Bee venom peptide-loaded platinum-ruthenium bimetallic nano preparation and preparation method thereof

Abstract

The invention relates to a melittin-loaded platinum-ruthenium bimetallic nano preparation and a preparation method thereof. The melittin-loaded platinum-ruthenium bimetallic nano preparation is prepared by taking chloroplatinic acid hexahydrate, ruthenium trichloride trihydrate, polyvinylpyrrolidone, ascorbic acid and melittin as basic materials. In-vitro basic property experiments show that the nano preparation has good active oxygen scavenging performance. Further cell experiments and animal experiments show that the nano preparation has good biocompatibility and oxidation resistance, can relieve oxidative stress of an ulcerative colon part, and has a wide application prospect in treatment of ulcerative colitis.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a platinum-ruthenium bimetallic nano preparation loaded with melittin and a preparation method thereof. Background Art

[0002] Ulcerative colitis is a chronic inflammatory bowel disease characterized by chronic inflammation and ulcer formation in the large intestine, which seriously affects the health and quality of life of patients. Currently, there is no cure for ulcerative colitis, and existing drug treatments can usually only relieve symptoms, and long-term use will bring certain side effects. Based on this, nanotechnology has shown great potential in the development of drug delivery systems, providing a new approach for the treatment of ulcerative colitis.

[0003] Reactive oxygen species (ROS) are defined as oxygen / nitrogen-containing reactive substances, mainly including OH, H2O2, etc., which are byproducts of many enzymatic reactions in various cell compartments and have strong oxidative capacity. Under the pathological conditions of ulcerative colitis, reactive oxygen species oxidize protein and lipid components in cells and damage DNA. Therefore, the scavenging of reactive oxygen species is essential for the treatment of intestinal inflammation. Nanomaterials with the function of scavenging reactive oxygen species can overcome the shortcomings of natural antioxidants, such as increased side effects due to multiple administrations, low absorption rate and bioavailability.

[0004] Melittin has different biological functions such as anti-inflammatory, anti-cancer, antibacterial, and antioxidant. Melittin has anti-inflammatory activity by inhibiting the release of inflammatory cytokines such as tumor necrosis factor (TNF-α), interleukin (IL-6), and prostaglandin E2 (PGE2). In addition, melittin induces its anti-inflammatory effect by reducing toll-like receptor 4 (TLR4), which is overexpressed in patients with ulcerative colitis.

[0005] Based on the above research background, we prepared a platinum-ruthenium bimetallic nanoformulation loaded with bee venom peptide, which has an appropriate particle size distribution, good reactive oxygen scavenging performance, and good cell biocompatibility and animal therapeutic effect. Therefore, it is expected to achieve good therapeutic effects on ulcerative colitis. Summary of the invention

[0006] The purpose of the present invention is to provide a platinum-ruthenium bimetallic nanoformulation loaded with melittin and a preparation method thereof. The platinum-ruthenium bimetallic nanoformulation loaded with melittin is prepared with chloroplatinic acid hexahydrate, ruthenium trichloride trihydrate, polyvinyl pyrrolidone, ascorbic acid and melittin as basic materials. The nanoformulation has good active oxygen scavenging performance, biocompatibility and animal treatment effect, and has broad application prospects in the treatment of ulcerative colitis.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] Step 1: dissolving ruthenium trichloride trihydrate and chloroplatinic acid hexahydrate in ultrapure water respectively, and stirring the two to obtain a platinum-ruthenium mixed solution;

[0009] Step 2: prepare polyvinyl pyrrolidone and ascorbic acid solution and add the platinum-ruthenium mixed solution obtained in step 1 in sequence, heat and stir, centrifuge and wash to obtain a platinum-ruthenium bimetallic nanoparticle preparation;

[0010] Step 3: Melittin is dissolved in ultrapure water;

[0011] Step 4: fully mix the platinum-ruthenium bimetallic nano-dispersion obtained in step 2 and the melittin solution obtained in step 3, stir at 4° C., centrifuge, and wash to obtain a platinum-ruthenium bimetallic nano-preparation loaded with melittin;

