Cobalt-methionine nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses cobalt-methionine nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of medicines. The cobalt and methionine in the cobalt-methionine nano-particles provided by the invention have dual effects after being combined, so that Fenton-like reaction can be promoted, high-activity hydroxyl radicals are generated, oxidative stress is further initiated, and apoptosis of tumor cells is induced; the activity of thioredoxin reductase can be inhibited, the protection effect of an antioxidant system is weakened, and the curative effect of CDT is further enhanced; therefore, the compound can be applied to tumor treatment, has important significance in improving the curative effect of CDT, and can further improve the accuracy of tumor treatment; moreover, the average particle size of the product prepared by the preparation method of the cobalt-methionine nanoparticles is in a proper range, and the cobalt-methionine nanoparticles have excellent stability and can play a better role.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology, and in particular relates to cobalt-methionine nanoparticles and a preparation method and application thereof. Background Art

[0002] Tumor is a disease that seriously threatens human health. Traditional tumor treatment methods such as chemotherapy, radiotherapy and surgery have certain limitations and side effects. Therefore, finding more effective tumor treatment methods has become a hot topic in current research.

[0003] Chemodynamic therapy (CDT) is an emerging anticancer therapeutic strategy that utilizes transition metal ions to catalyze the catalysis of endogenous hydrogen peroxide (H 2 O 2 ) generates highly toxic hydroxyl radicals (·OH), thereby inducing tumor cell death. However, the effectiveness of CDT is limited by a series of protective mechanisms present in the tumor microenvironment. Specifically, there is an abundance of the antioxidant molecule glutathione (GSH) in tumor cells, which can bind to ROS and reduce oxidative stress, reducing the efficacy of CDT. In addition, tumor cells also use antioxidant enzymes such as thioredoxin reductase (TrxR) to repair oxidative damage, thereby reducing the toxicity of hydroxyl radicals to cells. Therefore, how to provide a more effective drug for treating tumors is imminent. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a cobalt-methionine nanoparticle that can both enhance reactive oxygen and inhibit the activity of antioxidant enzymes, thereby effectively improving the efficacy of CDT and achieving the treatment of tumor-related diseases.

[0005] To achieve the above object, in a first aspect of the present invention, the present invention provides a cobalt-methionine nanoparticle, wherein the cobalt-methionine nanoparticle is spherical and has an average particle size of 70-190 nm.

[0006] The cobalt and methionine in the cobalt-methionine nanoparticles provided by the present invention have a dual effect after being combined; specifically, the nanoparticles release cobalt ions, and after entering the tumor cells, the cobalt ions can be reduced to active Co by glutathione in the cells. +Ions promote Fenton-like reactions in cells, produce highly active oxygen free radicals, and the produced oxygen free radicals can trigger oxidative stress reactions in cells, further enhancing the effect of chemotherapy and making tumor cells more easily killed; at the same time, cobalt-methionine nanoparticles have antioxidant enzyme inhibitory effects, and methionine in nanoparticles can inhibit the activity of antioxidant enzymes in tumor cells, weakening their ability to resist oxidative stress, and this inhibitory effect further strengthens the oxidative stress effect, making tumor cells more susceptible to damage and killing, which is of great significance for improving the efficacy of CDT, and can further enhance the effectiveness of tumor treatment. In addition, the average particle size of the cobalt-methionine nanoparticles provided by the present invention is within a suitable range, and it has excellent stability and can play a better role.

[0007] As a preferred embodiment of the nanoparticles of the present invention, the average particle size of the cobalt-methionine nanoparticles is 90-120 nm.

[0008] The present invention has found that the average particle size of cobalt-methionine nanoparticles affects the stability of the product. When the average particle size is further selected to be the above value, the stability of the obtained nanoparticles is better and can be stably present in a PBS solution with a pH value of 7.4 for more than one week.

[0009] In the second aspect of the present invention, the present invention provides a method for preparing the cobalt-methionine nanoparticles, which comprises the following steps: uniformly mixing a solution containing cobalt ions and a methionine solution, then dripping a sodium hydroxide solution and stirring, stopping dripping the sodium hydroxide solution after monitoring the pH value of the system to 8-9, continuing to stir the reaction, centrifuging after the reaction is completed, collecting and drying the precipitate to obtain the cobalt-methionine nanoparticles.

