Pro-drug for inhibiting tumor cell activity through double-enzyme response type intracellular polymerization and application thereof
By developing a dual-enzyme-responsive prodrug, it uses overexpressed enzymes in tumor cells to specifically activate them to generate fibrous polymers, solving the nonspecific and toxic problems of existing chemotherapy drugs in tumor treatment, and achieving efficient and specific tumor suppression effects.
Patent Information
- Application Number
- CN202510253803.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-05
- Publication Date
- 2025-06-06
AI Technical Summary
Existing chemotherapy drugs have problems such as nonspecific delivery, rapid clearance, insufficient tumor accumulation and toxicity in tumor treatment. Nanodies face limitations such as low drug loading efficiency, rapid clearance of liver and kidneys, or difficulty in tumor penetration, and the single enzyme targeting strategy is limited in selectivity.
A bienzyme-responsive prodrug, including 4-(4-nitropobenzo)resorcinol, trialdehyde-based resorcinol and scandium-line Lewis acid were developed to specifically activate azoreductase and nitroreductase overexpressed in tumor cells, and to generate fibrous polymers through in situ polymerization, inhibiting tumor cell activity.
It has achieved efficient and specific activation of prodrugs in tumor cells, and produced polymers that effectively inhibit tumor cell activity, improving the targeting and therapeutic effect of the drug, and reducing toxicity to normal tissues.
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Figure CN120093726A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a dual-enzyme responsive intracellular polymerization prodrug for inhibiting tumor cell activity and application thereof. Background Art
[0002] Tumor treatment is a major challenge in modern medicine. Chemotherapy, as the cornerstone of systemic treatment, relies on small molecule drugs to achieve rapid tumor penetration. However, these drugs have problems such as nonspecific delivery, rapid clearance, insufficient tumor accumulation and toxicity. Advances in nanoscience have promoted the development of nano drug delivery systems, which improve the delivery accuracy by coupling chemotherapeutic drugs to tumor-targeted nanocarriers. However, nano drugs still face limitations such as low drug loading efficiency (usually less than 20%), rapid clearance in the liver and kidneys (<200nm) or difficulty in tumor penetration (larger particles), which hinder their clinical application.
[0003] As an innovative method, in situ synthesis in cells has shown potential in prodrug synthesis, bioimaging and functional regulation. This method uses small molecules or biomolecules with high uptake efficiency and good biocompatibility as precursors to synthesize large-sized polymers or biomaterials in target cells to regulate cell functions. For example, light-driven free radical polymerization can induce apoptosis of tumor cells, but short-wave light activation and non-targetedness limit its application. In addition, although the in vivo self-assembly of small molecule drugs has anti-tumor activity, it lacks a clear targeting mechanism. Therefore, it is still challenging to develop an in situ synthesis strategy in cells without external stimulation and with tumor response characteristics.
[0004] Endogenous enzymes play a key role in many biochemical reactions, and abnormal expression of enzymes is closely related to diseases such as cancer. Among them, the widespread overexpression of azoreductase (AzoR) and nitroreductase (NTR) in solid tumors has become a research consensus. The expression of these two enzymes in tumors is usually associated with a hypoxic microenvironment, because rapid tumor growth can lead to local hypoxia, thereby inducing overexpression of reductases. Although studies have shown that the use of AzoR or NTR alone provides new ideas for targeted therapy of tumors, due to the characteristics of tumor heterogeneity, the selectivity of using a single AzoR or NTR targeting strategy is limited and prone to off-target effects, and its activation efficiency and specificity still need to be improved. In addition, how to achieve efficient in situ polymerization reactions in cells and have good solubility of drugs to further enhance the therapeutic effect of drugs is still a technical problem.
[0005] Therefore, the development of a prodrug with good solubility based on dual enzyme response, which can achieve efficient in situ polymerization reaction in tumor cells and form polymers with inhibitory tumor cell activity, has important scientific significance and application value. Summary of the invention
[0006] The purpose of the present invention is to provide a dual-enzyme responsive intracellular polymerization prodrug for inhibiting tumor cell activity and its application. The prodrug has good solubility, can be specifically activated in tumor cells, and generates a high molecular polymer through an in situ polymerization reaction, thereby inhibiting the activity of tumor cells.
