A nano-drug with a photosensitive effect, its preparation method and application

Abstract

The present invention relates to a nano-drug with a photosensitive effect, a preparation method thereof and an application. The preparation method includes the steps of: preparing a mixture of trametinib, polyethylene glycol 400, purified water and poloxamer; performing a normal temperature - low temperature freeze-thaw cycle; centrifuging, washing, and freeze-drying. By designing the raw material composition and using the normal temperature - low temperature freeze-thaw cycle method, each component can be firmly combined together through hydrophilic-hydrophobic layer-by-layer self-assembly without the need to additionally introduce a cross-linking agent. The obtained nano-drug has strong pH stability; has the effect of photothermal controlled release of chemical drugs, can control the release of chemical drugs at the tumor site for treating tumors; and has strong aqueous phase stability, overcoming the disadvantages that hydrophobic bismuthene and trametinib are insoluble in water and difficult to disperse in water. The nano-drug of the present invention can be used for the preparation of anti-tumor preparations.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and particularly to a nano-drug with a photosensitizing effect, its preparation method and application. Background Art

[0002] Nano-drugs refer to drugs prepared by using nano-preparation technologies to make active pharmaceutical ingredients, etc. into particles with nano-scale sizes, or by combining appropriate carrier materials with active pharmaceutical ingredients to form particles with nano-scale sizes and the finally prepared pharmaceutical preparations. The size of their active ingredients or carrier particles is the primary feature of nano-drugs and an important basis for the nano-effects presented by the drugs. Compared with traditional drugs, nano-drugs have obvious differences in terms of their physicochemical properties, pharmacodynamics (PD), and pharmacokinetics (PK). Therefore, it has been a research hotspot in recent years.

[0003] In the case of nano-antitumor drugs, compared with traditional antitumor drugs, nano-antitumor drugs have better targeting, sustained release, stability and functionality.

[0004] However, the currently common preparation methods for nano-drugs mainly include chemical cross-linking methods and mixing and stirring methods. However, the reaction conditions of chemical cross-linking methods are generally relatively harsh, usually requiring the introduction of cross-linking agents, and the introduction of cross-linking agents will bring certain toxicity to nano-drugs. The mixing and stirring method mainly prepares nano-drugs through physical adsorption or electrostatic interaction, but the nano-drugs prepared by this method usually have poor stability and are prone to aggregation, precipitation and degradation.

[0005] As a new type of tumor treatment method, photothermal therapy loads photosensitizers on carriers and then uses laser irradiation to convert light energy into heat energy to kill tumor cells or tissues. The excitation light used for irradiation generally uses near-infrared light, which is a non-invasive cancer treatment method that can effectively penetrate normal human tissues to reach the cancer site and greatly reduce the damage to normal tissues. Common photosensitizers include two-dimensional materials such as bismuthene, phosphorene, and antimonene.

[0006] Trametinib is a MEK1 / 2 inhibitor that mainly affects the MAPK pathway and inhibits cell proliferation by acting on the MEK protein. MEK is a downstream signal transduction protein of RAS and RAF. Therefore, trametinib may also be effective against cancer types with RAS or RAF mutations, such as non-small cell lung cancer, melanoma, thyroid cancer, etc. It has potential in the treatment of various tumor diseases. However, trametinib is an antitumor drug mainly based on hydrophobic interaction. During the preparation of nano-drugs, generally in order to better disperse in the carrier, cross-linking agents, etc. are usually introduced. Summary of the Invention

[0007] In order to overcome at least one problem existing in the above-mentioned prior art, the present invention provides a nano-drug with photosensitive effect, its preparation method and application through a one-step method of normal temperature-low temperature cycle.

