Preparation method and application of EGCG (epigallocatechin gallate) organic selenium nanoparticles with tumor cell growth inhibition activity

Abstract

The invention discloses a preparation method and application of EGCG (epigallocatechin gallate) organic selenium nanoparticles with tumor cell growth inhibition activity, and belongs to the field of functional nano materials. The nano-particles are organic nano-particles (ESNPs) which are formed by combining epigallocatechin gallate (EGCG) and organic selenium (selenocystine or selenomethionine) through chemical bonds by taking a C2-C6 aldehyde compound or hexamethylenetetramine (HMTA) as a cross-linking agent. The nanostructure has relatively high stability, and can induce oxidative stress in cells to further stimulate ferroptosis and apoptosis, so that the nanostructure shows remarkable inhibitory activity on various tumor cells. The invention provides a new technical approach and theoretical basis for the application of the natural polyphenol and organic selenium composite material in the field of tumor prevention and treatment.

Description

Technical Field

[0001] This invention relates to a method for preparing EGCG organoselenium nanoparticles with inhibitory activity against tumor cell growth and their application, belonging to the field of functional nanomaterials. Background Technology

[0002] The occurrence and development of tumors is a complex pathological process involving multiple factors and stages, usually accompanied by various biological mechanisms such as oxidative stress, inflammatory response, apoptosis imbalance, and abnormal ferroptosis pathway. In recent years, researchers have generally believed that regulating redox homeostasis, promoting programmed cell death, and inhibiting metabolic reprogramming of tumor cells are important strategies for developing novel anti-tumor drugs. Natural polyphenols have become a hot topic in tumor intervention research due to their multiple biological activities, including antioxidant, anti-inflammatory, and anti-cancer effects. Among them, epigallocatechin gallate (EGCG) is one of the most abundant and active natural polyphenols in tea, with the effects of scavenging free radicals, inhibiting tumor cell proliferation, and inducing apoptosis. However, EGCG contains multiple phenolic hydroxyl groups in its chemical structure, making it highly susceptible to oxidation or self-polymerization under alkaline conditions, leading to decreased biological activity and a shortened half-life. In addition, EGCG is rapidly metabolized in vivo, has limited water solubility, poor stability, and low oral absorption rate. These factors severely limit its clinical translation and application in the field of anti-tumor therapy.

[0003] Selenium (Se) is an essential trace element for the human body, playing a crucial role in the antioxidant defense system. Selenium maintains cellular redox balance and mitigates oxidative damage by participating in the synthesis of key antioxidant enzymes such as glutathione peroxidase (GPx), thioredoxin reductase (TrxR), and selenoprotein P. Recent studies have shown that organoselenium compounds (such as selenocysteine ​​SeCys2 and selenomethionine SeMet) possess higher bioactivity and safety compared to inorganic selenium, achieving antitumor effects by inducing apoptosis, regulating immune responses, and activating the ferroptosis pathway. Among these, selenocysteine ​​(SeCys2), as a structural unit of natural selenoproteins, exhibits good biocompatibility and controllable reactivity, making it a potential functional selenium source for constructing bioactive materials. However, the use of organoselenium alone still suffers from weak targeting and insufficient stability. Therefore, effectively combining the structural characteristics of natural polyphenols and organoselenium molecules to construct a composite system with both antioxidant and targeted antitumor capabilities has become a key research focus and challenge.

[0004] To improve the stability and bioavailability of EGCG, nanotechnology has become an effective means of carrier modification. Through intermolecular self-assembly, cross-linking, or chemical bonding, active ingredients can be formed into nanoparticles with stable structures and high bioavailability. Nanoparticles have a large specific surface area and good biopermeability, which can significantly enhance drug solubility, prolong its in vivo circulation time, and achieve sustained release and targeted delivery. Although there have been related studies on polyphenolic substances or selenium-based nanomaterials, most of them are still at the stage of physical adsorption or simple compounding, with poor particle stability and unclear synergistic mechanisms between the two. Existing technologies attempt to encapsulate EGCG or organoselenium in nanocarriers such as liposomes, PLGA, and chitosan to improve their stability. However, the above systems all belong to the category of "physical encapsulation", and there is a lack of chemical bonding between the carrier and the active ingredient, resulting in: (1) EGCG is still easily oxidized and degraded during storage and in vivo drug release; (2) organoselenium and EGCG have large steric hindrance, making it difficult to achieve synchronous targeted delivery; (3) the two only produce an additive effect, the synergistic anticancer mechanism is unclear, and the enhancement of antitumor activity is limited.

