Multi-module probe capable of being rapidly delivered to cell nucleus, preparation method and application thereof

Abstract

The present invention provides a multi-module probe capable of being rapidly delivered to the cell nucleus, its preparation method and application, belonging to the technical field of biological probes. The multi-module probe is mainly synthesized from an aggregation-induced emission photosensitizer and a polypeptide LK-M-N with the following structural formula: The multi-module probe simultaneously has strong cell membrane affinity and the ability to rapidly internalize and target the cell nucleus, and also has high reactive oxygen species generation ability, good biosafety and high tumor cell killing efficiency; it can be used as a fluorescence probe targeting the cell nucleus.

Description

Technical Field

[0001] The present invention relates to the technical field of the development and application of biological probes, specifically to the design and synthesis of probes with multiple functional modules, and particularly to a multi-module probe capable of being rapidly delivered to the cell nucleus, its preparation method and application. Background Art

[0002] Since tumor diseases are seriously threatening human health, the development and improvement of highly efficient tumor treatment probes have a long way to go. The cell nucleus is the largest and most important cell structure in eukaryotic cells and is the regulatory center of cell heredity and metabolism. Nucleus disruption has been widely regarded as a method to improve the efficiency of anti-cancer treatment because DNA or RNA damage in the cell nucleus induced by therapeutic agents is considered to be the most direct and serious damage to cells. Nowadays, the delivery of the cell nucleus is mainly mediated by nuclear import proteins in cells. However, as the first barrier of cells, the cell membrane protects living cells from the adjacent environment by restricting the entry and exit of foreign substances. Therefore, the binding efficiency of nuclear import proteins is often limited by the cell uptake efficiency, that is, the cell membrane transport efficiency. Therefore, the existence of the cell membrane makes the wide application of cell nucleus-targeted therapy difficult.

[0003] For most biological probes applied to diagnosis, imaging and treatment, cell membrane transport is a crucial process because they need to interact with intracellular targets. In fact, for most biological probes, cell membrane transport is a two-step process, that is, the probe initially binds to the cell membrane and then is internalized by the cell through various energy-dependent pathways. At present, researchers have proposed a variety of strategies to enhance the affinity of the probe for the cell membrane, thereby promoting the cell membrane binding step. For example, adjusting physical and chemical properties such as size, shape, charge, lipophilicity, or using ligand-protein binding and bioorthogonal reactions to promote the binding of the cell membrane, thereby enhancing cell membrane transport. However, blindly improving the cell membrane binding ability by enhancing the membrane affinity does not guarantee high membrane transport efficiency.

[0004] This is because if the membrane binding ability of the biological probe is too strong and lacks permeability, the biological probe will be anchored on the membrane for a long time and no subsequent internalization process will occur. For example, “Acell membrane-anchored nanoassembly with self-reporting property for enhanced second near-infrared photothermaltherapy” (Nano Today, 2021, 41, 101312) compared biological probes modified with cell membrane-binding peptides containing different numbers of Arg, and found that when the number of Arg was 9, the biological probe would be anchored on the cell membrane and stay for 8 hours. For biological probes whose targets are located inside the cell, failure to be effectively internalized by the cell means that they cannot function. Therefore, the problem that the cell membrane binding rate and the cell internalization rate cannot be taken into account at the same time will seriously affect the application of biological probes. It is urgent to develop a biological probe that has both high cell membrane affinity (that is, it can quickly anchor to the cell membrane) and can be quickly internalized. Summary of the Invention

[0005] To address the above-mentioned issues, the first objective of the present invention is to provide a multi-module probe that can be rapidly delivered to the cell nucleus. This multi-module probe exhibits different affinities during the two-step cell membrane transport process. During cell membrane binding, the probe has a stronger affinity for the cell membrane, while during internalization, the probe has a weaker affinity for the cell membrane. This tunable membrane binding ability maximizes the probe's targeting efficiency to the cell nucleus.

