A nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer and its preparation method and application

Abstract

The present invention provides a nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer, and a preparation method and application thereof. By modifying a Dasatinib derivative with triple-negative breast cancer targeting ability on the surface of polycyclodextrin and mixing and assembling it with an endoplasmic reticulum-targeted photosensitizer, the constructed novel dual-targeted nanomaterial can accurately target and identify tumor cells, increase the enrichment of photosensitizers at the tumor site, and can further achieve accumulation in the endoplasmic reticulum. The generated ROS can directly act on the endoplasmic reticulum, causing stronger endoplasmic reticulum stress, thereby significantly improving the anti-tumor effect of targeted photodynamic therapy. The novel dual-targeted functional nano-drug delivery system prepared by the present invention solves the problems of poor targeting, biological solubility and limited therapeutic effect of traditional photosensitizers, and provides an innovative strategy for achieving targeted photodynamic therapy of triple-negative breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of novel targeted nanomedicines, and specifically relates to a nano delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer, and a preparation method and application thereof. Background Art

[0002] Triple-negative breast cancer (TNBC) is a subtype of breast cancer with strong invasiveness, easy metastasis, and extremely poor prognosis, resulting in a low survival rate for its patients. Since the expression of estrogen receptor (ER), progesterone receptor (PR) and proto-oncogene Her-2 on the surface of TNBC tumors are all negative, very few anticancer drugs can enter tumor cells, greatly reducing their therapeutic effect. For this reason, increasing the dose of anticancer drugs is often used to achieve the desired therapeutic effect. Unfortunately, this inevitably increases the toxic side effects of the drugs and tumor resistance, which in turn reduces the treatment options for TNBC. At present, chemotherapy, immunotherapy, surgery and radiotherapy are often used to treat TNBC according to the different stages of TNBC in clinical practice. However, the above treatments can cause serious side effects, such as wound infection, bone marrow suppression, liver toxicity, skin and heart damage. In addition, resistance to chemotherapy and radiotherapy, metastasis and recurrence also often occur in TNBC. Therefore, finding targeted drugs that can effectively treat TNBC to improve the treatment effect of TNBC is the main challenge currently faced.

[0003] Tyrosine kinase inhibitors are highly selective and have few side effects. They can be used as competitive inhibitors of adenosine triphosphate (ATP) binding to tyrosine kinases, or as tyrosine analogs to block the activity of tyrosine kinases and inhibit cell proliferation. They have a wide range of targets, such as BCR-ABL kinases, SRC family kinases (SRC, LCK, YES, FYN), etc., and have become the first-line drugs for clinical treatment of tumors. In addition, many studies have shown that the level of SRC protein in TNBC is significantly higher than that in other breast cancer subtypes. Dasatinib, as a tyrosine kinase inhibitor, can accurately target SRC family proteins (including SRC, LCK, FYN, etc.), providing a feasible solution for achieving targeted treatment of TNBC. However, the existing Dasatinib targeted TNBC treatment is still used in preclinical studies in the form of chemotherapy or combined chemotherapy, which will inevitably bring about problems such as patient resistance and adverse reactions. Therefore, the combination of Dasatinib with other tumor treatments for targeted treatment of TNBC is expected to become a new tumor treatment strategy.

[0004] Photodynamic therapy (PDT) has become a new cancer treatment method due to its unique advantages such as minimally invasive, high efficiency and no significant drug resistance. However, due to the problems of insufficient tumor tissue targeting and poor biosolubility of traditional photosensitizers such as phthalocyanine and porphyrin, the therapeutic effect of PDT is limited. With the development and application of nanomaterial technology, the strategy of constructing nanodelivery systems as photosensitizer carriers has undoubtedly provided a new platform for the development of PDT and increased the selective enrichment of photosensitizers in tumor sites. Unfortunately, this strategy still faces two obstacles: 1) the poor biosolubility of nanocarriers and premature leakage of photosensitizers cause stronger toxic side effects; 2) due to the short half-life of ROS (<0.04μs) and limited action radius (<0.02μm), its effectiveness is undoubtedly limited to the vicinity of the production site, which leads to limited PDT therapeutic effect. Therefore, in response to these problems, it is urgent to develop a soluble nanodelivery carrier with tumor cell and subcellular organelle targeting function to achieve the purpose of effectively improving the photodynamic anti-tumor effect. Summary of the invention

