Nano-drug with photosensitive effect as well as preparation method and application of nano-drug

Abstract

The invention relates to a nano-drug with a photosensitive effect and a preparation method and application thereof, and the preparation method comprises the following steps: preparing a mixed solution of trametinib, polyethylene glycol 400, purified water and poloxamer; performing normal temperature-low temperature freezing and thawing circulation; and centrifuging, washing and freeze-drying. According to the preparation method disclosed by the invention, by designing the raw material composition and utilizing a normal-temperature-low-temperature freeze-thaw cycle method, all the components can be stably combined together through hydrophilic-hydrophobic layer-by-layer self-assembly, a cross-linking agent does not need to be additionally introduced, and the obtained nano-drug has very strong pH stability; the effect of photothermal controlled release of chemical drugs is achieved, and the chemical drugs can be controlled to be released at tumor positions to treat tumors; the bismuth alkene / trametinib compound has high water-phase stability, and overcomes the defects that hydrophobic bismuth alkene and trametinib are insoluble in water and are difficult to disperse in water. The nano-drug provided by the invention can be used for preparing an anti-tumor preparation.

Description

Technical Field

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

[0002] Nanomedicine refers to the use of nano-preparation technology to make APIs into nano-scale particles, or to combine APIs with appropriate carrier materials to form nano-scale particles and the final drug preparations. The size of its active ingredients or carrier particles is the primary feature of nanomedicine and an important basis for the nano-effects exhibited by the drug. Compared with traditional drugs, nanomedicines have obvious differences in physicochemical properties, pharmacodynamics (PD), and pharmacokinetics (PK). Therefore, it has been a hot topic of research in recent years.

[0003] As for nano-anti-tumor drugs, compared with traditional anti-tumor drugs, nano-anti-tumor drugs have better targeting, sustained release, stability and functionality.

[0004] However, the most common methods for preparing nanomedicines are chemical crosslinking and mixing. However, the chemical crosslinking reaction conditions are generally harsh and usually require the introduction of a crosslinking agent, which can cause certain toxicity to the nanomedicine. The mixing method mainly prepares nanomedicines through physical adsorption or electrostatic action, but the nanomedicines prepared by this method are usually less stable and are prone to aggregation, precipitation, and degradation.

[0005] Photothermal therapy is a new type of tumor treatment. It uses a carrier to load photosensitizers, 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. It is a non-invasive cancer treatment method that can effectively penetrate normal human tissue to reach the cancer site and greatly reduce damage to normal tissue. 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 MEK protein. MEK is a downstream signal transduction protein of RAS and RAF, so trametinib may also be effective against cancers 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, but trametinib is an anti-tumor drug mainly based on hydrophobic effects. In the process of preparing nanomedicines, cross-linking agents are generally introduced in order to better disperse in the carrier. 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 photosensitizing effect and a preparation method and application thereof through a normal temperature-low temperature cycle one-step method.

[0008] One aspect of the present invention provides a method for preparing a nano drug having a photosensitizing effect, comprising the steps of:

[0009] S1: preparing a mixed solution: adding trametinib to polyethylene glycol 400 and stirring to dissolve, then adding purified water and poloxamer and stirring to dissolve, and finally adding bismuthene nanosheets and mixing evenly to obtain the mixed solution;

[0010] S2 Normal temperature-low temperature freeze-thaw cycle: The mixed solution is stirred and dispersed at room temperature and protected from light, and then frozen. The frozen mixed solution is then transferred to room temperature and protected from light for natural thawing. After thawing, the normal temperature-low temperature freeze-thaw cycle is repeated several times;

[0011] S3 centrifuges, washes, and freeze-dries to obtain the nano drug with photosensitizing effect.

[0012] The preparation method of the present invention first coats trametinib with polyethylene glycol 400 to improve its dispersibility in water, and then mixes it with water, poloxamer, and bismuthene nanosheets. The resulting mixed solution undergoes several normal temperature-low temperature freeze-thaw cycles. Since the solubility of each substance in water is different, the hydrophobic substance and the hydrophilic substance will each self-assemble layer by layer as the temperature decreases. During the repeated normal temperature-low temperature freeze-thaw cycles, the hydrophobic segments of bismuthene, trametinib and poloxamer will self-assemble together through hydrophobic forces, while polyethylene glycol 400 will assemble together with the hydrophilic segments of poloxamer. Finally, the assembled hydrophilic structure and the 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, the polymer segments of poloxamer are entangled with each other to play a strong binding role, so that the nano drug has good pH stability. At the same time, since a lot of hydrophilic cores composed of (polyethylene glycol 400-poloxamer hydrophilic segments) are embedded 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 rises due to the photothermal effect of bismuthene. At high temperature, the hydrophobic core and the hydrophilic core formed by self-assembly are disassembled due to the intensified Brownian motion, which leads to the collapse of the structure of the entire nano drug, thereby releasing trametinib.

