Preparation method and application of ferroptosis regulating nanoparticles based on unsaturated fatty acid

Abstract

The invention relates to a preparation method of ferroptosis regulating nanoparticles based on unsaturated fatty acid, the lipid nanoparticles are prepared by compounding a model drug, monounsaturated fatty acid and phospholipid polyethylene glycol conjugates with different PEG molecular weights, and the method comprises the following steps: mixing the model drug, the monounsaturated fatty acid and the phospholipid polyethylene glycol conjugates with different PEG molecular weights; dissolving in ethanol, ultrasonically mixing uniformly, dropwise adding into deionized water, stirring, performing rotary evaporation, removing methanol and ethanol, centrifuging, removing supernate, and drying to obtain the product. The pharmaceutical composition is rich in raw materials, simple in preparation method, easy to produce, good in product quality and stable in property, polyphenol drugs can be effectively delivered to pancreatitis parts, the bioavailability of the drugs and the pancreatitis targeting capacity are greatly improved, ferroptosis of pancreatic beta cells is inhibited, and the effect of treating diabetes mellitus is achieved. The traditional Chinese medicine composition is a great innovation in medicines for treating diabetes and pancreatitis, and has huge social and economic benefits.

Description

Technical Field

[0001] The present invention relates to the medical field, and in particular to a preparation method and application of ferroptosis regulating nanoparticles based on unsaturated fatty acids. Background Art

[0002] Diabetes is a common chronic disease, including type 1 diabetes (T1DM) and type 2 diabetes (T2DM), which is a chronic disease caused by autoimmunity. In the early stage of diabetes, the body's immune system mistakenly identifies pancreatic β cells as foreign pathogens, thereby activating immune cells such as T lymphocytes, B lymphocytes and macrophages, and releasing a variety of inflammatory factors, such as interleukin-1β (IL-1β) and interferon-γ (IFN-γ). These inflammatory factors increase iron uptake by upregulating the expression of transferrin receptors in pancreatic β cells, while inhibiting the activity of antioxidant enzymes such as glutathione peroxidase 4 (GPX4), leading to increased levels of intracellular reactive oxygen species (ROS), thereby inducing lipid peroxidation and ferroptosis. These mechanisms ultimately lead to a decrease in the number of pancreatic β cells and impaired insulin secretion, causing continuous increase in blood sugar and accelerating the occurrence and progression of diabetes. Given the important role of ferroptosis in pancreatic β cell damage, regulating the ferroptosis signaling pathway is expected to become an effective technical approach for the treatment of diabetes.

[0003] In recent years, polyphenolic compounds such as curcumin, quercetin, catechins, rutin, etc. have shown the potential to inhibit ferroptosis and repair pancreatic β-cell function due to their multiple mechanisms such as regulating iron metabolism, anti-oxidation and inhibiting lipid peroxidation. However, in practical applications, these drugs are limited by low oral bioavailability and poor targeting efficiency, making it difficult to achieve effective accumulation at the site of pancreatic inflammation, resulting in limited efficacy. Therefore, improving the oral absorption efficiency of drugs and the ability of pancreatic targeted delivery is crucial for the treatment of diabetes. In response to these problems, the research experiment found that monounsaturated fatty acids can significantly improve the absorption efficiency of nanoparticles in the intestine by regulating the hardness of nanoparticles. The flexible structure enables nanoparticles to bind more tightly to the intestinal mucus layer, prolong their retention time in the intestine, and promote their passage through the intestinal barrier (such as intestinal microfold cells, M cells) through deformability, thereby improving the oral bioavailability of drugs. In addition, in order to further improve the accumulation efficiency of drugs in the pancreatic inflammation site, the targeted ligand-modified nanoparticles specifically target the integrin receptors of M cells and macrophages in Peyer's patches, and use the inflammatory chemotaxis of macrophages to precisely deliver drugs to the pancreatic inflammation site. After delivery to the pancreas, the nanoparticles reduce pancreatic β cell damage and protect pancreatic islet function by regulating the key pathway of ferroptosis.

[0004] Therefore, based on the dual optimization of hardness and softness regulation and targeted ligand modification, a nanoparticle loaded with polyphenol drugs was constructed to inhibit pancreatic β-cell ferroptosis and reduce its damage, providing a new and efficient solution for the treatment of diabetes, showing important application value, but no public reports have been seen so far. Summary of the invention

[0005] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a preparation method and application of ferroptosis regulating nanoparticles based on unsaturated fatty acids, which can effectively solve the problems of low bioavailability and poor targeting of polyphenol drugs, and greatly improve the bioavailability of drugs and the targeting ability of pancreatic inflammation.

