An EGCG-Fe-Cur spherical nanomolecule and its application in preventing radiation damage.

Abstract

The application relates to a nanomolecular synthesis technology, in particular to EGCG-Fe-Cur spherical nanomolecules and application thereof in preventing radiation damage. 3+ The EGCG-Fe-Cur spherical nanomolecules can be dissociated in a weak acid environment, release curcumin, Fe 3+ , and EGCG, respectively play the roles of removing free radicals, resisting oxidation, protecting DNA, and the like, significantly inhibit iron death and apoptosis induced by rays, and play the anti-radiation role.

Description

Technical Field

[0001] This invention relates to a nanomolecule synthesis technique, specifically to an EGCG-Fe-Cur spherical nanomolecule and its application in preventing radiation damage. Background Technology

[0002] The national economy's demand for energy has increased significantly, and there is a risk of exposure to high doses of ionizing radiation during nuclear energy generation and nuclear power plant accidents. Currently, patients with severe (6 Gy) or lower myelopathic radiation sickness can receive good treatment and have a high survival rate. However, there are still no effective treatments for patients with extremely severe (above 6 Gy) myelopathic radiation sickness. The development of anti-radiation drugs targeting high-dose ionizing radiation damage has been a key focus.

[0003] Epigallocatechin gallate (EGCG) is highly water-soluble and possesses various effects including free radical scavenging, antioxidant, anti-radiation, DNA protection, anti-apoptosis, and anti-tumor activity. EGCG is a major component of green tea polyphenols and is a catechin monomer isolated from tea leaves. It is an ester formed from 2-phenylbenzopyran and gallic acid, exhibiting the versatility of phenolic antioxidants. Due to the presence of six ortho- and ortho-phenolic hydroxyl groups in its structure, it possesses many properties superior to other catechins and exhibits a wide range of pharmacological activities. EGCG scavenge free radicals, resists free radical DNA damage, and its antioxidant activity is at least 100 times that of vitamin C and 25 times that of vitamin E. It can protect cells and DNA from damage; these effects are attributed to EGCG's ability to scavenge oxygen free radicals (antioxidant). EGCG has anti-radiation and anti-ultraviolet radiation properties, prevents lipid peroxidation, reduces serum levels of low-density cholesterol, very low-density cholesterol, and triglycerides, interferes with signal transduction required for cancer cell survival, inhibits carcinogens in the diet, and works with other enzymes and antioxidants in the intestines, liver, and lungs to inhibit the activity of certain carcinogens. In mice, before exposure to cobalt-60 radiation, the blood counts in the EGCG-orally administered group were significantly higher than those in the gamma-ray irradiation group alone; after irradiation, the blood counts in the EGCG-orally administered group significantly improved compared to the gamma-ray irradiation group alone, indicating that EGCG can alleviate or promote the recovery of blood system damage. Monzen and Kashiuakura also found that EGCG has a radioprotective effect on the hematopoietic system. Animal model studies showed that EGCG can alleviate gamma-ray-induced spleen cell damage in rats. It can increase the thymus index, spleen index, and cell number in tumor-bearing mice, indicating that EGCG promotes the immune function of tumor-bearing mice. However, studies on EGCG radiation resistance have mostly focused on cell irradiation and whole-body animal irradiation doses within the range of 0.05-4 Gy, with some studies exceeding this range for HaCaT cells in the skin. Even the 22 Gy irradiation of rat lungs was only localized, and there are very few reports of high-dose cell irradiation or whole-body animal irradiation.

