Manganese-phenylalanine nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses manganese-phenylalanine nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of medicines. Manganese and phenylalanine in the manganese-phenylalanine nano-particles provided by the invention have dual effects after being combined, and can enhance the generation of active oxygen and inhibit the activity of antioxidant enzyme, so that the manganese-phenylalanine nano-particles can be applied to tumor treatment, have important significance in improving the curative effect of CDT, and can further improve the accuracy of tumor treatment; moreover, the average particle size and Zeta potential of the product prepared by the manganese-phenylalanine nanoparticle preparation method provided by the invention are in proper ranges, and the product has 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 a manganese-phenylalanine nanoparticle and a preparation method and application thereof. Background Art

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

[0003] Chemodynamic therapy (CDT) is an emerging anticancer treatment strategy that uses transition metal ions to catalyze endogenous hydrogen peroxide (H2O2) to generate highly toxic hydroxyl radicals (·OH), thereby inducing tumor cell death. However, the effectiveness of CDT is limited to a certain extent by the presence of antioxidant molecules and enzymes in the tumor microenvironment. Specifically, glutathione (GSH), as a high-level antioxidant molecule, binds to intracellular ROS inside tumor cells, thereby alleviating oxidative stress and reducing the efficacy of CDT. On the other hand, antioxidant enzymes in tumor cells can also repair damaged biomolecules and protect cells from the toxic effects of hydroxyl radicals. Among them, superoxide dismutase (SOD) is an important antioxidant enzyme that can degrade superoxide anion radicals and reduce the toxicity of ROS. However, there is currently a lack of a method to simultaneously overcome the protective effects of GSH and SOD to achieve more effective CDT treatment. 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 manganese-phenylalanine 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 manganese-phenylalanine nanoparticle, wherein the manganese-phenylalanine nanoparticle is spherical, has an average particle size of 90-200 nm, and has a Zeta potential of (-35)-(-15) mV.

[0006] The manganese and phenylalanine in the manganese-phenylalanine nanoparticles provided by the present invention have a dual effect after being combined, which can not only enhance the generation of reactive oxygen species (ROS), but also inhibit the activity of antioxidant enzymes. 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. In addition, the average particle size and Zeta potential of the manganese-phenylalanine nanoparticles provided by the present invention are within a suitable range, and the nanoparticles have excellent stability and can play a better role.

[0007] Specifically, the manganese ions (Mn 2+ ) can be reduced to Mn by glutathione (GSH) in cells + , thereby promoting the Fenton-like reaction in the cell, rapidly generating hydroxyl radicals (·OH) and consuming GSH, thereby inducing an oxidative stress response in the cell; phenylalanine, as an amino acid, can effectively inhibit the activity of the antioxidant enzyme SOD, further enhancing the oxidative stress in tumor cells.

[0008] As a preferred embodiment of the nanoparticles of the present invention, the average particle size of the manganese-phenylalanine nanoparticles is 105 nm.

[0009] The present invention has found that the average particle size of manganese-phenylalanine 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.

[0010] In the second aspect of the present invention, the present invention provides a method for preparing the manganese-phenylalanine nanoparticles, which comprises the following steps: mixing a phenylalanine solution and a sodium hydroxide solution, then dripping a solution containing manganese ions, stirring the reaction during the dripping process, and dialyzing after the reaction is completed to obtain manganese-phenylalanine nanoparticles.

[0011] The present invention has found that the method for preparing manganese-phenylalanine nanoparticles provided by the present invention can obtain a product having an average particle size and a Zeta potential within the range given by the present invention, and can make the obtained product have excellent stability.

[0012] As a preferred embodiment of the preparation method of the present invention, the molar ratio of the manganese ion to phenylalanine is 1:(1-6).

[0013] Preferably, the molar ratio of the manganese ion to phenylalanine is 1:(2-3).

[0014] The present invention has found that the molar ratio of manganese ions to phenylalanine during the preparation process affects the stability of the prepared product. When the molar ratio of manganese ions to phenylalanine is further selected to be 1:(2-3), the stability of the obtained product is better.

[0015] As a preferred embodiment of the nanoparticles of the present invention, the solution containing manganese ions includes manganese chloride as a source of manganese ions.

[0016] As a preferred embodiment of the preparation method of the present invention, the reaction time is 25-35 minutes.

[0017] As a preferred embodiment of the preparation method of the present invention, the dialysis bag used for dialysis has a molecular weight cutoff of 10KDa, and the dialysis time is 70-74h.

[0018] The present invention has found that when the dialysis parameters are within the above range, the remaining small molecules and impurities in the system can be effectively removed.