[0007] In order to achieve the above object, the present invention provides a dual enzyme responsive intracellular polymerization prodrug for inhibiting tumor cell activity, wherein the prodrug comprises 4-(4-nitrophenylazo)resorcinol, trialdehyde phloroglucinol and a catalyst scandium Lewis acid.
[0008] As a preferred embodiment, the molar ratio of 4-(4-nitrobenzeneazo)resorcinol to trialdehyde phloroglucinol is 1:1-1:10, more preferably 1:1, 1:3, 1:4.5, 1:5.5, 1:8, 1:9, 1:10.
[0009] The molar concentration of 4-(4-nitrobenzeneazo)resorcinol is preferably 0.01mM-1mM, the molar concentration of trialdehyde phloroglucinol is preferably 0.01mM-1mM; and the molar concentration of the catalyst is preferably 0.01mM-1mM.
[0010] As a preferred embodiment, the scandium Lewis acid includes one or more of scandium trifluoromethanesulfonate, scandium nitrate, scandium sulfate, scandium p-toluenesulfonate, scandium trichloride, scandium tris(hexafluoroacetylacetonate), scandium trifluoroacetate, scandium tris(2-pyridone) and alkyl / aryloxy scandium. 3 +) as a typical rare earth metal ion, has strong Lewis acidity, can accept electron pairs and catalyze a variety of organic reactions including the Schiff base reaction between amino groups and aldehyde groups.
[0011] The present invention also discloses a high molecular polymer for inhibiting the activity of tumor cells. The polymer is prepared by the following method: a prodrug is co-incubated with tumor cells, wherein the tumor is a tumor that overexpresses endogenous azoreductase and nitroreductase, and the prodrug includes 4-(4-nitrobenzeneazo)resorcinol, trialdehyde phloroglucinol and a catalyst, a scandium-based Lewis acid. After the 4-(4-nitrobenzeneazo)resorcinol is activated by the azoreductase and nitroreductase, the activation product thereof and the trialdehyde phloroglucinol are polymerized and self-assembled in situ to form a fibrous polymer under the action of the catalyst, the scandium-based Lewis acid.
[0012] The present invention also discloses an in vitro preparation method of a high molecular polymer for inhibiting the activity of tumor cells. The preparation method comprises co-incubating a prodrug with tumor cells, wherein the tumor is a tumor overexpressing endogenous azoreductase and nitroreductase, and the prodrug comprises 4-(4-nitrobenzeneazo)resorcinol, trialdehyde phloroglucinol and a catalyst, a scandium-based Lewis acid. After the 4-(4-nitrobenzeneazo)resorcinol is activated by the azoreductase and nitroreductase, the activation product thereof and the trialdehyde phloroglucinol are polymerized and self-assembled in situ to form a fibrous polymer under the action of the catalyst, the scandium-based Lewis acid.
[0013] The present invention also discloses an in vitro method for inhibiting tumor cell activity by intracellular polymerization, wherein a prodrug is co-incubated with tumor cells, and a polymer is synthesized in situ in the tumor cells to induce tumor cell apoptosis. The tumor is a tumor that overexpresses endogenous azoreductase and nitroreductase. The prodrug includes 4-(4-nitrobenzeneazo)resorcinol, trialdehyde phloroglucinol and a catalyst, a scandium-based Lewis acid. After the 4-(4-nitrobenzeneazo)resorcinol is activated by the azoreductase and nitroreductase, its activation product polymerizes with the trialdehyde phloroglucinol under the action of the catalyst, the scandium-based Lewis acid, and self-assembles in situ to form a fibrous polymer.
[0014] The invention also discloses the use of the prodrug in synthesizing anti-tumor drugs, wherein the tumor is a tumor that over-expresses endogenous azoreductase and nitroreductase.
[0015] Azoreductase and nitroreductase are widely overexpressed in solid tumors, including but not limited to lung adenocarcinoma, breast cancer, colon cancer, prostate cancer, gastric adenocarcinoma, pancreatic cancer, liver cancer, cervical cancer, ovarian cancer and kidney cancer.
