Anti-tumor double-medicine nano preparation and preparation method and application thereof
阅读说明：本技术 一种抗肿瘤双药纳米制剂及其制备方法和应用 (Anti-tumor double-medicine nano preparation and preparation method and application thereof ) 是由 张强 柳懿桓 于 2020-06-12 设计创作，主要内容包括：本发明公开了一种抗肿瘤的双药纳米制剂,包括高分子材料、大蒜素、活性氧自由基产生剂等。该纳米药剂利用高分子材料包裹两种可诱导肿瘤细胞内活性氧自由基水平升高的药物,其中一种药物促进肿瘤细胞内生成更多的活性氧自由基,另一种药物抑制肿瘤细胞内的抗氧化系统功能,通过两种药物的协同效应,可显著提高肿瘤细胞内活性氧自由基水平,从而实现高效杀灭肿瘤细胞的效果。(The invention discloses an anti-tumor double-medicine nano preparation, which comprises a high polymer material, allicin, an active oxygen radical generator and the like. The nano medicament is prepared by wrapping two medicaments capable of inducing the increase of the level of active oxygen free radicals in tumor cells by using a high polymer material, wherein one medicament promotes the generation of more active oxygen free radicals in the tumor cells, the other medicament inhibits the function of an antioxidant system in the tumor cells, and the level of the active oxygen free radicals in the tumor cells can be remarkably increased through the synergistic effect of the two medicaments, so that the effect of efficiently killing the tumor cells is realized.)
1. A double-medicine nanometer preparation is characterized in that the nanometer preparation comprises a high polymer material, allicin and an active oxygen free radical generator.
2. The nano-preparation according to claim 1, wherein the polymer material comprises one or more of liposome, polyethylene glycol-poly (lactic-co-glycolic acid), polycaprolactone, and albumin.
3. The nano-formulation of claim 1, wherein the reactive oxygen species generator comprises one or more of cinnamaldehyde, fisetin, germacrone, lentinan, isoalantolactone, artesunate.
4. The nano-formulation according to claim 1, wherein the nano-agent comprises the following materials in parts by weight: 40-98 parts of high polymer material, 1-59 parts of active oxygen free radical generator and 1-59 parts of allicin.
5. The nanoformulation according to claim 1, further comprising a tumor targeting molecule comprising folic acid, RGD peptide, hyaluronic acid, bisphosphonate.
6. The nano-preparation according to claim 1, wherein the polymer material is used to encapsulate allicin and an active oxygen radical generator to prepare a double-drug nano-preparation.
7. A method for preparing double-medicine nanometer preparation is characterized in that allicin and active oxygen free radical generating agent are wrapped by polymer material to prepare the double-medicine nanometer preparation.
8. The method according to claim 7, wherein the polymer material comprises one or more of liposome, polyethylene glycol-poly lactic acid-glycolic acid, polycaprolactone, and albumin; and/or the active oxygen free radical generator comprises one or more of cinnamaldehyde, fisetin, germacrone, lentinan, isoalantolactone and artesunate.
9. The double-drug nano preparation prepared by the method according to claim 7 or 8.
10. A pharmaceutical composition comprising the dual drug nano formulation of any one of claims 1 to 6 and 9 and a carrier.
11. Use of a dual-drug nano-formulation according to any one of claims 1-6, 9, or a pharmaceutical composition according to claim 10, in the preparation of an anti-tumor medicament.
12. The use of claim 11, wherein the tumor comprises breast cancer, lung cancer, liver cancer, prostate cancer.
Technical Field
The invention belongs to the technical field of medicines, and relates to an anti-tumor double-medicine nano preparation (medicament) and a preparation method and application thereof.
Background
Cancer has now become one of the major diseases of human death. According to the reports of the American cancer society, the number of new cancer cases and cancer death cases in the world is up to 2760 ten thousands in 2018, and the number of new cancer cases and death cases in 2018 in 2 months, which is released by the national cancer center in China, shows that the number of new cancer cases and death cases in China accounts for more than 20% of the number of new cancer cases in the world, so that the current situation of cancer treatment is very severe. At present, the clinical therapies for cancer mainly include surgery, chemotherapy, radiotherapy, photothermal therapy, photodynamic therapy, immunotherapy, biomolecule targeted therapy, sonodynamic therapy and the like, and the therapies all have various defects, such as large toxic and side effects, poor curative effect on metastatic tumors, easy generation of drug resistance and the like. Therefore, development of novel antitumor agents is urgently required.
