides, and the preparation method forms a controllable drug release system. The microspheres are suitable for inflammatory microenvironment-responsive delivery, preferably for enhanced adhesion and adaptive controlled release at sites of intestinal inflammation. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of diseases characterized by chronic, relapsing inflammation of the intestines, primarily including ulcerative colitis and Crohn's disease. These diseases are often accompanied by damage to the intestinal mucosal barrier, increased local oxidative stress, and abnormal expression of inflammatory factors. Current treatments mostly involve oral or systemic administration, but the local accumulation efficiency of drugs at the site of inflamed intestinal lesions is limited, and some drugs are prone to premature release or degradation during gastrointestinal transport, thus affecting treatment efficacy. Therefore, developing drug delivery systems that can achieve stable delivery and local action at intestinal inflammation sites after oral administration is of great significance.

[0003] Ginsenosides (such as Rg3, Rb1, Rd and their metabolites / total saponins) are important pharmacologically active components of ginseng, possessing various pharmacological activities including anti-inflammatory, immunomodulatory, and antioxidant effects, and showing promising application prospects in the intervention of intestinal inflammation-related diseases (Phytomedicine, 2024, 130, 155476). However, direct oral administration of ginsenosides still suffers from insufficient stability in the gastrointestinal environment, limited local retention time, and insufficient effective exposure to lesion sites, thus limiting their full efficacy.

[0004] Currently, oral intestinal delivery carriers mainly include ionically cross-linked polysaccharide microspheres, hydrogel particles, pH-responsive carriers, and adhesive materials. Among them, carboxyl-containing polysaccharides such as alginate and Ca2+... 2+ Ion-crosslinked microspheres formed by isovalent cations are widely used in the construction of oral delivery systems due to their mild preparation conditions, good biocompatibility, and low cost. However, single ion-crosslinked networks usually have problems such as relatively loose structure, easy drug leakage and premature release in the gastric segment, limited ability to regulate release behavior, and lack of inflammatory microenvironment response, making it difficult to simultaneously meet the requirements of gastric segment protection, intestinal segment delivery, and on-demand release to the lesion site.

[0005] Furthermore, effective treatment of IBD lesions depends not only on drug release behavior but also on the local retention capacity of the carrier on the intestinal wall and mucus layer surface. The intestinal mucus layer is mainly composed of mucins, and its structure significantly affects particle adhesion, migration, and clearance. Existing adhesion strategies mostly rely on weak interactions such as electrostatic interactions and hydrogen bonding. Under conditions of intestinal fluid flushing, intestinal peristalsis, and continuous mucus renewal, the carrier is prone to detachment from the lesion surface, making stable retention difficult. Previous studies have shown that materials containing thiol groups or disulfide bond exchange capacity can enhance the interaction with mucins, thereby improving adhesion strength and retention capacity (Advanced drug delivery reviews, 2005, 57(11): 1569-1582). Therefore, introducing a dynamic disulfide bond-related adhesion mechanism into oral IBD delivery systems is expected to improve the problem of easy carrier flushing and clearance, and enhance its local retention effect at intestinal inflammatory sites.

[0006] Alpha-lipoic acid (LA), a small molecule containing cyclic disulfide bonds, possesses both antioxidant activity and disulfide dynamic exchange potential, and has been widely used in recent years to construct dynamic materials or drug carrier systems. However, existing LA-related technologies mainly focus on hydrogels, material adhesion, or dynamic material construction (CN120960169A, CN113061263A, and CN121379411A), with applications primarily in engineering materials or in vitro medical materials. There are still relatively few technical solutions that incorporate the polymerization / crosslinking capabilities of LA into oral ion-crosslinked polysaccharide microsphere systems and further utilize them for the oral delivery of ginsenosides to local intestinal inflammation, simultaneously achieving enhanced adhesion and functional synergy.

[0007] Therefore, there is an urgent need to develop a novel delivery system that combines gastric protection, intestinal release, local adhesion retention, and inflammatory microenvironment response capabilities to improve the local delivery efficiency and application effect of ginsenosides at sites of intestinal inflammation. Summary of the Invention

[0008] To address the problems of insufficient local retention, limited release regulation, and lack of inflammatory microenvironment response mechanisms in the oral delivery of ginsenosides for the treatment of inflammatory bowel disease (IBD) in existing technologies, the present invention aims to provide a structurally stable oral microsphere delivery system for ginsenosides that has dynamic adhesion capabilities and can achieve adaptive controlled release in an inflammatory microenvironment.

