Mitochondrial uncoupling inhibitor slow release nanodots, methods of making and use in type 2 diabetes

Abstract

The present application relates to a kind of mitochondrial uncoupling inhibitor sustained-release nanodots and its preparation method and application in type 2 diabetes, belong to nanopharmaceutical technical field.The preparation method of the present application, glycine and genipin occur Schiff reaction to prepare and obtain sustained-release nanodot, it is monodisperse spherical structure and has surface negative charge, it can slowly release genipin under physiological conditions, fully exert its mitochondrial protective effect.The mitochondrial uncoupling inhibitor sustained-release nanodot prepared in the present application can significantly improve mitochondrial membrane potential and increase cell ATP content, effectively inhibit IL-1 beta and mtDNA release under palmitic acid stimulation, effectively inhibit IL-1 beta and PA stimulate STING inflammatory pathway activation, significantly reduce islet beta cell apoptosis, significantly alleviate endoplasmic reticulum stress, significantly reduce T2DM mouse blood glucose improve its glucose tolerance and insulin sensitivity, with good biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, specifically to a sustained-release nanodot of a mitochondrial uncoupling inhibitor, its preparation method, and its application in type 2 diabetes. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by hyperglycemia caused by insulin resistance and damage to pancreatic beta cells. It currently affects nearly 500 million people worldwide and significantly increases the risk of cardiovascular disease, kidney disease, liver disease, cancer, and infections. Multiple studies have shown that T2DM is an inflammatory disease; pancreatic beta cells are not only affected by systemic inflammatory factors from the liver and adipose tissue, but also experience intense spontaneous inflammation. Currently, various interleukin (IL)-1β antibodies, IL-1β vaccines, and IL-1 receptor blockers are available to treat T2DM; however, these drugs are expensive, lack specific targeting, and are prone to causing immunosuppressive side effects, which greatly limits their clinical applicability.

[0003] Recent studies have shown that damaged mitochondria under stress release mitochondrial DNA (mtDNA) and cytochrome C. mtDNA activates the STING pathway, further exacerbating the inflammatory response, while cytochrome C induces endogenous apoptosis, both contributing to increased damage to pancreatic β-cells. Therefore, protecting mitochondria is crucial. Genipin, as a mitochondrial uncoupling inhibitor, effectively restores mitochondrial membrane potential and has been extensively studied in biomedical fields such as cancer treatment, hepatoprotection, and neuroprotection. However, genipin readily cross-links with proteins in the body, making it difficult to accurately reach lesion sites, resulting in blue pigment deposition as a side effect and affecting its therapeutic efficacy. Summary of the Invention

[0004] This invention aims to address the technical problem in existing technologies where the mitochondrial uncoupling inhibitor genipin is prone to cross-linking with proteins, making it difficult to accurately reach the lesion site and thus failing to fully exert its therapeutic effect. Therefore, this invention provides a sustained-release nanodot for the mitochondrial uncoupling inhibitor, its preparation method, and its application in type 2 diabetes. This invention uses a Schiff reaction between glycine and genipin to prepare sustained-release nanodots, which can slowly release genipin under physiological conditions, fully exerting its mitochondrial protective effect.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0006] A method for preparing sustained-release nanodots of mitochondrial uncoupling inhibitors includes the following steps:

[0007] The sustained-release nanodots of the mitochondrial uncoupling inhibitor were prepared by a Schiff reaction of glycine and genipin.

[0008] In the above technical solution, a further preferred embodiment of the preparation method includes the following steps:

[0009] Step (1) Synthesis:

[0010] Dissolve glycine in PBS buffer solution, add concentrated hydrochloric acid or saturated sodium hydroxide solution to adjust the reaction system, dissolve genipin in ethanol, mix the dissolved glycine and genipin solutions, and stir.

[0011] Step (2) Purification:

[0012] Centrifuge the sample obtained in step (1) to discard the supernatant, and dissolve the precipitate in ultrapure water and then dialyze to remove unreacted impurities;

[0013] Step (3) Drying:

[0014] The sample obtained in step (2) was freeze-dried to obtain the sustained-release nanoparticle powder of the mitochondrial uncoupling inhibitor.

