An arachidonic acid micelle prepared from amino acids, a preparation method thereof and an application thereof

Abstract

The present invention belongs to the field of biomedicine, and particularly relates to an arachidonic acid micelle prepared from amino acids, a preparation method thereof and an application. The present invention uses a variety of essential amino acids in the human body to prepare an arachidonic acid micelle. The components of the micelle are only amino acids and arachidonic acid, and the components contained in the micelle are all inherent substances in the human body, with excellent biocompatibility. The prepared micelle has excellent stability and dispersibility in physiological saline. The micelle preparation process is simple and convenient, and has low requirements for equipment. In terms of function, the micelle can induce significant lipid peroxidation in tumor cells, resulting in ferroptosis of tumor cells. At the same time, in combination with traditional tumor treatment methods, it also shows a significant anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to arachidonic acid micelles prepared from amino acids, and a preparation method and application thereof. Background Art

[0002] Ferroptosis is a type of programmed cell death mediated by excessive lipid peroxidation caused by an intracellular redox imbalance. This death process damages the polyunsaturated fatty acids on the cell membrane and mitochondrial membrane by oxidation, thereby damaging the cell membrane or mitochondrial membrane and killing the cell (Brent R. Cell, 2022, 185: 2401-2421). Currently, a large number of studies have shown that ferroptosis is mainly caused by the peroxidation of phospholipids containing polyunsaturated fatty acids, and arachidonic acid is one of the important components of polyunsaturated fatty acids in cells (Kagan V., et al. Nature Chemical Biology, 2017, 13: 81-90).

[0003] Currently, the main methods of inducing ferroptosis of tumor cells include: 1. Increasing the level of intracellular arachidonic acid through dietary intake and other means to provide sufficient polyunsaturated fatty acid phospholipids for tumor cell ferroptosis (Doll S., et al. Nature Chemical Biology, 2017, 13:91-98); 2. Delivering metal ions such as ferrous ions and manganese ions that are prone to Fenton reactions or Fenton-like reactions to increase the level of intracellular reactive oxygen species and promote the oxidation of polyunsaturated fatty acids in cell biomembranes (Y Liu, et al. Journal of the American Chemical Society, 2023, 145:8965-8978); 3. Using glutathione peroxidase 4 (GPX4) inhibitors to inhibit the synthesis of glutathione, thereby disrupting the redox balance in tumor cells and inducing ferroptosis of tumor cells (Shimada K., et al. Nature Chemical Biology, 2016, 12:497-503). In addition, some studies have also successfully induced the occurrence of tumor cell ferroptosis through ultrasound therapy (TNie, et al. Advanced Materials, 2022, 34: 2206286), laser or near-infrared light irradiation (YZhou, et al. Advanced Healthcare Materials, 2023, 12: 2370170), and X-ray irradiation (G Lei, et al. Cell Research, 2020, 30: 146-162).

[0004] However, there are many restrictions in the above-mentioned ways of inducing tumor ferroptosis. Metal elements such as iron and manganese are difficult to metabolize in the body and have potential biological toxicity; GPX4 inhibitors such as RSL3, FIN56, and erastin lack targeting to tumor cells and are prone to cause systemic drug toxicity, restricting their application; treatment methods such as ultrasound, light, and radiation have a series of problems such as complex treatment methods and poor treatment prognosis. Arachidonic acid, as an inherent component in the human body, has excellent biocompatibility and is one of the preferred strategies for inducing ferroptosis. However, due to the physical properties of arachidonic acid, it cannot be dispersed in physiological saline and there are difficulties in high-dose intravenous and intratumoral drug administration. Therefore, at present, only oral intake can be used to regulate the arachidonic acid level in patients, and the regulation process is slow and the effect is not good.

