Bee venom peptide targeting preparation and application thereof

Abstract

The invention discloses a melittin targeted preparation and application, and in the melittin targeted preparation, albumin nanoparticles are loaded with melittin and peptin. Experimental results show that the melittin targeting preparation provided by the invention can greatly reduce the hemolytic activity of melittin. An in-vivo anti-tumor experiment result shows that the melittin targeting preparation provided by the invention has an outstanding anti-tumor curative effect. The albumin nanoparticles in the melittin targeted preparation can jointly deliver melittin and peptin, and after the melittin targeted preparation reaches a tumor site, melittin and peptin can inhibit proliferation of oxidative phosphorylated tumor cells and glycolytic cells respectively; growth of breast cancer, lung cancer and colon cancer is effectively inhibited through the combined effect of melittin and peptin, and the in-vivo anti-tumor effect is obvious. In addition, the targeting effect of the albumin nanoparticles can further improve the anti-tumor effect. Therefore, the compound is expected to be used in preparation and development of antitumor drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a melittin targeting preparation and its application. Background Art

[0002] Melittin is a major active substance derived from bee venom, which has various physiological activities such as anti-tumor, anti-inflammatory and immunomodulatory effects. Especially in the field of anti-tumor, it shows a unique molecular mechanism and potential application value. Research shows that melittin can penetrate the mitochondrial membrane of tumor cells, induce the collapse of mitochondrial potential, and release pro-apoptotic factors such as cytochrome C to kill tumor cells. Therefore, it has a good effect on treating oxidative phosphorylation tumor cells. Hypoxia is a significant feature of the tumor microenvironment, and there is a large heterogeneity. Tumor cells proximal to blood vessels generally metabolize through oxidative phosphorylation, while tumor cells far from blood vessels usually maintain their own needs by taking up glucose for glycolysis. Therefore, using melittin and glycolysis inhibitors can simultaneously inhibit different types of tumor cells, which plays an important role in improving tumor treatment.

[0003] Glycolysis includes a series of metabolic reactions. In an aerobic environment, glucose can be decomposed into pyruvate, and in an anaerobic environment, it is decomposed into lactate. Glycolysis is crucial for the metabolism of tumor cells, and tumor cells have also evolved various mechanisms to enhance glycolysis. Existing research shows that tumor cell glycolysis is affected by various factors such as lactate dehydrogenase, pyruvate kinase, and hexokinase. Therefore, regulating the activity of the above enzymes is of great significance for inhibiting the glycolysis of tumor cells.

[0004] Phellodrin is a natural polyphenol inhibitor that can target lactate dehydrogenase and convert pyruvate to lactate at the end of glycolysis. Research shows that phellodrin can effectively disrupt glycolysis and reduce the viability of various tumor cells. Therefore, combining it with melittin is of great significance for treating heterogeneous tumors.

[0005] Existing research shows that melittin can cause relatively serious hemolytic reactions during injection and damage normal tissues. In addition, since phellodrin is a small molecule drug, it has disadvantages such as low targeting performance and fast metabolism in the body. Therefore, simultaneously solving the in vivo delivery problems of melittin and phellodrin is of great significance for treating tumors.

[0006] In recent years, with the continuous development of nanotechnology, materials such as albumin nanoparticles, liposomes, and nanospheres have been applied to biomaterials to achieve the effects of enhancing efficacy and reducing toxicity. Among them, albumin nanoparticles are a type of carrier with protein as the carrier, which have the advantages of high safety, easy preparation, and strong stability. And the delivery of paclitaxel through this carrier has been successfully marketed, showing extremely high application prospects and potential.

[0007] In view of this, the present invention co-delivers melittin and periflavone by using albumin nanoparticles, in order to achieve efficient treatment of tumors. Summary of the Invention

[0008] Aiming at the technical problems existing in the background art, the purpose of the present invention is to provide a melittin targeting preparation and its application.

[0009] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0010] The first aspect of the present invention provides a preparation method of a melittin targeting preparation, including the following steps:

[0011] S1. Prepare a melittin-periflavone complex:

[0012] S11. Dissolve melittin and periflavone in a first organic solvent, and then remove the first organic solvent by rotary evaporation under reduced pressure to obtain a crude melittin-periflavone complex;

[0013] S12. Dissolve the obtained crude melittin-periflavone complex in a second organic solvent for re-dissolution, then filter it using a microporous filter membrane, and then remove the second organic solvent by rotary evaporation under reduced pressure to obtain a melittin-periflavone complex;

[0014] S2. Add the melittin-periflavone complex to a solvent to obtain an organic phase;

[0015] S3. Add a PBS solution containing albumin nanoparticles to the organic phase, dissolve it by ultrasonic treatment, centrifuge to take the supernatant, and filter it using a microporous filter membrane to obtain melittin-periflavone-albumin nanoparticles.