The biological function of reactive oxygen species and their potential role in cancer development have been reported in recent years. Reactive oxygen radicals are small molecules with high reactivity produced by all aerobic organisms, including hydrogen peroxide, superoxide, hydroxyl radicals and the like. Active oxygen radicals are also important intracellular signaling molecules involved in the regulation of many cellular biological processes, such as immune defense mechanisms, cell signaling, cell development, drug metabolism, viral infection mechanisms, and cancer development. Reactive oxygen radicals are continuously produced in and removed from cells in organisms through various complex synthetic and degradation pathways as by-products of oxygen metabolism. The dynamic balance between production and clearance of reactive oxygen species is critical, and when this balance is disrupted, the presence of reactive oxygen species at high concentrations can cause a range of oxidative stress in cells, with increased oxidative stress causing senescence, degeneration or fatal pathologies in cells by altering intracellular redox states, oxidatively modifying key amino acid residues to destroy cellular proteins, lipids and DNA, which are associated with many human diseases, including cancer, cardiovascular and neuro-degenerative diseases, and the like.
Selective treatment is essential in the treatment of cancer. Reactive oxygen radicals exist within cells as "threshold concepts". The threshold concept suggests that a modest increase in reactive oxygen radicals can promote cell proliferation and survival. However, when the increase of reactive oxygen radicals reaches a certain level (toxicity threshold), it may overwhelm the antioxidant capacity of the cells and trigger cell death. Under physiological conditions, normal cells maintain redox homeostasis with low levels of reactive oxygen radicals by controlling a balance between reactive oxygen radical production (pro-oxidant) and elimination (antioxidant capacity). Normal cells can tolerate a certain level of exogenous reactive oxygen species stress by mobilizing "reserve" antioxidant capacity to prevent reactive oxygen species levels from reaching cell death thresholds. In cancer cells, however, reactive oxygen species caused by metabolic abnormalities and oncogenic signaling are maintained at a high level, and by up-regulation of antioxidant capacity and shift of redox kinetics, the reactive oxygen species level is maintained below the toxicity threshold. Therefore, cancer cells will rely more on antioxidant systems and are more susceptible to oxidative stress induced by exogenous substances. The use of exogenous regulators to alter the stress of reactive oxygen radicals in cancer cells may raise the reactive oxygen radicals above a toxicity threshold level, resulting in cell death. This provides a biochemical basis for designing therapeutic strategies to selectively kill cancer cells using reactive oxygen radical mediated mechanisms.
There are generally two approaches to increasing the level of reactive oxygen radicals in cancer cells. One method is to introduce agents that promote the production of reactive oxygen radicals, and the other method is to disrupt the cell's antioxidant system. Although reactive oxygen species generating agents are effective when used alone, in combination with agents that disrupt the antioxidant system, it may be easier to raise the reactive oxygen species in cancer cells to cytotoxic levels, while normal cells remain in an adaptive proliferative oscillation state.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention expects to improve the level of active oxygen free radicals in the tumor cells by a dual mechanism of improving the active oxygen free radicals of the tumor cells and consuming reducing agents in the tumor cells, thereby efficiently killing the cancer cells. Related research reports indicate that cinnamaldehyde, fisetin, germacrone and the like can increase the level of active oxygen free radicals in cells, so that the apoptosis pathway of the cells is stimulated. Allicin can up-regulate reactive oxygen radicals by consuming intracellular reduced glutathione via a thiol-disulfide exchange reaction pathway. In the invention, the double-drug (composite) nano drug-carrying particles for synergistically improving the active oxygen free radicals are provided, and the high-efficiency and low-toxicity tumor treatment is realized by synergistically improving the level of the active oxygen free radicals in tumor cells.
The invention provides a double-medicine nano preparation (novel nano medicament for resisting tumor by using active oxygen free radicals), which comprises a high molecular material, allicin and an active oxygen free radical generator.
Wherein the high molecular material comprises one or more of liposome, polyethylene glycol-polylactic acid-glycolic acid, polycaprolactone, albumin and the like; preferably, it is polyethylene glycol-poly (lactic-co-glycolic acid).
Wherein the active oxygen free radical generator can promote the generation of more active oxygen free radicals in tumor cells, and comprises one or more of cinnamaldehyde, fisetin, germacrone, lentinan, isoalantolactone, artesunate, etc.; preferably, cinnamaldehyde.
Wherein the allicin inhibits the function of an antioxidant system in the tumor cells.
Allicin is an organic sulfur compound extracted from the bulb of garlic, and is diallyl trisulfide (diallyl trisulfide).
Wherein the allicin and the active oxygen free radical generating agent cooperate to induce the increase of the level of active oxygen free radical in the tumor cell.
Wherein the nano medicament comprises the following substances in parts by weight: 40-98 parts of high polymer material, 1-59 parts of active oxygen free radical generator and 1-59 parts of allicin.
Preferably, the nano-medicament comprises the following substances in parts by weight: 40-60 parts of high polymer material, 20-40 parts of active oxygen free radical generator and 2-20 parts of allicin.