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

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

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

[0017] Furthermore, 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] Furthermore, in step S2, the room temperature is 10°C to 30°C, the room temperature stirring speed is 1000r / min to 3000r / min, and the stirring time is 20min to 40min; in a preferred embodiment, the room temperature is 20°C, the room temperature stirring speed is 1500r / min, and the stirring time is 30min.

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

[0020] Furthermore, in step S2, the normal temperature-low temperature freeze-thaw cycle process is repeated 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 nanomedicine preparation method of the present invention, by designing the raw material composition and utilizing the normal temperature-low temperature freeze-thaw cycle method, each component can be firmly combined together through hydrophilic-hydrophobic layer self-assembly, without the need for additional introduction of cross-linking agents, thereby effectively ensuring the safety of the nanomedicine.

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

[0024] 3) The bismuthene@trametinib nanodrug prepared by the preparation method of the present invention has the effect of photothermal controlled release of chemical drugs, and can control the release of chemical drugs at the tumor site to treat the tumor.

[0025] 4) The bismuthene@trametinib nanodrug prepared by the preparation method of the present invention has strong water phase stability, overcoming the shortcomings of hydrophobic bismuthene and trametinib being insoluble in water and difficult to disperse in water. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a photothermal curve diagram of the nanomedicine bismuthene@trametinib according to an embodiment of the present invention;

[0027] Figure 2 This is a drug release curve of the nanomedicine bismuthene@trametinib in an embodiment of the present invention. DETAILED DESCRIPTION

[0028] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings and specific embodiments. Preferred embodiments of the present invention are given 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, the purpose of providing these embodiments is 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, terms such as heating, cleaning, weighing, freezing, etc. should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in the present invention in combination with the specific content of the technical solution.

[0030] In the description of the present invention, the description with reference to the terms "some embodiments", "examples", etc. means that the specific methods and materials described in conjunction with the embodiments or examples 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 any one or more embodiments or examples in a suitable manner.

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

[0032] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0033] Example 1

[0034] This embodiment provides a nano drug with photosensitizing effect - bismuthene@trametinib. Specifically, the preparation method thereof comprises the steps of:

[0035] S1: prepare a mixed solution: add trametinib to polyethylene glycol 400 and stir to dissolve, then add purified water and poloxamer and stir to dissolve, finally add bismuthene nanosheets and mix evenly to obtain a mixed solution; in the mixed solution, the concentration of trametinib is 9 mg / mL; the concentration of bismuthene nanosheets is 2 mg / mL; the concentration of polyethylene glycol 400 is 20 mg / mL; and the concentration of poloxamer is 80 mg / mL;

[0036] S2 Normal temperature-low temperature freeze-thaw cycle: The mixed solution was stirred and dispersed at room temperature 20°C in the dark, with a stirring speed of 1500r / min and a stirring time of 30min; then frozen at -50°C for 2h, and then the frozen mixed solution was transferred to room temperature 20°C in the dark for natural thawing. After thawing, the normal temperature-low temperature freeze-thaw cycle was repeated 3 times;

[0037] S3 centrifuges, washes, and freeze-dries to obtain the nano drug with photosensitizing effect. Specifically, the mixed solution is centrifuged at a speed of 5000 r / min for 15 minutes, and the precipitate is resuspended and washed with purified water; the centrifugation and washing process is repeated 3 times. After the last centrifugation, the precipitate is dispersed with purified water and freeze-dried to obtain the bismuthene@trametinib nano drug.

[0038] Example 2

[0039] This embodiment provides a nano drug with photosensitizing effect - bismuthene@trametinib. Specifically, the preparation method thereof comprises the steps of:

[0040] S1 prepares a mixed solution: add trametinib to polyethylene glycol 400 and stir to dissolve, then add purified water and poloxamer and stir to dissolve, finally add bismuthene nanosheets and mix evenly to obtain a mixed solution; in the mixed solution, the concentration of trametinib is 6 mg / mL; the concentration of bismuthene nanosheets is 1 mg / mL; the concentration of polyethylene glycol 400 is 10 mg / mL; and the concentration of poloxamer is 60 mg / mL;

[0041] S2 Normal temperature-low temperature freeze-thaw cycle: The mixed solution was stirred and dispersed at room temperature 10°C in the dark, with a stirring speed of 1000r / min and a stirring time of 20min; then frozen at -80°C for 1h, and then the frozen mixed solution was transferred to room temperature 10°C in the dark for natural thawing. After thawing, the normal temperature-low temperature freeze-thaw cycle was repeated 3 times;

[0042] S3 centrifuges, washes, and freeze-dries to obtain the nano drug with photosensitizing effect. Specifically, the mixed solution is centrifuged at a speed of 5000 r / min for 15 minutes, and the precipitate is resuspended and washed with purified water; the centrifugation and washing process is repeated 3 times. After the last centrifugation, the precipitate is dispersed with purified water and freeze-dried to obtain the bismuthene@trametinib nano drug.