[0005] Therefore, how to design based on molecular structure to simultaneously solve the technical problems of poor stability of EGCG, weak targeting of organoselenium, and lack of synergistic mechanism between the two has become a difficult problem that urgently needs to be solved in this field. Summary of the Invention

[0006] To overcome the technical defects of existing EGCG formulations, such as poor pH stability and insufficient targeting of organoselenium monomers in antitumor activity, this invention provides EGCG organoselenium nanoparticles with inhibitory activity against tumor cell growth. The nanoparticles are characterized by being formed by the Mannich reaction of epigallocatechin gallate (EGCG) and organoselenium (SeCys2 or SeMet) into nanoparticles (ESNPs). The nanoparticles have a particle size of 100-500 nm, a zeta potential of -10 to -40 mV, and can inhibit tumor growth in tumor cells by inducing ferroptosis and apoptosis.

[0007] The first objective of this invention is to provide a method for preparing epigallocatechin gallate-organoselenium nanoparticles, characterized by comprising the following steps: (1) Epigallocatechin gallate and organic selenium are dissolved in the aqueous phase at a molar ratio of 5:1 to 2:1 to form a mixed solution; (2) Adjust the pH of the mixed solution to 5.0~7.0, add a crosslinking agent, and carry out the crosslinking reaction at 25~40℃; (3) The solution after the cross-linking reaction was purified to obtain epigallocatechin gallate-organoselenic nanoparticles (ESNPs).

[0008] Preferably, in step (1), EGCG and organic selenium are dissolved in the aqueous phase at a molar ratio of 3:1 to form a mixed solution.

[0009] In one embodiment of the present invention, in step (1), the organic selenium is selected from selenocysteine ​​dimer or selenomethionine.

[0010] In one embodiment of the present invention, in step (1), it is dissolved in deionized water.

[0011] In one embodiment of the present invention, in step (2), the crosslinking reaction is carried out under vigorous stirring at a speed of 400~600 rpm.

[0012] In one embodiment of the present invention, in step (2), the crosslinking agent is selected from C2-C6 aldehyde compounds or hexamethylenetetramine.

[0013] Preferably, the C2-C6 aldehyde compounds include one or more of glyoxal, butyraldehyde, and adipaldehyde.

[0014] In one embodiment of the present invention, in step (2), the total molar amount of EGCG and organoselenium is in a molar ratio of 1:1 to 1:2 with respect to the crosslinking agent.

[0015] In one embodiment of the present invention, in step (2), the reaction time is controlled to be 20 to 60 min.

[0016] In one embodiment of the present invention, the purification is dialysis purification.

[0017] Preferably, dialysis is performed using a dialysis bag with a molecular weight cutoff of 3–6 kDa.

[0018] In one embodiment of the present invention, the dialysis conditions are dialysis at 2–8 °C for 24–72 h.

[0019] A second objective of this invention is to provide epigallocatechin gallate-organoselenoseslenyl nanoparticles (ESNPs) prepared according to the method described above.

[0020] A third object of the present invention is to provide a pharmaceutical composition comprising epigallocatechin gallate-organoselenoseslenium nanoparticles.

[0021] In one embodiment of the invention, the pharmaceutical composition package includes pharmaceutically acceptable excipients.

[0022] In one embodiment of the present invention, the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0023] In one embodiment of the present invention, the carrier is one or more of a virus, liposomes, and nanoparticles.

[0024] In one embodiment of the present invention, the excipient is one or more of mannitol, lactose, fatty acids, and polyethylene glycol.

[0025] In one embodiment of the present invention, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, suppositories, enemas, aerosols, patches, or drops.

[0026] In one embodiment of the present invention, the route of administration of the drug includes oral administration, sublingual administration, rectal administration, skin and mucous membrane administration, inhalation administration, or injection administration.

[0027] A fourth object of the present invention is to provide the role of the epigallocatechin gallate-organoselenoseslenium nanoparticles in the preparation of a drug for inhibiting tumors.

[0028] Preferably, the tumor includes liver cancer, lung cancer, squamous cell carcinoma, breast cancer, prostate cancer, colorectal cancer, stomach cancer, pancreatic cancer, and esophageal cancer.

[0029] More preferably, the tumor includes liver cancer, lung cancer, and squamous cell carcinoma.

[0030] A fifth object of the present invention is to provide the use of the pharmaceutical composition in the preparation of a medicament having at least one of the following functions: (1) Reduce the proliferation and growth of tumor cells; (2) Induces ferroptosis in tumor cells.