[0016] Incubation refers to culturing the prodrug and tumor cells together under specific conditions to simulate the drug's action process in vivo. Conventional methods can be used for incubation in the present invention. The incubation time is preferably 0.5-96 hours; the incubation temperature is preferably 30-37°C.
[0017] The prodrug of the present invention comprises the following components:
[0018] 4-(4-Nitrophenylazo)resorcinol: Precursor 1 molecule, as an azo-nitro compound, can be reduced to amine compounds by azoreductase and nitroreductase in tumor cells.
[0019] Trialdehyde phloroglucinol: 2 precursor molecules, as a polyaldehyde compound, can undergo condensation reaction with the reduced amine compound.
[0020] Scandium series (Sc 3+) Lewis acid: acts as a catalyst to promote the condensation reaction of aldehydes and amines to form high molecular polymers.
[0021] The molecular structures of the prodrugs 4-(4-nitrophenylazo)resorcinol and trialdehyde phloroglucinol contain multiple phenolic groups that contribute to dissolution, so that the selected prodrug molecules have better solubility during the implementation process than other azo-nitro compounds and polyaldehyde compounds.
[0022] After the azo compound 4-(4-nitrophenylazo)resorcinol is activated by azoreductase and nitroreductase, its activation product reacts with trialdehyde phloroglucinol in the presence of a scandium catalyst (Sc 3+ ) Under the action of Lewis acid, polymerization and in situ self-assembly form fibrous polymers inside tumor cells. The strategy triggered by two tumor-related enzymes (AzoR, NTR) can provide a more accurate and safe tumor targeted treatment method.
[0023] The advantages of the present invention are that the present invention utilizes azoreductase and nitroreductase overexpressed in tumor cells to achieve specific activation of prodrugs, reduce toxicity to normal tissues, and significantly improve the targeting of drugs. Through the dual enzyme response mechanism and the catalytic action of scandium Lewis acid, efficient in situ polymerization is achieved, and the generated fibrous polymer can effectively inhibit the activity of tumor cells and enhance the therapeutic effect. The prodrug of the present invention has good solubility, is suitable for a variety of tumors that overexpress azoreductase and nitroreductase, and has broad application prospects. Since the prodrug is only activated and exerts its effect in tumor cells, it has little effect on normal tissues, thereby significantly reducing the side effects of traditional chemotherapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 This is a bright field micrograph of the polymer synthesized in situ within the cell in Example 1.
[0025] Figure 2 This is the cell activity result after the polymer is synthesized in situ in the cell in Example 1.
[0026] Figure 3 This is the cell activity result after the polymer is synthesized in situ in the cell in Example 2.
[0027] Figure 4 This is the cell activity result after the polymer is synthesized in situ in the cell in Example 3.
[0028] Figure 5 This is the cell activity result after the polymer is synthesized in situ in the cell in Example 4.
[0029] Figure 6 This is a confocal fluorescence image of live and dead cells after the in situ synthesis of polymers in the tumor cell spheres in Example 5.
[0030] Figure 7 This is a TEM image of the polymer synthesized in situ within the cell in Example 6.
[0031] Figure 8 This is a TEM image of a section of a cell after in situ synthesis of the polymer in the cell in Example 7.
[0032] Fig. 9 This is a graph showing the growth curve of mouse tumors in Example 8.
[0033] Fig.10 This is the anatomical diagram of the mouse tumor in Example 8. DETAILED DESCRIPTION
[0034] The technology of the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following specific embodiments are only used to help those skilled in the art understand the present invention, and are not intended to limit the present invention.
[0035] Example 1. In situ synthesis of polymers in mouse breast cancer 4T1 cells
[0036] Precursor 1 molecule: 4-(4-nitrophenylazo)resorcinol (CAS number 74-39-5);
[0037] Precursor 2 molecule: trialdehyde phloroglucinol (CAS number 34374-88-4);
[0038] Catalyst: Scandium trifluoromethanesulfonate Sc(OTf) 3 (CAS number is 144026-79-9).
