Application of polyphenol polymers in the preparation of anti-tumor drugs for starvation therapy

Abstract

The invention discloses an application of a polyphenol polymer in the preparation of a starvation therapy anti-tumor drug, wherein the polyphenol polymer is a polymer grafted with a polyphenol small molecule, or a copolymer of a polyphenol monomer and a carboxyl monomer obtained by reacting an unsaturated acid anhydride with dopamine hydrochloride. The polyphenol polymer can respond to active oxygen in the tumor tissue microenvironment and form a random cross-linked network around the tumor or cancer cells. The dense network structure can limit molecular diffusion, hinder the intake of nutrients by the tumor or cancer cells, disrupt the metabolic process, and achieve the purpose of starvation therapy for cancer. The polyphenol polymer of the invention shows excellent anti-tumor effects in models such as adherent cells, cell spheres and tumor-bearing mice, and has certain research significance and clinical transformation potential.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-tumor drugs, and specifically relates to the application of a polyphenol polymer in the preparation of a starvation therapy anti-tumor drug. Background Art

[0002] Cancer, a major disease that seriously threatens human life and health, has the characteristics of high morbidity and mortality, which not only brings great physical and mental pain and heavy medical burden to patients, but also consumes a large amount of medical and health resources. With the aggravation of population aging and the increase of unhealthy lifestyles such as irregular diet and staying up late, the incidence of malignant tumors has increased year by year.

[0003] With the continuous deepening of cancer research, more and more new anti-cancer treatment strategies have been developed. Among them, starvation therapy mainly causes tumor cells to be in a "starved" state of insufficient energy for a long time, causing metabolic disorders, thereby achieving necrosis or apoptosis. As a potential new strategy for cancer treatment, starvation therapy can selectively deprive the supply of nutrients and oxygen by inhibiting tumor angiogenesis, destroying or blocking tumor blood vessels, accelerating the depletion of substances such as glucose and oxygen in tumors, and other processes. For example: directly interfering with tumor blood vessels by injecting embolic agents (blocking tumor blood vessels), vascular inhibitors (inhibiting new angiogenesis) or vascular destroyers (destroying original blood vessels) to block their nutrient supply to tumor cells; or introducing exogenous glucose oxidase (GOx) into tumor cells to quickly consume glucose and O inside the cells. 2 , causing tumor cells to enter a state of excessive starvation and eventually die. Summary of the invention

[0004] The object of the present invention is to provide a new use of polyphenol polymers, namely a new application in pharmaceutical preparation.

[0005] In fact, the present invention relates to the use of polyphenol polymers in the preparation of anti-tumor drugs for starvation therapy. Polyphenol polymers are rich in a large number of phenolic hydroxyl groups, which can responsively undergo oxidative cross-linking reactions with active oxygen in the microenvironment of tumor tissues, forming a polymer network with a random cross-linked dense structure around tumors or cancer cells. The dense network structure can limit molecular diffusion, hinder the intake of nutrients by tumors or cancer cells, disrupt the normal metabolic process of cells, and make cells in a state of starvation for a long time and finally die, thereby achieving the purpose of starvation therapy for cancer.

[0006] The polyphenol polymer of the present invention is a polymer of grafted polyphenol small molecules obtained by grafting polyphenol small molecules onto a polymer rich in amino groups or carboxyl groups; or the polyphenol polymer is a copolymer obtained by copolymerizing a polyphenol monomer and a carboxyl monomer, wherein the polyphenol monomer is obtained by reacting an unsaturated acid anhydride with dopamine hydrochloride; the number average molecular weight of the copolymer is 5000 to 25000.

[0007] The above-mentioned amino-rich polymer is any one of ε-polylysine, polyethyleneimine, polyamide-amine dendrimer, etc.; the carboxyl-rich polymer is any one of chitosan, polyacrylic acid, hyaluronic acid, etc.; the polyphenol small molecule is any one of 3,4-dihydroxybenzoic acid, gallic acid, catechin, tea polyphenols, tannic acid, epicatechin gallate, epigallocatechin, dopamine, epigallocatechin gallate, etc.; the unsaturated acid anhydride is any one of methacrylic anhydride, acrylic anhydride, maleic anhydride (maleic anhydride), etc.; the carboxyl monomer is any one of acrylic acid, methacrylic acid, fumaric acid, maleic acid (maleic acid), etc.