[0009] Specifically, the present invention aims to construct a multifunctional microsphere structure that is stable in the stomach, has basic sustained-release capability in the intestine, and achieves accelerated release and enhanced local retention in the inflammatory area through microenvironment triggering, thereby improving the local exposure and therapeutic effect of ginsenosides at the site of intestinal inflammation.

[0010] To achieve the above objectives, the present invention provides an interpenetrating double-network cross-linked microsphere, characterized in that the microsphere comprises:

[0011] First network: an ionic cross-linked network formed by alginate and / or ionically cross-linkable carboxylated polysaccharides and multivalent cations;

[0012] Second network: a polymeric cross-linked network containing dynamic disulfide bonds formed by lipoic acid under light conditions;

[0013] The first network and the second network form an interpenetrating double network (IPN) structure in space. The first network is formed by cross-linking polysaccharides that can form ionogel structures with multivalent cations.

[0014] Preferably, the alginate is selected from one or more of sodium alginate, potassium alginate, and ammonium alginate.

[0015] Preferably, the carboxyl-containing polysaccharide is selected from one or more of pectin, hyaluronic acid, and carboxymethyl cellulose.

[0016] Preferably, the multivalent cation is selected from Ca. 2+ Ba 2+ 、Sr 2+ Zn 2+ One or more of the following, preferably Ca 2+ .

[0017] Preferably, the ionic crosslinking is achieved through exogenous diffusion crosslinking or endogenous release crosslinking.

[0018] The first network is used to provide microsphere forming capability and structural stability, and helps to reduce premature drug release in the gastric environment.

[0019] The second network is formed by the polymerization and / or cross-linking reaction of thioctic acid under the action of light and photoinitiator.

[0020] Preferably, the mass fraction of thioctic acid in the microsphere precursor system is 0.1–5 wt%, more preferably 0.5–3 wt%, and even more preferably 1–3 wt%.

[0021] Preferably, the photoinitiator is selected from one or more of LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphonate) and Irgacure 2959 (2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropionyl phenyl ketone); wherein, LAP is preferred.

[0022] Preferably, the photoinitiator has a mass fraction of 0.01–0.5 wt% in the microsphere precursor system, more preferably 0.05–0.2 wt%, and even more preferably 0.05–0.1 wt%.

[0023] Preferably, the illumination wavelength is 320-400 nm.

[0024] Preferably, the intensity of the ultraviolet light irradiation is 10–50 mW / cm². 2 More preferably 10-30 mW / cm 2 .

[0025] Preferably, the ultraviolet light irradiation time is 60–600 s, more preferably 120–600 s.

[0026] Preferably, the ultraviolet light irradiation is performed when the microspheres are in a wet or water-dispersed state.

[0027] Preferably, the second network includes a dynamic structure that can participate in disulfide bond exchange, thereby enhancing the interaction between the microspheres and the intestinal mucus layer and improving their local retention capacity at the lesion site.

[0028] Preferably, under an inflammatory-related redox abnormality environment, the second network undergoes dynamic rearrangement, thereby causing changes in the network structure and / or pore structure, promoting drug release.

[0029] Preferably, the microspheres maintain a basal sustained-release state in normal intestinal segments and exhibit a responsive release characteristic with an increased release rate in inflamed areas.

[0030] Drug components: Ginsenosides dispersed, encapsulated, or dissolved in the above-mentioned dual network structure.

[0031] Preferably, the ginsenoside can be one or more of Rb1, Rd, Rg1, and Rg3, but is not limited to the types listed above.

[0032] Preferably, the ginsenosides in the precursor solution have a mass fraction of 0.01–2 wt%, more preferably 0.05–0.5 wt%.

[0033] Preferably, for ginsenosides with low solubility, they can be introduced into the precursor solution using cyclodextrin inclusion complexes and / or pharmaceutically acceptable solubilizing forms.

[0034] Preferably, the microspheres have a particle size of 10–2000 μm, more preferably 50–1000 μm.