[0015] In the above technical solution, it is further preferred that the molar ratio of glycine to genipin in step (1) is 1:3 to 4:1.

[0016] In the above technical solution, it is further preferred that the reaction system in step (1) is a buffer solution with pH = 1 to 10.

[0017] In the above technical solution, it is further preferred that the reaction stirring time in step (1) is 12 to 36 hours and the temperature is 50°C.

[0018] In the above technical solution, it is further preferred that the dialysis time in step (2) is 24 hours, and the ultrapure water is changed every 4 hours; the centrifugation is 12000r for 10 minutes.

[0019] In the above technical solution, it is further preferred that the freeze-drying temperature in step (3) is -50 to -40°C and the duration is 72 to 76 hours.

[0020] A sustained-release nanodot of mitochondrial uncoupling inhibitor prepared by the above method has a monodisperse spherical structure with a particle size between 5 and 10 nm.

[0021] Application of a sustained-release nanodot containing a mitochondrial uncoupling inhibitor in the preparation of a drug for treating type 2 diabetes.

[0022] Furthermore, the application is the use of mitochondrial uncoupling inhibitor sustained-release nanodots in the preparation of drugs for treating pancreatic β-cell damage in type 2 diabetes.

[0023] The beneficial effects of this invention are:

[0024] The present invention provides a method for preparing mitochondrial uncoupling inhibitor sustained-release nanodots, which is a simple and green synthesis method. The obtained nanodots are capable of slowly releasing genipin. The raw materials used are inexpensive and readily available, and the synthesis method is simple.

[0025] The method for preparing sustained-release nanodots of mitochondrial uncoupling inhibitors provided by this invention is based on a Schiff reaction between genipin and glycine, resulting in nanodots with a monodisperse spherical structure. Figure 1 ), has a negative surface charge ( Figure 2 ) and the slow-release properties of genipin Figure 3 ).

[0026] The sustained-release nanodots of the mitochondrial uncoupling inhibitor prepared in this invention can significantly increase mitochondrial membrane potential and increase cellular ATP content. Figure 4 ).

[0027] The mitochondrial uncoupling inhibitor sustained-release nanodots prepared in this invention can effectively inhibit the release of mtDNA stimulated by IL-1β and palmitic acid (PA). Figure 5 ).

[0028] The sustained-release nanodots of the mitochondrial uncoupling inhibitor prepared in this invention can effectively inhibit the activation of the STING inflammatory pathway under IL-1β and PA stimulation. Figure 6 ).

[0029] The sustained-release nanodots of the mitochondrial uncoupling inhibitor prepared in this invention can significantly reduce pancreatic β-cell apoptosis. Figure 7 ).

[0030] The mitochondrial uncoupling inhibitor sustained-release nanodots prepared in this invention can significantly alleviate endoplasmic reticulum stress. Figure 8 ).

[0031] The sustained-release nanodots of the mitochondrial uncoupling inhibitor prepared in this invention can significantly reduce blood glucose in T2DM mice and improve their glucose tolerance and insulin sensitivity. Figure 9 ).

[0032] The sustained-release nanodots of the mitochondrial uncoupling inhibitor prepared in this invention have good biocompatibility and are non-toxic to rat islet cell tumor cells (INS-1 cells) at the dosage concentrations specified in this invention. Figure 10 Furthermore, long-term administration had no effect on the heart, liver, spleen, lungs, or kidneys of normal mice. Figure 11 ). Attached Figure Description

[0033] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0034] Figure 1Transmission electron microscopy image of the sustained-release nanodots (labeled: Mito-G) of the mitochondrial uncoupling inhibitor prepared in Example 4.

[0035] Figure 2 The Zeta potential diagram of the Mito-G prepared in Example 4.

[0036] Figure 3 This is a schematic diagram of the release of genipin slowly under physiological conditions by Mito-G prepared in Example 4, where (A) is a color change diagram and (B) is a quantitative diagram of genipin release.