[0006] The technical solution provided by the present invention is a method for preparing ferroptosis regulating nanoparticles based on unsaturated fatty acids, wherein the lipid nanoparticles are composited with a model drug, a monounsaturated fatty acid, and a phospholipid polyethylene glycol conjugate with different PEG molecular weights, and the method is: The model drug, monounsaturated fatty acid and phospholipid polyethylene glycol conjugates with different PEG molecular weights are mixed, wherein the mass ratio of the model drug to the monounsaturated fatty acid is 1: 5-15, and the mass ratio of the monounsaturated fatty acid to the phospholipid polyethylene glycol conjugates with different PEG molecular weights is 3.5-4.5: 1, and then dissolved in 3-5 mL of ethanol, and ultrasonically mixed at a frequency of 60-120 Hz to be uniform, and then at 25-60 ° C, under stirring, the mixed solution is slowly dripped into 5-15 mL of deionized water, and stirred at a speed of 500-1200 r / min for 0.5-2 h; then, rotary evaporated at 25-60 ° C for 15-40 min to remove ethanol, and centrifuged at 1500-3000 r / min for 5-20 min to remove the precipitate, so as to obtain ferroptosis regulating nanoparticles loaded with the model drug; The monounsaturated fatty acid is one of oleic acid (OA), erucic acid (EA), palmitoleic acid (PA), nervonic acid (NA), hexadecenoic acid or eicosenoic acid; The phospholipid polyethylene glycol conjugates with different PEG molecular weights are DSPE-PEG2000-RGD, DSPE-PEG3000-RGD, DSPE-PEG3400-RGD, DSPE-PEG4000-RGD, DSPE-PEG5000-RGD, DSPE-PEG2000-mannose (Man), DSPE-PEG3000-Man, DSPE-PEG3400-Man, DSPE-PEG4000-Man, DSPE-PEG5000-Man, DSPE-PEG2000-iRGD, DSPE-PEG3000-iRGD, DSPE-PEG3400-iRGD, DSPE-PEG4000- -iRGD, DSPE-PEG5000-iRGD, DSPE-PEG2000-folic acid (FA), DSPE-PEG3000-FA, DSPE-PEG3400-FA, DSPE-PEG4000-FA, DSPE-PEG5000-FA, DSPE-PEG2000-hyaluronic acid (HA), D One of SPE-PEG3000-HA, DSPE-PEG3400-HA, DSPE-PEG4000-HA, DSPE-PEG5000-HA, DSPE-PEG2000, DSPE-PEG3000, DSPE-PEG3400, DSPE-PEG4000 or DSPE-PEG5000; The model drug is one of the polyphenol compounds of curcumin (Cur), quercetin (Que), resveratrol, catechin, rutin, baicalein, nobiletin, chlorogenic acid, gallic acid, proanthocyanidin, epigallocatechin gallate, anthocyanidin, luteolin or rutinose.

[0007] The invention discloses an application of unsaturated fatty acid-based ferroptosis regulating nanoparticles prepared by the method of the present invention in preparing a drug for treating diabetes, wherein the diabetes is type 1 diabetes and type 2 diabetes.

[0008] Application of unsaturated fatty acid-based ferroptosis regulating nanoparticles prepared by the method of the present invention in the preparation of drugs for treating pancreatic inflammatory diseases.

[0009] Application of unsaturated fatty acid-based ferroptosis regulating nanoparticles prepared by the method of the present invention in oral drug delivery.

[0010] The invention has abundant raw materials, a simple preparation method, is easy to produce, has good product quality, and stable properties. The prepared lipid nanoparticles have high oral availability and good targeting efficiency. They can effectively deliver polyphenol drugs to the site of pancreatic inflammation, greatly improve drug bioavailability and pancreatic inflammation targeting ability, and inhibit ferroptosis of pancreatic beta cells to play a role in treating diabetes. This is a major innovation in drugs for treating diabetes and pancreatitis, and has huge social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 are atomic force microscopy images of different nanoparticles of the present invention; Figure 2 The Young's modulus diagram of different nanoparticles of the present invention; (Abstract attached) Figure 3 This is a transmission electron microscope image of the nanoparticles of the present invention. DETAILED DESCRIPTION

[0012] The specific implementation of the present invention is described in detail below with reference to the accompanying drawings and examples.