[0008] The above-mentioned polymer grafted with polyphenol small molecules is prepared by a free radical induction method. The specific preparation method is: weigh the polymer rich in amino or carboxyl groups and the polyphenol small molecules respectively, add them to the organic solvent, stir and ultrasonically dissolve them fully, and obtain a polymer solution and a polyphenol small molecule solution respectively; add ascorbic acid to hydrogen peroxide and stir until completely dissolved, add the resulting solution to the polymer solution and incubate for 2 to 3 hours, and finally add the polyphenol small molecule solution to continue the reaction for 1 to 7 days. After the reaction is completed, the organic solvent is removed by rotary evaporation of the reaction solution, and after redispersing with ethanol, the solution is added to ethyl acetate for washing, and then the solution is centrifuged and the solid-liquid separation is performed, ethanol is added to the solid to disperse evenly, and then added to ether for sedimentation, centrifuged and the solid-liquid separation is performed, and water is added to the solid for freeze-drying to obtain a polymer grafted with polyphenol small molecules. Wherein, the weight ratio of the polymer rich in amino or carboxyl groups, the polyphenol small molecules, ascorbic acid and hydrogen peroxide is 1: 0.5-10: 0.5-10: 10-60.

[0009] The preparation method of the copolymer of the polyphenol monomer and the carboxyl monomer comprises the following steps:

[0010] (1) Add the protective agent and salt to deionized water, continue to pass argon to remove the dissolved oxygen in the solution, then add dopamine hydrochloride, stir and dissolve to obtain a reaction solution; dissolve the unsaturated acid anhydride in an organic solvent, add it dropwise to the reaction solution, stir at room temperature for 12 to 16 hours under the protection of argon, and continuously add sodium hydroxide to adjust the pH during the reaction so that the pH of the entire reaction system is maintained at >8. After the reaction is completed, the solid and liquid are separated to obtain a filtrate, and ethyl acetate is added to the filtrate for extraction. During the extraction process, hydrochloric acid is used to acidify to pH ≤ 2. After separation, the upper organic solution is taken, anhydrous magnesium sulfate is added, and refrigerated and dried overnight to remove excess water in the organic solution, and then the refrigerated and dried solution is freed of anhydrous magnesium sulfate, concentrated by rotary evaporation, added to n-hexane, and vigorously stirred until a brown solid appears. After refrigeration and recrystallization, after solid-liquid separation, the solid is taken and dried to obtain a polyphenol monomer. Wherein, the weight ratio of the protective agent, salt, dopamine hydrochloride and unsaturated acid anhydride is 10 to 20: 1 to 5: 5 to 10: 5 to 10. The protective agent is any one or more of sodium metaborate, borax decahydrate, borax pentahydrate, anhydrous borax, and potassium tetraborate; the salt is any one or more of sodium carbonate, potassium carbonate, sodium acetate, sodium sulfite, sodium bisulfite, sodium hydrogen sulfide, sodium phosphate, and ammonium bicarbonate.

[0011] (2) Weigh polyphenol monomer, carboxyl monomer, and azobisisobutyronitrile (AIBN) and add them to an organic solvent, stir to fully dissolve them, bubble with argon for a period of time to remove all oxygen in the solution, and then continue to react the reaction solution in an oil bath at 60-70°C for 10-12 hours. After the reaction is completed, the reaction solution is dialyzed with a dialysis bag with a molecular weight cutoff of 3kD to remove by-products. After dialysis, the retentate is freeze-dried. The weight ratio of the polyphenol monomer, carboxyl monomer, and AIBN is 1:1.5-2.5:0.1-0.2.

