Strong transmembrane photothermal conversion material based on St-L17E and Mo154 and application of strong transmembrane photothermal conversion material in preparation of antitumor drugs

Abstract

The invention discloses a strong transmembrane photothermal conversion material based on St-L17E and Mo154 and application of the strong transmembrane photothermal conversion material in preparation of antitumor drugs, and belongs to the technical field of photothermal antitumor materials. The strong transmembrane efficient photothermal conversion material constructed by the invention has the advantages of simple preparation, good biocompatibility, strong transmembrane, high photothermal conversion efficiency and the like. The stability of the Mo154 is improved by adding the St-L17E, and meanwhile, due to the synergistic effect of promoting the macropinocytosis effect of cells by the transmembrane peptide L17E and the super-chaotropic effect of the Mo154, the assembly has higher cell transmembrane capacity; a photo-thermal anti-tumor experiment is carried out through near-infrared light 808nm (1.4 W / cm < 2 >) irradiation, the survival rates of HeLa and A549 cells are respectively reduced to 46.62% and 3.73% under the concentration of 3.2 mu M, and the killing efficiency on tumor cells is remarkably improved, so that the compound can be used for preparing anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of photothermal anti-tumor materials, and particularly relates to a strong transmembrane photothermal conversion material based on St-L17E and Mo 154 and its application in the preparation of anti-tumor drugs. Background Art

[0002] Tumors seriously threaten human health and are one of the main causes of human death. At present, the main treatment methods for tumors include: surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy, interventional therapy, etc. Among them, photothermal therapy, as an emerging treatment method, has the advantages of precision, high efficiency, minimally invasive, etc. The key to photothermal therapy lies in the photothermal conversion material, which can convert light energy into heat energy to kill tumor cells. However, the existing photothermal conversion materials still have deficiencies in cell transmembrane and photothermal conversion efficiency, which limits their application effects in tumor photothermal therapy.

[0003] Polyoxometalate Na 15 [Mo VI 126 Mo V 28 O 462 H 14 (H2O) 70 ·400H2O (Mo 154 ) is an inorganic cyclic compound with a special structure, which has strong absorption in the near-infrared region and good photothermal conversion ability, and is the main source of heat in photothermal therapy. In addition, it can interact with biomolecules (amino acids, peptides, proteins, etc.) - this non-specific interaction is interpreted as super-chaotropic properties, and these properties can promote its binding with small peptides and further promote the transmembrane of small peptides. Due to its unique physical, chemical and biological properties, it has received extensive attention in the field of biochemistry.

[0004] St-L17E is a small peptide obtained by fusing SpyTag with the cell transmembrane peptide L17E, which can carry drugs or biomolecules into the cell interior and improve the cell uptake efficiency of drugs. Assembling St-L17E and Mo 154 to form a complex is expected to improve its cell transmembrane ability, stability and photothermal conversion efficiency, providing a new method for photothermal anti-tumor therapy. Summary of the Invention

[0005] The purpose of the present invention is to provide a strong transmembrane photothermal conversion material (St-L17E@Mo 154 ) based on St-L17E and Mo 154 and its application in the preparation of anti-tumor drugs. This strong transmembrane photothermal conversion material (St-L17E@Mo 154) is a binary assembly that can effectively improve the photothermal conversion efficiency and synergistically promote the cell transmembrane efficiency of the assembly through the macropinocytosis of cells promoted by L17E and the chaotropic effect of Mo 154 , thereby enhancing the photothermal therapy effect on tumor cells in vitro.

[0006] Construction of Strong Transmembrane Photothermal Conversion Material (St-L17E@Mo 154 ): First, prepare a 1 mg / mL St-L17E solution and a 600 μM Mo 154 solution using distilled water; then add the St-L17E solution and Mo 154 solution to distilled water so that the molar ratio of St-L17E to Mo 154 is 1-8:1; after slowly pipetting and mixing evenly, place it on ice for 0.5-2 h to prepare the strong transmembrane photothermal conversion material (St-L17E@Mo 154 ).

