Nanoparticles entrapped with sodium aescinate as well as preparation method and application of nanoparticles

Abstract

The invention discloses sodium aescinate-entrapped nanoparticles and a preparation method and application thereof, and the preparation method comprises the following steps: sodium aescinate and a cationic monomer are subjected to a free radical polymerization reaction under the action of an initiator and a cross-linking agent to obtain the sodium aescinate-entrapped nanoparticles. According to the sodium aescinate-entrapped nanoparticles prepared by the invention, the sodium aescinate is combined with ROS (reactive oxygen species) responsive nanoparticles to construct an intestinal targeting preparation based on a nano delivery system, so that drugs can be responsively released at intestinal parts with high inflammation incidence and high ROS level, the local concentration and treatment effect of the drugs are improved, and the bioavailability of the drugs is improved. The sodium aescinate effectively solves the problem of low oral bioavailability of sodium aescinate, also can effectively reduce the toxicity of sodium aescinate, improves the safety of clinical application, and shows a wide prospect in the aspect of preparing medicines for treating ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomedicine and nanomaterials, and particularly relates to a nanoparticle loaded with sodium aescinate, a preparation method thereof, and an application thereof. Background Art

[0002] Ulcerative Colitis (UC) is a chronic, non-specific inflammatory disease that affects the colon and rectum. Its inflammatory process usually begins in the rectum and gradually spreads upward to the entire colon. The main clinical manifestations of this disease are bloody stools, diarrhea, abdominal pain, weight loss, etc., which greatly affect the quality of life of patients. In recent years, the global incidence of ulcerative colitis has been continuously increasing, and it has become a public health problem that urgently needs to be solved. 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 treatment methods can relieve the condition to a certain extent, there are still many limitations, such as obvious drug side effects and unstable long-term efficacy. Therefore, the development of highly effective and low-side-effect therapeutic drugs, especially drugs that can target and drive the potential mechanisms of inflammation, achieve long-term control of inflammation, and maintain intestinal homeostasis, has become a research hotspot.

[0003] Due to their unique physicochemical properties, nanoparticles can overcome biological barriers, protect drugs from degradation, and achieve lesion-specific delivery through a responsive release mechanism, thereby improving the bioavailability and therapeutic effect of drugs. Free Radical Polymerization (FRP), as a synthetic method with simple operation, wide monomer applicability, and mild reaction conditions, provides a powerful tool for preparing nanoparticles with specific properties.

[0004] Sodium aescinate (SA) is an active ingredient extracted from plants of the Hippocastanaceae family and has significant anti-inflammatory effects. In the treatment of ulcerative colitis, sodium aescinate can inhibit the expression of inflammation-related genes, reduce the activation and infiltration of inflammatory cells, and thus effectively relieve symptoms such as abdominal pain, diarrhea, and mucopurulent bloody stools. However, the oral bioavailability of sodium aescinate is low, and it is easily degraded by various enzymes in the gastrointestinal tract, resulting in limited drug efficacy and possible large toxic side effects.

[0005] Therefore, constructing a nanoparticle that can load sodium aescinate and developing an intestinal-targeted preparation based on a nanodelivery system have important research value and clinical significance. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a nanometer particle loaded with sodium aescinate, a preparation method and an application thereof. The nanometer particle can correspondingly release sodium aescinate in a part with high levels of reactive oxygen species (ROS) in inflammation.

[0007] The present invention is realized through the following technical solutions:

[0008] In a first aspect of the present invention, a preparation method of a nanometer particle loaded with sodium aescinate is provided, including the following steps: sodium aescinate and a cationic monomer undergo a free radical polymerization reaction under the action of an initiator and a crosslinking agent to obtain the nanometer particle loaded with sodium aescinate;

[0009] The cationic monomer is selected from one or more of methacryloyloxyethyl trimethyl ammonium 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 dithioacetal-based diacrylate (Thioketal diacrylate, abbreviated as DAT).