[0006] Specifically, the multi-module probe is mainly obtained by reacting an aggregation-induced emission photosensitizer with a polypeptide LK-MN represented by the following structural formula:

[0007]

[0008] The molecular structure of the multi-module probe is as follows Figure 14 As shown, its molecular structure includes a cell membrane anchoring module, an affinity regulation module, and a cell nucleus targeting module. Therefore, this multi-module probe has both high cell membrane affinity and rapid internalization during cellular transport. The combination of these two can significantly improve cell membrane transport efficiency. This multi-module probe also has efficient reactive oxygen species generation capacity, good biosafety, and high tumor cell toxicity, and can be used for targeted treatment of tumor diseases under white light irradiation. Figure 14 In the figure, AIEPS represents a molecular group of an aggregation-induced emission photosensitizer, wherein R is a group of a molecular structure of TPA (triphenylamine), TPE (tetraphenylethylene), or QM (quinolinemalononitrile).

[0009] In an optional embodiment, the aggregation-induced emission photosensitizer is PyTPA-N3, and the structural formula of the multi-module probe is shown in Formula (III):

[0010]

[0011] The second object of the present invention is to provide a preparation method of the multi-module probe, comprising the following steps:

[0012] Add the aggregation-induced emission photosensitizer and the polypeptide LK-M-N into a mixed solvent of an organic solvent and water, then add sodium ascorbate and copper bromide, and obtain the multi-module probe after reaction, separation of the product, and purification of the product. The organic solvent is an organic solvent miscible with water, for example, DMSO, acetonitrile, ethanol, methanol, etc. The organic solvent is preferably DMSO, and more preferably the volume ratio of DMSO to water is 1:1.

[0013] The main equations involved in the reaction process are as follows:

[0014]

[0015] In an optional embodiment, the step of separating the product is to obtain the product by freeze-drying the solvent.

[0016] In an optional embodiment, the purification of the product means that the product is purified by at least one of column chromatography, reverse liquid chromatography separation, or liquid-liquid extraction separation; preferably reverse liquid chromatography separation. In the reverse liquid chromatography separation, gradient or isocratic elution is carried out with acetonitrile and water or methanol and water; preferably gradient elution is carried out with acetonitrile and water according to the volume ratio of acetonitrile being 20% to 100%.

[0017] In an optional embodiment, the molar ratio of the aggregation-induced emission photosensitizer to the polypeptide LK-M-N is 5:1.

[0018] In an optional embodiment, the preparation method of the aggregation-induced emission photosensitizer PyTPA-N3 comprises the following steps:

[0019] React TPA-Br and sodium azide in a polar organic solvent, and obtain the aggregation-induced emission photosensitizer PyTPA-N3 after extraction, rotary evaporation, and purification after the reaction ends.

[0020] The main reaction equations involved in the reaction process are as follows:

[0021]

[0022] Preferably, the polar organic solvent includes at least one of acetonitrile, methanol, and ethanol.

[0023] Preferably, the molar ratio of the TPA-Br to the sodium azide is 0.09:1.

[0024] Preferably, the purification refers to purification by column chromatography column separation method. More preferably, the elution solvent for purification by column chromatography column separation method is a mixed solvent prepared by mixing dichloromethane and methanol in a volume ratio of (40-60):1; most preferably 50:1.

[0025] The third object of the present invention is to provide the application of the multi-module probe as a fluorescent probe targeting the cell nucleus, and the application is for non-disease diagnosis or treatment.

[0026] Preferably, the multi-module probe is used to prepare products for treating or / and diagnosing tumors; when using the product containing the multi-module probe, light stimulation is required.

[0027] Further preferably, the light stimulation uses 200mW / cm 2 white light irradiation.

[0028] Further preferably, the tumor includes breast cancer.

[0029] Preferably, the multi-module probe is used for fluorescence imaging of the cell nucleus.

[0030] The method for fluorescence imaging of the cell nucleus using the multi-module probe includes the following steps:

[0031] Co-culture the multi-module probe with cells, then co-stain with Hoechst33258, extract the cell nucleus with a cell nucleus extraction kit, and image the obtained cell nucleus with a confocal fluorescence microscope.

[0032] Further preferably, the fluorescence imaging mode is single-photon imaging or two-photon imaging.

[0033] Even more preferably, the fluorescence imaging mode is single-photon imaging.