[0005] The purpose of the present invention is to provide a nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer and a preparation method thereof. First, p-Toluenesulfonamide ligand with endoplasmic reticulum targeting function is covalently coupled with photosensitizer Ce 6 to obtain photosensitizer CET with endoplasmic reticulum localization ability; then, Dasatinib derivative is modified on the surface of polycyclodextrin material through host-guest interaction to obtain nano-delivery material Da-CD with TNBC targeting ability; finally, photosensitizer CET and Da-CD are mixed and assembled into nano-material Da-CD@CET with tumor cell targeting and endoplasmic reticulum targeting functions. The high loading rate and high biocompatibility of polycyclodextrin can reduce biological toxicity. In addition, the Dasatinib derivatives loaded on its surface can accurately target and identify tumor cells, thereby promoting more enrichment of photosensitizers in the tumor site. The endoplasmic reticulum targeted photosensitizer CET wrapped in the material can further achieve more photosensitizers Accumulation in the endoplasmic reticulum, the generated ROS can directly act on the endoplasmic reticulum, causing stronger endoplasmic reticulum stress, thereby significantly improving the effect of targeted photodynamic therapy for TNBC. The dual-targeted functional nano drug delivery system prepared by the present invention has high biocompatibility and significant tumor cell and endoplasmic reticulum targeting, and exhibits negligible toxic side effects and significantly improves the photodynamic anti-tumor effect, providing an innovative strategy for achieving targeted photodynamic therapy of TNBC. At the same time, the present invention also provides a nano delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer obtained by the above method, and the application of the nano delivery material.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention discloses a method for preparing a nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer, comprising the following steps:

[0008] (1) First, accurately weigh β-CD and dissolve it in a 15 wt% NaOH aqueous solution, stirring at room temperature. Subsequently, add toluene to the mixture and continue stirring, then slowly add epichlorohydrin as a cross-linking agent to continue the reaction. After the reaction is completed, pre-precipitate the crude product with isopropanol, transfer the precipitate into an appropriate amount of water and adjust it to neutral with dilute hydrochloric acid. Dialyze it with a dialysis bag to completely remove monomers and oligomers, and finally freeze-dry it in vacuum to obtain a white solid product Poly-β-CD.

[0009] (2) Add anhydrous DMF to accurately weigh Dasatinib and succinic anhydride, stir evenly, and slowly add accurately weighed triethylamine dropwise to the mixed reaction. Finally, introduce nitrogen protection and continue the reaction by stirring at room temperature. After the reaction is completed, the reaction system is vacuum-rotated and evaporated to remove DMF, and the mixture is purified by column chromatography. Finally, the collected organic solvents are mixed and evaporated to obtain a white solid compound Da-COOH;

[0010] (3) Accurately weigh Da-COOH obtained in step (2), add anhydrous DMF solution to stir and mix evenly, then add accurately weighed NHS and EDCI to the mixed reaction solution, and react in an ice-water bath. Subsequently, accurately weighed adamantane amine is added to the mixed solution, and stirred at room temperature for reaction. After the reaction is completed, the reaction solution is transferred to a mixture of acetone and water to form a precipitate, and vacuum filtered. Vacuum drying is performed to obtain a white solid compound Da-Ad;

[0011] (4) Dissolve accurately weighed ethylenediamine in anhydrous dichloromethane solution, stir in an ice bath, then dissolve weighed p-toluenesulfonyl chloride in anhydrous dichloromethane solution, then slowly add dropwise to the reaction system, and continue the reaction at room temperature. 2 After the reaction was completed, the mixture was purified by column chromatography to obtain a light yellow solid compound ET;

[0012] (5) Dissolve accurately weighed Ce 6, HOBT and EDCI in anhydrous DMF solution, react at room temperature in the dark, then add ET obtained in step (4) to continue the reaction. 2 After the reaction, the mixture was purified by column chromatography to obtain a brown-black solid compound CET;

[0013] (6) Dissolve the Poly-β-CD obtained in step (1) and the CET obtained in step (5) in a mixed solution of DMSO and water, and react at room temperature in the dark for 2 hours. Then dissolve the Da-Ad obtained in step (3) in a mixed solution of DMSO and water, and then slowly add it dropwise to the reaction system. Continue the reaction at room temperature in the dark. After the reaction is completed, transfer the reaction mixture to a dialysis bag for dialysis. Finally, freeze-dry the reaction solution to obtain a brown-black solid powder Da-CD@CETNPs.

[0014] Furthermore, the dialysis condition in step (1) is to dialyze in a dialysis bag with MW=8000 for 24 hours, and to change the water every 3 hours.

[0015] Furthermore, in step (3), the ratio of the mixed solution of acetone and water is 1:4.

[0016] Furthermore, in step (6), the ratio of the mixed solution of DMSO and water is 1:3. The dialysis condition is to dialyze in a dialysis bag of MW=3000 for 24 hours, and change the water every 3 hours.

[0017] The purpose of the second aspect of the present invention is to disclose a nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer, which is prepared by the above method.

[0018] The third aspect of the present invention aims to disclose the application of the nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer in a dual-targeted functional nano-drug delivery system.

[0019] The significant advantages of the present invention are:

[0020] (1) Polycyclodextrin-based nano-delivery carriers have good biocompatibility and stability under physiological conditions, and have the ability to release drugs in response to the disintegration of the tumor microenvironment. They can significantly reduce the toxic side effects and adverse reactions caused by premature leakage and poor solubility of nano-drugs during transportation, and can serve as ideal nano-drug carriers.

[0021] (2) The nano-delivery materials modified with small molecule targeted drug Dasatinib derivatives showed significant targeting recognition ability for triple-negative breast cancer cells, providing a promising targeted treatment strategy for triple-negative breast cancer.

