WO2021196086A1 - 一种细胞内聚合抑制细胞活性的方法及实现该方法的前体药物 - Google Patents

一种细胞内聚合抑制细胞活性的方法及实现该方法的前体药物 Download PDF

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WO2021196086A1
WO2021196086A1 PCT/CN2020/082806 CN2020082806W WO2021196086A1 WO 2021196086 A1 WO2021196086 A1 WO 2021196086A1 CN 2020082806 W CN2020082806 W CN 2020082806W WO 2021196086 A1 WO2021196086 A1 WO 2021196086A1
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cells
cell
dimethylacrylamide
prodrug
propionic acid
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PCT/CN2020/082806
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French (fr)
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耿晋
张一川
高权
连前进
何荣坤
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深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the present invention relates to the technical field of medicines, in particular to a method for inhibiting cell activity and a prodrug for realizing the method. Specifically, it relates to a method for in situ synthesis of a linear high molecular polymer in a cell to cause toxicity to cells. So as to inhibit cell activity and realize the prodrug of this method.
  • Prodrugs are low-active substances that can be activated in situ (to generate highly active drug molecules) in organisms through enzymatic or chemical stimulation. Through targeted modification of drug molecules, the water solubility of many traditional drugs can be significantly improved. Chemical/ There are many clinical problems such as biological stability, oral absorption efficiency, internal circulation time, and systemic toxicity. At present, 5-7% of all drugs on the market can be identified as prodrugs, and this proportion still maintains an increasing trend, which shows that it has an important position in the development and application of medicine.
  • prodrug molecules have been developed and used in antibacterial, antiviral, and antitumor treatments. Most of the prodrug molecules are achieved by specifically modifying the active sites of known active drug molecules. There are few examples of other mechanisms for in-situ generation of macromolecular drugs. There are no relevant reports about prodrugs that use in-situ generation of macromolecular polymers in cells to inhibit cell activity.
  • cancerous tissues that cannot be removed by surgery (such as the brain, heart, trachea, etc.), currently only radiotherapy and chemotherapy can be used to control the rate of cancer deterioration, but it is difficult to completely cure.
  • radiotherapy and chemotherapy can be used to control the rate of cancer deterioration, but it is difficult to completely cure.
  • cancer cells are easy to metastasize and once they spread. It will be difficult to be cured, and the above reasons all contribute to the extremely high mortality rate of cancer.
  • high molecular weight polymers produced in cells through free radical polymerization can significantly change the growth of cytoskeleton, viscosity and cell structure, etc., and can affect physiological properties such as cell cycle. It is generally believed that high-molecular-weight polymers have higher biocompatibility than small molecules. This is because high-molecular-weight polymer molecules are difficult to be directly taken up by cells, and the larger the molecular weight, the lower the uptake efficiency, so it must be achieved through polymers. The impact on cells requires the synthesis of polymer molecules in situ in the cell, but so far there is no relevant report on whether intracellular polymerization can cause toxicity to tumor cells.
  • the polymer produced in the cell Due to the high activity of the free radical reaction itself, the polymer produced in the cell has a small molecular weight and a wide molecular weight distribution, which has a small impact on the cell structure and function, so the cytotoxicity is also low, and the new type Light-controlled reversible addition fragmentation chain transfer (RAFT) polymerization has stronger time/space controllability and more stable active intermediates due to its unique polymerization reaction mechanism, so it can be used in relatively complex reaction systems. Controlled realization of the synthesis of higher molecular weight polymers.
  • RAFT Light-controlled reversible addition fragmentation chain transfer
  • the initiation efficiency of the RAFT polymerization used is significantly higher than that of traditional free radical polymerization, it can greatly reduce the amount of monomers and initiators while ensuring the generation of polymer molecules, and reduce the toxicity of other cells due to molecular diffusion. .
  • stimulus response capabilities are usually introduced into the cancer treatment system, so that cytotoxicity occurs only at a specified time and location.
  • the system that uses light as the stimulus signal has higher temporal/spatial controllability, and can actively control the activation of drugs, thereby realizing non-invasive treatment of the organism.
  • the RAFT polymerization reaction initiated by light energy has the characteristics of mild reaction conditions, fewer side reactions, and high polymerization efficiency. It can accurately achieve high-efficiency polymerization in a designated area under the conditions of water, aerobic, and various biomolecules.
  • the polymerization reaction itself has little effect on cells, and it is not easy to cause systemic toxicity due to the diffusion of reactants. Therefore, it is suitable for in-situ synthesis of high molecular polymers in cells and selective treatment of tumor tissues.
  • the present invention provides a prodrug based on in-situ light-controlled RAFT polymerization and a preparation method thereof.
  • the prodrug can synthesize a high-toxicity polymer under light conditions, thereby destroying tumor cells and inhibiting tumor growth .
  • the present invention discloses a method for inhibiting cell activity and a prodrug for realizing the method.
  • the prodrug in the present invention includes: N,N-dimethylacrylamide (DMA), 2-(butylthiocarbonyl) Thiothiothio) propionic acid (RAFTCTA) and Eosin Y.
  • DMA N,N-dimethylacrylamide
  • RAFTCTA 2-(butylthiocarbonyl) Thiothiothio) propionic acid
  • Eosin Y Eosin Y.
  • the above-mentioned prodrug is incubated with the cell together, and the polymerization reaction in the cell is triggered by light to realize the in-situ synthesis of the high molecular polymer in the cell.
  • High molecular polymers synthesized in situ in cells can effectively affect cell activity, induce cell apoptosis and autophagy, thereby achieving the purpose of inhibiting tumor growth.
  • a prodrug for inhibiting cell activity by intracellular polymerization including N,N-dimethylacrylamide, 2-(butylthiocarbonylthiothiothio)propionic acid, and Eosin Y.
  • N,N-dimethylacrylamide is used as a monomer
  • 2-(butylthiocarbonylthiothiothio)propionic acid is a chain transfer agent
  • Eosin Y is a photoinitiator.
  • the amount of monomer N,N-dimethylacrylamide is controlled at the maximum allowable dose of 5mM for toxicity;
  • the molecular weight of the polymer is achieved by adjusting the amount of 2-(butylthiocarbonylthiothiothio)propionic acid and Eosin Y.
  • the molar ratio of 2-(butylthiocarbonylthiothio) propionic acid and eosin Y is 10:1; N,N-dimethylacrylamide and 2-(butylthiocarbonylthiothio)
  • the molar ratio of (sulfanyl) propionic acid is (50-500):1.
  • a high-molecular polymer that inhibits cell activity prepared by the following method:
  • the prodrug includes N,N-dimethylacrylamide and 2-(butylthiocarbonylthiothio) Propionic acid, Eosin Y;
  • the intracellular polymerization reaction is realized through illumination.
  • Using light as a stimulus signal has higher temporal/spatial controllability, and can actively control the activation of drugs, thereby realizing non-invasive treatment of organisms.
  • step (1) living cells are seeded in a cell culture plate for incubation, N,N-dimethylacrylamide, 2-(butylthiocarbonylthiothio)propionic acid, and Red Y is dissolved in the cell culture medium in proportion, and the washed live cells are added for incubation; in the step (2), the cells obtained in the step (1) are added to the cell culture medium, and the polymerization reaction is realized by light. .