[0039] The specific steps are as follows:
[0040] The density is 10 6 A suspension of mouse breast cancer 4T1 cell line with a concentration of 100 units / mL was placed in a 1640 culture flask or culture well plate containing 10% (v / v) fetal bovine serum, penicillin (100 units / mL), streptomycin (100 μg / mL), and cultured at 37°C, 5% carbon dioxide, and 95% humidity in an incubator. After the cells adhered to the wall and grew to a density of 50%-80%, they were washed three times with PBS (0.01M, pH 7.4). Precursor 1 and Precursor 2 molecules were pre-dissolved in acetonitrile to prepare a 24mM stock solution, and Sc(OTf) 3 The 24 mM solution was dissolved in PBS (0.01 M, pH 7.4). Before incubation with cells, the pre-prepared solution was filtered through a 0.22 μm filter membrane. Subsequently, the cells were placed in a medium containing precursor molecule 1 (480 μM), precursor molecule 2 (480 μM) and Sc(OTf) 3(480 μM) in 1640 medium, and cultured in an incubator at 37°C, 5% carbon dioxide, and 95% humidity for 6 hours. Subsequently, the cells were washed three times with PBS (0.01 M, pH 7.4), and finally the cells were cultured in 1640 medium supplemented with 10% (V / V) fetal bovine serum, penicillin (100 units / mL), and streptomycin (100 μg / mL) for subsequent microscopic observation and cell activity analysis.
[0041] Figure 1 The cell and the precursor molecule 1, the precursor molecule 2, Sc(OTf) 3 Bright field microscopic imaging after co-incubation. It can be seen from this figure that compared with the control group, a large amount of yellow polymers are generated in the cells after co-incubation. Figure 2 The cell and the precursor molecule 1, the precursor molecule 2, Sc(OTf) 3 The cell activity results after co-incubation were determined using a CCK-8 kit, which showed that the polymer generated in situ within the cells could effectively inhibit the activity of the cells.
[0042] Example 2. In situ synthesis of polymers in human breast cancer MCF-7 cells
[0043] Precursor 1 molecule: 4-(4-nitrophenylazo)resorcinol (CAS number 74-39-5);
[0044] Precursor 2 molecule: trialdehyde phloroglucinol (CAS number 34374-88-4);
[0045] Catalyst: Scandium trifluoromethanesulfonate Sc(OTf) 3 (CAS number is 144026-79-9).
[0046] The density is 10 6 The suspension of human breast cancer MCF-7 cell line with 100 units / mL was placed in a DMEM culture bottle or culture plate containing 10% (v / v) fetal bovine serum, penicillin (100 units / mL), streptomycin (100 μg / mL), and cultured at 37°C, 5% carbon dioxide, and 95% humidity in an incubator. After the cells adhered to the wall and grew to a density of 50%-80%, they were washed three times with PBS (0.01M, pH 7.4). Precursor 1 and Precursor 2 molecules were pre-dissolved in acetonitrile to prepare a 24mM stock solution, and Sc(OTf) 3The solution was dissolved in PBS (0.01 M, pH 7.4) to form a 24 mM solution. Before incubation with cells, the pre-prepared solution was filtered through a 0.22 μm filter membrane. Subsequently, the cells were placed in the presence of precursor molecule 1 (0, 60, 120, 240, 480, 960 μM), precursor molecule 2 (0, 60, 120, 240, 480, 960 μM) and Sc(OTf) 3 The cells were cultured in 1640 medium with 5% (0, 60, 120, 240, 480, 960 μM) at 37°C, 5% carbon dioxide, and 95% humidity in an incubator for 6 hours. The cells were then washed three times with PBS (0.01 M, pH 7.4), and finally the cells were cultured in DMEM medium supplemented with 10% (v / v) fetal bovine serum, penicillin (100 units / mL), and streptomycin (100 μg / mL) for subsequent cell activity analysis.
[0047] Figure 3 The cells are exposed to different concentrations of precursor molecule 1, precursor molecule 2, Sc(OTf) 3 The cell activity results after co-incubation were determined using a CCK-8 kit, which showed that the polymer generated in situ within the cells could effectively inhibit the activity of the cells.