[0005] In response to the current difficulties in the delivery of arachidonic acid, a large number of researchers have developed delivery systems based on arachidonic acid for inducing ferroptosis in tumor cells, which mainly include loading with amphiphilic liposome molecules, self-assembling micelles conjugated with amphiphilic polymers, and loading with hydrogel carriers, etc. Regarding the loading of amphiphilic liposome molecules, there are literature reports on the use of amphiphilic polyethylene glycol-phospholipid liposomes modified with disulfide bonds to load arachidonic acid and the ferroptosis inducer FIN56 for tumor treatment (X Sun, et al. Chemical Engineering Journal, 2023, 451: 138991). There are also literature reports on the use of amphiphilic polyethylene glycol-phospholipid molecules to simultaneously load arachidonic acid, the photosensitizer pyropheophorbide A, and heme, and the singlet oxygen released by the photosensitizer under near-infrared light irradiation is used to oxidize polyunsaturated fatty acids to induce ferroptosis in tumor cells (S Zhang, et al. ACS Applied Materials & Interfaces, 2022, 14: 38497-38505). Regarding the self-assembling micelles conjugated with amphiphilic polymers, there are literature reports on conjugating polyethylene glycol-poly(lysine) polymers on arachidonic acid molecules to self-assemble the micelles, and then delivering arachidonic acid in vivo to achieve the effect of tumor treatment (M Gao, et al. Biomaterials, 2019, 223: 119486). There are also literature reports on conjugating amphiphilic dextran on arachidonic acid molecules to self-assemble the conjugated polymer micelles to deliver arachidonic acid and induce ferroptosis in tumor cells (R Guo, et al. Nano Letter, 2023, 23: 3401-3411). In addition, there are literature reports on the use of injectable thermosensitive hydrogels to load and deliver arachidonic acid and the ferroptosis inducer RSL3 to induce ferroptosis in tumor cells (X Chen, et al. Biomaterials, 2023, 298: 122139).

[0006] Although these carriers have successfully achieved the in - vivo delivery of arachidonic acid, the introduction of non - functional carriers and the modification of arachidonic acid molecules have greatly increased the preparation difficulty of the drug - loaded system and have had a certain impact on the therapeutic effect and biocompatibility of arachidonic acid. There is no relevant report in the existing literature on the preparation of arachidonic acid micelles using amino acids. Therefore, preparing a micelle delivery system for arachidonic acid using two natural components inherent in the human body, amino acids and arachidonic acid, can greatly ensure the therapeutic effect of the drug while not changing the properties of the components. Summary of the Invention

[0007] The following problems exist in the prior art:

[0008] 1. Currently, conventional means of inducing ferroptosis in tumor cells, such as dietary regulation of arachidonic acid intake, delivery of metal ions, delivery of GPX4 inhibitors, light, sound, radiotherapy, etc., have defects such as low treatment efficiency, complex treatment methods, poor biocompatibility, and obvious toxic and side effects;

[0009] 2. Arachidonic acid is difficult to disperse in physiological saline and is difficult to administer in large doses through intravenous and intratumoral routes;

[0010] 3. Existing arachidonic acid delivery systems need to introduce non - functional drug carriers, increasing the toxic and side effects of the drug delivery system and the metabolic burden on the body;

[0011] 4. Polymer conjugation modification of arachidonic acid molecules changes the molecular structure of arachidonic acid, affecting the function of arachidonic acid to a certain extent, and it is extremely easy to cause oxidation of unsaturated bonds in arachidonic acid molecules during the conjugation modification process, with a large preparation difficulty.

[0012] To solve the above - mentioned existing technical problems, the present application provides the following technical solutions:

[0013] One of the objectives of the present invention is to provide a preparation method of arachidonic acid micelles prepared from amino acids, comprising the following steps:

[0014] S1: Dissolve arachidonic acid in an organic solvent, add an aqueous solution of amino acids after rotary evaporation, and obtain a clear solution;

[0015] S2: Concentrate and purify the clear solution to obtain the arachidonic acid micelles prepared from amino acids; in the arachidonic acid micelles prepared from amino acids, the diameter of the micelles is 80 - 150 nm, and the molar ratio of amino acids to arachidonic acid is 1:1.75 - 2.

[0016] Preferably, the amino acid is selected from one or more of arginine, lysine, taurine, glutamine, glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, threonine, aspartic acid, glutamic acid, and histidine.

[0017] Further, the amino acid is selected from arginine, lysine, histidine, taurine or glutamine.

[0018] Preferably, in the step S1, the mass ratio of the amino acid to arachidonic acid is 2 - 10:3.

[0019] Preferably, in the step S1, the temperature of rotary evaporation is 30 - 40 °C.

[0020] Preferably, in the step S1, the mixing method is ultrasonic oscillation for 5 - 15 min; the temperature of the ultrasonic oscillation is lower than 30 °C, and the ultrasonic power is 100 W.

[0021] Preferably, in the aqueous solution of the amino acid, the pH value is 10 - 12, and the concentration of the amino acid is 20 - 100 mg / mL.

[0022] Further, the pH of the aqueous solution of the amino acid is adjusted with a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 1 mol / L.

[0023] Preferably, in the step S2, filtration is performed with a filter membrane before and after concentration and purification.

[0024] Further, the filter membrane is a mixed cellulose filter membrane with a pore size of 0.22 μm.