  • the amount of monomer N,N-dimethylacrylamide (DMA) is controlled at the maximum allowable dose of toxicity, 5mM;
  • the molecular weight of the polymer is achieved by adjusting the amount of 2-(butylthiocarbonylthiothiothio)propionic acid (RAFTCTA) and Eosin Y.
  • the molar ratio of 2-(butylthiocarbonylthiothio) propionic acid and eosin Y is 10:1; N,N-dimethylacrylamide and 2-(butylthiocarbonylthiothio)
  • the molar ratio of (sulfanyl) propionic acid is (50-500):1.
  • the maximum excitation wavelength of the light source is 470 nm, and blue light irradiation causes less damage to the cells.
  • the said high molecular polymer which inhibits cell activity has the following structure:
  • the compound is a high-molecular polymer with high toxicity.
  • step (1) living cells were seeded in a cell culture plate at a density of 2 ⁇ 10 5 , and incubated at 37° C. and 4% carbon dioxide for 18 hours; N,N-dimethylacrylamide, 2- (Butylthiocarbonylthiothiothio)propionic acid and Eosin Y are dissolved in the cell culture medium in proportion, and 2mL per well is added to the cells washed with PBS, and incubated at 37°C for 4h;
  • step (2) the cells obtained in the step (1) were washed with PBS, and 1 mL of fresh cell culture medium was added to each well, and the polymerization reaction was achieved by vertical irradiation with a light source from the bottom of the culture plate for 10 minutes.
  • a method for preparing a high molecular weight polymer that inhibits cell activity includes the following steps:
  • the prodrug includes N,N-dimethylacrylamide and 2-(butylthiocarbonylthiothio) Propionic acid, Eosin Y;
  • the intracellular polymerization reaction is realized through illumination.
  • Using light as a stimulus signal has higher temporal/spatial controllability, and can actively control the activation of drugs, thereby realizing non-invasive treatment of organisms.
  • step (1) living cells are seeded in a cell culture plate for incubation, N,N-dimethylacrylamide, 2-(butylthiocarbonylthiothio)propionic acid, and Red Y is dissolved in the cell culture medium in proportion, and the washed live cells are added for incubation; in the step (2), the cells obtained in the step (1) are added to the cell culture medium, and the polymerization reaction is realized by light. .
  • the amount of monomer N,N-dimethylacrylamide (DMA) is controlled at the maximum allowable dose of toxicity, 5mM;
  • the molecular weight of the polymer is achieved by adjusting the amount of 2-(butylthiocarbonylthiothiothio)propionic acid (RAFTCTA) and Eosin Y.
  • the molar ratio of 2-(butylthiocarbonylthiothio) propionic acid and eosin Y is 10:1; N,N-dimethylacrylamide and 2-(butylthiocarbonylthiothio)
  • the molar ratio of (sulfanyl) propionic acid is (50-500):1.
  • the maximum excitation wavelength of the light source is 470 nm, and blue light irradiation causes less damage to the cells.
  • step (1) living cells were seeded in a cell culture plate at a density of 2 ⁇ 10 5 , and incubated at 37° C. and 4% carbon dioxide for 18 hours; N,N-dimethylacrylamide, 2- (Butylthiocarbonylthiothiothio)propionic acid and Eosin Y are dissolved in the cell culture medium in proportion, and 2mL per well is added to the cells washed with PBS, and incubated at 37°C for 4h;
  • step (2) the cells obtained in the step (1) were washed with PBS, and 1 mL of fresh cell culture medium was added to each well, and the polymerization reaction was achieved by vertical irradiation with a light source from the bottom of the culture plate for 10 minutes.
  • a method for intracellular polymerization to inhibit cell activity including the following steps:
  • the prodrug includes N,N-dimethylacrylamide and 2-(butylthiocarbonylthiothio) Propionic acid, Eosin Y;
  • the intracellular polymerization reaction is realized through illumination.
  • Using light as a stimulus signal has higher temporal/spatial controllability, and can actively control the activation of drugs, thereby realizing non-invasive treatment of organisms.
  • step (1) living cells are seeded in a cell culture plate for incubation, N,N-dimethylacrylamide, 2-(butylthiocarbonylthiothio)propionic acid, and Red Y is dissolved in the cell culture medium in proportion, and the washed live cells are added for incubation; in the step (2), the cells obtained in the step (1) are added to the cell culture medium, and the polymerization reaction is realized by light. .
  • the amount of monomer N,N-dimethylacrylamide (DMA) is controlled at the maximum allowable dose of toxicity, 5mM;
  • the molecular weight of the polymer is achieved by adjusting the amount of 2-(butylthiocarbonylthiothiothio)propionic acid (RAFTCTA) and Eosin Y.
  • the molar ratio of 2-(butylthiocarbonylthiothio) propionic acid and eosin Y is 10:1; N,N-dimethylacrylamide and 2-(butylthiocarbonylthiothio)
  • the molar ratio of (sulfanyl) propionic acid is (50-500):1.
  • the maximum excitation wavelength of the light source is 470 nm, and blue light irradiation causes less damage to the cells.
  • step (1) living cells were seeded in a cell culture plate at a density of 2 ⁇ 10 5 , and incubated at 37° C. and 4% carbon dioxide for 18 hours; N,N-dimethylacrylamide, 2- (Butylthiocarbonylthiothiothio)propionic acid and Eosin Y are dissolved in the cell culture medium in proportion, and 2mL per well is added to the cells washed with PBS, and incubated at 37°C for 4h;
  • step (2) the cells obtained in the step (1) were washed with PBS, and 1 mL of fresh cell culture medium was added to each well, and the polymerization reaction was achieved by vertical irradiation with a light source from the bottom of the culture plate for 10 minutes.
  • the application of the prodrug in inhibiting cell activity includes HeLa, 1205Lu, and A375 cell lines.
  • the present invention realizes the construction of a controllable RAFT polymer in a cell for the first time.
  • the prodrug of the present invention synthesizes the RAFT polymer in situ in the cell, and the polymer is directly toxic to the cell and can reduce the cell activity.
  • the present invention realizes the inhibition of cell activity through the mechanism of new prodrugs synthesize high molecular polymers in cells, and provides a new kind of cell drug resistance. Ideas.
  • the monomers, chain transfer agents and photosensitizers used in the present invention are all relatively low in toxicity, so when the prodrug is used, systemic toxicity will not be caused by the diffusion of the prodrug.
  • the present invention initiates the polymerization reaction by light stimulation, and realizes the high controllability and selectivity of the synthesis of toxic high molecular polymers for prodrugs at the time/space level.
  • Figure 1 is a schematic diagram of the intracellular polymerization of prodrugs.
  • Figure 3 shows 5mM N,N-dimethylacrylamide (DMA) and different concentrations of 2-(butylthiocarbonylthiothiothio)propionic acid (RAFTCTA) and Eosin Y solution (RAFTCTA and Eosin Y).
  • DMA N,N-dimethylacrylamide
  • RAFTCTA 2-(butylthiocarbonylthiothiothiothio)propionic acid
  • RAFTCTA and Eosin Y solution Eosin Y
  • the molar ratio is 10:1, and the concentration of RAFTCTA is 0.1, 0.05 and 0.01 mM).
