Use of a chiral manganese nanoparticle in the preparation of a medicament for treating inflammatory bowel disease

Abstract

The application discloses application of chiral manganese nanoparticles in preparation of a drug for treating inflammatory bowel disease, and the chiral manganese nanoparticles are L-type chiral Mn3O4 nanoparticles. By regulating the chiral configuration of the chiral manganese nanoparticles, the subpopulation transformation of intestinal inherent lymphoid-like cells (ILC) is regulated, the differentiation of protective ILC3 is promoted, and the proliferation of proinflammatory ILC1 is inhibited, so that the colon inflammation is effectively relieved, and the intestinal barrier function is repaired.

Description

Technical Field

[0001] This invention relates to the field of nanobiomedicine technology, specifically to the application of chiral manganese nanoparticles in the preparation of drugs for treating inflammatory bowel disease. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, relapsing, nonspecific inflammatory bowel disease, primarily including ulcerative colitis and Crohn's disease. IBD is characterized by intestinal mucosal immune dysregulation, intestinal barrier dysfunction, and microbiome disruption. Currently, clinical treatment for IBD is mainly based on drug control, with mainstream medications including aminosalicylic acids, glucocorticoids, immunosuppressants, and biologics. However, these treatments have limitations, significant side effects, and the potential for drug resistance with long-term use.

[0003] Innate lymphoid cells (ILCs) are a core component of the innate immune system. Based on their characteristic transcription factors and secreted effector cytokines, ILCs can be divided into three major subsets: ILC1 (expressing T-bet, secreting IFN-γ, pro-inflammatory), ILC2 (expressing GATA3, secreting IL-5 and IL-13, involved in allergic reactions), and ILC3 (expressing RORγt, secreting IL-22 and IL-17, maintaining intestinal barrier and homeostasis). In the intestines of IBD patients, there is a significant imbalance in ILC subsets, mainly manifested as an increase in pro-inflammatory ILC1 and a decrease in protective ILC3, leading to persistent inflammation and tissue damage. Therefore, regulating ILC subset transformation and restoring the ILC1 / ILC3 balance has become a novel strategy for IBD treatment.

[0004] However, existing strategies generally suffer from the following common core bottlenecks: a lack of delivery systems capable of precisely targeting the gut, especially ILC subgroups; an inability to simultaneously achieve bidirectional synergistic regulation that inhibits pro-inflammatory ILC1 and promotes protective ILC3; difficulty in reversing the already imbalanced pathological plasticity of ILC; and poor material biocompatibility and unclear in vivo metabolism, leading to significant challenges in clinical translation. These shortcomings severely restrict the long-term, safe, and effective treatment of IBD. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned technical problems and provide an application of chiral manganese nanoparticles in the preparation of drugs for treating inflammatory bowel disease. Chiral manganese nanoparticles can precisely target the intestinal immune microenvironment, bidirectionally regulate the ILC1 / ILC3 balance, reverse pathological plasticity, and have good biodegradability and safety.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] The application of chiral manganese nanoparticles in the preparation of drugs for treating inflammatory bowel disease, wherein the chiral manganese nanoparticles are L-type chiral Mn3O4 nanoparticles.

[0008] Manganese tetroxide (Mn3O4) nanoparticles possess excellent superoxide dismutase (SOD) and catalase (CAT)-like activities, which can efficiently scavenge reactive oxygen species and inhibit oxidative stress damage. They also exhibit good biocompatibility and biodegradability. Among them, L-type chiral Mn3O4 nanoparticles have stronger cell-cell interaction capabilities and bioactivity. Due to their unique stereoconfiguration, chiral nanomaterials can achieve specific recognition and interaction with biomolecules and immune cells, significantly improving drug targeting, bioavailability, and bioactivity.

[0009] This invention applies chiral manganese nanoparticles to the preparation of drugs for treating inflammatory bowel disease. Through chiral ligand modification, the interaction between these nanoparticles and intestinal immune cells is enhanced. The nanoparticles exert anti-inflammatory effects by regulating the transformation of intestinal lymphoid cell (ILC) subsets, thereby restoring intestinal immune homeostasis through a bidirectional regulatory effect that promotes ILC3 differentiation and inhibits ILC1 proliferation. The specific mechanisms include:

[0010] (1) Promote ILC3 differentiation: L-type chiral Mn3O4 nanoparticles can upregulate the levels of metabolites such as retinoic acid and aryl hydrocarbon receptor (AhR) ligand in the intestinal microenvironment, activate RORγt transcription factor, promote the differentiation of ILC precursors into ILC3, increase the number of ILC3 and the secretion of its effector molecule IL-22, thereby enhancing the intestinal barrier function.

