A nano-particle for encapsulating esculin sodium, a preparation method and application thereof
Sodium aescinate nanoparticles prepared by free radical polymerization undergo responsive dissociation under ROS conditions, solving the problem of low oral bioavailability of sodium aescinate and achieving effective treatment and enhanced safety for ulcerative colitis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2025-04-07
- Publication Date
- 2026-07-31
AI Technical Summary
Sodium aescinate has low oral bioavailability and is easily degraded by gastrointestinal enzymes, resulting in limited efficacy and potentially significant toxic side effects. Existing drugs for treating ulcerative colitis have limitations.
Nanoparticles loaded with sodium aescinate were prepared by free radical polymerization. A stable three-dimensional network structure was formed by using cationic monomers and cross-linking agents to encapsulate sodium aescinate, and the drug was released in response to reactive oxygen species (ROS) conditions.
It improves drug bioavailability and local concentration, enhances therapeutic efficacy, reduces toxicity, and provides safety and tolerability for intestinal-targeted agents, making it suitable for the treatment of ulcerative colitis.
Smart Images
Figure CN120284906B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of biomedicine and nanomaterials technology, specifically to a nanoparticle loaded with sodium aescinate, its preparation method, and its application. Background Technology
[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory disease affecting the colon and rectum. The inflammatory process typically begins in the rectum and gradually spreads upwards throughout the colon. The main clinical manifestations include hematochezia, diarrhea, abdominal pain, and weight loss, significantly impacting patients' quality of life. In recent years, the global incidence of ulcerative colitis has been steadily rising, becoming a pressing public health issue. The pathogenesis of ulcerative colitis is complex, involving genetic susceptibility, environmental factors, and abnormal interactions between the gut microbiota and the host immune system. Although current clinical treatments can alleviate symptoms to some extent, they still have many limitations, such as significant drug side effects and unstable long-term efficacy. Therefore, developing highly effective and low-side-effect therapeutic drugs, especially those that can target the underlying mechanisms of inflammation, achieve long-term inflammation control, and maintain intestinal homeostasis, has become a current research hotspot.
[0003] Nanoparticles, due to their unique physicochemical properties, can overcome biological barriers, protect drugs from degradation, and achieve lesion-specific delivery through responsive release mechanisms, thereby improving drug bioavailability and therapeutic efficacy. Free radical polymerization (FRP), as a simple, widely applicable, and mild synthetic method, provides a powerful tool for preparing nanoparticles with specific properties.
[0004] Sodium aescinate (SA) is an active ingredient extracted from plants in the Aesculus family (horsehortaceae family) and possesses significant anti-inflammatory effects. In the treatment of ulcerative colitis, sodium aescinate can inhibit the expression of inflammation-related genes and reduce the activation and infiltration of inflammatory cells, thereby effectively relieving symptoms such as abdominal pain, diarrhea, and bloody mucus stools. However, sodium aescinate has low oral bioavailability and is easily degraded by various enzymes in the gastrointestinal tract, leading to limited efficacy and potentially causing significant toxic side effects.
[0005] Therefore, constructing nanoparticles capable of encapsulating sodium aescinate and developing intestinal-targeted formulations based on nanodelivery systems has significant research value and clinical implications. Summary of the Invention
[0006] To address the above technical problems, this invention provides nanoparticles loaded with sodium aescinate, their preparation method, and their applications. These nanoparticles are capable of releasing sodium aescinate in response to highly reactive oxygen species (ROS) in inflammation.
[0007] This invention is achieved through the following technical solution:
[0008] The first aspect of the present invention provides a method for preparing nanoparticles loaded with sodium aescinate, comprising the following steps: sodium aescinate and cationic monomer undergoing free radical polymerization reaction under the action of an initiator and a crosslinking agent to obtain the nanoparticles loaded with sodium aescinate.