[0012] The above-mentioned organic solvent is dimethyl sulfoxide, N,N-dimethylformamide, tetrahydrofuran, ethanol and the like.

[0013] The anti-tumor drug of the present invention comprises a therapeutically effective amount of polyphenol polymer and a pharmaceutically acceptable carrier. The drug is an injection agent and is used for treatment through tail vein injection or in situ injection.

[0014] The tumor targeted by the anti-tumor drug of the present invention is a solid tumor.

[0015] The working principle of the polyphenol polymer used for tumor starvation therapy of the present invention is that the polyphenol structure in the polymer will responsively undergo cross-linking reaction with the active oxygen around the tumor tissue in the tumor microenvironment, forming a random cross-linking network near the cancer cells and adhering to the cell surface. The dense network structure can limit the diffusion of molecules, hinder the cell's intake of nutrients, and can retain and enrich for a long time, disrupting the cell's metabolic process, thereby achieving the effect of starvation therapy on tumor tissue.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The present invention prepares the polyphenol polymer by grafting reaction or free radical copolymerization, and the polyphenol polymer has the characteristic of cross-linking in response to hydrogen peroxide overexpressed in the acid environment of tumor.

[0018] 2. The polyphenol polymer of the present invention can be randomly cross-linked in a tumor environment rich in hydrogen peroxide, presenting an obvious gel fiber network structure on the cell surface, and can achieve a good starvation treatment effect by inhibiting the cell's absorption of nutrients such as glucose.

[0019] 3. The polyphenol polymers of the present invention can be responsively cross-linked after reaching the tumor to achieve the anti-tumor effect of starvation therapy, which will provide new ideas for the development of new anti-tumor agents, and will also be of great significance to the clinical treatment of tumors and the exploration of new tumor treatment schemes. It has certain research significance and clinical transformation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are the hydrogen NMR spectra of EPL, TA, and EPL-TA.

[0021] Figure 2 It is the Fourier transform infrared spectra of EPL and EPL-TA.

[0022] Figure 3 It is the hydrogen nuclear magnetic resonance spectrum of DMA.

[0023] Figure 4 This is the H NMR spectrum of PMAA-DMA.

[0024] Figure 5 It is the Fourier transform infrared spectrum of PMAA-DMA.

[0025] Figure 6 It is the hydrogen NMR spectrum of EPL, GTP, and EPL-GTP.

[0026] Figure 7 It is the Fourier transform infrared spectrum of EPL and EPL-GTP.

[0027] Figure 8 These are the hydrogen NMR spectra of EPL, GA, and EPL-GA.

[0028] Fig. 9 It is the Fourier transform infrared spectra of EPL and EPL-GA.

[0029] Fig.10 These are scanning electron micrographs of EPL-TA (a), PMAA-DMA (b), and oxidized EPL-TA (c).

[0030] Fig.11 The results of reactive oxygen species fluorescence imaging of HeLa multicellular tumor spheres without any treatment (a), HeLa multicellular tumor spheres co-incubated with EPL-TA (b), and the curve of reactive oxygen species fluorescence changing with the line distance (c).

[0031] Fig.12 These are scanning electron microscopy photos of the control group (top) and EPL-TA (bottom) after co-incubation with HeLa multicellular tumor spheres.

[0032] Fig.13 It is a graph of glucose concentration in the culture medium of HeLa multicellular tumor spheres treated with PMAA-DMA or PBS.

[0033] Fig.14 It is a graph of glucose concentration in the culture medium of HeLa multicellular tumor spheres treated with EPL-TA or PBS.

[0034] Fig.15 It is a curve of GSH concentration in the culture medium of HeLa multicellular tumor spheres treated with EPL-TA or PBS.

[0035] Fig.16 This is the cell survival rate result after PMAA-DMA was co-incubated with HeLa adherent cells.

[0036] Fig.17 This is the cell survival rate result after EPL-TA was co-incubated with HeLa adherent cells.

[0037] Fig.18 This is the cell survival rate result after EPL-GTP was co-incubated with HeLa adherent cells.