[0010] The present invention prepares the nanometer particle loaded with sodium aescinate based on the principle of free radical polymerization. Free radical polymerization reaction refers to the process in which monomer molecules generate high molecular polymers through free radical reactions. This polymerization reaction generally involves three basic steps: chain initiation, chain growth, and chain termination. The cationic monomer is first adsorbed on the surface of sodium aescinate through electrostatic interaction. Subsequently, the initiator generates free radicals under anaerobic conditions, thereby initiating the chain polymerization reaction of the monomer. A covalently crosslinked polymer network is formed through the crosslinking agent to form a stable three-dimensional network structure to effectively encapsulate and stabilize sodium aescinate. The nanometer particle loaded with sodium aescinate provided by the present invention can undergo responsive dissociation under reactive oxygen species (ROS) conditions to achieve the release of sodium aescinate.

[0011] Further, the structural formula of the dithioacetal-based diacrylate is

[0012]

[0013] Further, the initiator is a thermal initiator or a redox initiator.

[0014] Further, the thermal initiator is azobisisobutyronitrile (AIBN), and the redox initiator is ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TMEDA).

[0015] Further, the mass ratio of the cationic monomer to sodium aescinate is (2-10):1.

[0016] Further, the mass ratio of the cationic monomer to the crosslinking agent is 10:(3 - 4).

[0017] Further, the molar ratio of the cationic monomer to the initiator is (9 - 24):1.

[0018] Further, the particle size of the nanoparticles encapsulating sodium aescinate is 100 - 500 nm.

[0019] Further, the method for preparing the nanoparticles encapsulating sodium aescinate includes the following steps: under an inert atmosphere, dissolving sodium aescinate, a cationic monomer, a crosslinking agent and an initiator in water, performing a free radical polymerization reaction, and centrifuging, resuspending, washing and drying the resulting emulsion to obtain the nanoparticles encapsulating sodium aescinate.

[0020] In a specific embodiment, the method for preparing the nanoparticles encapsulating sodium aescinate includes the following steps: under an inert atmosphere, dissolving sodium aescinate, a cationic monomer, a crosslinking agent and APS in water, then adding a TMEDA solution with oxygen removed by introducing argon, performing a free radical polymerization reaction, centrifuging the resulting emulsion and resuspending it with water, washing it multiple times and then performing freeze-drying treatment to obtain the nanoparticles encapsulating sodium aescinate.

[0021] The second aspect of the present invention provides nanoparticles encapsulating sodium aescinate prepared by the preparation method described in the first aspect.

[0022] The nanoparticles encapsulating sodium aescinate provided by the present invention are reactive oxygen species (ROS)-responsive nanoparticles, which dissociate under ROS conditions to release sodium aescinate, and sodium aescinate exerts its effects through mechanisms such as inhibiting the release of inflammatory factors and promoting the repair of the intestinal barrier.

[0023] The third aspect of the present invention provides an application of the nanoparticles encapsulating sodium aescinate described in the second aspect in the preparation of a drug for treating ulcerative colitis.

[0024] The present invention verifies the application at the cellular level and in a mouse model, and proves that the nanoparticles encapsulating sodium aescinate can effectively promote the repair of the intestinal barrier and can inhibit the inflammatory response, and have good safety and tolerance.

[0025] The beneficial effects of the present invention are as follows:

[0026] 1. The method for preparing the nanoparticles encapsulating sodium aescinate provided by the present invention is simple and fast, and the required cost is relatively low, which is conducive to realizing industrial production.

[0027] 2. The nano-particles loaded with sodium aescinate prepared by the present invention combine sodium aescinate with ROS-responsive nano-particles to construct an intestinal-targeted preparation based on a nano-delivery system, which can responsively release the drug at the intestinal site with high inflammation and elevated ROS levels, improve the local concentration and therapeutic effect of the drug, effectively solve the problem of low oral bioavailability of sodium aescinate, and can also effectively reduce its toxicity and improve the safety of clinical application, showing broad prospects in the preparation of drugs for treating ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 FIGURES are the DLS diagram, SEM diagram and TEM diagram of the nano-particles loaded with sodium aescinate prepared in Example 1; wherein, (a) is the DLS diagram, (b) is the SEM diagram, and (c) is the TEM diagram.

