Photo-thermal agent, preparation method thereof and application of photo-thermal agent in treatment of tumors by combining mild photo-thermal therapy with abelsiril

Abstract

The invention relates to a photo-thermal agent, a preparation method thereof and application of the photo-thermal agent in treating tumors by combining a mild photo-thermal therapy with abesilib. The photo-thermal agent has excellent photo-thermal effect and light stability, and when the photo-thermal agent is used for mild photo-thermal-abelsilib combined treatment, tumor cells are induced to age through abelsilib, then fragile and aged cancer cells are accurately hit by using a mild photo-thermal therapy, and a double-hit effect is formed. According to the therapy, the treatment effect is remarkably improved, meanwhile, side effects are reduced, and the combination therapy has higher targeting property due to the fact that mild photo-thermal energy is low; in addition, treatment is carried out step by step, aging is induced firstly, then photo-thermal treatment is carried out, the scheme can be adjusted according to the reaction of a patient, safety and controllability are improved, and the acute toxicity risk is reduced. According to the invention, drug-induced aging and physical treatment are innovatively combined, and a new treatment approach is provided for refractory or recurrent cancers.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceuticals, and particularly relates to a photothermal agent and its application in the treatment of tumors by combining mild photothermal therapy with abemaciclib; the present invention also provides a method for preparing the photothermal agent. Background Art

[0002] Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma in adults, accounting for about 30% to 40% of all cases. Although existing treatment methods such as chemotherapy, targeted therapy, and immunotherapy have shown good effects in some patients, many patients still respond poorly to these treatments or experience disease recurrence. Therefore, developing new effective treatment methods is crucial for improving the survival rate and quality of life of such patients.

[0003] Abemaciclib is a selective CDK4 / 6 inhibitor that has been approved for the treatment of certain types of breast cancer. It acts by blocking the progression of the cancer cell cycle from the G1 phase to the S phase, thereby causing cancer cells to stagnate or even enter a senescent state. However, the use of abemaciclib alone has limitations: on the one hand, senescent cells may remain in the tumor microenvironment, and their metabolic reprogramming and pro-inflammatory phenotypes may promote the survival of residual cancer cells through paracrine mechanisms; on the other hand, long-term drug use is prone to drug-resistant mutations, and the penetration of deep lesions in solid tumors is insufficient.

[0004] Photothermal Therapy (PTT) uses near-infrared light to excite a photothermal conversion agent (such as indocyanine green IR820) to generate local high temperatures (usually >50°C) to kill cancer cells, but its clinical translation is limited by the risk of thermal damage to normal tissues. However, traditional photothermal therapy may cause significant side effects because it not only affects cancer cells but may also damage surrounding normal tissues.

[0005] In existing studies, photothermal therapy is often combined with other treatment methods to enhance the treatment effect. However, most current studies focus on combining chemotherapy or immunotherapy with PTT and do not optimize for the characteristics of senescent cells. For example, some protocols attempt to first induce cell senescence with drugs and then implement interventions, but in terms of drug selection and timing design, the synergistic effect between senescence-related pathways and thermotherapy sensitivity has not been fully realized.

[0006] In summary, combining multiple treatment methods to develop a combined treatment method that can not only significantly improve the treatment effect but also effectively reduce side effects and enhance treatment safety has extremely important practical significance. Summary of the Invention

[0007] In view of this, the object of the present invention is to provide photothermal agents, as well as their preparation methods and applications. Different from the existing single therapy or combined therapy, the present invention proposes a brand-new treatment idea, that is, combining drug-induced senescence and mild photothermal therapy, which not only overcomes the limitations of single treatment methods, but also provides a new direction for tumor treatment, especially showing potential advantages in dealing with refractory and recurrent cases.

[0008] Specifically, the present invention provides a photothermal agent, which comprises nanoparticles PO NPs prepared from compound PYIT-OD and DSPE-PEG2000-COOH.

[0009] The CAS number of the said PYIT-OD is 2747095-31-2; the CAS number of the said DSPE-PEG2000-COOH is 474922-20-8.

[0010] In some specific embodiments of the present invention, the specific preparation steps of the said nanoparticles PO NPs are as follows: Dissolve compound PYIT-OD in an organic solvent, and after ultrasonic assistance for dissolution, dropwise add it to the solution of DSPE-PEG 2000 -COOH drop by drop while maintaining a high-speed rotation state during the dropping process. After dropping, continue to stir at this speed until the mixed solution becomes transparent, then remove the organic solvent by reduced pressure distillation, and freeze-dry the remaining aqueous solution. The freeze-dried powdery solid is the nanoparticles PO NPs.