[0031] Beneficial effects: This invention leverages the chemical properties of EGCG with selenocysteine ​​(SeCys2) or selenomethionine (SeMet). Through a Mannich reaction initiated by aldehydes (such as glyoxal) or hexamethylenetetramine, EGCG forms stable chemical bonds with SeCys2 or SeMet, thereby constructing polyphenol-organoselenium nanoparticles (ESNPs) with well-defined molecular binding structures. These nanoparticles not only improve the chemical stability and dispersibility of EGCG but also enhance its antioxidant capacity and cell-regulating effects through the participation of selenium, exhibiting synergistic antitumor activity. The preparation process is mild and highly controllable, and the product possesses good biocompatibility and storage stability, making it suitable for development into antitumor drugs or functional nutritional preparations. This invention provides a new technical approach and theoretical basis for the application of natural polyphenol-organoselenium composite materials in the field of tumor prevention and treatment. (1) Improved stability and delivery efficiency: EGCG and organic selenium form an organic cross-linked network in the nanoparticle structure, which effectively improves the antioxidant stability and cell uptake efficiency of both.

[0032] (2) Synergistic effect: The introduction of organic selenium achieves synergistic anti-tumor effect with EGCG, induces ferroptosis and enhances tumor cell apoptosis; cell survival rate is reduced to below 37%.

[0033] (3) The preparation method is simple and controllable: the entire reaction process is mild, easy to operate, easy to scale up industrially, and the product has good biocompatibility, making it suitable for development into a new type of natural anti-tumor drug or functional nutritional preparation. Attached Figure Description

[0034] Figure 1 STEM image of ESNP nanoparticles; Figure 2 (A) is the EDS energy dispersive spectroscopy (EDS) spectrum of ESNP nanoparticles; (D) is the STEM image of the corresponding ESNPs; (B), (C), (E), and (F) are the elemental distribution diagrams of C, N, O, and Se, respectively. Figure 3 The spectra of EGCG, ESNPs nanoparticles, and SeCys2 are shown; (A) UV-Vis spectrum; (B) FTIR spectrum; (C) 1 H NMR (EGCG and ESNPs).

[0035] Figure 4 LPO fluorescence intensity diagrams of the effects of EGCG, ESNPs nanoparticles and EGCG+SeCys2 on Hepa1-6 and SCC-7 tumor cells; (A) Hepa1-6; (B) SCC-7; Figure 5Different nanoparticle size distributions were obtained by adjusting the temperature, pH, reactant ratio, and reaction time in the reaction conditions; (A) pH; (B) reaction time; (C) n EGCG :n SeCys2 (D) Reaction temperature; Figure 6 Study on pH stability of ESNP nanoparticles; (A) UV-Vis spectrum; (B) Zeta potential spectrum; (C) Particle size distribution map. Detailed Implementation

[0036] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0037] Raw materials used in the examples: Epigallocatechin gallate (EGCG): CAS No.: 989-51-5.

[0038] Selenocysteine ​​dimer (SeCys2): purchased from Adamas.

[0039] Selenomethionine: CAS No.: 3211-76-5.

[0040] Glyoxal: CAS No.: 107-22-2.

[0041] Adipaldehyde: CAS No.: 1072-21-5.

[0042] Hexamethylenetetramine: CAS No.: 100-97-0.

[0043] The measurement methods involved in the examples are as follows: The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0044] In the following embodiments, the measurements were performed according to the following methods: 1. Method for determining total selenium content Take 0.1–0.2 g of sample into a microwave digestion tube, add 3.5 mL of concentrated nitric acid, and digest overnight in a cold environment. Then add 5.0 mL of ultrapure water, seal the tube with both inner and outer stoppers, and proceed with microwave digestion. Dilute the digested solution to the appropriate volume with ultrapure water and mix thoroughly. Take approximately 15 mL of the solution, pass it through a 0.22 μm nylon membrane, and transfer it to a 15 mL centrifuge tube. Perform a reagent blank simultaneously.

[0045] Total selenium was determined using inductively coupled plasma mass spectrometry in TQ-O2 reaction mode. 80Se isotopes were monitored by the instrument, and the sampling cone was a nickel cone. A pig liver standard (selenium content 1.54 ± 0.29 mg / kg) was used as the quality control sample for total selenium determination. A series of standard solutions were prepared, and the selenium content was calculated using the external standard method.

[0046] The formula for calculating selenium content is:

[0047] in X Selenium content of the sample (mg / kg); C Selenium concentration (μg / L) measured after sample digestion and volume adjustment; K： Dilution factor; V : The volume (mL) of the sample after digestion and final volume adjustment; M : Amount of sample digested (g).

[0048] 2. MTT Method This study investigated the effect of ESNP nanoparticles on tumor cell survival. A control group and a sample group were set up during cell culture, and the cells were treated as follows: (1) Culture of tumor cells Hepa1-6, LLC, and SCC-7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and then in a cell culture incubator at 37°C containing 5% CO2. The medium was changed every 2 days, and cells were passaged at a ratio of 1:5. When the cells covered more than 80% of the bottom of the culture flask, they were digested with 0.25% trypsin and ready for use.