[0019] As a preferred embodiment of the preparation method of the present invention, the volume ratio of the phenylalanine solution, the sodium hydroxide solution and the solution containing manganese ions is 1: (0.8-1.2): (0.5-0.8).

[0020] As a preferred embodiment of the preparation method of the present invention, the molar concentration of phenylalanine in the phenylalanine solution is 4.5-5.5 mmol / L.

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

[0022] As a preferred embodiment of the preparation method of the present invention, the molar concentration of sodium hydroxide in the sodium hydroxide solution is 4.5-5.5 mmol / L.

[0023] Preferably, in the sodium hydroxide solution, the solvent is water.

[0024] As a preferred embodiment of the preparation method of the present invention, the molar concentration of manganese ions in the solution containing manganese ions is 2-3 mmol / L.

[0025] Preferably, in the solution containing manganese ions, the solvent is water.

[0026] As a preferred embodiment of the preparation method of the present invention, after the dialysis is completed, the process further includes storing the product at -4°C.

[0027] In the third aspect of the present invention, the present invention also provides the use of the manganese-phenylalanine nanoparticles in the preparation of anti-tumor drugs.

[0028] The manganese-phenylalanine nanoparticles provided by the present invention can not only promote the Fenton reaction in cells to quickly generate hydroxyl radicals and consume GSH, but also effectively inhibit the activity of superoxide dismutase (SOD) in tumor cells, reduce the scavenging ability of SOD to ROS, and increase oxidative stress and cell damage; therefore, they can be widely used in the preparation of anti-tumor drugs.

[0029] As a preferred embodiment of the application of the present invention, the tumor includes any one of breast cancer, glioblastoma, and lung cancer.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The manganese and phenylalanine in the manganese-phenylalanine nanoparticles provided by the present invention have a dual effect after being combined, which can not only enhance the generation of reactive oxygen species (ROS), but also inhibit the activity of antioxidant enzymes. 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. Moreover, the average particle size and Zeta potential of the product prepared by the method for preparing the manganese-phenylalanine nanoparticles provided by the present invention are within a suitable range, and it has excellent stability and can play a better role. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a TEM observation result of the nanoparticles prepared in Example 1;

[0033] Figure 2 This is a particle size distribution diagram of the nanoparticles prepared in Example 1;

[0034] Figure 3 This is the result of cell apoptosis displayed by flow cytometry in effect example 3;

[0035] Figure 4 This is a photo of the tumor tissue in effect example 4;

[0036] Figure 5 This is the result of TUNEL staining of tumor sections in Effect Example 4. DETAILED DESCRIPTION

[0037] 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.

[0038] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0039] Example 1

[0040] The embodiment of the present invention provides a manganese-phenylalanine nanoparticle, and the preparation method of the manganese-phenylalanine nanoparticle comprises the following steps:

[0041] (1) mixing 15 mL of a 5 mmol / L sodium hydroxide aqueous solution and 15 mL of a 5 mmol / L phenylalanine aqueous solution to obtain a mixed solution;

[0042] (2) adding 10 mL of a 2.5 mmol / L aqueous solution of manganese chloride (the molar ratio of manganese ion to phenylalanine is 1:3) dropwise to the mixed solution of step (1), stirring while adding dropwise;

[0043] (3) After the addition is completed, stirring is continued for 30 minutes, and then the system is dialyzed. The molecular weight cutoff of the dialysis bag used for dialysis is 10 KDa, and the dialysis time is 72 hours. After the dialysis is completed, the system is stored at -4°C to obtain manganese-phenylalanine nanoparticles.

[0044] Example 2

[0045] The embodiment of the present invention provides a manganese-phenylalanine nanoparticle, and the only difference between the manganese-phenylalanine nanoparticle and the embodiment 1 is that the molar ratio of manganese ions to phenylalanine is 1:2.

[0046] Example 3

[0047] The embodiment of the present invention provides a manganese-phenylalanine nanoparticle, and the only difference between the manganese-phenylalanine nanoparticle and the embodiment 1 is that the molar ratio of manganese ions to phenylalanine is 1:1.

[0048] Example 4

[0049] The embodiment of the present invention provides a manganese-phenylalanine nanoparticle, and the only difference between the manganese-phenylalanine nanoparticle and the embodiment 1 is that the molar ratio of manganese ions to phenylalanine is 1:6.

[0050] Comparative Example 1

[0051] The comparative example of the present invention provides a nanoparticle, the only difference between the nanoparticle and Example 1 is that zinc chloride is used instead of manganese chloride.

[0052] Comparative Example 2

[0053] The comparative example of the present invention provides a nanoparticle, the only difference between the nanoparticle and Example 1 is that phenylalanine is replaced by lysine.