[0008] One aspect of the present invention provides a preparation method of a nano-drug with photosensitive effect, including the steps:

[0009] S1 Prepare a mixed solution: Put trametinib into polyethylene glycol 400 and stir to dissolve, then add purified water and poloxamer and stir to dissolve together, and finally add bismuthene nanosheets and mix evenly to obtain the mixed solution;

[0010] S2 Normal temperature-low temperature freeze-thaw cycle: Stir and disperse the mixed solution in the dark at room temperature, then perform freezing treatment, and then transfer the frozen mixed solution to room temperature and let it thaw naturally in the dark. Repeat the above normal temperature-low temperature freeze-thaw cycle process several times;

[0011] S3 Centrifuge, wash, and freeze-dry to obtain the nano-drug with photosensitive effect.

[0012] In the preparation method of the present invention, trametinib is first coated with polyethylene glycol 400, which can improve its dispersion performance in water, and then mixed with water, poloxamer, and bismuthene nanosheets. The obtained mixed solution undergoes several normal temperature-low temperature freeze-thaw cycles. Due to the differences in the solubility of each substance in water, hydrophobic substances and hydrophilic substances will each show a phenomenon of layer-by-layer self-assembly as the temperature decreases. During the repeated normal temperature-low temperature freeze-thaw cycle process, the hydrophobic segments of bismuthene, trametinib, and poloxamer will self-assemble together through hydrophobic interactions, while polyethylene glycol 400 will assemble with the hydrophilic segments of poloxamer. Finally, the assembled hydrophilic structure and hydrophobic structure will be tightly and disorderly combined together through the polymer segments of poloxamer to obtain the nano-drug of the present invention - bismuthene@trametinib.

[0013] In the obtained nano-drug, due to the strong binding effect of the intertwined polymer segments of poloxamer, the nano-drug has good pH stability. At the same time, due to the embedding of many hydrophilic cores composed of (polyethylene glycol 400-poloxamer hydrophilic segments) inside the nano-drug, the nano-drug has strong water dispersibility. When the nano-drug is exposed to 808nm laser irradiation, the temperature of the nano-drug increases due to the photothermal effect of bismuthene. At high temperatures, the hydrophobic core and hydrophilic core formed by self-assembly disassemble due to the increased Brownian motion, and then the structure of the entire nano-drug collapses, thereby releasing trametinib.

[0014] Further, in the mixed solution, the concentration of trametinib is: 6mg / mL to 12mg / mL. In a preferred embodiment, the concentration of trametinib is 9mg / mL.

[0015] Further, in the mixed solution, the concentration of bismuthene nanosheets is 1 mg / mL to 3 mg / mL. In a preferred embodiment, the concentration of bismuthene nanosheets is 2 mg / mL.

[0016] Further, in the mixed solution, the concentration of polyethylene glycol 400 is 10 mg / mL to 30 mg / mL. In a preferred embodiment, it is preferably 20 mg / mL.

[0017] Further, in the mixed solution, the concentration of poloxamer is 60 mg / mL to 100 mg / mL. In a preferred embodiment, the concentration of poloxamer is 80 mg / mL.

[0018] Further, in step S2, the room temperature is 10°C to 30°C, the stirring speed at room temperature is 1000 r / min to 3000 r / min, and the stirring time is 20 min to 40 min. In a preferred embodiment, the room temperature is 20°C, the stirring speed at room temperature is 1500 r / min, and the stirring time is 30 min.

[0019] Further, in step S2, the temperature for cryogenic treatment is -80°C to -20°C, and the freezing time is 1 h to 3 h. In a preferred embodiment, the temperature for cryogenic treatment is -50°C, and the freezing time is 2 h.

[0020] Further, in step S2, the number of repetitions of the normal temperature - low temperature freeze-thaw cycle process is 2 to 5 times.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1) Compared with the prior art, the method for preparing the nano-drug of the present invention, by designing the raw material composition and using the normal temperature - low temperature freeze-thaw cycle method, each component can be firmly combined together through hydrophilic-hydrophobic layer-by-layer self-assembly without the need to additionally introduce a cross-linking agent, effectively ensuring the safety of the nano-drug.

[0023] 2) The bismuthene@trametinib nano-drug prepared by the preparation method of the present invention has strong pH stability.