[0049] (2) Cell experiments Control group: Cultured cells were inoculated at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of 100 μL / mL in 96-well plates. After cell attachment (24 h), 100 μL of complete culture medium was added and the cells were cultured for another 24 h. 50 µL of MTT was added to each well, and after incubation for 4 h, the MTT was carefully removed, and 150 µL of DMSO was added. The absorbance of the cells was measured at 570 nm using a microplate reader.

[0050] Sample group: Cultured cells were mixed at a rate of 1×10⁻⁶. 5Cells were seeded at a density of 100 μL / mL in 96-well plates. After cell attachment (24 h), 100 μL of EGCG, ESNPs nanoparticles (60, 100, 200, 300, and 400 μg / mL), and EGCG+SeCys2 solution were prepared in each well of the 96-well plate. Cells were cultured for 24 h. 50 µL of MTT was added to each well, and after incubation for 4 h, the MTT was carefully removed, and 150 µL of DMSO was added. The absorbance of the cells was measured at 570 nm using a microplate reader.

[0051] Cell viability is calculated using the following formula:

[0052] In the formula, the experimental group contains cell culture medium and sample (i.e., model group or sample group); the control group contains only cell culture medium (i.e., control group); and the blank group contains only 250 μL PBS buffer.

[0053] 3. Determination methods for lipid peroxides To investigate the effect of ESNP nanoparticles on ferroptosis in tumor cells, a lipid peroxide detection kit was used to detect intracellular ferroptosis markers—lipid peroxides (LPO). The specific procedures are as follows: Control group: Cultured cells were inoculated at a rate of 2 × 10⁻⁶. 6 Cells were seeded at a density of 100 cells / mL in 6-well plates, with a volume of 2.5 mL per well. After cell attachment (24 h), 2.5 mL of complete culture medium was added and the cells were cultured for another 24 h. After culture, the culture medium was discarded, the cells were washed once with PBS, and 1 mL of BODIPY 581 / 591 C11 staining working solution was added to each well. The cells were incubated at 37°C for 10 min, the supernatant was aspirated, and the cells were washed twice with PBS. The reduced red fluorescence and oxidized green fluorescence were observed using an inverted fluorescence microscope.

[0054] Sample group: Cultured cells were subjected to a 2×10⁻⁶ incubation period. 6 Cells were seeded at a density of 10 cells / mL in 6-well plates, with a volume of 2.5 mL per well. After cell attachment (24 h), 2.5 mL of EGCG, ESNPs nanoparticles (20 μg / mL), and EGCG+SeCys2 culture medium (20 μg / mL and 200 μg / mL, respectively) were prepared in each well. Cells were cultured for 24 h. After culture, the culture medium was discarded, and the cells were washed once with PBS. 1 mL of BODIPY 581 / 591 C11 staining working solution was added to each well, and the cells were incubated at 37°C for 10 min. The supernatant was then aspirated, and the cells were washed twice with PBS. The reduced red fluorescence and oxidized green fluorescence were observed using an inverted fluorescence microscope.

[0055] 4. Methods for determining particle size By adjusting the temperature, pH, reactant ratio, and reaction time, nanoparticles formed under different conditions can be obtained. 10 mg of the obtained nanoparticle sample was dispersed in 20 mL of deionized water to prepare a nanoparticle dispersion with a concentration of 0.5 mg / mL. The dispersion was then subjected to particle size analysis using a Nano&Zeta potentiometer; specific test conditions are shown in Table 1.

[0056] Table 1. Parameters of Nano & Zeta Potentiometer

[0057] Example 1 This embodiment provides a method for preparing EGCG organoselenium nanoparticles with inhibitory activity against tumor cell growth, as detailed below: Weigh 100 mg of epigallocatechin gallate (EGCG) and 21.8 mg of selenocysteine ​​dimer (SeCys2), add them to 40 mL of deionized water, and treat them under ultrasonic conditions of 40 Hz and 360 W for 5 min to completely dissolve the raw materials and obtain a mixed solution. After adjusting the pH of the mixed solution to 6.0, 42 μL of 40% glyoxal solution was added to the mixed solution, and the mixture was stirred vigorously at 30°C for 20 min. After the reaction was completed, the resulting solution was placed in a dialysis bag with a molecular weight cutoff of 3 kDa, and dialyzed at 4°C for 48 h with water as the dialysate to remove unreacted small molecule impurities.