[0035] This invention also provides a double-crosslinked microsphere for oral delivery of ginsenosides, its preparation method, and its application, characterized by comprising the following two crosslinking methods and process steps:

[0036] (A) Combination of exogenous diffused ion crosslinking and ultraviolet secondary crosslinking

[0037] (1) Preparation of a multivalent cation crosslinking bath

[0038] Weigh anhydrous calcium chloride (CaCl2) to prepare a CaCl2 aqueous solution with a mass fraction of 0.5–5 wt% (preferably 1–3 wt%), add a magnetic stir bar, and stir at a speed of 200–600 rpm for later use. The polyvalent cation may also be selected from Ba... 2+ 、Sr 2+ Zn 2+ One or more of the following; the corresponding salt solution concentration can be 0.1–3 wt%.

[0039] (2) Preparation of polysaccharide solution (first network precursor solution)

[0040] Sodium alginate (SA) is weighed and dissolved in deionized water or PBS to prepare an SA solution with a mass fraction of 0.5–5 wt% (preferably 1–3 wt%), and stirred at room temperature until clear and free of visible particles. The polysaccharide may be alginate and / or carboxyl-containing polysaccharide, selected from one or more of potassium alginate, ammonium alginate, pectin, hyaluronic acid, and sodium carboxymethyl cellulose; the total polysaccharide concentration is maintained at 0.5–5 wt%.

[0041] (3) Preparation of ginsenoside solubilizing drug-carrying phase

[0042] Weigh out ginsenosides (such as Rb1, Rg3, Rd, or total saponins) and mix them with hydroxypropyl-β-cyclodextrin (HP-β-CD) at a mass ratio of 1:(2–20), preferably 1:(5–15), more preferably 1:10; add deionized water and stir for 0.5–4 h to form an inclusion solution; or use ultrasonic dispersion for 5–30 min to obtain a clear / uniformly dispersed saponin inclusion solution. Alternatively, the saponins can be directly dispersed / dissolved in the polysaccharide solution of step 2; or a pharmaceutically acceptable solubilizing system can be used, neither of which will affect subsequent spheroidization.

[0043] (4) Add thioctic acid and photoinitiator to prepare a double crosslinking precursor solution.

[0044] Lipoic acid (LA) is added to the polysaccharide solution obtained in step 2, such that the mass fraction of LA in the precursor solution is 0.1–5 wt% (preferably 0.5–3 wt%). A photoinitiator, preferably LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphonate) or Irgacure 2959, is added, such that the mass fraction of LA in the precursor solution is 0.01–0.5 wt% (preferably 0.05–0.2 wt%). The saponin inclusion solution (or saponin dispersion) obtained in step 3 is added to the above mixture, such that the mass fraction of saponin in the precursor solution is 0.01–2 wt% (preferably 0.05–0.5 wt%). The mixture is stirred for 10–60 min until homogeneous; if necessary, it is sonicated for 1–10 min to remove agglomerates; thus, a bicrosslinked microsphere precursor solution is obtained. To reduce oxygen inhibition, the precursor solution can be degassed under vacuum for 5–20 min or allowed to stand for 10–60 min.

[0045] (5) Drop / spray droplets to form spheres and complete the first cross-linking (ionic cross-linking network).

[0046] The precursor solution obtained in step 4 is added to the CaCl2 crosslinking bath in step 1 by dripping. The dripping method can be a syringe needle, a burette, or a peristaltic pump. The needle specification can be 18–30 G. The dripping height is 1–10 cm. The dripping speed is 0.1–5 mL / min. After dripping, continue stirring and crosslinking for 3–30 min (preferably 5–15 min) to form ionic crosslinked microspheres, thus obtaining the "first crosslinked microsphere".

[0047] (6) Washing removes free ions and unreacted small molecules

[0048] Collect the microspheres obtained in step 5 using a filter or centrifugation (e.g., 500–2000 rpm, 1–5 min), and wash them 1–5 times (preferably 3 times) with deionized water or PBS to obtain wet ion-crosslinked microspheres.

[0049] (7) Secondary cross-linking under ultraviolet light to form LA dynamic disulfide network (second cross-linking network).