[0043] Example 3

[0044] This embodiment provides a nano drug with photosensitizing effect - bismuthene@trametinib. Specifically, the preparation method thereof comprises the steps of:

[0045] S1: prepare a mixed solution: add trametinib to polyethylene glycol 400 and stir to dissolve, then add purified water and poloxamer and stir to dissolve, finally add bismuthene nanosheets and mix evenly to obtain a 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; and the concentration of poloxamer is 100 mg / mL;

[0046] S2 Normal temperature-low temperature freeze-thaw cycle: The mixed solution was stirred and dispersed at room temperature 30°C in the dark, with a stirring speed of 3000r / min and a stirring time of 40min; then frozen at -20°C for 3h, and then the frozen mixed solution was transferred to room temperature 30°C in the dark for natural thawing. After thawing, the normal temperature-low temperature freeze-thaw cycle was repeated 3 times;

[0047] S3 centrifuges, washes, and freeze-dries to obtain the nano drug with photosensitizing effect. Specifically, the mixed solution is centrifuged at a speed of 5000 r / min for 15 minutes, and the precipitate is resuspended and washed with purified water; the centrifugation and washing process is repeated 3 times. After the last centrifugation, the precipitate is dispersed with purified water and freeze-dried to obtain the bismuthene@trametinib nano drug.

[0048] Example 4

[0049] This embodiment provides a nano drug with photosensitizing effect - bismuthene@trametinib. Specifically, the preparation method thereof comprises the steps of:

[0050] S1 prepares a mixed solution: add trametinib to polyethylene glycol 400 and stir to dissolve, then add purified water and poloxamer and stir to dissolve, finally add bismuthene nanosheets and mix evenly to obtain a 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; and the concentration of poloxamer is 80 mg / mL;

[0051] S2 Normal temperature-low temperature freeze-thaw cycle: The mixed solution was stirred and dispersed at room temperature 20°C in the dark, with a stirring speed of 1000r / min and a stirring time of 20min; then frozen at -20°C for 1h, and then the frozen mixed solution was transferred to room temperature 20°C in the dark for natural thawing. After thawing, the normal temperature-low temperature freeze-thaw cycle was repeated 3 times;

[0052] S3 centrifuges, washes, and freeze-dries to obtain the nano drug with photosensitizing effect. Specifically, the mixed solution is centrifuged at a speed of 5000 r / min for 15 minutes, and the precipitate is resuspended and washed with purified water; the centrifugation and washing process is repeated 3 times. After the last centrifugation, the precipitate is dispersed with purified water and freeze-dried to obtain the bismuthene@trametinib nano drug.

[0053] Comparative Example 1

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

[0055] Comparative Example 2

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

[0057] Comparative Example 3

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

[0059] Comparative Example 4

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

[0061] Comparative Example 5

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

[0063] Comparative Example 6

[0064] The same as Example 1, the only difference is that in step S2, the freezing environment temperature is: -15°C, the freezing time is: 30 minutes, and the rest is the same as Example 1.

[0065] Comparative Example 7

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

[0067] Comparative Example 8

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

[0069] Performance Testing

[0070] Test Example 1: The nanomedicine prepared in Example 1 was selected to perform the following tests:

[0071] (1) Photothermal performance test

[0072] The bismuthene@trametinib nanodrug prepared in Example 1 was prepared with purified water to the following concentrations: 10 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL. Then it was irradiated with 808 nm laser for 5 min, and the temperature rise curve of the nanodrug at each concentration was observed. The test results are detailed in Figure 1 .

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

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

[0075] Depend on Figure 1 It can be seen that the bismuthene@trametinib nanodrug has the property of responding to heat with 808nm laser, and the photothermal effect becomes more obvious with the increase of concentration. Figure 2 It can be seen that the prepared bismuthene@trametinib nanodrug has the ability to release drugs in response to photothermal reaction, and the release rate of trametinib drug can reach more than 80% in the first photothermal reaction of 5 minutes.

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

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

[0078] Nanodrug size: The size of nanodrugs is detected by Malvern laser scattering particle size analyzer (DLS). The mean of the normal distribution of nanodrug size should be between 50nm and 200nm.

[0079] Photothermal effect: The nanodrug was prepared into a concentration of 200 μg / mL, exposed to 808 nm laser irradiation for 5 min, and the temperature change △T was detected. △T should be between 20℃ and 40℃.