[0013] However, it should be noted that those skilled in the art will appreciate that the specific embodiments and examples provided are intended to illustrate the present invention, rather than to limit the present invention. Throughout the specification, unless otherwise specifically stated, the terms used are to be understood as the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used have the meanings commonly understood by those skilled in the art. If there is a conflict, this specification takes precedence. Various raw materials, reagents, instruments and equipment used in the present invention, etc., can be purchased from the market, or can be prepared by existing methods. Example

[0014] A method for preparing ferroptosis regulating nanoparticles based on unsaturated fatty acids is as follows: Cur, OA and DSPE-PEG2000-RGD are mixed, wherein the mass ratio of Cur to OA is 1:10, and the mass ratio of OA to DSPE-PEG2000-RGD is 4:1, and then the mixture is dissolved in 4.5 mL of ethanol, and ultrasonically mixed at a frequency of 90 Hz to obtain uniform mixture, and then the mixed solution is slowly dropped into 10 mL of deionized water at 45°C while stirring, and stirred at a speed of 800 r / min for 1 hour; finally, the mixture is rotary evaporated at 45°C for 30 minutes to remove the ethanol, and centrifuged at 2200 r / min for 10 minutes to remove the precipitate, thereby obtaining nanoparticles loaded with Cur. Example

[0015] A method for preparing ferroptosis regulating nanoparticles based on unsaturated fatty acids is as follows: Que, EA and DSPE-PEG3000-Man are mixed, wherein the mass ratio of Que to EA is 1:5, and the mass ratio of EA to DSPE-PEG3000-Man is 3.5:1, and then the mixture is dissolved in 4 mL of ethanol, and ultrasonically mixed at a frequency of 60 Hz for uniform mixing, and then the mixed solution is slowly dripped into 5 mL of deionized water at 25°C while stirring, and stirred at a speed of 500 r / min for 0.5 h; finally, the mixture is rotary evaporated at 40°C for 15 min to remove ethanol, and centrifuged at 1500 r / min for 5 min to remove the precipitate, thereby obtaining nanoparticles loaded with Que. Example

[0016] A method for preparing ferroptosis-regulating nanoparticles based on unsaturated fatty acids is as follows: catechin, PA and phospholipid polyethylene glycol conjugates with different PEG molecular weights are mixed, wherein the mass ratio of catechin to PA is 1:15, and the mass ratio of PA to DSPE-PEG3400-iRGD is 4.5:1, and then they are dissolved in 5 mL of ethanol, and ultrasonically mixed at a frequency of 120 Hz for uniform mixing, and then at 60°C, under stirring, the mixed solution is slowly dripped into 15 mL of deionized water, and stirred at a speed of 1200 r / min for 2 h; then, the ethanol is removed by rotary evaporation at 60°C for 40 min, and the precipitate is removed by centrifugation at 3000 r / min for 20 min to obtain nanoparticles loaded with catechins. Example

[0017] A method for preparing ferroptosis regulating nanoparticles based on unsaturated fatty acids is as follows: resveratrol, NA and DSPE-PEG4000-FA are mixed, wherein the ratio of resveratrol to NA is 1:7, and the mass ratio of NA to DSPE-PEG4000-FA is 4.2:1, and then the mixture is dissolved in 3.5 mL of ethanol, and ultrasonically mixed at a frequency of 105 Hz for uniform mixing, and then the mixed solution is slowly dripped into 12 mL of deionized water at 35° C. while stirring, and stirred at a speed of 700 r / min for 1.5 h; then the mixture is rotary evaporated at 35° C. for 30 min to remove ethanol, and centrifuged at 2400 r / min for 16 min to remove the precipitate, thereby obtaining nanoparticles loaded with resveratrol. Example

[0018] A method for preparing ferroptosis regulating nanoparticles based on unsaturated fatty acids is as follows: rutin, hexadecenoic acid and DSPE-PEG5000-HA are mixed, wherein the ratio of rutin to hexadecenoic acid is 1: 12, and the mass ratio of hexadecenoic acid to DSPE-PEG5000-HA is 3.7: 1, and then the mixture is dissolved in 4.5 mL of ethanol, and ultrasonically mixed at a frequency of 75 Hz for uniform mixing, and then the mixed solution is slowly dripped into 7.5 mL of deionized water at 50°C under stirring, and stirred at a speed of 900 r / min for 0.8 h; then the mixture is rotary evaporated at 50°C for 20 min to remove ethanol, and centrifuged at 1800 r / min for 8 min to remove the precipitate, thereby obtaining nanoparticles loaded with rutin. Example