[0025] Preferably, in the step S2, centrifugal ultrafiltration is performed before concentration and purification; during centrifugal ultrafiltration, the molecular weight cut-off of the ultrafiltration tube is 10 - 100 kDa, the centrifugal force is 1500 - 2000 g, and the centrifugation time is 15 - 45 min.

[0026] Specifically, the preparation method of the arachidonic acid micelle prepared from an amino acid includes the following steps:

[0027] Dissolve arachidonic acid in dichloromethane, remove the solvent by rotary evaporation, and disperse arachidonic acid in a thin film on the bottle wall. Add the amino acid solution, ultrasonically oscillate until the solution is clear, remove impurities using a filter membrane, then use an ultrafiltration tube to remove excess raw materials and concentrate the micelle solution, and filter the concentrated solution again with a filter membrane to obtain the arachidonic acid micelle prepared from an amino acid.

[0028] The present invention also provides an arachidonic acid micelle prepared from an amino acid by the above preparation method, which can induce ferroptosis of tumor cells.

[0029] The present invention also provides the use of the above-mentioned arachidonic acid micelles prepared from amino acids in the preparation of antitumor drugs.

[0030] Existing ferroptosis induction methods, such as increasing the level of arachidonic acid in the body through diet, delivering metal elements, taking GPX4 inhibitors, light, sound, radiation therapy, etc., all have certain defects.

[0031] The present invention uses a variety of essential amino acids in the human body to prepare arachidonic acid micelles. The components of the micelles are only amino acids and arachidonic acid. The components contained in the micelles are all inherent substances in the human body, and have excellent biocompatibility. The prepared micelles have excellent stability and dispersibility in physiological saline. The process of preparing the micelles is simple and convenient, and the requirements for equipment are relatively low. In terms of function, the micelles can induce significant lipid peroxidation in tumor cells, leading to ferroptosis of tumor cells. At the same time, combined with traditional tumor treatment methods, they also show significant antitumor effects.

[0032] The technical solution of the present invention has the following advantages compared with the prior art:

[0033] To solve the problems of low treatment efficiency, complex treatment methods, poor biocompatibility, large toxic and side effects, etc. existing in the existing methods of inducing ferroptosis in tumor cells, the present invention prepares an arachidonic acid micelle with excellent stability and dispersibility in a physiological environment, and induces ferroptosis of tumor cells by injecting the micelle intravenously and into the tumor, so as to achieve a significant inhibitory effect on tumors. At the same time, the amino acids used to prepare the micelles have certain functions, can enhance the function of immune cells after antitumor treatment, and have a certain inhibitory effect on metastatic tumors and recurrent tumors.

[0034] Arachidonic acid is a fatty acid that is liquid at room temperature. This fatty acid is poorly soluble in water and cannot be dispersed in physiological saline for injection, which greatly limits its application in clinical treatment. Currently, the commonly used arachidonic acid delivery strategies include liposome delivery, polymer conjugate self-assembly, hydrogel delivery and other routes. The above routes introduce some non-functional carriers or change the molecular structure of arachidonic acid, increase the preparation difficulty of the drug delivery system, and at the same time increase the toxic and side effects of the drug and the metabolic burden of the body, and to a certain extent affect the therapeutic effect of arachidonic acid.

[0035] The present invention only assembles amino acids and arachidonic acid to form micelles. The micelles have excellent dispersibility and stability in physiological saline. At the same time, the micelle system only contains polyunsaturated fatty acids and amino acid components, and both components are inherent substances in the human body and have excellent biocompatibility. In addition, the present invention verifies the excellent antitumor effect and biocompatibility of the prepared micelles by combining with traditional tumor treatment methods through cell and in vivo experiments. Description of the Drawings

[0036] Figure 1 Transmission electron microscopy image of arachidonic acid micelles prepared from arginine, lysine, taurine, and glutamine;

[0037] Figure 2 High performance liquid chromatography analysis chart of arachidonic acid micelles prepared from taurine;

[0038] Figure 3 Lipid peroxidation map of tumor cells induced by arachidonic acid micelles combined with X-ray irradiation;

[0039] Figure 4 Monitoring chart of the therapeutic effect of arachidonic acid micelles prepared from taurine combined with X-ray irradiation on tumor-bearing mice. Detailed implementation mode

[0040] As introduced in the background art, there is no relevant record in the prior art of preparing micelles by using amino acids and arachidonic acid. To solve the above technical problems, the present application proposes an arachidonic acid micelle prepared from amino acids and its application in tumor treatment.