[0054] Comparative Example 3

[0055] The comparative example of the present invention provides a manganese-phenylalanine nanoparticle, and the only difference between the manganese-phenylalanine nanoparticle and Example 1 is that the molar ratio of manganese ions to phenylalanine is 1:0.5.

[0056] Comparative Example 4

[0057] The comparative example of the present invention provides a manganese-phenylalanine nanoparticle, and the only difference between the manganese-phenylalanine nanoparticle and Example 1 is that the molar ratio of manganese ions to phenylalanine is 1:8.

[0058] Effect Example 1

[0059] Effect Examples of the Invention The average particle size, Zeta potential and stability of the nanoparticles prepared in Examples 1-4 and Comparative Examples 1-4 were investigated, specifically including the following aspects:

[0060] 1. Average particle size and Zeta potential: measured using dynamic light scattering (DLS) technology;

[0061] 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.

[0062] The obtained results are shown in Table 1;

[0063] Table 1

[0064]

[0065]

[0066] 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 105 and 200 nm, and the obtained nanoparticles have excellent stability and will not aggregate or settle after being placed for more than 7 days; when the molar ratio of manganese ion to phenylalanine is not within the range given by the present invention, the corresponding nanoparticle structure cannot be formed; when zinc is used to replace manganese, the stability of the obtained nanoparticles is too poor, and obvious aggregation and sedimentation phenomena are observed after being placed for 1 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.

[0067] Effect Example 2

[0068] 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 1-4 and Comparative Example 2 on the in vitro cell survival rate of MCF-7 breast cancer cells, including the following steps:

[0069] (1) Evenly suspending MCF-7 breast cancer cells in a culture dish, and culturing the cells under appropriate culture conditions (e.g., 37° C., 5% CO 2 ) until the cells reach an appropriate growth state;

[0070] (2) The nanoparticles were added to the MCF-7 cell culture at a final concentration of 20 μg / mL and co-cultured with the cells. 2+The solution and phenylalanine solution were used as control treatments, and PBS solution was used as a blank control. The incubation time was 12 hours;

[0071] (3) Remove the treated cells from the culture dish and transfer them to a new culture dish. Use the CCK-8 reagent to perform the experiment according to the manufacturer's instructions. Add an appropriate amount of CCK-8 reagent to the treated cells, mix it with the cells thoroughly, and continue to incubate for a period of time, generally 2 hours, to allow the CCK-8 reagent to react with the living cells. Finally, use an ELISA reader or a multi-function plate reader to measure the absorbance value at a wavelength of 450nm. Calculate the cell viability based on the absorbance value. The calculation method is as follows:

[0072] S1. Calculation of cell survival rate:

[0073] a. The absorbance value (OD value) of cells in each experimental group was determined using the MTT method;

[0074] 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:

[0075] Survival rate (%) = OD value of experimental group / OD value of PBS group × 100%;

[0076] S2. Data processing and statistical analysis:

[0077] a. The results of cell viability are represented by the mean and standard deviation of multiple parallel experiments;

[0078] b. Use statistical software to perform variance analysis to determine the significant differences between groups. The significance level was set at p < 0.05.

[0079] Through the above calculation method, the cell survival rate data obtained are shown in Table 2;

[0080] Table 2

[0081]

[0082] 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 MCF-7 breast cancer cell viability, with a survival rate of less than 55.71%, and have a significant inhibitory effect compared to the use of manganese ions and phenylalanine alone (*p<0.05); and, compared with Comparative Example 2, it also shows a better inhibitory ability ( # p<0.05).

[0083] Effect Example 3

[0084] This effect example explores the effect of the nanoparticles prepared in Example 1 on the apoptosis of MCF-7 breast cancer cells, and specifically includes the following steps:

[0085] Manganese-phenylalanine nanoparticles were co-cultured with tumor cell lines to evaluate their pro-apoptotic effect on tumor cells. The specific operation steps are as follows: MCF-7 cells were cultured in RPMI-1640 / Dulbecco'smodified Eagle's medium (DMEM) containing FBS / penicillin / streptomycin (37°C, 5% CO2). Apoptosis analysis of MCF-7 cells was performed by flow cytometry. In a 6-well plate, MCF-7 cells were co-cultured with PBS and PBS solution of manganese-phenylalanine nanoparticles (manganese-phenylalanine nanoparticles group), PBS solution of phenylalanine (phenylalanine solution group), and Mn 2+ PBS solution (manganese ion group, manganese chloride was used in the manganese ion group), wherein the test solution contained manganese-phenylalanine nanoparticles, phenylalanine and Mn 2+ The final concentration of each was 35 μg / mL. After 4 hours of incubation, the cells were washed several times with PBS, digested with trypsin, resuspended with PBS and counted. Subsequently, the cells were stained with 5 μL of annexin V-FITC and 10 μL of PI at 4°C for 15 minutes and 5 minutes, respectively. Finally, the cells were analyzed using flow cytometry;