[0037] Figure 4 The diagram shows the effect of Mito-G prepared in Example 4 on mitochondrial membrane potential and ATP content. (A) shows the effect of genipin (GP) or Mito-G placed for different times on IL-1β-induced changes in mitochondrial membrane potential. (B) shows the relative fluorescence intensity of TMRE in different groups of cells after 10 minutes of Mito-G treatment. (C) shows the changes in ATP content in cells after Mito-G treatment in different groups.

[0038] Figure 5 Immunofluorescence results of Mito-G treatment affecting mtDNA release after IL-1β and PA stimulation of INS-1 cells, prepared in Example 4, are shown. (A) shows the immunofluorescence results of Mito-G affecting mtDNA release after IL-1β modeling, and (B) shows the immunofluorescence results of Mito-G affecting mtDNA release after PA modeling.

[0039] Figure 6 WB images of STING, P-NF-κB, NF-κB, P-IRF3 and IRF3 proteins after Mito-G treatment were prepared in Example 4 after IL-1β and PA stimulation of INS-1 cells. (A) is the WB image of the above proteins after IL-1β stimulation, and (B) is the WB image of the above proteins after PA stimulation.

[0040] Figure 7 The results of dual immunofluorescence of pancreatic cytochrome C and insulin in normal mice and type 2 diabetic mice after treatment with Mito-G and metformin prepared in Example 4 are shown in Figure 4, and their quantification are also shown. (A) shows the results of dual immunofluorescence of pancreatic cytochrome C and insulin in each group of mice, and (B) shows the quantification of the positive area of ​​cytochrome C in each islet in Figure (A).

[0041] Figure 8The graph shows the relative content of endoplasmic reticulum stress-related genes in the pancreatic tissue of normal mice and type 2 diabetic mice after treatment with Mito-G and metformin prepared in Example 4. (A)-(H) are the quantitative graphs of the relative mRNA levels of Bip, Perk, eIF-2α, Atf-4, Atf-6, Chop, Ero1-α, and Ire-1α in the pancreatic tissue of each group, respectively.

[0042] Figure 9 The therapeutic effects of different concentrations of Mito-G prepared in Example 4 on T2DM mice are shown in the figures. (A)-(C) show the changes in blood glucose in mice during 6 weeks of drug treatment, the results of the intraperitoneal glucose tolerance test, and the results of the insulin tolerance test, respectively.

[0043] Figure 10 The effect of different concentrations of Mito-G prepared in Example 4 on the viability of INS-1 cells.

[0044] Figure 11 H&E staining images of major organs in normal mice after continuous intravenous injection of Mito-G (5 mg / kg) prepared in Example 4 for 30 days. Detailed Implementation

[0045] The technical solution of the present invention will be clearly and completely described below through embodiments. However, it should be understood that the following embodiments do not limit the scope of protection of the present invention.

[0046] All reagents used in the following examples are commercially available, including glycine-Macklin, G800880; and genipin-Macklin, G810337.

[0047] Example 1

[0048] (1) Synthesis of sustained-release nanodots

[0049] Dissolve 0.1 g glycine in 70 mL PBS, then add concentrated hydrochloric acid to adjust the pH to 2; dissolve 0.675 g genipin in 30 mL ethanol; mix the two solutions in a 150 mL round-bottom flask and add a magnetic rotor; then place the round-bottom flask in a water bath and magnetically stir the reaction at 50 °C for 24 hours.

[0050] (2) Purification

[0051] The sample obtained in step (1) was centrifuged at 12000r for 10 minutes to precipitate the active ingredients. After discarding the supernatant, the precipitate was redissolved in ultrapure water. The dissolved solution was placed in a dialysis bag, and then the dialysis bag was placed in 2L of ultrapure water. Dialysis was performed under magnetic stirring. The ultrapure water was changed every 4 hours, and dialysis was performed for a total of 24 hours to remove unreacted impurities.

[0052] (3) Drying

[0053] Take the sample obtained in step (2), dispense it into 50 mL centrifuge tubes (15 mL sample per tube), and then pre-freeze it in a -20°C freezer for 2 hours. After 2 hours, place the sample in a freeze dryer and freeze-dry it at -50°C under vacuum for 72 hours to obtain sustained-release nanoparticle powder 1.