[0050] Spread the wet microspheres obtained in step 6 evenly in a quartz dish or UV-transparent container, add a small amount of deionized water to keep them moist (to prevent drying and cracking), and irradiate them under a UV light source: wavelength 320–400 nm (preferably 365 nm); light intensity 10–50 mW / cm². 2 Irradiation time: 60–600 s; after irradiation, dual-network (IPN) microspheres were obtained.

[0051] (8) Post-processing and storage

[0052] The resulting double-crosslinked microspheres can be directly dispersed in PBS in a wet state for later use; or they can be lyophilized / vacuum dried to obtain dried microspheres (lyophilization may involve adding protective agents such as trehalose / mannitol, with a mass fraction of 1–10 wt%) to improve storage stability.

[0053] (B) Combination of endogenous ion crosslinking and ultraviolet secondary crosslinking

[0054] (9) Preparation of endogenous crosslinking precursor solution

[0055] Prepare a precursor solution containing polysaccharide, LA, photoinitiator and saponin according to steps 2-4;

[0056] Additionally, insoluble calcium salts are added as endogenous calcium. 2+ The source (e.g., pharmaceutically acceptable calcium salts such as calcium carbonate or calcium phosphate) is added in an amount of 0.1–10 wt% (preferably 0.5–5 wt%) of the polysaccharide mass, and stirred until homogeneous.

[0057] (10) Spheroidization and endogenous crosslinking

[0058] Add the precursor solution from step 9 dropwise into the aqueous phase containing a mild acid release / complexation regulation system (to promote the slow release of calcium salt Ca). 2+ (and achieve more uniform cross-linking), forming microspheres and maintaining them for 5–60 min to complete the endogenous ion cross-linking; then wash according to step 6.

[0059] (11) Ultraviolet secondary crosslinking

[0060] Follow step 7 to perform ultraviolet irradiation to obtain interpenetrating double network microspheres.

[0061] The beneficial effects of this invention are:

[0062] (1) Enhanced structural stability: The double cross-linked network improves the resistance to swelling and ion exchange;

[0063] (2) Reduce explosive release: Ion network and dynamic network jointly regulate pore structure;

[0064] (3) Enhanced adhesion: The dynamic disulfide bond structure helps to enhance the interaction between microspheres and the intestinal mucus layer, and improve local retention capacity;

[0065] (4) Inflammation-adaptive release: The release rate increases in an inflammation-related environment;

[0066] (5) Simple process: No complex reducing agents or high-temperature melting are required; it can be completed simply by conventional ion crosslinking and ultraviolet light treatment. Detailed Implementation

[0067] The technical solutions adopted in this invention will be further explained and described below with reference to specific embodiments.

[0068] Example 1

[0069] This embodiment provides a method for preparing thioctic acid photocrosslinked double crosslinked calcium alginate microspheres.

[0070] Weigh 2.0 g of anhydrous calcium chloride, dissolve it in deionized water, and bring the volume to 100 mL to prepare a 2 wt% CaCl2 solution. Stir magnetically at 300 rpm at room temperature for later use. Separately weigh 2.0 g of sodium alginate and add it to 100 mL of deionized water to prepare a 2 wt% sodium alginate solution. Stir magnetically at room temperature for 4 h until completely dissolved, and allow to stand for 30 min to remove bubbles.

[0071] The drug loading test was conducted using Rb1 as a representative ginsenoside. 0.1 g of ginsenoside Rb1 and 1.0 g of hydroxypropyl-β-cyclodextrin were weighed and mixed, then added to 10 mL of deionized water and stirred at room temperature for 2 h to form an inclusion solution. Subsequently, 1.0 g of lipoic acid was added to the sodium alginate solution to achieve a mass fraction of 1 wt% in the precursor solution; 0.1 g of photoinitiator LAP was added to achieve a mass fraction of 0.1 wt% in the precursor solution; then the above ginsenoside inclusion solution was added, and the mixture was stirred for 30 min and sonicated for 5 min to remove air bubbles, resulting in a homogeneous precursor solution.

[0072] The precursor solution was loaded into a 10 mL syringe (22G needle) and added dropwise to a CaCl2 solution at a rate of 1 mL / min, to a height of approximately 5 cm. The solution was stirred continuously for 10 min to form ionicly cross-linked microspheres. The microspheres were collected, centrifuged at 1000 rpm for 2 min, and washed three times with deionized water.

