Mitochondrial uncoupling inhibitor sustained-release nanodot, preparation method thereof and application of nanodot in type 2 diabetes mellitus

Abstract

The invention relates to a mitochondrial uncoupling inhibitor sustained-release nanodot, a preparation method thereof and application of the nanodot in type 2 diabetes mellitus, and belongs to the technical field of nano-drugs. According to the preparation method disclosed by the invention, glycine and genipin are subjected to Schiff reaction to prepare the slow-release nanodot, and the slow-release nanodot is of a monodisperse spherical structure, has surface negative charges, and can slowly release genipin under physiological conditions, so that the mitochondrial protection effect of the genipin is fully exerted. The mitochondrial uncoupling inhibitor slow-release nanodot prepared by the invention can obviously improve the mitochondrial membrane potential, increase the cell ATP content, effectively inhibit mtDNA release under the stimulation of IL-1beta and palmitic acid, effectively inhibit STING inflammation pathway activation under the stimulation of IL-1beta and PA, obviously reduce pancreatic beta cell apoptosis, obviously relieve endoplasmic reticulum stress, and can be used for preparing the mitochondrial uncoupling inhibitor slow-release nanodot. The T2DM mouse blood sugar is obviously reduced, the glucose tolerance dose and the insulin sensitivity are improved, and good biocompatibility is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and in particular to a mitochondrial uncoupling inhibitor sustained-release nanodot, a preparation method thereof, and an application thereof in type 2 diabetes. Background Art

[0002] Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by hyperglycemia caused by insulin resistance and pancreatic β-cell damage. It currently affects nearly 500 million people worldwide and significantly increases the risk of cardiovascular disease, kidney disease, liver disease, cancer, and infection. Many studies have shown that T2DM is an inflammatory disease. Pancreatic β-cells are not only affected by systemic inflammatory factors such as the liver and adipose tissue, but also undergo strong 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 targeting, and are prone to immunosuppressive side effects, which greatly limits their clinical applicability.

[0003] The latest research shows that damaged mitochondria under stress conditions release mitochondrial DNA (mtDNA) and cytochrome C, among which mtDNA activates the STING pathway to further aggravate the inflammatory response, while cytochrome C induces the endogenous cell apoptosis pathway, both of which lead to aggravated damage to pancreatic beta cells. Therefore, it is extremely important to protect mitochondria. As a mitochondrial uncoupling inhibitor, genipin can well restore mitochondrial membrane potential, and has been studied in many biomedical fields such as cancer treatment, liver protection and choleresis, and neuroprotection. However, genipin is easily cross-linked with proteins in the body, making it difficult to accurately reach the lesion site, resulting in the side effect of blue pigment deposition, and affecting its efficacy. Summary of the invention

[0004] The present invention aims to solve the technical problem in the prior art that the mitochondrial uncoupling inhibitor genipin is easily cross-linked with proteins, making it difficult to accurately reach the lesion site and thus unable to fully exert its therapeutic effect, and further provides a mitochondrial uncoupling inhibitor sustained-release nanodots and a preparation method thereof and an application in type 2 diabetes. The present invention adopts glycine and genipin to undergo Schiff reaction to prepare sustained-release nanodots, which can slowly release genipin under physiological conditions and fully exert its mitochondrial protective effect.

[0005] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0006] A method for preparing mitochondrial uncoupling inhibitor sustained-release nanodots, comprising the following steps:

[0007] The mitochondrial uncoupling inhibitor sustained-release nanodots are prepared by using glycine and genipin to undergo Schiff reaction.

[0008] In the above technical solution, it is further preferred that the preparation method comprises the following steps:

[0009] Step (1) Synthesis:

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

[0011] Step (2) purification:

[0012] The sample obtained in step (1) is centrifuged to discard the supernatant, and the precipitate is dissolved in ultrapure water and dialyzed to remove unreacted impurities;

[0013] Step (3) Drying:

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

[0015] In the above technical solution, it is further preferred that the molar concentration 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 a pH of 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, the ultrapure water is replaced every 4 hours, and the centrifugation is performed at 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] The mitochondrial uncoupling inhibitor sustained-release nanodots prepared by the above method have a monodisperse spherical structure and a particle size between 5 and 10 nm.