Further preferably, the nano-medicament comprises the following substances in parts by weight: 59 parts of high polymer material, 39 parts of active oxygen free radical generator and 2 parts of allicin.
Wherein, the nano preparation can also comprise tumor targeting molecules, and the tumor targeting molecules comprise folic acid, RGD peptide, hyaluronic acid and diphosphate, but are not limited thereto.
The weight portion of the tumor targeting molecules in the nano medicament is 0-2 parts calculated by the total weight of all the components of the nano medicament being 100 parts; preferably, it is 0 to 1 part.
The invention also provides a preparation method of the double-medicine nano preparation, which is prepared by wrapping allicin and an active oxygen free radical generator with a high polymer material.
Wherein the high molecular material comprises one or more of liposome, polyethylene glycol-polylactic acid-glycolic acid, polycaprolactone, albumin and the like; preferably, it is polyethylene glycol-poly (lactic-co-glycolic acid).
Wherein the allicin and the active oxygen free radical generating agent cooperate to induce the increase of the level of active oxygen free radical in the tumor cell.
Wherein the active oxygen free radical generator can promote the generation of more active oxygen free radicals in tumor cells, and comprises one or more of cinnamaldehyde, fisetin, germacrone, lentinan, isoalantolactone, artesunate, etc.; preferably, cinnamaldehyde.
Wherein the allicin inhibits the function of an antioxidant system in the tumor cells.
Furthermore, the tumor targeting molecule can be modified on the surface of the high molecular material through chemical reaction.
Wherein the tumor targeting molecule includes folic acid, RGD peptide, hyaluronic acid, and diphosphate, but is not limited thereto.
Wherein the method for preparing the nano preparation comprises one or more of a nano coprecipitation method, a single emulsion method, a double emulsion method, a chemical linking method and the like; preferably, a nano-coprecipitation process.
The preparation method of the nano preparation takes a nano coprecipitation method as an example, and specifically comprises the following steps:
(1) according to the weight ratio of the raw material components, respectively weighing 20-40 parts of active oxygen free radical generator, 2-20 parts of allicin and 40-60 parts of high polymer material (drug-loaded material).
(2) And (2) dissolving the medicines (active oxygen free radical generator and allicin) and the medicine-carrying material measured in the step (1) in 1 ml of organic solvent to form a solution.
(3) Slowly dripping the solution obtained in the step (2) into a certain volume of phosphate buffer solution under the condition of stirring, wherein the volume ratio of the phosphate buffer solution to the organic solvent is 3:1, and forming a liquid.
(4) Removing the organic solvent in the liquid in the step (3) by means of high-speed centrifugation, and washing 5 times by using a phosphate buffer solution.
The innovation point of the invention is that two natural medicines which can induce the level of active oxygen free radicals in tumor cells to increase are prepared into a double-medicine (composite) nano medicament by utilizing a nano technology, wherein one kind of medicinal active oxygen free radical generator promotes the generation of more active oxygen free radicals in the tumor cells, and the other kind of medicament allicin inhibits the function of an antioxidant system in the tumor cells; the nano medicament can improve the level of active oxygen free radicals in tumor cells by improving the dual mechanisms of the active oxygen free radicals in the tumor cells and consuming reducing agents in the tumor cells, thereby efficiently killing cancer cells.
The invention also provides the double-medicine nano medicament prepared by the method.
The invention also provides a pharmaceutical composition, which comprises the double-drug nano medicament and a carrier.
The carrier is a nanoparticle constructed by one or more of liposome, polyethylene glycol-polylactic acid-glycolic acid, polycaprolactone, albumin and the like.
The invention also provides application of the double-drug nano-medicament or the pharmaceutical composition in preparing anti-tumor medicaments.
The double-drug nano medicament or the medicinal composition is used for inducing the rising of the active oxygen free radical level in the tumor cells.
The tumors include but are not limited to breast cancer, lung cancer, liver cancer, prostate cancer and the like.
The invention has the beneficial effects that: the double-drug nano drug-loaded particles with the synergistic effect of improving the active oxygen free radicals prepared by the invention can improve the level of the active oxygen free radicals in tumor cells by synergy, thereby realizing high-efficiency and low-toxicity tumor treatment. In the double-drug nano-drug particles, the cinnamaldehyde and the allicin both keep a slow release rate, and the double-drug nano-drug has good stability at room temperature. The treatment effect of the nano medicament can be further improved by modifying the tumor targeting molecules on the surface of the treatment carrier.
Drawings
Fig. 1 is a schematic diagram of preparation of a double-drug nano drug-loaded particle for resisting breast cancer.
Fig. 2 is a transmission electron microscope image photograph of the prepared nano-particles of cinnamaldehyde and allicin.