[0011] In some specific embodiments of the present invention, the specific preparation steps of the said nanoparticles PO NPs are as follows: Dissolve compound PYIT-OD in tetrahydrofuran, and then slowly dropwise add the tetrahydrofuran solution of PYIT-OD to the ddH2O solution of DSPE-PEG 2000 -COOH while stirring. Continue to stir the mixture until the mixed solution becomes transparent, then remove the organic solvent by reduced pressure distillation, and freeze-dry the remaining aqueous solution. The freeze-dried powdery solid is the nanoparticles PO NPs; the mass ratio of the said PYIT-OD to DSPE-PEG 2000 -COOH is 1:(1-5).

[0012] The present invention also provides a preparation method of a photothermal agent, which comprises the following preparation steps:

[0013] Dissolve compound PYIT-OD in tetrahydrofuran, and then slowly dropwise add the tetrahydrofuran solution of PYIT-OD to the ddH2O solution of DSPE-PEG 2000 -COOH while stirring. Continue to stir the mixture until the mixed solution becomes transparent, then remove the organic solvent by reduced pressure distillation, and freeze-dry the remaining aqueous solution. The freeze-dried powdery solid is the nanoparticles PONPs.

[0014] In some specific embodiments of the present invention, the following preparation steps are included:

[0015] The compound PYIT-OD was dissolved in tetrahydrofuran to a concentration of 250 μg / mL. The tetrahydrofuran solution of PYIT-OD was slowly added dropwise to the DSPE-PEG solution having a concentration of 1 mg / mL while stirring. 2000 -COOH in ddH2O solution, continue to stir the mixture until the mixed solution becomes transparent, then remove the organic solvent by reduced pressure distillation, and freeze-dry the remaining aqueous solution. The freeze-dried powdered solid is the nanoparticles PO NPs.

[0016] In some specific embodiments of the present invention, the PYIT-OD and DSPE-PEG 2000 The mass ratio of -COOH is 1:(1-5).

[0017] The present invention also provides a pharmaceutical composition for treating tumors by mild photothermal-abecib combination therapy, comprising the photothermal agent described in any one of the above items or the photothermal agent prepared by any one of the above preparation methods.

[0018] In some specific embodiments of the present invention, abemaciclib is also included.

[0019] The CAS number of abemaciclib is 1231929-97-7.

[0020] In some specific embodiments of the present invention, a pharmaceutically acceptable carrier or excipient is further included.

[0021] The present invention also provides the use of any of the above-mentioned photothermal agents, or the photothermal agents prepared by any of the above-mentioned preparation methods, or the pharmaceutical composition for treating tumors by the mild photothermal-abecib combination therapy described in any of the above-mentioned items in tumor treatment.

[0022] In some specific embodiments of the invention, the tumor is diffuse large B-cell lymphoma.

[0023] Compared with the prior art, the present invention has the following significant advantages and effects:

[0024] 1. Improve treatment effect

[0025] Double-strike strategy: A "double-strike" treatment strategy is formed by first inducing diffuse large B-cell lymphoma cells into a senescent state with abemaciclib, and then using the photothermal agent and mild photothermal therapy provided by the present invention to precisely strike these more fragile aging cancer cells. This combination method significantly improves the treatment effect and is more effective than using any one treatment method alone.

[0026] 2. Reducing Side Effects

[0027] Advantages of mild photothermal therapy: Compared with traditional high-intensity photothermal therapy, mild photothermal therapy uses lower energy, can reduce damage to surrounding healthy tissues, and thus significantly reduce treatment-related side effects.

[0028] Precision treatment: Abemaciclib mainly acts on cancer cells to make them enter a senescent state, and mild photothermal therapy further targets these senescent cancer cells. This combination can act more precisely on target cells and reduce the impact on normal cells.

[0029] 3. Enhancing Treatment Safety

[0030] Based on the photothermal agent or pharmaceutical composition provided by the present invention, step-by-step treatment of tumors can be achieved: first inducing cancer cell senescence and then performing photothermal treatment; this enables the adjustment of treatment plans according to the specific reactions of patients clinically, improving the safety and controllability of treatment.

[0031] Reducing the risk of acute toxicity: Due to the adoption of mild photothermal therapy, the risk of acute toxicity caused by high temperature is reduced, which helps to improve the tolerance of patients to treatment.

[0032] 4. Innovative Treatment Approaches

[0033] Combined treatment strategy: Combining drug-induced senescence with physical treatment methods provides a new idea and method for cancer treatment, overcoming the limitations of single treatment methods.

[0034] Breakthrough progress: Especially in the treatment of refractory or recurrent diffuse large B-cell lymphoma, this innovative method may bring breakthrough progress and provide a new direction for cancer treatment.

[0035] In summary, the photothermal agent and pharmaceutical composition provided by the present invention, by combining drug-induced senescence and mild photothermal therapy, not only significantly improve the treatment effect, but also reduce side effects, enhance treatment safety, and provide an innovative treatment approach, with broad application prospects. Description of the Drawings

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0037] Figure 1Schematic diagram of the process for preparing PO NPs nanoparticles by a typical nanoprecipitation method.