[0016] Preferably, the mass ratio of melittin to periflavone is 1:5 to 5:1.

[0017] Preferably, the mass-volume concentration of albumin nanoparticles in the PBS solution containing albumin nanoparticles is 0.2 to 5 mg / mL.

[0018] Preferably, the concentrations of both melittin and periflavone are 0.05 to 0.5 mg / mL.

[0019] Preferably, the ultrasonic treatment is probe ultrasonic treatment, the ultrasonic time is 10 to 50 min, and the power is 100 to 300 W.

[0020] Preferably, the particle size of the melittin-periflavone-albumin nanoparticles (hereinafter simply referred to as nano-drug) is 100 to 200 nm, and the Zeta potential is 0 to -20 mV.

[0021] The second aspect of the present invention provides the melittin targeting preparation prepared by the above preparation method.

[0022] The third aspect of the present invention provides the application of the above-mentioned melittin-targeted preparation in the preparation of anti-tumor drugs.

[0023] The present invention has the following beneficial effects:

[0024] (1) The melittin-targeted preparation prepared by the present invention is specifically melittin curcumin albumin nanoparticles, wherein the albumin nanoparticles are loaded with melittin and curcumin. This melittin-targeted preparation can deliver melittin and curcumin simultaneously. After reaching the tumor treatment site through the EPR effect, the hemolytic property of melittin is significantly reduced. At the same time, the phenolic acid group of curcumin increases the binding effect between melittin and albumin, further reducing the hemolytic property of melittin.

[0025] (2) The melittin-targeted preparation prepared by the present invention can deliver melittin and curcumin simultaneously. After reaching the tumor site, melittin and curcumin will respectively inhibit the proliferation (activity) of tumor cells by oxidative phosphorylation and glycolytic cells. Through the combined action of melittin and curcumin, it can effectively inhibit the growth of breast cancer, lung cancer and colon cancer, achieve efficient treatment of lung tumors, and have obvious anti-tumor effects in vivo. In addition, the targeting effect of albumin nanoparticles can further improve its anti-tumor effect.

[0026] (3) The method for preparing the melittin-targeted preparation of the present invention has a simple process, can be scaled up and is suitable for subsequent large-scale production.

[0027] (4) The preparation method of the melittin-targeted preparation of the present invention is simple and controllable, and the physical properties such as the particle size and potential of the nano-preparation can be adjusted by adjusting experimental conditions (raw material quality, temperature, feeding ratio, etc.). Therefore, it is expected to be used in the development of anti-tumor drugs. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0029] Figure 1 It is the particle size distribution result diagram of the melittin curcumin albumin nanoparticles prepared in Example 1;

[0030] Figure 2 It is; the morphology structure diagram of the melittin curcumin albumin nanoparticles prepared in Example 1;

[0031] Figure 3 It is the morphology structure diagram of the melittin curcumin albumin nanoparticles prepared in Example 1;

[0032] Figure 4 Result 1 for the growth and development of mouse lung cancer cells;

[0033] Figure 5 Result 2 for the growth and development of mouse lung cancer cells;

[0034] Figure 6 Result for the growth and development of mouse lung cancer cells;

[0035] Figure 7 Result for the growth and development of mouse colon cancer cells. Detailed implementation method

[0036] In the following description, specific details such as specific system structures and technologies are presented for the purpose of illustration rather than limitation, so as to thoroughly understand the embodiments of the present invention. However, those skilled in the art should clearly understand that the present invention can also be implemented in other embodiments without these specific details.

[0037] Example 1

[0038] In this example, the melittin-targeted preparation was prepared by the thin film dispersion method, and the specific steps are as follows:

[0039] S1. Prepare the melittin-purpurin complex:

[0040] (1) Weigh 2 mg of melittin and 2 mg of purpurin, add them to 50 mL of tetrahydrofuran to completely dissolve, and then remove the tetrahydrofuran by rotary evaporation under reduced pressure to obtain the crude melittin-purpurin complex;

[0041] (2) Dissolve the obtained crude melittin-purpurin complex in 20 mL of dichloromethane to redissolve it, filter it through a microporous filter membrane, and then remove the dichloromethane by rotary evaporation under reduced pressure to obtain the melittin-purpurin complex;

[0042] (3) Weigh 2 mg of the obtained melittin-purpurin complex and add it to 20 mL of dichloromethane to dissolve it to obtain the organic phase;

[0043] (4) Weigh 40 mg of albumin nanoparticles and dissolve them in 20 mL of PBS solution to obtain the PBS solution containing albumin nanoparticles, and the mass-volume concentration of albumin nanoparticles is 2 mg / mL;

[0044] (5) Slowly drop the organic phase into the PBS solution containing albumin nanoparticles, stir rapidly for 10 min, finally remove the dichloromethane in the system by evaporation under reduced pressure, and filter through a microporous filter membrane to obtain the melittin-purpurin-albumin nanoparticles, that is, the melittin-targeted preparation, denoted as melittin-purpurin-albumin nanoparticles.