[0004] Curcumin (Cur), as a nonsteroidal anti-inflammatory drug, possesses broad-spectrum preventative properties against diseases, exhibiting various pharmacological effects including anti-infection, anti-tumor, antiviral, antibacterial, antioxidant, anticoagulant, and anti-liver fibrosis. Studies on its anti-radiation effects have primarily focused on cell irradiation and whole-body animal irradiation doses within the 1.15-4 Gy range, with some studies showing 9.6-10 Gy doses in mice and rats, but the anti-radiation effects were unsatisfactory. Even 13.6 Gy irradiation of the mouse chest was only localized, mimicking a radiation-induced lung injury model. Curcumin also has certain drawbacks, such as low water solubility, poor stability, and low absorption rate. It is easily converted into complexes like glucuronic acid and sulfonic acid in the intestine, and its rapid metabolism and short half-life contribute to its low bioavailability, limiting its application in the food and pharmaceutical fields. Summary of the Invention

[0005] The purpose of this invention is to improve an EGCG-Fe-Cur spherical nanomolecule and its application in preventing radiation damage. This invention utilizes nano-self-assembly technology to integrate curcumin and Fe... 3+ By combining EGCG with other molecules, a stable spherical nanomolecule, EGCG-Fe-Cur, was synthesized. This nanomolecule not only has good biosafety but also significant anti-radiation effects, providing a new approach to prevent radiation damage.

[0006] The technical solution of this invention is:

[0007] The EGCG-Fe-Cur spherical nanomolecules synthesized by nano-self-assembly optimization contain: epigallocatechin gallate, curcumin and ferric chloride hexahydrate.

[0008] This invention first utilizes nano-self-assembly technology to integrate curcumin and Fe... 3+ By combining EGCG and Fe-Cur, a stable spherical nanomolecule EGCG-Fe-Cur was synthesized. The spherical nanomolecule EGCG-Fe-Cur described in this invention exhibits significant anti-radiation effects both in vitro and in vivo. The synthesis method of the EGCG-Fe-Cur spherical nanomolecule described in this invention is simple, the reaction conditions are mild, it is easy to operate, and it shows good prospects for anti-radiation.

[0009] The EGCG-Fe-Cur spherical nanomolecules of this invention can be prepared by the following method:

[0010] 1) Prepared from mother liquor containing epigallocatechin gallate, curcumin, and ferric chloride hexahydrate;

[0011] 2) Synthesis of EGCG-Fe-Cur spherical nanomolecules: Epigallocatechin gallate (EGCG), curcumin (Cur), and ferric chloride hexahydrate mother liquor were mixed, and Tris-HCl pH 8.8 buffer was added dropwise to adjust the pH of the solution to 8.2. The reaction was carried out in a sealed environment for 3 hours. After dialysis, the resulting EGCG-Fe-Cur spherical nanomolecules were obtained.

[0012] The preparation method described in step 1) is to dissolve epigallocatechin gallate, curcumin and ferric chloride hexahydrate in ethanol to prepare a stock solution, wherein the concentration of the epigallocatechin gallate solution is 1.5-4.5 mg / mL, the concentration of the curcumin solution is 2-6 mg / mL, and the concentration of the ferric chloride solution is 0.3-0.7 mg / mL.

[0013] In step 2), the molar ratio of epigallocatechin gallate, curcumin, and ferric chloride hexahydrate in the mother liquor mixture is 2–6:3–9:1.

[0014] The EGCG-Fe-Cur spherical nanomolecules described in this invention can be dissociated in a weakly acidic environment, releasing curcumin and Fe. 3+ EGCG and Fe-Cur exert their respective functions of scavenging free radicals, antioxidation, and DNA protection, significantly inhibiting radiation-induced ferroptosis and apoptosis to exert anti-radiation effects. Therefore, the EGCG-Fe-Cur spherical nanomolecules of this invention can be used to prevent radiation damage. The EGCG-Fe-Cur spherical nanomolecules of this invention can also be used to inhibit ferroptosis and apoptosis in cells.

[0015] The present invention also provides a formulation for preventing radiation damage, comprising the aforementioned EGCG-Fe-Cur spherical nanomolecules.

[0016] The present invention also provides an inhibitor of ferroptosis and apoptosis, comprising the aforementioned EGCG-Fe-Cur spherical nanomolecules.

[0017] The present invention also provides a reactive oxygen species scavenging agent comprising the aforementioned EGCG-Fe-Cur spherical nanomolecules.