[0029] Figure 2 FIGURES are the results diagrams of the cell-level tests of sodium aescinate and the nano-particles loaded with sodium aescinate prepared in Example 1; wherein, (a) is the cell viability data diagram of the CCK-8 experiment, (b) is the expression data diagram of iNOS detected by real-time fluorescence quantitative PCR, and (c) is the expression data diagram of COX-2 detected by real-time fluorescence quantitative PCR.

[0030] Figure 3 FIGURES are the colon tissue section diagrams (H&E staining) of each group of mice in Test Example 3.

[0031] Figure 4 FIGURES are the pathological score data diagrams of the colon tissues of each group of mice in Test Example 3.

[0032] Figure 5 FIGURES are the detection result diagrams of the colon inflammatory factors of each group of mice in Test Example 3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0034] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments given are not intended to limit the present invention.

[0035] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0036] Example 1

[0037] A method for preparing nanoparticles loaded with sodium aescinate, comprising the following steps:

[0038] Add 100 mg of APMA, 30 mg of crosslinking agent DAT and 7.1 mg of initiator APS into a round-bottom flask, then add purified water and disperse by ultrasonic, add an aqueous solution containing 10 mg of sodium aescinate to make the total volume of the mixed solution 10 mL. Then add a 2 mg / mL TMEDA solution deoxygenated by passing argon, with the mass of TMEDA being 3.6 mg, to initiate a free radical polymerization reaction. The reaction is carried out in argon all the time for 65 min. Centrifuge the obtained emulsion and resuspend it with purified water, repeat the washing three times, and freeze-dry to obtain nanoparticles loaded with sodium aescinate.

[0039] Example 2

[0040] A method for preparing nanoparticles loaded with sodium aescinate, comprising the following steps:

[0041] Add 100 mg of DMC, 30 mg of crosslinking agent DAT and 7.1 mg of initiator APS into a round-bottom flask, then add purified water and disperse by ultrasonic, add an aqueous solution containing 10 mg of sodium aescinate to make the total volume of the mixed solution 10 mL. Then add a TMEDA solution deoxygenated by passing argon, with the mass of TMEDA being 3.6 mg, to initiate a free radical polymerization reaction. The reaction is carried out in argon all the time for 65 min. Centrifuge the obtained emulsion and resuspend it with purified water, repeat the washing three times, and freeze-dry to obtain nanoparticles loaded with sodium aescinate.

[0042] Test Example 1

[0043] Particle size testing and morphology characterization of the nanoparticles loaded with sodium aescinate prepared in Example 1 and Example 2 were carried out by dynamic light scattering (DLS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The test results are as Figure 1 shown. It can be seen from Figure 1 that the nanoparticles loaded with sodium aescinate are spherical, evenly distributed, and the particle size is 180.7 ± 37.90 nm. The particle size distribution of the nanoparticles loaded with sodium aescinate prepared in Example 2 is uneven, showing a multi-peak distribution, which are 161.43 ± 26.29 nm and 866.50 ± 74.40 nm respectively.

[0044] Test Example 2

[0045] Cell-level experiments were conducted on sodium aescinate (SA) and the sodium aescinate-loaded nanoparticles (SA-NPs) prepared in Example 1, including the detection of cell viability and the verification of related mechanisms. The test method was as follows: (1) In vitro cell experiments were carried out to evaluate the biocompatibility of SA and SA-NPs. The CCK-8 method was used to detect the effects of SA and SA-NPs on the viability of RAW264.7 cells in the concentration range of 0 - 150 μg / mL.

[0046] (2) Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by real-time fluorescence quantitative PCR to understand the changes in the expression of inflammation-related genes. iNOS and COX-2 are key enzymes in the inflammatory response, and the changes in their mRNA expression levels can reflect the intensity of the inflammatory response. The control group was RAW264.7 cells without any treatment, serving as a control for normal cells. The LPS group was RAW264.7 cells treated with 1 μg / mL lipopolysaccharide (LPS) solution for 18 hours to simulate the situation of intestinal infection by pathogens or endotoxin stimulation, inducing intestinal barrier damage and inflammatory response. SA group: RAW264.7 cells were pretreated with 10 μg / mL SA solution for 2 h and then treated with 1 μg / mL LPS solution for 18 hours. SA-NPs group: RAW264.7 cells were pretreated with 10 μg / mL SA-NPs solution for 2 h and then treated with 1 μg / mL LPS solution for 18 hours.