[0009] The cationic monomer is selected from one or more of methacryloyloxyethyltrimethylammonium chloride (DMC), N-(3-aminopropyl)methacrylamide hydrochloride (APMA), dimethylaminoethyl methacrylate (DMAEMA), N-[(3-(dimethylamino)propyl)]methacrylamide (DMAPMA), and diethylaminoethyl methacrylate (DEAEMA); the crosslinking agent is a thioketal diacrylate (DAT).
[0010] This invention prepares sodium aescinate-encapsulated nanoparticles based on the principle of free radical polymerization. Free radical polymerization refers to the process by which monomer molecules generate high-molecular-weight polymers through free radical reactions under the action of free radical initiators. This polymerization reaction typically involves three basic steps: chain initiation, chain propagation, and chain termination. Cationic monomers are first adsorbed onto the surface of sodium aescinate through electrostatic interactions. Subsequently, the initiator generates free radicals under anaerobic conditions, thereby initiating the chain polymerization reaction of the monomers. A covalently cross-linked polymer network is formed through a cross-linking agent, creating a stable three-dimensional network structure to effectively encapsulate and stabilize sodium aescinate. The sodium aescinate-encapsulated nanoparticles provided by this invention can undergo responsive dissociation under reactive oxygen species (ROS) conditions, realizing the release of sodium aescinate.
[0011] Furthermore, the structural formula of the diacrylate containing the thioketal bond is as follows:
[0012]
[0013] Furthermore, the initiator is a thermal initiator or a redox initiator.
[0014] Furthermore, the thermal initiator is azobisisobutyronitrile (AIBN), and the redox initiator is ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TMEDA).
[0015] Furthermore, the mass ratio of the cationic monomer to sodium aescinate is (2-10):1.
[0016] Furthermore, the mass ratio of the cationic monomer to the crosslinking agent is 10:(3-4).
[0017] Furthermore, the molar ratio of the cationic monomer to the initiator is (9-24):1.
[0018] Furthermore, the nanoparticles loaded with sodium aescinate have a particle size of 100-500 nm.
[0019] Furthermore, the preparation method of the nanoparticles loaded with sodium aescinate includes the following steps: under an inert atmosphere, sodium aescinate, cationic monomer, crosslinking agent and initiator are dissolved in water and subjected to free radical polymerization reaction; the resulting emulsion is centrifuged, resuspended, washed and dried to obtain the nanoparticles loaded with sodium aescinate.
[0020] In a specific embodiment, the preparation method of the nanoparticles loaded with sodium aescinate includes the following steps: under an inert atmosphere, sodium aescinate, cationic monomer, crosslinking agent and APS are dissolved in water, and then TMEDA solution with oxygen removed by argon gas is added to carry out a free radical polymerization reaction. The resulting emulsion is centrifuged and resuspended in water, washed multiple times and then freeze-dried to obtain the nanoparticles loaded with sodium aescinate.
[0021] The second aspect of the present invention provides nanoparticles loaded with sodium aescinate prepared by the preparation method described in the first aspect.
[0022] The nanoparticles loaded with sodium aescinate provided by this invention are reactive oxygen species (ROS) responsive nanoparticles. They dissociate under ROS conditions and release sodium aescinate. Sodium aescinate works by inhibiting the release of inflammatory factors and promoting the repair of the intestinal barrier.
[0023] The third aspect of this invention provides the application of the nanoparticles containing sodium aescinate as described in the second aspect in the preparation of a drug for treating ulcerative colitis.
[0024] This invention demonstrates, through application verification at the cellular level and in mouse models, that nanoparticles encapsulated with sodium aescinate can effectively promote the repair of the intestinal barrier and inhibit inflammatory responses, exhibiting good safety and tolerability.
[0025] The beneficial effects of this invention are:
[0026] 1. The preparation method of nanoparticles loaded with sodium aescinate provided by the present invention is simple and quick, and the required cost is relatively low, which is conducive to realizing industrial production.