[0011] (2) Inhibit ILC1 proliferation: By downregulating the expression of pro-inflammatory cytokines (such as IL-12 and IL-15), the amplification of T-bet+ILC1 is reduced, the production of IFN-γ is decreased, and the inflammatory cascade reaction is inhibited.

[0012] (3) Restoring the ILC1 / ILC3 balance: L-type chiral Mn3O4 nanoparticles significantly reduce the ILC1 / ILC3 ratio in colon tissue through the above bidirectional regulatory effects, rebuild intestinal immune homeostasis, and alleviate chronic inflammation.

[0013] Furthermore, the chiral manganese nanoparticles are chiral manganese nanoparticles synthesized using amino acids or monosaccharides as chiral ligands.

[0014] Furthermore, the amino acid is selected from one or more of cysteine, leucine, proline, phenylalanine, lysine, arginine, aspartic acid, alanine, and penicillamine; the monosaccharide is selected from one or more of glucose, fructose, galactose, and mannose.

[0015] Further, the chiral manganese nanoparticles are prepared by the following method: manganese source is dissolved in an organic solvent, oleylamine and oleic acid are added, water is added, and the reaction is carried out at 85-95 °C for 3-5 h. Then, a chiral ligand and potassium hydroxide aqueous solution are added to adjust the pH of the system to 9-10, and the reaction is continued for 11-13 h. The resulting reaction product is post-processed to obtain the chiral manganese nanoparticles.

[0016] Furthermore, the manganese source is manganese acetate (Mn(AC)2); the organic solvent is xylene.

[0017] Further, the molar ratio of the manganese source, oleylamine and oleic acid is 1:(8-10):(2-4); the molar ratio of the manganese source to the chiral ligand is 1:(0.5-2).

[0018] Furthermore, the post-processing includes washing the reaction product and performing solid-liquid separation, with the resulting solid phase being the chiral manganese nanoparticles.

[0019] Furthermore, the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

[0020] Furthermore, the chiral manganese nanoparticles have a particle size of 5-15 nm.

[0021] Furthermore, the dosage of the chiral manganese nanoparticles is 50-200 mg / kg / day.

[0022] Furthermore, the chiral manganese nanoparticles are administered once every 1-2 days for 2 consecutive weeks.

[0023] Furthermore, the circular dichroism chromatographic signal of the chiral manganese nanoparticles is 400-900 nm, and the chiral characteristic peak position is 550-700 nm.

[0024] Furthermore, the dosage form of the drug is an injection, an oral preparation, or an enema.

[0025] Furthermore, the drug also includes pharmaceutically acceptable salts, esters, hydrates, solvates, enantiomers, stereoisomers, metabolites, and prodrugs.

[0026] Furthermore, the drug also includes a pharmaceutically acceptable carrier selected from one or more of disintegrants, diluents, lubricants, binders, humectants, flavoring agents, suspending agents, surfactants, and preservatives.

[0027] The above-described technical solution of the present invention has the following beneficial effects:

[0028] This invention provides an application of chiral manganese nanoparticles in the preparation of drugs for treating inflammatory bowel disease. By regulating the chiral conformation of the chiral manganese nanoparticles, the transformation of intestinal lymphoid cell (ILC) subsets is regulated, promoting the differentiation of protective ILC3 and inhibiting the proliferation of pro-inflammatory ILC1, thereby effectively alleviating colitis and repairing intestinal barrier function. Animal experiments show that these chiral manganese nanoparticles can significantly reduce the disease activity index and inflammatory factor levels in colitis mice, and improve symptoms such as weight loss and colonic shortening. Attached Figure Description

[0029] Figure 1 The graph shows the effects of L-type chiral Mn3O4 nanoparticles from Example 1, D-type chiral Mn3O4 nanoparticles from Comparative Example 1, and achiral Mn3O4 nanoparticles on the relative body weight changes of DSS-induced acute colitis mice.

