Tumor stimulation response type photosensitizer and camptothecin coupled prodrug compound, supramolecular assembly, preparation method and application thereof

Abstract

The invention belongs to the technical field of chemical pharmacy, and particularly relates to a tumor stimulation response type photosensitizer and camptothecin coupled prodrug compound, a supramolecular assembly, a preparation method and application thereof. The invention provides a prodrug compound I-1 coupled by a tumor stimulation response type photosensitizer and camptothecin and pharmaceutically acceptable salt thereof, and provides a micro-nano structure formed by self-assembly of the prodrug compound I-1 and application of the compound in preparation of drugs for diagnosis and / or cancer treatment. Through the combined effect of photo-thermal, photodynamic therapy and chemotherapy, the tumor treatment efficiency can be remarkably improved, and side effects are reduced; not only is targeted killing of tumor cells enhanced, but also MPR1 protein expression related to tumor drug resistance is inhibited. Therefore, the combined treatment generates a synergistic effect, the drug resistance is reduced, the tumor treatment effect is obviously enhanced, and the treatment effect of 1 + 1 > 2 is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical pharmaceuticals, and particularly relates to a prodrug compound in which a tumor-stimulus-responsive photosensitizer is conjugated with camptothecin, a supramolecular assembly, a preparation method thereof, and an application thereof. Background Art

[0002] Traditional cancer treatment methods have always faced the problems of limited efficacy and large side effects. For example, surgical, chemotherapy, and radiotherapy methods, although effective, have challenges such as drug resistance and toxic side effects. Therefore, finding new, more selective, and low-side-effect treatment methods has become the current research focus.

[0003] Photothermal therapy (PTT) and photodynamic therapy (PDT) have received extensive attention in tumor treatment in recent years due to their high selectivity, non-invasiveness, and relatively low side effects. Photosensitizers are the core components of these two treatment methods and can generate a thermal effect or singlet oxygen under laser irradiation, thereby effectively killing tumor cells. However, the low targeting and instability of photosensitizers are still the main obstacles affecting their clinical applications. To improve their targeting, researchers have used nanocarriers to achieve drug enrichment through the enhanced permeability and retention (EPR) effect of tumors. However, how to precisely control drug release remains a challenge.

[0004] Camptothecin, as an important anti-cancer drug, has significant anti-tumor activity, but its low bioavailability and poor solubility in vivo limit its clinical application.

[0005] Based on this, the present invention conjugates a photosensitizer with camptothecin and uses a self-assembled nanostructure for drug delivery, which can improve the targeting of the drug, simultaneously achieve the synergistic effect of multiple treatment modes, and enhance the overall effect of tumor treatment. Thus, a composite prodrug system that can self-assemble into a nanostructure, enrich at the tumor site through the EPR effect, and release the drug under specific conditions is developed, which becomes the key technology for achieving efficient and multimodal cancer treatment. Summary of the Invention

[0006] The object of the present invention is to solve the above problems existing in the prior art, and a prodrug compound in which a tumor-stimuli responsive photosensitizer is conjugated with camptothecin, a supramolecular assembly, a preparation method thereof and an application are provided. The prodrug system provided by the present invention has a significant synergistic effect in multimodal therapy, and can significantly improve the efficiency of tumor treatment and reduce side effects through the combined effects of photothermal, photodynamic therapy and chemotherapy. After the combined phototherapy-chemotherapy treatment, the expression of the multidrug resistance-related protein (MPR1 protein) is significantly decreased, indicating that the treatment not only enhances the targeted killing of tumor cells, but also inhibits the expression of the MPR1 protein related to tumor drug resistance. Therefore, the combined phototherapy and chemotherapy treatment produces a synergistic effect, reduces drug resistance, and significantly enhances the tumor treatment effect, thus achieving a treatment effect of 1 + 1 > 2.

[0007] The technical solution of the present invention is as follows:

[0008] The present invention provides a prodrug compound in which a tumor-stimuli responsive photosensitizer is conjugated with camptothecin, having a structure shown in the following formula (I-1):

[0009]

[0010] The present invention also provides a supramolecular assembly based on the above-mentioned prodrug compound, and the supramolecular assembly is a micro-nano structure self-assembled by the prodrug compound in an aqueous solution. The preparation method of the supramolecular assembly includes the following steps:

[0011] Dissolve the prodrug compound having the structure shown in formula (I-1) using dimethyl sulfoxide as a solvent;

[0012] Add the obtained solution to water to obtain a final concentration of the prodrug compound of 0.2 μM to 500 μM;

[0013] The prodrug compound self-assembles in this solution to form an I-1 micro-nano structure.

[0014] Wherein the content of dimethyl sulfoxide is ≤1%.

[0015] The above preparation method is simple and convenient, and is suitable for large-scale production.

[0016] Design and Synthesis of Prodrug Compounds: A photosensitizer and camptothecin are chemically conjugated to form a composite prodrug compound, which can self-assemble into micro-nano structures. Due to the rich blood vessels and lack of lymphatic drainage system in tumor (especially solid tumor) tissues, the micro-nano structures described in the present invention have passive high permeability and retention at the tumor site. This high permeability effect and retention effect of the micro-nano structures in solid tumor tissues are called the EPR effect. This ability to passively target tumors gives such small molecule compounds that can form micro-nano structures through supramolecular assembly significant advantages over other reported small molecule photosensitizers.