[0024] 3) The bismuthene@trametinib nano-drug prepared by the preparation method of the present invention has the effect of controlling the release of chemical drugs by photothermal means, and can control the release of chemical drugs at the tumor site for tumor treatment.

[0025] 4) The bismuthene@trametinib nano-drug prepared by the preparation method of the present invention has strong aqueous phase stability, overcoming the disadvantages that hydrophobic bismuthene and trametinib are insoluble in water and difficult to disperse in water. Description of the Drawings

[0026] Figure 1 The photothermal curve of the nano-drug bismuthene@trametinib according to the embodiment of the present invention;

[0027] Figure 2 The drug release curve of the nano-drug bismuthene@trametinib according to the embodiment of the present invention. Specific embodiments

[0028] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings and specific embodiments. The preferred embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0029] In the description of the present invention, unless otherwise clearly defined, words such as heating, cleaning, weighing, freezing, etc. should be understood in a broad sense, and those skilled in the art can reasonably determine the specific meanings of the above words in the present invention in combination with the specific content of the technical solution.

[0030] In the description of the present invention, the descriptions referring to terms such as "some embodiments", "examples", etc. mean that the specific methods and materials described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific methods and materials described can be combined in a suitable manner in any one or more embodiments or examples.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art belonging to the technical field of the present invention. The terms used in the description of the present invention in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0032] In the following embodiments and comparative examples, the experimental methods used are all conventional methods unless otherwise specified, and the materials, reagents, etc. used can all be obtained from commercial sources unless otherwise specified.

[0033] Example 1

[0034] This example provides a nano-drug with photosensitive effect - bismuthene@trametinib. Specifically, its preparation method includes the steps:

[0035] S1 Prepare the mixed solution: Add trametinib into polyethylene glycol 400 and stir to dissolve, then add purified water and poloxamer and stir to dissolve together. Finally, add bismuthine nanosheets and mix evenly to obtain the mixed solution. In the mixed solution, the concentration of trametinib is: 9 mg / mL; the concentration of bismuthine nanosheets is: 2 mg / mL; the concentration of polyethylene glycol 400 is: 20 mg / mL; the concentration of poloxamer is: 80 mg / mL;

[0036] S2 Room temperature - low temperature freeze-thaw cycle: Disperse the mixed solution under light protection at room temperature of 20 °C with a stirring speed of: 1500 r / min and a stirring time of 30 min; then freeze at -50 °C for 2 h, and then transfer the frozen mixed solution to room temperature of 20 °C under light protection to thaw naturally. After thawing, repeat the above room temperature - low temperature freeze-thaw cycle process 3 times;

[0037] S3 Centrifuge, wash, and freeze-dry to obtain the nanodrug with photosensitizing effect. Specifically, centrifuge the mixed solution at a speed of 5000 r / min for 15 min, take the precipitate, resuspend and wash it with purified water; repeat the centrifuge and wash process 3 times. After the last centrifugation, take the precipitate, disperse it with purified water, and freeze-dry to obtain the bismuthine@trametinib nanodrug.

[0038] Example 2

[0039] This example provides a nanodrug with photosensitizing effect - bismuthine@trametinib. Specifically, its preparation method includes the steps:

[0040] S1 Prepare the mixed solution: Add trametinib into polyethylene glycol 400 and stir to dissolve, then add purified water and poloxamer and stir to dissolve together. Finally, add bismuthine nanosheets and mix evenly to obtain the mixed solution. In the mixed solution, the concentration of trametinib is: 6 mg / mL; the concentration of bismuthine nanosheets is: 1 mg / mL; the concentration of polyethylene glycol 400 is: 10 mg / mL; the concentration of poloxamer is: 60 mg / mL;

[0041] S2 Room temperature - low temperature freeze-thaw cycle: Disperse the mixed solution under light protection at room temperature of 10 °C with a stirring speed of: 1000 r / min and a stirring time of 20 min; then freeze at -80 °C for 1 h, and then transfer the frozen mixed solution to room temperature of 10 °C under light protection to thaw naturally. After thawing, repeat the above room temperature - low temperature freeze-thaw cycle process 3 times;

[0042] S3 Centrifuge, wash, and freeze-dry to obtain the nanodrug with photosensitizing effect. Specifically, centrifuge the mixed solution at a speed of 5000 r / min for 15 min, take the precipitate, resuspend and wash it with purified water; repeat the centrifuge and wash process 3 times. After the last centrifugation, take the precipitate, disperse it with purified water, and freeze-dry to obtain the bismuthine@trametinib nanodrug.