[0054] Example 2

[0055] (1) Synthesis of sustained-release nanodots

[0056] Dissolve 0.1 g of glycine in 70 mL of PBS, then add an appropriate amount of saturated NaOH solution to adjust the pH to 7; dissolve 0.675 g of genipin in 30 mL of ethanol; mix the two solutions in a 150 mL round-bottom flask and add a magnetic stirrer. Then place the round-bottom flask in a water bath and magnetically stir the reaction at 50 °C for 24 hours.

[0057] (2) Purification is the same as in Example 1.

[0058] (3) Dry the sustained-release nanoparticle powder 2 as in Example 1.

[0059] Example 3

[0060] (1) Synthesis of sustained-release nanodots

[0061] Dissolve 0.1 g of glycine in 70 mL of PBS, then add an appropriate amount of saturated NaOH solution to adjust the pH to 10; dissolve 0.675 g of genipin in 30 mL of ethanol; mix the two solutions in a 150 mL round-bottom flask and add a magnetic stirrer. Then place the round-bottom flask in a water bath and magnetically stir the reaction at 50 °C for 24 hours.

[0062] (2) Purification is the same as in Example 1.

[0063] (3) Dry the same as in Example 1 to obtain sustained-release nanoparticle powder 3.

[0064] Example 4

[0065] (1) Synthesis of sustained-release nanodots

[0066] Dissolve 0.3 g of glycine in 70 mL of PBS, then add an appropriate amount of concentrated hydrochloric acid solution to adjust the pH to 2; dissolve 0.675 g of genipin in 30 mL of ethanol; mix the two solutions in a 150 mL round-bottom flask and add a magnetic stirrer. Then place the round-bottom flask in a water bath and magnetically stir the reaction at 50 °C for 24 hours.

[0067] (2) Purification is the same as in Example 1.

[0068] (3) Dry the same as in Example 1 to obtain sustained-release nanoparticle powder 4, which is referred to as Mito-G.

[0069] Example 5

[0070] (1) Synthesis of sustained-release nanodots

[0071] Dissolve 0.3 g of glycine in 70 mL of PBS, then add an appropriate amount of saturated NaOH solution to adjust the pH to 7; dissolve 0.675 g of genipin in 30 mL of ethanol; mix the two solutions in a 150 mL round-bottom flask and add a magnetic stirrer. Then place the round-bottom flask in a water bath and magnetically stir the reaction at 50 °C for 24 hours.

[0072] (2) Purification is the same as in Example 1.

[0073] (3) Dry the same as in Example 1 to obtain sustained-release nanoparticle powder 5.

[0074] Example 6

[0075] (1) Synthesis of sustained-release nanodots

[0076] Dissolve 0.3 g of glycine in 70 mL of PBS, then add an appropriate amount of saturated NaOH solution to adjust the pH to 10; dissolve 0.675 g of genipin in 30 mL of ethanol; mix the two solutions in a 150 mL round-bottom flask and add a magnetic stirrer. Then place the round-bottom flask in a water bath and magnetically stir the reaction at 50 °C for 24 hours.

[0077] (2) Purification is the same as in Example 1.

[0078] (3) Dry the powder as in Example 1 to obtain sustained-release nanoparticle powder 6.

[0079] Example 7

[0080] (1) Synthesis of sustained-release nanodots

[0081] Dissolve 0.9 g of glycine in 70 mL of PBS, then add an appropriate amount of concentrated hydrochloric acid solution to adjust the pH to 2; dissolve 0.675 g of genipin in 30 mL of ethanol; mix the two solutions in a 150 mL round-bottom flask and add a magnetic stirrer. Then place the round-bottom flask in a water bath and magnetically stir the reaction at 50 °C for 24 hours.

[0082] (2) Purification is the same as in Example 1.

[0083] (3) Dry the powder as in Example 1 to obtain sustained-release nanoparticle powder 7.

[0084] Example 8

[0085] (1) Synthesis of sustained-release nanodots

[0086] Dissolve 0.9 g of glycine in 70 mL of PBS, then add an appropriate amount of saturated NaOH solution to adjust the pH to 7; dissolve 0.675 g of genipin in 30 mL of ethanol; mix the two solutions in a 150 mL round-bottom flask and add a magnetic stirrer. Then place the round-bottom flask in a water bath and magnetically stir the reaction at 50 °C for 24 hours.