[0007] The strong transmembrane photothermal conversion material (St-L17E@Mo 154 ) prepared by the present invention was labeled with a FITC fluorescent probe, and after co-incubation with the human cervical cancer HeLa cell line, the intracellular green fluorescence intensity and distribution were observed by a fluorescence microscope, and the uptake of tumor cells was quantitatively detected by a flow cytometer, and then the cell transmembrane ability of the strong transmembrane photothermal conversion material (St-L17E@Mo 154 ) was evaluated. The results showed that the cell uptake efficiency of the strong transmembrane photothermal conversion material (St-L17E@Mo 154 ) prepared by the present invention was greatly improved.

[0008] The photothermal conversion ability of St-L17E@Mo 2 was measured by irradiation with near-infrared light at 808 nm (1.4 W / cm 154 ) in vitro. The absorption at 808 nm increased by 6.07% compared to Mo 154 . And after irradiation with an 808 nm laser (1.4 W / cm 2 ) for 5 minutes in vitro, the temperature of the St-L17E@Mo 154 solution (the molar ratio of St-L17E to Mo 154 was 4:1) increased by 35.9 °C, and the temperature of Mo 154 alone increased by 31.8 °C. The photothermal conversion efficiency of St-L17E@Mo 154 was 7.35% higher than that of Mo 154 . It shows that the strong transmembrane photothermal conversion material (St-L17E@Mo 154 ) prepared by the present invention has enhanced photothermal performance.

[0009] The strong transmembrane photothermal conversion material (St-L17E@Mo prepared by the present invention 154 ) has the advantages of simple preparation, good biocompatibility, strong transmembrane property, high photothermal conversion efficiency, etc. In vitro, the strong transmembrane photothermal conversion material (St-L17E@Mo prepared by the present invention 154 ) was used to conduct photothermal anti-tumor cell (HeLa, A549 cells) experiments by irradiating with near-infrared light at 808 nm (1.4 W / cm 2 ). At a concentration of 3.2 μM, the survival rates of HeLa and A549 cells decreased to 46.62% and 3.73% respectively, significantly improving the killing rate of tumor cells.

[0010] The cell uptake efficiency of the strong transmembrane photothermal conversion material (St-L17E@Mo prepared by the present invention 154 ) has been significantly enhanced, and it can be enriched in the cytoplasm in a short time. Moreover, the in vitro photothermal conversion efficiency has been improved. Finally, high-efficient killing of tumor cells has been achieved through photothermal therapy. Therefore, the strong transmembrane photothermal conversion material (St-L17E@Mo prepared by the present invention 154 ) can be used to prepare anti-tumor drugs. Description of the Drawings

[0011] Figure 1 : UV-visible absorption spectra (A), absorption results at 808 nm (B), DLS particle size distribution results (C), Zeta-potential results (D), TEM image of Mo 154 solution (5 μM) and binary assemblies prepared with different concentrations of St-L17E solution (5 - 40 μM), TEM image of Mo 154 (E) and TEM image of St-L17E@Mo 154 (F);

[0012] Figure 2 : Heating curve (A) of Mo 154 solution, St-L17E@Mo 154 (molar ratio of St-L17E and Mo 154 is 4:1) binary assembly solution and distilled water irradiated with 808 nm (1.4 W / cm 2 ) laser for 5 min; Infrared thermal images (B) of Mo 154 solution (the first row) and St-L17E@Mo 154 solution (the second row) irradiated with 808 nm (1.4 W / cm 2 ) laser for different times; Infrared thermal images (C) of Mo 154 solution and (D) of St-L17E@Mo 154 solution irradiated with 808 nm (1.4 W / cm 2) Heating and cooling curves under laser irradiation (the abscissa is the heating and cooling time, and the ordinate ΔT represents the temperature change); Mo 154 Graph (E) of the cooling time of Mo varying with -lnθ (-lnθ is calculated based on the cooling data in (C)), and its slope is the time constant τ = 531.01; St-L17E@Mo 154 Graph (F) of the cooling time of Mo varying with -lnθ (-lnθ is calculated according to the cooling data in (D)), and its slope is the time constant τ = 493.74. In all test solutions, the concentration of Mo used 154 is 5 μM;