[0038] Fig.19 This is the cell survival rate result after EPL-GA was co-incubated with HeLa adherent cells.

[0039] Fig. 20 This is the cell survival result after PMAA-DMA was co-incubated with HeLa multicellular tumor spheres.

[0040] Fig.21 This is the cell survival result after EPL-TA was co-incubated with HeLa multicellular tumor spheres.

[0041] Fig. 22 This is the cell survival rate result after EPL-GTP was co-incubated with HeLa multicellular tumor spheres.

[0042] Fig.23 This is the cell survival rate result after EPL-GA was co-incubated with HeLa multicellular tumor spheres.

[0043] Fig.24 It is the result of hemolysis rate after PMAA-DMA was co-incubated with rabbit red blood cells.

[0044] Fig.25 The results of cell invasion experiments of HeLa multicellular tumor spheres without any treatment (a), EPL-TA and HeLa multicellular tumor spheres co-cultured (b), and the statistical chart of the number of invasion cells in the control group and the experimental group (c).

[0045] Fig.26 It is a statistical chart of body weight of tumor-bearing mice in the control group, PMAA-DMA group, and after EPL-TA treatment.

[0046] Fig. 27 This is the curve of tumor volume changes in the control group, PMAA-DMA group, and tumor-bearing mice after EPL-TA treatment.

[0047] Fig.28 It is a statistical chart of the survival rates of tumor-bearing mice in the control group, PMAA-DMA group, and after EPL-TA treatment.

[0048] Fig.29 These are the H&E staining images of the main organs (heart, liver, spleen, lung, and kidney) and tumors of tumor-bearing mice in the control group, PMAA-DMA group, and EPL-TA-treated group. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical scheme and advantages of the present invention more clear, the content of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It is necessary to point out that the following embodiments only explain and illustrate the present invention and are not intended to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still belong to the protection scope of the present invention.

[0050] Example 1

[0051] 72 mg of ε-polylysine (EPL) and 450 mg of tannic acid (TA) were weighed and added to 30 mL of dimethyl sulfoxide (DMSO), stirred and ultrasonicated to fully dissolve each; 50 mg of ascorbic acid was added to 1 mL of hydrogen peroxide and stirred until completely dissolved, and then the solution was added to the ε-polylysine solution and incubated for 2 hours, and finally the TA solution was added to continue the reaction for 5 days. After the reaction, the reaction solution was evaporated to remove DMSO, and after redispersing with ethanol, the solution was added to ethyl acetate for washing, the solution was centrifuged and the solid-liquid separation was performed, ethanol was added to the solid to disperse it evenly, and then it was added to ether for sedimentation, centrifuged and the solid-liquid separation was performed, and an appropriate amount of water was added to the solid for freeze-drying to obtain a polymer grafted with TA (referred to as EPL-TA), and the structural characterization results are shown in Figure 1 and Figure 2 .

[0052] Example 2

[0053] 1. Weigh 5g of sodium bicarbonate and 20g of sodium tetraborate decahydrate and add them to 100mL of deionized water. Bubble with argon for 30 minutes to remove oxygen from the solution. Then, add 6g of dopamine hydrochloride to the solution and stir for 5 minutes to dissolve it to obtain a reaction solution. Dissolve 6g of methacrylic anhydride in 15mL of tetrahydrofuran and add it dropwise to the reaction solution. Stir and react at room temperature for 14 hours under argon protection. During the reaction, adjust the pH by continuously adding 1mol / L NaOH aqueous solution to keep the pH of the entire reaction system>8. After the reaction, the mixed solution was filtered under reduced pressure to remove sodium bicarbonate and sodium tetraborate decahydrate solids. The clear and transparent filtrate was extracted 3 times with ethyl acetate. During the extraction process, concentrated hydrochloric acid was used to adjust the pH to ≤2. Anhydrous MgSO 4 Seal the solution in a refrigerator and dry overnight to remove excess water from the organic solution. Then filter the dried solution to remove MgSO 4 The product was concentrated by rotary evaporation to about 50 mL, and then dripped into 450 mL of icy n-hexane under vigorous stirring, and then vigorously stirred until a brown solid appeared. The product was placed in a refrigerator for overnight recrystallization, and then filtered to obtain the final solid product, which was dried in a vacuum drying oven at room temperature to obtain 3-methacryloyl dopamine (denoted as DMA). The structural characterization results are shown in Figure 3 .