[0019] A method for preparing ferroptosis regulating nanoparticles based on unsaturated fatty acids is as follows: baicalein, eicosenoic acid and DSPE-PEG2000 are mixed, wherein the mass ratio of baicalein to eicosenoic acid is 1: 6, and the mass ratio of eicosenoic acid to DSPE-PEG2000 is 4.3: 1, and then the mixture is dissolved in 3.8 mL of ethanol, and ultrasonically mixed at a frequency of 85 Hz for uniform mixing, and then the mixed solution is slowly dripped into 13 mL of deionized water at 35°C while stirring, and stirred at a speed of 650 r / min for 1.7 h; then the mixture is rotary evaporated at 35°C for 19 min to remove ethanol, and centrifuged at 2750 r / min for 9 min to remove the precipitate, thereby obtaining nanoparticles loaded with baicalein.

[0020] In addition to the embodiments given above, the given raw materials can be mixed in different amounts and many other embodiments can be given under different process conditions, which are not listed here one by one.

[0021] The preparation method of the present invention is simple, and effectively solves the problems of low pancreatic targeting efficiency and poor oral bioavailability of drugs. It not only overcomes the barrier of drug gastrointestinal transport by optimizing the ratio of unsaturated fatty acids, but also promotes intestinal lymphatic transport by modifying the targeting ligand, thereby increasing the enrichment of the preparation in the pancreas. In vitro experiments have shown that the preparation has the effect of inhibiting ferroptosis of mouse pancreatic β cells (such as Min-6 cells), and in vivo experiments have shown that it has a significant ability to inhibit ferroptosis, can repair pancreatic β cell function and effectively reduce blood sugar levels. Taking Example 1 as an example, after repeated experiments for many times, the results are consistent, and the test data are as follows: Curcumin (Cur) is used as a model drug. It can regulate iron metabolism, anti-oxidation and inhibit lipid peroxidation. In vitro and in vivo, Cur reverses the oxidative damage of pancreatic β cells by inhibiting ferroptosis, ultimately achieving the purpose of treating T1DM. However, due to the poor water solubility of Cur, low oral bioavailability, and lack of pancreatic inflammation targeting, oral Cur is difficult to achieve effective therapeutic effects. In order to better exert the therapeutic advantages of Cur, relevant experiments were conducted, as follows:

[0022] The present invention found in the research process that the physical and chemical properties of nanoparticles, such as rigidity, have a great influence on the nanoparticles. Therefore, six nanoparticles with different hardness were prepared for experiments: OA and DSPE-PEG2000-RGD were dissolved in 3-6 mL of ethanol solution at a mass ratio of 0.5:1-1.5:1, 1.5:1-2.5:1, 2.5:1-3.5:1, 3.5:1-4.5:1, 4.5:1-5.5:1, 5.5:1-6.5:1, respectively, and mixed under ultrasonic conditions (60-120 Hz). Then, the mixed solution was slowly dripped into 5-15 mL of deionized water at 25-60 °C and stirred at a speed of 500-1200 r / min for 0.5-2h. Finally, the ethanol was removed by rotary evaporation at 40-60 °C for 15-40 min to obtain 6 nanoparticles with different hardness. According to the mass ratio of the feed materials, they are named nanoparticle 1 (NP1), nanoparticle 2 (NP2), nanoparticle 3 (NP3), nanoparticle 4 (NP4), nanoparticle 5 (NP5) and nanoparticle 6 (NP6).

[0023] Nanoparticle solutions of different hardness were directly dropped onto the cleaned silicon wafer surface and PeakForce Tapping was used to measure the surface of the wafer. ™ The two-dimensional morphology of the sample was measured in the mode, and the force-displacement curve was recorded simultaneously. During imaging, the scanning rate was set to 1 Hz, and a cantilever probe with a polarization sensitivity of 99 nm / V and an elastic modulus of 0.16 N / m was selected. Image processing and data analysis were completed using Nanoscope Analysis software (Bruker). In addition, during the scanning process, an additional external force of 100 pN was applied to observe the deformation of the particles. Finally, the fitted force curve data was analyzed using NanoScope Analysis software, and the Young's modulus of the sample was calculated based on the elastic mechanics contact model.

[0024] The results are as follows Figure 1As shown: Under the condition of applying the same external force, nanoparticles of different hardness exhibit different degrees of deformation. Figure 2 The Young's modulus distribution of the six nanoparticles is shown, with the maximum value being about 900 MPa and the minimum value being about 450 MPa.