[0045] Characterization and performance evaluation

[0046] 1. Particle size and potential detection

[0047] The particle size and potential of the melittin - perflavin - albumin nanoparticles prepared in Example 1 were detected using a nano - particle size analyzer, and the morphological structure of the melittin - perflavin - albumin nanoparticles was observed using a transmission electron microscope. The results are shown in Figure 1 and Figure 2 .

[0048] From Figure 1 the results, it can be seen that the particle size distribution of the melittin - perflavin - albumin nanoparticles prepared in Example 1 of the present invention is concentrated, the particle size range is 100 - 200 nm, and the Zeta potential is - 13.4 mV. This indicates that the melittin - perflavin complex has been loaded onto the albumin nanoparticles, and the melittin - perflavin - albumin nanoparticles have been successfully prepared.

[0049] From Figure 2 the results, it can be seen that the morphology of the melittin - perflavin - albumin nanoparticles prepared in Example 1 of the present invention is nanospherical, and the particle size range is 100 - 200 nm.

[0050] 2. Evaluation of the hemolytic behavior of melittin - perflavin - albumin nanoparticles

[0051] Specific steps: Take 2 mL of fresh mouse blood, centrifuge to separate red blood cells, and after washing thoroughly with PBS, prepare a series of melittin solutions with different concentrations and a series of melittin - perflavin - albumin nanoparticle solutions with the same corresponding concentrations as the melittin concentrations in centrifuge tubes as experimental groups, and use Triton as a positive control. The volume of the solution in each centrifuge tube is 500 μL. Subsequently, add 20 μL of red blood cells to each of the above - mentioned centrifuge tubes and mix well, incubate at room temperature for 4 h, and after the incubation, use an ultraviolet spectrophotometer to calculate the hemolysis rate. The calculation formula is as follows:

[0052] Hemolysis rate (%)=(absorbance of experimental group - absorbance of blank control group) / (absorbance value of positive control - absorbance of blank control group)*100%, and the results are as shown in Figure 3 .

[0053] From Figure 3 the results, it can be seen that the melittin - perflavin - albumin nanoparticles can significantly reduce the hemolytic property of melittin. This may be because in the melittin - perflavin - albumin nanoparticles, the phenolic acid groups in perflavin loaded on the albumin nanoparticles can enhance the binding between melittin and albumin nanoparticles, thereby significantly reducing the hemolytic property of melittin.

[0054] 3. Evaluation of the inhibitory ability of melittin - perflavin - albumin nanoparticles against mouse breast cancer using an in - vivo anti - breast cancer model

[0055] The specific steps are as follows: When the tumor of breast cancer 4T1 mice grows to about 100 mm 3 , the mice are randomly divided into a model group, a melittin (5 mg / kg) group, a melittin (5 mg / kg) + periflavin (5 mg / kg) group, and a melittin periflavin albumin nanoparticle drug group, and tail vein injection is performed according to the melittin dose of 5 mg / kg. After the first round of drug administration, at an interval of 48 h, all the mice are sacrificed after a total of 5 times of drug administration, and the tumors in each group are detected. The results are shown in Figure 4 and Figure 5 .

[0056] As can be seen from Figure 4 and Figure 5 , after 5 times of drug administration, it is found that the tumor growth in the melittin periflavin albumin nanoparticle drug group is the slowest. Compared with the melittin group and the melittin + periflavin group, the anti-tumor efficacy of melittin periflavin albumin nanoparticles against breast cancer is the most prominent. This is because albumin nanoparticles can co-deliver melittin and periflavin to the tumor site for targeted therapy. After reaching the tumor site, melittin and periflavin will respectively inhibit the proliferation (activity) of oxidative phosphorylation tumor cells and glycolytic cells, thereby achieving efficient treatment of breast tumors. In addition, the targeting effect of albumin nanoparticles can further improve its anti-tumor effect.