[0034] Further preferably, the concentration of the multi-module probe in the co-culture system is 5 μmol / L.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In the present invention, a multi-module probe LK-M-NA that exhibits different affinities in the two-step process of cell membrane transport is synthesized. This multi-module probe simultaneously has a strong cell membrane affinity and the ability to rapidly internalize and target the cell nucleus. Specifically, when the multi-module probe approaches the cell, the α-helical polypeptide LK increases the affinity for the cell membrane to obtain a higher membrane binding energy, thereby accelerating the binding process with the cell membrane; during the cell internalization process, the multi-module probe is cleaved, resulting in a decrease in lipophilicity, which accelerates the endocytosis mediated by nuclear import proteins; the combination of the two improves the efficiency of cell membrane transport. (2) The multi-module probe synthesized in the present invention has a fluorescence emission in the near-infrared region, and under white light irradiation, reactive oxygen species with cytotoxicity can be generated within 3 minutes. (3) This multi-module probe has good biosafety and tumor cell targeting, and can be directly used for the treatment or / and diagnosis of tumor diseases under white light irradiation; or used for preparing products for the treatment or / and diagnosis of tumors, improving the targeting efficiency and treatment efficiency of the products for tumors; it can also be used for fluorescence imaging of cell nuclei. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Mass spectrometry characterization diagram of PyTPA-N3 prepared in Example 1;

[0037] Figure 2 Mass spectrometry characterization diagram of the multi-module probe M-NA prepared in Example 1;

[0038] Figure 3 Mass spectrometry characterization diagram of the multi-module probe LK-NA prepared in Example 1;

[0039] Figure 4 Mass spectrometry characterization diagram of the multi-module probe LK-M-NA prepared in Example 1;

[0040] Figure 5 UV-visible absorption spectra of PyTPA-N3, M-NA, LK-NA, and LK-M-NA prepared in Example 1;

[0041] Figure 6 Fluorescence spectra of PyTPA-N3, M-NA, LK-NA, and LK-M-NA prepared in Example 1;

[0042] Figure 7 UV absorption spectra of the multi-module probes M-NA, LK-NA, and LK-M-NA measured under light irradiation conditions;

[0043] Figure 8 Circular dichroism spectra of the multi-module probes M-NA, LK-NA, and LK-M-NA;

[0044] Figure 9 Confocal fluorescence microscopy images of multi-module probes M-NA, LK-NA, and LK-M-NA located in the cell nucleus after co-culture with tumor cells;

[0045] Figure 10 Fluorescence spectra of cell nuclei extracted after co-culture of multi-module probes M-NA, LK-NA, and LK-M-NA with tumor cells;

[0046] Figure 11 Graph showing the relationship between the cell survival rate and the probe concentration after co-culture of multi-module probes M-NA, LK-NA, and LK-M-NA with tumor cells under no light condition;

[0047] Figure 12 Graph showing the relationship between the cell survival rate and the probe concentration after co-culture of multi-module probes M-NA, LK-NA, and LK-M-NA with tumor cells under light condition;

[0048] Figure 13 Molecular structure diagram of the multi-module probe LK-M-NA synthesized in Example 1;

[0049] Figure 14 Molecular structure diagram of the multi-module probe that can be rapidly delivered to the cell nucleus in the present invention. Detailed implementation manners

[0050] The technical solutions of the present invention are clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, any equivalent transformation or substitution made by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention according to the following implementation manners.

[0051] The method of reverse high performance liquid chromatography gradient elution in the following embodiments is as follows: The sample is dissolved in water or acetonitrile, applied to a Kromasil C18 column (Teknokroma, 10μm, 250×4.6mm), and eluted at a rate of 2 mL / min, with the gradient from 20% to 100% (20% at 0.01 min, 40% at 20 min, 60% at 25 min, 90% at 30 min, 100% solvent B at 50 min). The elution solvent A is water containing 0.1 wt% trifluoroacetic acid (TFA), and the elution solvent B is acetonitrile containing 0.1 wt% trifluoroacetic acid (TFA).

[0052] In the following embodiments, the M-N polypeptide, LK-N polypeptide, LK-M-N polypeptide, and α-helical polypeptide LK are all purchased from GL Biochem (Shanghai) Ltd. Among them, the structural formula of the α-helical polypeptide LK is shown as follows:

[0053]

[0054] Example 1

[0055] In this example, the aggregation-induced emission photosensitizer PyTPA-N3 was first synthesized, and then various multi-module probes were synthesized by reacting PyTPA-N3 with M-N polypeptide, LK-N polypeptide, and LK-M-N polypeptide respectively.