[0012] As a further improvement of the present invention, step 1 is specifically to dissolve 10-20 mg of ruthenium trichloride trihydrate and 20-40 mg of chloroplatinic acid hexahydrate in 1-10 mL and 1-10 mL of ultrapure water respectively, and stir the two to obtain a platinum-ruthenium mixed solution;

[0013] As a further improvement of the present invention, step 2 specifically comprises dissolving 50-200 mg of polyvinyl pyrrolidone and 50-200 mg of ascorbic acid in 1-10 mL and 2-10 mL of ultrapure water, respectively, and sequentially adding the platinum-ruthenium mixed solution obtained in step 1, heating and stirring for 4-8 hours, centrifuging, and washing to obtain a platinum-ruthenium bimetallic nanoformulation;

[0014] As a further improvement of the present invention, step 3 is specifically dissolving 2-20 mg of melittin in 1-10 mL of ultrapure water to obtain a melittin solution;

[0015] As a further improvement of the present invention, step 4 specifically comprises fully mixing the platinum-ruthenium bimetallic nano-dispersion obtained in step 2 with the melittin solution obtained in step 3, stirring at 4° C. for 6-24 hours, centrifuging, and washing to obtain a platinum-ruthenium bimetallic nano-preparation loaded with melittin;

[0016] The innovations of the present invention are as follows: The present invention uses platinum-ruthenium bimetallic nanoformulation to load bee venom peptide for the first time to prepare a composite nanoformulation for scavenging hydroxyl free radicals and hydrogen peroxide. The nanoformulation has good performance in scavenging active oxygen and has good biocompatibility and animal treatment effect, and is expected to achieve better therapeutic effect on ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the appearance of the platinum-ruthenium bimetallic nanoformulation loaded with melittin;

[0018] Figure 2 is the particle size distribution diagram of platinum-ruthenium bimetallic nanoformulation loaded with melittin;

[0019] Figure 3 Transmission electron microscopy image of platinum-ruthenium bimetallic nanoformulation loaded with melittin;

[0020] Figure 4 This is the ·OH scavenging performance diagram of the platinum-ruthenium bimetallic nanoformulation loaded with melittin;

[0021] Figure 5 This is a graph showing the H2O2 removal performance of platinum-ruthenium bimetallic nanoformulation loaded with melittin;

[0022] Figure 6 The biocompatibility diagram of platinum-ruthenium bimetallic nanoformulation loaded with melittin;

[0023] Figure 7 The figure shows the cellular antioxidant properties of platinum-ruthenium bimetallic nanoformulation loaded with melittin;

[0024] Figure 8 Figure 2 shows the colon of mice treated with melittin-loaded platinum-ruthenium bimetallic nanoparticles.

[0025] Fig. 9 A graph showing the colon length of mice after treatment with melittin-loaded platinum-ruthenium bimetallic nanoformulation. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below by examples:

[0027] Example 1

[0028] This embodiment is a method for preparing a platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin, comprising the following steps:

[0029] Weigh 10 mg of ruthenium trichloride trihydrate and 20 mg of chloroplatinic acid hexahydrate and dissolve them in 3 mL and 2.5 mL of ultrapure water respectively, stir the two to obtain a platinum-ruthenium mixed solution, then add polyvinyl pyrrolidone (20 mg / mL, 5 mL) and ascorbic acid (25 mg / mL, 4 mL) in sequence, stir at 80 ° C for 4 hours, centrifuge and wash to obtain a platinum-ruthenium bimetallic nanoformulation. Weigh 2.5 mg of bee venom peptide and dissolve it in 2.5 mL of ultrapure water. Disperse 2.5 mg of platinum-ruthenium bimetallic nanoformulation in 2.5 mL of pure water, add bee venom peptide solution, stir at 4 ° C for 12 hours, centrifuge and wash to obtain a platinum-ruthenium bimetallic nanoformulation loaded with bee venom peptide.