[0030] Figure 2 The graph shows the effects of L-type chiral Mn3O4 nanoparticles from Example 1, D-type chiral Mn3O4 nanoparticles from Comparative Example 1, and achiral Mn3O4 nanoparticles on the disease activity index of DSS-induced acute colitis mice.

[0031] Figure 3 The bar chart shows the effects of L-type chiral Mn3O4 nanoparticles from Example 1, D-type chiral Mn3O4 nanoparticles from Comparative Example 1, and achiral Mn3O4 nanoparticles on colon length in mice with DSS-induced acute colitis.

[0032] Figure 4 This is a flow cytometry analysis of the proportion of ILC subsets in the colon tissue of mice in each group of test case 2.

[0033] Figure 5 The bar chart shows the effects of different groups of mice in Test Example 3 on the expression levels of colonic cytokines (TNF-α, IL-1β, IL-6, and IL-10 from left to right).

[0034] Figure 6 H&E staining images of colon tissue from mice in each group of test case 4. Detailed Implementation

[0035] 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 specification of this 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.

[0036] 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.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0038] Example 1

[0039] An L-type chiral Mn3O4 nanoparticle was prepared by the following method:

[0040] 1 mmol Mn(AC)₂ was dissolved in 15 mL xylene, and 9 mmol oleylamine and 3 mmol oleic acid were added and stirred. 1 mL ultrapure water was added, and the mixture was reacted at 90 °C for 4 h. After the reaction was completed, 1 mL of 1 mol / L L-type cysteine ​​aqueous solution and 1 mL of 1 mol / L potassium hydroxide aqueous solution were added to adjust the pH to 9, and the reaction was continued for 12 h. The product was washed with ethanol, centrifuged to remove unreacted matter, and the precipitate was resuspended in ultrapure water to obtain L-type chiral Mn₃O₄ nanoparticles with an average particle size of 8 nm.

[0041] Comparative Example 1

[0042] A type D chiral Mn3O4 nanoparticle was prepared by the following method:

[0043] 1 mmol Mn(AC)₂ was dissolved in 15 mL xylene, and 9 mmol oleylamine and 3 mmol oleic acid were added and stirred. 1 mL ultrapure water was added, and the mixture was reacted at 90 °C for 4 h. After the reaction was completed, 1 mL of 1 mol / L D-type cysteine ​​aqueous solution and 1 mL of 1 mol / L potassium hydroxide aqueous solution were added to adjust the pH to 9, and the reaction was continued for 12 h. The product was washed with ethanol, centrifuged to remove unreacted matter, and the precipitate was resuspended in ultrapure water to obtain D-type chiral Mn₃O₄ nanoparticles with an average particle size of 8 nm.

[0044] Test Example 1

[0045] Eight-week-old Balb / c mice were used to establish an acute colitis mouse model by allowing them free access to 3% (w / v) sodium dextran sulfate (DSS) solution for 7 days. From the date of model establishment, the treatment group mice were administered an aqueous solution of L-type chiral Mn3O4 nanoparticles (Example 1), D-type chiral Mn3O4 nanoparticles (Comparative Example 1), or achiral Mn3O4 nanoparticles by gavage at a dose of 100 mg / kg once a day for 14 consecutive days. The normal control group (which did not drink DSS solution) and the colitis model group were administered an equal volume of physiological saline by gavage. During the experiment, mouse body weight, fecal characteristics, and fecal blood loss were recorded daily. The Disease Activity Index (DAI) was calculated based on the following quantitative standards: weight loss rate (0: no weight loss; 1: 1-5%; 2: 5-10%; 3: 10-15%; 4: >15%); fecal characteristics (0: normal; 2: loose stool; 4: diarrhea); degree of fecal blood loss (0: no blood; 2: occult blood positive; 4: gross bloody stool); and the total score was the average of the three scores (0-4). At the end of the experiment, mice were sacrificed, and colon length was measured to evaluate the therapeutic effect of the drug on colitis.