[0038] Figure 2 In the middle is an image of an aqueous solution of PO NPs nanoparticles.

[0039] Figure 3 Is the ultraviolet absorption spectra of PO and PO NPs nanoparticles.

[0040] Figure 4 In (A) is the TEM image and high-magnification TEM image (insert) of PO NPs nanoparticles; (B) is the particle size distribution of PO nanoparticles in PBS measured by the DLS method.

[0041] Figure 5 In (A) is the ultraviolet absorption spectra of PO nanoparticles irradiated by 808 nm laser at different times; (B) is the DLS data of PO NPs nanoparticles after irradiation by 808 nm laser for different times (n = 3, mean ± SD).

[0042] Figure 6 In (A) is the photothermal heating image of PO NPs in water at different concentrations (0 - 100 μg / ml) under 808 nm laser irradiation (1.0 W / cm 2 , 808 nm); (B) is the photothermal heating curve of PO nanoparticles at different concentrations (0 - 100 μg / ml) under 808 nm laser (1.0 W / cm 2 ) irradiation.

[0043] Figure 7 Is the photothermal performance of PO NPs (25 μg / ml) under 808 nm light irradiation at different power densities (0.5 - 1.5 W / cm 2 ).

[0044] Figure 8 Is the temperature change curve of PO NPs during four on / off cycles under 808 nm laser (1.0 W / cm 2 ) action.

[0045] Figure 9 In (A) is the heating / cooling curve of the PO-NPs solution (100 μg / mL) under laser irradiation (808 nm, 1.0 W / cm 2 ); (B) is the calculated photothermal conversion efficiency under 808 nm irradiation.

[0046] Figure 10(A) shows the PAI of PO-NPs at different concentrations (0 - 100 μg / m1); (B) shows the cross-sectional images of PO NPs at different concentrations (0 - 100 μg / ml); (C) shows the quantitative photoacoustic imaging of PO NPs at different concentrations (n = 3, mean ± SD).

[0047] Figure 11 It is a schematic diagram of the induced cellular senescence process.

[0048] Figure 12 It is the cell viability of A20 cancer cells co-cultured with different concentrations of abemaciclib.

[0049] Figure 13 (A) shows the staining map of β-galactosidase on normal A20 cells; (B) shows the β-galactosidase staining map of senescent A20 cells.

[0050] Figure 14 (A) shows the expression of p21, CDK4, and p53 proteins in normal and senescent A20 cancer cells treated with abemaciclib detected by Western blotting. β-actin was used as a loading control. Here, N represents normal and S represents senescent; (B) shows the expression of CDK4 protein detected by Western blot in normal cells and senescent lymphoma cells. β-actin was used as a loading control; (C) shows the quantitative results of p21, CDK4, and p53 expression; (D) shows the quantitative results of CDK4 expression.

[0051] Figure 15 It is a schematic diagram for validating the mouse aging model.

[0052] Figure 16 It is the staining images of p21, p53, β-Gal, and Ki67 in normal tumors and senescent tumors. All data are expressed as mean ± SEM (n = 3) ***p < 0.001, ****p < 0.0001.

[0053] Figure 17 (A) shows the expression of HSP70 protein detected by Western blot in normal and senescent A20 cells treated at different temperatures, with GADPH as an internal reference control; (B) shows the quantitative results of HSP70 expression in normal A20 cells; (C) shows the quantitative results of HSP70 expression in senescent A20 cells.

[0054] Figure 18 It is the CLSM images of Ce6@PONPs in normal A20 cells and senescent A20 cells (scale bar = 20 μm).

[0055] Figure 19Red fluorescence represents Ce6@PO. NPs intensity was calculated by ImageJ. Normal represents normal A20 cells, and Senescence represents senescent A20 cells.

[0056] Figure 20 After treatment with different concentrations and different groups, cell viability was detected by CCK-8 assay (808 nm, 1.0 W / cm 2 , 5 min). Among them, N+PO NPs represents the group of normal A20 cells without near-infrared laser irradiation, S+PO NPs represents the group of senescent A20 cells without near-infrared laser irradiation, N+PO NPs+Laser represents the group of normal A20 cells irradiated with near-infrared laser, and S+PO NPs+Laser represents the group of senescent A20 cells irradiated with near-infrared laser.

[0057] Figure 21 After laser irradiation (808 nm, 1.0 W / cm 2 ), fluorescence images of live cells (green) and dead cells (red) were captured by CLSM (scale bar = 50 μm). Among them, Control represents the control group, N+PO NPs represents the group of normal A20 cells, S+PO NPs represents the group of senescent A20 cells, Laser(-) represents no near-infrared laser irradiation, and Laser(+) represents near-infrared laser irradiation.