[0087] (2) Purification is the same as in Example 1.

[0088] (3) Dry the powder as in Example 1 to obtain sustained-release nanoparticle powder 8.

[0089] Example 9

[0090] (1) Synthesis of sustained-release nanodots

[0091] Dissolve 0.9 g of glycine in 70 mL of PBS, then add an appropriate amount of saturated NaOH solution to adjust the pH to 10; dissolve 0.675 g of genipin in 30 mL of ethanol; mix the two solutions in a 150 mL round-bottom flask and add a magnetic stirrer. Then place the round-bottom flask in a water bath and magnetically stir the reaction at 50 °C for 24 hours.

[0092] (2) Purification is the same as in Example 1.

[0093] (3) Dry the powder as in Example 1 to obtain sustained-release nanoparticle powder 9.

[0094] In the above examples, the molar ratio of glycine to genipin, pH value, magnetic stirring reaction time, and freeze-drying temperature and time can be any values ​​within the aforementioned limits, and will not be listed here.

[0095] The structure and properties of the sustained-release nanodots synthesized in Example 4 are characterized below. In the accompanying drawings, the sustained-release nanodot powders synthesized in Example 4 are all represented by Mito-G.

[0096] Example 10

[0097] Taking the sustained-release nanodots Mito-G synthesized in Example 4 as an example, we investigated their particle size, surface charge, and the degree of genipin release from the particles. The specific steps are as follows:

[0098] (1) Transmission electron microscopy and surface charge characterization: Transmission electron microscopy images were captured using a TECNAI G2 high-resolution transmission electron microscope. The results are as follows: Figure 1 As shown, Mito-G exhibits good dispersibility in water, a small size, and a spherical structure, with particle sizes ranging from 5 to 10 nm. Surface charge was measured using a Zeta potentiometer, and the results are as follows: Figure 2 As shown, the surface charge of Mito-G is -29.8mV.

[0099] (3) The release of genipin in Mito-G was measured using an Agilent / 1290 Infinity II analytical liquid chromatography purification system. Figure 3 As shown in the figure, the color of the Mito-G solution gradually lightens as the water bath time increases, indicating that genipin in Mito-G has a slow release characteristic.

[0100] Example 11

[0101] After stimulating INS-1 cells with 30 ng / mL IL-1β and 400 μM PA, respectively, they were treated with 10 mM genipin or 5 mM Mito-G (the sustained-release nanodot Mito-G synthesized in Example 4). The protective effect of Mito-G on mitochondria was investigated by dual immunofluorescence staining with TMRE kit, ATP kit, dsDNA antibody and TOM20 antibody.

[0102] Mitochondrial membrane potential detection: INS-1 cells were seeded in 96-well plates and incubated with the mitochondrial membrane potential probe TMRE for 30 min. Then, cell culture medium containing 30 ng / mL IL-1β was added and the cells were cultured for 20 min. Subsequently, cell culture medium containing different drugs (10 μM genipin or 5 μM Mito-G with different degradation times (0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h)) and 30 ng / mL IL-1β was added and the cells were cultured for another 10 min. Finally, cell culture medium containing 2 μM mitochondrial electron transport chain inhibitor CCCP and 30 ng / mL IL-1β was added and the cells were cultured for another 10 min. The results were then detected using a multi-functional fluorescent microplate reader. Figure 4 (AB).

[0103] ATP content detection: INS-1 cells were seeded in 6-well plates. Different groups of drugs dissolved in cell culture medium containing 30 ng / mL IL-1β were co-incubated with the cells for 1 h. The old culture medium was discarded, and the cells were washed with pre-cooled PBS. 200 μL of lysis buffer (Beyotime, P0013B) was added to each well to lyse the cells. After lysis, the cell supernatant from different treatment groups was collected by centrifugation (14000 rpm, 4℃, 5 min) for subsequent measurement. The ATP detection working solution was prepared and temporarily stored on ice. 100 μL of ATP detection working solution was added, and the ATP content was measured according to the instructions of the enhanced ATP detection kit. The ATP concentration was calculated based on the luminescent signal and then standardized. Figure 4 (C) by Figure 4 It can be seen that the sustained-release nanodots Mito-G prepared in Example 4 can significantly improve mitochondrial membrane potential and increase cellular ATP content.