[0021] Application of mitochondrial uncoupling inhibitor sustained-release nanodots in the preparation of drugs 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 the present invention are:

[0024] The preparation method of the mitochondrial uncoupling inhibitor sustained-release nanodots provided by the present invention is a simple and green synthesis method, and the obtained nanodots are capable of slowly releasing genipin. The raw materials used are inexpensive and easily available, and the synthesis method is simple.

[0025] The invention provides a method for preparing the mitochondrial uncoupling inhibitor sustained-release nanodots, which are prepared by Schiff reaction of genipin and glycine, and have a monodisperse spherical structure ( Figure 1 ), with a negative surface charge ( Figure 2 ) and the slow release properties of genipin ( Figure 3 ).

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

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

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

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

[0030] The mitochondrial uncoupling inhibitor sustained-release nanodots prepared by the present invention can significantly alleviate endoplasmic reticulum stress ( Figure 8 ).

[0031] The mitochondrial uncoupling inhibitor sustained-release nanodots prepared by the present invention can significantly reduce blood sugar in T2DM mice and improve their glucose tolerance and insulin sensitivity ( Fig. 9 ).

[0032] The mitochondrial uncoupling inhibitor sustained-release nanodots prepared by the present invention have good biocompatibility and are non-toxic to rat islet cell tumor cells (INS-1 cells) at the administration concentrations involved in the present invention. Fig.10 ), and long-term administration had no effect on the heart, liver, spleen, lung, and kidney of normal mice ( Fig.11 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

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

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

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

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

[0038] Figure 5 These are immunofluorescence results of the effect of Mito-G treatment prepared in Example 4 on mtDNA release after IL-1β and PA stimulation of INS-1 cells, wherein (A) is an immunofluorescence result of the effect of Mito-G on mtDNA release after IL-1β modeling, and (B) is an immunofluorescence result of the effect of Mito-G on mtDNA release after PA modeling.

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

[0040] Figure 7 The double immunofluorescence results of pancreatic cytochrome C and insulin in normal mice, type 2 diabetic mice, and mice after injection of Mito-G prepared in Example 4 and metformin treatment and their quantification, wherein (A) is the double immunofluorescence results of pancreatic cytochrome C and insulin in each group of mice, and (B) is the quantitative graph of the cytochrome C-positive area of ​​each islet in Figure (A).

[0041] Figure 8The figures are the relative contents of endoplasmic reticulum stress-related genes in pancreatic tissue of normal mice, type 2 diabetic mice, and mice after injection of Mito-G prepared in Example 4 and treatment with metformin, wherein (A)-(H) are 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] Fig. 9 The figures are diagrams showing the therapeutic effects of different concentrations of Mito-G prepared in Example 4 on T2DM mice, wherein (A)-(C) are diagrams showing the changes in blood glucose in mice within 6 weeks of drug treatment, the results of intraperitoneal glucose tolerance test in mice, and the results of insulin tolerance test in mice, respectively.

[0043] Fig.10 This is a graph showing the effect of different concentrations of Mito-G prepared in Example 4 on the viability of INS-1 cells.

[0044] Fig.11 H&E staining of the main organs of normal mice after intravenous injection of Mito-G (5 mg / kg) prepared in Example 4 for 30 consecutive days. DETAILED DESCRIPTION

[0045] The technical scheme of the present invention is clearly and completely described below through examples, but it should be understood that the following examples do not limit the protection scope of the present invention.

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

[0047] Example 1

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

[0049] Take 0.1g of glycine and dissolve it in 70mL of PBS, then add concentrated hydrochloric acid to adjust the pH to 2; take 0.675g of genipin and dissolve it in 30mL of ethanol; mix the two solutions in a 150mL round-bottom flask and add a magnetic rotor; then place the round-bottom flask in a water bath and react with magnetic stirring 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 ingredient. The supernatant was discarded and 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 ultrapure water. Dialysis was performed under magnetic stirring, and the ultrapure water was changed every 4 hours for a total of 24 hours to remove unreacted impurities.