[0022] (3) The nanodrug delivery system has excellent stability and uniformity, which is conducive to use and long-term storage.

[0023] (4) The preparation method is simple, with few side reactions, readily available raw materials, and low cost, which is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1Transmission electron microscopy and particle size distribution diagrams of different nano drug delivery systems;

[0025] Figure 2 The stability and particle size change diagram of different nano drug delivery systems in deionized water and culture medium;

[0026] Figure 3 Figure 2 shows the tumor cell targeting of Da-CD@CETNPs characterized by flow cytometry.

[0027] Figure 4 :The endoplasmic reticulum targeting ability of Da-CD@CETNPs was characterized by laser confocal microscopy;

[0028] Figure 5 :The cell activity inhibition ability of different drug groups detected by MTT method;

[0029] Figure 6 :The inhibitory ability of different drug groups on triple-negative breast cancer mouse tumor model; DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0031] The present invention provides a method for preparing a nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer, comprising the following steps:

[0032] (1) First, accurately weigh 10.0g of β-CD and dissolve it in 15mL of NaOH (15WT%) aqueous solution, and stir at room temperature for 2h. Then, add 2mL of toluene to the mixture and continue stirring for 2h, then slowly add 3.8mL of epichlorohydrin as a cross-linking agent and continue the reaction for 3h. After the reaction is completed, pre-precipitate the crude product with 200mL of isopropanol, transfer the precipitate into an appropriate amount of water and adjust it to neutral with dilute hydrochloric acid. Finally, dialyze it with a dialysis bag of MW=8000 for 2 days, changing the water every 3h to completely remove the monomers and oligomers, and finally freeze-dry it in vacuum to obtain a white solid product Poly-β-CD.

[0033] (2) Accurately weighed Dasatinib (1.60 g, 3.27 mmol) and succinic anhydride (0.37 g, 3.70 mmol) were placed in a 100 mL double-necked bottle. Then, 15 mL of anhydrous DMF was added to the double-necked bottle and stirred evenly, and accurately weighed triethylamine (0.70 g, 6.90 mmol) was slowly added dropwise to the mixed reaction. Finally, nitrogen protection was introduced and the reaction was continued for 48 h at room temperature with stirring. After the reaction was completed, the reaction system was vacuum-rotated and DMF was removed. Column chromatography (CH 2 Cl 2 / CH3 OH30:1 v / v) was used to purify the mixture. Finally, the collected organic solvents were mixed and evaporated to obtain 1.73 g of white solid compound Da-COOH with a yield of 90%. 1 H NMR (400 MHz, DMSO-d 6 )δ=11.45 (s, 1H), 9.89 (s, 1H), 8.23 ​​(s, 1H), 7.38 (d, J=7.4Hz, 1H), 7.27 (dd, J=10.6, 7.5Hz, 2H), 6.06 (s , 1H), 4.15 (t, J=5.7Hz, 2H), 3.50 (s, 4H), 2.58 (t, J=5.7Hz, 2H), 2.49 (s, 8H), 2.40 (s, 3H), 2.23 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 )δ=172.60, 165.63, 162.93, 160.41, 157.43, 141.34, 139.30, 134.03, 132.93, 129.48, 128. 62, 127.47, 126.18, 83.12, 61.97, 58.95, 56.42, 53.18, 52.86, 44.04, 29.29, 26.05, 18.79. HRMS(ESI):m / z calculated for C 26 H 30 C1N 7 O 5 S[M+H] + :588.1790;found:588.1781.

[0034] (3) Accurately weigh Da-COOH (0.9 g, 2.0 mmol) obtained in step (2) and place it in a 50 mL round-bottom flask, add 30 mL of anhydrous DMF solution and stir to mix evenly, then accurately weigh NHS (0.3 g, 2.4 mmol) and EDCI (0.5 g, 2.4 mmol) to the mixed reaction solution, and react in an ice-water bath for 1 h. Subsequently, accurately weigh adamantane amine (0.3 g, 2.1 mmol) is added to the mixed solution, and the reaction is stirred at room temperature for 12 h. After the reaction is completed, the reaction solution is transferred to a mixture of 300 mL of acetone and water (1:4) to form a precipitate, which is then vacuum filtered. Finally, vacuum dried at 50°C for 24 h to obtain 0.72 g of a white solid compound Da-Ad with a yield of 65%. 1 H NMR (400 MHz, DMSO-d 6) δ = 10.21 (s, 1H), 8.79 (s, 1H), 8.46 (s, 1H), 7.35 (d, J = 7.4Hz, 1H), 7.32 (dd, J = 10.6, 7.5Hz, 2H), 5.68 (s, 1H), 4.35 (t, J = 5.7Hz, 2H), 3.6 2 (s, 4H), 3.44 (s, 4H), 2.97 (t, J=5.7Hz, 2H), 2.52 (s, 4H), 2.40 (s, 3H), 2.23 (s, 3H), 2.21 (s, 2H), 1.98 (s, 4H), 1.87 (s, 3H), 1.76 (s, 6H). 13 C NMR (101 MHz, DMSO-d 6 )δ=173.60, 172.70, 169.23, 165.43, 161.31, 158.43, 143.58, 138.25, 135.63, 130.73, 128.93, 127.78, 127.47, 126.18, 1 21.52, 79.32, 62.15, 57.65, 56.36, 52.08, 51.26, 47.89, 43.94, 41.16, 36.48, 32.12, 29.29, 26.05, 24.65, 18.79, 16.58. HRMS(ESI):m / z calculated forC 36 H 45 C1N 8 O 4 S[M+H] + :721.3257;found:721.3296.