[0048] Example 3. In situ synthesis of polymers in human liver cancer HepG2 cells
[0049] Precursor 1 molecule: 4-(4-nitrophenylazo)resorcinol (CAS number 74-39-5);
[0050] Precursor 2 molecule: trialdehyde phloroglucinol (CAS number 34374-88-4);
[0051] Catalyst: Scandium trifluoromethanesulfonate Sc(OTf) 3 (CAS number is 144026-79-9).
[0052] The specific steps are as follows:
[0053] The density is 10 6 The suspension of HepG2 human liver cancer cell line with 100 units / mL was placed in a DMEM culture bottle or culture plate containing 10% (v / v) fetal bovine serum, penicillin (100 units / mL), streptomycin (100 μg / mL), and cultured at 37°C, 5% carbon dioxide, and 95% humidity in an incubator. After the cells adhered to the wall and grew to a density of 50%-80%, they were washed three times with PBS (0.01M, pH 7.4). Precursor 1 and Precursor 2 molecules were pre-dissolved in acetonitrile to prepare a 24mM stock solution, and Sc(OTf) 3The solution was dissolved in PBS (0.01 M, pH 7.4) to form a 24 mM solution. Before incubation with cells, the pre-prepared solution was filtered through a 0.22 μm filter membrane. Subsequently, the cells were placed in the presence of precursor molecule 1 (0, 60, 120, 240, 480, 960 μM), precursor molecule 2 (0, 60, 120, 240, 480, 960 μM) and Sc(OTf) 3 (0, 60, 120, 240, 480, 960 μM) in DMEM medium, and cultured in an incubator at 37°C, 5% carbon dioxide, and 95% humidity for 6 hours. Subsequently, the cells were washed three times with PBS (0.01 M, pH 7.4), and finally the cells were cultured in DMEM medium supplemented with 10% (V / V) fetal bovine serum, penicillin (100 units / mL), and streptomycin (100 μg / mL) for subsequent cell activity analysis.
[0054] Figure 4 The cells are exposed to different concentrations of precursor molecule 1, precursor molecule 2, Sc(OTf) 3 The cell activity results after co-incubation were measured using a CCK-8 kit, which showed that the polymer generated in situ within the cells could effectively inhibit the activity of the cells.
[0055] Example 4. In situ synthesis of polymers in human normal breast cells MCF-10A
[0056] Precursor 1 molecule: 4-(4-nitrophenylazo)resorcinol (CAS number 74-39-5);
[0057] Precursor 2 molecule: trialdehyde phloroglucinol (CAS number 34374-88-4);
[0058] Catalyst: Scandium trifluoromethanesulfonate Sc(OTf) 3 (CAS number is 144026-79-9).
[0059] The specific steps are as follows:
[0060] The density is 10 6 The suspension of normal human breast cell MCF-10A strain at 100 units / mL was placed in a DMEM culture bottle or culture plate containing 10% (v / v) fetal bovine serum, penicillin (100 units / mL), streptomycin (100 μg / mL), and cultured at 37°C, 5% carbon dioxide, and 95% humidity in an incubator. After the cells adhered to the wall and grew to a density of 50%-80%, they were washed three times with PBS (0.01M, pH 7.4). Precursor 1 and Precursor 2 molecules were pre-dissolved in acetonitrile to prepare a 24mM stock solution, and Sc(OTf) 3The solution was dissolved in PBS (0.01 M, pH 7.4) to form a 24 mM solution. Before incubation with cells, the pre-prepared solution was filtered through a 0.22 μm filter membrane. Subsequently, the cells were placed in the presence of precursor molecule 1 (0, 60, 120, 240, 480, 960 μM), precursor molecule 2 (0, 60, 120, 240, 480, 960 μM) and Sc(OTf) 3 (0, 60, 120, 240, 480, 960 μM) in DMEM medium, and cultured in an incubator at 37°C, 5% carbon dioxide, and 95% humidity for 6 hours. Subsequently, the cells were washed three times with PBS (0.01 M, pH 7.4), and finally the cells were cultured in DMEM medium supplemented with 10% (V / V) fetal bovine serum, penicillin (100 units / mL), and streptomycin (100 μg / mL) for subsequent cell activity analysis.