[0052] (3) Drying

[0053] The sample obtained in step (2) was divided into 50 mL centrifuge tubes (15 mL sample per tube), and then placed in a -20°C refrigerator for pre-freezing for 2 hours. After 2 hours, the sample was placed in a freeze dryer and freeze-dried at -50°C under vacuum for 72 hours to obtain a sustained-release nanodot powder 1.

[0054] Example 2

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

[0056] Take 0.1g of glycine and dissolve it in 70mL of PBS, then add an appropriate amount of saturated NaOH solution to adjust the pH to 7; take 0.675g of genipin and dissolve it in 30mL of ethanol; mix the two solutions in a 150mL round-bottom flask and add a magnetic rotor. Then place the round-bottom flask in a water bath and react with magnetic stirring at 50℃ for 24 hours.

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

[0058] (3) Drying is the same as in Example 1 to obtain the sustained-release nanodot powder 2.

[0059] Example 3

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

[0061] Take 0.1g of glycine and dissolve it in 70mL of PBS, then add an appropriate amount of saturated NaOH solution to adjust the pH to 10; take 0.675g of genipin and dissolve it in 30mL of ethanol; mix the two solutions in a 150mL round-bottom flask and add a magnetic rotor. Then place the round-bottom flask in a water bath and react with magnetic stirring at 50℃ for 24 hours.

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

[0063] (3) Drying is the same as in Example 1 to obtain sustained-release nanodot powder 3.

[0064] Example 4

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

[0066] Take 0.3g of glycine and dissolve it in 70mL of PBS, then add an appropriate amount of concentrated hydrochloric acid solution to adjust the pH to 2; take 0.675g of genipin and dissolve it in 30mL of ethanol; mix the two solutions in a 150mL round-bottom flask and add a magnetic rotor. Then place the round-bottom flask in a water bath and react with magnetic stirring at 50℃ for 24 hours.

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

[0068] (3) Drying is the same as in Example 1 to obtain a sustained-release nanodot powder 4, which is denoted as Mito-G.

[0069] Example 5

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

[0071] 0.3 g of glycine was dissolved in 70 mL of PBS, and then an appropriate amount of saturated NaOH solution was added to adjust the pH to 7; 0.675 g of genipin was dissolved in 30 mL of ethanol; the two solutions were mixed in a 150 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at 50 °C for 24 hours.

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

[0073] (3) Drying is the same as in Example 1 to obtain a sustained-release nanodot powder 5.

[0074] Example 6

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

[0076] 0.3 g of glycine was dissolved in 70 mL of PBS, and then an appropriate amount of saturated NaOH solution was added to adjust the pH to 10; 0.675 g of genipin was dissolved in 30 mL of ethanol; the two solutions were mixed in a 150 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at 50 °C for 24 hours.

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

[0078] (3) Drying is the same as in Example 1 to obtain a sustained-release nanodot powder 6.

[0079] Example 7

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

[0081] Take 0.9g of glycine and dissolve it in 70mL of PBS, then add an appropriate amount of concentrated hydrochloric acid solution to adjust the pH to 2; take 0.675g of genipin and dissolve it in 30mL of ethanol; mix the two solutions in a 150mL round-bottom flask and add a magnetic rotor. Then place the round-bottom flask in a water bath and react with magnetic stirring at 50℃ for 24 hours.

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

[0083] (3) Drying is the same as in Example 1 to obtain sustained-release nanodot powder 7.

[0084] Example 8

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

[0086] 0.9 g of glycine was dissolved in 70 mL of PBS, and then an appropriate amount of saturated NaOH solution was added to adjust the pH to 7; 0.675 g of genipin was dissolved in 30 mL of ethanol; the two solutions were mixed in a 150 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at 50 °C for 24 hours.

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

[0088] (3) Drying is the same as in Example 1 to obtain sustained-release nanodot powder 8.

[0089] Example 9

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

[0091] 0.9 g of glycine was dissolved in 70 mL of PBS, and then an appropriate amount of saturated NaOH solution was added to adjust the pH to 10; 0.675 g of genipin was dissolved in 30 mL of ethanol; the two solutions were mixed in a 150 mL round-bottom flask and a magnetic rotor was added. The round-bottom flask was then placed in a water bath and reacted with magnetic stirring at 50 °C for 24 hours.