[0058] Figure 22 (A) is a schematic diagram of the expression and effect of HSP70 in normal A20 cells and senescent A20 cells under MTT; (B) Western blot was used to detect the expression of HSP70 protein in normal and senescent A20 cells, with β-actin as an internal reference control. Among them, Control represents the control group without near-infrared laser irradiation, Control+Laser represents the control group irradiated with laser, N+PO NPs represents the group of normal A20 cells without near-infrared laser irradiation, N+PO NPs+Laser represents the group of normal A20 cells irradiated with near-infrared laser, S+PO NPs represents the group of senescent A20 cells without near-infrared laser irradiation, and S+PO NPs+Laser represents the group of senescent A20 cells irradiated with near-infrared laser.

[0059] Figure 23 After irradiation with different doses of laser (808 nm, 1.0 W / cm 2 , 5 min), flow cytometry was used to detect cell apoptosis. Among them, Control represents the control group, N+PO NPs represents the group of normal A20 cells, S+PONPs represents the group of senescent A20 cells, Laser(-) represents no near-infrared laser irradiation, and Laser(+) represents near-infrared laser irradiation.

[0060] Figure 24 After laser irradiation (808 nm, 1.0 W / cm 2 , 5 min), cell apoptosis was detected using a cell cycle and apoptosis detection kit. All data represent the mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, where Control represents the control group, N+PO NPs represents the normal A20 cell group, S+PO NPs represents the senescent A20 cell group, Laser(-) represents no near-infrared laser irradiation, and Laser(+) represents near-infrared laser irradiation.

[0061] Figure 25 Schematic diagram of the treatment protocol for subcutaneous tumor-bearing mice.

[0062] Figure 26 Tumor infrared thermograms of normal and senescent mice after intravenous injection of PBS and PO NPs for 5 minutes. Among them, Control represents the control group, N+PO NPs represents the normal A20 cell group, and S+PO NPs represents the senescent A20 cell group.

[0063] Figure 27 Tumor temperature changes after 808 nm laser irradiation for 5 minutes in normal and senescent mice after intravenous injection of PBS and PO NPs (n = 3, mean ± standard deviation). Among them, Control represents the control group, N+PO NPs represents the normal A20 cell group, and S+PO NPs represents the senescent A20 cell group.

[0064] Figure 28 (A) shows the body weight change curves of tumor-bearing mice after different treatments; (B) shows the tumor volume change curves of tumor-bearing mice over time in different treatment groups. Among them, Control represents the control group, N+PO NPs represents the PO NPs treatment group of normal A20 cells without near-infrared laser irradiation, N+PO NPs+Laser represents the PO NPs treatment group of normal A20 cells with near-infrared laser irradiation, Senescence represents the untreated senescent A20 cell group, S+PO NPs represents the PO NPs treatment group of senescent A20 cells without near-infrared laser irradiation, and S+PO NPs+Laser represents the PO NPs treatment group of senescent A20 cells with near-infrared laser irradiation.

[0065] Figure 29Digital photographs of the corresponding tumors collected after treatment, where Control represents the control group, N+PO NPs represents the PO NPs treatment group of normal A20 cells without near-infrared laser irradiation, N+PO NPs+Laser represents the PO NPs treatment group of normal A20 cells with near-infrared laser irradiation, Senescence represents the untreated group of senescent A20 cells, S+PO NPs represents the PO NPs treatment group of senescent A20 cells without near-infrared laser irradiation, and S+PO NPs+Laser represents the PO NPs treatment group of senescent A20 cells with near-infrared laser irradiation.

[0066] Figure 30 Images of H&E, Tunel, Ki67, HSP70, p53, and p21 at the tumor site for different groups. All data are expressed as mean ± SEM (n = 5). **p < 0.01, ***p < 0.0001, where Control represents the control group, N+PONPs represents the PO NPs treatment group of normal A20 cells without near-infrared laser irradiation, N+PO NPs+Laser represents the PO NPs treatment group of normal A20 cells with near-infrared laser irradiation, Senescence represents the untreated group of senescent A20 cells, S+PO NPs represents the PO NPs treatment group of senescent A20 cells without near-infrared laser irradiation, and S+PO NPs+Laser represents the PO NPs treatment group of senescent A20 cells with near-infrared laser irradiation. Detailed implementation mode

[0067] 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 and should not be used to limit the protection scope of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the protection scope of the present invention.

[0068] Example 1: Preparation of PO NPs nanoparticles

[0069] The specific operation steps are as follows:

[0070] To prepare PYIT-OD (PO) into nanoparticles, the compound was dissolved in tetrahydrofuran (THF), and then ultrasonic treatment was carried out in an ice bath for 30 minutes to assist dissolution. On a magnetic stirrer set at 1200 rpm, 4 mL of PO (250 μg / mL) was slowly added dropwise to 5 mL of DSPE-PEG 2000-COOH (in 1 mg / mL ddH2O). Stir the mixture for 1 hour until the solution becomes transparent. Then remove THF using a rotary evaporator, and lyophilize the remaining aqueous solution. The lyophilized powdery solid is the nanoparticle PO NPs.