[0017] Drug Release Mechanism: In the tumor microenvironment, excessive glutathione (GSH) can trigger the disassembly of the prodrug compound, promoting the release of the photosensitizer and camptothecin. Since the concentration of glutathione in normal cells is relatively low, the nanostructures remain stable in normal cells and do not undergo disassembly, thus avoiding drug release and damage to normal cells. Only within tumor cells, the increase in glutathione concentration triggers the disassembly of the nanostructures, enabling the photosensitizer and camptothecin to be specifically released to the tumor site, enhancing tumor targeting and therapeutic effects while reducing side effects on normal tissues.

[0018] Application of Photosensitizer: The released photosensitizer can be used for tumor imaging, photothermal therapy, and photodynamic therapy. The photosensitizer has high photothermal conversion efficiency, high singlet oxygen yield, and good photothermal stability, and can effectively kill tumor cells.

[0019] Application of Camptothecin: The released camptothecin can exert its anti-tumor effect, perform chemotherapy, and enhance the comprehensive treatment effect.

[0020] Advantages of the Composite Prodrug System: The composite prodrug system of the present invention is enriched at the tumor site through the EPR effect, combining the multiple effects of photothermal therapy, photodynamic therapy, and chemotherapy, which can effectively improve the efficiency of tumor treatment while reducing side effects. Through the combined application of phototherapy and chemotherapy, the photosensitizer and camptothecin can act synergistically and play their respective advantages. Photothermal therapy and photodynamic therapy can directly kill tumor cells and promote the heat stress of tumor cells, thereby enhancing the chemotherapy effect of camptothecin and breaking through the drug resistance mechanism of tumor cells. Camptothecin, on the other hand, further inhibits the growth and spread of tumors through the chemotherapy mechanism, enhancing the anti-tumor effect of phototherapy. The combined treatment of the two produces an effect of 1 + 1 > 2, significantly improving the treatment effect through synergistic action and reducing the side effects and drug resistance problems that may be brought about by single treatment methods. This combined treatment strategy provides a more efficient and safe tumor treatment plan.

[0021] The present invention also provides pharmaceutically acceptable salts of the above-mentioned prodrug compounds.

[0022] The present invention further provides a supramolecular assembly of a pharmaceutically acceptable salt of the prodrug compound described above, and the supramolecular assembly is a micro-nano structure self-assembled from the pharmaceutically acceptable salt of the prodrug compound in an aqueous solution. The preparation method of the supramolecular assembly comprises the following steps:

[0023] Dissolve the pharmaceutically acceptable salt of the prodrug compound having the structure shown in formula (I-1) using dimethyl sulfoxide as a solvent;

[0024] Add the obtained solution to water to obtain a final concentration of the compound (i.e., the pharmaceutically acceptable salt of the prodrug compound) of 0.2 μM to 500 μM;

[0025] The compound self-assembles in the aqueous solution to form a micro-nano structure. The content of dimethyl sulfoxide is ≤1%.

[0026] The above preparation method is simple and convenient, and suitable for large-scale production.

[0027] Furthermore, the particle size of the micro-nano structure is 30 nm to 200 nm. Preferably, the particle size of the micro-nano structure is 30 to 120 nm.

[0028] The present invention also provides a pharmaceutical composition, comprising any one or several of the above-mentioned prodrug compound, the pharmaceutically acceptable salt of the prodrug compound, the supramolecular assembly of the prodrug compound, and the supramolecular assembly of the pharmaceutically acceptable salt of the prodrug compound; and further comprising a pharmaceutically acceptable carrier.

[0029] The present invention also provides the use of the above-mentioned prodrug compound or its supramolecular assembly in the preparation of a drug for diagnosing and / or treating cancer.

[0030] The present invention also provides the use of the pharmaceutically acceptable salt of the above-mentioned prodrug compound or its supramolecular assembly in the preparation of a drug for diagnosing and / or treating cancer.

[0031] Furthermore, the cancer includes breast cancer; the treatment is a combined treatment, specifically, including the combined treatment of phototherapy and chemotherapy, or including the combined treatment of any two or three of photothermal therapy, photodynamic therapy, and chemotherapy.

[0032] Advantages of the present invention:

[0033] (1) Multimodal therapeutic effect: The prodrug system of the present invention combines the multiple effects of photothermal therapy, photodynamic therapy, and chemotherapy. Through multimodal therapy, this system can synergistically exert its effects during the same treatment process, acting synergistically on tumor treatment, thereby significantly improving the efficiency of tumor treatment while reducing side effects. With its tumor-specific drug release, excellent biocompatibility, and controllable drug release characteristics, the prodrug system and micro / nano structure of the present invention have shown broad application prospects in multimodal imaging-guided cancer combination therapy, especially having important technical value in the field of chemical pharmaceuticals.