[0058] After dialysis, the target nanoparticles ESNPs were obtained, with an average particle size of 178.9 nm and a selenium content of 6.48 g / 100 g.

[0059] Example 2 This embodiment systematically evaluated the effects of pH, reaction time, reactant molar ratio, and temperature on the formation of EGCG-SeCys2 nanoparticles. Figure 5 ), as detailed below: (1) pH of the reaction To investigate the effect of the pH of the reaction solution on the nanoparticles, the pH of the mixed solution was adjusted to 5.0, 6.0, and 7.0, respectively.

[0060] The results showed that well-dispersible particles could be formed in the pH range of 5 to 7, with the smallest particle size and best uniformity at pH 6.0.

[0061] (2) Reaction time To investigate the effect of reaction time on nanoparticles, the reaction time was adjusted to 30, 40, 50, and 60 min, respectively, by vigorous stirring.

[0062] Although the reaction time has little effect on the particle size, the nanoparticle structure tends to stabilize after 20 min of reaction, and 20–60 min can be regarded as a suitable reaction time.

[0063] (3) Molar ratio of reactants To demonstrate the effect of the molar ratio of gallocatechin gallate (EGCG) and 21.8 mg selenocysteine ​​dimer (SeCys2) on nanoparticles, the molar ratio was adjusted to 5:1, 4:1, and 2:1, respectively.

[0064] The results showed that the nanostructures could be stably formed when the molar ratio of epigallocatechin gallate (EGCG) and 21.8 mg selenocysteine ​​dimer (SeCys2) was 5:1 to 2:1.

[0065] (4) Reaction temperature To investigate the effect of reaction temperature on nanoparticles, the temperature of the vigorously stirred reaction was adjusted to 25, 35, and 40°C, respectively.

[0066] The results showed that nanostructures could be formed at temperatures ranging from 25 to 40°C.

[0067] Example 3 This embodiment provides a method for preparing EGCG organoselenium nanoparticles with inhibitory activity against tumor cell growth, as detailed below: Weigh 100 mg of epigallocatechin gallate (EGCG) and 21.8 mg of selenocysteine ​​dimer (SeCys2), add them to 40 mL of deionized water, and treat them under ultrasonic conditions of 40 Hz and 360 W for 5 min to completely dissolve the raw materials and obtain a mixed solution. After adjusting the pH of the mixed solution to 5.0, 29.1 mg of hexadialdehyde was added to the solution, and the mixture was stirred vigorously at 30°C for 30 min. After the reaction was completed, the resulting solution was placed in a dialysis bag with a molecular weight cutoff of 5 kDa, and dialyzed at 4°C for 60 h with water as the dialysate to remove unreacted small molecule impurities. After dialysis, the target nanoparticles ESNPs were obtained, with an average particle size of 261.3 nm and a selenium content of 7.37 g / 100 g.

[0068] Example 4 This embodiment provides a method for preparing EGCG organoselenium nanoparticles with inhibitory activity against tumor cell growth, as detailed below: Weigh 100 mg of epigallocatechin gallate (EGCG) and 21.4 mg of selenomethionine (SeMet), add them to 40 mL of deionized water, and treat them under ultrasonic conditions of 40 Hz and 400 W for 5 min to completely dissolve the raw materials and obtain a mixed solution. After adjusting the pH of the mixed solution to 7.0, 95 μL of 40% glyoxal solution was added to the solution, and the mixture was stirred vigorously at 40°C for 30 min. After the reaction was completed, the resulting solution was placed in a dialysis bag with a molecular weight cutoff of 5 kDa, and dialyzed at 4°C for 60 h with water as the dialysate to remove unreacted small molecule impurities. After dialysis, the target nanoparticles ESNPs were obtained, with an average particle size of 231.3 nm and a selenium content of 5.68 g / 100 g.

[0069] Example 5 This embodiment provides a method for preparing EGCG organoselenium nanoparticles with inhibitory activity against tumor cell growth, as detailed below: Weigh 100 mg of epigallocatechin gallate (EGCG) and 10.7 mg of selenomethionine (SeMet), add them to 40 mL of deionized water, and treat them under ultrasonic conditions of 35 Hz and 400 W for 5 min to completely dissolve the raw materials and obtain a mixed solution. After adjusting the pH of the mixed solution to 5.0, 76.5 mg of hexamethylenetetramine was added to the solution, and the mixture was stirred vigorously at 30°C for 40 min. After the reaction was completed, the resulting solution was placed in a dialysis bag with a molecular weight cutoff of 4 kDa, and dialyzed at 4 °C for 48 h with water as the dialysate to remove unreacted small molecule impurities. After dialysis, the target nanoparticles ESNPs were obtained, with an average particle size of 191.3 nm and a selenium content of 6.81 g / 100 g.