[0013] Figure 3 : Fluorescence emission result graphs of FITC and FITC-labeled St-L17E (FITC-St-L17E); indicating that the characteristic emission peak of FITC is located at about 517 nm;

[0014] Figure 4 : Fluorescence microscope images of fluorescently labeled HeLa cells (the control group is untreated cells); HeLa cells were incubated with FITC-St-L17E and FITC-St-L17E@Mo 154 (the molar ratio of St-L17E and Mo 154 is 4:1) at pH = 6.0 for 4 hours, and the concentration of FITC-St-L17E used is 5 μM; further, the nucleus was stained with DAPI fluorescent dye, and the stained nucleus showed blue fluorescence under the fluorescence microscope. (DAPI: shows blue fluorescence, for the stained nucleus; FITC: shows green fluorescence, for the material labeled by FITC; Merge: is the result of overlapping DAPI and FITC, the blue fluorescence shows the position of the nucleus, and the green fluorescence shows the distribution of the material labeled by FITC in the cell; Bright: is the cell image result under white light);

[0015] Figure 5 : Line graph of the summary information statistics of flow cytometry results: HeLa cells were incubated with FITC-St-L17E and FITC-St-L17E@Mo 154 (the molar ratio of St-L17E and Mo 154 is 4:1) at pH = 6.0 for 1, 2, 4, and 6 hours respectively, and then the cell uptake rate was measured using a flow cytometer (the concentration of FITC-St-L17E used is 5 μM). Further, a line graph was plotted with the cell uptake rate and uptake time, where the abscissa is the uptake time and the ordinate is the cell uptake efficiency (%) at the corresponding time;

[0016] Figure 6:Cell photothermal therapy results: HeLa (A) and A549 (B) with different concentrations of St-L17E, Mo 154 as well as St-L17E@Mo 154 (St-L17E and Mo 154 molar ratio 4:1) under the condition of pH = 6.0 with or without NIR light irradiation (808 nm, 1.4 w / cm 2 ) for 5 min treatment, cell viability graphs after incubation for 24 h; where the abscissa is the concentration of Mo 154 (St-L17E), "+" and "-" represent with or without laser treatment respectively, and the ordinate represents the cell survival rate (%). Each experiment was repeated three times, and the data was shown in the form of standard deviation ± mean value. Specific implementation mode

[0017] The chemical reagents such as Na2MoO4·2H2O, Na2S2O4 and anhydrous calcium chloride used in the present invention were all purchased from Sinopharm Chemical Reagent Co., Ltd., hydrochloric acid was purchased from Xilong Science Co., Ltd., St-L17E was purchased from Shanghai Chutai Biotechnology Co., Ltd., FITC was purchased from Shanghai Macklin Biochemical Co., Ltd., DMEM, trypsin and penicillin-streptomycin mixture were purchased from Wuhan Sevier Biotechnology Co., Ltd., serum was purchased from Anhui Kangyuan Biotechnology Co., Ltd., CCK-8 was purchased from Bimake Biotechnology Co., Ltd. in the United States, DAPI was purchased from Beijing Solarbio Science & Technology Co., Ltd., and 4% paraformaldehyde was purchased from Beijing Lanjeck Technology Co., Ltd.

[0018] Example 1

[0019] Preparation and characterization of Mo 154 :

[0020] For the synthesis of Mo 154 , referring to the method reported in the literature (J Mater Chem B. 2018, 6(2): 241-248), first dissolve 3.04 g of Na2MoO4·2H2O in 25 mL of distilled water, then add 2.7 mL of hydrochloric acid with a mass fraction of 32% and stir continuously. Then add 0.15 g of NaS2O4 to obtain a dark blue solution, which is stored at room temperature in a closed container for 24 h. Filter to obtain blue crystals, and quickly rinse with cold water, and finally dry with CaCl2 at room temperature; then dissolve the dried Mo 154 in distilled water for testing, with a concentration of 5 μM. As shown in Figure 1 A, Mo 154 showed an absorption peak at 748 nm, proving the synthesis of Mo 154 .