[0080] Drug release capacity: 200 μg / mL nanodrug solution was exposed to 808 nm laser irradiation for 5 min, and the release ratio of trametinib was detected and analyzed by UV spectrophotometer, which should be greater than 70%.

[0081] Water stability: Disperse the nanodrug in PBS with pH = 7.4 and prepare it to 200 μg / mL. Place it in an environment of 20℃~30℃ for 24 hours. The nanodrug should be evenly dispersed without aggregation and precipitation.

[0082] pH stability: Disperse the nanodrug in PBS at pH = 4.0 and pH = 10.0, respectively, prepare it into 200 μg / mL, and place it in an environment of 20℃~30℃ for 24 hours. The nanodrug should not aggregate or precipitate.

[0083] Killing ability against B16 (melanoma cells): The nanodrug was prepared into 200 μg / mL, and the nanodrug was co-cultured with B16 melanoma cells according to the MTT cytotoxicity test operation method in the standard "GB / T16886.5-2017". At the 12th hour, 808 nm laser irradiation was given for 5 minutes, and the killing ability of the nanodrug on the cells was evaluated at the 24th hour. The killing ability should be greater than 90%.

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

[0085] Table 1: Results of drug performance evaluation 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 that are stably dispersed in water, have good pH stability, strong drug release ability, and good therapeutic effect.

[0088] In the preparation method of the present invention, the concentrations of the components affect each other and work synergistically, and the reaction parameter conditions, especially the temperature conditions of the room temperature-low temperature freeze-thaw cycle, affect the stability and drug release capacity of the final product.

[0089] The concentrations of trametinib and bismuthene nanosheets will affect the photothermal effect and drug release capacity, thereby affecting the anti-tumor ability; if the concentrations of both are too low, the photothermal effect will be too poor, and if the concentrations of both are too high, the photothermal effect will be too strong, which will easily damage normal tissues.

[0090] The concentrations of polyethylene glycol 400 and poloxamer will affect the size of nanoparticles, photothermal effect, drug release capacity, water stability and pH stability; too low concentrations of both will lead to poor stability of nanodrugs, while too high concentrations will easily 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, or the time is too long, the size of the nanoparticles may be too large, which is not conducive to drug release; if the room temperature is too high, the stirring speed is too fast, which is not conducive to self-assembly and causes the stability of the nanomedicine to decrease. If the freezing temperature is too low or the freezing time is too long, the stability of the nanoparticles may be destroyed; if the freezing temperature is not enough or the freezing time is too short, the self-assembly degree may be insufficient, and the stability of the nanomedicine may be easily destroyed.

[0092] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, 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, they should be considered to be within the scope of this specification.

[0093] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a nano drug having a photosensitizing effect, characterized in that: Includes steps: S1: preparing a mixed solution: adding trametinib to polyethylene glycol 400 and stirring to dissolve, then adding purified water and poloxamer and stirring to dissolve, and finally adding bismuthene nanosheets and mixing evenly to obtain the mixed solution; S2 Normal temperature-low temperature freeze-thaw cycle: The mixed solution is stirred and dispersed at room temperature and protected from light, and then frozen. The frozen mixed solution is then transferred to room temperature and protected from light for natural thawing. After thawing, the normal temperature-low temperature freeze-thaw cycle is repeated several times; S3 centrifuges, washes, and freeze-dries to obtain the nano drug with photosensitizing effect.

2. The preparation method according to claim 1, characterized in that: In the mixed solution, the concentration of trametinib is 6 mg / mL to 12 mg / mL.

3. The preparation method according to claim 1, characterized in that: In the mixed solution, the concentration of bismuthene nanosheets is 1 mg / mL to 3 mg / mL.

4. The preparation method according to claim 1, characterized in that: In the mixed solution, the concentration of polyethylene glycol 400 is 10 mg / mL to 30 mg / mL.

5. The preparation method according to claim 1, characterized in that: In the mixed solution, the concentration of poloxamer is 60 mg / mL to 100 mg / mL.

6. The preparation method according to claim 1, characterized in that: In the step S2, the room temperature is 10°C to 30°C, the stirring speed is 1000 r / min to 3000 r / min, and the stirring time is 20 min to 40 min.

7. The preparation method according to claim 1, characterized in that: In step S2, the freezing treatment temperature is -80°C to -20°C, and the freezing time is 1h to 3h.

8. The preparation method according to claim 1, characterized in that: In step S2, the normal temperature-low temperature cycle process is repeated 2 to 5 times.

9. The nanomedicine with photosensitizing effect prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the nano drug with photosensitizing effect as claimed in claim 9 in the preparation of anti-tumor drugs.

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