  • Figure 4 is a mouse tumor growth curve, where the 470nm light time is 10 minutes, the prodrug contains 5mM DMA, 0.05mM RAFTCTA and 0.005mM Eosin Y, and the dosage is 5000 ⁇ L/kg.
  • Figure 5 is a picture of the tumors taken out of the killed mice 14 days after the administration (corresponding to the 14th day in the tumor growth curve in Figure 4), and each tumor is taken from a different mouse.
  • Fig. 8 is a 1 HNMR spectrum measured after the poly(DMA) polymer obtained by the intracellular polymerization of the simulated prodrug is dissolved in deuterated chloroform.
  • Figure 9 is a GPC profile measured after the poly(DMA) polymer obtained by the intracellular polymerization of the simulated prodrug is dissolved in DMF.
  • the method for inhibiting cell activity of the present invention can effectively generate acrylamide polymer inside the cell. Due to the special linear chain structure of such polymer, it can interact with a variety of biological molecules in the cell non-specifically. Physical adsorption, non-covalent bond forms and other interactions, so it can induce autophagy and inhibit cell proliferation by inhibiting PI3K/Akt/mTOR and AMPK signaling pathways, and by activating endoplasmic reticulum stress response and mitochondrial outer membrane permeation It induces apoptosis and programmed necrosis and other types of cell death methods to achieve the effect of inhibiting cell viability, and this effect has a general effect on cells, not only for a certain type of cell.
  • a method for inhibiting cell activity by intracellular polymerization which mainly includes the following steps:
  • the prodrug includes N,N-dimethylacrylamide (DMA), 2-(butylthiocarbonylthiothio) Thio) propionic acid (RAFTCTA), photoinitiator eosin Y;
  • DMA N,N-dimethylacrylamide
  • RAFTCTA 2-(butylthiocarbonylthiothio) Thio) propionic acid
  • eosin Y photoinitiator eosin Y
  • Viable cells at a density of 1x10 4 per well were seeded in 96 cell culture plate wells, and incubation temperature 18h at 37 °C, 4% carbon dioxide conditions to ensure cell attachment; DMA, RAFTCTA photoinitiator Eosin Y scale dissolution In high-sugar DMEM cell culture medium (containing 10% calf serum, 100uni/mL penicillin and streptomycin), and add 0.1mL per well to cells washed with PBS (3 times), constant temperature at 37°C Incubate for 4h to achieve the uptake of the prodrug by the cells.
  • DMA RAFTCTA photoinitiator Eosin Y scale dissolution
  • high-sugar DMEM cell culture medium containing 10% calf serum, 100uni/mL penicillin and streptomycin
  • the amount of monomer N,N-dimethylacrylamide (DMA) is controlled at the maximum allowable dose of toxicity, 5mM;
  • the molecular weight of the polymer is achieved by adjusting the dosage of 2-(butylthiocarbonylthiothiothio)propionic acid (RAFTCTA) and Eosin Y.
  • the molar ratio of 2-(butylthiocarbonylthiothio) propionic acid and eosin Y is 10:1; N,N-dimethylacrylamide and 2-(butylthiocarbonylthiothio)
  • the molar ratio of (sulfanyl) propionic acid is (50-500):1.
  • the intracellular polymerization reaction is realized through illumination.
  • Using light as a stimulus signal has higher temporal/spatial controllability, and can actively control the activation of drugs, thereby realizing non-invasive treatment of organisms.
  • the tumor area can be irradiated so that polymerization occurs only in the tumor, so as to achieve the purpose of selectively inhibiting the activity of tumor cells.
  • the cells ingested the prodrug were washed 3 times with PBS to remove the surface-adhesive reactants, and 0.1 mL of fresh DMEM medium was added to each well, using a 470nm LED blue light source (power: 260mW/cm 2 ) from the bottom of the culture plate vertically Irradiate for 10 minutes to achieve polymerization.
  • the light source of the wavelength band causes less damage to the cells, and experiments prove that neither the 405nm nor the 530nm light source can initiate the intracellular polymerization reaction.
  • THF solution containing 16.0g of n-butanol was added dropwise to 150mL THF suspension containing 9.0g of KOH, stirred at room temperature for 30min, and then 50mL of THF solution containing 17.0g of CS 2 was added dropwise, and stirred at room temperature After 24h, it was concentrated to 50mL under reduced pressure. 50mL of THF solution containing 22.4g of n-propylammonium bromide was added dropwise to the concentrated solution under stirring. After stirring for 24h at room temperature, the solvent was removed under reduced pressure to obtain the target crude product, which was further passed through a silica gel column. Chromatographic purification yields bright yellow crystals of the target product.
  • CCK-8 test HeLa cells were first seeded at a density of 1x10 4 cells in 96-well culture plates and incubated for 18h at thermostat 37 °C, 4% carbon dioxide conditions to ensure adherent cells; the cells were then administered different concentrations Or light (5-20min, 260mW/cm 2 ) and other treatments, and continue to incubate at a constant temperature for 24h; after 24h, aspirate the original medium and wash the cells with PBS 3 times, add diluted CCK-8 solution to each well (the ratio of the medium is 1:10) 100 ⁇ L, incubated at a constant temperature for 4h, detect the ultraviolet absorption at 450nm in each well with a microplate reader, compare the absorbance of the treated cells and untreated cells to obtain the cell viability value.
  • Example 3 Influence of 470nm light at different time on HeLa cell activity
  • the cell viability before and after light can be quantitatively characterized by the CCK-8 test.
  • (2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt It can be catalyzed and reduced by enzymes in cell mitochondria, and the amount of reduction is proportional to the number of cells, so it is widely used to test cell viability).
  • the cell viability remained above 88% after 5-10min of blue light irradiation, indicating that blue light irradiation caused less damage to the cells and could be used as a light source for intracellular polymerization; and after 20min of irradiation, the cell viability decreased to 76%, therefore, the illumination time for activating the prodrug of the present invention is selected to be 10 minutes.
  • HeLa cells were seeded at a density of 1x10 4 cells in 96-well culture plate, and incubated for 18h at thermostat 37 °C, 4% carbon dioxide condition; 5mM DMA, 0.1mM RAFTCTA 0.01mM and Eosin Y, or 5mM DMA, 0.05mM RAFTCTA And 0.005mM Eosin Y, or 5mM DMA, 0.01mM RAFTCTA and 0.001mM Eosin Y are dissolved in proportion in high glucose DMEM cell culture medium (containing 10% calf serum, 100uni/mL penicillin and streptomycin), HeLa cells washed with PBS (3 times) were added according to the amount of 0.1mL per well, incubated at 37°C for 4h, HeLa cells were washed 3 times with PBS to remove surface-adhesive reactants, and 0.1mL freshly added to each well DMEM medium was irradiated vertically with a 470nm LED blue
  • the cell viability before and after light can be quantitatively characterized by the CCK-8 test. As shown in Figure 3, no polymer is produced in the cell before light, so the cell viability is high, indicating that the reactant itself is not cytotoxic before polymerization, and 470nm light does not affect cell viability (Example 3), and cell viability after light Significantly lower, indicating that the polymer produced in the cell has a direct impact on cell viability.
  • Example 5 Using mice as a model to verify the inhibitory effect of intracellular polymerization on tumor growth
  • Tumors were subcutaneously inoculated into the outer thighs of 3-week-old mice at a density of 1 ⁇ 10 6 HeLa cells per mouse, and they were reared for 7 days.