[0035] (4) Dissolve accurately weighed ethylenediamine (1.5 g, 25 mmol) in 50 mL of anhydrous dichloromethane solution and stir in an ice bath. Then, dissolve weighed p-toluenesulfonyl chloride (0.95 g, 5 mmol) in 5 mL of anhydrous dichloromethane solution and slowly add dropwise to the reaction system. Continue the reaction at room temperature for 24 h. 2 After the reaction, column chromatography (CH 2 Cl 2 / CH 3 OH30:1 v / v) was used to purify the mixture, and finally 0.75 g of light yellow solid compound ET was obtained with a yield of 72%. 1 H NMR (400 MHz, DMSO-d 6 ) δ = 7.66 (s, 2H), 7.39 (s, 2H), 3.37 (s, 2H), 2.76 (t, J = 5.7Hz, 2H), 2.43 (s, 3H). 13C NMR (101 MHz, DMSO-d 6 )δ=137.86, 137.25, 129.78, 129.43, 128.31, 127.28, 44.13, 39.28, 27.39. HRMS(ESI):m / z calculated for C 9 H 14 N 2 O 2 S[M+H] + :214.0846;found:214.0913.

[0036] (5) Accurately weighed Ce 6 (48.0 mg, 0.08 mmol), HOBT (16.0 mg, 0.12 mmol) and EDCI (23.0 mg, 0.12 mmol) were dissolved in 3 mL of anhydrous DMF solution and reacted at room temperature in the dark for 30 min. Then, ET (21.4 mg, 0.10 mmol) obtained in step (4) was added and the reaction was continued for 24 h. 2 After the reaction, column chromatography (CH 2 Cl 2 / CH 3 OH30:1 v / v) was used to purify the mixture, and finally 18.3 mg of brown-black solid compound CET was obtained with a yield of 29%. 1 H NMR (400 MHz, DMSO-d 6 )δ=7.86(s, 2H), 7.54(s, 2H), 6.65(s, 1H), 6.46(d, J=6.4Hz, 1H), 5.32(dd , J=10.6, 7.5Hz, 2H), 5.16 (s, 1H), 4.35 (t, J=5.7Hz, 2H), 3.66 (s, 2H), 3.37 (s, 2H), 2.92 (t, J=5.7Hz, 2H), 2.44 (s, 2H), 2.40 (s, 3H), 2.35 (s, 3H), 2.1 6(s, 6H), 2.05(s, 2H), 2.01(s, 1H), 1.55(s, 2H), 0.87(s, 3H), 0.76(s, 3H). 13 C NMR (101 MHz, DMSO-d 6)δ=172.24, 171.46, 169.58, 164.79, 150.28, 148.65, 147.58, 146.72, 145.75, 144.5 8, 142.15, 139.68, 138.48, 137.47, 136.63, 134.56, 131.84, 130.53, 129.92, 129.23, 128.78, 128.37, 125.69, 123.38, 119.32, 113.15, 97.68, 96.56, 95.28, 54.36, 42.49, 38.64, 38.18, 34.58, 30.15, 28.89, 21.65, 19.75, 17.65, 15.69, 12.69, 11.58, 10.79. HRMS(ESI):m / z calculated forC 43 H 48 N 6 O 7 S[M+H] + :792.3248;found:792.3286.

[0037] (6) Dissolve the Poly-β-CD (21.0 mg) obtained in step (1) and the CET (2.0 mg) obtained in step (5) in a mixed solution of DMSO and water (1:3) and react at room temperature in the dark for 2 h. Subsequently, dissolve 0.5 mg of Da-Ad obtained in step (3) in a mixed solution of DMSO and water (1:3) and then slowly add it dropwise to the reaction system. Continue to react for 12 h at room temperature in the dark. After the reaction is completed, transfer the reaction mixture to a dialysis bag with MW = 3000 and dialyze for 24 h, changing the water every 3 h. Finally, freeze-dry the reaction solution to obtain a brown-black solid powder Da-CD@CETNPs.

[0038] Nanomaterial characterization (see Appendix Figure 1 )

[0039] The morphology of Da-CD NPs and Da-CD@CET NPs was characterized by transmission electron microscopy (TEM) at room temperature. Secondly, the hydrodynamic size of Da-CD NPs and Da-CD@CETNPs in aqueous solution was measured by Malvern Nano-ZS ZEN3600. TEM images showed that both nanomaterials presented spherical structures with sizes of about 60nm. Then, the hydrodynamic diameter and stability of Da-CD NPs and Da-CD@CET NPs were further confirmed by dynamic light scattering (DLS). The average hydrodynamic diameters were about 61nm, which was almost consistent with the TEM data, and the polydispersity indexes were 0.23 and 0.22, respectively, indicating that the preparation of Da-CD@CET NPs did not change the original morphology of Da-CD NPs, and also showed a smaller particle size, which was more conducive to tumor cell endocytosis.