[0073] Wet microspheres were uniformly spread in a quartz dish and incubated under 365 nm ultraviolet light at a concentration of 20 mW / cm². 2 Irradiation for 180 s forms a second cross-linked network, yielding double-cross-linked microspheres. If necessary, 5 wt% trehalose can be added as a protective agent. After pre-freezing at -80℃ for 4 h, the microspheres are freeze-dried under vacuum for 48 h to obtain dried microspheres.

[0074] Example 2

[0075] This embodiment investigates the effect of the amount of thioctic acid added on the preparation of double-crosslinked microspheres.

[0076] Except for the amount of lipoic acid added, the other conditions were the same as in Example 1. The mass fraction of lipoic acid in the precursor solution was set to 0.5 wt%, 1 wt%, 3 wt%, and 5 wt%, respectively. The microspheres were prepared by forming, washing, and UV irradiation according to the steps described in Example 1, and thus obtained double cross-linked microspheres with different lipoic acid contents.

[0077] Example 3

[0078] This embodiment is used to expand the scope of the polysaccharide system in the first cross-linked network.

[0079] In Example 1, sodium alginate was replaced with a mixture of sodium alginate and hyaluronic acid at a mass ratio of 8:2, with a total polysaccharide concentration of 2 wt%; or a mixture of sodium alginate and sodium carboxymethyl cellulose at a mass ratio of 7:3, with a total polysaccharide concentration of 2 wt%. The remaining steps were the same as in Example 1, and the corresponding double-crosslinked microspheres were prepared.

[0080] Example 4

[0081] This embodiment is used to expand the range of types of polyvalent cations.

[0082] The CaCl2 solution in Example 1 was replaced with 2 wt% BaCl2 solution or 1 wt% ZnCl2 solution, while the other steps remained unchanged. The microspheres were then prepared by dropping, washing, and UV irradiation.

[0083] Example 5

[0084] This embodiment is used to evaluate the in vitro release behavior of double-crosslinked microspheres and their responsive release characteristics under inflammatory simulation conditions.

[0085] Equal amounts of drug-loaded single-network microspheres (SA MSs), drug-loaded double-crosslinked microspheres (SA / LA MSs), and drug-loaded double-crosslinked microspheres under oxidative stress conditions were placed in release media containing simulated gastric fluid (SGF, pH 1.2) and incubated with shaking at 37°C and 100 rpm. The simulated gastric fluid release phase lasted for 2 h, and samples were taken at preset time points to determine the drug release amount. After 2 h, the microspheres of each group were collected by centrifugation and lightly washed with an appropriate amount of buffer solution, and then transferred to simulated intestinal fluid conditions for further release. Among them, the control group and the example group were placed in pH 6.8 PBS; the inflammation simulation group was placed in pH 6.8 PBS containing H2O2 to simulate an inflammation-related oxidative stress environment. The samples of each group continued to be released at 37°C and 100 rpm for 24 h, and samples were taken for analysis at preset time points. The total volume of the release media was 10 mL, and the volume of each sample was 1 mL, with an equal volume of fresh corresponding media added simultaneously to maintain a constant total volume of the release system. Sampling time points included 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, and 24 hours. After centrifugation or filtration, the drug concentration in the release medium was determined by ultraviolet spectrophotometry, and the cumulative release percentage at each time point was calculated according to the cumulative release rate formula.

[0086] Example 6

[0087] This embodiment is used to evaluate the in vitro adhesion and retention ability of double cross-linked microspheres.

[0088] Mucin was prepared as a 1.0 wt% aqueous solution and coated onto the substrate surface to form a mucin coating at 37°C. Then, an equal amount of microspheres were placed on the surface of the mucin coating and incubated in pH 6.8 PBS at 37°C for 60 min. After incubation, the microspheres were gently rinsed three times with PBS, and the proportion of residual microspheres was recorded to evaluate the adhesion and retention capacity of different microspheres in a simulated intestinal mucus environment.

[0089] Example 7

[0090] This embodiment illustrates an implementation method for further introducing a photocrosslinkable precursor into a dual-crosslinked microsphere system.