  • the administration was started when the tumor volume was about 25 mm 3.
  • the prodrug was dissolved in PBS at a concentration of 5mM DMA, 0.05mM RAFTCTA, 0.005mM Eosin Y, and intratumorally administered at a dose of 5000 ⁇ L/kg. After 24 hours of administration, it was administered with 470nm blue light (power: 260mW/cm 2 )
  • the tumor is irradiated for 10 minutes to achieve polymerization.
  • the administration and illumination were carried out every 7 days.
  • the tumor volume was measured at 7 and 14 days after the illumination.
  • mice were sacrificed on the 14th day and the tumors were taken and photographed.
  • A375 cells were seeded at a density of 1x10 4 cells in 96-well culture plate, and incubated for 18h at thermostat 37 °C, 4% carbon dioxide condition; 5mM DMA, 0.1mM RAFTCTA 0.01mM and Eosin Y dissolved in a proportionally high glucose DMEM cell
  • the medium containing 10% calf serum, 100uni/mL penicillin and streptomycin
  • HeLa cells washed with PBS (3 times) according to the amount of 0.1mL per well, and incubate at 37°C for 4h, HeLa
  • the cells were washed 3 times with PBS to remove the reactants adhered to the surface, and 0.1 mL of fresh DMEM medium was added to each well, and the polymerization reaction was achieved by vertical irradiation with a 470nm LED blue light source from the bottom of the culture plate for 10 minutes.
  • Example 7 Inhibition of intracellular polymerization on 1205Lu cells
  • Example 8 Structural analysis of high molecular polymer produced by prodrugs in cells

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Abstract

一种细胞内聚合抑制细胞活性的方法及实现该方法的前体药物。所述前体药物包括：N,N-二甲基丙烯酰胺、RAFT链转移剂2-(丁基硫代羰基硫代硫基硫基)丙酸以及光引发剂曙红Y。把上述前体药物与细胞共同孵育，并以光照引发细胞内的聚合反应实现细胞内高分子聚合物的原位合成。细胞内原位合成的高分子聚合物能够有效影响细胞活性，诱发细胞凋亡及自噬，从而实现抑制肿瘤生长。

Description

一种细胞内聚合抑制细胞活性的方法及实现该方法的前体药物 技术领域
本发明涉及药物技术领域，特别涉及一种抑制细胞活性的方法及实现该方法的前体药物，具体地说，是涉及一种通过在细胞内原位合成线性高分子聚合物对细胞造成毒性，从而抑制细胞活性及实现该方法的前体药物。
背景技术
前体药物，即可以通过酶或化学刺激在生物体内原位激活(生成高活性药物分子)的低活性物质，通过对药物分子的针对性修饰，能够显著改善许多传统药物的水溶性，化学/生物稳定性、口服吸收效率、体内循环时间、全身毒性等诸多临床上出现的问题。目前市场上所有药物中的5-7％可以被认定为前体药物，并且此比例目前仍保持着增长的趋势，可见其在医药发展及应用中具有重要地位。
目前许多前体药物分子被研发出来并用于抗菌、抗病毒、抗肿瘤治疗当中，其中大部分前体药物分子都是通过对已知活性药物分子的活性位点进行特异性修饰实现的，而通过其他机理原位生成大分子药物的实例却较少，利用在细胞内原位生成高分子聚合物结构来实现细胞活性抑制的前体药物暂时还没有相关报道。
癌症作为一种严重威胁人类健康的恶性疾病，长期困扰着肿瘤患者和相关医护人员。抗癌药物的研发可以追溯到20世纪四十年代，虽然针对抗癌药物的研究至今已经超过70年，然而可以有效治愈癌症的治疗手段尚极为有限，成功案例也较少。这主要是由于癌症的内源性、遗传异质性、个体差异性、耐药性等特点。早期癌症在确诊后通过手术治疗一般能达到较好的治疗效果，但由于检测手段的局限性，早期癌症检测难度较高，并且手术切除有时不能完全去除癌变组织，从而造成较高的复发率。对于一些手术无法切除的癌变组织(如大脑，心脏，气管等部位)，目前只能通过放射治疗和化学治疗的方法来控制癌症恶化速率，但难以彻底治愈，同时癌细胞容易转移且一旦转移扩散将难以被治愈，以上原因都造成癌症死亡率极高的特点。
传统基于有机小分子的抗癌药物由于其高效的肿瘤抑制作用被广泛应用于各种癌症治疗中，例如2010年在国内批准上市的艾力替尼可用于治疗肺癌和乳腺癌；2011年批准上市的埃克替尼可以用于治疗非小细胞肺癌；以及2013年批准上市的氟马替尼可用 于治疗慢性粒细胞白血病等。然而由于肿瘤细胞突变概率高，接受化学治疗的患者大部分都会出现耐药性因而降低治疗效果，目前普遍采用的对抗耐药性的方案多为结合多种抗肿瘤机制的药物进行同时给药，因此开发基于新型毒性机理的抗肿瘤治疗方法对治疗肿瘤，特别是耐药性肿瘤有着重要意义。
研究发现通过自由基聚合在细胞内生成的高分子聚合物能够显著改变如细胞骨架生长、粘度和细胞结构等，并且能够影响细胞周期等生理性质。目前普遍认为高分子量聚合物相比于小分子具有更高的生物相容性，这是由于高分子量聚合物分子难以直接被细胞摄取，且分子量越大摄取效率越低，因此要通过聚合物实现对细胞的影响就需要在细胞内原位合成聚合物分子，但是到目前为止是否可以通过细胞内聚合对肿瘤细胞产生毒性尚无相关报道。