[0027] 2. The nanoparticles loaded with sodium aescinate prepared in this invention combine sodium aescinate with ROS-responsive nanoparticles to construct an intestinal-targeted formulation based on a nanodelivery system. This formulation can responsively release the drug in the intestinal region where inflammation is high and ROS levels are elevated, thereby improving the local concentration and therapeutic effect of the drug. It effectively solves the problem of low oral bioavailability of sodium aescinate, and can also effectively reduce its toxicity and improve the safety of clinical application. It shows broad prospects in the preparation of drugs for the treatment of ulcerative colitis. Attached Figure Description
[0028] Figure 1 The images shown are DLS, SEM, and TEM images of the nanoparticles loaded with sodium aescinate prepared in Example 1; where (a) is the DLS image, (b) is the SEM image, and (c) is the TEM image.
[0029] Figure 2 The figures show the results of cell-level experiments on sodium aescinate and the sodium aescinate nanoparticles prepared in Example 1; where (a) is the cell viability data of the CCK-8 experiment, (b) is the expression data of iNOS detected by real-time quantitative PCR, and (c) is the expression data of COX-2 detected by real-time quantitative PCR.
[0030] Figure 3 Images of colon tissue sections (H&E staining) from mice in each group of test case 3.
[0031] Figure 4 This is a graph showing the histopathological scoring data of the colon tissue of mice in each group in test case 3.
[0032] Figure 5 The image shows the detection results of colonic inflammatory factors in each group of mice in test case 3. Detailed Implementation
[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.
[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.
[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.
[0036] Example 1
[0037] A method for preparing nanoparticles loaded with sodium aescinate includes the following steps:
[0038] 100 mg APMA, 30 mg crosslinking agent DAT, and 7.1 mg initiator APS were added to a round-bottom flask, followed by the addition of purified water and ultrasonic dispersion. An aqueous solution containing 10 mg sodium aescinate was then added to bring the total volume of the mixture to 10 mL. Next, a 2 mg / mL TMEDA solution (3.6 mg TMEDA) was added under argon gas to initiate free radical polymerization. The reaction was carried out continuously under argon gas for 65 min. The resulting emulsion was centrifuged and resuspended in purified water, washed three times, and then freeze-dried to obtain sodium aescinate-encapsulated nanoparticles.
[0039] Example 2
[0040] A method for preparing nanoparticles loaded with sodium aescinate includes the following steps:
[0041] 100 mg DMC, 30 mg crosslinking agent DAT, and 7.1 mg initiator APS were added to a round-bottom flask, followed by the addition of purified water and ultrasonic dispersion. An aqueous solution containing 10 mg sodium aescinate was then added to bring the total volume of the mixture to 10 mL. Next, a TMEDA solution (3.6 mg) purged with argon gas was added to initiate a free radical polymerization reaction, which was carried out continuously under argon gas for 65 min. The resulting emulsion was centrifuged and resuspended in purified water, washed three times, and then freeze-dried to obtain sodium aescinate-encapsulated nanoparticles.
[0042] Test Example 1
[0043] The particle size and morphology of the sodium aescinate nanoparticles prepared in Examples 1 and 2 were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The test results are as follows: Figure 1 As shown, from Figure 1 As can be seen, the nanoparticles loaded with sodium aescinate are spherical, uniformly distributed, and have a particle size of 180.7±37.90 nm. The nanoparticles loaded with sodium aescinate prepared in Example 2 have an uneven particle size distribution, exhibiting a multi-peak distribution with sizes of 161.43±26.29 nm and 866.50±74.40 nm, respectively.
[0044] Test Example 2
[0045] Cellular experiments were conducted on sodium aescinate (SA) and sodium aescinate nanoparticles (SA-NPs) prepared in Example 1, including cell viability detection and verification of related mechanisms. The test methods were as follows: (1) In vitro cell experiments were conducted to evaluate the biocompatibility of SA and SA-NPs. The CCK-8 method was used to detect the effect of SA and SA-NPs on the viability of RAW264.7 cells in the concentration range of 0-150 μg / mL.