[0018] Using nano-self-assembly technology, curcumin and Fe 3+By combining curcumin and EGCG, a stable spherical nanomolecule EGCG-Fe-Cur is formed. This self-assembled molecule has the following advantages: ① Improves the water solubility of curcumin and EGCG; ② Improves bioavailability; ③ It is stable in the blood and will not be metabolized into other substances, but will be released only after reaching the damaged target organ; ④ In addition to the pharmacological effects of curcumin and EGCG in scavenging free radicals and anti-oxidation, it can also significantly increase the anti-radiation effect of the complex and increase the anti-radiation effect against high doses of irradiation.

[0019] To make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the autonomous assembly and synthesis of EGCG-Fe-Cur spherical nanomolecules.

[0021] Figures 2A-2F Characterized as EGCG-Fe-Cur spherical nanomolecules, among which Figure 2A This is a transmission electron microscope (TEM) image. Figure 2B This is a hydrated particle size distribution diagram. Figure 2C This is a Zeta potential diagram. Figure 2D This is a UV absorption spectrum. Figure 2E This is an infrared spectrum. Figure 2F To qualitatively determine iron ions using methylene blue.

[0022] Figures 3A-3D The effects of different doses of gamma-ray irradiation and different concentrations of EGCG-Fe-Cur spherical nanomolecules on the survival of AHH-1 cell line were investigated. Figure 3A To determine cell survival after 24 hours of irradiation with different doses of gamma rays, Figure 3B To determine cell survival after 48 hours of irradiation with different doses of gamma rays, Figure 3C Figure D shows cell survival after treatment with different concentrations of EGCG-Fe-Cur for 24 hours. Figure D shows cell survival after treatment with different concentrations of EGCG-Fe-Cur for 48 hours.

[0023] Figure 4 To evaluate the radiation protection effect of 12 Gy γ-ray radiation after 12 h of pretreatment with different drugs and 48 h of post-treatment with CCK8.

[0024] Figure 5 shows the ROS and lipid peroxidation levels of AHH-1 cells after 12 h of different drug treatments and 48 h of 12 Gy γ-ray irradiation.

[0025] Figures 6A-6B EGCG-Fe-Cur spherical nanomolecules promote cell survival by inhibiting ferroptosis. Figure 6AAHH-1 cells were treated with EGCG-Fe-Cur for 12 h, and with different concentrations of the ferroptosis inducer FIN56 for 48 h. CCK8 assay was used to detect cell viability. Figure 6B Pretreatment with 8 μM MEGCG-Fe-Cur for 12 h, followed by treatment with FIN56 (400 nM) for 6 h, and flow cytometry was used to detect lipid peroxidation levels in AHH-1 cells.

[0026] Figure 7 EGCG-Fe-Cur spherical nanomolecules promote cell survival by inhibiting apoptosis. Detailed Implementation

[0027] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0028] The specific embodiments of the present invention will be further described below. Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0029] Example 1: Self-assembly synthesis of EGCG-Fe-Cur spherical nanomolecules

[0030] Combination Figure 1 A schematic diagram of the self-assembly synthesis of EGCG-Fe-Cur spherical nanomolecules, mainly including the following steps:

[0031] 1) Solution preparation: Accurately weigh 25 mg of EGCG and dissolve it in 10 mL of 50% ethanol to prepare a 2.5 mg / mL EGCG solution; separately dissolve 30 mg of Cur in 10 mL of 100% ethanol to prepare a 3 mg / mL solution; dissolve 5 mg of FeCl3 in 10 mL of 50% ethanol to prepare a 0.5 mg / mL solution for later use. Store all solutions in a refrigerator at 4°C protected from light.