[0025] The T1DM mouse model was established by intraperitoneal injection of streptozotocin (STZ) for 5 consecutive days. To study the in vivo distribution characteristics of the drug, IR780 was used as a tracer, and the distribution of NP1, NP2, NP3, NP4, NP5, and NP6 was observed. The mice were fasted 12 h in advance (water was not prohibited), the drug was administered at 0 h, and the pancreas was removed 8 h later, and its accumulation behavior in the pancreatic inflammation site was analyzed by ex vivo imaging.

[0026] Results: The in vivo experimental results showed that NP4 had the strongest accumulation ability at the inflammatory site, so NP4 was selected for the preparation of drug-loaded nanoparticles in subsequent experiments.

[0027] Prepare NP4 of appropriate concentration, take 15 μL and drop it on the surface of pure carbon film copper mesh. After standing for 30 min, absorb the excess sample and observe it under transmission electron microscope after standing.

[0028] The results are as follows Figure 3 As shown: NP4 is spherical, with a particle size of about 200nm, a smooth surface, uniform distribution, and no obvious aggregation phenomenon.

[0029] Weigh 3 portions of 2 mL of NP4 solution, put them into 5-1500KDa dialysis bags, and place them in beakers containing 30 mL phosphate buffer solution (PBS), 30 mL simulated gastric fluid (SGF), and 30 mL simulated intestinal fluid (SIF). Incubate at 5-150r / min in a 37°C constant temperature shaker, take out samples at the specified time of 0, 0.5, 1, 2, 4, and 8h, and measure their particle size and potential changes.

[0030] Results: After NP4 was co-incubated with different solutions, the particle size did not change significantly and remained at around 200 nm, indicating that NP4 had good gastrointestinal stability.

[0031] Pretreatment was performed according to the method of Experiment 2, and the specific experiment was divided into: free group (IR780) and preparation group (NP4@IR780). The mice were fasted 12 h in advance (but not water), and the drug was administered at 0 h. The intestine, lymph nodes, and pancreas were removed at the specified time points (2 h, 4 h, and 8 h), and in vitro imaging was performed to observe its accumulation in different organs.

[0032] Results: The results of in vitro imaging of small animals showed that NP4@IR780 had a longer intestinal retention time. Compared with free IR780, the fluorescence signal of NP4@IR780 was stronger in the intestine, mesenteric lymph nodes (MLN) and pancreas, which were 3 times, 5 times and 10 times that of the free IR780 group, respectively.

[0033] (1) Assessment of ROS levels After culturing Min-6 cells for 24 hours, culture medium containing 100 nM glucose solution was added to induce cell damage. After incubation for 12 hours, the supernatant was discarded, and free Cur, NP4 and Cur@NP4 solutions were added to the set wells, respectively. The experiment was divided into 5 groups: blank carrier group (NP4), free Cur group (Cur), preparation group (Cur@NP4), high glucose damage group (T1DM) and normal group (Normal). After 12 hours, 1 mL of reactive oxygen species fluorescent probe (DCFH-DA) was added to each well, incubated in the dark for 30 minutes, the probe working solution was discarded, PBS was added to wash 3 times, and the fluorescence signal of the probe was observed under a microscope.

[0034] Results: The ROS level can be judged according to the intensity of green fluorescence. The fluorescence of the Normal group was the weakest, and the fluorescence of the T1DM group was the strongest, indicating that the high glucose environment can induce pancreatic β-cell damage and increase the intracellular ROS level. The Cur and Cur@NP4 groups have a good effect of clearing ROS. Among them, the intracellular ROS level of the Cur@NP4 group is equivalent to that of the Normal group, which proves that it can effectively repair damaged mouse Min-6 cells.

[0035] (2) Evaluation of ferrous ion content After culturing Min-6 cells for 24 hours, a medium containing 100 nM glucose solution was added to induce cell damage. After incubation for 12 hours, the supernatant was discarded, and free Cur, NP4 and Cur@NP4 solutions were added to the set wells respectively. The experiment was divided into 5 groups: blank carrier group (NP), free Cur group (Cur), preparation group (Cur@NP4), high glucose damage group (T1DM) and normal group (Normal). After 12 hours, the experimental operation was carried out according to the ferrous ion detection kit. Specifically, 1×106 cells were taken from each group and 0.2 mL of the kit buffer was added. After mixing, they were placed at 4 ℃ for lysis for 30 minutes, and the supernatant was taken for use after centrifugation at 15000g for 30 minutes. After that, 60-120 μL of different concentrations of standard products were added to the standard wells, and 60-120 μL of the test samples were added to the measurement wells and control wells. Add 60-120 μL of reagent 2 to the control well, and add 60-120 μL of reagent 3 to the assay well and standard well. Mix well and incubate at 37°C for 30 min, and measure the OD value of each well at 593 nm on a microplate reader.