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

[0093] (3) Drying is the same as in Example 1 to obtain sustained-release nanodot powder 9.

[0094] The molar ratio of glycine to genipin, pH value, magnetic stirring reaction time, freeze-drying temperature and time of the reaction system in the above embodiments can also be any values ​​within the aforementioned limited ranges, and examples are not given here one by one.

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

[0096] Example 10

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

[0098] (1) Transmission electron microscopy and surface charge characterization: Transmission electron microscopy images were taken using a TECNAI G2 high-resolution transmission electron microscope. The results are shown in Figure 1 As shown in Figure 1, Mito-G has good dispersibility, small size and spherical structure in water, and the particle size is between 5 and 10 nm. The surface charge was detected by Zeta potential instrument, and the results are shown in Figure 1. Figure 2 As shown, the surface charge of Mito-G is -29.8 mV.

[0099] (3) The release of genipin from Mito-G was measured using the Agilent / 1290 InfinityⅡ analytical liquid chromatography purification system ( Figure 3 ), it can be seen from the figure that as the water bath time increases, the color of the Mito-G solution gradually becomes lighter, indicating that genipin in Mito-G has a slow release characteristic.

[0100] Embodiment 11

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

[0102] Mitochondrial membrane potential detection: INS-1 cells were seeded in a 96-well plate and incubated in the mitochondrial membrane potential probe TMRE for 30 min. Then, cell culture medium containing 30 ng / mL IL-1β was added and 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 were added and cultured for 10 min. Finally, cell culture medium containing 2 μM mitochondrial electron transport chain inhibitor CCCP and 30 ng / mL IL-1β were added and cultured for 10 min. The cells were detected using a multifunctional fluorescence microplate reader ( Figure 4 (middle AB).

[0103] ATP content detection: INS-1 cells were seeded in 6-well plates, and different groups of drugs dissolved in cell culture medium containing 30 ng / mL IL-1β were 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 supernatants of different treatment groups were collected by centrifugation (14000 rpm, 4°C, 5 minutes) for subsequent determination. The ATP detection working solution was prepared and stored on ice. 100 μL of ATP detection working solution was added to determine the ATP content according to the instructions of the enhanced ATP detection kit. The ATP concentration was calculated according to the luminescent signal and standardized ( Figure 4 C). 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 on a 24-well plate cell slide, and 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 was incubated with the cells for 24 hours. The 24-well plate was removed, the culture medium was discarded, and the plate was washed three times with PBS preheated at 37°C. After discarding PBS, add 500 μL of universal tissue fixative (Servicebio, G1101) to each well and fix at room temperature for 10 minutes; after washing with PBS, add 0.1% triton-pbs to break the membrane at room temperature for 10 minutes; after washing with PBS, add 10% goat serum to each well and block at room temperature for 1 hour; incubate with primary antibody (TOM20+dsDNA antibody) at four degrees overnight, wash with PBS, add corresponding fluorescent secondary antibody (Alexa Fluor488+Alexa Fluor 555) and incubate in dark for 1 hour, wash with PBS, incubate with DAPI in dark for 10 minutes, wash with PBS, seal the slide, and observe with laser confocal microscope. The results show that the release of free dsDNA in the model group increased, while it recovered after Mito-G treatment ( Figure 5 (middle AB).

[0105] Example 12

[0106] The sustained-release nanodots Mito-G synthesized in Example 4 were used to quantify the protein content 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 30ng / mL IL-1β or 400μM PA and treated with Mito-G after modeling, and the improvement effect of Mito-G on INS-1 spontaneous inflammation induced by IL-1β or PA was evaluated. The specific steps are as follows:

[0107] The INS-1 cell modeling and administration process was the same as in Example 11, and proteins were extracted from the cells using RIPA buffer containing PMSF and phosphatase inhibitors. The supernatant of the mixture was further determined at 4°C using a BCA protein assay kit for total protein concentration. The protein extracts were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted on a PVDF membrane. After blocking with fat-free milk, the membrane was incubated in the primary antibody (STING, P-NF-κB, NF-κB, P-IRF3, and IRF3) at 4°C overnight. The bound antibodies were detected with horseradish peroxidase (HRP)-coupled IgG and observed with enhanced chemiluminescence detection reagents. Protein bands were evaluated using Image J software.