[0071] The schematic diagram of the preparation steps is as Figure 1 shown.

[0072] Example 2: Characterization of PO NPs nanoparticles

[0073] The gray aqueous solution of the PO NPs nanoparticles prepared in Example 1 is uniformly transparent (as Figure 2 shown). The UV-Vis spectrum of free PYIT-OD in THF shows a peak at 760 nm, which redshifts to 820 nm upon the formation of PO NPs (as Figure 3 shown). According to TEM and DLS analyses, PO NPs have nanoparticles with a diameter of 70 nm (as shown in A and B of Figure 4 ). Under 808 nm laser irradiation, the UV-visible absorption spectrum or particle size of PO NPs does not change significantly over time, indicating its strong resistance to laser irradiation and temperature changes (as shown in A and B of Figure 5 ). This stability makes PO NPs suitable for subsequent biological experiments.

[0074] Example 3: Measurement and characterization of the photothermal performance and photostability of PO NPs nanoparticles

[0075] To evaluate the photothermal performance, solutions containing PO NPs nanoparticles (1 mL) with different concentrations (0, 6.25, 12.5, 25, 50, 100 μg / mL) were prepared and placed in centrifuge tubes. Then, they were irradiated with an 808 nm laser with a power density of 1.0 W / cm 2 for 10 minutes, and the temperature change was monitored using an infrared thermal imager. In addition, 25 μg / mL PO NPs (1 mL) was added to the centrifuge tube and irradiated with an 808 nm laser at different power densities (0.5, 0.75, 1.0, 1.5 W / cm 2 ). To further determine the photothermal stability of PO NPs, four cycles of on-off irradiation were performed using an 808 nm laser (1.0 W / cm 2 ). The dispersion (100 μg / mL, 1 mL) was measured, and during irradiation, the solution temperature was detected using an infrared thermal imager, and the real-time temperature was recorded every 30 seconds for 10 minutes.

[0076] The temperature rise of PO NPs nanoparticles under 808 nm laser irradiation was measured using an infrared thermal imager, and the results are as Figure 6As shown in (A), the temperature increases with the increase in the concentration of PO NPs; at a concentration of 100 μg / mL, the temperature reaches 50.1 °C, confirming the significant photothermal conversion ability of PO NPs. The photothermal heating curve further confirms the superior photothermal effect of PO NPs [as shown in Figure 6 (B)]. Similarly, the temperature increases with the increase in laser power ( Figure 7 ). As shown in Figure 8 , after four consecutive laser irradiations (808 nm, 1.0 W / cm 2 , 10 min), the photothermal conversion performance of PO NPs remains unchanged, showing excellent photostability. In addition, the complete cooling curve of the nano-solution under 808 nm laser irradiation was measured and plotted [as shown in Figure 9 (A)], and then the calculation results [as shown in Figure 9 (B)] indicate that under 808 nm laser irradiation, the calculated high photothermal conversion efficiency (40.403%) of PO nanoparticles is obtained. In addition, to verify the feasibility of PO-NPs for photoacoustic imaging (PAI), the in vitro photoacoustic effect of PO-NPs solution under 808 nm laser irradiation was evaluated. The results show that under 808 nm laser irradiation, the PAI signal intensity increases with the increase in the concentration of PONPs [as shown in Figure 10 (A)], and the cross-sectional photoacoustic imaging shows consistent results [as shown in Figure 10 (B)]. The quantitative analysis is as shown in Figure 10 (C).

[0077] Example 4: Establishment of an aging model

[0078] To prove that abemaciclib can promote A20 cells to enter the senescent state, first, the biological safety of A20 cells was evaluated by performing a CCK-8 assay on A20 cells: the prepared abemaciclib solution was diluted to different concentrations and applied to A20 cells according to the experimental procedures outlined in Figure 11 . The detection results are as shown in Figure 12 , when the concentration of abemaciclib is below 0.05 μg / mL, no significant change in cell viability is observed, indicating that the cytotoxicity is negligible at these concentrations.