[0036] Results: Compared with the T1DM group, CUR@NP4 significantly reduced the intracellular ferrous ion content by about 66.3%. In addition, the intracellular ferrous ion level of Min-6 cells after CUR@NP4 treatment was comparable to that of the Normal group.

[0037] (3) Assessment of lipid peroxidation levels After culturing Min-6 cells for 24 hours, a medium containing 100 nM glucose solution was added to induce cell damage. After incubation for 12 hours, the supernatant was discarded, and free Cur, NP4, and Cur@NP4 solutions were added to the set wells, respectively. The experiment was divided into 5 groups: blank carrier group (NP), free Cur group (Cur), preparation group (Cur@NP4), high glucose damage group (T1DM), and normal group (Normal). After 12 hours, 1 mL of BODIPY 581 / 591 C11 staining working solution was added to each well and incubated at 37ºC for 30 min. Then, the supernatant was discarded, washed twice with PBS, and the intracellular fluorescence signal was observed under a laser confocal microscope.

[0038] Results: The degree of oxidation of the plasma membrane can be determined based on the fluorescence signal. The reduced product of BODIPY 581 / 591 C11 is mainly red fluorescence, and after lipid peroxide oxidation, it is mainly green fluorescence. The Normal group had the strongest red fluorescence and the weakest green fluorescence, while the T1DM group had the weakest red fluorescence and the strongest green fluorescence, indicating that a high-sugar environment can seriously damage the plasma membrane of pancreatic β cells. The Cur@NP4 group can effectively reduce the level of lipid peroxidation and reverse the damage of Min-6 cells.

[0039] The STZ-induced diabetic C57BL / 6 mouse model was used as the research object, and the healthy control group (Normal), high glucose damage group (T1DM), Cur group, NP4 group, Cur@NP4 group and subcutaneous insulin group (INS) were set up. All groups were given oral gavage of Cur at a dose of 5-150 mg / kg, and the subcutaneous insulin group received insulin treatment, and the administration scheme lasted for 12 weeks.

[0040] The weight of mice at the time of successful modeling was recorded as the result of week 0. The blood glucose and weight data of mice were recorded every week. After the treatment cycle, the pancreatic tissue of each group of mice was taken, fixed with 4% paraformaldehyde, embedded in paraffin, sliced ​​and stained, the tissue morphology was observed under a microscope and images were taken, and the remaining tissue was tested for ferrous ions and malondialdehyde (MDA) content in the mouse tissue using a kit.

[0041] Results: After administration of Cur@NP4, blood glucose levels were significantly reduced, and blood glucose showed a steady downward trend. By the 12th week, it gradually stabilized and reached normal levels. The blood glucose level was about 7.1 mmol / L, which was not significantly different from the Normal group and the INS group. The morphological observation of islet cells showed that the islet cells of the mice in the normal group had complete structure, smooth and clear edges, and a large number of cells with uniform distribution; while the islet cells of the mice in the high glucose damage group (T1DM) were significantly atrophied and deformed, with blurred boundaries, irregular morphology, and a significant decrease in the number of nuclei. In contrast, the morphology, number, and nuclear distribution of islet cells in the Cur@NP4 group were significantly improved, similar to those in the Normal group. In addition, Cur@NP4 treatment can effectively reduce the content of ferrous ions and MDA in tissues. The above results show that Cur@NP4 has a good therapeutic effect on TIDM mice.

[0042] While conducting experiments on Example 1, the present invention also conducted the same experiments on other embodiments, and also obtained the same or similar experimental results, which will not be listed one by one here.

[0043] In summary, compared with the prior art, the present invention has the following significant advantages: 1. The lipid nanoparticles prepared by the present invention effectively solve the problems of poor pancreatic targeting efficiency and low oral bioavailability of polyphenol drugs through the dual optimization strategy of regulating hardness and softness by unsaturated fatty acids and modifying targeting ligands, and build a platform for efficient drug delivery for oral drug preparations.

[0044] 2. The lipid nanoparticles prepared by the present invention inhibit the ferroptosis of pancreatic β cells and restore their insulin secretion function, laying a foundation for oral drug treatment of diabetes in a unique way, and are an important innovation in the treatment of diabetes.