[0104] Immunofluorescence detection of mtDNA release: INS-1 cells were seeded onto 24-well cell culture slides and incubated with Mito-G (concentrations of 1 μg / mL and 3 μg / mL, respectively, both incubated at 37°C for 7 days) dissolved in cell culture medium containing 30 ng / mL IL-1β and 400 μM PA for 24 h. After incubation, the 24-well plates were removed, the culture medium was discarded, and the cells were washed three times with preheated PBS at 37°C. After discarding PBS, 500 μL of general tissue fixative (Servicebio, G1101) was added to each well and fixed at room temperature for 10 min. After washing with PBS, 0.1% Triton-PBS was added to permeate the membrane at room temperature for 10 min. After washing with PBS, 10% goat serum was added to each well for blocking at room temperature for 1 h. The membrane was incubated overnight at °C with primary antibody (TOM20 + dsDNA antibody). After washing with PBS, the membrane was incubated with the corresponding fluorescent secondary antibody (Alexa Fluor 488 + Alexa Fluor 555) in the dark for 1 h. After washing with PBS, DAPI was incubated in the dark for 10 min. After washing with PBS, the membrane was mounted and observed under a laser confocal microscope. The results showed that the release of free dsDNA in the model group increased, but recovered somewhat after Mito-G treatment. Figure 5 (AB).

[0105] Example 12

[0106] Using the sustained-release nanodots Mito-G synthesized in Example 4, Western blotting was used to quantify the protein levels of STING inflammatory pathway-related proteins (STING, P-NF-κB, NF-κB, P-IRF3, and IRF3) in normal INS-1 cells, INS-1 cells stimulated with 30 ng / mL IL-1β or 400 μM PA, and INS-1 cells treated with Mito-G after modeling. The ameliorative effect of Mito-G on IL-1β or PA-induced spontaneous inflammation in INS-1 cells was evaluated. The specific steps are as follows:

[0107] The INS-1 cell modeling and drug administration process was the same as in Example 11, using RIPA buffer containing PMSF and a phosphatase inhibitor to extract proteins from the cells. The supernatant of the mixture was further analyzed at 4°C using a BCA protein assay kit to determine the total protein concentration. The protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto a PVDF membrane. After blocking with fat-free milk, the membrane was incubated overnight at 4°C with the first antibody (STING, P-NF-κB, NF-κB, P-IRF3, and IRF3). The bound antibodies were detected using horseradish peroxidase (HRP)-conjugated IgG and observed using an enhanced chemiluminescence assay. Protein bands were evaluated using ImageJ software.

[0108] The results are as follows Figure 6As shown in Figure AB, the results indicate that co-incubation with Mito-G can effectively reduce intracellular inflammatory activation through the STING pathway.

[0109] Example 13

[0110] C57BL / 6 mice (male, 8 weeks old, 23-25g) were fed a standard diet and water for 7 days in a clean environment with a 12-hour light / dark cycle at 24±2℃, followed by a high-fat diet (HFD) for 4 weeks. Then, streptozotocin (STZ, 60mg / kg) was injected intraperitoneally for 5 consecutive days to establish an HFD-STZ-induced type 2 diabetes model. The mice were divided into four groups: Control group, T2DM group, 2.5mg / kg Mito-G treatment group, 5mg / kg Mito-G treatment group, and metformin group, with 6 mice in each group. Using Mito-G synthesized in Example 4 as an example, Mito-G was administered via tail vein injection every 3 days for one month. Five days after the end of treatment, the mice were euthanized, and a portion of pancreatic tissue was collected for paraffin sectioning. Another portion of pancreatic tissue was frozen for RNA extraction.