[0091] Based on Example 1, PEGDA was further added to the precursor solution to achieve a mass fraction of 0.5 wt%, while the remaining components and their amounts remained unchanged. After adding PEGDA, the mixture was stirred until homogeneous, and then the steps described in Example 1 were followed to drop-form microspheres, wash, and treat with ultraviolet light to prepare PEGDA-containing double-crosslinked microspheres. Attached image description:

[0092] Figure 1 Morphological characterization of double cross-linked microspheres (SA / LA MSs) by SEM

[0093] Figure 2 Comparison of cumulative release curves of different microspheres under simulated gastric and intestinal fluid conditions

[0094] Figure 3 Experiments on the simulated adhesion enhancement of different microspheres in vitro

[0095] Figure 4 Free radical scavenging properties of different microspheres

[0096] Figure 5 The alleviating effects of different microspheres on LPS-induced cellular oxidative stress

[0097] Effect Verification Instructions

[0098] The morphology of the double-crosslinked microspheres (SA / LA MSs) obtained in the examples was observed using SEM, and the results are as follows: Figure 1As shown in the figure, the results indicate that the obtained microspheres can form micron-sized particles, with an overall spherical or near-spherical shape, demonstrating that the precursor system can successfully form stable microspheres after cross-linking with multivalent cation ions and subsequent light treatment. The microsphere surface has certain wrinkles and rough structures, suggesting that its surface has a large specific surface area and rich interfacial morphology features. No large-area collapse, obvious breakage, or fusion into blocks was observed in the figure, indicating that the dual-crosslinked system has good sphericity and structure retention capabilities. These results verify that the technical solution of this invention can successfully construct dual-crosslinked microsphere carriers.

[0099] The drug release performance of the microspheres was evaluated using a two-stage release experiment simulating gastric and intestinal fluids. The results are as follows: Figure 2 As shown, single-network microspheres (SA MSs) released the drug more rapidly in the early stage, while the cumulative release of double-crosslinked microspheres (SA / LA MSs) was lower in the simulated gastric fluid stage (2h), indicating that the introduction of the second crosslinking network can effectively inhibit premature drug release. After being transferred to simulated intestinal fluid, SA / LA MSs continued to release the drug, demonstrating good intestinal segment drug release capability. Furthermore, under oxidative stress conditions containing H2O2, the release rate of SA / LA MSs was higher than that under ordinary simulated intestinal fluid conditions, indicating that this double-crosslinked system has certain inflammatory microenvironment response drug release characteristics. The above results verify the application potential of the double-crosslinked microspheres of the present invention in oral intestinal local delivery.

[0100] The adhesion and retention capacity of different microspheres was evaluated using a mucin model, and the results are as follows: Figure 3 As shown, the residue rate of single-network microspheres (SA MSs) was approximately 20%, while the residue rate of double-crosslinked microspheres (SA / LA MSs) increased to approximately 40%, significantly higher than that of single-network microspheres. This result indicates that the introduction of a dynamic disulfide network of lipoic acid enhances the interfacial interaction between the microspheres and the mucus layer, thereby improving the microspheres' resistance to flushing and retention in a simulated intestinal mucus environment. These results demonstrate that the double-crosslinked microspheres constructed in this invention exhibit better adhesion and retention performance in the intestinal mucus layer environment, which is beneficial for prolonging the local residence time of the carrier at the lesion site, providing a basis for improving local delivery efficiency.

[0101] The antioxidant capacity of different microsphere systems was evaluated using a free radical scavenging experiment, and the results are as follows: Figure 4 As shown, the free radical scavenging efficiency of single-network microspheres (SA MSs) was relatively low. After introducing a dynamic lipoic acid network, the free radical scavenging efficiency of SA / LA MSs significantly increased to approximately 60%. Further loading with ginsenoside Rb1 further enhanced the free radical scavenging efficiency of SA / LA / Rb1 MSs to over 80%. These results indicate that the dynamic disulfide network of lipoic acid itself can endow microspheres with strong free radical scavenging ability; based on this, loading with ginseng active ingredients further enhances the antioxidant performance of the microsphere system.