[0061] Figure 5 The cell and the precursor molecule 1, the precursor molecule 2, Sc(OTf) 3 The cell activity results after co-incubation were determined using the CCK-8 kit, indicating that this method was non-toxic to normal cells and the precursor molecules used had good biocompatibility.
[0062] Example 5. In situ synthesis of polymers in mouse breast cancer 4T1 cells kills tumor cell spheres
[0063] Precursor 1 molecule: 4-(4-nitrophenylazo)resorcinol (CAS number 74-39-5);
[0064] Precursor 2 molecule: trialdehyde phloroglucinol (CAS number 34374-88-4);
[0065] Catalyst: Scandium trifluoromethanesulfonate Sc(OTf) 3 (CAS number is 144026-79-9).
[0066] The specific steps are as follows:
[0067] The mouse breast cancer 4T1 cell line suspension was inoculated in a 96-well round-bottom culture plate at a density of 200 cells per well and cultured in an incubator containing 10% (v / v) fetal bovine serum, penicillin (100 units / mL), streptomycin (100 μg / mL), and 1640 at 37°C, 5% carbon dioxide, and 95% humidity. After 3 days of culture, tumor cell spheres with a diameter of approximately 100-200 μm were selected for the experiment. Precursor 1 and Precursor 2 molecules were pre-dissolved in acetonitrile to prepare a 24 mM stock solution, while Sc(OTf)3 was dissolved in PBS (0.01 M, pH 7.4) to form a 24 mM solution. Before incubation with the tumor cell spheres, the pre-configured solution was filtered through a filter membrane with a diameter of 0.22 μm. Subsequently, the tumor cell spheres were placed in DMEM medium containing precursor molecule 1 (480 μM), precursor molecule 2 (480 μM) and Sc(OTf)3 (480 μM) and cultured in an incubator at 37°C, 5% carbon dioxide, and 95% humidity for 46 hours. Subsequently, the cells were washed three times with PBS (0.01 M, pH 7.4), and finally the cells were cultured in 1640 medium supplemented with 10% (v / v) fetal bovine serum, penicillin (100 units / mL), and streptomycin (100 μg / mL) to prepare for the subsequent characterization of cell live and dead conditions using confocal fluorescence microscopy.
[0068] Figure 6 The tumor cell sphere and the precursor molecule 1, the precursor molecule 2, Sc(OTf) 3 Confocal fluorescence images of live and dead cells after co-incubation, using AM-PI staining kit to determine cell live and dead (green represents live cells, red represents dead cells), indicating that the polymers generated in situ within the cells can effectively kill tumor cell spheres.
[0069] Example 6. TEM images of polymers
[0070] Precursor 1 molecule: 4-(4-nitrophenylazo)resorcinol (CAS number 74-39-5);
[0071] Precursor 2 molecule: trialdehyde phloroglucinol (CAS number 34374-88-4);
[0072] Catalyst: Scandium trifluoromethanesulfonate Sc(OTf) 3 (CAS number is 144026-79-9).
[0073] The density is 10 6A suspension of mouse breast cancer 4T1 cell line with a concentration of 100 units / mL was placed in a 1640 culture flask or culture well plate containing 10% (v / v) fetal bovine serum, penicillin (100 units / mL), streptomycin (100 μg / mL), and cultured in an incubator at 37°C, 5% carbon dioxide, and 95% humidity. After the cells adhered to the wall and grew to a density of 50%-80%, they were washed three times with PBS (0.01M, pH 7.4). Precursor 1 and Precursor 2 molecules were pre-dissolved in acetonitrile to prepare a 24mM stock solution, and Sc(OTf) 3 The 24 mM solution was dissolved in PBS (0.01 M, pH 7.4). Before incubation with cells, the pre-prepared solution was filtered through a 0.22 μm filter membrane. Subsequently, the cells were placed in a medium containing precursor molecule 1 (480 μM), precursor molecule 2 (480 μM) and Sc(OTf) 3 The cells were cultured in 1640 medium (480 μM) at 37°C, 5% carbon dioxide, and 95% humidity in an incubator for 6 hours. The cells were then washed three times with PBS (0.01 M, pH 7.4), collected by centrifugation (1000 rpm, 5 min), added with 0.01% SDS cell lysis buffer for 5 minutes, and centrifuged (5000 rpm, 5 min) to collect the precipitate for TEM analysis.