[0046] Figure 1 The graph shows the effect of L-type chiral Mn3O4 nanoparticles of Example 1, D-type chiral Mn3O4 nanoparticles and non-chiral Mn3O4 nanoparticles of Comparative Example 1 on the relative body weight changes of DSS-induced acute colitis mice. Figure 2 The graph shows the effects of L-type chiral Mn3O4 nanoparticles from Example 1, D-type chiral Mn3O4 nanoparticles from Comparative Example 1, and achiral Mn3O4 nanoparticles on the disease activity index of DSS-induced acute colitis mice. Figure 3 The bar chart shows the effects of L-chiral Mn3O4 nanoparticles (Example 1), D-chiral Mn3O4 nanoparticles (Comparative Example 1), and achiral Mn3O4 nanoparticles on colon length in DSS-induced acute colitis mice. Control group represents the normal control group, DSS group represents the colitis model group, L-Mn3O4 group represents the L-chiral Mn3O4 nanoparticle treatment group, D-Mn3O4 group represents the D-chiral Mn3O4 nanoparticle treatment group, and Mn3O4 group represents the achiral Mn3O4 nanoparticle treatment group. The results showed that compared with the colitis model group, the L-chiral Mn3O4 nanoparticle treatment group significantly reduced the rate of weight loss, had a significantly lower DAI score, and showed a significant recovery in colon length at the experimental endpoint (P < 0.05). The above results fully demonstrate that the L-type chiral Mn3O4 nanoparticles of the present invention can effectively improve the clinical symptoms of DSS-induced acute colitis in mice, significantly alleviate colonic inflammatory damage, and have excellent therapeutic effects on inflammatory bowel disease.

[0047] Test Example 2

[0048] After the treatment cycle in Test Example 1 was completed, colonic tissues from mice in the normal control group, colitis model group, and L-type chiral Mn3O4 nanoparticle treatment group were aseptically isolated. Colonic lamina propria lymphocytes (LPL) were obtained through collagenase digestion and density gradient centrifugation. Flow cytometry was used to quantitatively analyze the intestinal lymphoid cell (ILC) subsets. The specific staining protocol was as follows: ILC populations were delineated using antibodies such as CD45, Lineage, CD127, and CD90.2. ILC1, ILC2, and ILC3 subsets were further distinguished using T-bet, GATA3, and RORγt. The proportions of each subset and changes in the ILC1 / ILC3 ratio were quantitatively analyzed.

[0049] Figure 4 The image shows a flow cytometry analysis of the proportion of ILC subsets in the colon tissue of mice in each group of test example 2. Control represents the normal control group, DSS represents the colitis model group, and L-Mn3O4 represents the L-chiral Mn3O4 nanoparticle treatment group. The results showed that, compared with the normal control group, the proportion of pro-inflammatory ILC1 subsets in the colon tissue of mice in the colitis model group was significantly increased, while the proportion of protective ILC3 subsets was significantly decreased, and the ILC1 / ILC3 ratio was abnormally high, exhibiting typical pathological characteristics of intestinal ILC subset imbalance. In the L-chiral Mn3O4 nanoparticle treatment group, the proportion of pro-inflammatory ILC1 in the colon was significantly decreased, the proportion of protective ILC3 was significantly increased, and the abnormally high ILC1 / ILC3 ratio was significantly restored (P<0.01). This confirms that the L-chiral Mn3O4 nanoparticles of this invention can exert a therapeutic effect by regulating ILC subset transformation and rebuilding intestinal immune homeostasis.

[0050] Test Example 3

[0051] After the treatment cycle in Test Example 1 was completed, colonic tissues from mice in the normal control group, colitis model group, and L-type chiral Mn3O4 nanoparticle treatment group were aseptically isolated, weighed, and added to RIPA lysis buffer containing protease inhibitors. The tissue was then homogenized thoroughly using a tissue homogenizer. The homogenate was centrifuged at 4 ℃ and 12000 rpm for 15 min, and the supernatant was collected. The expression levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10) in the supernatant were quantitatively detected by enzyme-linked immunosorbent assay (ELISA). The results were expressed as the cytokine content per milligram of colonic tissue homogenate (pg / mg).