[0034] (2) Precise targeting and efficient drug release: Through the enhanced permeability and retention (EPR) effect at the tumor site, the nanostructures of the present invention can be effectively enriched in the tumor region, reducing the impact on normal tissues. The nanostructures disassemble through the trigger of excessive glutathione in the tumor microenvironment, achieving the precise release of photosensitizers and camptothecin, and optimizing the treatment timing. In normal cells, due to the low content of glutathione, it is difficult to break the bond of I-1, and almost no CPT and photosensitizers are released, thus avoiding damage to normal cells.

[0035] (3) Safety and controllability: The prodrug system has good biocompatibility and degradability, can stably circulate in the body, and controls the drug release rate, reducing the risks of side effects and toxicity.

[0036] (4) High-performance photosensitizers: The photosensitizers have high photothermal conversion efficiency, high singlet oxygen yield, good photothermal stability, strong photothermal effect, and photodynamic effect. The release of photosensitizers can not only be used for tumor imaging but also play a powerful therapeutic role in photothermal therapy and photodynamic therapy, enhancing the killing effect on tumor cells.

[0037] (5) Improving the cellular uptake rate of CPT: In traditional chemotherapy, the drug resistance of CPT is usually related to the overexpression of drug efflux pumps (such as P-gp) on the tumor cell membrane; these efflux pumps pump the drug out of the tumor cells, resulting in too low drug concentration and reducing the treatment effect. However, the prodrug system designed in the present invention can enhance the endocytosis of drugs through self-assembled nanostructures. Under the activation of the photothermal effect, the membrane structure of tumor cells changes to a certain extent, leading to an increase in the cell membrane permeability, and CPT can enter the cells more efficiently, thereby significantly increasing the effective drug concentration. In addition, the release of photosensitizers not only provides imaging and phototherapy effects but also can accelerate the drug release through the photothermal effect, further enhancing the effect of CPT in tumor cells.

[0038] (6) Synergistic therapy reduces drug resistance: Camptothecin (CPT), as an effective chemotherapeutic drug, exerts its antitumor effect by inhibiting DNA topoisomerase I in tumor cells, thereby preventing DNA replication and repair. However, during the course of treatment, tumor cells gradually develop resistance to CPT, mainly manifested as overexpression of drug efflux pumps, changes in targets, and enhancement of repair systems. By combining a photosensitizer with CPT and utilizing the auxiliary effect of photothermal therapy, the targeted accumulation of CPT in tumor tissues can be increased, thereby enhancing the concentration of the drug within tumor cells and effectively overcoming drug resistance; in addition, photothermal therapy can, to a certain extent, trigger the heat shock response of tumor cells, promote the endocytosis of drugs, increase the cellular uptake of CPT, reduce the effect of efflux pumps, and achieve a "1 + 1 > 2" therapeutic effect. Description of the Drawings

[0039] Figure 1 It is the synthetic route diagram of compound I-1 provided by the present invention;

[0040] Figure 2 It is the ultraviolet absorption spectrum diagram of compound I-1 in acetonitrile and water;

[0041] Figure 3 It is the fluorescence emission spectrum diagram of compound I-1 in acetonitrile and water;

[0042] Figure 4 It is the dynamic light scattering (DLS) spectrum diagrams of compound I-1 in aqueous solution at 0 hour and 48 hours;

[0043] Figure 5 It is the transmission electron microscopy (TEM) image of the micro-nano structure formed by self-assembly of compound I-1 in aqueous solution;

[0044] Figure 6 It is the atomic force microscopy (AFM) image of the micro-nano structure formed by self-assembly of compound I-1 in aqueous solution;

[0045] Figure 7 It is the temperature change curve of the micro-nano structure of compound I-1 with different concentrations under 808 nm laser irradiation within 10 minutes;

[0046] Figure 8 It is the test result diagram of the photothermal stability of the micro-nano structure of compound I-1;

[0047] Figure 9 It is the reactive oxygen species (ROS) generation spectrum diagram of the micro-nano structure of compound I-1;

[0048] Figure 10 It is the camptothecin (CPT) release curve of the micro-nano structure of compound I-1 under different concentrations of glutathione or hydrogen peroxide conditions;

[0049] Figure 11 Fluorescence images of reactive oxygen species production by the I-1 compound micro / nano structure in L929 cells and 4T1 cells;

[0050] Figure 12 Dark toxicity and phototoxicity images of the I-1 compound micro / nano structure against 4T1 cells;

[0051] Figure 13 Photoacoustic imaging images of tumor-bearing mice at different time points after intravenous injection of the I-1 compound micro / nano structure;

[0052] Figure 14 Fluorescence imaging images of tumor-bearing mice at different time points after intravenous injection of the I-1 compound micro / nano structure;

[0053] Figure 15 Photographs of tumor-bearing mice within 20 days after intravenous injection of the I-1 compound micro / nano structure and combined treatment;

[0054] Figure 16 Changes in tumor volume of tumor-bearing mice within 20 days after intravenous injection of the I-1 compound micro / nano structure and combined treatment;

[0055] Figure 17 Expression of MPR1 (multidrug resistance-related protein) in tumors of different groups of tumor-bearing mice after 20 days. Detailed implementation mode

[0056] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0057] To further understand the present invention, the present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0058] Example 1 Synthesis of Compound I-1 and Its Fluorescent Properties

[0059] As Figure 1 shown, the synthesis of compound I-1 includes the following steps:

[0060] 1) Synthesis of compound 1:

[0061] 1.05 g of malononitrile (Compound 6) and 0.75 g of magnesium ethoxide were added to 60 mL of ethanol solution and stirred evenly. Then, 0.6 mL of 3-hydroxy-3-methylbutan-2-one (Compound 5) was added, and the reaction system was heated to 65 °C and reacted for 10 hours. After the reaction was completed, the solvent was evaporated under vacuum to obtain the solid after reaction. The solid was separated and purified by column chromatography and recrystallized with ethanol to finally obtain the target compound 1.