[0045] 3. The lipid nanoparticles prepared by the present invention are simple to synthesize and have good stability. The results have been verified by multiple experiments and remain consistent. The raw materials used are low in cost, easy to process and have good biocompatibility, and have huge economic and social benefits.

[0046] It should also be pointed out that the above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession can make changes or modify the technical contents disclosed above into equivalent embodiments with equivalent changes without departing from the scope of the technical solution of the present invention, which are all within the protection scope of the present invention.

Claims

1. A method for preparing ferroptosis-regulating nanoparticles based on unsaturated fatty acids, wherein the lipid nanoparticles are composited with a model drug, a monounsaturated fatty acid, and a phospholipid polyethylene glycol conjugate with different PEG molecular weights, and the method is: The model drug, monounsaturated fatty acid and phospholipid polyethylene glycol conjugates with different PEG molecular weights are mixed, wherein the mass ratio of the model drug to the monounsaturated fatty acid is 1: 5-15, and the mass ratio of the monounsaturated fatty acid to the phospholipid polyethylene glycol conjugates with different PEG molecular weights is 3.5-4.5: 1, and then dissolved in 3-5 mL of ethanol, and ultrasonically mixed at a frequency of 60-120 Hz to be uniform, and then at 25-60 ° C, under stirring, the mixed solution is slowly dripped into 5-15 mL of deionized water, and stirred at a speed of 500-1200 r / min for 0.5-2 h; then, rotary evaporated at 25-60 ° C for 15-40 min to remove ethanol, and centrifuged at 1500-3000 r / min for 5-20 min to remove the precipitate, so as to obtain ferroptosis regulating nanoparticles loaded with the model drug; The monounsaturated fatty acid is one of oleic acid (OA), erucic acid (EA), palmitoleic acid (PA), nervonic acid (NA), hexadecenoic acid or eicosenoic acid; The phospholipid polyethylene glycol conjugates with different PEG molecular weights are DSPE-PEG2000-RGD, DSPE-PEG3000-RGD, DSPE-PEG3400-RGD, DSPE-PEG4000-RGD, DSPE-PEG5000-RGD, DSPE-PEG2000-mannose (Man), DSPE-PEG3000-Man, DSPE-PEG3400-Man, DSPE-PEG4000-Man, DSPE-PEG5000-Man, DSPE-PEG2000-iRGD, DSPE-PEG3000-iRGD, DSPE-PEG3400-iRGD, DSPE-PEG4000- -iRGD, DSPE-PEG5000-iRGD, DSPE-PEG2000-folic acid (FA), DSPE-PEG3000-FA, DSPE-PEG3400-FA, DSPE-PEG4000-FA, DSPE-PEG5000-FA, DSPE-PEG2000-hyaluronic acid (HA), D One of SPE-PEG3000-HA, DSPE-PEG3400-HA, DSPE-PEG4000-HA, DSPE-PEG5000-HA, DSPE-PEG2000, DSPE-PEG3000, DSPE-PEG3400, DSPE-PEG4000 or DSPE-PEG5000; The model drug is one of the polyphenol compounds of curcumin (Cur), quercetin (Que), resveratrol, catechin, rutin, baicalein, nobiletin, chlorogenic acid, gallic acid, proanthocyanidin, epigallocatechin gallate, anthocyanidin, luteolin or rutinose.

2. The method for preparing ferroptosis regulating nanoparticles based on unsaturated fatty acids according to claim 1, characterized in that: The preparation method is as follows: Cur, OA and DSPE-PEG2000-RGD are mixed, wherein the mass ratio of Cur to OA is 1:10, and the mass ratio of OA to DSPE-PEG2000-RGD is 4:1, and then they are dissolved in 4.5 mL of ethanol, and ultrasonically mixed at a frequency of 90 Hz to make them uniform, and then at 45 ° C, under stirring, the mixed solution is slowly dripped into 10 mL of deionized water, and stirred at a speed of 800 r / min for 1 hour; finally, the ethanol is removed by rotary evaporation at 45 ° C for 30 min, and the precipitate is removed by centrifugation at 2200 r / min for 10 min to obtain Cur-loaded nanoparticles.