[0030] Example 2

[0031] This embodiment is a method for preparing a platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin, comprising the following steps:

[0032] Weigh 20 mg of ruthenium trichloride trihydrate and 40 mg of chloroplatinic acid hexahydrate and dissolve them in 3 mL and 2.5 mL of ultrapure water respectively, stir the two to obtain a platinum-ruthenium mixed solution, then add polyvinyl pyrrolidone (20 mg / mL, 5 mL) and ascorbic acid (25 mg / mL, 4 mL) in sequence, stir at 80 ° C for 4 hours, centrifuge and wash to obtain a platinum-ruthenium bimetallic nanoformulation. Weigh 2.5 mg of bee venom peptide and dissolve it in 2.5 mL of ultrapure water. Disperse 2.5 mg of platinum-ruthenium bimetallic nanoformulation in 2.5 mL of pure water, add bee venom peptide solution, stir at 4 ° C for 12 hours, centrifuge and wash to obtain a platinum-ruthenium bimetallic nanoformulation loaded with bee venom peptide.

[0033] Example 3

[0034] This embodiment is a method for preparing a platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin, comprising the following steps:

[0035] Weigh 20 mg of ruthenium trichloride trihydrate and 40 mg of chloroplatinic acid hexahydrate and dissolve them in 3 mL and 2.5 mL of ultrapure water respectively, stir the two to obtain a platinum-ruthenium mixed solution, then add polyvinyl pyrrolidone (30 mg / mL, 5 mL) and ascorbic acid (40 mg / mL, 4 mL) in sequence, stir at 80 ° C for 4 hours, centrifuge and wash to obtain a platinum-ruthenium bimetallic nanoformulation. Weigh 2.5 mg of bee venom peptide and dissolve it in 2.5 mL of ultrapure water. Disperse 2.5 mg of platinum-ruthenium bimetallic nanoformulation in 2.5 mL of pure water, add bee venom peptide solution, stir at 4 ° C for 12 hours, centrifuge and wash to obtain a platinum-ruthenium bimetallic nanoformulation loaded with bee venom peptide.

[0036] Example 4

[0037] This embodiment is a method for preparing a platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin, comprising the following steps:

[0038] Weigh 20 mg of ruthenium trichloride trihydrate and 40 mg of chloroplatinic acid hexahydrate and dissolve them in 3 mL and 2.5 mL of ultrapure water respectively, stir the two to obtain a platinum-ruthenium mixed solution, then add polyvinyl pyrrolidone (40 mg / mL, 5 mL) and ascorbic acid (50 mg / mL, 4 mL) in sequence, stir at 80 ° C for 4 hours, centrifuge and wash to obtain a platinum-ruthenium bimetallic nanoformulation. Weigh 2.5 mg of bee venom peptide and dissolve it in 2.5 mL of ultrapure water. Disperse 2.5 mg of platinum-ruthenium bimetallic nanoformulation in 2.5 mL of pure water, add bee venom peptide solution, stir at 4 ° C for 12 hours, centrifuge and wash to obtain a platinum-ruthenium bimetallic nanoformulation loaded with bee venom peptide.

[0039] Example 5

[0040] This embodiment is a method for preparing a platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin, comprising the following steps:

[0041] Weigh 20 mg of ruthenium trichloride trihydrate and 40 mg of chloroplatinic acid hexahydrate and dissolve them in 3 mL and 2.5 mL of ultrapure water respectively, stir the two to obtain a platinum-ruthenium mixed solution, then add polyvinyl pyrrolidone (40 mg / mL, 5 mL) and ascorbic acid (50 mg / mL, 4 mL) in sequence, stir at 80 ° C for 6 hours, centrifuge and wash to obtain a platinum-ruthenium bimetallic nanoformulation. Weigh 2.5 mg of bee venom peptide and dissolve it in 2.5 mL of ultrapure water. Disperse 2.5 mg of platinum-ruthenium bimetallic nanoformulation in 2.5 mL of pure water, add bee venom peptide solution, stir at 4 ° C for 12 hours, centrifuge and wash to obtain a platinum-ruthenium bimetallic nanoformulation loaded with bee venom peptide.