[0062] 1 H NMR (400 MHz, CDCl3): δ (ppm): 2.36 (s, 3H), 1.63 (s, 6H).

[0063] 2) Synthesis of Compound 2:

[0064] 2,3,3-Trimethylindoline (Compound 7, 318.46 mg, 2.00 mmol) and 2-iodoethanol (Compound 8, 467.10 mg, 2.60 mmol) were dissolved in 40 mL of acetonitrile. The resulting mixed solution was stirred and reacted at 90 °C for 48 hours under a nitrogen protection environment. After the reaction was completed, the obtained mixture was cooled to room temperature, and the precipitate was collected by filtration and sedimented three times from diethyl ether to obtain Compound 2 (296.86 mg), yield: 72.7%.

[0065] 1 H NMR (400 MHz, CDCl3): δ (ppm): 7.98 (m, 1H), 7.82 (m, 1H), 7.62 (m, 2H), 4.62 (t, 2H), 3.88 (t, 2H), 2.84 (s, 3H), 1.56 (s, 6H).

[0066] 3) Synthesis of Compound 3:

[0067] Under ice bath conditions, 20 mL of dichloromethane and 20 mL of DMF were mixed evenly; then, 17.5 mL of phosphorus oxychloride was slowly and evenly added to the solution and continuously stirred; then, 5.3 mL of cyclohexanone was added, the temperature was adjusted to 80 °C, and this temperature was maintained for 3 hours until the reaction was completed. After the reaction was completed, the mixture was quickly poured into ice water to terminate the reaction and left standing in the refrigerator overnight. Finally, the solvent was removed by vacuum evaporation to obtain the unpurified crude product Compound 3, which was directly used for the subsequent reaction.

[0068] 4) Synthesis of Compound 4:

[0069] Dissolve 1.00 g of Compound 1 and 1.27 g of Compound 3 in 50 mL of ethanol, and heat the mixed solution to 90 °C, then reflux for 12 hours. After the reaction is completed, allow the reaction system to cool to room temperature, and then separate the crude product Compound 4 by suction filtration. This crude product is directly used in the next reaction without purification.

[0070] 5) Synthesis of Compound Cy7-TCF-OH: Mix Compound 4 (200.00 mg, 0.52 mmol) and Compound 2 (117.70 mg, 0.58 mmol) in ethanol. Add a few drops of pyridine to this mixture. Stir the resulting mixture under nitrogen at 90 °C overnight. After the reaction is completed, cool the obtained mixture to room temperature, and then dry it under vacuum. Then purify it by column chromatography (dichloromethane:methanol = 50:1 volume ratio) to obtain Cy7-TCF-OH (128.16 mg). Yield: 45.39%. 1 H NMR (400 MHz, DMSO-d6): δ (ppm): 8.30 (d, J = 14.8 Hz, 1H), 8.24 (d, J = 14.4 Hz, 1H), 7.63 (d, J = 7.2 Hz, 1H), 7.44 (m, 2H), 6.46 (d, J = 14.0 Hz, 1H), 6.08 (d, J = 14.0 Hz, 1H), 5.06 (s, 1H), 4.30 (t, J = 5.2 Hz, 2H), 3.80 (t, J = 5.2 Hz, 2H), 2.66 (m, 4H), 1.81 (m, 2H), 1.67 (s, 6H), 1.60 (s, 6H).

[0071] 6) Synthesis of Compound Cy7-TCF-SS-COOH:

[0072] In anhydrous dichloromethane (25 mL), add Cy7-TCF-OH (250.00 mg, 0.47 mmol), 4,4'-dithiobutyric acid (221.04 mg, 0.93 mmol), EDC (287.97 mg, 1.86 mmol) and 4-dimethylaminopyridine (12.22 mg, 0.10 mmol) and mix them. Stir the mixture at room temperature for 24 hours. After the reaction is completed, directly add 100 mL of saturated ammonium chloride solution to the reaction-completed solution, and then extract with dichloromethane (100 mL). Subsequently, purify the crude product by column chromatography (dichloromethane:methanol = 30:1 volume ratio) to obtain Cy7-TCF-SS-COOH. Yield: 56.3%.

[0073] 7) Synthesis of Compound I-1:

[0074] Cy7-TCF-SS-COOH (250.00 mg, 0.33 mmol), CPT (159.72 mg, 0.40 mmol), EDC (51.08 mg, 0.33 mmol) and 4-dimethylaminopyridine (6.72 mg, 0.06 mmol) were added to 25 mL of anhydrous dichloromethane. After stirring evenly, the reaction was carried out at room temperature for 10 hours. After the reaction was completed, the reaction mixture was poured into 100 mL of ammonium chloride solution, and then extracted with dichloromethane (150 mL). The extract was purified by column chromatography using dichloromethane and methanol (30:1 v / v) as the eluent, and finally the target compound I-1 was obtained with a yield of 52.1%.