[0070] Test Example 1: Product Structure Characterization To verify the structural characteristics and formation mechanism of the EGCG–SeCys2 nanoparticles prepared in this invention, the product of Example 1 was subjected to morphological observation, elemental analysis and spectroscopic characterization.

[0071] (1) Morphology and particle size analysis like Figure 1As shown, the transmission electron microscope (STEM) images of the ESNPs reveal a particle size of approximately 96.3 nm. The particles are regularly spherical, uniformly distributed, and have smooth surfaces without significant agglomeration, indicating that the nanosystem possesses good dispersibility and shapeability. These results demonstrate that the formaldehyde crosslinking reaction between EGCG and SeCys2 in this invention can effectively control the particle size and morphology, achieving a stable nanostructure.

[0072] (2) Elemental composition and distribution analysis ICP-MS and EDS energy dispersive spectroscopy analyses were performed on the ESNPs, and the results are as follows: Figure 2 As shown in the figure. ICP-MS analysis revealed a Se content of 6.48 g / 100 g, corresponding to a total atomic percentage of 1.47%; C accounted for 36.39%, O for 29.74%, and N for 32.40%. EDS surface scanning results indicated that the elements were uniformly distributed within the nanoparticles, demonstrating the uniform embedding of SeCys2 into the network structure. The excellent consistency between the elemental proportions in the product and those in the raw materials further confirms the effective introduction of Se during the synthesis process and the reliability of the ESNP composition.

[0073] (3) Ultraviolet-vis spectroscopy analysis (UV-vis) like Figure 3 As shown in Figure A, the UV-Vis absorption spectrum of ESNPs exhibits a characteristic absorption peak at 276 nm consistent with that of free EGCG, indicating that the main aromatic structure of EGCG is preserved during the reaction. Simultaneously, the absorption enhancement and redshift observed near 300 nm suggest that SeCys2 incorporation caused a redistribution of the system's electronic structure, further confirming the chemical interaction and energy level coupling between EGCG and SeCys2.

[0074] (4) FTIR (Full-Time Infrared Spectroscopy) like Figure 3 As shown in Figure B, the characteristic peaks of carbonyl, amide, and ether bonds in the infrared spectra of ESNPs undergo significant changes. The original ester carbonyl absorption peak of EGCG changes from 1690 cm⁻¹. -1 Shift to lower wavenumbers to 1683 cm -1 The broadening and weakening of the peak shape indicate that some carbonyl groups participated in the formaldehyde bridging reaction with SeCys2 or formed a new Schiff base (C=N) structure, leading to a decrease in vibrational frequency. The amide peak of SeCys2 decreased from 1575 cm⁻¹. -1 Moved to 1520 cm -1 The decrease in absorption intensity, accompanied by a reduction in absorption strength, indicates that its –NH2 or –NH– groups participated in the reaction and formed cross-linking bonds. At 1250–1150 cm⁻¹ -1The region exhibits wider and more complex multiple absorption shoulders, which can be attributed to newly formed C–O–C and C–N–C bridging bond vibrations. This is in contrast to EGCG (≈1245 cm⁻¹). -1 ) and SeCys2 (≈1250 cm) -1 Single peak positions of ESNPs at ~1260, 1220 and 1180 cm⁻¹ -1 The presence of multiple peaks further confirms that the two components formed a complex covalently cross-linked structure through a formaldehyde bridging reaction. Additionally, the aromatic C=C vibration peak (approximately 1610 cm⁻¹) was observed. -1 The intensity of the peaks decreased and the peak shape broadened in ESNPs, suggesting the existence of hydrogen bonds or weak π–Se interactions between the aromatic ring and SeCys2.

[0075] (5) Nuclear magnetic resonance spectrum ( 1 H NMR analysis like Figure 3 As shown in Figure C, the ESNPs show a significant signal enhancement in the 3.4–4.2 ppm region. The corresponding –C2H5–O bridge and –C2H5–Se– signals in this region provide direct evidence of successful acetaldehyde crosslinking. The flavan skeleton hydrogen signals in the 4.5–5.4 ppm region are still detectable, but the peaks are blunted or slightly broadened, indicating that the C-ring structure of EGCG is largely preserved and only affected by the local chemical environment. In the 5.7–7.0 ppm region, the aromatic hydrogen signals of the original EGCG are significantly weakened or even diffused, indicating that aromatic hydroxyl groups may have participated in the crosslinking reaction, leading to changes in the aromatic electron cloud density. In the 8.5–9.5 ppm region, the hydroxyl (–OH) signal of the original EGCG almost completely disappears, indicating that the hydroxyl groups have participated in the formation of new chemical bonds such as ether bonds (C–O–C), thioethers (C–S–C), and selenide bonds (C–Se–C). ​​Overall, the blunting and broadening of the ESNP peaks reflect the characteristics of restricted molecular motion and shortened relaxation time in the system, consistent with the NMR characteristics of macromolecules or nanocomposite systems.