由于自由基类反应本身的高活性，在细胞内所产生的聚合物分子量较小且分子量分布较宽，对细胞结构及功能所产生的影响较小，因此造成的细胞毒性也较低，而新型光控可逆加成断裂链转移(RAFT)聚合由于其独特的聚合反应机理，使其具有更强的时间/空间可控性、更稳定的活性中间体，因此可以在相对复杂的反应体系中可控的实现较高分子量聚合物的合成。同时由于所采用的RAFT聚合的引发效率显著高于传统自由基聚合，因此可以在保证生成聚合物分子的同时大幅减少单体及引发剂的用量，降低由于分子扩散对周围的其他细胞产生的毒性。
为了增加药物选择性，降低对于健康细胞和组织的伤害，通常会将刺激响应能力引入癌症治疗体系中，使细胞毒性仅在指定时间及位置产生。其中以光作为刺激信号的体系具有更高的时间/空间可调控性，能够主动控制药物激活，从而实现对生物体的无创治疗。通过光能引发实现的RAFT聚合反应具有反应条件温和、副反应少以及聚合反应效率高等特点，且可以在水相、有氧及多种生物分子存在的条件下在指定区域精准实现高效聚合反应，并且聚合反应本身对细胞影响较小，不容易由于反应物扩散引发全身毒性，因此适合用于在细胞内原位合成高分子聚合物并实现针对肿瘤组织的选择性治疗。
发明内容
本发明提供了一种基于原位光控RAFT聚合的前体药物及其制备方法，所述前体药物在光照条件下能够合成较高毒性的高分子聚合物，从而消灭肿瘤细胞、抑制肿瘤生长。
本发明公开了一种抑制细胞活性的方法及实现该方法的前体药物，本发明中的前体 药物包括：N,N-二甲基丙烯酰胺(DMA)、2-(丁基硫代羰基硫代硫基硫基)丙酸(RAFTCTA)以及曙红Y。把上述前体药物与细胞共同孵育，并以光照引发细胞内的聚合反应实现细胞内高分子聚合物的原位合成。细胞内原位合成的高分子聚合物能够有效影响细胞活性，诱发细胞凋亡及自噬，从而实现抑制肿瘤生长的目的。
一种细胞内聚合抑制细胞活性的前体药物，包括N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y。
其中，N,N-二甲基丙烯酰胺作为单体，2-(丁基硫代羰基硫代硫基硫基)丙酸为链转移剂，曙红Y为光引发剂。
为了保证聚合反应效率及聚合物的生成量，单体N,N-二甲基丙烯酰胺的用量控制在毒性允许的最大剂量5mM；
聚合物分子量通过调控2-(丁基硫代羰基硫代硫基硫基)丙酸及曙红Y的用量来实现。2-(丁基硫代羰基硫代硫基硫基)丙酸与曙红Y的摩尔比为10：1；N,N-二甲基丙烯酰胺与2-(丁基硫代羰基硫代硫基硫基)丙酸的摩尔比为(50-500)：1。
一种抑制细胞活性的高分子聚合物，由如下方法制备：
(1)将前体药物与细胞共同孵育，使细胞摄取所述前体药物，前体药物包括N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y；
(2)以光作为刺激信号，通过光照实现细胞内聚合反应。以光作为刺激信号，具有更高的时间/空间可调控性，能够主动控制药物激活，从而实现对生物体的无创治疗。
进一步的，所述步骤(1)中活细胞接种在细胞培养板中进行孵育，N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y按比例溶解于细胞培养基中，并加入清洗过的所述活细胞进行孵育；所述步骤(2)中用所述步骤(1)获得的细胞，加入细胞培养基，光照实现聚合反应。
为了保证聚合反应效率及聚合物的生成量，单体N,N-二甲基丙烯酰胺(DMA)的用量控制在毒性允许的最大剂量5mM；
聚合物分子量通过调控2-(丁基硫代羰基硫代硫基硫基)丙酸(RAFTCTA)及曙红Y的用量来实现。2-(丁基硫代羰基硫代硫基硫基)丙酸与曙红Y的摩尔比为10：1；N,N-二甲基丙烯酰胺与2-(丁基硫代羰基硫代硫基硫基)丙酸的摩尔比为(50-500)： 1。
进一步的，所述步骤(2)中光源的最大激发波长为470nm，蓝光照射对细胞损伤较小。
所述的抑制细胞活性的高分子聚合物，具有如下结构：
Figure PCTCN2020082806-appb-000001
该化合物是具有较高毒性的高分子聚合物。
进一步的，所述步骤(1)中活细胞以2x10 5的密度接种于细胞培养板中，并在37℃，4％二氧化碳条件下恒温孵育18h；N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y按比例溶解于细胞培养基中，并按每孔2mL的用量加入用PBS清洗过的细胞，37℃恒温孵育4h；所述步骤(2)中用PBS清洗所述步骤(1)获得的细胞，并于每孔加入1mL新鲜细胞培养基，从培养板下方用光源垂直照射10min实现聚合反应。
一种抑制细胞活性的高分子聚合物的制备方法，包括如下步骤：
(1)将前体药物与细胞共同孵育，使细胞摄取所述前体药物，前体药物包括N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y；
(2)以光作为刺激信号，通过光照实现细胞内聚合反应。以光作为刺激信号，具有更高的时间/空间可调控性，能够主动控制药物激活，从而实现对生物体的无创治疗。
进一步的，所述步骤(1)中活细胞接种在细胞培养板中进行孵育，N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y按比例溶解于细胞培养基中，并加入清洗过的所述活细胞进行孵育；所述步骤(2)中用所述步骤(1)获得的细胞，加入细胞培养基，光照实现聚合反应。
为了保证聚合反应效率及聚合物的生成量，单体N,N-二甲基丙烯酰胺(DMA)的用量控制在毒性允许的最大剂量5mM；
聚合物分子量通过调控2-(丁基硫代羰基硫代硫基硫基)丙酸(RAFTCTA)及曙红Y的用量来实现。2-(丁基硫代羰基硫代硫基硫基)丙酸与曙红Y的摩尔比为10：1；N,N-二甲基丙烯酰胺与2-(丁基硫代羰基硫代硫基硫基)丙酸的摩尔比为(50-500)： 1。
进一步的，所述步骤(2)中光源的最大激发波长为470nm，蓝光照射对细胞损伤较小。
进一步的，所述步骤(1)中活细胞以2x10 5的密度接种于细胞培养板中，并在37℃，4％二氧化碳条件下恒温孵育18h；N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y按比例溶解于细胞培养基中，并按每孔2mL的用量加入用PBS清洗过的细胞，37℃恒温孵育4h；所述步骤(2)中用PBS清洗所述步骤(1)获得的细胞，并于每孔加入1mL新鲜细胞培养基，从培养板下方用光源垂直照射10min实现聚合反应。
一种细胞内聚合抑制细胞活性的方法，包括如下步骤：
(1)将前体药物与细胞共同孵育，使细胞摄取所述前体药物，前体药物包括N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y；
(2)以光作为刺激信号，通过光照实现细胞内聚合反应。以光作为刺激信号，具有更高的时间/空间可调控性，能够主动控制药物激活，从而实现对生物体的无创治疗。
进一步的，所述步骤(1)中活细胞接种在细胞培养板中进行孵育，N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y按比例溶解于细胞培养基中，并加入清洗过的所述活细胞进行孵育；所述步骤(2)中用所述步骤(1)获得的细胞，加入细胞培养基，光照实现聚合反应。
为了保证聚合反应效率及聚合物的生成量，单体N,N-二甲基丙烯酰胺(DMA)的用量控制在毒性允许的最大剂量5mM；