[0010] The present invention has found that the method for preparing cobalt-methionine nanoparticles provided by the present invention can obtain a product with an average particle size within the range given by the present invention, and can make the obtained product have excellent stability, and the subsequently obtained nanoparticles have a stronger ability to kill tumor cells.

[0011] As a preferred embodiment of the preparation method of the present invention, the molar ratio of the cobalt ion to methionine is (0.5-2):1.

[0012] Preferably, the molar ratio of the manganese ion to phenylalanine is 1:1.

[0013] The present invention has found that the molar ratio of cobalt ions to methionine during the preparation process affects the stability of the prepared product and its killing effect on tumor cells. When the molar ratio of cobalt ions to methionine is further selected to be (0.5-2):1, especially 1:1, the stability of the obtained product is better and the killing effect on tumor cells is also stronger.

[0014] As a preferred embodiment of the preparation method of the present invention, the cobalt ions provided in the solution containing cobalt ions include cobalt chloride.

[0015] As a preferred embodiment of the preparation method of the present invention, the stirring reaction time is 1.5-2.5h.

[0016] As a preferred embodiment of the preparation method of the present invention, in the solution containing cobalt ions, the molar concentration of cobalt ions is 0.08-0.12 mol / L.

[0017] Preferably, in the solution containing cobalt ions, the solvent is water.

[0018] As a preferred embodiment of the preparation method of the present invention, the molar concentration of methionine in the methionine solution is 0.08-0.12 mol / L.

[0019] Preferably, in the methionine solution, the solvent is water.

[0020] As a preferred embodiment of the preparation method of the present invention, the molar concentration of sodium hydroxide in the sodium hydroxide solution is 0.48-0.52 mol / L.

[0021] Preferably, in the sodium hydroxide solution, the solvent is water.

[0022] As a preferred embodiment of the preparation method of the present invention, the drying temperature is (-55)-(-45)°C, and the drying time is 22-26 hours.

[0023] In the third aspect of the present invention, the present invention also provides the use of the cobalt-methionine nanoparticles in the preparation of anti-tumor drugs.

[0024] The cobalt ions in the cobalt-methionine nanoparticles provided by the present invention can be reduced to active Co by glutathione in cells. + ions, promoting the Fenton-like reaction and producing highly active hydroxyl radicals (·OH). These hydroxyl radicals can induce oxidative stress and induce apoptosis of tumor cells. At the same time, the methionine gradually released by the nanoparticles can inhibit the activity of thioredoxin reductase (TrxR), weaken the protective effect of the antioxidant system, and further enhance the efficacy of CDT; therefore, it can be widely used in the preparation of anti-tumor drugs.

[0025] As a preferred embodiment of the application of the present invention, the tumor includes any one of breast cancer, glioblastoma, and lung cancer.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The cobalt and methionine in the cobalt-methionine nanoparticles provided by the present invention have a dual effect after being combined, which can not only promote the Fenton-like reaction to produce highly active hydroxyl free radicals, thereby triggering oxidative stress and inducing apoptosis of tumor cells; at the same time, the nanoparticles can also inhibit the activity of thioredoxin reductase, weaken the protective effect of the antioxidant system, and further enhance the efficacy of CDT; therefore, it can be applied to the treatment of tumors, which is of great significance for improving the efficacy of CDT and can further improve the accuracy of tumor treatment; and the average particle size of the product prepared by the cobalt-methionine nanoparticle preparation method provided by the present invention is within a suitable range, it has excellent stability, and can play a better role. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a TEM observation result of the nanoparticles prepared in Example 1;

[0029] Figure 2 This is a particle size distribution diagram of the nanoparticles prepared in Example 1;

[0030] Figure 3 This is the result of cell apoptosis displayed by flow cytometry in effect example 3;

[0031] Figure 4 This is a photo of the tumor tissue in effect example 4;

[0032] Figure 5 This is the result of TUNEL staining of tumor sections in Effect Example 4. DETAILED DESCRIPTION

[0033] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0034] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0035] Example 1

[0036] An embodiment of the present invention provides a cobalt-methionine nanoparticle, and a method for preparing the cobalt-methionine nanoparticle comprises the following steps:

[0037] (1) Take 10 mL of 0.1 mol / L cobalt salt (CoCl 2 Solution) and 10mL 0.1mol / L methionine solution, and mix the two evenly;

[0038] (2) slowly adding 0.5 mol / L NaOH solution to the mixed solution of step (1) while gently stirring until the pH value of the solution stabilizes between 8 and 9, and continuing to stir the reaction for 2 h;

[0039] (3) After the reaction is completed, the mixture is centrifuged at 5000 rpm for 10 min, the precipitate is collected and washed with deionized water, and then dried at -50°C for 24 h to obtain cobalt-methionine nanoparticles.