3. The method for preparing ferroptosis regulating nanoparticles based on unsaturated fatty acids according to claim 1, characterized in that: The preparation method is as follows: Que, EA and DSPE-PEG3000-Man are mixed, wherein the mass ratio of Que to EA is 1:5, and the mass ratio of EA to DSPE-PEG3000-Man is 3.5:1, and then they are dissolved in 4 mL of ethanol, and ultrasonically mixed at a frequency of 60 Hz for uniform mixing, and then the mixed solution is slowly dropped into 5 mL of deionized water at 25 °C while stirring, and stirred at a speed of 500 r / min for 0.5 h; finally, the mixture is rotary evaporated at 40 °C for 15 min to remove the ethanol, and centrifuged at 1500 r / min for 5 min to remove the precipitate, thereby obtaining nanoparticles loaded with Que.

4. The method for preparing ferroptosis regulating nanoparticles based on unsaturated fatty acids according to claim 1, characterized in that: The preparation method is as follows: catechin, PA and phospholipid polyethylene glycol conjugates with different PEG molecular weights are mixed, wherein the mass ratio of catechin to PA is 1:15, and the mass ratio of PA to DSPE-PEG3400-iRGD is 4.5:1, and then they are dissolved in 5 mL of ethanol, and ultrasonically mixed at a frequency of 120 Hz for uniform mixing, and then at 60 ° C, under stirring, the mixed solution is slowly dripped into 15 mL of deionized water, and stirred at a speed of 1200 r / min for 2 h; then, the ethanol is removed by rotary evaporation at 60 ° C for 40 min, and the precipitate is removed by centrifugation at 3000 r / min for 20 min to obtain catechin-loaded nanoparticles.

5. The method for preparing ferroptosis regulating nanoparticles based on unsaturated fatty acids according to claim 1, characterized in that: The preparation method is as follows: resveratrol, NA and DSPE-PEG4000-FA are mixed, wherein the ratio of resveratrol to NA is 1: 7, and the mass ratio of NA to DSPE-PEG4000-FA is 4.2: 1, and then they are dissolved in 3.5 mL of ethanol, and ultrasonically mixed at a frequency of 105 Hz for uniform mixing, and then the mixed solution is slowly dripped into 12 mL of deionized water at 35 ° C with stirring, and stirred at a speed of 700 r / min for 1.5 h; then, the mixture is rotary evaporated at 35 ° C for 30 min to remove ethanol, and centrifuged at 2400 r / min for 16 min to remove the precipitate, thereby obtaining nanoparticles loaded with resveratrol.

6. The method for preparing ferroptosis regulating nanoparticles based on unsaturated fatty acids according to claim 1, characterized in that: The preparation method is as follows: rutin, hexadecenoic acid and DSPE-PEG5000-HA are mixed, wherein the ratio of rutin to hexadecenoic acid is 1:12, and the mass ratio of hexadecenoic acid to DSPE-PEG5000-HA is 3.7:1, and then dissolved in 4.5 mL of ethanol, and ultrasonically mixed at a frequency of 75 Hz to make it uniform, and then the mixed solution is slowly dripped into 7.5 mL of deionized water at 50 ° C under stirring, and stirred at a speed of 900 r / min for 0.8 h; The mixture was then rotary evaporated at 50°C for 20 min to remove the ethanol, and centrifuged at 1800 r / min for 8 min to remove the precipitate to obtain the rutin-loaded nanoparticles.

7. The method for preparing ferroptosis regulating nanoparticles based on unsaturated fatty acids according to claim 1, characterized in that: The preparation method is as follows: baicalein, eicosenoic acid and DSPE-PEG2000 are mixed, wherein the mass ratio of baicalein to eicosenoic acid is 1: 6, and the mass ratio of eicosenoic acid to DSPE-PEG2000 is 4.3: 1, and then dissolved in 3.8 mL of ethanol, ultrasonically mixed at a frequency of 85 Hz for uniform mixing, and then slowly dripped into 13 mL of deionized water at 35 ° C with stirring, and stirred at a speed of 650 r / min for 1.7 hours; then rotary evaporated at 35 ° C for 19 minutes to remove ethanol, and centrifuged at 2750 r / min for 9 minutes to remove the precipitate, thereby obtaining nanoparticles loaded with baicalein.

8. Use of unsaturated fatty acid-based ferroptosis regulating nanoparticles prepared by the preparation method according to any one of claims 1 to 6 in the preparation of a drug for treating diabetes, wherein the diabetes is type 1 diabetes and type 2 diabetes.

9. Use of unsaturated fatty acid-based ferroptosis-regulating nanoparticles prepared by the preparation method of any one of claims 1 to 6 in the preparation of drugs for treating pancreatic inflammatory diseases.

10. Use of unsaturated fatty acid-based ferroptosis-regulating nanoparticles prepared by the preparation method of any one of claims 1 to 6 in oral drug delivery.

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