[0056] S1. Synthesis of PyTPA-N3

[0057] Weigh 2 g of TPA-Br and 2 g of sodium azide (NaN3) respectively and add them into 100 mL of acetonitrile. Stir and reflux under condensation at 80 °C for 24 h. After the reaction, extract with a mixed solvent of 50 mL of dichloromethane and water (volume ratio 1:1). Collect the organic phase and spin-dry the solvent with a rotary evaporator. The crude product is eluted and purified by a silica gel column to obtain PyTPA-N3. The elution solvent is a mixed solvent prepared by mixing dichloromethane and methanol at a volume ratio of 50:1. The yield of PyTPA-N3 is 34.3%.

[0058] The main reaction equations involved in the reaction process are as follows:

[0059]

[0060] Figure 1 is the mass spectrometry characterization diagram of PyTPA-N3 prepared in step S1, which proves that the PyTPA-N3 molecule was successfully synthesized in this example.

[0061] S2. Preparation of multi-module probe M-NA

[0062] Weigh PyTPA-N3 (50 mg) and polypeptide M-N respectively, and add them to a mixed solvent of DMSO (dimethyl sulfoxide) and water (volume ratio 1:1) at a molar ratio of 5:1. Then add 2 mg of sodium ascorbate and 1 mg of copper bromide (CuBr) respectively, and stir well at room temperature for 24 h. After the reaction, perform reverse high-performance liquid chromatography gradient elution on the reaction solution, and separate and purify to obtain the multi-module probe M-NA, with a yield of 27.4%.

[0063] The main reaction equations involved in the reaction process are as follows:

[0064]

[0065] Figure 2 is the mass spectrometry characterization diagram of M-NA prepared in step S2, which proves that the M-NA molecule was successfully synthesized in this example.

[0066] S3. Preparation of multi-module probe LK-NA

[0067] Weigh PyTPA-N3 (50 mg) and polypeptide LK-N separately, and add the two into a mixed solvent of DMSO (dimethyl sulfoxide) and water (volume ratio 1:1) at a molar ratio of 5:1; then add 2 mg of sodium ascorbate and 1 mg of copper(I) bromide (CuBr) respectively, and stir well at room temperature for 24 h. After the reaction is completed, the reaction solution is subjected to reverse high performance liquid chromatography gradient elution and separation and purification to obtain the multi-module probe LK-NA, with a yield of 34.7%.

[0068] The main reaction equations involved in the reaction process are as follows:

[0069]

[0070] Figure 3 is the mass spectrometry characterization diagram of LK-NA prepared in step S3, which proves that the LK-NA molecule was successfully synthesized in this example.

[0071] S4. Preparation of multi-module probe LK-M-NA

[0072] Weigh PyTPA-N3 (50 mg) and polypeptide LK-M-N separately, and add the two into a mixed solvent of DMSO (dimethyl sulfoxide) and water (volume ratio 1:1) at a molar ratio of 5:1; then add 2 mg of sodium ascorbate and 1 mg of copper(I) bromide (CuBr) respectively, and stir well at room temperature for 24 h. After the reaction is completed, the reaction solution is subjected to reverse high performance liquid chromatography gradient elution and separation and purification to obtain the multi-module probe LK-M-NA, with a yield of 48.3%.

[0073] The main reaction equations involved in the reaction process are as follows:

[0074]

[0075] Figure 4 is the mass spectrometry characterization diagram of LK-M-NA prepared in step S4, which proves that the LK-M-NA molecule was successfully synthesized in this example.

[0076] Figure 5 is the ultraviolet-visible absorption spectrogram of PyTPA-N3, M-NA, LK-NA and LK-M-NA in the range of 300 - 600 nm. It can be seen from the figure that the maximum absorption wavelength of each multi-module probe is 425 nm, and the absorbance is basically the same as that of PyTPA-N3.