[0075] 1 H NMR (400 MHz, CDCl3): δ (ppm): 8.42 (s, 1H), 8.21 (d, J = 16 Hz, 1H), 8.17 (d, J = 16 Hz, 1H), 7.92 (d, J = 8 Hz, 1H), 7.86 (m, 2H), 7.68 (m, 1H), 7.21 (s, 2H), 7.03 (t, 1H), 6.8 (d, J = 8 Hz, 1H), 6.34 (d, J = 16 Hz, 1H), 5.75 (d, J = 12 Hz, 1H), 5.70 (d, J = 16 Hz, 1H), 5.36 (d, J = 16 Hz, 1H), 5.27 (s, 2H), 4.43 (m, 2H), 4.06 (m, 2H), 2.71 (t, 2H), 2.65 (t, 4H), 2.60 (t, 4H), 2.35 (m, 4H), 2.08 (m, 2H), 1.96 (m, 4H), 1.70 (s, 6H), 1.65 (S, 6H), 1.58 (m, 2H), 0.98 (m, 3H).

[0076] Figure 2 and Figure 3 respectively show the ultraviolet absorption spectra and fluorescence emission spectra of compound I-1 in water and acetonitrile. It can be observed that there are significant differences in the absorption and emission characteristics of I-1 in these two solvents. Especially in aqueous solution, the absorption band of compound I-1 is wider and the emission intensity is weaker, indicating that the optical behavior of this compound is different in water and acetonitrile, which may be due to the physical property differences caused by solvent effects or intermolecular interactions, indicating that compound I-1 aggregates in aqueous solution and its assembly behavior in aqueous solution can be further characterized.

[0077] Example 2 Preparation method of supramolecular assembly

[0078] First, compound I-1 was dissolved in DMSO at a concentration of 2 mM (other suitable organic solvents such as ethanol can also be used) to obtain a storage solution. To prepare nanoparticles, 10 μL of the storage solution was added to 2 mL of deionized water, stirred vigorously, and treated with ultrasound for 1 hour to ensure that the solution was evenly mixed. With the difference in polarity between the aqueous phase and the solvent, I-1 molecules will spontaneously assemble in the aqueous solution to form micro-nanoscale particles.

[0079] Example 3 Characterization method of micro-nanostructure

[0080] In order to characterize the self-assembled micro-nanostructures, the dynamic light scattering (DLS) technique was first used to measure their particle size distribution. Figure 4 As shown in the figure, the particle size of the self-assembled micro-nanostructure of I-1 is about 100nm, which indicates that the prepared nanoparticles have a relatively uniform size. As can be seen from the figure, the particle size range is between 30 and 200nm; further, the size range of the nanoparticles is between 30 and 120nm. In addition, TEM and AFM were used to further observe its morphology. TEM image (see Figure 5 ) shows that the nanostructure has a distinct nanoparticle shape. AFM images (see Figure 6 ) further confirmed this nanomorphology. These results show that compound I-1 can form a stable micro-nanostructure through self-assembly and has good morphology control.

[0081] Example 4 In vitro photothermal effect and photothermal stability of the micro-nanostructure of compound I-1

[0082] In order to evaluate the photothermal effect and photothermal stability of the micro-nanostructure of compound I-1, four groups of I-1 solutions with different concentrations (0 μM, 10 μM, 20 μM, and 40 μM) were designed for testing.

[0083] First, prepare 3 mL of each sample solution, including: 3 mL of deionized water for the control group, and 10 μM, 20 μM, and 40 μM I-1 micro-nanostructure solutions for the sample groups, which are prepared by adding different volumes of I-1 stock solution (2 mM, dissolved in DMSO) to deionized water. These solutions are added to the cuvettes and sealed with lids to ensure sealing to avoid interference from the external environment.

[0084] Each group of samples was irradiated with 808nm laser for 5 minutes, and the power and duration of laser irradiation were kept consistent. During the irradiation process, the temperature change of the samples was monitored in real time using a thermal imager, and the data was recorded every 10 seconds. Subsequently, these data were input into the Origin software to draw a curve of temperature change over time (see Figure 7)。The experimental results showed that for the 0 μM sample (control group, water), the temperature change was minimal, only increasing by 1.6 °C; for the 10 μM sample, the temperature rose from room temperature (30 °C) to 51 °C, with a temperature increase of 21 °C; for the 20 μM sample, the temperature rose from 30 °C to 57.2 °C, with a temperature increase of 27.2 °C; for the 40 μM sample, the temperature rose from 30 °C to 61.2 °C, with a temperature increase of 31.2 °C. These results indicate that as the concentration of I-1 micro-nano structures increases, the photothermal effect is significantly enhanced.

[0085] To further verify the photothermal stability of compound I-1 micro-nano structures, a 40 μM sample was selected for repeated light irradiation experiments. After the 40 μM sample was irradiated with an 808 nm laser for 10 minutes, its temperature rose from room temperature of 30 °C to 61.2 °C, and then naturally cooled down to room temperature. This process was repeated 5 times. Each time after laser irradiation, the temperature increased by 25 °C, and a stable temperature increase effect was maintained throughout. The experimental results show that the 40 μM sample exhibits good photothermal stability under multiple laser irradiations, with a consistent temperature increase amplitude and no obvious attenuation phenomenon (see Figure 8 ).