[0046] (2) Real-time quantitative PCR was used to detect inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) to understand the expression changes of inflammation-related genes. iNOS and COX-2 are key enzymes in the inflammatory response, and changes in their mRNA expression levels can reflect the intensity of the inflammatory response. The control group consisted of untreated RAW264.7 cells as a control for normal cells. The LPS group was treated with 1 μg / mL lipopolysaccharide (LPS) solution for 18 hours to simulate the intestinal infection by pathogens or endotoxin stimulation, inducing intestinal barrier damage and inflammatory response. The SA group was pretreated with 10 μg / mL SA solution for 2 hours, followed by treatment with 1 μg / mL LPS solution for 18 hours. The SA-NPs group was pretreated with 10 μg / mL SA-NPs solution for 2 hours, followed by treatment with 1 μg / mL LPS solution for 18 hours.
[0047] Test results are as follows Figure 2 As shown, from Figure 2 As shown in (a), the CCK-8 assay indicates that sodium aescinate has good cell safety at concentrations of 0-40 μg / mL, but at high concentrations, it exhibits some cytotoxicity, inhibiting cell activity. In contrast, nanoparticles encapsulating sodium aescinate show relatively low cytotoxicity, especially at high concentrations, exhibiting weaker inhibitory effects on cell activity, allowing cells to better maintain their viability. Real-time quantitative PCR analysis revealed that, as... Figure 2 As shown in (b) and (c), both sodium aescinate and nanoparticles loaded with sodium aescinate can significantly inhibit the expression of LPS-induced iNOS mRNA and COX-2 mRNA. The nanoparticles loaded with sodium aescinate have a stronger inhibitory effect on expression than sodium aescinate, showing an advantage.
[0048] Test Example 3
[0049] Sodium aescinate (SA) and sodium aescinate-loaded nanoparticles (SA-NPs) prepared in Example 1 were used in mouse experiments. The experiments included observing morphological changes in the colon, histopathological scoring of the colon, and detecting relevant inflammatory factors. The testing method was as follows: 6-8 week old female C57BL / 6 mice were selected, and the ulcerative colitis model was induced using the DSS method. C57BL / 6 mice were randomly divided into four groups: G1 (Control), G2 (Model), G3 (Sodium aescinate group, SA), and G4 (Sodium aescinate-loaded nanoparticle group, SA-NPs), with four mice in each group. Modeling and drug administration: From day 1 to day 7, mice in groups G2, G3, and G4 had free access to a 3% DSS aqueous solution for 7 days (from day 1 to day 7), while mice in group G1 had free access to normal drinking water. On day 1, mice G3 and G4 were administered drugs at doses of 10 mg / kg (days 1-7) and 25 mg / kg (days 8-13), respectively, while the control and model groups were given an equal volume of physiological saline by gavage. On day 8, one mouse from each group was randomly selected for endpoint sampling. On day 13, the remaining three mice were sampled for endpoint sampling. If any mouse reached the experimental endpoint during the period, endpoint sampling was performed on that day.
[0050] Evaluation indicators: After the experiment, mice were euthanized, the abdominal cavity was opened, the colon and rectum were harvested, the tissue around the colon was removed, and the longitudinal length from the ileocecal valve to the anus was measured and photographed. The intestinal contents were cleaned, weighed, and recorded. Colonic tissue was fixed, sectioned, stained with H&E, and histopathological changes were observed. The pathological condition of the colon was scored from 0 to 4 points based on the severity of inflammation (0 points for none, 1 point for mild, 2 points for moderate, and 3 points for severe), the extent of inflammatory lesions (0 points for none, 1 point for limited to the mucosa, 2 points for extending to the submucosa, and 3 points for transmural (full thickness) lesions), crypt damage (0 points for no damage, 1 point for damage in the basal 1 / 3 region, 2 points for damage in the basal 2 / 3 region, 3 points for loss of crypt structure but presence of surface epithelium, and 4 points for loss of both crypt structure and surface epithelium), and the percentage of lesion extent (0 points for 0%, 1%-25% for 1 point, 26%-50% for 2 points, 51%-75% for 3 points, and 76%-100% for 4 points). The scores for each lesion were summed to obtain the mouse colonic histopathological score. Cytokine (IL-6) levels were detected using an ELISA kit.