[0040] Material stability characterization (see Appendix Figure 2 )

[0041] The stability of materials has always been the premise and key to their application. For this reason, we studied the stability of various nanomaterials. Take 2 mg of Da-CD NPs, CD@CETNPs, and Da-CD@CETNPs solid powders, dissolve them in 3 mL of deionized water, and observe their solution states. After 7 days in deionized water, Da-CD NPs, CD@CET NPs, and Da-CD@CET NPs did not show obvious turbidity and precipitation, which was consistent with the initial state of the solution on the first day, and all nanomaterials showed a small PDI, which remained basically unchanged within 7 days. At the same time, after being treated in deionized water for 7 days, its average hydrodynamic diameter was stable without obvious size changes, and the size of the target drug Da-CD@CET NPs in deionized water and culture medium hardly changed within 7 days, indicating that these poly-β-CD-based nanomaterials have long-term stability and uniformity. The drug loading capacity (DLC) and drug loading efficiency (DLE) of CET calculated by UV-visible spectrophotometry and based on the drug standard curve were 5.02±0.13% and 79.90±0.35%, respectively, which also indicates that Da-CD NPs have good drug loading capacity.

[0042] Cell targeting characterization (see Appendix Figure 3 )

[0043] (1) Cell plating: First, select 4T1 cells in good growth condition, remove the old culture medium, wash three times with sterile PBS, and then digest with 0.25% trypsin. After centrifugation, add fresh culture medium and resuspend. Count the cell suspension with a cell counter, and then dilute the cell suspension to 2.0×10 5 Finally, all confocal dishes were placed in a 37°C, 5% CO 2 Incubate overnight in a cell culture incubator.

[0044] (2) Adding drugs: After the cells are completely attached, the culture medium in the laser confocal dish is removed, and then sterile PBS is used to wash three times. Finally, 1 mL of fresh culture medium with a concentration of 20 μg / mL Da-CD@CET NPs or CD@CET NPs is added to the laser confocal dish and placed in a 37°C, 5% CO 2 Incubate in a cell culture incubator for 12 h.

[0045] (3) Test: After the incubation, remove the drug-containing culture medium in all laser confocal dishes, wash them three times with sterile PBS, and then add 1 mL of sterile PBS and wait for photography. At the same time, add 0.5 mL of 0.25% trypsin to other washed confocal dishes for digestion, centrifuge and resuspend in 1 mL of sterile PBS. Finally, laser confocal electron microscopy and flow cytometer were used for intracellular drug fluorescence testing (excitation wavelength: 633 nm, emission wavelength: 650-750 nm). In the experiment, all cell blank control groups were incubated with drug-free culture medium, and other operations were consistent with the experimental group.

[0046] The fluorescence intensity of Da-CD@CET NPs in cells was detected by laser confocal microscopy and flow cytometry. Da-CD@CET NPs showed a stronger fluorescence signal intensity than Da-CD@CET NPs, about 3 times that of Da-CD@CET NPs, indicating that Da-CD@CETNPs exhibited stronger cell-targeted uptake ability. This shows that the introduction of Dashatinib greatly enhanced the specific recognition of nanomaterials to 4T1 cells, thereby promoting cell uptake. Targeted competitive studies were performed to further reveal whether the enhanced uptake of Da-CD@CET NPs in 4T1 cells was mediated by Dashatinib. Before incubation with Da-CD@CET NPs, different concentrations of free Dashatinib (0, 5, 10 and 20 nM) were pre-incubated with 4T1 cells for 12 h, which would result in a certain amount of SRC protein being occupied and then reduced in number. The results of laser confocal microscopy and flow cytometry showed that the intracellular fluorescence intensity of Da-CD@CETNPs decreased with the increase of free Dashatinib concentration, indicating that more Da-CD@CETNPs can be accumulated in 4T1 cells through SRC receptor-mediated endocytosis due to the presence of the targeting unit Dashatinib. Therefore, all the results of cell uptake experiments indicate that Dashatinib-modified nanocarriers can significantly improve the targeting ability of Da-CD@CETNPs to triple-negative breast cancer cells overexpressing SRC protein.

[0047] ER targeting characterization (see Appendix Figure 4 )

[0048] (1) Plate plating: First, select 4T1 cells in good growth condition, remove the old culture medium, wash three times with sterile PBS, and then digest with 0.25% trypsin. After centrifugation, add fresh culture medium and resuspend. Count the cell suspension with a cell counter, and then dilute the cell suspension to 1.0×10 4 Finally, all confocal dishes were placed in a 37°C, 5% CO 2 Incubate overnight in a cell culture incubator.