[0042] Example 6

[0043] This embodiment is a method for preparing a platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin, comprising the following steps:

[0044] Weigh 20 mg of ruthenium trichloride trihydrate and 40 mg of chloroplatinic acid hexahydrate and dissolve them in 3 mL and 2.5 mL of ultrapure water respectively, stir the two to obtain a platinum-ruthenium mixed solution, then add polyvinyl pyrrolidone (40 mg / mL, 5 mL) and ascorbic acid (50 mg / mL, 4 mL) in sequence, stir at 80 ° C for 6 hours, centrifuge and wash to obtain a platinum-ruthenium bimetallic nanoformulation. Weigh 2.5 mg of bee venom peptide and dissolve it in 2.5 mL of ultrapure water. Disperse 2.5 mg of platinum-ruthenium bimetallic nanoformulation in 2.5 mL of pure water, add bee venom peptide solution, stir at 4 ° C for 24 hours, centrifuge and wash to obtain a platinum-ruthenium bimetallic nanoformulation loaded with bee venom peptide.

[0045] Example 7

[0046] This example is an appearance inspection of a platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin, and includes the following steps:

[0047] Take photos to record the appearance of the platinum-ruthenium bimetallic nanoparticles loaded with melittin. Figure 1 As shown, the nanoformulation is black and has good dispersibility.

[0048] Example 8

[0049] This example is a particle size study of a platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin, and includes the following steps:

[0050] The particle size of the platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin at an appropriate concentration was measured using a Malvern particle size analyzer. Figure 2 As shown, the nanoformulation has a suitable particle size.

[0051] Example 9

[0052] This example is a transmission electron microscopy characterization of a platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin, comprising the following steps:

[0053] The nanoparticles dropped on the copper grid were photographed using a low voltage transmission electron microscope. Figure 3 As shown, the prepared platinum-ruthenium bimetallic nanoformulation loaded with melittin is in the shape of a black sphere.

[0054] Example 10

[0055] This example is to investigate the ROS scavenging performance of the platinum-ruthenium bimetallic nanoformulation loaded with melittin. Two representative ROS are selected: OH and H2O2 to study the ROS scavenging performance of the platinum-ruthenium bimetallic nanoformulation loaded with melittin, and the steps are as follows:

[0056] The ·OH scavenging performance experiment was conducted using the TMB colorimetric method to detect whether the platinum-ruthenium bimetallic nanoformulation loaded with melittin can scavenging ·OH. The specific method is: 6mM TMB, 3mM H2O2, 1mM FeSO4 and the platinum-ruthenium bimetallic nanoformulation loaded with melittin are placed in the dark for different periods of time, and the absorbance of the test solution at 652nm is monitored. Figure 4 As shown, the platinum-ruthenium bimetallic nanoformulation loaded with melittin can scavenge ·OH.

[0057] The TiSO4 colorimetric method was used to conduct a H2O2 scavenging performance experiment to detect whether the platinum-ruthenium bimetallic nanoformulation loaded with melittin could scavenging H2O2. The specific method is: a platinum-ruthenium bimetallic nanoformulation containing 6mM H2O2, 5% TiSO4 and melittin was placed in the dark for different periods of time, and the absorbance of the test solution at 415nm was monitored. Figure 5 As shown, the platinum-ruthenium bimetallic nanoformulation loaded with melittin can scavenge H2O2.

[0058] Embodiment 11

[0059] This example is a biocompatibility study of a platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin, and includes the following steps:

[0060] Macrophages (RAW264.7) were used to investigate the biocompatibility of platinum-ruthenium bimetallic nanoformulations loaded with bee venom peptide. RAW264.7 cells were first inoculated in a 96-well plate. After the cells grew to an appropriate density, different concentrations of platinum-ruthenium bimetallic nanoformulations loaded with bee venom peptide were given. The culture was continued for 24 hours. After the nanodispersion was aspirated, 100 μL of MTT solution was added to each well. After another 4 hours of culture, dimethyl sulfoxide solution was added and the absorbance of the cells at 490 nm was detected. The results are shown in Figure 2. Figure 6 As shown, the cell group treated with the platinum-ruthenium bimetallic nanoformulation loaded with bee venom peptide still had a high survival rate, indicating that the nanoformulation had good biocompatibility.