[0076] Integrating STEM, ICP-MS, FTIR and 1 ¹H NMR results show that EGCG and SeCys2 undergo a condensation crosslinking reaction induced by aldehyde groups under weakly acidic conditions, generating a three-dimensional crosslinked network structure containing C–O–C, C–N–C, and C–Se–C bridging bonds. This structure endows the particles with good dispersibility and stability, while effectively fixing and distributing Se in the nanosystem. Therefore, it can be inferred that the ESNPs nanoparticles prepared in this invention are covalently crosslinked nanonetworks constructed from EGCG–SeCys2 through aldehyde bridging.

[0077] Test Example 2: Inhibitory effect of nanoparticles on tumor cell growth The inhibitory effects of EGCG, SeCys2, EGCG+SeCys2 (EGCG and SeCys2 were mixed in a molar ratio of 4:1), nanoparticles of Example 1, nanoparticles of Example 3, nanoparticles of Example 4, and nanoparticles of Example 5 on the proliferation of three types of tumor cells, namely Hepa1-6, LLC and SCC-7, were investigated respectively, and a blank control group was set up.

[0078] The results showed that all three treatment groups exhibited a concentration-dependent decrease in cell activity, indicating that EGCG and its complex system have a certain inhibitory effect on tumor cells.

[0079] At all tested concentrations, ESNPs showed significantly better inhibitory effects on tumor cells than free EGCG and the EGCG+SeCys2 mixture. For example, at 30 μg / mL, cell viability decreased significantly to below 40% after ESNP treatment, while the EGCG and EGCG+SeCys2 groups maintained above 80%. This indicates that nanoparticles formed by the reaction of EGCG and SeCys2 can significantly enhance the bioactivity of EGCG, thereby improving its inhibitory effect on the growth of various tumor cells. The construction of ESNPs achieved stronger antitumor activity, suggesting that this nanoparticle system has potential application value in antitumor drug development and targeted tumor therapy.

[0080] Table 2. Cell survival rate of nanoparticles inhibiting Hepa 1-6.

[0081] Table 3 Cell survival rate of nanoparticles inhibiting LLC

[0082] Table 4. Cell survival rate of nanoparticles inhibiting SCC-7

[0083] Test Example 3: Nanoparticles induce ferroptosis in tumor cells Hepa1-6 cells were tested using the BODIPY 581 / 591 C11 lipid peroxide detection reagent. Figure 4 A) and SCC-7 cells ( Figure 4B) Fluorescent labeling was performed, where red fluorescence represented reduced lipids and green fluorescence represented oxidized lipids. The normal control group mainly showed red fluorescence, indicating a low level of intracellular lipid peroxidation. After different sample treatments, the lipid peroxidation signal increased in a dose-dependent manner. Under low-dose conditions (10 μg / mL), EGCG, SeCys2, and their complex EGCG+SeCys2 all showed a certain degree of enhanced green fluorescence, suggesting that they could induce lipid oxidation; while the ESNPs group showed the most obvious green fluorescence, indicating a stronger lipid peroxidation capacity. Under high-dose conditions (100 μg / mL), this trend was further enhanced, especially in the ESNPs group where green fluorescence increased significantly and red fluorescence decreased significantly, indicating a large accumulation of intracellular lipid peroxides and a significantly higher degree of ferroptosis than other groups. The results indicate that the nanoparticles (ESNPs) formed by formaldehyde cross-linking of EGCG and SeCys2 have higher reactivity in cells and can effectively promote ferroptosis-related lipid peroxidation. Therefore, ESNPs can significantly enhance the level of lipid peroxidation in tumor cells, and their ferroptosis induction effect is significantly better than that of single components EGCG or SeCys2, suggesting that the nanocomposite structure has a synergistic effect in regulating cellular oxidative stress and anti-tumor mechanisms.

[0084] Table 5. Relative fluorescence intensity of LPO in Hepa 1-6 cells treated by nanoparticles

[0085] Table 6. Relative fluorescence intensity of LPO in SCC-7 cells treated by nanoparticles

[0086] Test Example 4: Investigation of pH Stability of Nanoparticles The ESNPs prepared in Example 1 were dispersed in PBS buffer solutions of different pH values ​​(3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5) to prepare 100 μg / mL nanoparticle solutions at different pH values, and the structure and colloidal stability of the ESNPs under different pH conditions were investigated.