[0041] A typical implementation mode of the present application provides an arachidonic acid micelle, which is a liquid micelle with a diameter of 80 - 150 nm. The micelle includes amino acids and arachidonic acid. In the prepared micelle, the molar ratio of amino acids to arachidonic acid is 1:1.75 - 2.

[0042] Through the research of the present application, it is found that by combining different types and concentrations of amino acids with arachidonic acid, arachidonic acid micelles with different morphologies can be prepared.

[0043] Preferably, the types of amino acids are: arginine, lysine, taurine, and glutamine, and the concentration used in the preparation process is 25 mg / mL. The amount of arachidonic acid used in the preparation is 60 mg, and the molar ratio of amino acids to arachidonic acid in the prepared micelle is 1:1.75 - 2.

[0044] Another implementation mode of the present application provides a preparation method of the above arachidonic acid micelle. According to a predetermined material ratio, dissolve arachidonic acid in dichloromethane, and remove the solvent through a rotary evaporator. Add a certain volume of a pre-prepared amino acid solution with a certain concentration, and perform ultrasonic dispersion. Then use a filter membrane for filtration. After filtration, use an ultrafiltration tube for purification and concentration, and finally filter the concentrated solution again with a filter membrane. Further preferably, the water bath temperature during the rotary evaporation process is 40 °C, the filter membrane used for filtration is a 0.22 μm mixed cellulose filter membrane, the molecular weight cut-off of the ultrafiltration tube is 100 kDa, and the ultrafiltration centrifugal force is 2000 g.

[0045] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in combination with specific embodiments.

[0046] Material source

[0047] Arginine and lysine were purchased from Shanghai Titan Technology Co., Ltd., taurine, glutamine, and arachidonic acid were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., ultrafiltration tubes were purchased from Merck Group, Germany, and mixed cellulose filter membranes were purchased from Suzhou Suker Precision Instruments Co., Ltd.

[0048] Example 1

[0049] Dissolve 60 mg of arachidonic acid in 2 mL of dichloromethane, and evaporate the solvent at 40 °C using a rotary evaporator. Prepare an aqueous solution of taurine at 25 mg / mL, and adjust the pH value of the solution to 10 using 1 M sodium hydroxide. Take 2 mL of the taurine solution after adjusting the alkali, add it to the arachidonic acid after rotary evaporation, ultrasonically oscillate for 10 minutes until the solution is clear and transparent, and filter the obtained product using a 0.22 μm mixed cellulose filter membrane. After filtration, dilute with water to 15 mL, add it to a 100 kDa ultrafiltration tube, centrifuge at a centrifugal force of 2000 g for 20 minutes, collect the concentrated solution, and filter it again using a 0.22 μm mixed cellulose filter membrane. The collected liquid is the aqueous dispersion of taurine arachidonic acid micelles.

[0050] Example 2

[0051] Dissolve 60 mg of arachidonic acid in 2 mL of dichloromethane, and evaporate the solvent at 40 °C using a rotary evaporator. Prepare an aqueous solution of arginine at 25 mg / mL. Take 2 mL of the arginine solution, add it to the arachidonic acid after rotary evaporation, ultrasonically oscillate for 10 minutes until the solution is clear and transparent, and filter the obtained product using a 0.22 μm mixed cellulose filter membrane. After filtration, dilute with water to 15 mL, add it to a 100 kDa ultrafiltration tube, centrifuge at a centrifugal force of 2000 g for 20 minutes, collect the concentrated solution, and filter it again using a 0.22 μm mixed cellulose filter membrane. The collected liquid is the aqueous dispersion of arginine arachidonic acid micelles.

[0052] Example 3

[0053] Dissolve 60 mg of arachidonic acid in 2 mL of dichloromethane, and evaporate the solvent at 40 °C using a rotary evaporator. Prepare an aqueous lysine solution with a concentration of 25 mg / mL. Take 2 mL of the lysine solution and add it to the arachidonic acid after rotary evaporation. Ultrasonically oscillate for 10 minutes until the solution becomes clear and transparent, and filter the resulting product using a 0.22 μm mixed cellulose filter membrane. After filtration, dilute with water to 15 mL, add it to a 100 kDa ultrafiltration tube, centrifuge at a centrifugal force of 2000 g for 20 minutes, collect the concentrated solution, and filter it again using a 0.22 μm mixed cellulose filter membrane. The collected liquid is the aqueous dispersion of lysine arachidonic acid micelles.