[0040] Example 2

[0041] The embodiment of the present invention provides a cobalt-methionine nanoparticle. The only difference between the cobalt-methionine nanoparticle and the embodiment 1 is that the molar ratio of cobalt ions to phenylalanine is 0.5:1.

[0042] Example 3

[0043] The embodiment of the present invention provides a cobalt-methionine nanoparticle, and the only difference between the cobalt-methionine nanoparticle and the embodiment 1 is that the molar ratio of cobalt ions to phenylalanine is 2:1.

[0044] Comparative Example 1

[0045] The comparative example of the present invention provides a nanoparticle, the only difference between the nanoparticle and Example 1 is that the cobalt ions are replaced by iron ions.

[0046] Comparative Example 2

[0047] The comparative example of the present invention provides a nanoparticle, the only difference between the nanoparticle and Example 1 is that methionine is replaced by arginine.

[0048] Comparative Example 3

[0049] The comparative example of the present invention provides a nanoparticle, the only difference between the nanoparticle and Example 1 is that the molar ratio of cobalt ions to phenylalanine is 3:1.

[0050] Comparative Example 4

[0051] The comparative example of the present invention provides a nanoparticle, the only difference between the nanoparticle and Example 1 is that the molar ratio of cobalt ions to phenylalanine is 0.3:1.

[0052] Effect Example 1

[0053] Effect Examples of the Invention The average particle size and stability of the nanoparticles prepared in Examples 1-3 and Comparative Examples 1-4 were investigated, specifically including the following aspects:

[0054] 1. Average particle size: measured using dynamic light scattering (DLS) technology;

[0055] 2. Stability: 10 mg of nanoparticles were placed in 10 mL of PBS buffer with a pH value of 7.4 and observed for aggregation and sedimentation. If there was no aggregation or sedimentation after 7 days, the time was recorded as >7 days.

[0056] The obtained results are shown in Table 1;

[0057] Table 1

[0058]

[0059] As can be seen from Table 1, the average particle size of the nanoparticles prepared by the preparation method of the present invention is between 68 and 184 nm, and the obtained nanoparticles have excellent stability and will not aggregate or precipitate after being placed for more than 7 days; when the molar ratio of cobalt ions to methionine is not within the range given by the present invention, the corresponding nanoparticles cannot be prepared; when iron is used to replace cobalt, the stability of the obtained nanoparticles is too poor, and a large amount of aggregation and precipitation are observed after being placed for one day; the nanoparticles prepared in Example 1 are further observed by transmission electron microscopy (TEM), and the obtained picture is as follows: Figure 1 As shown, from Figure 1 As can be seen from the figure, the prepared product is spherical; the particle size distribution of the nanoparticles prepared in Example 1 is as follows Figure 2 As shown, from Figure 2 It can be seen that the prepared nanoparticles have excellent dispersibility.

[0060] Effect Example 2

[0061] Since the stability of the nanoparticles prepared in Comparative Example 1 is too poor, nanoparticles cannot be formed in Comparative Examples 3-4. Therefore, this effect example does not further explore Comparative Examples 1 and Comparative Examples 3-4. This effect explores the effect of the nanoparticles prepared in Examples 1-3 and Comparative Example 2 on the in vitro cell survival rate of U87 tumor cells, including the following steps:

[0062] (1) U87 tumor cells were evenly suspended in a culture dish and incubated at 37°C and 5% CO 2 Cultivate cells under appropriate culture conditions to allow them to grow and attach;