[0021] Example 2

[0022] St-L17E@Mo 154 Construction and Characterization of Binary Assemblies:

[0023] Prepare 1 mg / mL St-L17E solution (stored at 4 °C) and 600 μM Mo 154 solution as stock solutions. Then add 1 mg / mL St-L17E solution and 600 μM Mo 154 solution to distilled water to make the final concentrations of St-L17E be 5 μM, 10 μM, 20 μM, 30 μM, and 40 μM respectively, and the final concentration of Mo 154 be 5.0 μM. Slowly pipette and mix evenly with a 1 mL pipette tip, and then incubate on ice for 1 h to obtain strong transmembrane photothermal conversion materials (St-L17E@Mo 154 ) with different molar ratios (the molar ratios of St-L17E to Mo 154 are 1:1, 2:1, 4:1, 6:1, and 8:1 respectively). The ultraviolet-visible absorption is shown in Figure 1 Figures A and 1B. As the concentration of St-L17E increases, the absorption peak intensities of St-L17E@Mo 154 at 748 nm and 808 nm gradually increase; compared with Mo alone 154 , the absorption of St-L17E@Mo 154 (the molar ratio of St-L17E to Mo 154 is 4:1) binary assembly at 808 nm increases by 6.07%, indicating that this component can be an effective photothermal agent in the near-infrared region. The DLS results are shown in Figure 1 Figure C. As St-L17E is added, the particle size gradually increases. When the molar ratio of St-L17E to Mo 154 is 4:1, the particle size is 27.8 nm, and when the molar ratio of St-L17E to Mo 154 is 8:1, the particle size reaches 50.8 nm, indicating the formation of St-L17E@Mo 154 assemblies. The surface potential is shown in Figure 1 Figure D. As the St-L17E peptide is added, the degree of negative potential gradually decreases, indicating that St-L17E and Mo 154 may bind together through electrostatic interactions, which can reduce the cytotoxicity caused by the exposure of Mo 154 . Further, the TEM results of Mo 154 ( Figure 1 ) show that it is evenly distributed in water with a particle size of about 3.6 nm, while the TEM image results of St-L17E@Mo 154 (the molar ratio of St-L17E to Mo 154 is 4:1) ( Figure 1F) It shows that the addition of St-L17E promotes the assembly of Mo 154 , forming larger nanostructures with a particle size of about 25 nm, which is consistent with the results of DLS( Figure 1 B), further confirming the successful construction of the St-L17E@Mo 154 binary assembly.

[0024] Example 3

[0025] St-L17E@Mo 154 In vitro photothermal conversion performance detection:

[0026] As shown in Figure 2 A and 2B, after 5 μM Mo 154 solution and St-L17E@Mo 154 solution (molar ratio 4:1) are irradiated with near-infrared (NIR) light (1.4 W / cm 2 , 808 nm) for 5 minutes, the temperature of the St-L17E@Mo 154 solution (molar ratio of St-L17E to Mo 154 is 4:1) increases by 35.9 °C, the temperature of the individual Mo 154 increases by 31.8 °C, and the temperature of H2O as the control group only increases by 3.6 °C. Under the same conditions, the temperature change of the St-L17E@Mo 154 solution is 4.1 °C higher than that of Mo 154 . This indicates that the St-L17E@Mo 154 binary assembly has enhanced photothermal performance, which can be directly attributed to the higher A 154 of St-L17E@Mo 808 than that of Mo 154 . Then, according to Figure 2 C and D, the -lnθ is calculated from the data of the cooling part in the heating-cooling curves of the Mo 154 solution and the St-L17E@Mo 154 solution under 808 nm (1.4 W / cm 2 ) laser irradiation; further, by plotting the curve of cooling time versus -lnθ, as shown in Figure 2 E and F, according to the slopes of the two curves, i.e., the time constants (Mo 154 : τ = 531.01; St-L17E@Mo 154 : τ = 493.74), the photothermal conversion efficiency of Mo 154 is calculated to be 37.28%, while the photothermal conversion efficiency of St-L17E@Mo 154 is 44.63%, which is 7.35% higher than that of Mo 154 .