[0054] 2. Take 0.3g DMA, 0.6g methacrylic acid (MAA) and 0.045g AIBN and add them to 5mL dimethyl sulfoxide (DMSO), stir to fully dissolve, and bubble with argon for 30 minutes to ensure that all oxygen in the solution is removed. Then continue to react the reaction solution in an oil bath at 65°C for 12 hours. After the reaction is completed, the reaction solution is dialyzed with a 3KD dialysis bag for 48 hours, and the water is changed every 12 hours to remove by-products. After dialysis, the retentate is frozen with liquid nitrogen and then freeze-dried in a freeze dryer to obtain a copolymer of DMA and methacrylic acid (denoted as PMAA-DMA). The structural characterization results are shown in Figure 4 and Figure 5 .

[0055] Example 3

[0056] 36 mg of ε-polylysine (EPL) and 200 mg of tea polyphenols (GTP) were weighed and added to 20 mL of dimethyl sulfoxide (DMSO), stirred and ultrasonicated to fully dissolve each; 80 mg of ascorbic acid was added to 2 g of hydrogen peroxide and stirred until completely dissolved, and then the solution was added to the ε-polylysine solution and incubated for 2 hours, and finally the GTP solution was added to continue the reaction for 7 days. After the reaction was completed, the reaction solution was evaporated to remove DMSO, and after redispersing with ethanol, the solution was added to ethyl acetate for washing, the solution was centrifuged and the solid-liquid separation was performed, ethanol was added to the solid to disperse it evenly, and then it was added to ether for precipitation, centrifuged and the solid-liquid separation was performed, and an appropriate amount of water was added to the solid for freeze-drying to obtain a polymer grafted with GTP (referred to as EPL-GTP), and the structural characterization results are shown in Figure 6 and Figure 7 .

[0057] Example 4

[0058] 80 mg of ε-polylysine (EPL) and 58 mg of gallic acid (GA) were weighed and added to 15 mL of dimethyl sulfoxide (DMSO), stirred and ultrasonicated to fully dissolve each; 50 mg of ascorbic acid was added to 1 g of hydrogen peroxide and stirred until completely dissolved, and then the solution was added to the ε-polylysine solution and incubated for 2 hours, and finally the GA solution was added to continue the reaction for 24 hours. After the reaction, the reaction solution was evaporated to remove DMSO, and after redispersing with ethanol, the solution was added to ethyl acetate for washing, the solution was centrifuged and the solid-liquid separation was performed, ethanol was added to the solid to disperse it evenly, and then it was added to ether for sedimentation, centrifuged and the solid-liquid separation was performed, and an appropriate amount of water was added to the solid for freeze-drying to obtain a GTP-grafted polymer (referred to as EPL-GA), and the structural characterization results are shown in Figure 8 and Fig. 9 .

[0059] Fig.10 The SEM images show that the nanoparticles of PMAA-DMA and EPL-TA are around 100 nm, and EPL-TA forms a random polymer network after being oxidized and cross-linked by active oxygen.

[0060] Application Example 1

[0061] In vitro antitumor activity of polyphenol polymers prepared in Examples 1 to 4

[0062] HeLa multicellular tumor spheres were incubated with a medium solution of PMAA-DMA (500 μg / mL) for 24 hours, and then a 20 μM DCFH-DA fluorescent probe solution in D-PBS was added for another 30 minutes. Untreated HeLa multicellular tumor spheres were used as controls. Fluorescence images were taken and analyzed using a confocal microscope under 492 nm laser excitation. Fig.11The control group of HeLa multicellular tumor spheres without any treatment had strong green fluorescence, indicating that the content of reactive oxygen species was high (see Fig.11 a), while the green fluorescence of HeLa multicellular tumor spheres after co-incubation with PMAA-DMA was much weakened (see Fig.11 b), indicating that PMAA-DMA consumes a certain amount of reactive oxygen species. The fluorescence intensity analysis of the streaking line starting from the left side shows more clearly the weakening of the fluorescence intensity of HeLa multicellular tumor spheres after co-incubation with PMAA-DMA.