[0086] In summary, compound I-1 micro-nano structures not only possess excellent photothermal effects but also exhibit strong photothermal stability, overcoming the limitations of many organic small molecule fluorescent compounds in terms of photothermal stability and having potential clinical application prospects.

[0087] Example 5 Generation of Reactive Oxygen Species In Vitro by Compound I-1 Micro-Nano Structures

[0088] To verify whether compound I-1 micro-nano structures can generate reactive oxygen species (ROS) under 808 nm laser irradiation, experiments were conducted using the ROS-specific probe 1,4-diphenyl-2,3-benzofuran (DPBF).

[0089] First, compound I-1 micro-nano structures were mixed with the DPBF solution to form a solution with a final concentration of 10 μM for compound I-1 micro-nano structures and 50 μM for DPBF. Then, the sample was irradiated with an 808 nm laser (0.5 W cm -2 ) for 5 minutes, and the absorption spectrum of the sample was regularly recorded during this period. Figure 9 The results showed that after the DPBF solution was irradiated with the laser, the absorbance at 420 nm decreased significantly, proving that DPBF reacted with the generated ROS. For the same sample without laser irradiation, the absorbance did not change significantly, further verifying the ROS generation caused by laser irradiation. The experimental results indicate that compound I-1 micro-nano structures can effectively generate ROS under 808 nm laser irradiation, supporting its potential as a photothermal agent and for application in potential photodynamic therapy (PDT).

[0090] In vitro CPT release of the compound I-1 micro-nanostructure in Example 6

[0091] This experiment aimed to study the CPT release characteristics of the compound I-1 micro-nanostructure under different concentrations of glutathione (GSH) and hydrogen peroxide (H2O2) to evaluate its drug release potential in the tumor microenvironment.

[0092] By means of high performance liquid chromatography (HPLC) technology, the CPT release of the compound I-1 micro-nanostructure was analyzed at different time points (0, 2, 4, 6, 8, 12, 24 hours). Multiple reaction conditions were set in the experiment: 1 mM and 10 mM glutathione, 1 mM and 10 mM hydrogen peroxide. Figure 10 The results showed that under the conditions of 1 mM glutathione or 1 mM hydrogen peroxide, the CPT release rate of the compound I-1 was 20%-30%, indicating that the release of CPT was relatively limited under lower concentrations of reducing and oxidizing conditions. On the contrary, when the H2O2 concentration was increased to 10 mM, the CPT release rate could reach 50% within 24 hours, and when the GSH concentration was increased to 10 mM, the CPT release rate increased significantly, reaching 78.5%, indicating that the concentrations of GSH and H2O2 had a significant impact on the drug release of the compound I-1 micro-nanostructure, and the compound I-1 micro-nanostructure was more sensitive to GSH than to H2O2. These results indicated that the compound I-1 micro-nanostructure could effectively release CPT in the simulated tumor microenvironment, especially under the conditions of excessive GSH and H2O2, and had good targeted drug release characteristics.

[0093] Reactive oxygen species generation of the compound I-1 micro-nanostructure in tumor cells in Example 6

[0094] A high level of GSH is a significant feature of tumor cells, which is significantly different from healthy cells. Therefore, in this invention, 4T1 mouse cancer cells and normal L929 fibroblast cells were selected as model cells. To verify the specificity that the compound I-1 micro-nanostructure induced the release of photosensitizer and generated ROS in tumor cells and did not generate ROS in normal cells, cell experiments were carried out using the SOSG probe.

[0095] First, these cells were treated with 10 μM of the compound I-1 micro-nanostructure, then 100 μM of the SOSG probe was added and the cells were cultured for another 1 hour. After that, the cells were irradiated with an 808 nm laser for 5 minutes, and the fluorescence intensity change of the SOSG probe was detected by a laser confocal microscope. In the tumor cells 4T1, the fluorescence intensity of the SOSG probe increased significantly after laser irradiation, indicating that the photosensitizer was released under the action of excessive GSH and ROS was generated; while in the normal cells L929, due to the lack of sufficient GSH, the photosensitizer was not released, and the fluorescence intensity change of the SOSG probe was small and no reactive oxygen species were generated. Figure 11)。This indicates that the I-1 micro-nano structure of the compound can specifically release photosensitizer in tumor cells and generate ROS under laser irradiation, showing potential for PDT applications. However, no ROS is generated in normal cells, demonstrating its safety for normal cells.

[0096] Example 7 Intracellular Phototoxicity and Dark Toxicity Experiments

[0097] To evaluate the effects of phototherapy, chemotherapy, and combined photochemotherapy on cell proliferation inhibition, the MTT method was used in this experiment to detect cell viability.