聚合物分子量通过调控2-(丁基硫代羰基硫代硫基硫基)丙酸(RAFTCTA)及曙红Y的用量来实现。2-(丁基硫代羰基硫代硫基硫基)丙酸与曙红Y的摩尔比为10：1；N,N-二甲基丙烯酰胺与2-(丁基硫代羰基硫代硫基硫基)丙酸的摩尔比为(50-500)：1。
进一步的，所述步骤(2)中光源的最大激发波长为470nm，蓝光照射对细胞损伤较小。
进一步的，所述步骤(1)中活细胞以2x10 5的密度接种于细胞培养板中，并在37℃， 4％二氧化碳条件下恒温孵育18h；N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y按比例溶解于细胞培养基中，并按每孔2mL的用量加入用PBS清洗过的细胞，37℃恒温孵育4h；所述步骤(2)中用PBS清洗所述步骤(1)获得的细胞，并于每孔加入1mL新鲜细胞培养基，从培养板下方用光源垂直照射10min实现聚合反应。
所述的前体药物在抑制细胞活性方面的应用，所述细胞包括HeLa、1205Lu、A375细胞系。
所述的前体药物在诱发细胞凋亡及细胞自噬方面的应用。
所述的前体药物在抑制肿瘤组织生长方面的应用。
所述的前体药物在制备抗肿瘤药物中的应用。
所述的前体药物在光控靶向肿瘤治疗方面的应用。
所述的高分子聚合物在诱发细胞凋亡及细胞自噬方面的应用。
所述的高分子聚合物在抑制肿瘤组织生长方面的应用。
所述的高分子聚合物在制备抗肿瘤药物中的应用。
所述的高分子聚合物在光控靶向肿瘤治疗方面的应用。
综上，与现有技术相比，本发明达到了以下技术效果：
1.本发明首次实现了细胞内可控RAFT聚合物的构建。
2.本发明的前体药物在细胞内原位合成RAFT聚合物，该高分子聚合物对细胞有直接毒性，能够降低细胞活性。
3.相比传统的抑制细胞活性的前体药物，本发明通过新型前体药物在细胞内合成高分子聚合物的机理实现了对细胞活性的抑制，对细胞耐药性提供了一种新的思路。
4.本发明所应用的单体、链转移剂以及光敏剂毒性均较低，因此在使用该前体药物时不会因前体药物的扩散造成全身毒性。
5.本发明通过光刺激引发聚合反应，在时间/空间层面实现了针对前体药物合成毒性高分子聚合物的高度可控性以及选择性。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为前体药物在细胞内聚合反应示意图。
图2为470nm光照不同时间对HeLa细胞活性产生的影响(测试结果通过CCK-8测试得到，n＝6)，ns：不显著，**：p<0.05。
图3为5mM N,N-二甲基丙烯酰胺(DMA)与不同浓度2-(丁基硫代羰基硫代硫基硫基)丙酸(RAFTCTA)及曙红Y溶液(RAFTCTA与曙红Y的摩尔比为10：1，RAFTCTA浓度为0.1，0.05和0.01mM)加入HeLa细胞后，光照10min/不光照后37℃孵育24小时后细胞活性的变化(测试结果通过CCK-8测试得到，n＝6)，ns：不显著，****：p<0.0001。
图4为小鼠肿瘤生长曲线，其中470nm光照时间为10min，前体药物中含有5mM DMA、0.05mM RAFTCTA及0.005mM曙红Y，给药剂量为5000μL/kg。
图5为给药14天后(与图4中肿瘤生长曲线中第14天相对应)处死小鼠取出的肿瘤照片，每个肿瘤均取自不同小鼠。
图6为5mM DMA与不同浓度RAFTCTA及曙红Y溶液(RAFTCTA与曙红Y的摩尔比为10：1，RAFTCTA浓度为0.1，0.05和0.01mM)加入A375细胞后，光照10min/不光照后37℃孵育24小时后细胞活性的变化(测试结果通过CCK-8测试得到，n＝6)，ns：不显著，****：p<0.0001。
图7为5mM DMA与不同浓度RAFTCTA及曙红Y溶液(RAFTCTA与曙红Y的摩尔比为10：1，RAFTCTA浓度为0.1，0.05和0.01mM)加入1205Lu细胞后，光照10min/不光照后37℃孵育24小时后细胞活性的变化(测试结果通过CCK-8测试得到，n＝6)，ns：不显著，***：p<0.001，****：p<0.0001。
图8为模拟前体药物细胞内聚合得到的poly(DMA)聚合物溶解于氘代氯仿后测得的 1HNMR图谱。
图9为模拟前体药物细胞内聚合得到的poly(DMA)聚合物溶解于DMF后测得的GPC图谱。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
本发明的方法能够抑制细胞活性的原理
本发明所述抑制细胞活性的方法能够有效在细胞内部生成丙烯酰胺类高分子聚合物，由于此类聚合物特殊的线性链状结构，使其能与细胞内多种生物分子发生非特异性的如物理吸附、非共价键形式等相互作用，因此能通过抑制PI3K/Akt/mTOR及AMPK信号通路，诱导细胞自噬并抑制细胞增殖，并通过激活内质网应激反应、线粒体外膜的透化，诱导细胞凋亡及程序性坏死等多种类型细胞死亡方式，达到抑制细胞活性的效果，而且这种效果对细胞具有普遍效果，并非只针对某一种细胞有效。
一种细胞内聚合抑制细胞活性的方法，主要包括如下步骤：
(1)将前体药物与细胞共同孵育，使细胞摄取所述前体药物，前体药物包括N,N-二甲基丙烯酰胺(DMA)、2-(丁基硫代羰基硫代硫基硫基)丙酸(RAFTCTA)、光引发剂曙红Y；
活细胞以1x10 4每孔的密度接种于96孔细胞培养板中，并在37℃，4％二氧化碳条件下恒温孵育18h以保证细胞贴壁；DMA、RAFTCTA以及光引发剂曙红Y按比例溶解于高糖DMEM细胞培养基中(含有10％小牛血清、100uni/mL的青霉素和链霉素)，并按每孔0.1mL的用量加入用PBS清洗过的(3次)细胞，37℃恒温孵育4h，实现细胞对前体药物的摄取。
其中，为了保证聚合反应效率及聚合物的生成量，单体N,N-二甲基丙烯酰胺(DMA)的用量控制在毒性允许的最大剂量5mM；
聚合物分子量通过调控2-(丁基硫代羰基硫代硫基硫基)丙酸(RAFTCTA)及曙 红Y的用量来实现。2-(丁基硫代羰基硫代硫基硫基)丙酸与曙红Y的摩尔比为10：1；N,N-二甲基丙烯酰胺与2-(丁基硫代羰基硫代硫基硫基)丙酸的摩尔比为(50-500)：1。
(2)以光作为刺激信号，通过光照实现细胞内聚合反应。以光作为刺激信号，具有更高的时间/空间可调控性，能够主动控制药物激活，从而实现对生物体的无创治疗。比如可以针对肿瘤区域照射，使聚合仅发生在肿瘤内，达到选择性抑制肿瘤细胞活性的目的。
摄取了前体药物的细胞用PBS清洗3次以除去表面黏附的反应物，并于每孔加入0.1mL新鲜DMEM培养基，以470nm LED蓝光光源(功率：260mW/cm 2)从培养板下方垂直照射10min实现聚合反应。所述波段的光源对细胞损伤较小，通过实验证明用405nm和530nm的光源都不能引发所述细胞内聚合反应。
实施例1：RAFT链转移剂2-(丁基硫代羰基硫代硫基硫基)丙酸(RAFTCTA)的合成步骤
在30min内，向含有9.0g KOH的150mL THF悬浊液中逐滴加入50mL含有16.0g正丁醇的THF溶液，室温搅拌30min，再逐滴加入50mL含有17.0g CS 2的THF溶液，室温搅拌24h后减压浓缩至50mL，搅拌下将50mL含有22.4g正丙基溴化铵的THF溶液逐滴加入上述浓缩液中，室温搅拌24h后减压去除溶剂即得目标粗产物，进一步通过硅胶柱色谱纯化即得亮黄色晶体状目标产物。