[0032] 2) Synthesis of EGCG-Fe-Cur: Add 2 mL of the prepared Cur solution to a 20 mL reaction flask, turn on low-speed magnetic stirring, add 2 mL of the prepared EGCG solution dropwise to the reaction flask and stir for 5 min, then slowly add FeCl₂. 3 Add 2 mL of the prepared solution to the reaction flask and stir magnetically for 5 min. Then, slowly add 300 μL of Tris-HCl pH 8.8 buffer solution to the reaction flask to adjust the pH of the mixture to 8.2. Seal the reaction flask and react for 3 h. Finally, add 2 mL of deionized water to the reaction flask and stir for 10 min to stop the reaction. Transfer the reaction solution to a dialysis bag and dialyze in water for 9 h, changing the water every 3 h during dialysis. After dialysis, store the obtained liquid away from light.

[0033] 3) Characterization of EGCG-Fe-Cur: The morphology of the prepared nanoparticles was analyzed using transmission electron microscopy (TEM). Particle size and zeta potential were analyzed using a laser particle size analyzer. 7 μM samples of Cur, EGCG, and EGCG-Fe-Cur materials were taken. Separately, EGCG-Fe-Cur nanoparticles were freeze-dried and then compressed with 5 mg each of Cur and EGCG using KBr before infrared spectroscopy analysis. The iron ions in the nanomaterials were qualitatively analyzed using methylene blue catalytic spectrophotometry.

[0034] The size of the synthesized EGCG-Fe-Cur nanoparticles was analyzed using transmission electron microscopy, such as... Figure 2A As shown, its size is approximately 40 nm, forming a spherical nanostructure with good uniformity; laser particle size analyzer analysis results indicate that its hydrated particle size is approximately 77 nm, with good uniformity. Figure 2B As shown; Zeta potential analysis shows (e.g.) Figure 2C Its average Zeta potential is -23.2 mV, and it exhibits good stability in aqueous phase; ultraviolet-visible spectrophotometry (e.g.) Figure 2D This indicates that the UV absorption peak of the synthesized EGCG-Fe-Cur nanoparticles is significantly enhanced at 423 nm; infrared spectroscopy analysis results show (e.g.) Figure 2E EGCG-Fe-Cur nanoparticles contain characteristic hydroxyl and ketone peaks of EGCG and Cur, at 1500 cm⁻¹. -1 The characteristic peak of the C=C stretching vibration of the benzene ring skeleton is obvious, at 1245 cm⁻¹. -1 A methoxyl peak appears at [location]. Methylene blue catalysis experiments show (e.g.) Figure 2F The nanoparticles prepared contain iron ions.

[0035] Example 2: Effects of different doses of gamma ray irradiation and different concentrations of EGCG-Fe-Cur spherical nanomolecules on the survival of AHH-1 cell line.

[0036] The experimental procedure is as follows:

[0037] 1) AHH-1 cells (ATCC) were cultured in a constant temperature incubator (Queue, USA) on RPMI 1640 medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) and 20 μg / mL penicillin and streptomycin (Beyotime, China).

[0038] 2) Collect cells in the logarithmic growth phase, at a concentration of 1×10⁻⁶. 4The cells were uniformly seeded in 96-well plates (Corning, USA) and then irradiated with 0, 4, 8, and 12 Gy of gamma rays. The effect of different doses of gamma ray irradiation on the survival of AHH-1 cell lines was detected by CCK-8 assay at 24 h and 48 h after irradiation.

[0039] 3) Collect cells in the logarithmic growth phase, at a concentration of 1×10⁻⁶. 4 The cells were evenly seeded in 96-well plates (Corning, USA), and then different concentrations (0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16 μM) of EGCG-Fe-Cur were added to the cells and incubated for 24 h and 48 h. The effect of different concentrations of EGCG-Fe-Cur spherical nanomolecules on the survival of AHH-1 cell line was detected by CCK-8 assay.