[0063] (2) 1×10 4 U87 cells were seeded in 96-well plates and divided into the following groups: PBS group, cobalt ion group, methionine solution group, and cobalt-methionine nanoparticle group described in Example 1-3. 2 Under the conditions, the cells were pre-cultured for 24 hours. Then, the cells in each group were treated with PBS and 20 μg / mL cobalt ions, 20 μg / mL methionine solution, and 20 μg / mL cobalt-methionine nanoparticles;

[0064] (3) The survival rate of each group of cells was determined using the MTT method, and the absorbance of the generated formazol blue crystals was read using a microplate reader (BiochromAnthos 2020 microplate reader) at a wavelength of 570 nm;

[0065] The calculation method is as follows:

[0066] S1. Calculation of cell survival rate:

[0067] a. The absorbance value (OD value) of cells in each experimental group was determined using the MTT method;

[0068] b. Take the absorbance value of the PBS group as 100% survival rate, compare the absorbance values ​​of the other groups, and calculate their survival rates. The formula is as follows:

[0069] Survival rate (%) = OD value of experimental group / OD value of PBS group × 100%;

[0070] S2. Data processing and statistical analysis:

[0071] a. The results of cell viability are represented by the mean and standard deviation of multiple parallel experiments;

[0072] b. Use statistical software to perform variance analysis to determine the significant differences between groups. The significance level was set at p < 0.05.

[0073] Through the above calculation method, the cell survival rate data obtained are shown in Table 2;

[0074] Table 2

[0075]

[0076] As can be seen from Table 2, the nanoparticles prepared by the technical solution provided by the present invention have excellent in vitro inhibition of U87 tumor cell viability, with a survival rate of less than 46.91%, which has a significant inhibitory effect compared to the use of cobalt ions and methionine alone (*p<0.05); and, compared with Comparative Example 2, it also shows a better inhibitory ability ( # p<0.05).

[0077] Effect Example 3

[0078] This effect example explores the apoptosis of U87 tumor cells in vitro by the nanoparticles prepared in Example 1, and specifically includes the following steps:

[0079] (1) U87 cells were treated with equal concentrations of cobalt ion solution, methionine solution, and cobalt-methionine nanoparticle solution for 24 hours, and PBS was used as a blank control group;

[0080] (2) After the treatment, the supernatant was removed and replaced with fresh medium, and the culture was continued for 24 hours, and then the cell pellet was collected and washed by centrifugation (300g, 5 minutes). The cell pellet was resuspended in 500 μL binding buffer, 5 μL Annexin V and PI solution (Lianke Technology, Hangzhou, China) was added, and incubated in the dark for 15 minutes. Cell apoptosis was analyzed by flow cytometry (BD Biosciences);

[0081] The results are as follows Figure 3 As shown, from Figure 3 It can be seen that the nanoparticles prepared in Example 1 of the present invention can significantly increase cell apoptosis, that is, have excellent anti-tumor effects. Specifically, the cell apoptosis rate of the PBS group was the lowest, about 3.04%, and as a control group, there was almost no effect of inducing apoptosis. The apoptosis rate of the cobalt ion group increased to 9.24%, indicating that cobalt ions have a certain pro-apoptotic effect. The apoptosis rate of the methionine solution group further increased to 14.87%, showing a more obvious pro-apoptotic effect. The most significant was the cobalt-methionine nanoparticle group, with a cell apoptosis rate of up to 41.08%, significantly exceeding that of other groups, indicating that cobalt-methionine nanoparticles have a very strong effect in inducing tumor cell apoptosis.

[0082] Effect Example 4

[0083] This effect example explores the nanoparticles prepared in Examples 1-3 and conducts in vivo animal experiments, including the following steps:

[0084] (1) In the in vivo animal experiment, BALB / c female mice (5 weeks old) were used as research subjects to simulate the inhibition of glioblastoma. First, U87 cells (1×10 6Cells, 100 μL PBS) were implanted into the right posterior thigh of BALB / c nude mice. On the seventh day after tumor implantation, all mice were randomly divided into four treatment groups (n=5). The doses of the four treatment groups were equivalent to 10 mg of the drug per kilogram of body weight: (Group 1) PBS group (0.9% saline injection); (Group 2) cobalt ion group (10 mg / kg, dissolved in 0.9% saline injection); (Group 3) methionine solution group (10 mg / kg, dissolved in 0.9% saline injection); (Group 4) cobalt-methionine nanoparticle group in Example 1 (equivalent to 10 mg / kg of cobalt-methionine nanoparticles, dissolved in 0.9% saline injection); (Group 5) cobalt-methionine nanoparticle group in Example 2 (equivalent to 10 mg / kg of cobalt-methionine nanoparticles, dissolved in 0.9% saline injection) (Group 6) cobalt-methionine nanoparticle group in Example 3 (equivalent to 10 mg / kg of cobalt-methionine nanoparticles, dissolved in 0.9% saline injection). Drug treatment was injected into the tail vein once a week for four weeks; after 4 weeks, the nude mice were euthanized and dissected, the tumors were removed, and fixed with 4% paraformaldehyde;

[0085] (2) Measure and record tumor volume and evaluate the inhibitory effect of cobalt-methionine nanoparticles on tumor growth;

[0086] (3) The apoptosis of tumor cells was evaluated by TUNEL staining to reveal the apoptosis-inducing effect of cobalt-methionine nanoparticles on tumor cells;

[0087] Calculation method of tumor tissue reduction

[0088] In the in vivo experiment, the tumor volume before and after treatment was measured to calculate the reduction in tumor tissue. The specific steps are as follows:

[0089] S1. Tumor volume measurement:

[0090] a. Use calipers to measure the long diameter (L) and short diameter (W) of the tumor, and calculate the tumor volume (V) according to the following formula: V = (L × W 2 ) / 2;

[0091] b. Record the tumor volume of each experimental group before and after treatment;

[0092] S2. Calculation of tumor reduction:

[0093] a. Calculate the tumor volume difference before and after treatment (ΔV): ΔV = V 治疗前 -V 治疗后 ;

[0094] b. Calculate the reduction rate of tumor volume (Reduction Rate), expressed as a percentage:

[0095] Reduction Rate (%) = (ΔV / V 治疗前 )×100;

[0096] S3. The results are expressed as the average reduction in each experimental group.

[0097] By using the above method, the detailed data of tumor volume reduction in each group in Table 3 can be obtained and used to compare the anti-tumor effects of different groups;

[0098] Table 3

[0099]

[0100] It can be seen from Table 3 that the nanoparticles obtained by the technical solution of the present invention can also significantly reduce the tumor volume in vivo, especially the nanoparticles prepared at a mass ratio of 1:1; among them, the tumor tissues obtained in groups 1-4 are as follows Figure 4 As shown, TUNEL staining of tumor tissue sections from groups 1-4 was performed. Figure 5 As shown, from Figure 4-5 It can be seen that the nanoparticles provided by the present invention can significantly reduce the volume of tumors in vivo and achieve excellent anti-tumor effects.

[0101] Finally, it should be noted that the above embodiments are intended to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A cobalt-methionine nanoparticle, characterized in that: The cobalt-methionine nanoparticles are spherical and have an average particle size of 70-190 nm.

2. The method for preparing cobalt-methionine nanoparticles according to claim 1, characterized in that: The preparation method comprises the following steps: uniformly mixing a solution containing cobalt ions and a methionine solution, then dripping a sodium hydroxide solution and stirring, stopping dripping the sodium hydroxide solution after monitoring the pH value of the system to 8-9, continuing to stir the reaction, centrifuging after the reaction is completed, collecting and drying the precipitate, and obtaining cobalt-methionine nanoparticles.

3. The preparation method according to claim 2, characterized in that: The molar ratio of the cobalt ion to methionine is (0.5-2):

1.

4. The preparation method according to claim 2, characterized in that: The stirring reaction time is 1.5-2.5h.

5. The preparation method according to claim 2, characterized in that: In the solution containing cobalt ions, the molar concentration of cobalt ions is 0.08-0.12 mol / L; and / or, in the methionine solution, the molar concentration of methionine is 0.08-0.12 mol / L; And / or, in the sodium hydroxide solution, the molar concentration of sodium hydroxide is 0.48-0.52 mol / L.

6. The preparation method according to claim 2, characterized in that: The drying temperature is (-55)-(-45)°C, and the drying time is 22-26 hours.

7. Use of the cobalt-methionine nanoparticles according to claim 1 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that: The tumor includes any one of breast cancer, glioblastoma, and lung cancer.

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