[0111] Cytochrome C (CytC) and insulin immunofluorescence double staining: Pancreatic tissue, fixed in 4% paraformaldehyde for 24 hours after collection, was sequentially immersed in 70%, 80%, 85%, 90%, and 95% ethanol for 1 hour each, then immersed in anhydrous ethanol for 0.5 hours, replaced with anhydrous ethanol, and immersed for another 0.5 hours. The tissue was then transferred to xylene for 0.5 hours, replaced with xylene, and immersed again for 0.5 hours to complete the dehydration process. The dehydrated tissue was then immersed in paraffin solution overnight, rapidly cooled on an embedding machine, and the largest cross-section of the tissue block was removed for embedding. The embedded paraffin blocks were labeled and numbered and stored at -20℃. The procedure involved slide preparation, dewaxing, antigen retrieval, tissue permeation, blocking, primary antibody incubation (Cyt C + Insulin antibody, overnight at 4°C), secondary antibody incubation (Alexa Flour 488 + Alexa Flour 555), mounting, observation under a fluorescence microscope, and imaging. Figure 7 AB in the middle, by Figure 7 It can be seen that the sustained-release nanodots prepared in Example 4 can significantly reduce pancreatic β-cell apoptosis.

[0112] Detection of endoplasmic reticulum stress-related gene levels: Total RNA was extracted from frozen pancreatic tissue using TRIzo and reverse transcribed to obtain cDNA. Quantitative real-time PCR was performed using an Applied Biosystems Step One Plus instrument and TB Green Premix ExTaq™ (Tli RNaseH Plus). Gene expression was assessed by comparative Ct assay using β-actin as a reference gene. Primers used for quantitative real-time PCR are as follows:

[0113]

[0114]

[0115] The results are as follows Figure 8 As shown in Figure AH, the sustained-release nanodots prepared in Example 4 can effectively alleviate endoplasmic reticulum stress in type 2 diabetic mice.

[0116] Example 14

[0117] Using Mito-G synthesized in Example 4, fasting blood glucose levels were monitored in normal mice, type 2 diabetic mice, and type 2 diabetic mice treated with Mito-G. Glucose tolerance and insulin sensitivity tests were performed to evaluate the therapeutic effect of Mito-G on type 2 diabetic mice. The mouse modeling and administration process was the same as in Example 13. Fasting blood glucose levels in mice were measured weekly from the start of STZ injection until the end of Mito-G administration (fasting with free access to water for 12 hours).

[0118] Intraperitoneal glucose tolerance test (IPGTT): After 3 weeks of drug administration, mice were fasted but allowed free access to water for 12 hours. Blood was collected from the tail of the mice to measure blood glucose once, and the blood glucose at 0 was recorded. Glucose solution (1g / kg) was injected intraperitoneally using a 1mL syringe. Blood glucose levels were measured by collecting blood from the tail of the mice at 15, 30, 60, 90 and 120 minutes after the glucose load.

[0119] Insulin tolerance test (ITT): After 4 weeks of drug administration, mice were fasted but allowed free access to water for 4 hours. Blood was collected from the tail of the mice to measure blood glucose, which was recorded as the blood glucose at 0. Insulin (1U / kg) was injected intraperitoneally using a 1mL insulin syringe. Blood glucose levels were measured by collecting blood from the tail of the mice at 15, 30, 45, 60, 75, 105, and 120 minutes after insulin injection.

[0120] like Figure 9 As shown in the AC study, Mito-G treatment significantly reduced elevated blood glucose levels in T2DM mice, improved their glucose tolerance, and enhanced their insulin sensitivity.

[0121] Example 15

[0122] The biocompatibility of Mito-G synthesized in Example 4 was evaluated at both the cellular and animal levels. The specific steps are as follows:

[0123] Cellular level: INS-1 cells were seeded into 96-well plates and incubated for 24 hours. Mito-G was dispersed in culture medium to prepare cell culture media of different concentrations (0, 0.31, 1.25, 5, 20, 80, and 320 μg / mL, respectively). INS-1 cells were added to the above cell culture media and incubated for another 24 hours. Then, 10 μL of CCK-8 reagent was added to each well for cell staining, and cell viability was detected by measuring absorbance at 450 nm.