[0043] Example 3

[0044] This example provides a nano-drug with photosensitive effect - bismuthene@trametinib. Specifically, its preparation method includes the steps:

[0045] S1 Prepare the mixed solution: Put trametinib into polyethylene glycol 400 and stir to dissolve it. Then add purified water and poloxamer and stir to dissolve them together. Finally, add bismuthene nanosheets and mix evenly to obtain the mixed solution. In the mixed solution, the concentration of trametinib is: 12 mg / mL; the concentration of bismuthene nanosheets is: 3 mg / mL; the concentration of polyethylene glycol 400 is: 30 mg / mL; the concentration of poloxamer is: 100 mg / mL;

[0046] S2 Room temperature - low temperature freeze-thaw cycle: Disperse the mixed solution at 30°C in the dark with a stirring speed of: 3000 r / min and a stirring time of 40 min. Then freeze it at -20°C for 3 h. After that, transfer the frozen mixed solution to room temperature of 30°C and let it thaw naturally in the dark. Repeat the above room temperature - low temperature freeze-thaw cycle process 3 times;

[0047] S3 Centrifuge, wash, and freeze-dry to obtain the nano-drug with photosensitive effect. Specifically, centrifuge the mixed solution at a speed of 5000 r / min for 15 min, take the precipitate, resuspend and wash it with purified water; repeat the centrifugation and washing process 3 times. After the last centrifugation, take the precipitate, disperse it with purified water, and freeze-dry to obtain the bismuthene@trametinib nano-drug.

[0048] Example 4

[0049] This example provides a nano-drug with photosensitive effect - bismuthene@trametinib. Specifically, its preparation method includes the steps:

[0050] S1 Prepare the mixed solution: Put trametinib into polyethylene glycol 400 and stir to dissolve it. Then add purified water and poloxamer and stir to dissolve them together. Finally, add bismuthene nanosheets and mix evenly to obtain the mixed solution. In the mixed solution, the concentration of trametinib is: 9 mg / mL; the concentration of bismuthene nanosheets is: 3 mg / mL; the concentration of polyethylene glycol 400 is: 10 mg / mL; the concentration of poloxamer is: 80 mg / mL;

[0051] S2 Room temperature - low temperature freeze-thaw cycle: Disperse the mixed solution at 20°C in the dark with a stirring speed of: 1000 r / min and a stirring time of 20 min. Then freeze it at -20°C for 1 h. After that, transfer the frozen mixed solution to room temperature of 20°C and let it thaw naturally in the dark. Repeat the above room temperature - low temperature freeze-thaw cycle process 3 times;

[0052] Centrifuge, wash, and freeze-dry the mixture to obtain the nanomedicine with photosensitizing effect. Specifically, centrifuge the mixture at a speed of 5000 r / min for 15 min, take the precipitate, resuspend and wash it with purified water; repeat the centrifugation and washing process three times. After the last centrifugation, take the precipitate, disperse it with purified water, and freeze-dry it to obtain the bismuthene@trametinib nanomedicine.

[0053] Comparative Example 1

[0054] It is the same as Example 1, except that in step S1, the concentration of trametinib is 5 mg / mL, and the concentration of bismuthene nanosheets is 0.5 mg / mL, and the rest is the same as Example 1.

[0055] Comparative Example 2

[0056] It is the same as Example 1, except that in step S1, the concentration of polyethylene glycol 400 is 5 mg / mL, and the concentration of poloxamer is 50 mg / mL, and the rest is the same as Example 1.