[0054] Example 4

[0055] Dissolve 60 mg of arachidonic acid in 2 mL of dichloromethane, and evaporate the solvent at 40 °C using a rotary evaporator. Prepare an aqueous glutamine solution with a concentration of 25 mg / mL, and adjust the pH value of the solution to 10 using 1 M sodium hydroxide. Take 2 mL of the glutamine solution after adjusting the alkali and add it to the arachidonic acid after rotary evaporation. Ultrasonically oscillate for 10 minutes until the solution becomes clear and transparent, and filter the resulting product using a 0.22 μm mixed cellulose filter membrane. After filtration, dilute with water to 15 mL, add it to a 100 kDa ultrafiltration tube, centrifuge at a centrifugal force of 2000 g for 20 minutes, collect the concentrated solution, and filter it again using a 0.22 μm mixed cellulose filter membrane. The collected liquid is the aqueous dispersion of glutamine arachidonic acid micelles.

[0056] Effect evaluation 1

[0057] Transmission electron microscope image

[0058] The transmission electron microscope pictures of the samples prepared in Examples 1-4 are as Figure 1 shown. The arachidonic acid micelles prepared from arginine, lysine, taurine, and glutamine all have a diameter of 80-150 nm, and a clear micelle structure can be seen from the transmission electron microscope image;

[0059] High performance liquid chromatography analysis

[0060] High performance liquid chromatography analysis of the arachidonic acid micelles prepared from taurine in Example 1 ( Figure 2 ) shows that before and after the preparation of the micelles, the taurine component and the arachidonic acid component in the micelles have not changed, ensuring the functionality of the micelles;

[0061] Experiment on the lipid peroxidation of tumor cells induced by the combination of micelles and X-rays

[0062] After incubating the prepared arachidonic acid micelles with mouse colon cancer cells for 6 hours, irradiate them with 6 Gy using an X-ray irradiator. After irradiation, continue incubating for 24 hours, stain the cells with a lipid peroxide probe, and analyze the lipid peroxidation level of the cells using a flow cytometer. As Figure 3 can be seen, arachidonic acid micelles can significantly induce lipid peroxidation in cells. At the same time, under the induction of radiation, the lipid peroxidation level of the micelles has increased significantly.

[0063] Antitumor treatment experiment on tumor-bearing mice

[0064] Treat the tumors of tumor-bearing mice with the prepared arachidonic acid micelles by intratumoral injection. The micelles are injected into the mice 3 times at a dose of 5 mg / kg on days 0, 2, and 4. After each administration, irradiate the mice with X-rays for 6 hours. The dose of each radiotherapy is 2 Gy. As Figure 4 can be seen, the radiotherapy effect of the mice after administration is significantly improved compared with that of the mice without administration.

[0065] Obviously, the above embodiments are only examples clearly described and not limitations on the implementation manners. For those of ordinary skill 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 manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A preparation method of arachidonic acid micelles prepared from taurine, characterized in that, It includes the following steps: S1: Dissolve arachidonic acid in an organic solvent, and after rotary evaporation, add an aqueous solution of taurine and mix to obtain a clear solution; S2: Concentrate and purify the clear solution to obtain the arachidonic acid micelles prepared from taurine; in the arachidonic acid micelles prepared from taurine, the diameter of the micelles is 80 - 150 nm, and the molar ratio of taurine to arachidonic acid is 1:1.75 - 2.

2. The preparation method according to claim 1, wherein In the step S1, the mass ratio of taurine to arachidonic acid is 2 - 10:

3.

3. The preparation method according to claim 1, characterized in that, In the step S1, the temperature of rotary evaporation is 30 - 40 °C.

4. The preparation method according to claim 1, wherein In the step S1, the mixing method is ultrasonic oscillation for 5 - 15 min; the temperature of the ultrasonic oscillation is lower than 30 °C, and the ultrasonic power is 100 W.

5. The preparation method according to claim 1, characterized in that, In the aqueous solution of taurine, the pH value is 10 - 12, and the concentration of taurine is 20 - 100 mg / mL.

6. The preparation method according to claim 1, characterized in that, In the step S2, filtration is performed using a filter membrane before and after concentration and purification.

7. The preparation method according to claim 1, characterized in that In the step S2, ultrafiltration by centrifugation is performed before concentration and purification; during the ultrafiltration by centrifugation, the molecular weight cut-off of the ultrafiltration tube is 10 - 100 kDa, the centrifugal force is 1500 - 2000 g, and the centrifugation time is 15 - 45 min.

8. Arachidonic acid micelles prepared from taurine prepared by the preparation method according to any one of claims 1 - 7.

9. Use of the arachidonic acid micelles prepared from taurine according to claim 8 in the preparation of a drug for treating colon cancer.

Images