[0098] The experiment was divided into four groups: a control group (untreated), a phototherapy group (treated with light alone), a chemotherapy group (treated with chemotherapy alone), and a combined photochemotherapy group (treated with both light and chemotherapy simultaneously). First, tumor cells were inoculated into 96-well plates. When the cells grew to approximately 70% confluence, different treatments were given to each group. The phototherapy group was added with the Cy7-TCF-OH photosensitizer and irradiated with near-infrared light of a specific wavelength. The chemotherapy group was incubated with the I-1 micro-nano structure of the compound for 24 hours. After incubating with the I-1 micro-nano structure of the compound for 24 hours in the combined photochemotherapy group, it was irradiated with near-infrared light of a specific wavelength. After 48 hours of treatment, MTT reagent was added and incubated for 4 hours. Then, the solubilization solution was added to dissolve the Formazan crystals, and the absorbance value (OD value) was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader to calculate the cell viability of each group. According to the cell viability measured by the MTT method, the OD values of the phototherapy group, chemotherapy group, and combined photochemotherapy group were lower than those of the control group, indicating that both phototherapy and chemotherapy can effectively inhibit the proliferation of tumor cells. Among all the treatment groups, the combined photochemotherapy group had the lowest cell viability, indicating that the combined treatment with the I-1 micro-nano structure of the compound had a synergistic effect in enhancing the inhibitory effect on tumor cell proliferation ( Figure 12 ).

[0099] Example 8 Photoacoustic Imaging Test in Mice

[0100] To evaluate the photoacoustic imaging effect of the I-1 micro-nano structure in mouse tumors, the I-1 micro-nano structure was injected via the tail vein in this experiment, and its distribution and dynamic changes at the tumor site were tracked in real time using photoacoustic imaging technology.

[0101] First, an appropriate amount of tumor cells was inoculated into the right axilla of the mouse to form a tumor model. When the tumor grew to a certain size (about 100 mm 3 ), the I-1 micro-nano structure was injected via the tail vein. After injection, photoacoustic imaging monitoring was performed at 2, 4, 6, 8, 10, and 24 hours. A laser source with a specific wavelength was used for excitation, and the signal intensity at the tumor site was obtained through a photoacoustic imaging system. Images were collected at each time point and the signal changes were analyzed.

[0102] The results are asFigure 13 As shown, at the 4th hour, obvious photoacoustic signals were visible at the tumor site, indicating that the I-1 micro-nano structure began to accumulate in vivo and formed strong signals in the tumor area; at the 10th hour, the photoacoustic signal reached its peak, showing the maximum concentration of the I-1 micro-nano structure at the tumor site, indicating that it achieved the best imaging effect at this time point; by the 24th hour, the photoacoustic signal at the tumor site decreased significantly, suggesting that the I-1 micro-nano structure might have undergone a metabolic or clearance process.

[0103] This example demonstrated the excellent performance of the compound I-1 micro-nano structure in tumor targeting, which could effectively accumulate in the tumor area through the EPR effect and provide strong photoacoustic imaging signals. Meanwhile, during the 24-hour observation period, no adverse reactions such as spasm or twitch occurred in the experimental mice, indicating that the compound I-1 micro-nano structure had almost no toxicity and extremely good safety.

[0104] Example 9: Fluorescence imaging test of mice

[0105] To evaluate the fluorescence distribution and dynamic changes of the I-1 micro-nano structure in mouse tumors, tumor cells were inoculated on the chest of mice in this experiment to form a tumor model. When the tumor grew to a certain size (about 100 mm 3 ), the I-1 micro-nano structure was injected via the tail vein. After injection, fluorescence imaging was performed at 0, 1, 2, 4, 6, and 8 days. The fluorescence signal was excited with a specific wavelength and monitored through a fluorescence imaging system. Whole-body imaging of the mice was carried out at each time point, with a focus on the signal changes at the tumor site and other important organs such as the liver and kidneys.

[0106] As Figure 14 shown, on the 1st day (D1) after injection, obvious fluorescence signals could be detected at the tumor site, but the signals in other body parts such as the mouse liver and kidneys were also strong, indicating the rapid distribution of the I-1 micro-nano structure in vivo. However, over time, the fluorescence signal at the tumor site gradually increased, showing the accumulation effect of the I-1 micro-nano structure; meanwhile, the fluorescence signals in the liver and kidneys gradually decreased, suggesting that the I-1 micro-nano structure might be gradually cleared through metabolic and excretion pathways. By the 8th day (D8), the fluorescence signal at the tumor site increased significantly, while the signals in other organs further decreased, indicating a more significant enrichment effect of the I-1 micro-nano structure at the tumor site.

[0107] Example 10: Experimental study on combined phototherapy and chemotherapy for mouse tumors

[0108] This experiment aimed to evaluate the effects of phototherapy, chemotherapy, and combined phototherapy-chemotherapy in the treatment of mouse tumors. In the experiment, tumor cells were subcutaneously inoculated on the right hind leg of mice. When the tumor grew to about 100 mm 3When, they were randomly divided into four groups: control group, phototherapy group, chemotherapy group, and phototherapy-chemotherapy combined treatment group. The control group received no treatment. The phototherapy group received light treatment at a specific wavelength (808 nm) 24 hours after injecting the Cy7-TCF-OH photosensitizer. The chemotherapy group only injected the I-1 nanostructure without light treatment. The phototherapy-chemotherapy combined treatment group received light treatment at a specific wavelength (808 nm) 24 hours after injecting the I-1 nanostructure. During the experiment, the tumor volume of the mice was recorded every 2 days ( Figure 16 ), and a photo of the mice was taken every 5 days ( Figure 15 ).