[0079] After determining the safe concentration of abemaciclib, further senescence verification experiments were conducted at this concentration. To detect whether the cells treated with abemaciclib enter the senescent state, a senescence-associated β-galactosidase staining kit was used to measure the β-galactosidase expression in the treated and control cells. By observing and comparing the relative number of blue cells with the total number of cells at the same magnification, it was demonstrated that the A20 cells treated with abemaciclib entered the senescent state (the detection results are respectively as shown in Figure 13As shown in A and B). Senescence depends on several signaling pathways that together lead to permanent and irreversible cell cycle arrest. Among them, p53 can inhibit CDK2 by activating p21, ultimately preventing Rb phosphorylation and inducing senescence. The phosphorylation of Rb is attributed to the combined action of cyclin D-CDK4 / 6 and cyclin E-CDK2 complexes. Abemaciclib inhibits CDK4 / 6 activity, thereby also inhibiting Rb phosphorylation. When Rb is in the dephosphorylated state, it inhibits the activity of the E2F transcription factor, thus preventing the transition from the G1 phase to the S phase, leading to cell cycle arrest and ultimately cell senescence. Key proteins such as p53, p21, and CDK4 are crucial for cell senescence. Western blot analysis of these key proteins was used to verify whether abemaciclib induces cell senescence. As Figure 14 shown in A and B, compared with normal A20 cells, the expression levels of p21 and p53 proteins were upregulated in A20 cells treated with abemaciclib, confirming the aforementioned senescence signaling pathway. The upregulation of CDK4 can be attributed to compensatory upregulation, in which cells attempt to compensate for the inhibitory activity of CDK4 / 6 by increasing the expression of CDK4 to restore the cell cycle process - a common phenomenon in tumor cells. Despite the increase in CDK4 protein, due to the upregulation of p21, it does not affect cell senescence, and p21 continues to inhibit Rb phosphorylation, ultimately leading to senescence. To verify that abemaciclib only affects tumor cells and not normal cells, L929 and A20 cells were first treated with abemaciclib, and then the expression of CDK4 protein was evaluated by Western blot. The results showed that abemaciclib did not affect normal L929 cells, but it caused compensatory upregulation in the tumor cell line A20 (as Figure 14 shown in C and D). The schematic diagram of the in vivo senescence verification experiment is as Figure 15 shown. In addition, immunohistochemical staining was performed on tumors induced by abemaciclib, and the results are as Figure 16 shown. Comparing the results with the normal group, the expression of p21 and p53 was observed, which was consistent with the Western blot verification of the expression of p21 and p53 in vitro cell experiments. The expression of Ki67 was higher in normal tumor mice than in senescent tumor mice, indicating that tumor cells inhibited the proliferation of tumor cells to a certain extent, but not completely. This indirectly indicated that the senescence model in this example was successfully established, and the staining results of β-galactosidase directly indicated the successful establishment of the senescence model.

[0080] Example 5: Cell Uptake and In Vitro Therapy Experiments

[0081] To achieve the combination of the cell senescence promotion strategy and mild phototherapy, first, the effects of different temperatures on normal and senescent A20 cells were simulated in vitro. The temperature was controlled by water bath heating, and its effect on HSP70 expression was detected to verify the accuracy of the mild photothermal-abemaciclib combination method. AsFigure 17 As shown in (A), when the temperature exceeds the physiological normal value of 37 °C, the expression of HSP70 in normal A20 cells increases. This increase reflects the inherent heat stress response of the cells, and they upregulate HSP70 as a protective mechanism against stress. However, when senescent A20 cells are exposed to temperatures higher than 37 °C, the expression of HSP70 decreases rather than increases. Especially at 43 °C, compared with other temperatures, the expression of HSP70 is the lowest (as shown in Figure 17 B and C), which indicates that at 43 °C, the ability of the cells to withstand stress becomes very fragile and is more vulnerable to damage. Importantly, when exposed to mild hyperthermia conditions of 43 °C, the protective mechanism of the cells cannot provide sufficient defense against external heat stress, ultimately leading to apoptosis or necrosis of the cells.

[0082] To observe the uptake of cells at different time points, confocal laser scanning microscopy (CLSM) was used and the nuclei were stained with Hoechst 33342 (emitting blue fluorescence). Ce6 (CAS number: 19660-77-6) with red absorption and emission characteristics was selected to prepare Ce6@PONPs. To verify the uptake ability of senescent A20 cells compared with normal A20 cells, a similar uptake experiment was conducted on senescent tumor cells as on normal A20 cells. The results are as Figure 18 shown. The results show that over time, the red fluorescence intensity of Ce6 gradually increases, and according to the relative position of the Hoechst blue nuclear staining, the nanoparticles gradually accumulate inside the cells. To confirm whether Ce6@PONPs truly enter the cells, rather than simply attaching to the cell surface or remaining in the extracellular environment, it was labeled and localized with Dio. Dio has good membrane permeability and stable fluorescence characteristics and can be observed under a microscope, so it is usually used to label cell membranes. As Figure 18 shown, when CLSM observes Ce6@PONPs in normal and senescent A20 cells, the green fluorescence from Dio overlaps with the red fluorescence from Ce6@PO, indicating that Ce6@PONPs have completely entered the cells. Importantly, compared with normal A20 cells, senescent A20 cells have a certain trend for Ce6@PONPs. This trend was further quantified by Image J software (as shown in Figure 19 ), supporting these observations. This result can be attributed to the fact that the cell membranes of senescent cells become more relaxed and permeable, thus increasing the chance of nanoparticles entering the cells. In addition, by Hoechst staining of the nuclei, it was observed that the nuclei of senescent cells were slightly larger than those of normal cells in the same field of view, indirectly indicating that the pro-senescence strategy was successfully established. This result is due to the accumulation of more DNA damage in senescent cells, which needs to be processed by DNA repair mechanisms, involving protein complexes that occupy additional space, resulting in nuclear expansion.