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

[0109] Embodiment 13

[0110] C57BL / 6 mice (male, 8 weeks, 23-25g) were fed with a standard diet and water for 7 days in a clean environment with a 12-hour light / dark cycle at 24±2°C, and then fed with a high-fat diet (HFD) for 4 weeks, followed by intraperitoneal injection of streptozotocin (STZ, 60 mg / kg) for 5 consecutive days to construct a HFD-STZ-induced type 2 diabetes model. The mice were divided into: Control group, T2DM group, 2.5 mg / kg Mito-G treatment group, 5 mg / kg Mito-G treatment group and metformin group, with 6 mice in each group. Taking the synthesized Mito-G in Example 4 as an example, Mito-G was injected into the tail vein once every 3 days for a continuous treatment of one month. The mice were euthanized 5 days after the end of the treatment, and a part of the pancreatic tissue was prepared for paraffin sections, and another part of the pancreatic tissue was frozen to extract RNA.

[0111] Immunofluorescence double staining of cytochrome C (CytC) and insulin: The pancreatic tissue fixed in 4% paraformaldehyde for 24 hours was placed in 70%, 80%, 85%, 90%, and 95% ethanol for 1 hour, and then soaked in anhydrous ethanol for 0.5 hours. After replacing anhydrous ethanol, continue to soak for 0.5 hours, and then transfer to xylene for 0.5 hours. After replacing xylene, soak again for 0.5 hours to complete the dehydration process. The dehydrated tissue was soaked in paraffin solution overnight, quickly condensed on the embedding machine, and the largest cross-section of the tissue block was taken out for embedding. The embedded wax block was marked with numbers and stored at -20°C. The dehydrated tissue was soaked in paraffin solution overnight, quickly condensed on the embedding machine, and the largest cross-section of the tissue block was taken out for embedding. The embedded wax block was marked with numbers and stored at -20°C. After baking, dewaxing, antigen repair, tissue permeabilization, blocking, primary antibody incubation (Cyt C + Insulin antibody, overnight at 4 °C), fluorescent secondary antibody incubation (Alexa Flour 488 + Alexa Flour 555), sealing, fluorescence microscope observation and filming ( Figure 7 AB in , 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 ER 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 ExTaqTM (Tli RNaseH Plus). Gene expression was assessed by the comparative Ct method using β-actin as a reference gene. The primers used for quantitative real-time PCR are as follows:

[0113]

[0114]

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

[0116] Embodiment 14

[0117] The Mito-G synthesized in Example 4 was used to monitor the fasting blood glucose of normal mice, type 2 diabetic mice and type 2 diabetic mice treated with Mito-G, and 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. From the beginning of STZ injection to the end of Mito-G administration, the fasting blood glucose of the mice was tested once a week (fasting but not water for 12 hours).

[0118] Intraperitoneal glucose tolerance test (IPGTT): After 3 weeks of administration, the mice were fasted but not watered for 12 h, and blood glucose was measured once by cutting the tails of the mice, which was recorded as the blood glucose at time 0. Glucose solution (1 g / kg) was injected intraperitoneally with a 1 mL syringe, and blood glucose content was measured by cutting the tails of the mice at 15, 30, 60, 90, and 120 minutes after the glucose load.

[0119] Insulin tolerance test (ITT): After 4 weeks of administration, the mice were fasted but not watered for 4 hours, and blood glucose was measured once by cutting the tails of the mice and recorded as the blood glucose at time 0. Insulin (1U / kg) was injected intraperitoneally using a 1mL insulin syringe, and blood glucose content was measured by cutting the tails of the mice at 15, 30, 45, 60, 75, 105, and 120 minutes after the injection of insulin.