[0027] Example 4

[0028] St-L17E@Mo 154 Detection of cell transmembrane efficiency:

[0029] Fluorescent labeling of St-L17E: Dissolve 1 mg of FITC in 1 mL of DMSO to obtain a FITC solution; dissolve St-L17E in distilled water to obtain a St-L17E solution with a concentration of 1 mg / mL; drop 65 μL of the FITC solution into 1 mL of the St-L17E solution under stirring, and all operations are carried out at 4 °C. Invert the solution up and down every two hours. After reacting for 8 hours, transfer the solution to a dialysis bag (16 kDa) and dialyze it in water for 24 h to remove free FITC. Finally, collect the solution in the dialysis bag to obtain FITC-St-L17E. As Figure 3 shown, after FITC labeling, a characteristic emission peak of FITC appears at 517.9 nm, indicating that FITC is successfully labeled on the St-L17E peptide. Further mix the FITC-St-L17E and Mo 154 solutions evenly at a molar ratio of 4:1 and place them on ice for 1 h to obtain FITC-St-L17E@Mo 154 .

[0030] Qualitative evaluation was carried out with a fluorescence microscope. Incubate HeLa cells with 5 μM FITC-St-L17E and FITC-St-L17E@Mo 154 (molar ratio 4:1) at pH = 6.0 for 4 hours respectively, wash them three times with PBS solution, then fix the cells with 4% paraformaldehyde for 15 min. After washing the cells twice with PBS solution, stain the cell nuclei with DAPI (10 μg / mL), wash them three times with PBS solution, and analyze the cell uptake rate of FITC-St-L17E@Mo 154 . The results are as Figure 4 shown. After DAPI staining, the cell nuclei show blue fluorescence in the fluorescence photographs; in addition, HeLa cells incubated with FITC-St-L17E show characteristic green fluorescence of FITC in the cytoplasm, indicating that a large amount of the peptide enters the cells and is dispersed in the cytoplasm (as Figure 4 shown in the second row). However, the cells incubated with FITC-St-L17E@Mo 154 show stronger green fluorescence (as Figure 4 shown in the third row), which indicates that under the same conditions, the amount of St-L17E@Mo 154 entering the cells is more than that of St-L17E, which also more intuitively shows that St-L17E and Mo 154 synergistically promote the cell transmembrane of the assembly.

[0031] After HeLa cells were incubated with FITC-St-L17E for different times (1, 2, 4, and 6 h) respectively, flow cytometry was used to quantitatively analyze cell uptake. As Figure 5 shown, at 1 h, 2 h, and 4 h, compared with St-L17E alone, the uptake rates of St-L17E@Mo 154 cells increased by 21.07%, 20.97%, and 2.93% respectively, further indicating that after the formation of the binary assembly of Mo 154 and St-L17E, the cell uptake efficiency was significantly enhanced. At 6 h, the uptake rates of both St-L17E and St-L17E@Mo 154 cells reached 100%, indicating that at 6 h, all the detected cells showed fluorescence signals and cell uptake had reached a saturated state at this concentration.

[0032] Example 5

[0033] Determination of the photothermal therapy effect on tumor cells:

[0034] HeLa and A549 cells were incubated with Mo 154 or St-L17E@Mo 154 (the molar ratio of St-L17E to Mo 154 was 4:1) at pH = 6.0 and irradiated with an 808 nm laser (1.4 W / cm 2 ) for 5 minutes. Subsequently, the cells were placed in an incubator at 37 °C for 24 h (the culture medium was DMEM medium, 10% FBS). Then the original culture medium was replaced with FBS-free DMEM medium containing 10% (v / v) CCK-8 and co-incubated for another 2 h. The cell viability (%) of each group of cells was calculated by measuring the absorbance at 450 nm.