[0057] Comparative Example 3

[0058] It is the same as Example 1, except that in step S1, the concentration of trametinib is 15 mg / mL, and the concentration of bismuthene nanosheets is 4 mg / mL, and the rest is the same as Example 1.

[0059] Comparative Example 4

[0060] It is the same as Example 1, except that in step S1, the concentration of polyethylene glycol 400 is 40 mg / mL, and the concentration of poloxamer is 150 mg / mL, and the rest is the same as Example 1.

[0061] Comparative Example 5

[0062] It is the same as Example 1, except that in step S2, the room temperature is 5°C, the stirring speed is 800 r / min, and the room temperature stirring time is 50 min, and the rest is the same as Example 1.

[0063] Comparative Example 6

[0064] It is the same as Example 1, except that in step S2, the freezing temperature is -15°C, and the freezing time is 30 min, and the rest is the same as Example 1.

[0065] Comparative Example 7

[0066] It is the same as Example 1, except that in step S2, the room temperature is 35°C, the stirring speed is 3500 r / min, and the room temperature stirring time is 50 min, and the rest is the same as Example 1.

[0067] Comparative Example 8

[0068] It is the same as Example 1, except that in step S2, the freezing environment temperature is: -90 °C, and the freezing time is: 4 h, and the rest is the same as Example 1.

[0069] Performance Test

[0070] Test Example 1: Select the nano-drug prepared in Example 1 for the following tests:

[0071] (1) Photothermal performance test

[0072] The bismuthene@trametinib nano-drug prepared in Example 1 was formulated with purified water into the following concentrations: 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL. Then it was irradiated with an 808 nm laser for 5 min, and the heating curves of the nano-drug at each concentration were observed. The test results are shown in Figure 1 .

[0073] (2) Drug release ability of bismuthene@trametinib

[0074] The sample of Example 1 was formulated into bismuthene@trametinib with a concentration of 500 μg / mL as the research object. The control group was dispersed in purified water without 808 nm laser irradiation, and the drug release curve for 24 h was detected and analyzed by an ultraviolet spectrophotometer. The experimental group was irradiated with an 808 nm laser for 5 min at 6 h and 18 h respectively, and the drug release curve for 24 h was observed. The experimental results are shown in Figure 2 .

[0075] It can be seen from Figure 1 that the bismuthene@trametinib nano-drug has the property of heat generation in response to 808 nm laser, and with the increase of concentration, the photothermal effect becomes more obvious. It can be seen from Figure 2 that the prepared bismuthene@trametinib nano-drug has the ability to release drugs in response to photothermal, and trametinib drug can be released by more than 80% after the first photothermal treatment for 5 min.

[0076] Test Example 2: Select the samples of Examples 1-4 and Comparative Examples 1-8 to evaluate the size, photothermal effect, drug release ability, water stability, pH stability, and killing ability against B16 (melanoma cells) of the nano-drugs.

[0077] The specific evaluation methods and criteria are as follows:

[0078] Size of nano-drug: The size of the nano-drug was detected by a Malvern laser scattering particle size analyzer (DLS), and the normal distribution mean of the nano-drug size should be between 50 nm and 200 nm;

[0079] Photothermal effect: The nano-drug was formulated at a concentration of 200 μg / mL and exposed to 808 nm laser irradiation for 5 min. The temperature change ΔT was detected, and ΔT should be between 20 °C and 40 °C.

[0080] Drug release ability: The 200 μg / mL nano-drug solution was exposed to 808 nm laser irradiation for 5 min. The release ratio of trametinib was detected and analyzed by ultraviolet spectrophotometer, and it should be greater than 70%.

[0081] Water stability: The nano-drug was dispersed in PBS with pH = 7.4 and formulated at 200 μg / mL. It was placed at 20 °C - 30 °C for 24 h. The nano-drug should be evenly dispersed without aggregation and precipitation.