[0049] (2) Adding drugs: After the cells are completely attached to the wall, the culture medium in the laser confocal dish is removed, and then sterile PBS is used to wash three times. Finally, 1 mL of fresh culture medium containing Ce6, CET or Da-CD@CET NPs at a concentration of 20 μg / mL is added to the laser confocal dish and placed in a 37°C, 5% CO 2 Incubate in a cell culture incubator for 12 h.

[0050] (3) Staining: First, take out 2 μL of the mother solution of the endoplasmic reticulum green probe with a concentration of 1 mM and mix it with the new culture medium to finally make the concentration of the probe solution 2 μM. Then, remove the drug-containing culture medium in the laser confocal dish, wash it three times by centrifugation with sterile PBS, and then resuspend the cells in 1 mL of the culture medium containing the probe dye and transfer them to the corresponding laser confocal dish. Finally, place it at 37°C and 5% CO 2 Incubate in a cell culture incubator for 20 min.

[0051] (4) Photography: After the co-incubation is completed, the corresponding probe dye in the dish is removed by centrifugation, and sterile PBS is used for centrifugation and washing three times to remove the excess probe dye to prevent interference with the photography background. Then a small amount of PBS is added to resuspend the cells, and the cells are transferred to a new confocal dish before taking pictures. Finally, the cells are photographed using a laser confocal microscope equipped with a 405nm or 488nm multi-argon laser and a 633nm diode laser. Among them, the excitation and emission wavelength parameters of the photosensitizer compound are: Ex / Em=633 / 650-750nm; the excitation and emission wavelength parameters of the endoplasmic reticulum probe dye are: Ex / Em=488 / 510-570nm.

[0052] Laser confocal microscopy was used to observe the red fluorescence of Ce6 and CET and the green fluorescence of the endoplasmic reticulum probe. There was almost no overlap between the red fluorescence of free Ce6 and the green fluorescence signal representing the endoplasmic reticulum. Further qualitative analysis of the line scanning spectrum of fluorescence intensity showed that the Ce6 group and the endoplasmic reticulum dye had significant separation signals ( Figure 4 b). By introducing p-toluenesulfonamide, the red fluorescence signal of the free CET group showed obvious fluorescence overlap with the endoplasmic reticulum dye after incubation for 12 hours, and yellow fluorescent spots appeared in the endoplasmic reticulum. The qualitative analysis of the line scanning spectrum of fluorescence intensity also showed that the CET group and the endoplasmic reticulum dye had partially overlapping signals, indicating that the free CET has shown visible endoplasmic reticulum targeting ability. Compared with free CET, strong yellow fluorescent spots appeared in the endoplasmic reticulum in the Da-CD@CET NPs group. At the same time, the qualitative analysis of the line scanning spectrum of fluorescence intensity also showed a high degree of overlap between CET and the endoplasmic reticulum dye. Therefore, these data strongly prove that Da-CD@CET NPs can significantly enhance the enrichment of photosensitizers in the endoplasmic reticulum and achieve the purpose of targeting the endoplasmic reticulum.

[0053] In vitro photodynamic activity characterization (see Appendix Figure 5 )

[0054] During photodynamic therapy, the cytotoxicity exhibited by photosensitizers is one of the main research contents for evaluating their properties. The MTT method is currently a widely used experimental method for evaluating the ability of drugs to induce cell apoptosis. MTT (3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide) is a yellow dye, and its trade name is thiazolyl blue. The detection principle of MTT can be summarized as follows: the mitochondria of living cells contain a reducing succinate dehydrogenase, which can reduce the yellow MTT to an insoluble blue-purple crystalline substance formazan and deposit it in the cells. The mitochondria in dead cells do not contain this reducing succinate dehydrogenase, and thus cannot reduce MTT or generate blue-purple crystalline formazan. Related experiments have shown that the organic solvent dimethyl sulfoxide (DMSO) can dissolve the formazan deposited in the cells, and the light absorption value (OD value) at a wavelength of 490nm or 570nm is measured by a microplate reader, thereby indirectly obtaining the corresponding number of living cells. The formula for calculating cell viability (I%) is as follows:

[0055] I%=[(A-A0) / (As-A0)]×100%

[0056] Among them, A, A 0 and As are the OD values ​​of the cell drug group, blank solvent control group, and cell solvent control group, respectively. During the experiment, the logarithm of the corresponding drug concentration was taken as the horizontal axis, and the cell survival rate (%) was taken as the vertical axis. The experimental data were expressed as the mean value of three independent experiments (Mean±SD). Finally, the experimental data were plotted using GraphPad Prism 6.0 software to obtain the relationship curve between cytotoxicity and drug dosage.

[0057] Experimental methods

[0058] (1) Plating: Select 4T1 cells with good growth status, remove the old culture medium, and wash three times with sterile PBS. Then, add 1 mL of trypsin (containing 0.25% EDTA) for digestion and centrifugation. Resuspend the cells in fresh cell culture medium, count them using a cell counter, and dilute them to a density of 8.0×10 4 Then, 100 μL of the diluted cell suspension was added to each well of the 96-well plate. Different drug concentration groups and all control groups were set up with 6 replicate wells to reduce experimental errors. Finally, all 96-well plates were placed in a 37°C, 5% CO 2 The cells were cultured adherently in a cell culture incubator.