[0083] To demonstrate the synergistic effect between cellular senescence promotion and mild phototherapy, this example evaluated the cytotoxicity of near-infrared laser-irradiated and non-irradiated PO NPs. First, the standard CCK-8 method was used to evaluate the cytotoxicity of PO NPs on cells. The results are as Figure 20 shown. Senescent A20 cells showed lower viability under laser irradiation conditions, effectively killing most cancer cells. This indicates that there is a synergistic effect between cellular senescence promotion and mild phototherapy. Calcein AM (green fluorescence) and PI (red fluorescence) were used to further distinguish live cells and dead cells. The results are as Figure 21 shown. In the absence of laser irradiation, both normal and senescent A20 cells treated with PO NPs showed strong green fluorescence, and the red fluorescence was negligible, indicating little effect on cell viability. In the absence of PO-NPs, laser irradiation alone had no obvious effect on cells. After 808 nm laser irradiation (5 min, 1.0 W / cm 2 ), a small amount of red fluorescence appeared in normal A20 cells, and a large amount of red fluorescence appeared in senescent A20 cells.

[0084] To verify the expression and apoptosis of HSP70 in cell therapy experiments, Figure 22 the relevant biological schematic diagram is provided in (A) below. The expression of HSP70 in cells after different treatments was studied, and the expression level of HSP70 in cells after different treatments was detected by Western blot analysis. The results are as Figure 22 shown in (B) below. Compared with other groups, senescent cells under mild photothermal conditions showed a decrease in HSP70 expression. These results indicate that this synergistic effect is beneficial for killing a large number of cancer cells.

[0085] In addition, this example also used the Annexin V-FITC / PI cell apoptosis detection method to verify its cytotoxic effect. The results are as Figure 23 shown. The apoptosis rate of the control group cells was only 4.77%, which was negligible. In addition, compared with normal A20 cells (16.13%), the apoptosis rate (55.71%) was higher after laser irradiation of senescent A20 cells, which was consistent with the previous results, confirming the superiority of the combined treatment. At the same time, each group of A20 cells was pretreated with a cell cycle and apoptosis detection kit, and flow cytometry analysis was performed. The results are as Figure 24 shown. Similarly, the results showed that the S+PO NPs+laser group showed stronger apoptosis induction than the N+PO NPs+laser group under laser irradiation. This further confirmed that mild phototherapy has a good killing effect on senescent A20 cells, indicating the feasibility of combining mild phototherapy with promoting cellular senescence.

[0086] Example 6: Anti-tumor effect experiment of promoting senescence combined with MPTT (mild photothermal therapy)

[0087] After validating and establishing the senescence model and understanding the distribution of the drug in mice, this example initiated a mouse tumor treatment experiment. As Figure 25 shown, the schematic diagram illustrates the process of mouse tumor treatment. Subsequently, the photothermal properties of PO NPs in vivo were studied using an infrared thermal imager, and the results are as Figure 26 shown.

[0088] In addition, to achieve MPTT, the temperature was controlled using an infrared thermal imager. After injecting PBS and PO NPs, the temperature of the tumor region in mice treated with PO NPs reached 45 °C after 5 minutes of NIR laser irradiation at 1.0 W / cm 2 , which was significantly higher than that of the control group (37.2 °C) (as Figure 27 shown). This indicates that PO NPs have an enhanced photothermal effect after accumulation in vivo. Compared with the normal tumor region, the temperature increase in the senescent tumor region is slightly higher, corresponding to the in vivo biodistribution of PO NPs.