[0124] Animal Level: Healthy C57BL / 6 mice were injected intravenously with 100 μL Mito-G (5 mg / kg dissolved in 1×PBS) every 4 days for 30 days, after which the mice were euthanized. Kidney tissue from each group of mice was then cut into small pieces and fixed in 4% paraformaldehyde solution. The kidney tissue was then placed in an embedding cassette and rinsed with running water to remove any remaining fixative. The embedding cassette was then immersed in a gradient of ethanol for dehydration, followed by immersion in xylene to ensure tissue miscibility with the embedding medium. The cleared tissue blocks were placed in melted paraffin and incubated overnight in a paraffin bath. The paraffin-impregnated tissue material was placed in the center of a metal embedding frame containing paraffin solution. After adding paraffin, the frame was placed on ice. Once the surface of the paraffin solidified, the pre-embedded tissue was held with forceps, its orientation adjusted, and inserted into the paraffin solution. An open embedding cassette was placed flat on the mold surface to cover it, and paraffin solution was added until it completely covered the mold. The frame was gently pressed and placed in the solidification zone until the paraffin block was completely solidified. After cooling at 0℃ for about 30 minutes, fix the embedded paraffin block onto a microtome and cut it into 4-micrometer thick sections. Flatten the sections in heated water, then attach them to glass slides and dry them in a 65℃ oven. Next, dewax the sections in xylene. After dewaxing, add hematoxylin staining solution to the tissue, ensuring complete coverage, and stain for 10-15 minutes. Rinse with running water to remove excess staining solution. Differentiate with 1% hydrochloric acid-alcohol solution, soak in water for 2 minutes to regain blue color, then add eosin staining solution to the tissue for about 10 seconds. After eosin staining, immediately immerse in anhydrous ethanol twice (2 minutes each time) for dehydration. Immerse twice in xylene (2 minutes each time), air dry in a fume hood for 10-30 minutes, and mount with neutral resin.

[0125] The results are as follows Figure 10 As shown, Mito-G remained non-toxic to INS-1 cells even at a concentration of 320 μg / mL. Furthermore, H&E staining results indicated that normal mice showed no significant damage to the heart, liver, spleen, lungs, or kidneys after 30 days of intravenous injection of Mito-G. Figure 11 ).

[0126] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of a sustained-release nanodot of a mitochondrial uncoupling inhibitor in the preparation of a drug for treating type 2 diabetes, wherein the sustained-release nanodot of the mitochondrial uncoupling inhibitor is prepared by a Schiff reaction of glycine and genipin.

2. The application according to claim 1, characterized in that, The preparation of the sustained-release nanodots of the mitochondrial uncoupling inhibitor includes the following steps: Step (1) Synthesis: Dissolve glycine in PBS buffer solution, add concentrated hydrochloric acid or saturated sodium hydroxide solution to adjust the reaction system, dissolve genipin in ethanol, mix the dissolved glycine and genipin solutions, and stir. Step (2) Purification: Centrifuge the sample obtained in step (1) to discard the supernatant, and dissolve the precipitate in ultrapure water and then dialyze to remove unreacted impurities; Step (3) Drying: The sample obtained in step (2) was freeze-dried to obtain the sustained-release nanoparticle powder of the mitochondrial uncoupling inhibitor.

3. The application according to claim 2, characterized in that, The molar ratio of glycine to genipin in step (1) is 1:3 to 4:

1.

4. The application according to claim 2, characterized in that, In step (1), the reaction system is a buffer solution with pH = 1 to 10.

5. The application according to claim 2, characterized in that, In step (1), the reaction stirring time is 12 to 36 hours and the temperature is 50°C.

6. The application according to claim 2, characterized in that, In step (2), the dialysis time is 24 hours, and the ultrapure water is changed every 4 hours; the centrifugation is 12000r for 10 minutes.

7. The application according to claim 2, characterized in that, In step (3), the freeze-drying temperature is -50 to -40°C and the duration is 72 to 76 hours.

8. The application according to claim 2, characterized in that, The prepared mitochondrial uncoupling inhibitor sustained-release nanodots have a monodisperse spherical structure with a particle size between 5 and 10 nm.

9. The application according to any one of claims 1-8, characterized in that, Application of the sustained-release nanodots of the mitochondrial uncoupling inhibitor in the preparation of drugs for treating pancreatic β-cell damage in type 2 diabetes.

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