[0102] Intracellular ROS levels were detected using a reactive oxygen species (ROS) fluorescent probe method, and the results are as follows: Figure 5 As shown in the figure, compared with the control group, the model group cells showed a significantly enhanced green fluorescence signal, indicating a significant increase in intracellular reactive oxygen species levels and a state of oxidative stress. After treatment with different microspheres, the intensity of intracellular green fluorescence decreased to varying degrees. The decrease was limited in the SA MSs group, while the decrease was more significant in the SA / LA MSs group. The fluorescence signal in the SA / LA / Rb1 MSs group was the weakest, closest to the level of the normal control group.

Claims

1. A dual-network cross-linked microsphere loaded with ginsenosides, characterized in that, The microspheres comprise: First network: an ionic cross-linked network formed by alginate and / or ionically cross-linkable carboxylated polysaccharides and multivalent cations; Second network: a polymeric cross-linked network containing dynamic disulfide bonds formed by lipoic acid under light conditions; Drug components: Ginsenosides dispersed, encapsulated, or dissolved in the above-mentioned double network structure; The first network and the second network form an interpenetrating dual network (IPN) structure in space.

2. The dual-network cross-linked microspheres according to claim 1, characterized in that, The alginate is selected from one or more of sodium alginate, potassium alginate, and ammonium alginate; the carboxyl-containing polysaccharide is selected from one or more of pectin, hyaluronic acid, and carboxymethyl cellulose; the polyvalent cation is selected from Ca... 2+ Ba 2+ 、Sr 2+ Zn 2+ One or more of them.

3. The dual-network cross-linked microspheres according to claim 1, characterized in that, The second crosslinking network is formed by photo-induced polymerization and / or crosslinking reaction of thioctic acid; the light wavelength is 320–400 nm; the microsphere precursor system contains a photoinitiator selected from LAP, Irgacure 2959, and combinations thereof; the mass fraction of thioctic acid in the microsphere precursor system is 0.1–5 wt%, the mass fraction of the photoinitiator in the microsphere precursor system is 0.01–0.5 wt%, the light intensity is 10–50 mW / cm², the irradiation time is 60–600 s, and the irradiation is carried out when the microspheres are in a wet or aqueous dispersion state.

4. The dual-network cross-linked microspheres according to claim 1, characterized in that, The second cross-linking network contains a dynamic disulfide bond structure, thereby enhancing the interaction between the microspheres and the intestinal mucus layer, and the dual-network cross-linked microspheres exhibit a responsive characteristic of increased release rate under inflammatory-related redox abnormalities.

5. The dual-network cross-linked microspheres according to claim 1, characterized in that, The ginsenosides comprise one or more ginsenoside monomers; the mass fraction of the ginsenosides in the microsphere precursor system is 0.01–2 wt%; the ginsenosides can exist directly in the precursor system in a dissolved form; for ginsenosides with low solubility, cyclodextrin inclusion complexes and / or pharmaceutically acceptable solubilizing forms can be introduced into the precursor system; the particle size of the microspheres is 10–2000 μm.

6. A method for preparing the double-crosslinked microspheres according to any one of claims 1-5, characterized in that, Includes the following steps: A) Formulate a microsphere precursor system containing alginate and / or carboxylated polysaccharides, lipoic acid, photoinitiator and ginsenosides; B) Contact the microsphere precursor system with multivalent cations to form ionically cross-linked microspheres, thereby obtaining a first cross-linked network; C) The microspheres obtained in step B are subjected to light treatment to form a dynamic disulfide crosslinking network of lipoic acid, thereby obtaining interpenetrating double-network double-crosslinked microspheres.

7. The method according to claim 6, characterized in that, The ionic crosslinking in step B is achieved through exogenous diffusion crosslinking or endogenous release crosslinking; when exogenous diffusion crosslinking is used, the microsphere precursor system is added to a CaCl2 aqueous solution to form ionicly crosslinked microspheres, and the mass fraction of the CaCl2 aqueous solution is 0.5–5 wt%.

8. The use of the double-crosslinked microspheres according to any one of claims 1-5 in the preparation of oral delivery formulations of ginsenosides, characterized in that, The formulation is for local delivery to sites of intestinal inflammation and / or delivery in response to the inflammatory microenvironment, wherein the inflammatory microenvironment is the intestinal inflammatory microenvironment, and the microspheres are suitable for the intestinal inflammatory microenvironment associated with inflammatory bowel disease.

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