[0063] 0.5 mg / mL of EPL-TA was incubated with HeLa multicellular tumor spheres in agarose gel for 48 hours. The agarose gel was then soaked in a 2.5% glutaraldehyde aqueous solution for 1 hour, washed 5 times with deionized water (5 minutes each time), and then dehydrated with 20%, 40%, 60%, 80%, 85%, 90%, 95% and 100% ethanol aqueous solutions (incubated for 15 minutes at each concentration). Next, the sample was placed in a mixture of hexamethyldisilazane and ethanol (1:3 volume ratio) for 15 minutes, then immersed in pure hexamethyldisilazane for 15 minutes and dried in air overnight. The dried HeLa multicellular tumor spheres in agarose gel were photographed by SEM, with the HeLa multicellular tumor spheres without any treatment as a reference, see. Fig.12 As can be seen from the figure, the surface of the HeLa multicellular tumor spheres not co-incubated with EPL-TA is smooth and neat, while the surface of the HeLa multicellular tumor spheres co-incubated with EPL-TA is covered with a thick and dense polymer network.

[0064] The cultured HeLa multicellular tumor spheres were co-incubated with PMAA-DMA, and the glucose content in the cell culture medium was detected by a blood glucose meter at 24 hours, 48 ​​hours, and 72 hours, respectively. The untreated HeLa multicellular tumor spheres were used as a control. Fig.13 As can be seen from the figure, the glucose content in the culture medium of the control group decreased significantly faster than that of the PMAA-DMA group, thus proving that the absorption of glucose by the coated HeLa multicellular tumor spheres was reduced.

[0065] The cultured HeLa adherent cells were co-incubated with EPL-TA, and the contents of nutrients such as glucose and glutathione (GSH) in the cells were detected by the kit at different time points. Untreated HeLa adherent cells were used as the control. Figures 14-15 As can be seen from the figure, the glucose content in the control group culture medium decreased significantly faster than that in the EPL-TA group, and the GSH content decreased significantly slower than that in the EPL-TA group. This proves that the absorption of glucose by the coated HeLa multicellular tumor spheres is reduced.

[0066] The cultured HeLa adherent cells were washed once with PBS buffer, and then the culture medium solutions containing PMAA-DMA, EPL-TA, EPL-GTP and EPL-GA were added and incubated. Then the culture medium was discarded, and the cells were washed once with fresh DMEM culture medium to remove the residual materials. After washing, 100 μL Alamar Blue dye solution (prepared with fresh DMEM culture medium to 10%) was added to each well and incubated at 37°C, 5% CO 2 After 4 hours of incubation in the cell culture incubator, the fluorescence intensity of each group was measured with an enzyme-labeled instrument at 530 nm excitation and 590 nm emission, and the cell survival rate of each group was calculated. Figures 16 to 19 The experimental results show that EPL-TA, EPL-GTP, and EPL-GA have certain anti-tumor effects on HeLa adherent cells.

[0067] Multicellular tumor spheroids are very useful for studying tumor behavior and evaluating the response to pharmacologically active drugs. We used a commercial mold 3D Petri Dish to prepare HeLa multicellular tumor spheroids. In this method, cells settle into the small grooves of the micro-molded non-sticky agarose gel prepared in 3DPetri Dish and aggregate into multicellular spheroids. The prepared HeLa multicellular tumor spheroids were gently blown off the agarose gel with culture medium solutions containing PMAA-DMA, EPL-TA, EPL-GTP and EPL-GA, and then continued to be incubated at 37°C, 5% CO 2 After replacing the fresh culture medium, add 100 μL of 10% Alarm Bule solution to each well and continue incubation for 4 hours. Use an ELISA reader to measure the excitation at 530 nm and the emission at 590 nm of each well solution. Calculate the cell survival rate of each group. Figures 20-23 The experimental results show that PMAA-DMA has strong toxicity to HeLa multicellular tumor spheroids, producing a starvation therapy effect. At the same time, PMAA-DMA has a certain therapeutic effect on adherent multicellular spheroids, but the therapeutic effect on adherent cells is not obvious because the three-dimensional structure of the multicellular spheroids makes it easier for the polymer to cover the multicellular spheroids and achieve starvation therapy.