[0074] Figure 7 is a TEM image of the polymer, indicating that the polymer generated in situ within the cell presents a fiber network structure.
[0075] Example 7. TEM image of a cell section after polymer formation
[0076] Precursor 1 molecule: 4-(4-nitrophenylazo)resorcinol (CAS number 74-39-5);
[0077] Precursor 2 molecule: trialdehyde phloroglucinol (CAS number 34374-88-4);
[0078] Catalyst: Scandium trifluoromethanesulfonate Sc(OTf) 3 (CAS number is 144026-79-9).
[0079] The density is 10 6A suspension of mouse breast cancer 4T1 cell line with a concentration of 100 units / mL was placed in a 1640 culture flask or culture well plate containing 10% (v / v) fetal bovine serum, penicillin (100 units / mL), streptomycin (100 μg / mL), and cultured in an incubator at 37°C, 5% carbon dioxide, and 95% humidity. After the cells adhered to the wall and grew to a density of 50%-80%, they were washed three times with PBS (0.01M, pH 7.4). Precursor 1 and Precursor 2 molecules were pre-dissolved in acetonitrile to prepare a 24mM stock solution, and Sc(OTf) 3 The 24 mM solution was dissolved in PBS (0.01 M, pH 7.4). Before incubation with cells, the pre-prepared solution was filtered through a 0.22 μm filter membrane. Subsequently, the cells were placed in a medium containing precursor molecule 1 (480 μM), precursor molecule 2 (480 μM) and Sc(OTf) 3 The cells were cultured in 1640 medium (480 μM) at 37°C, 5% carbon dioxide, and 95% humidity in an incubator for 6 hours. The cells were then washed three times with PBS (0.01 M, pH 7.4), collected by centrifugation (1000 rpm, 5 min), and fixed, dehydrated, resin-embedded, sectioned, and stained for biological electron microscopy.
[0080] Figure 8 is a TEM image of a section of the cell after the polymer is formed, indicating that the fibrous polymer is successfully formed in the cell.
[0081] Example 8. Mouse Experiment
[0082] Precursor 1 molecule: 4-(4-nitrophenylazo)resorcinol (CAS number 74-39-5);
[0083] Precursor 2 molecule: trialdehyde phloroglucinol (CAS number 34374-88-4);
[0084] Catalyst: Scandium trifluoromethanesulfonate Sc(OTf) 3 (CAS number is 144026-79-9).
[0085] First, 20 g female BALB / c mice, aged 4 to 6 weeks, were injected subcutaneously with 1 × 10 6 4T1 cells were used to form tumors in mice. When the tumor volume reached about 80-100 mm 3 The mice were randomly divided into 8 groups, with 4 mice in each group: Group G1 (normal saline); Group G2 (precursor molecule 1); Group G3 (precursor molecule 2); Group G4 (Sc(OTf) 3 ); Group G5 (precursor molecule 1+Sc(OTf) 3); Group G6 (precursor molecule 2 + Sc(OTf)3); Group G7 (precursor molecule 1 + precursor molecule 2); Group G8 (precursor molecule 1 + precursor molecule 2 + Sc(OTf) 3 ). Then, precursor molecule 1, precursor molecule 2, Sc(OTf) 3 The drug was administered by tail vein injection, with an injection every 1 day for 12 days. The dosage was: precursor molecule 1 (24.8 mg / kg), precursor molecule 2 (20.2 mg / kg), Sc(OTf) 3 (14.4mg / kg). During the treatment, the body weight of mice in each group was monitored, and the tumor size was measured using a digital caliper to calculate the tumor volume. After the treatment cycle, the mice were killed under anesthesia, and the tumors were removed and photographed.