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

[0121] Embodiment 15

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

[0123] Cell level: INS-1 cells were inoculated into 96-well plates and incubated for 24 hours. Mito-G was dispersed in the culture medium to prepare cell culture fluids of different concentrations (0, 0.31, 1.25, 5, 20, 80 and 320 μg / mL, respectively). The above cell culture fluids were added to INS-1 cells and incubated for another 24 hours, and 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 with 100 μL Mito-G (5 mg / kg dissolved in 1× PBS) through the tail vein every 4 days, and the mice were euthanized after 30 days. Then the kidney tissues of each group of mice were cut into small pieces and fixed in 4% paraformaldehyde solution. Then the kidney tissues were placed in an embedding box and the fixative left in the tissue was washed away with running water. The embedding box was immersed in gradient ethanol for dehydration, and then immersed in xylene to make the tissue and embedding medium dissolve and infiltrate. The transparent tissue blocks were placed in the melted paraffin and placed in a wax melting box for overnight insulation. The wax-impregnated tissue material was placed in the center of the metal embedding frame containing wax liquid, and after dripping wax, it was moved to ice. When the surface of the wax liquid solidified, the pre-embedded tissue was clamped with tweezers, the tissue direction was adjusted, and it was inserted into the wax liquid. The uncovered embedding box was placed flat on the surface of the mold to cover it, and the wax liquid was dripped to cover the mold. It was gently pressed and placed in the solidification area until the wax block was completely solidified. Cool at 0℃ for about 30 minutes, fix the embedded wax block on the slicer, and cut into 4-micron thick slices. Put the cut slices into heated water to flatten them, then stick them on the slide, put them in a 65℃ constant temperature box to dry, and then put the slices into xylene for dewaxing. After the slices are dewaxed, add hematoxylin staining solution to the tissue to ensure that the tissue is completely covered, and stain for 10-15 minutes. Rinse with running water to wash away the excess stain. Differentiate with 1% hydrochloric acid alcohol solution, soak in water to turn blue for 2 minutes, add eosin staining solution to the tissue for about 10s, and after eosin staining is completed, immediately immerse in anhydrous ethanol for dehydration twice (2 minutes each time). Soak in xylene twice (2 minutes each time), dry naturally in a fume hood for 10-30 minutes, and seal with neutral resin.

[0125] The results are as follows Fig.10 As shown in Figure 2, Mito-G was nontoxic to INS-1 cells even at 320 μg / mL. In addition, H&E staining results showed that normal mice had no obvious damage to the heart, liver, spleen, lung, and kidney after intravenous injection of Mito-G for 30 days ( Fig.11 ).

[0126] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing mitochondrial uncoupling inhibitor sustained-release nanodots, characterized in that: The following steps are involved: The mitochondrial uncoupling inhibitor sustained-release nanodots are prepared by using glycine and genipin to undergo Schiff reaction.

2. The preparation method according to claim 1, characterized in that: The following steps are involved: Step (1) Synthesis: Dissolve glycine in a PBS buffer solution, add concentrated hydrochloric acid or a saturated sodium hydroxide solution to adjust the reaction system, dissolve genipin in ethanol, mix the dissolved glycine and genipin solution, and stir; Step (2) purification: The sample obtained in step (1) is centrifuged to discard the supernatant, and the precipitate is dissolved in ultrapure water and dialyzed to remove unreacted impurities; Step (3) Drying: The sample obtained in step (2) is freeze-dried to obtain the mitochondrial uncoupling inhibitor sustained-release nanodot powder.

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

1.

4. The preparation method according to claim 2, characterized in that: The reaction system in step (1) is a buffer solution with a pH of 1 to 10.

5. The preparation method according to claim 2, characterized in that: In step (1), the reaction stirring time is 12 to 36 hours at a temperature of 50°C.

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

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

8. A mitochondrial uncoupling inhibitor sustained-release nanodot prepared by the preparation method according to any one of claims 1 to 7, which has a monodisperse spherical structure and a particle size between 5 and 10 nm.

9. Use of the mitochondrial uncoupling inhibitor sustained-release nanodots according to claim 8 in the preparation of a drug for treating type 2 diabetes.

10. The use according to claim 9, characterized in that: The application of the mitochondrial uncoupling inhibitor sustained-release nanodots in the preparation of a drug for treating pancreatic β cell damage in type 2 diabetes.

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