[0052] Figure 5The bar chart shows the effects of different groups of mice in Example 3 on the expression levels of colonic cytokines (TNF-α, IL-1β, IL-6, and IL-10 from left to right). Control represents the normal control group, DSS represents the colitis model group, and L-Mn3O4 represents the L-chiral Mn3O4 nanoparticle treatment group. The results showed that compared with the colitis model group, the expression levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the colonic tissue of mice treated with L-chiral Mn3O4 nanoparticles were significantly reduced, while the expression level of the anti-inflammatory cytokine IL-10 was significantly increased. This confirms that the L-chiral Mn3O4 nanoparticles of this invention can effectively regulate the local inflammatory microenvironment of the colon and alleviate intestinal inflammatory damage.

[0053] Test Example 4

[0054] After the treatment cycle in Test Example 1 was completed, colon tissues of mice in the normal control group, colitis model group, and L-type chiral Mn3O4 nanoparticle treatment group were aseptically isolated. After fixation with 4% paraformaldehyde solution and paraffin embedding, tissue sections with a thickness of 5 μm were prepared. Histopathological staining was performed using hematoxylin-eosin (H&E) staining. Colonic mucosal integrity, crypt structure, degree of inflammatory cell infiltration, and epithelial damage were observed and evaluated under an optical microscope.

[0055] Figure 6 The images show H&E staining of colonic tissue from mice in each group of test example 4; where Control represents the normal control group, DSS represents the colitis model group, and L-Mn3O4 represents the L-type chiral Mn3O4 nanoparticle treatment group. The colonic structure of mice in the normal control group was intact; the colonic mucosa of mice in the colitis model group was extensively damaged, crypts disappeared, and a large number of inflammatory cells infiltrated; the colonic structure of mice in the L-type chiral Mn3O4 nanoparticle treatment group was basically restored, crypt morphology was intact, and inflammatory cell infiltration was significantly reduced, confirming that the chiral Mn3O4 nanoparticles of this invention can effectively protect colonic tissue and repair intestinal barrier damage.

[0056] 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. The application of chiral manganese nanoparticles in the preparation of drugs for treating inflammatory bowel disease, characterized in that, The chiral manganese nanoparticles are L-type chiral Mn3O4 nanoparticles.

2. The application according to claim 1, characterized in that, The chiral manganese nanoparticles are chiral manganese nanoparticles synthesized using amino acids or monosaccharides as chiral ligands; the amino acids are selected from one or more of cysteine, leucine, proline, phenylalanine, lysine, arginine, aspartic acid, alanine, and penicillamine; the monosaccharides are selected from one or more of glucose, fructose, galactose, and mannose.

3. The application according to claim 2, characterized in that, The chiral manganese nanoparticles were prepared by the following method: manganese source was dissolved in an organic solvent, oleylamine and oleic acid were added, water was added, and the reaction was carried out at 85-95 °C for 3-5 h. Then, a chiral ligand and potassium hydroxide aqueous solution were added to adjust the pH of the system to 9-10, and the reaction was continued for 11-13 h. The resulting reaction product was post-processed to obtain the chiral manganese nanoparticles.

4. The application according to claim 3, characterized in that, The manganese source is manganese acetate; the molar ratio of the manganese source, oleylamine and oleic acid is 1:(8-10):(2-4); the molar ratio of the manganese source to the chiral ligand is 1:(0.5-2).

5. The application according to claim 1, characterized in that, The inflammatory bowel disease mentioned refers to ulcerative colitis or Crohn's disease.

6. The application according to claim 1, characterized in that, The chiral manganese nanoparticles are administered at a dose of 50-200 mg / kg / day; the administration frequency of the chiral manganese nanoparticles is once every 1-2 days for 2 consecutive weeks.

7. The application according to claim 1, characterized in that, The chiral manganese nanoparticles have a particle size of 5-15 nm; the circular dichroism chromatographic signal of the chiral manganese nanoparticles is 400-900 nm, and the chiral characteristic peak position is 550-700 nm.

8. The application according to claim 1, characterized in that, The drug is available in the form of an injection, an oral preparation, or an enema.

9. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable salts, esters, hydrates, solvates, enantiomers, stereoisomers, metabolites, and prodrugs.

10. The application according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier selected from one or more of disintegrants, diluents, lubricants, binders, humectants, flavoring agents, suspending agents, surfactants, and preservatives.

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