[0087] Depend on Figure 6 As can be seen, the UV-Vis absorption spectra of ESNPs show significant differences with pH changes: under acidic conditions (pH 3.5~5.0), the intensity of the characteristic absorption peak decreases and is accompanied by a slight red shift, indicating that the particle structure has become loose or aggregated to some extent; while in the neutral to weakly alkaline range (pH 6.5~7.5), the absorption peak intensity is significantly enhanced and the peak shape is sharper, indicating that the particle structure is dense and stable.

[0088] Figure 6The Zeta potential results for B further support the above conclusions: below pH 4.0, the absolute value of the particle potential is low, indicating insufficient system stability; when the pH is increased to above 6.5, the Zeta potential rises rapidly and turns positive (>15 mV), showing that a stable surface charge layer is formed in the system. This potential increase may originate from the gradual ionization of the amino and thiol groups of SeCys2, constructing a more stable electric double layer structure on the particle surface, thereby enhancing colloidal dispersibility.

[0089] At the same time, such as Figure 6 As shown in Figure C, the particle size decreases significantly with increasing pH, and the distribution curve narrows, indicating that particle aggregation gradually decreases and the system tends to become more homogeneous and stable. Combining the changes in UV, Zeta potential, and particle size, it can be seen that the pH stability of ESNPs is mainly regulated by surface charge. The increase in Zeta potential is consistent with the enhancement of the absorption peak and the decrease in particle size, indicating that moderate alkaline conditions can effectively maintain the structural integrity and dispersion stability of nanoparticles.

[0090] Comparative Example 1 The specific implementation method is the same as in Example 1, except that the pH of the solution is adjusted to 4.0 or 8.0: The results showed that the nanoparticles could form well-dispersed particles in the pH range of 5 to 7. When the pH was <5 or >7, the average particle size of the prepared nanoparticles increased.

[0091] Comparative Example 2 The specific implementation method is the same as in Example 1, except that the time for vigorous stirring reaction is adjusted to 10 min: The results showed that the reaction was incomplete at 10 min, and a stable structure could not be formed.

[0092] Comparative Example 3 The specific implementation method is the same as in Example 1, except that the molar ratio of EGCG and SeCys2 is adjusted to 1:1 or 1:3. The results showed that the average particle size increased and there was a problem with poor stability.

[0093] Comparative Example 4 The specific implementation method is the same as in Example 1, except that the reaction temperature is adjusted to 50°C: The results showed that the high-temperature reaction caused polyphenol oxidation, making it impossible to obtain the desired nanoparticles.

[0094] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing epigallocatechin gallate-organoselenium nanoparticles, characterized in that, Includes the following steps: (1) Epigallocatechin gallate and organic selenium are dissolved in the aqueous phase at a molar ratio of 5:1 to 2:1 to form a mixed solution; (2) Adjust the pH of the mixed solution to 5.0~7.0, add a crosslinking agent, and carry out the crosslinking reaction at 25~40℃; (3) The solution after the cross-linking reaction was purified to obtain epigallocatechin gallate-organoselenium nanoparticles.

2. The method according to claim 1, characterized in that, In step (1), the organic selenium is selected from selenocysteine ​​dimer or selenomethionine.

3. The method according to claim 1, characterized in that, In step (2), the crosslinking reaction is carried out under vigorous stirring at a speed of 400~600 rpm.

4. The method according to claim 1, characterized in that, In step (2), the crosslinking agent is selected from C2-C6 aldehyde compounds or hexamethylenetetramine; Preferably, the C2-C6 aldehyde compounds include one or more of glyoxal, butyraldehyde, and adipaldehyde.

5. The method according to claim 1, characterized in that, In step (2), the total molar amount of epigallocatechin gallate and organic selenium is in a molar ratio of 1:1 to 1:2 to the crosslinking agent.

6. The method according to claim 1, characterized in that, In step (2), the reaction time is controlled between 20 and 60 min.

7. The method according to claim 1, characterized in that, In step (3), the purification is dialysis purification; Preferably, dialysis is performed using a dialysis bag with a molecular weight cutoff of 3–6 kDa.

8. Epigallocatechin gallate-organoselenium nanoparticles prepared by the method according to any one of claims 1 to 7.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains epigallocatechin gallate-organoselenium nanoparticles as described in claim 8.

10. The role of the epigallocatechin gallate-organoselenoseslenium nanoparticles according to claim 8 in the preparation of a drug for inhibiting tumor cell growth; Preferably, the tumor includes liver cancer, lung cancer, squamous cell carcinoma, breast cancer, prostate cancer, colorectal cancer, stomach cancer, pancreatic cancer, and esophageal cancer.

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