[0077] Figure 6Fluorescence spectra of PyTPA-N3, M-NA, LK-NA, and LK-M-NA in the range of 500 - 850 nm. As can be seen from the figure, the maximum emission wavelength of each multi-module probe is 600 nm. Under the action of the polypeptide, the degree of aggregation weakens, and thus the fluorescence intensity also weakens.

[0078] Performance testing and application of multi-module probes

[0079] The preparation method of the multi-module probe mother liquor used in the following experiments is as follows:

[0080] Respectively take 1.0 μmol of each of the multi-module probes prepared in steps S2 - S4 of Example 1, and dissolve them in 1 mL of DMSO respectively to obtain three probe mother liquors with a concentration of 1 mmol / L each. When other concentrations are needed, dilute them with DMSO to the required concentration.

[0081] 1. Testing the ability of multi-module probes to generate reactive oxygen species

[0082] The specific method for studying the ability of various multi-module probes prepared in Examples 2 - 4 to generate reactive oxygen species (ROS) under white light irradiation is as follows:

[0083] Weigh 5.0 μmol of the reactive oxygen species detection reagent 9,10 - anthryl - bis(methylene)dipropionic acid (ABDA) and dissolve it in 1 mL of DMSO to obtain a 5 mmol / L ABDA solution. Take 10 μL of the ABDA solution and add it to 1 mL of deionized water, and prepare four portions. Among them, three portions are respectively added with 5 μL of the 1 mmol / L multi-module probe mother liquors in Examples 2 - 4, mix well, and record the ultraviolet absorption spectra of the mixed solutions at 300 - 500 nm with an ultraviolet spectrophotometer, and record once every 30 s of illumination. Use the ABDA solution without adding the probe mother liquor as the blank control.

[0084] The detection results are as Figure 7 shown. As can be seen from Figure 7 , when the probe mother liquor is not added, the ultraviolet absorption value of the ABDA solution under white light (50 mW / cm 2 ) irradiation hardly changes; while after adding the probe molecules, under white light irradiation, the ultraviolet absorption values of the mixed solutions of each probe and ABDA all rapidly decrease within 3 min. This shows that the multi-module probes prepared in Examples 2 - 4 all have excellent reactive oxygen species generation ability.

[0085] 2. Testing the ability of multi-module probes to bind to cell membranes

[0086] The specific method for studying the binding ability of various multi-module probes prepared in Examples 2 - 4 to cell membranes is as follows:

[0087] 50 μL of each 1 mmol / L probe mother liquor and α-helical polypeptide LK were respectively added to a 1 mL mixed solvent prepared by mixing PBS solution (50 mM, pH = 7.4) and trifluoroethanol at a volume ratio of 1:1. The spectrum of the mixed solution in the range of 180 - 280 nm was detected and recorded using a circular dichroism spectrometer. The mixed solvent prepared by mixing PBS solution (50 mM, pH = 7.4) and trifluoroethanol at a volume ratio of 1:1 was used as the blank control. The test results are as Figure 8 shown. As can be seen from Figure 8 , α-helical polypeptide LK, multi-module probe LK-M-NA, and multi-module probe LK-NA showed higher negative absorption at 208 nm and 222 nm than multi-module probe M-NA. This indicates that both multi-module probe LK-M-NA and multi-module probe LK-NA have a higher α-helix ratio than multi-module probe M-NA, and thus have a greater cell membrane binding ability.

[0088] 3. Targeting efficiency test of multi-module probes to the nucleus

[0089] The specific methods for studying the targeting efficiency of various multi-module probes prepared in Examples 2 - 4 to the nucleus are as follows:

[0090] 3.1. MCF-7 cells (purchased from Wuhan Punosai Life Science Co., Ltd.) were cultured in a cell culture medium (containing 10 wt% fetal bovine serum, 1 wt% penicillin, and 1 wt% streptomycin) in an oven at 37°C.

[0091] 3.2. Then, the cells were seeded into four confocal imaging dishes at 2.0×10 5 cells / well. After 24 h, 20 μmol / mL nuclear import protein inhibitor Ivermectin was added to one of the imaging dishes, and Ivermectin was not added to the other three wells. Incubation was carried out for 12 h.