[0082] pH stability: The nano-drug was respectively dispersed in PBS with pH = 4.0 and pH = 10.0 and formulated at 200 μg / mL. It was placed at 20 °C - 30 °C for 24 h. The nano-drug should not aggregate and precipitate.

[0083] Killing ability against B16 (melanoma cells): The nano-drug was formulated at 200 μg / mL. According to the MTT cytotoxicity test operation method in the standard "GB / T 16886.5 - 2017", the nano-drug was co-cultured with B16 melanoma cells. At the 12th h, it was exposed to 808 nm laser irradiation for 5 min. The killing ability of the nano-drug against cells at the 24th h was evaluated, and the killing ability should be greater than 90%.

[0084] The evaluation results are shown in Table 1.

[0085] Table 1: Evaluation results of drug performance of examples and comparative examples

[0086]

[0087] It can be seen from the above results that the preparation method of the present invention can obtain nano-drugs with stable dispersion in water, good pH stability, strong drug release ability, and good therapeutic effect.

[0088] In the preparation method of the present invention, the concentrations of each component affect each other and act synergistically. The parameter conditions of the reaction, especially the temperature conditions of normal temperature - low temperature freeze-thaw cycle, affect the stability and drug release ability of the final product.

[0089] The concentrations of trametinib and bismuthene nanosheets will affect the photothermal effect and drug release ability, and thus affect the anti-tumor ability; too low concentrations of the two lead to too poor photothermal effect, and too high concentrations of the two lead to too strong photothermal effect, which is likely to damage normal tissues.

[0090] The concentrations of polyethylene glycol 400 and poloxamer can affect the nanoparticle size, photothermal effect, drug release ability, water stability, and pH stability. Too low concentrations of both lead to poor stability of the nano-drug, while too high concentrations are likely to block the laser and cause a decrease in the photothermal effect.

[0091] During freeze-thaw cycles, if the room temperature is too low, the stirring speed is too slow, and the time is too long, it is easy to cause too large a size, which is not conducive to drug release. If the room temperature is too high and the stirring speed is too fast, it is not conducive to self-assembly and causes a decrease in the stability of the nano-drug. If the freezing temperature is too low and the freezing time is too long, it may damage the stability of the nanoparticles. If the freezing temperature is insufficient and the freezing time is too short, it may result in insufficient self-assembly and the stability of the nano-drug is easily damaged.

[0092] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0093] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A preparation method of a nano-drug with photosensitive effect, characterized in that, Including the steps: S1 Prepare the mixed solution: Put trametinib into polyethylene glycol 400 and stir to dissolve, then add purified water and poloxamer and stir to dissolve together. Finally, add bismuthene nanosheets and mix evenly to obtain the mixed solution; In the mixed solution, the concentration of trametinib is: 6 mg / mL - 12 mg / mL; the concentration of bismuthene nanosheets is: 1 mg / mL - 3 mg / mL; the concentration of polyethylene glycol 400 is: 10 mg / mL - 30 mg / mL; the concentration of poloxamer is: 60 mg / mL - 100 mg / mL; S2 Room temperature - low temperature freeze - thaw cycle: Stir and disperse the mixed solution in the dark at room temperature, then perform freezing treatment. After that, transfer the frozen mixed solution to room temperature and let it thaw naturally in the dark. Repeat the above room temperature - low temperature freeze - thaw cycle process several times; The room temperature environment temperature is: 10°C - 30°C, the room temperature stirring speed is: 1000 r / min - 3000 r / min, and the stirring time is: 20 min - 40 min; the temperature for freezing treatment is -80°C - -20°C, and the freezing time is: 1 h - 3 h; the number of repetitions of the room temperature - low temperature cycle process is 2 - 5 times; S3 Centrifuge, wash, and freeze - dry to obtain the nano - drug with photosensitizing effect.

2. The nano - drug with photosensitizing effect prepared by the preparation method according to claim 1.

3. Use of the nano - drug with photosensitizing effect according to claim 2 in the preparation of anti - tumor agents.

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