[0057] 4. Evaluate the inhibitory ability of melittin periflavin albumin nanoparticles on mouse lung cancer cells using an in vivo anti-lung cancer model

[0058] Specific steps: When the tumor of lung cancer-bearing Lewis mice grows to about 100 mm 3 , the mice are randomly divided into a model group, a melittin group, a melittin + periflavin group, and a melittin periflavin albumin nanoparticle drug group, and tail vein injection is performed according to the melittin dose of 5 mg / kg. After the first round of drug administration, at an interval of 48 h, all the mice are sacrificed after a total of 5 times of treatment, and the tumor growth is observed. The results are shown in Figure 6 .

[0059] As can be seen from Figure 6 the results, the tumor growth in the melittin periflavin albumin nanoparticle drug group is the slowest, and it has the most prominent anti-tumor efficacy. This is because albumin nanoparticles can co-deliver melittin and periflavin to the tumor site for targeted therapy. After reaching the tumor site, melittin and periflavin will respectively inhibit the proliferation (activity) of oxidative phosphorylation tumor cells and glycolytic cells, thereby achieving efficient treatment of lung tumors. In addition, the targeting effect of albumin nanoparticles can further improve its anti-tumor effect.

[0060] 5. Evaluate the inhibitory ability of melittin periflavin albumin nanoparticles on mouse colon cancer cells using an in vivo anti-colon cancer model

[0061] Specific steps: When the tumor of CT26 mice bearing colon cancer grows to about 100mm 3 The mice were randomly divided into a model group, a melittin group, a melittin + peflavin group, and a melittin peflavin albumin nanomedicine group. They were injected with 5 mg / kg melittin by tail vein. After the first round of administration, all mice were killed after 5 treatments at intervals of 48 hours to observe the tumor growth. The results are shown in Table 1. Figure 7 .

[0062] Depend on Figure 7 The results show that the tumor growth of the bee venom peptide and peflavin albumin nanoparticles group is the slowest and has the most outstanding anti-tumor effect. This is because albumin nanoparticles can co-deliver bee venom peptide and peflavin to the tumor site for targeted treatment. After reaching the tumor site, bee venom peptide and peflavin will inhibit the proliferation (activity) of oxidative phosphorylation tumor cells and glycolytic cells respectively, thereby achieving efficient treatment of breast tumors. In addition, the targeting effect of albumin nanoparticles can further improve its anti-tumor effect.

[0063] In summary, the results of the in vivo anti-tumor effect test show that the bee venom peptide-peflavin albumin nanoparticles provided by the present invention can effectively inhibit the growth of breast cancer, lung cancer and colon cancer through the combined action of bee venom peptide and pflavin, and therefore are expected to be used in the development of the preparation of therapeutic anti-tumor drugs.

[0064] The present invention is not limited to the above-mentioned specific implementation modes. Various changes made by ordinary technicians in this field based on the above-mentioned concepts without creative work are all within the protection scope of the present invention.

Claims

1. A method for preparing a bee venom peptide targeting preparation, characterized in that: The following steps are involved: S1. Preparation of Melittin-Peflavin Complex: S11, dissolving melittin and pemetrexed in a first organic solvent, and then removing the first organic solvent by rotary evaporation under reduced pressure to obtain a crude melittin-pemetrexed complex; S12, dissolving the obtained crude melittin-peitangin complex in a second organic solvent to redissolve it, filtering it through a microporous filter membrane, and then removing the second organic solvent through reduced pressure rotary evaporation to obtain a melittin-peitangin complex; S2, adding the melittin-penetranthus complex into a solvent to obtain an organic phase; S3. Add PBS solution containing albumin nanoparticles to the organic phase, dissolve by ultrasonication, take out the supernatant by centrifugation, and filter with a microporous filter membrane to obtain bee venom peptide and flavin albumin nanoparticles.

2. The method for preparing the melittin targeting preparation according to claim 1, characterized in that: The mass ratio of melittin to pemetrexedin is 1:5 to 5:

1.

3. The method for preparing the melittin targeting preparation according to claim 1, characterized in that: The mass volume concentration of the albumin nanoparticles in the PBS solution containing the albumin nanoparticles is 0.2-5 mg / mL.

4. The method for preparing the melittin targeting preparation according to claim 1, characterized in that: The concentrations of melittin and pemetrexedin are both 0.05-0.5 mg / mL.

5. The method for preparing the melittin targeting preparation according to claim 1, characterized in that: The particle size of the melittin-peutin albumin nanoparticles is 100-200 nm, and the Zeta potential is 0--20 mV.

6. A melittin targeting preparation prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the melittin targeting preparation according to claim 6 in the preparation of anti-tumor drugs.

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