Fig. 3 and 4 are in vitro release curves of cinnamaldehyde and allicin in the double-drug nanoparticle of cinnamaldehyde and allicin of the present invention under room temperature conditions.
Fig. 5 is an in vitro stability test of the dual drug nanoparticles of cinnamaldehyde and allicin under room temperature conditions.
FIG. 6 is an evaluation of the in vitro synergistic anti-tumor effect of the combination of cinnamaldehyde and allicin of the present invention on breast cancer cells.
Fig. 7 is an evaluation of the in vitro synergistic anti-tumor effect of the dual drug nanoparticles of cinnamaldehyde and allicin of the present invention on breast cancer cells.
Detailed Description
The present invention will be further described with reference to the following examples. The following detailed description is not to be construed as limiting the invention.
Example 1:
the invention further discloses a preferable scheme, which comprises the following components in parts by weight: is prepared by mixing 39 parts of cinnamyl aldehyde, 2 parts of allicin and 59 parts of medicine-carrying materials. Firstly, respectively weighing 39 parts of cinnamyl aldehyde, 2 parts of garlicin and 59 parts of a medicine-carrying material according to the weight ratio of the raw material components, wherein the medicine-carrying material is polyethylene glycol-polylactic acid glycolic acid. And step two, dissolving the cinnamaldehyde, the garlicin and the medicine carrying material measured in the step one in 1 ml of organic solvent. And step three, slowly dripping the organic solvent dissolved with the cinnamyl aldehyde, the allicin and the medicine carrying materials obtained in the step two into phosphate buffer solution with a certain volume under the condition of stirring, wherein the volume ratio of the phosphate buffer solution to the organic solvent is 3: 1. And step four, removing the organic solvent from the liquid obtained in the step three in a high-speed centrifugation mode, and washing the liquid for 5 times by using a phosphate buffer solution.
The preparation examples of the cinnamaldehyde and allicin nanoparticle of the present invention are further described with reference to the accompanying drawings. With reference to the attached drawing 1, all the raw materials such as cinnamaldehyde, garlicin, a drug-carrying material and the like adopted for preparing the finished product of the anti-tumor double-drug nano-medicament particle provided by the invention are commercially available medical raw materials, and the specific specifications are as follows:
1. cinnamaldehyde: molecular formula C9H8O, molecular weight 132.16, purity 98%.
2. Allicin; molecular formula C6H10S3Molecular weight is 178.34, purity is 98%.
3. Medicine carrying material: the polyethylene glycol-polylactic acid-glycolic acid is subjected to nuclear magnetic verification.
4. Solvent:
(1) acetonitrile, HPLC grade.
(2) Distilled water and chromatographic purity.
The invention obtains satisfactory effect through repeated verification. Taking a double-medicine nano drug-carrying particle prepared by mixing cinnamyl aldehyde, garlicin and polyethylene glycol-polylactic acid-glycolic acid as an example:
and (5) displaying the transmission electron microscope image photo. The double-drug nano-medicament particles containing the cinnamaldehyde and the garlicin are relatively regular round particles with smooth surfaces and uniform particle diameters of about 180 nanometers (figure 2). In the double-drug nano-drug particles, both cinnamaldehyde and allicin maintained a slower release rate (fig. 3 and 4). In addition, the double-drug nano-drug particles containing cinnamaldehyde and garlicin keep good stability in a slow-release solution and 10% fetal calf serum, and the particle size of the double-drug nano-drug particles is not obviously changed in a long period of time (figure 5).
In an in vitro experiment, the killing effect of the medicament on breast cancer cells is determined by adopting an MTT method, the experiment is divided into two groups, namely a cinnamaldehyde single-component group, a cinnamaldehyde and allicin combined group, wherein the medicament concentration of the allicin is 75 micromole, and the concentration of the cinnamaldehyde is 0-200 micromole; fig. 6 shows that the combined group of cinnamaldehyde and allicin has a significantly better therapeutic effect than the group of cinnamaldehyde unit drugs, and the optimal drug concentration of cinnamaldehyde was determined to be 50 μm by this experiment.
Further, a single medicine or a combined medicine is coated by polyethylene glycol-polylactic acid-glycolic acid to prepare a nano medicament, the breast cancer cell killing effect of the nano medicament is tested, and the experiment is divided into 4 groups: control group (PBS buffer), cinnamaldehyde-only group (50 micromolar), allicin-only group (75 micromolar), cinnamaldehyde-50 micromolar, and allicin-75 micromolar combinations. The experimental result shows that the cancer cell killing effect of the two-medicine combination treatment group of the cinnamaldehyde (50 micromole) and the allicin (75 micromole) is better than the treatment effect of the single medicine group.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.