[0092] 3.3. The mother liquors of multi-module probes LK-M-NA, LK-NA, and M-NA were respectively added to the imaging dishes without Ivermectin, and the mother liquor of multi-module probe LK-M-NA was added to the imaging dish with Ivermectin, so that the final concentration of the multi-module probe mother liquor in each imaging dish was 5 μM. The four confocal imaging dishes were incubated in an environment of 37°C and 5% CO2 for 5 h. 10 μL of 1 mM / L Hoechst 33258 staining solution was added to each imaging dish for staining for 30 min. The culture medium was discarded, and after washing with PBS (10 mol / L, pH = 7.4), it was used for confocal fluorescence imaging.

[0093] 3.4. The cells were seeded at 2.0×10 5Four wells of a 6-well plate were implanted with cells / holes, and 20 μmol / mL of the nuclear import protein inhibitor ivermectin was added to one of the wells, while ivermectin was not added to the other three wells. After incubation for 12 h, the mother solutions of the multi-module probes LK-M-NA, LK-NA, and M-NA were added separately to the wells without ivermectin, and the mother solution of the multi-module probe LK-M-NA was added to the well with ivermectin, so that the final concentration of the multi-module probe mother solution in each well was 5 μM. The 6-well plate was incubated in an environment of 37 °C and 5% CO2 for 5 h. Subsequently, the cells were lysed, and the cell nuclei were extracted using a cell nucleus extraction kit (produced by Abbkine Scientific Co., Ltd). The obtained cell nuclei were dissolved in 100 μL of PBS (10 mol / L, pH = 7.4), and the fluorescence intensity was measured using a fluorescence spectrometer.

[0094] The results of confocal fluorescence microscopy imaging are as Figure 9 shown, and the fluorescence spectrogram of the cell nucleus solution is as Figure 10 shown. It can be seen from Figure 9 that among the three wells without ivermectin, the overlapping coefficient of the fluorescence of the photosensitizer PyTPA-N3 in the cells of the multi-module probe LK-M-NA group with the nuclear dye Hoechst 33258 was the highest, indicating that LK-M-NA has the highest cell membrane anchoring ability and rapid internalization ability, and LK-M-NA has the highest targeting efficiency for the cell nucleus. In the well with ivermectin added, the overlapping coefficient of the fluorescence of the photosensitizer PyTPA-N3 in the cells with the nuclear dye Hoechst 33258 decreased significantly, indicating that the nuclear import protein inhibitor can significantly inhibit the nuclear targeting of the probe, and further indicating that the multi-module probe enters the cell nucleus through the nuclear import protein-mediated manner. It can be seen from Figure 10 that the fluorescence intensity of the cell nucleus solution incubated with the probe LK-M-NA was the highest, indicating that the aggregation-induced emission photosensitizer PyTPA accumulated the most in the cell nuclei of the cells treated with LK-M-NA. Therefore, the nuclear delivery efficiency of LK-M-NA was the highest, and this conclusion was consistent with the conclusion obtained from Figure 9 .

[0095] 4. Bio-safety of the multi-module probe and toxicity test on tumor cells

[0096] The specific methods for studying the bio-safety of various multi-module probes prepared in Research Examples 2-4 and their toxicity to tumor cells are as follows:

[0097] 4.1. MCF-7 cells (purchased from Wuhan Punosai Life Science Co., Ltd) were cultured in a cell culture medium (containing 10 wt% fetal bovine serum, 1 wt% penicillin, and 1 wt% streptomycin) in an oven at 37 °C.

[0098] 4.2 Digest the cells with trypsin (purchased from Wuhan Procell Life Science & Technology Co., Ltd.), centrifuge at 1000 rpm for 4 min, discard the supernatant, and dilute the cells to 8000 - 10000 cells / 100 μL with cell culture medium (containing 10 wt% fetal bovine serum, 1 wt% penicillin, and 1 wt% streptomycin).

[0099] 4.3 Take 100 μL of the cell suspension and add it to a 96 - well plate, and continue culturing for 24 h.

[0100] 4.4 Add multi - module probe mother liquor with different concentrations (0 μM, 1 μM, 2 μM, 3 μM, 5 μM) to the 96 - well plate, and continue culturing for 24 h.