[0086] The results are as follows Figure 3 As shown, from Figure 3 As can be seen from the flow cytometry analysis results, there are significant differences in the cell apoptosis rates among the groups. The PBS group had the lowest cell apoptosis rate, about 2.45%, and as a control group, it had almost no effect of inducing apoptosis. The apoptosis rate in the manganese ion group increased to 8.12%, indicating that manganese ions have a certain pro-apoptotic effect. The apoptosis rate in the phenylalanine solution group further increased to 13.57%, showing a more obvious pro-apoptotic effect. The most significant was the cobalt-phenylalanine nanoparticle group, with a cell apoptosis rate of up to 38.49%, significantly exceeding that of other groups, indicating that cobalt-phenylalanine nanoparticles have a very strong effect in inducing tumor cell apoptosis.

[0087] Effect Example 4

[0088] This effect example investigates the nanoparticles prepared in Example 1 for in vivo animal experiments, including the following steps: 6-week-old female BALB / c mice were used to test the therapeutic effect of manganese-phenylalanine nanoparticles. To establish a xenograft tumor model, MCF-7 cells (1×10 7 ) was injected subcutaneously into the right side of the back of the mouse hind leg. When the tumor volume reached about 50 mm 3At the time of treatment, mice with MCF-7 tumors were randomly divided into 4 groups (n=5), namely PBS group, manganese ion solution group (prepared with manganese chloride), phenylalanine solution group and manganese-phenylalanine nanoparticle group for treatment. The drug concentration was 200μg / mL, and each mouse was injected with an appropriate amount of drug solution according to its body weight. The injection volume was 10μL / g (PBS group used PBS solution, and the other groups were prepared with PBS of the corresponding solution). After 18 days of treatment, all mice were killed by pulling the neck, and the tumor tissues were collected. After 10% formalin fixation and paraffin embedding, the tumor tissues were sectioned and TUNEL staining was performed to detect the apoptosis of tumor tissues induced by manganese-phenylalanine nanoparticles.

[0089] Obtain tumor tissue Figure 4 As shown, the experimental results show that manganese-phenylalanine nanoparticles are most effective in tumor treatment. Compared with the PBS group, the tumor volume in the manganese ion group decreased by about 25%-33%, the phenylalanine solution group decreased by about 40%-47%, and the manganese-phenylalanine nanoparticle group significantly decreased by about 60%-80%. These data fully demonstrate that manganese-phenylalanine nanoparticles have obvious advantages in inhibiting tumor volume and show extremely strong anti-tumor effects, which are far superior to the treatment methods using manganese ions or phenylalanine alone. TUNEL staining after tumor tissue sectioning is shown in Figure 2. 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.

[0090] 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 manganese-phenylalanine nanoparticle, characterized in that: The manganese-phenylalanine nanoparticles are spherical, have an average particle size of 90-200 nm, and a Zeta potential of (-35)-(-15) mV.

2. The method for preparing manganese-phenylalanine nanoparticles according to claim 1, characterized in that: The preparation method comprises the following steps: mixing a phenylalanine solution and a sodium hydroxide solution, then dripping a solution containing manganese ions, stirring the solution for reaction during the dripping process, and dialyzing after the reaction is completed to obtain manganese-phenylalanine nanoparticles.

3. The preparation method according to claim 2, characterized in that: The molar ratio of the manganese ion to phenylalanine is 1:(1-6).

4. The preparation method according to claim 2, characterized in that: The reaction time is 25-35 min.

5. The preparation method according to claim 2, characterized in that: The dialysis bag used in the dialysis has a molecular weight cutoff of 10 KDa, and the dialysis time is 70-74 hours.

6. The preparation method according to claim 2, characterized in that: The volume ratio of the phenylalanine solution, the sodium hydroxide solution and the solution containing manganese ions is 1:(0.8-1.2):(0.5-0.8).

7. The preparation method according to claim 2, characterized in that: In the phenylalanine solution, the molar concentration of phenylalanine is 4.5-5.5 mmol / L; And / or, in the sodium hydroxide solution, the molar concentration of sodium hydroxide is 4.5-5.5 mmol / L; And / or, in the solution containing manganese ions, the molar concentration of manganese ions is 2-3 mmol / L.

8. Use of the manganese-phenylalanine nanoparticles according to claim 1 in the preparation of anti-tumor drugs.

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

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