[0109] According to the changes in tumor volume measured every two days during the experiment, the tumor growth of the four groups of mice will show different trends. The tumor volume of the mice in the control group will increase rapidly after treatment, and the tumor growth is relatively obvious. The tumor growth rate of the mice in the phototherapy group and the chemotherapy group will slow down compared with the control group, but the tumor will still continue to grow, showing a certain therapeutic effect. The tumor volume of the phototherapy-chemotherapy combined treatment group will show a significant inhibitory effect, and the obvious reduction in tumor volume indicates that the combined treatment has a synergistic effect in inhibiting tumor growth.

[0110] Example 11 Experiment on the Overcoming of Drug Resistance by Phototherapy-Chemotherapy Combined Treatment of Mouse Tumors

[0111] This experiment aimed to study the effect of phototherapy-chemotherapy combined treatment on the expression of MPR1 protein in tumors. First, the 4T1 tumor cell line was inoculated and divided into four groups: control group, phototherapy group, chemotherapy group, and phototherapy-chemotherapy combined treatment group. The phototherapy group treated the cells with the Cy7-TCF-OH photosensitizer, and after 2 hours of incubation, ultraviolet light or laser irradiation was carried out to simulate the photothermal effect. The chemotherapy group treated the cells with the chemotherapeutic drug (CPT) for 24 hours. The combined treatment group carried out phototherapy and chemotherapy simultaneously, that is, first added the compound I-1 nanostructure for incubation, and then carried out light irradiation. After treatment, the cells were lysed with lysis buffer to extract total protein, and the expression level of MPR1 protein was detected by Western Blot. The experimental results showed (see Figure 17 ) that neither phototherapy nor chemotherapy alone could reduce the expression of MPR1 protein in tumor tissues, and the MPR1 expression in the combined treatment group was significantly lower than that in other groups. It was shown that the combined treatment synergistically inhibited the expression of MPR1 protein through the dual effects of photothermal effect and chemotherapy, further enhancing the anti-tumor effect. This result indicated that the combined treatment of phototherapy and chemotherapy had a synergistic effect in tumor cells, could jointly inhibit the expression of MPR1 through multiple mechanisms, and then inhibit the transport and endocytosis functions of tumor cells, achieving a therapeutic effect of "1 + 1 > 2", providing a new strategy for tumor treatment.

[0112] The above description is only a preferred embodiment of the present invention and is not a limitation on the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent replacements for some of the technical features. Any modifications, equivalent replacements, modifications, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A prodrug compound in which a tumor-stimulus-responsive photosensitizer is conjugated with camptothecin, characterized in that, It has the structure shown in the following formula (I-1):

2. A supramolecular assembly of the prodrug compound according to claim 1, characterized in that, The supramolecular assembly is a micro-nano structure formed by the self-assembly of a prodrug compound in an aqueous solution; the preparation method of the supramolecular assembly includes the following steps: Dissolve the prodrug compound with the structure shown in formula (I-1) in dimethyl sulfoxide solvent, add the obtained solution to water to obtain a prodrug compound solution with a final concentration of 0.2 μM to 500 μM, and the prodrug compound self-assembles into a micro-nano structure in this solution; wherein the content of dimethyl sulfoxide ≤ 1%.

3. The pharmaceutically acceptable salt of the prodrug compound according to claim 1.

4. A supramolecular assembly of a pharmaceutically acceptable salt of the prodrug compound according to claim 3, characterized in that, The supramolecular assembly is a micro-nano structure formed by the self-assembly of a pharmaceutically acceptable salt of a prodrug compound in an aqueous solution; the preparation method of the supramolecular assembly includes the following steps: Dissolve the pharmaceutically acceptable salt of the prodrug compound using dimethyl sulfoxide solvent, add the obtained solution to water to obtain a solution with a final concentration of 0.2 μM to 500 μM of this salt, and the pharmaceutically acceptable salt of the prodrug compound self-assembles into a micro-nano structure in this solution; wherein the content of dimethyl sulfoxide ≤ 1%.

5. The supramolecular assembly according to claim 2 or 4, characterized in that, The particle size of the micro-nano structure is 30 nm to 200 nm.

6. A pharmaceutical composition, characterized in that, It includes any one or several of the prodrug compound according to claim 1, the supramolecular assembly according to claim 2, the pharmaceutically acceptable salt of the prodrug compound according to claim 3, and the supramolecular assembly according to claim 4; it also includes a pharmaceutically acceptable carrier.

7. Use of the prodrug compound according to claim 1 or the supramolecular assembly according to claim 2 in the preparation of a drug for diagnosing and / or treating cancer.

8. Use of the pharmaceutically acceptable salt of the prodrug compound according to claim 3 or the supramolecular assembly according to claim 4 in the preparation of a drug for diagnosing and / or treating cancer.

9. The application according to claim 7 or 8, characterized in that The cancer includes breast cancer; the treatment is a combination treatment including photodynamic therapy, chemotherapy or photothermal therapy.

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