[0061] Example 12

[0062] This example is to investigate the cellular antioxidant properties of the platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin, and includes the following steps:

[0063] RAW264.7 cells were seeded in a 96-well plate. After the cells grew to an appropriate density, different concentrations of platinum-ruthenium bimetallic nanoparticles loaded with bee venom peptide were administered. After 4 hours of culture, a certain concentration of hydrogen peroxide solution was administered. The culture was continued for 4 hours. After the nanodispersion was aspirated, 100 μL of MTT solution was added to each well. After another 4 hours of culture, dimethyl sulfoxide solution was added and the absorbance of the cells at 490 nm was detected. The results are shown in Figure 7 As shown, the platinum-ruthenium bimetallic nanoformulation loaded with bee venom peptide has good cellular antioxidant properties.

[0064] Embodiment 13

[0065] This example is an investigation of the animal treatment effect of a platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin, and includes the following steps:

[0066] Male Balb / C mice aged 6-8 weeks and weighing 15-20g were selected as animal experimental modeling subjects. The mice were randomly divided into two groups, with 3 mice in each group, including a positive control group and a platinum-ruthenium bimetallic nanoformulation group loaded with bee venom peptide. The two groups of mice were treated differently. The positive control group added 3.3% sodium dextran sulfate to drinking water for 7 consecutive days to induce ulcerative colitis in the mice; the platinum-ruthenium bimetallic nanoformulation group loaded with bee venom peptide added 3.3% sodium dextran sulfate to drinking water for 7 consecutive days. During this period, the platinum-ruthenium bimetallic nanoformulation group loaded with bee venom peptide was administered through the tail vein every other day. After 7 days, the mice were euthanized, the colon tissues were collected, the length of the mouse colon was measured and photographed for record. The results are as follows Figure 8 and Fig. 9 As shown, the colon length of mice in the platinum-ruthenium bimetallic nanoformulation group loaded with bee venom peptide was significantly higher than that of mice in the positive control group, indicating that the platinum-ruthenium bimetallic nanoformulation loaded with bee venom peptide alleviated the shortening of the mouse colon and had a therapeutic effect on ulcerative colitis in mice.

[0067] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A platinum-ruthenium bimetallic nanoparticle preparation loaded with bee venom peptide, It is characterized in that The nano preparation is prepared from chloroplatinic acid hexahydrate, ruthenium trichloride trihydrate, polyvinyl pyrrolidone, ascorbic acid and bee venom peptide, and the preparation method thereof comprises the following steps: (1) dissolving 10-20 mg of ruthenium trichloride trihydrate and 20-40 mg of chloroplatinic acid hexahydrate in 1-10 mL and 1-10 mL of ultrapure water respectively, and stirring the two to obtain a platinum-ruthenium mixed solution; (2) dissolving 50-200 mg of polyvinyl pyrrolidone and 50-200 mg of ascorbic acid in 1-10 mL and 2-10 mL of ultrapure water, respectively, and sequentially adding the platinum-ruthenium mixed solution obtained in step (1), heating and stirring for 4-8 h, centrifuging, and washing to obtain a platinum-ruthenium bimetallic nanoparticle preparation; (3) 2-20 mg of melittin is dissolved in 1-10 mL of ultrapure water to obtain a melittin solution; (4) The platinum-ruthenium bimetallic nanoparticle preparation obtained in step (2) is fully mixed with the bee venom peptide solution obtained in step (3), stirred at 4° C. for 6-24 hours, centrifuged, and washed to obtain a platinum-ruthenium bimetallic nanoparticle preparation loaded with bee venom peptide.

2. The platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin according to claim 1, It is characterized in that The heating temperature in the preparation step (2) is 60-100°C.

3. The platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin according to claim 1, It is characterized in that The particle size of the platinum-ruthenium bimetallic nanoparticle preparation loaded with melittin is 50-300nm.

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