实施例2：CCK-8测试表征细胞活性
CCK-8测试中，首先将HeLa细胞以1x10 4的密度接种于96孔细胞培养板中并在37℃，4％二氧化碳条件下恒温孵育18h，保证细胞贴壁；然后对细胞进行不同浓度给药或光照(5–20min，260mW/cm 2)等处理，并继续恒温孵育24h；24h后将原培养基吸出并用PBS清洗细胞3次，每孔加入稀释的CCK-8溶液(与培养基比例为1：10)100μL，恒温孵育4h后用酶标仪检测各孔内450nm处的紫外吸收，处理过的细胞与未处理细胞进行吸光度比较即可得到细胞活性值。
实施例3：470nm光照不同时间对HeLa细胞活性产生的影响
HeLa细胞以1x10 4的密度分别接种于三块96孔细胞培养板中，并在37℃，4％二氧化碳条件下恒温孵育18h；然后以470nm LED蓝光光源分别从三块培养板下方垂直 照射5、10、20min。
按上述方法，光照前后的细胞活力可以用CCK-8测试进行定量表征。(2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺酸苯)-2H-四唑单钠盐，可被细胞线粒体内酶催化还原，还原量与细胞数量成正比，因此被广泛用于细胞活性的测试)。
如图2所示，对细胞进行5-10min蓝光照射后细胞活力均保持在88％以上，说明蓝光照射对细胞损伤较小，可以作为细胞内聚合的光源使用；而照射20min后细胞活性降低至76％，因此本发明激活前体药物的光照时间选择在10min。
以下以肿瘤细胞为例，展示细胞内聚合对细胞活性的抑制作用。
实施例4：细胞内聚合对HeLa细胞的抑制作用
HeLa细胞以1x10 4的密度接种于96孔细胞培养板中，并在37℃，4％二氧化碳条件下恒温孵育18h；5mM DMA、0.1mM RAFTCTA以及0.01mM曙红Y，或5mM DMA、0.05mM RAFTCTA以及0.005mM曙红Y，或5mM DMA、0.01mM RAFTCTA以及0.001mM曙红Y按比例溶解于高糖DMEM细胞培养基中(含有10％小牛血清、100uni/mL的青霉素和链霉素)，并按每孔0.1mL的用量加入用PBS清洗过(3次)的HeLa细胞，37℃恒温孵育4h，HeLa细胞用PBS清洗3次以除去表面黏附的反应物，并于每孔加入0.1mL新鲜DMEM培养基，以470nm LED蓝光光源从培养板下方垂直照射10min实现聚合反应。
按上述方法，光照前后的细胞活力可以用CCK-8测试进行定量表征。如图3所示，光照前细胞内不生成聚合物，因此细胞活性高，说明聚合之前反应物本身不具有细胞毒性，并且470nm光照不会影响细胞活性(实施例3)，而光照后细胞活力显著降低，说明细胞内生成的聚合物对细胞活性产生了直接影响。
从CCK-8测试结果(图3)可以看出，光照引发聚合反应前细胞活性保持在95％以上(灰色柱)，说明前体药物毒性低；而加入0.1mM和0.05mM RAFTCTA的实验组中，通过光照聚合后细胞活性分别降低至48％和50％，说明两种条件配比下细胞内都生成了聚合物，且聚合物对细胞活性产生了显著抑制作用。
而在加入0.01mM RAFTCTA的实验组中，通过光照聚合后细胞活性仅降低至92％，说明由于链转移剂及光敏剂浓度较低，细胞内产生的聚合物较少，并没有显著影响细胞活性。
实施例5：以小鼠为模型验证细胞内聚合对肿瘤生长的抑制作用
以1x10 6HeLa细胞每鼠的密度在3周龄小鼠大腿外侧皮下接种肿瘤，并饲养7天，待肿瘤体积约为25mm 3时开始给药。前体药物按5mM DMA、0.05mM RAFTCTA、0.005mM曙红Y的浓度溶解于PBS中，并按5000μL/kg的剂量瘤内给药，给药24小时后以470nm蓝光(功率：260mW/cm 2)对肿瘤进行10min光照实现聚合。给药及光照每7天进行一次，光照后7天及14天时对肿瘤体积进行测量，在14天时处死小鼠，取肿瘤拍照。其中对照组仅向肿瘤注入PBS，光照组注射PBS后24小时进行10min光照(470nm)，前体药物组仅向瘤内注射80μL前体药物溶液，不进行光照(重复组n＝4)。
从肿瘤生长曲线及去除肿瘤的照片(图4、图5)可以看出加入前体药物或光照10min对肿瘤生长速度影响较小，而同时实施两者使肿瘤内生成聚合物则可以非常显著的抑制肿瘤生长速率，实现抑制肿瘤生长的目的。
实施例6：细胞内聚合对A375细胞的抑制作用
A375细胞以1x10 4的密度接种于96孔细胞培养板中，并在37℃，4％二氧化碳条件下恒温孵育18h；5mM DMA、0.1mM RAFTCTA以及0.01mM曙红Y按比例溶解于高糖DMEM细胞培养基中(含有10％小牛血清、100uni/mL的青霉素和链霉素)，并按每孔0.1mL的用量加入用PBS清洗过(3次)的HeLa细胞，37℃恒温孵育4h，HeLa细胞用PBS清洗3次以除去表面黏附的反应物，并于每孔加入0.1mL新鲜DMEM培养基，以470nm LED蓝光光源从培养板下方垂直照射10min实现聚合反应。
从CCK-8测试结果(图4)可以看出，光照引发聚合反应前细胞活性保持在90％以上，说明前体药物反应物毒性低，而聚合后细胞活性降低至22％，说明细胞内发生了聚合反应且生成的高分子聚合物显著抑制了细胞活性。与实施例3相比，聚合后实施例6中细胞活力降低比实施例3降低更为显著，说明本方法在不同细胞内都能生成聚合物，且聚合物对不同细胞活性产生了显著抑制。
实施例7：细胞内聚合对1205Lu细胞的抑制作用
1205Lu细胞以1x10 4的密度接种于96孔细胞培养板中，并在37℃，4％二氧化碳条件下恒温孵育18h；5mM DMA、0.1mM RAFTCTA以及0.01mM曙红Y按比例溶解于高糖DMEM细胞培养基中(含有10％小牛血清、100uni/mL的青霉素和链霉素)，并按每孔0.1mL的用量加入用PBS清洗过(3次)的HeLa细胞，37℃恒温孵育4h， HeLa细胞用PBS清洗3次以除去表面黏附的反应物，并于每孔加入0.1mL新鲜DMEM培养基，以470nm LED蓝光光源从培养板下方垂直照射10min实现聚合反应。
从CCK-8测试结果(图5)可以看出，光照引发聚合反应前细胞活性保持在90％以上，说明前体药物毒性低，而聚合后细胞活性降低至61％，说明细胞内发生了聚合反应且生成的聚合物显著抑制了细胞活性。与实施例3相比，聚合后实施例7中细胞活力降低与实施例3降低程度类似，说明本方法在不同细胞内都能生成聚合物，且聚合物对不同细胞活性产生了显著抑制。
实施例8：细胞内前体药物生成高分子聚合物的结构分析
通过紫外可见吸收光谱定量分析细胞裂解液的吸收光谱并与对应化合物的标准进行比较可得被摄取到细胞内的前体药物各组分的浓度，其中DMA为28.1mM，RAFTCTA为1.6mM，曙红Y为0.82mM。
按照细胞内浓度在体外以PBS为溶剂，通过透析法(MWCO＝1000Da)对聚合物进行提纯后，通过 1HNMR测试分析组成(如图8所示)，并通过GPC测试分析聚合物相对分子质量(Mn＝26kDa)及分子量分布
Figure PCTCN2020082806-appb-000002
如图9所示。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (27)