[0068] Fresh whole blood anticoagulated with sodium heparin was obtained from the ear vein of New Zealand rabbits. After standing at room temperature for 2 hours, it was centrifuged at 1000 rpm for 10 minutes to obtain a red blood cell-rich solution, and washed with sterile saline until the supernatant was colorless. Then, it was prepared into a 5% (v / v) red blood cell solution with saline for standby use; equal volumes of 500 μL PMAA-DMA saline solution and red blood cell solution were added to a 1.5 mL sterile EP tube and gently shaken to mix (the final concentration of PMAA-DMA was 0, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mg / mL), and saline was used as a negative control. 0.1% (v / v) Triton X-100) as a positive control, and treated in the same way; all EP tubes were placed in a 37°C shaker at 150 rpm for 1 hour; after incubation, the EP tubes were placed in a centrifuge at 1000 rpm for 10 minutes, and 80 μL of the upper clear liquid from each EP tube was added to a 96-well plate, and 80 μL of saline was added to each well until the volume of each well was 160 μL, and mixed evenly. Finally, the absorbance of each well solution at 545 nm was measured with an ELISA instrument, and the hemolysis rate of each group was calculated. The results are shown in the figure. Fig.24 As shown. As can be seen from the figure, after the red blood cells were incubated with the negative control saline and different concentrations of PMAA-DMA for 1 hour, there was no obvious hemolysis. However, after incubating with the positive control group 0.1% TritonX-100 for 1 hour, the solution in the EP tube showed a bright red color, and it was still a uniform red solution after centrifugation, showing obvious hemolysis. This is because 0.1% TritonX-100 ruptured the cell membrane of red blood cells and released a large amount of hemoglobin. The hemolysis rate of PMAA-DMA on red blood cells at concentrations of 0, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg / mL was all lower than 5%, which was much lower than the hemolysis rate of 0.1% TritonX-100 in the positive control group (about 100%). The above results show that PMAA-DMA has good blood compatibility and can circulate in vivo for a long time.

[0069] Tumor invasiveness was mainly analyzed by Transwell and Matrigel. Matrigel stored at -20℃ was placed in a 4℃ refrigerator overnight for dissolution. The pipette tip and centrifuge tube were stored at -20℃ before use to ensure that Matrigel would not solidify prematurely during the coating process. 8.1mg / mL Matrigel and serum-free DMEM medium were diluted 1:8, and 50μL was taken to coat the upper chamber surface of the bottom membrane of the Transwell chamber, air-dried at 4℃, and placed in a 37℃ incubator for 30 minutes to allow Matrigel to polymerize into gel. Before adding HeLa multicellular tumor spheres, the cell spheres were starved with serum-free DMEM medium for 24 hours to further remove the influence of serum. The Transwell coated with Matrigel was washed twice with PBS buffer, and EPL-TA was dissolved with 200μL DMEM medium to a concentration of 300mg / mL. After the HeLa multicellular tumor spheres were dispersed, they were added to the upper chamber, and DMEM medium with FBS was added to the lower chamber. Cells with invasive ability began to move through the membrane under serum induction, and the results were observed after 24 hours. Most of the cells that passed through the filter membrane adhered to the lower surface of the filter membrane. The cells on the upper surface were wiped off with a cotton swab, and then the filter membrane was fixed with a 2.5% glutaraldehyde aqueous solution for 20 minutes. After rinsing with sterile D-PBS twice, it was stained with a sterile D-PBS solution with a concentration of 0.1% crystal violet for 20 minutes, and then rinsed with sterile D-PBS three times. Gently wipe the upper membrane of Transwell with a cotton swab to wipe off the upper cells. The lower membrane of Transwell was then photographed with an intelligent fully automatic live cell fluorescence microscopy imaging system, and then the cells that passed through the membrane were counted using ImageJ software. The results are shown in Fig.25 After crystal violet staining, it can be seen that the number of tumor cell invasion in the control group without any treatment (25a) is significantly higher than that in the EPL-TA group ( Fig.25 b) more, the number of cells was analyzed by ImageJ software, the number of tumor cells in the control group (80) was 4 times that in the EPL-TA group (20) ( Fig.25 c), indicating that the cell invasion ability of HeLa multicellular tumor spheres decreased by 3 times after co-incubation with EPL-TA.