[0047] The test results are as Figure 2 shown. As can be seen from Figure 2 (a), the CCK-8 experiment showed that sodium aescinate had good cell safety at 0 - 40 μg / mL, but at high concentrations, sodium aescinate had a certain toxic effect on cells, inhibiting cell activity; while the sodium aescinate-loaded nanoparticles had relatively low toxicity to cells, especially at high concentrations, with a weaker inhibitory effect on cell activity, and cells could better maintain their viability. Through real-time fluorescence quantitative PCR analysis, as shown in Figure 2 (b) and (c), both sodium aescinate and the sodium aescinate-loaded nanoparticles could significantly inhibit the expression of iNOS mRNA and COX-2 mRNA induced by LPS, and the degree of inhibition of the sodium aescinate-loaded nanoparticles was stronger than that of sodium aescinate, showing an advantage.

[0048] Test Example 3

[0049] Mouse experiments were conducted on sodium aescinate (SA) and the sodium aescinate-loaded nanoparticles (SA-NPs) prepared in Example 1, including observing the morphological changes of the colon, colon histopathological scoring, and verifying the detection of related inflammatory factors. The test method was as follows: C57BL / 6 female mice aged 6-8 weeks were selected, and the ulcerative colitis model was induced by the DSS method. The C57BL / 6 mice were randomly divided into 4 groups, namely G1 - control group (Control), G2 - model group (Model), G3 - sodium aescinate group (SA), and G4 - sodium aescinate-loaded nanoparticle group (SA-NPs), with 4 mice in each group. Modeling and administration: From the 1st day to the 7th day, the mice in G2, G3, and G4 freely drank an aqueous solution containing 3% DSS for 7 days (from the 1st day to the 7th day), and the mice in G1 freely drank normal water. On the 1st day, G3 and G4 were administered according to the experimental design at 10 mg / kg (from the 1st day to the 7th day) and 25 mg / kg (from the 8th day to the 13th day) respectively, and the control group and the model group were given intragastric administration of the same volume of normal saline. On the 8th day, 1 mouse was randomly selected from each group for terminal sampling, and on the 13th day, the remaining 3 mice were sampled at the end point. If any mouse reached the experimental end point during this period, it was sampled at the end point on the same day.

[0050] Evaluation indicators: After the experiment, the mice were euthanized, the abdominal cavity was opened, the colorectal part of the mice was taken, the tissues around the colon were removed, and the longitudinal length from the ileocecal valve to the anus was measured and photographed as a whole. The intestinal contents were washed, weighed, and recorded. The colon tissues were fixed, sectioned, and stained with H&E to observe the histopathological changes. According to the severity of inflammation (none was 0 points, mild was 1 point, moderate was 2 points, severe was 3 points), the scope of inflammatory lesions (none was 0 points, limited to the mucosal layer was 1 point, extended to the submucosa was 2 points, transmural (full layer) was 3 points), crypt damage (no damage was 0 points, damage in the basal 1 / 3 area was 1 point, damage in the basal 2 / 3 area was 2 points, crypt structure disappeared and surface epithelium existed was 3 points, both crypt structure and surface epithelium disappeared was 4 points), and the proportion of the lesion area (0% was 0 points, 1%-25% was 1 point, 26%-50% was 2 points, 51%-75% was 3 points, 76%-100% was 4 points), the pathological condition of the colon was scored from 0 to 4 points, and the scores of each item were added up to obtain the histopathological score of the mouse colon tissue. An Elisa kit was used to detect the cytokine (IL-6).