[0101] 4.5 Remove the culture medium, add 10 μL of 1 mM MTT (3 - (4,5 - dimethyl - 2 - thiazolyl) - 2,5 - diphenyltetrazolium bromide) and 100 μL of fresh cell culture medium (containing 10 wt% fetal bovine serum, 1 wt% penicillin, and 1 wt% streptomycin) to each well, and continue culturing for 4 h.

[0102] 4.6 Aspirate the culture medium, add 100 μL of DMSO to each well, shake on a shaker for 10 min, measure the absorbance value of each well at 570 nm with an enzyme - labeled instrument, and calculate the cell survival rate in each group of samples according to the absorbance value.

[0103] Cell survival rate = A S / A0×100%, where A0 represents the absorbance value of the well without adding the multi - module probe, and As represents the absorbance value of the wells with different concentrations of the multi - module probe added.

[0104] The absorbance detection results are as Figure 11 shown. Under non - light conditions, when adding 5 μM of the multi - module probe M - NA, the cell survival rate is above 90%; when adding 5 μM of the multi - module probe LK - NA, the cell survival rate is above 80%; when adding 5 μM of the multi - module probe LK - M - NA, the cell survival rate is above 75%. This shows that under non - light conditions, the toxicity of each multi - module probe prepared by the present invention to tumor cells is very small, the interference to cell growth is very small, and it has good biosafety.

[0105] Repeat steps 4.1 - 4.6, except that after adding multi - module probes with different concentrations in step 4.4, place the 96 - well plate under white light illumination conditions of 200 mW / cm 2 and culture for 24 h.

[0106] The absorbance values measured under light - culture conditions are as Figure 12 shown. From Figure 12It can be seen that when adding 5 μM of the multi-module probe M-NA, the cell survival rate is above 80%; when adding 5 μM of the multi-module probe LK-NA, the cell survival rate is above 50%; when adding 5 μM of the multi-module probe LK-M-NA, the cell survival rate is above 30%. This shows that with the improvement of the endocytosis efficiency, the therapeutic effect is significantly enhanced.

[0107] In summary, as Figure 13 shown, the molecular structure of the multi-module probe LK-M-NA synthesized in the present invention contains a cell membrane anchoring module, an affinity regulation module, and a nucleus targeting module at the same time. Therefore, this multi-module probe has both a high cell membrane affinity ability and a rapid internalization ability during cell transport, and the combination of the two can significantly improve the transport efficiency of the cell membrane. This multi-module probe also has a high ability to generate reactive oxygen species, good biosafety, and high tumor cell toxicity, and can be used for targeted treatment of tumor diseases under white light irradiation. It should be noted that multi-module probes synthesized using other aggregation-induced emission photosensitizers (such as aggregation-induced emission photosensitizers containing molecular structures such as TPE and QM) and the polypeptide LK-M-N can also achieve similar or the same technical effects as the multi-module probe LK-M-NA.

[0108] The above are only the preferred embodiments of the present invention and are not used to limit the protection scope of the present invention. For any person skilled in the art, various changes and modifications can be made to the present invention. Any simple equivalent changes and modifications made according to the protection scope of the present invention application and the content of the specification shall be included in the protection scope of the present invention.

Claims

1. A multi-module probe capable of rapid delivery to the cell nucleus, characterized in that: The structural formula of the multi-module probe is shown in formula (III): (III)。 2. The method for preparing a multi-module probe capable of rapid delivery to the cell nucleus according to claim 1, characterized in that: The aggregation-induced emission photosensitizer and the polypeptide LK-MN are added to a mixed solvent of an organic solvent and water, and then sodium ascorbate and cuprous bromide are added. After reaction, separation of products, and purification of the products, the multi-module probe is obtained. The organic solvent is a water-miscible organic solvent. The structural formula of the polypeptide LK-MN is as follows: 。 3. The preparation method according to claim 2, characterized in that The molar ratio of the aggregation-induced emission photosensitizer to the polypeptide LK-MN is 5:

1.

4. The use of the multi-module probe according to claim 1, characterized in that: The multi-module probe is used to prepare a product for treating and / or diagnosing tumors; when using the product containing the multi-module probe, light stimulation is required.

5. The use according to claim 4, characterized in that The light stimulation adopts 200mW / cm 2 White light exposure.

Images