[0070] The above proves that polyphenol polymers can achieve anti-tumor effects by starving cancer cells, hindering the cell's absorption of nutrients, and inhibiting cell invasion.

[0071] Application Example 2

[0072] In vivo antitumor activity of polyphenol polymers prepared in Examples 1 to 4

[0073] HeLa tumor-bearing mice were randomly divided into three groups. 3At the same time, the mice were injected with: (1) 200 μL sterile D-PBS into the tail vein; (2) 200 μL PMAA-DMA (1 mg / mL) into the tail vein; (3) 200 μL EPL-TA (1 mg / mL) into the tail vein. After different treatments, the weight, tumor volume and mortality of the nude mice were counted every two days. Figures 26-28 The results showed that PMAA-DMA and EPL-TA had good anti-tumor ability.

[0074] HeLa tumor-bearing mice were randomly divided into three groups. 3 At the same time, (1) 200 μL sterile D-PBS was injected into the tail vein; (2) 200 μL PMAA-DMA (1 mg / mL) was injected into the tail vein; (3) 200 μL EPL-TA (1 mg / mL) was injected into the tail vein. The pathological analysis of the tumor and major organs was performed by hematoxylin and eosin (H&E) staining. Fig.29 The results showed that there were no obvious lesions or inflammation in the mouse organs, indicating that PMAA-DMA and EPL-TA had low biological toxicity in mice. As for tumor tissue, it can be observed that the tumor cells in the control group were not damaged at all; some tumor cells in the PMAA-DMA group were damaged, while most of the tumor cells in the EPL-TA group died, which was consistent with the statistical results of tumor growth.

[0075] The above proves that polyphenol polymers have good anti-tumor effects in animal models.

Claims

1. The use of polyphenol polymers in the preparation of anti-tumor drugs for starvation therapy, characterized in that: The polyphenol polymer is obtained by grafting polyphenol small molecules onto an amino-rich polymer; wherein the amino-rich polymer is ε-polylysine; the polyphenol small molecules are any one of gallic acid, tea polyphenols, and tannic acid; and the tumor is a cervical tumor.

2. The use of polyphenol polymers in the preparation of anti-tumor drugs for starvation therapy, characterized in that: The polyphenol polymer is a copolymer obtained by copolymerizing a polyphenol small molecule obtained by the reaction of an unsaturated acid anhydride and dopamine with a carboxyl monomer, and the number average molecular weight of the copolymer is 5000-25000; the unsaturated acid anhydride is any one of methacrylic anhydride and acrylic anhydride; the carboxyl monomer is any one of acrylic acid and methacrylic acid; and the tumor is a cervical tumor.

3. The use of the polyphenol polymer according to claim 1 or 2 in the preparation of anti-tumor drugs for starvation therapy, characterized in that: The drug comprises a therapeutically effective amount of a polyphenol polymer and a pharmaceutically acceptable carrier.

4. The use of the polyphenol polymer according to claim 3 in the preparation of anti-tumor drugs for starvation therapy, characterized in that: The drug is an injectable agent, and treatment is performed via tail vein injection or in situ injection.

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