[0051] The test results are as Figure 3 、 Figure 4 and Figure 5 shown. By staining the colon tissue with H&E, as Figure 3As shown, from left to right are the colon tissue section diagrams of four mice in each group. In the model group, there was a large amount of inflammatory cell infiltration in the colon mucosa layer of the mice, the colon tissue epithelium was loose, and the intestinal crypt structure was damaged, indicating that DSS could cause inflammation in the mice and damage the colon tissue, while the group of nanoparticles loaded with sodium aescinate could improve this situation.

[0052] As can be seen Figure 4 from the results of scoring the colon tissues of each group of mice, it shows that the colon tissue score of the model group increased, while the scores of the mice treated with the sodium aescinate group and the group of nanoparticles loaded with sodium aescinate could be reduced. Among them, the score of the group of nanoparticles loaded with sodium aescinate was lower than that of the sodium aescinate group, indicating that the effect of the nanoparticles loaded with sodium aescinate was better.

[0053] As can be seen Figure 5 from the results that the cytokine IL-6 in the colon of DSS-induced colitis mice was significantly increased, while administration of sodium aescinate and nanoparticles loaded with sodium aescinate could significantly reduce the level of this cytokine. The IL-6 level was significantly lower than that of G2 (p<0.05) and was closer to G1, indicating that the nanoparticles loaded with sodium aescinate could effectively inhibit the expression of IL-6, relieve the inflammatory symptoms of DSS-induced colitis, and had a certain degree of therapeutic improvement effect on DSS-induced colitis mice.

[0054] In summary, the nanoparticles loaded with sodium aescinate provided by the present invention have significant advantages in the treatment of ulcerative colitis. The nanoparticles loaded with sodium aescinate prepared by free radical polymerization can effectively promote the repair of the intestinal barrier, inhibit the inflammatory response, and have good safety and tolerance. The present invention provides a new option and idea for the treatment of ulcerative colitis.

[0055] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the implementation manners of the present invention. Those skilled in the art should understand that other different forms of changes or variations can be made on the basis of the above description. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A preparation method of nanoparticles loaded with sodium aescinate, characterized in that, It includes the following steps: Sodium aescinate and a cationic monomer undergo a free radical polymerization reaction under the action of an initiator and a crosslinking agent to obtain the nanoparticles encapsulating sodium aescinate; The cationic monomer is selected from one or more of methacryloyloxyethyl trimethyl ammonium chloride, N-(3-aminopropyl) methacrylamide hydrochloride, dimethylaminoethyl methacrylate, N-[(3-(dimethylamino)propyl)] methacrylamide, and diethylaminoethyl methacrylate; the crosslinking agent is a bisacrylate containing a sulfur ketal bond.

2. The preparation method according to claim 1, characterized in that, The initiator is a thermal initiator or a redox initiator.

3. The preparation method according to claim 2, wherein, The thermal initiator is azobisisobutyronitrile; the redox initiator is ammonium persulfate and N,N,N',N'-tetramethylethylenediamine.

4. The preparation method according to claim 1, characterized in that, The mass ratio of the cationic monomer to sodium aescinate is (2-10):

1.

5. The preparation method according to claim 1, characterized in that, The mass ratio of the cationic monomer to the crosslinking agent is 10:(3-4).

6. The preparation method according to claim 1, characterized in that, The molar ratio of the cationic monomer to the initiator is (9-24):

1.

7. The preparation method according to claim 1, characterized in that, The particle size of the nanoparticles encapsulating sodium aescinate is 100-500 nm.

8. The preparation method according to claim 1, characterized in that, The preparation method of the nanoparticles encapsulating sodium aescinate includes the following steps: under an inert atmosphere, sodium aescinate, a cationic monomer, a crosslinking agent, and an initiator are dissolved in water, and a free radical polymerization reaction is carried out. The resulting emulsion is centrifuged, resuspended, washed, and dried to obtain the nanoparticles encapsulating sodium aescinate.

9. Nanoparticles encapsulating sodium aescinate prepared by the preparation method according to any one of claims 1-8.

10. Use of the nanoparticles encapsulating sodium aescinate according to claim 9 in the preparation of a drug for treating ulcerative colitis.

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