[0086] Fig. 9 This is a graph of the tumor growth curves of mice in groups G1-G8, in which the tumor volumes of groups G1-G7 continued to increase, while the tumors of group G8 were significantly suppressed. Fig.10 The tumor anatomy of mice in groups G1-G8 after the treatment cycle was completed. The tumor size of group G8 was significantly smaller than that of other groups, indicating that only in the presence of precursor molecule 1+precursor molecule 2+Sc(OTf) 3 Only when the three work together can they have the ability to treat tumors.
[0087] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A dual enzyme-responsive intracellular polymerization prodrug that inhibits tumor cell activity, characterized in that: The prodrug comprises 4-(4-nitrobenzeneazo)resorcinol, trialdehyde phloroglucinol and a catalyst, a scandium-based Lewis acid.
2. A dual enzyme-responsive intracellular polymerization prodrug for inhibiting tumor cell activity according to claim 1, characterized in that: The molar ratio of the 4-(4-nitrobenzeneazo)resorcinol to the trialdehyde phloroglucinol is 1:1-1:
10.
3. A dual enzyme-responsive intracellular polymerization prodrug for inhibiting tumor cell activity according to claim 1, characterized in that: The scandium-based Lewis acid includes one or more of scandium trifluoromethanesulfonate, scandium nitrate, scandium trichloride, scandium tris(hexafluoroacetylacetonate), scandium trifluoroacetate and scandium tris(2-pyridone).
4. A high molecular weight polymer for inhibiting tumor cell activity, characterized in that: The polymer is prepared by the following method: a prodrug is co-incubated with tumor cells, wherein the tumor is a tumor that overexpresses endogenous azoreductase and nitroreductase, the prodrug includes 4-(4-nitrophenylazo)resorcinol, trialdehyde phloroglucinol and a catalyst, a scandium-based Lewis acid; after the 4-(4-nitrophenylazo)resorcinol is activated by the azoreductase and nitroreductase, the activation product thereof and the trialdehyde phloroglucinol are polymerized and self-assembled in situ to form a fibrous polymer under the action of the catalyst, the scandium-based Lewis acid.
5. An in vitro method for preparing a high molecular weight polymer that inhibits tumor cell activity, characterized in that: The preparation method comprises the following steps: co-incubating a prodrug with tumor cells, wherein the tumor is a tumor that overexpresses endogenous azoreductase and nitroreductase, and the prodrug comprises 4-(4-nitrobenzeneazo)resorcinol, trialdehyde phloroglucinol and a catalyst, a scandium-based Lewis acid. After the 4-(4-nitrobenzeneazo)resorcinol is activated by the azoreductase and nitroreductase, the activated product thereof and the trialdehyde phloroglucinol are polymerized and self-assembled in situ to form a fibrous polymer under the action of the catalyst, the scandium-based Lewis acid.
6. An in vitro method for inhibiting tumor cell activity by intracellular polymerization, characterized in that: The prodrug is co-incubated with tumor cells, and a polymer is synthesized in situ in the tumor cells to induce tumor cell apoptosis. The tumor is a tumor that overexpresses endogenous azoreductase and nitroreductase. The prodrug includes 4-(4-nitrobenzeneazo)resorcinol, trialdehyde phloroglucinol and a catalyst, a scandium-based Lewis acid. After the 4-(4-nitrobenzeneazo)resorcinol is activated by the azoreductase and nitroreductase, its activation product polymerizes with the trialdehyde phloroglucinol under the action of the catalyst, the scandium-based Lewis acid, and self-assembles in situ to form a fibrous polymer.
7. Use of the prodrug according to any one of claims 1 to 3 in the synthesis of anti-tumor drugs, characterized in that: The tumor is a tumor that overexpresses endogenous azoreductase and nitroreductase.
8. The use according to claim 7, characterized in that: The tumors include lung adenocarcinoma, breast cancer, colon cancer, prostate cancer, gastric adenocarcinoma, pancreatic cancer, liver cancer, cervical cancer, ovarian cancer, and kidney cancer.