[0035] As Figure 6 shown, Blank (Figure A and B, black) was the untreated cells with a viability of 100%, while in the presence of 3.2 μM Mo 154 , 12.8 μM St-L17E, and the same concentration of St-L17E@Mo 154 without laser irradiation, as well as under the irradiation of 808 nm NIR light alone (Figure A and B, gray, Control), the cell viability was higher than 80%, indicating that the above treatments had no killing effect on the two cancer cell lines. As Figure 6 shown in Figure A, under the irradiation of an 808 nm laser, Mo 154 and St-L17E@Mo 154The inhibition of the growth of HeLa cells by the two materials was dose-dependent, while the cell viability of St-L17E( Figure 6 A, blue) was higher than 80% after combined laser treatment at all concentrations, indicating that St-L17E had no effect on cells under 808 nm laser irradiation. Among them, without laser irradiation, the viability of HeLa cells treated with 3.2 μM Mo 154 ( Figure 6 A, green) was 85.96%. After co-treatment with 3.2 μM Mo 154 and 808 nm laser irradiation, the cell viability decreased to 55.88%, confirming the photothermal effect of Mo 154 . Similar to Mo 154 , without laser irradiation, the viability of HeLa cells treated with 3.2 μM St-L17E@Mo 154 ( Figure 6 A, red) was 94.6%. After co-treatment with 3.2 μM St-L17E@Mo 154 and 808 nm laser irradiation, the viability decreased to 46.62%. Compared with the Mo 154 group with laser at the same concentration, the inhibition rate of St-L17E@Mo 154 on HeLa cells was 9.26% higher. Therefore, after adding St-L17E@Mo 154 , it had stronger inhibition on tumor cells in the presence of 808 nm, which was due to the enhanced absorption and improved photothermal conversion efficiency of St-L17E@Mo 154 at 808 nm. Similarly, as shown in Figure 6 B, similar results were also obtained on the tumor cell line A549. Under 808 nm irradiation, the viabilities of cells treated with 3.2 μM Mo 154 and St-L17E@Mo 154 were 6.12% and 3.73% respectively. Therefore, the killing rates of Mo 154 and St-L17E@Mo 154 on A549 cells were significantly improved by 808 nm laser irradiation.

[0036] Through the above examples, it is shown that we successfully constructed a strong transmembrane and highly efficient photothermal conversion material St-L17E@Mo 154 based on the binary assembly of St-L17E and Mo 154 . By assembling St-L17E and Mo 154 , the cell transmembrane ability and photothermal conversion efficiency of the binary assembly were significantly improved, thereby enhancing the photothermal therapy effect and effectively promoting tumor cell apoptosis in vitro.

[0037] It should also be noted that the specific embodiments of the present invention are only used for illustrative purposes and do not limit the protection scope of the present invention in any way. Those skilled in the relevant art can make improvements or changes based on the above descriptions, but all such improvements and changes should fall within the protection scope of the claims of the present invention.

Claims

1. A method based on St-L17E and Mo 154 The strong transmembrane photothermal conversion material is characterized by: First, 1 mg / mL St-L17E solution and 600 μM Mo were prepared in distilled water. 154 solution; then add St-L17E solution and Mo to distilled water 154 solution, St-L17E and Mo 154 The molar ratio of St-L17E and Mo is 1 to 8:1; the mixture is mixed by slowly blowing and suction and then placed on ice for 0.5 to 2 hours, thereby preparing the 154 Strong transmembrane photothermal conversion material.

2. The method according to claim 1 based on St-L17E and Mo 154 The application of strong transmembrane photothermal conversion materials in promoting cell transmembrane.

3. A method based on St-L17E and Mo as described in claim 1 154 Application of strong transmembrane photothermal conversion materials in photothermal conversion.

4. The method according to claim 1 based on St-L17E and Mo 154 Application of strong transmembrane photothermal conversion materials in the preparation of anti-tumor drugs.

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