[0040] AHH-1 cells were treated with different doses of gamma rays, and cell viability was assessed using a CCK8 assay. The results showed that at 24 and 48 hours post-irradiation, 4, 8, and 12 Gy irradiations significantly inhibited cell viability compared to the control group. Furthermore, the survival rate at 48 hours post-irradiation was lower for all doses than at 24 hours, and the survival rate at the 12 Gy dose was only about 50% at 48 hours post-irradiation. Figure 3A and Figure 3B Treatment of AHH-1 cells with different concentrations of EGCG-Fe-Cur spherical nanomolecules for 24 h and 48 h followed by CCK8 assay revealed that EGCG-Fe-Cur had a certain promoting effect on AHH-1 cell survival in the short term, and the effect increased with increasing concentration. Figure 3C This promoting effect decreases over time. Figure 3D Therefore, the median lethal dose (LD50) of AHH-1 was 12 Gy 48 h after gamma irradiation; overall, different concentrations of EGCG-Fe-Cur showed relatively low cytotoxicity to AHH-1 cells.

[0041] Example 3: Radiation-resistant effect of EGCG-Fe-Cur spherical nanomolecules

[0042] The experimental procedure is as follows:

[0043] 1) AHH-1 cells (ATCC) were cultured in a constant temperature incubator (Queue, USA) on RPMI 1640 medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) and 20 μg / mL penicillin and streptomycin (Beyotime, China).

[0044] 2) Collect cells in the logarithmic growth phase, at a concentration of 1×10⁻⁶. 4Cells were evenly seeded in 96-well plates (Corning, USA), and then pretreated with different concentrations (0, 2, 8, 16 μM) of EGCG-Fe-Cur, 8 μM WR2721, 8 μM EGCG, 8 μM Cur, and 8 μM FeCl3 for 12 h. After 48 h of 12 Gy γ-ray irradiation, the relative viability of cells was determined by CCK-8 assay.

[0045] Figure 4 The results showed that EGCG-Fe-Cur had a significant anti-radiation effect on AHH-1 cells. Compared with the blank control group, the cell survival rate in the irradiation-only group (12 Gy) was approximately 69%. Pretreatment with 2, 8, and 16 μM EGCG-Fe-Cur spherical nanomolecules resulted in cell survival rates of 89%, 138%, and 86%, respectively, with 8 μM EGCG-Fe-Cur showing the most significant effect. Pretreatment with 8 μM MWR2721, 8 μM EGCG, 8 μM Cur, and 8 μM FeCl3 resulted in cell survival rates of approximately 80%, 110%, 56%, and 107%, respectively. Therefore, compared with other drugs at the 8 μM concentration, EGCG-Fe-Cur exhibited superior anti-radiation effects.

[0046] Example 4: ROS scavenging effect of EGCG-Fe-Cur spherical nanomolecules in cells

[0047] AHH-1 cells were pretreated with 8 μM MEGCG-Fe-Cur, WR2721, EGCG, Cur, and FeCl3 for 12 h, followed by 12 Gy γ-ray irradiation for 48 h. Flow cytometry analysis revealed that the total ROS in the cells was... Figure 5A Compared with the blank control group, the total ROS levels in cells were increased by 21%, 3%, 5%, and 14% in the simple irradiation (12 Gy), EGCG, Cur, and FeCl3 pretreatment groups, respectively, while they were decreased by 10% and 18% in the WR2721 and EGCG-Fe-Cur pretreatment groups, respectively. Flow cytometry analysis of cellular lipid peroxidation levels (Lip-ROS) revealed ( Figure 5B Compared with the blank control group, the lipid peroxidation level (Lip-ROS) in cells treated with simple irradiation (12 Gy), WR2721, EGCG-Fe-Cur, EGCG, Cur, and FeCl3 increased by 84%, 75%, 54%, 79%, 92%, and 79%, respectively. The lipid peroxidation level in the EGCG-Fe-Cur treatment group was reduced by 30% compared with the simple irradiation (12 Gy) group. Therefore, EGCG-Fe-Cur showed a significant effect in scavenging ROS and Lip-ROS induced by γ-rays in cells.