[0059] (2) Adding drugs: First, remove the old culture medium in the 96-well plate and wash each well three times with sterile PBS. Then, add the drugs prepared in fresh cell culture medium to different 96-well plates in turn and place them in a 37°C, 5% CO 2 Incubate in a cell culture incubator for 12 h.

[0060] (3) Illumination: After incubation, remove the old medium containing drugs in the 96-well plate and wash it three times with sterile PBS to completely remove the unabsorbed drugs. Then, add 100 μL of new cell culture medium to each well, and place the 96-well plate for phototoxicity experiment under the LED laser board (λ = 670 nm, 4 mW / cm 2 , 1.2J / cm 2 Finally, place in 37°C, 5% CO 2 The cells were incubated in a cell culture incubator for 24 h. The dark toxicity experiment was conducted without light treatment, and the other operation steps were consistent with the light toxicity experiment and were performed in a light-proof environment.

[0061] (4) Test: After 24 hours of culture, the 96-well plates of the two cells were taken out of the incubator, and 10 μL of the pre-prepared MTT solution was added to each well, and then the plates were placed in the cell incubator for another 4 hours. Then, the supernatant in the 96-well plates was removed, and 100 μL of DMSO solvent was added to each well, and the plates were placed in a 37°C shaker for 30 minutes to fully dissolve the formazan that might have been generated. Finally, the OD value of the 96-well plates at 570 nm was measured using an ELISA reader.

[0062] The experimental data show that in the absence of laser irradiation, no obvious cytotoxicity was observed in all drug groups on 4T1 cells, indicating that different drug groups have negligible dark toxicity. 2 , 1.2J / cm 2 ) irradiated for 5 min, the photocytotoxicity of the Da-CD@CET NPs group was significantly higher than that of the single-targeted CET, CD@CETNPs group and the blank control group, indicating that within a smaller concentration range, the Da-CD@CETNPs with dual-targeting function exhibited a more significant killing ability against 4T1 cells, thereby achieving a stronger anti-tumor cell proliferation ability.

[0063] Characterization of photodynamic activity in vivo (see Appendix Figure 6 )

[0064] 200 μL of 4T1 cells (2×10 6 The tumor model was established by subcutaneously inoculating the mouse right hind limb with saline solution containing 100 cells. The growth of the tumor was closely observed. After 5 days, when the tumor grew to 50-100 mm 3Size (length × width 2 / 2 to calculate the tumor volume), and all mice were randomly divided into five groups (Control, Ce6, CET, CD@CETNPs, Da-CD@CETNPs), with five mice in each group (n=5) for the experiment.

[0065] Before intravenous injection of drugs, the mice were weighed and the tumor size was measured. Then, each mouse was intravenously injected with different drugs (100ug / mL, 200μL) and saline solution according to the group. After 12h of administration, the tumor area was irradiated with a 670nm laser for 10min, and the tumor volume was monitored every other day. The growth status of the mice and their tumor size showed that the single-targeted material groups including free Ce6, CET and CD@CET NPs groups could only partially inhibit tumor growth, while the Da-CD@CET NPs group almost completely inhibited tumor growth, which fully demonstrated that the dual-targeted nanomaterial photosensitizer improved the tumor inhibition ability of PDT. At the same time, the tumor in the Da-CD NPs group grew rapidly, similar to the control group. According to the relative tumor growth curve drawn by the measurement data, it can be clearly observed that the Da-CD@CET NPs group can almost completely inhibit tumor growth. In addition, the weight of the tumor removed after 14 days obtained the same tumor inhibition results. All experimental results indicate that Da-CD@CET NPs with dual targeting function can accurately deliver photosensitizers to triple-negative breast cancer cells and further deliver them to the endoplasmic reticulum, thereby achieving targeted photodynamic therapy for triple-negative breast cancer and exhibiting significant in vivo tumor inhibition efficacy.

Claims

1. A method for preparing a nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer, comprising the following steps: (1) β-CD is dissolved in a NaOH aqueous solution and reacted under the action of a cross-linking agent to obtain Poly-β-CD; (2) adding anhydrous DMF to Dasatinib and succinic anhydride, and reacting with triethylamine to obtain Da-COOH; (3) adding the Da-COOH obtained in step (2) to anhydrous DMF solution, then adding NHS and EDCI to the mixed reaction solution, and reacting in an ice-water bath to obtain Da-Ad; (4) dissolving ethylenediamine in an anhydrous dichloromethane solution, dissolving p-toluenesulfonyl chloride in an anhydrous dichloromethane solution, slowly adding the two solutions dropwise to the reaction system, and reacting at room temperature to obtain ET; (5) dissolving Ce 6, HOBT and EDCI in anhydrous DMF solution, adding ET obtained in step (4) to continue the reaction to obtain CET; (6) dissolving the Poly-β-CD obtained in step (1) and the CET obtained in step (5) in a mixed solution of DMSO and water, dissolving the Da-Ad obtained in step (3) in a mixed solution of DMSO and water, and then slowly adding them dropwise to the reaction system to react to obtain the final product Da-CD@CETNPs; In step (3), 0.9 g, 2.0 mmol of Da-COOH obtained in step (2) was accurately weighed and placed in a 50 mL round-bottom flask, 30 mL of anhydrous DMF solution was added to stir and mix evenly, and then 0.3 g, 2.4 mmol of NHS and 0.5 g, 2.4 mmol of EDCI were accurately weighed and added to the mixed reaction solution, and the mixture was reacted in an ice-water bath for 1 h. Subsequently, 0.3 g, 2.1 mmol of adamantane amine was accurately weighed and added to the mixed solution, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, the reaction solution was transferred to 300 mL of a mixture of acetone and water in a dosage ratio of 1:4 to form a precipitate, which was vacuum filtered and finally vacuum dried at 50° C. for 24 h to obtain 0.72 g of a white solid compound Da-Ad.