[0089] To study the in vivo anti-tumor effect and biotoxicity of PO NPs, nude mice carrying A20 tumors were randomly divided into six groups, and the growth trend of tumors was observed after treatment. At the same time, the body weights of each group of mice were monitored. The results are as Figure 28 (A) shown. All groups showed similar body weight characteristics with no significant changes, indicating low systemic toxicity of PO NPs. During laser treatment, the tumor volume (V) was measured and recorded every other day, and the results are as Figure 28 (B) shown. One group was used as the control group, which had no inhibitory effect on tumor growth. In contrast, the combination of PO NPs and laser irradiation mildly inhibited tumor proliferation. Notably, after the tumor entered the senescent state, the combination of PO NP and laser irradiation showed a significant anti-tumor effect. In addition, compared with the control group, the senescent tumors in mice without laser irradiation only showed slight tumor inhibition, which may be due to the small inhibitory effect of abemaciclib on tumor growth. At the end of the treatment period, all mice were euthanized, and the tumors were collected and photographed. As Figure 29 shown, the results indicate that compared with other groups, MPTT almost controlled the growth of senescent tumors treated with PO NPs under 808 nm laser irradiation. After treatment, tumor tissues from each group were collected for hematoxylin and eosin (H&E) staining and immunohistochemical analysis, as Figure 30As shown, compared with other groups, the S+PO NPs+laser group showed relatively loose tumor cells and severe tumor destruction. The PO NPs+laser group showed slight damage, while the other groups showed no effect. TUNEL (green) staining showed that although there were a small number of apoptotic cells in the PO NPs+laser group, the S+PO NPs+laser group showed extensive apoptosis of tumor cells in the tumor tissue. Consistent with the staining results, Ki67 staining in the tumor showed that S+PO NPs+laser significantly reduced the increase in Ki67-positive tumor cells. These findings highlight the specific anti-tumor effect of the combination therapy. To explore the mechanism of MPTT in this synergistic therapy, this example used immunofluorescence staining to analyze the expression of HSP70 (green) in tumors of six groups. The PO NPs+laser group showed high expression of HSP70, indicating that hyperthermia induced the upregulation of HSP70, which activated the self-protection mechanism of tumor cells against heat and hindered cell death. In contrast, the S+PO NPs+laser group showed significantly lower fluorescence, indicating downregulation of HSP70. These results suggest that tumor cell senescence plays a key role in enhancing PTT, which is consistent with the results of western blot analysis. In addition, to study the expression of p53 (brown) and p21 (brown) in tumors, this example used immunohistochemistry to detect their presence. Regardless of laser irradiation, the senescent group expressed p53 and p21, which was consistent with the previous verification of senescence and was particularly obvious in the laser irradiation group, probably due to the damaging effect of laser on tumor tissue.

Claims

1. A photothermal agent, characterized in that: The nanoparticles PO NPs are prepared from the compounds PYIT-OD and DSPE-PEG2000-COOH.

2. The photothermal agent according to claim 1, characterized in that: The specific preparation steps of the nanoparticles PO NPs are as follows: dissolve the compound PYIT-OD in an organic solvent, add DSPE-PEG dropwise after ultrasonic dissolution 2000 -COOH solution, the dropping process is maintained in a high-speed rotating state, and after the dropping is completed, the stirring speed is continued until the mixed solution becomes transparent, and then the organic solvent is removed by reduced pressure distillation, and the remaining aqueous solution is freeze-dried. The freeze-dried powdered solid is the nanoparticle PO NPs.

3. The photothermal agent according to claim 2, characterized in that: The PYIT-OD and DSPE-PEG 2000 The mass ratio of -COOH is 1:(1-5).

4. A method for preparing a photothermal agent, characterized in that: The method comprises the following preparation steps: The compound PYIT-OD was dissolved in an organic solvent, and DSPE-PEG was added dropwise after ultrasonic dissolution. 2000 -COOH solution, the dropping process is maintained in a high-speed rotating state, and after the dropping is completed, the stirring speed is continued until the mixed solution becomes transparent, and then the organic solvent is removed by vacuum distillation, and the remaining aqueous solution is freeze-dried. The freeze-dried powdered solid is the nanoparticle PONPs.

5. The method for preparing the photothermal agent according to claim 4, characterized in that: The method comprises the following preparation steps: The compound PYIT-OD was dissolved in tetrahydrofuran to a concentration of 250 μg / mL. The tetrahydrofuran solution of PYIT-OD was slowly added dropwise to the DSPE-PEG solution having a concentration of 1 mg / mL while stirring. 2000 -COOH in ddH2O solution, continue to stir the mixture until the mixed solution becomes transparent, then remove the organic solvent by reduced pressure distillation, and freeze-dry the remaining aqueous solution. The freeze-dried powdered solid is the nanoparticles PO NPs.

6. A pharmaceutical composition for treating tumors by mild photothermal-abecib combined therapy, characterized in that: The invention comprises the photothermal agent described in any one of claims 1 to 3 or the photothermal agent prepared by the preparation method described in any one of claims 3 to 5.

7. The pharmaceutical composition for treating tumors by mild photothermal-abecib combination therapy according to claim 6, characterized in that: Also contains abemaciclib.

8. The pharmaceutical composition for treating tumors by mild photothermal-abecib combination therapy according to claim 7, characterized in that: It also contains pharmaceutically acceptable carriers or excipients.

9. Use of the photothermal agent according to any one of claims 1 to 3, or the photothermal agent prepared by the preparation method according to any one of claims 3 to 5, or the pharmaceutical composition for treating tumors by mild photothermal-abecib combination therapy according to any one of claims 6 to 8 in tumor treatment.

10. The use according to claim 9, characterized in that: The tumor was diffuse large B-cell lymphoma.

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