[0048] Example 5: Inhibition of ferroptosis by EGCG-Fe-Cur spherical nanomolecules

[0049] To verify whether the anti-radiation effect of EGCG-Fe-Cur is achieved by inhibiting the ferroptosis signaling pathway, AHH-1 cells were first pretreated with 8 μM EGCG-Fe-Cur for 12 h, followed by treatment with the ferroptosis inducer FIN56 at final concentrations of 0, 50, 100, 200, and 400 nM for 48 h. Cell viability was then assessed using CCK8 assay. The results showed that ( Figure 6A Compared with the blank control group, the cell viability rates of the ferroptosis inducer treatment groups at 50, 100, 200, and 400 nM were approximately 91%, 83%, 67%, and 64%, respectively; after pretreatment with 8 μM EGCG-Fe-Cur, the cell viability rates of the ferroptosis inducer treatment groups at 50, 100, 200, and 400 nM were 91%, 89%, 89%, and 81%, respectively. Therefore, EGCG-Fe-Cur can significantly improve the cell viability induced by the ferroptosis inducer FIN56, especially at 200 and 400 nM, where the viability rate can be increased by approximately 22% and 17%, respectively. Furthermore, after pretreatment of AHH-1 cells with 8 μM EGCG-Fe-Cur for 12 h, followed by treatment with FIN56 (400 nM) for 6 h, flow cytometry analysis revealed that the level of lipid peroxidation in the cells... Figure 6B After 6 hours of induction with 400 nMFIN56, the lipid peroxidation level of AHH-1 cells increased by 10%, and EGCG-Fe-Cur reduced cellular lipid peroxidation induced by ferroptosis inducers (by 14%). These results suggest that EGCG-Fe-Cur can act as an inhibitor of ferroptosis.

[0050] Example 6: Inhibition of Apoptosis by EGCG-Fe-Cur Spherical Nanomolecules

[0051] To investigate the effect of EGCG-Fe-Cur on apoptosis in AHH-1 cells after 12 Gy γ-ray irradiation, Annexin-V and PI double staining were used, and flow cytometry was employed to detect apoptosis. The results showed that 12 Gy γ-ray irradiation induced apoptosis at a rate of 14.8%, while EGCG-Fe-Cur pretreatment resulted in an apoptosis rate of 9.4%. Therefore, EGCG-Fe-Cur pretreatment of AHH-1 cells can reduce the apoptosis rate induced by 12 Gy γ-ray irradiation by up to 5.4%. These results suggest that EGCG-Fe-Cur can act as an apoptosis inhibitor.

[0052] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of EGCG-Fe-Cur spherical nanomolecules in preparation of a medicine for preventing radiation damage, wherein the concentration of the medicine is 8 μM. The EGCG-Fe-Cur spherical nanomolecules are prepared by the following steps: 1) Epigallocatechin gallate, curcumin and ferric chloride hexahydrate stock solution preparation, the method of preparation is, respectively, epigallocatechin gallate, curcumin and ferric chloride hexahydrate are dissolved in ethanol to prepare a stock solution, wherein, The concentration of the epigallocatechin gallate solution is 1.5-4.5 mg / mL, the concentration of the curcumin solution is 2-6 mg / mL, and the concentration of the ferric chloride solution is 0.3-0.7 mg / mL. 2) EGCG-Fe-Cur spherical nanomolecule synthesis: epigallocatechin gallate, curcumin and ferric chloride hexahydrate mother liquor are mixed, and Tris-HCl pH 8.8 buffer solution is added dropwise to adjust the pH of the solution to 8.2, and then the mixture is sealed and reacted for 3 h. After dialysis, the obtained product is EGCG-Fe-Cur spherical nanomolecules.

2. Use according to claim 1, characterized in that: In step 2), the molar ratio of epigallocatechin gallate: curcumin: ferric chloride hexahydrate in the mother liquor mixture is 2-6: 3-9:

1.

3. Use according to claim 1, characterized in that, The EGCG-Fe-Cur spherical nanomolecule comprises epigallocatechin gallate, curcumin, and iron trichloride hexahydrate; curcumin, Fe 3+ EGCG self-assembly synthesis of spherical nanomolecule EGCG-Fe-Cur.

Images