[0051] Test results are as follows Figure 3 , Figure 4 and Figure 5 As shown, H&E staining of colon tissue reveals... Figure 3As shown, from left to right, the colon tissue sections of four mice in each group are shown. The colonic mucosa of the mice in the model group has a large number of inflammatory cells infiltrating, the colonic epithelium is loose, and the intestinal crypt structure is destroyed. This indicates that DSS will cause inflammation and damage to the colonic tissue in mice, while the nanoparticle group loaded with sodium aescinate can improve this situation.
[0052] from Figure 4 The results show that by scoring the colon tissue of mice in each group, the colon tissue score of the model group was increased, while the scores of mice treated with sodium aescinate group and sodium aescinate-loaded nanoparticle group were reduced. Among them, the score of sodium aescinate-loaded nanoparticle group was lower than that of sodium aescinate group, indicating that the effect of sodium aescinate-loaded nanoparticles was better.
[0053] from Figure 5 The results showed that the cytokine IL-6 in the colon of DSS-induced colitis mice was significantly elevated, while the administration of sodium aescinate and nanoparticles loaded with sodium aescinate significantly reduced the level of this cytokine. The IL-6 level was significantly lower than that of G2 (p<0.05) and closer to that of G1, indicating that the nanoparticles loaded with sodium aescinate can effectively inhibit IL-6 expression, alleviate the inflammatory symptoms of DSS-induced colitis, and have a certain degree of therapeutic improvement effect on DSS-induced colitis mice.
[0054] In summary, the sodium aescinate nanoparticles provided by this invention have significant advantages in the treatment of ulcerative colitis. The sodium aescinate nanoparticles prepared by free radical polymerization can effectively promote the repair of the intestinal barrier and inhibit inflammatory responses, exhibiting good safety and tolerability. This invention provides a new option and approach for the treatment of ulcerative colitis.
[0055] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art should understand that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. A method for preparing nanoparticles encapsulating sodium aescinate, characterized in that, Includes the following steps: Under an inert atmosphere, sodium aescinate, cationic monomer, crosslinking agent and initiator are dissolved in water and subjected to free radical polymerization. The resulting emulsion is centrifuged, resuspended, washed and dried to obtain the nanoparticles loaded with sodium aescinate. The cationic monomer is selected from one or more of methacryloyloxyethyltrimethylammonium chloride, N-(3-aminopropyl)methacrylamide hydrochloride, dimethylaminoethyl methacrylate, N-[(3-(dimethylamino)propyl)]methacrylamide, and diethylaminoethyl methacrylate; the crosslinking agent is a diacrylate containing a thioketal bond; The mass ratio of the cationic monomer to sodium aescinate is (2-10):1; the mass ratio of the cationic monomer to the crosslinking agent is 10:(3-4); and the molar ratio of the cationic monomer to the initiator is (9-24):
1.
2. The production method according to claim 1, characterized by, The initiator is a thermal initiator or a redox initiator.
3. The production method according to claim 2, characterized by, The thermal initiator is azobisisobutyronitrile; the redox initiator is ammonium persulfate and N,N,N',N'-tetramethylethylenediamine.
4. The production method according to claim 1, characterized by, The nanoparticles loaded with sodium aescinate have a particle size of 100-500 nm.
5. Nanoparticles loaded with sodium aescinate prepared by the preparation method according to any one of claims 1-4.
6. The use of the nanoparticles containing sodium aescinate as described in claim 5 in the preparation of a medicament for treating ulcerative colitis.