2. The method for preparing a nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer according to claim 1, characterized in that: In step (1), firstly, 10.0 g of β-CD is accurately weighed and dissolved in 15 mL of 15 WT% NaOH aqueous solution, and stirred at room temperature for 2 h. Subsequently, 2 mL of toluene is added dropwise to the mixture and stirring is continued for 2 h. Then, 3.8 mL of epichlorohydrin as a cross-linking agent is slowly added dropwise and the reaction is continued for 3 h. After the reaction is completed, the crude product is pre-precipitated with 200 mL of isopropanol, the precipitate is transferred to an appropriate amount of water and adjusted to neutral with dilute hydrochloric acid, and finally, it is dialyzed with a dialysis bag of MW=8000 for 2 days, and the water is changed every 3 h to completely remove monomers and oligomers, and finally vacuum freeze-dried to obtain a white solid product Poly-β-CD.

3. The method for preparing a nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer according to claim 1, characterized in that: In step (2), 1.60 g, 3.27 mmol of Dasatinib and 0.37 g, 3.70 mmol of succinic anhydride are accurately weighed and placed in a 100 mL double-necked flask, then 15 mL of anhydrous DMF is added to the double-necked flask to stir evenly, and 0.70 g, 6.90 mmol of triethylamine is accurately weighed and slowly added dropwise to the mixed reaction, and finally, nitrogen protection is introduced, and the reaction is continued for 48 hours under stirring at room temperature. After the reaction is completed, the reaction system is vacuum-rotated and evaporated to remove DMF, and the mixture is purified by column chromatography, and finally the collected organic solvents are mixed and evaporated to obtain 1.73 g of white solid compound Da-COOH; The volume ratio of CH2Cl2 / CH3OH in column chromatography was 30:

1.

4. The method for preparing a nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer according to claim 1, characterized in that: In step (4), 1.5 g, 25 mmol of ethylenediamine accurately weighed was dissolved in 50 mL of anhydrous dichloromethane solution, and stirred in an ice bath. Subsequently, 0.95 g, 5 mmol of p-toluenesulfonyl chloride weighed was dissolved in 5 mL of anhydrous dichloromethane solution, and then slowly added dropwise to the reaction system. The reaction was continued at room temperature for 24 h, with N2 protection. After the reaction was completed, the mixture was purified by column chromatography to obtain 0.75 g of a light yellow solid compound ET. The volume ratio of CH2Cl2 / CH3OH in column chromatography was 30:

1.

5. The method for preparing a nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer according to claim 1, characterized in that: In step (5), 48.0 mg, 0.08 mmol of Ce 6, 16.0 mg, 0.12 mmol of HOBT and 23.0 mg, 0.12 mmol of EDCI were accurately weighed and dissolved in 3 mL of anhydrous DMF solution, and reacted at room temperature in the dark for 30 min, and then 21.4 mg, 0.10 mmol of ET obtained in step (4) was added, and the reaction was continued for 24 h, with N2 protection. After the reaction was completed, the mixture was purified by column chromatography to obtain 18.3 mg of a brown-black solid compound CET; The volume ratio of CH2Cl2 / CH3OH in column chromatography was 30:

1.

6. The method for preparing a nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer according to claim 1, characterized in that: In step (6), 21.0 mg of Poly-β-CD obtained in step (1) and 2.0 mg of CET obtained in step (5) are dissolved in a mixed solution of DMSO and water in a dosage ratio of 1:3, and reacted at room temperature in the dark for 2 hours. Subsequently, 0.5 mg of Da-Ad obtained in step (3) is dissolved in a mixed solution of DMSO and water in a dosage ratio of 1:3, and then slowly added dropwise to the reaction system, and the reaction is continued at room temperature in the dark for 12 hours. After the reaction is completed, the reaction mixture is transferred to a dialysis bag with MW = 3000 and dialyzed for 24 hours, and the water is changed every 3 hours. Finally, the reaction solution is freeze-dried to obtain a brown-black solid powder Da-CD@CETNPs.

7. A nano-delivery material for dual-targeted photodynamic therapy of triple-negative breast cancer, characterized by: It is prepared by the method according to any one of claims 1 to 6.

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