  1. 一种细胞内聚合抑制细胞活性的前体药物，其特征在于，包括N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y。
  2. 根据权利要求1所述的前体药物，其特征在于，
    N,N-二甲基丙烯酰胺浓度为5mM；
    2-(丁基硫代羰基硫代硫基硫基)丙酸与曙红Y摩尔比为10：1；
    N,N-二甲基丙烯酰胺与2-(丁基硫代羰基硫代硫基硫基)丙酸的摩尔比为(50-500)：1。
  3. 一种抑制细胞活性的高分子聚合物，其特征在于，由如下方法制备：
    (1)将前体药物与细胞共同孵育，使细胞摄取所述前体药物，前体药物包括N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y；
    (2)以光作为刺激信号，通过光照实现细胞内聚合反应。
  4. 根据权利要求3所述的高分子聚合物，其特征在于，所述步骤(1)中活细胞接种在细胞培养板中进行孵育，N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y按比例溶解于细胞培养基中，并加入清洗过的所述活细胞进行孵育；所述步骤(2)中用所述步骤(1)获得的细胞，加入细胞培养基，光照实现聚合反应。
  5. 根据权利要求3所述的高分子聚合物，其特征在于，所述步骤(1)中N,N-二甲基丙烯酰胺浓度为5mM，2-(丁基硫代羰基硫代硫基硫基)丙酸与曙红Y的摩尔比为10：1，N,N-二甲基丙烯酰胺与2-(丁基硫代羰基硫代硫基硫基)丙酸的摩尔比为(50-500)：1。
  6. 根据权利要求3所述的高分子聚合物，其特征在于，所述步骤(2)中光源的最大激发波长为470nm。
  7. 根据权利要求3所述的高分子聚合物，其特征在于，具有如下结构：
    Figure PCTCN2020082806-appb-100001
  8. 根据权利要求3所述的高分子聚合物，其特征在于，所述步骤(1)中活细胞以2 x 10 5的密度接种于细胞培养板中，并在37℃，4％二氧化碳条件下恒温孵育18h；N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y按比例溶解于细胞培养基中，并按每孔2mL的用量加入用PBS清洗过的细胞，37℃恒温孵育4h；所述步骤(2)中用PBS清洗所述步骤(1)获得的细胞，并于每孔加入1mL新鲜细胞培养基，从培养板下方用光源垂直照射10min实现聚合反应。
  9. 一种抑制细胞活性的高分子聚合物的制备方法，其特征在于，包括如下步骤：
    (1)将前体药物与细胞共同孵育，使细胞摄取所述前体药物，前体药物包括N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y；
    (2)以光作为刺激信号，通过光照实现细胞内聚合反应。
  10. 根据权利要求9所述的制备方法，其特征在于，其特征在于，所述步骤(1)中活细胞接种在细胞培养板中进行孵育，N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y按比例溶解于细胞培养基中，并加入清洗过的所述活细胞进行孵育；所述步骤(2)中用所述步骤(1)获得的细胞，加入细胞培养基，光照实现聚合反应。
  11. 根据权利要求9所述的制备方法，其特征在于，所述步骤(1)中N,N-二甲基丙烯酰胺浓度为5mM，2-(丁基硫代羰基硫代硫基硫基)丙酸与曙红Y的摩尔比为10：1，N,N-二甲基丙烯酰胺与2-(丁基硫代羰基硫代硫基硫基)丙酸的摩尔比为(50-500)：1。
  12. 根据权利要求9所述的制备方法，其特征在于，所述步骤(2)中光源的最大激发波长为470nm。
  13. 根据权利要求9所述的制备方法，其特征在于，所述步骤(1)中活细胞以2 x 10 5的密度接种于细胞培养板中，并在37℃，4％二氧化碳条件下恒温孵育18h；N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y按比例溶解于细胞培养基 中，并按每孔2mL的用量加入用PBS清洗过的细胞，37℃恒温孵育4h；所述步骤(2)中用PBS清洗所述步骤(1)获得的细胞，并于每孔加入1mL新鲜细胞培养基，从培养板下方用光源垂直照射10min实现聚合反应。
  14. 一种细胞内聚合抑制细胞活性的方法，其特征在于，包括如下步骤：
    (1)将前体药物与细胞共同孵育，使细胞摄取所述前体药物，前体药物包括N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y；
    (2)以光作为刺激信号，通过光照实现细胞内聚合反应。
  15. 根据权利要求14所述的抑制细胞活性的方法，其特征在于，所述步骤(1)中活细胞接种在细胞培养板中进行孵育，N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y按比例溶解于细胞培养基中，并加入清洗过的所述活细胞进行孵育；所述步骤(2)中用所述步骤(1)获得的细胞，加入细胞培养基，光照实现聚合反应。
  16. 根据权利要求14所述的抑制细胞活性的方法，其特征在于，所述步骤(1)中N,N-二甲基丙烯酰胺浓度为5mM，2-(丁基硫代羰基硫代硫基硫基)丙酸与曙红Y的摩尔比为10：1，N,N-二甲基丙烯酰胺与2-(丁基硫代羰基硫代硫基硫基)丙酸的摩尔比为(50-500)：1。
  17. 根据权利要求14所述的抑制细胞活性的方法，其特征在于，所述步骤(2)中光源的最大激发波长为470nm。
  18. 根据权利要求14所述的抑制细胞活性的方法，其特征在于，所述步骤(1)中活细胞以2 x 10 5的密度接种于细胞培养板中，并在37℃，4％二氧化碳条件下恒温孵育18h；N,N-二甲基丙烯酰胺、2-(丁基硫代羰基硫代硫基硫基)丙酸、曙红Y按比例溶解于细胞培养基中，并按每孔2mL的用量加入用PBS清洗过的细胞，37℃恒温孵育4h；所述步骤(2)中用PBS清洗所述步骤(1)获得的细胞，并于每孔加入1mL新鲜细胞培养基，从培养板下方用光源垂直照射10min实现聚合反应。
  19. 权利要求1-2任一项所述的前体药物在抑制细胞活性方面的应用，所述细胞包括HeLa、1205Lu、A375细胞系。
  20. 权利要求1-2任一项所述的前体药物在诱发细胞凋亡及细胞自噬方面的应用。
  21. 权利要求1-2任一项所述的前体药物在抑制肿瘤组织生长方面的应用。
  22. 权利要求1-2任一项所述的前体药物在制备抗肿瘤药物中的应用。
  23. 权利要求1-2任一项所述的前体药物在光控靶向肿瘤治疗方面的应用。
  24. 权利要求3-8任一项所述的高分子聚合物在诱发细胞凋亡及细胞自噬方面的应用。
  25. 权利要求3-8任一项所述的高分子聚合物在抑制肿瘤组织生长方面的应用。
  26. 权利要求3-8任一项所述的高分子聚合物在制备抗肿瘤药物中的应用。
  27. 权利要求3-8任一项所述的高分子聚合物在光控靶向肿瘤治疗方面的应用。
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