Preparation method and application of mitochondrion-targeting drug-loaded micelles for chemotherapy-photothermal combined treatment of tumors

Abstract

The purpose of this invention is to provide a method for preparing and applying mitochondrial-targeted drug-loaded micelles for chemotherapy-photothermal combined therapy of tumors, belonging to the field of antitumor drug technology. This nanomedicine uses the chemotherapy drug mitoxantrone as its core and incorporates the mitochondrial-targeting functionalized polymer distearate phosphatidylethanolamine-polyethylene glycol. 2000 The triphenylphosphine drug delivery system serves as the outer shell. This invention employs a thin-film hydration method to prepare the nanomedicine MTO@DSPE-PEG. 2000 -TPP, this nanomedicine, based on the dual function of MTO chemotherapy and photothermal therapy, can be used for synergistic effects in chemotherapy and photothermal therapy for breast cancer. Furthermore, triphenylphosphine induces the nanomedicine to target tumor cell mitochondria, enhancing drug accumulation in tumor cells and promoting tumor cell death. This nanomedicine possesses excellent biocompatibility, solving problems such as severe toxic side effects and drug resistance associated with chemotherapy in clinical practice, and shows promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drug technology, specifically relating to a method for preparing and applying mitochondrial targeted drug-loaded micelles for chemotherapy-photothermal combined therapy of tumors. Background Technology

[0002] Malignant tumors seriously endanger human health and pose a severe challenge to human well-being. Chemotherapy is a routine strategy for treating malignant tumors. Mitoxantrone (MTO), a chemotherapy drug, induces apoptosis by intercalating into DNA and inhibiting topoisomerase II, and is used clinically to treat leukemia, sarcoma, prostate cancer, and breast cancer. However, as a chemotherapy drug, MTO faces challenges such as low selectivity, drug resistance, and cardiotoxicity and even systemic toxicity.

[0003] Traditional treatments have limitations. For example, chemotherapy easily leads to drug resistance in tumor cells and causes severe toxic side effects; radiotherapy causes irreversible damage to normal tissues, affecting prognosis. Therefore, exploring new treatment methods has significant research and clinical value. Photothermal therapy (PTT) uses laser irradiation of the tumor site, converting light energy into heat energy, inducing tumor cell death through local high temperature. High temperature can increase the sensitivity of tumor cells to chemotherapy drugs. Simultaneously, its spatiotemporal controllability addresses the severe toxic side effects caused by nonspecific chemotherapy. Combining chemotherapy with PTT provides an effective strategy for treating malignant tumors. However, the key to PTT lies in achieving the conversion of light and heat energy through a photothermal converter. Existing photothermal converters face drawbacks such as potential toxicity and poor biodegradability in clinical translation. Studies have shown that the chemotherapy drug MTO has good photothermal conversion effects and can act as a photothermal converter, overcoming the limitations of existing photothermal converters. This fully demonstrates the feasibility of combining chemotherapy and photothermal therapy based on the photothermal effect of MTO.

[0004] In recent years, nanomedicine has provided an effective treatment method for the precision treatment of malignant tumors. Nanomaterials effectively overcome the limitations of traditional chemotherapy, which has significant systemic toxicity, by enhancing permeability and retention (EPR) effects. Furthermore, surface functionalization enables precise drug delivery, thereby overcoming drug resistance, improving the specificity of malignant tumor treatment, and ultimately enhancing efficacy.

[0005] Distearate phosphatidylethanolamine-polyethylene glycol 2000 -Triphenylphosphine (1,2-distaroyl-sn-glycero-3-phosphoethanolamine-poly(ethyleneglycol) 2000-triphenylphosphine,DSPE-PEG 2000 Triphenylphosphine (TPP) passively targets tumor tissue through the EPR effect, enters cells via endocytosis, and increases intracellular drug concentration. Furthermore, TPP induces drug targeting of mitochondria, concentrating the drug in the energy metabolism center and thus efficiently promoting tumor cell apoptosis.

[0006] Existing reports on chemotherapy-photothermal combined therapy using nanomedicine delivery systems rarely cover the synergistic effect of chemotherapy-PTT on malignant tumors based on the dual chemotherapy-PTT function of chemotherapeutic drugs. Therefore, mitochondrial-targeted nanomedicines based on the photothermal conversion properties of mitochondrial toxins (MTO) for chemotherapy-photothermal combined therapy of tumors overcome the limitations of photothermal converters in existing combined therapies, providing a novel multifunctional nanoplatform for the treatment of malignant tumors. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing mitochondrial-targeted drug-loaded micelles for chemotherapy-photothermal combined therapy of tumors and their application, wherein MTO@DSPE-PEG is prepared by thin-film hydration method. 2000 -TPP, this nanomedicine has good photothermal conversion properties and mitochondrial targeting function, and can be used for chemotherapy-photothermal synergistic therapy, and can be applied to anti-tumor treatment.

[0008] The present invention adopts the following technical solution: A method for preparing mitochondrial-targeted drug-loaded micelles for chemotherapy-photothermal combined therapy of tumors includes the following steps: S1. Weigh out MTO and dissolve it in methanol. Sonicate for 15 min until completely dissolved to obtain solution one. S2, Weigh out DSPE-PEG 2000 - TPP was placed in a round-bottom flask and dissolved in a mixed solvent of methanol and dichloromethane in a volume ratio of 1:1. The solution was sonicated for 30 min until the solute was completely dissolved, resulting in solution two. S3. Mix solution one and solution two by ultrasonication to obtain a homogeneous and clear solution. Place the clear solution in a rotary evaporator and set the temperature to 35°C. Wait for the organic solvent to evaporate completely to form a thin and uniform blue film. S4. Add the membrane to ultrapure water for hydration, stir at room temperature for 8 h, and then place it in an ice bath for sonication for 30 min to obtain a micelle dispersion. S5. The micelle dispersion was freeze-dried in a freeze dryer for 24 h to obtain a solid nanomedicine, denoted as MTO@DSPE-PEG. 2000 -TPP nanomedicines should be stored at -20°C for later use.

[0009] Furthermore, in S1, the amount of MTO used is 1.5 mg, and the amount of methanol used is 2 mL.

[0010] Furthermore, in S2, the DSPE-PEG 2000 The dosage of TPP is 10-15 mg, and the dosage of the mixed solvent is 25 mL.

[0011] Furthermore, in S3, the amount of ultrapure water used is 1 mL.

[0012] Application of mitochondrial-targeted drug-carrying micelles for chemotherapy-photothermal combined therapy of tumors in the preparation of antitumor drugs.

[0013] The beneficial effects of this invention are as follows: The preparation process of this invention is simple, and the resulting nanomedicine dispersion exhibits excellent uniformity and dispersibility. This nanomedicine utilizes the dual chemotherapy-PTT effect of MTO to achieve synergistic enhancement of tumor chemotherapy-PTT. Furthermore, TPP causes drug aggregation in mitochondria, achieving targeted therapy and anti-tumor drug resistance effects. Moreover, under laser irradiation, mitochondria become more sensitive to high temperatures, effectively disrupting mitochondrial energy metabolism and further enhancing the tumor-killing effect.

[0014] The nanomedicine of this invention exhibits excellent sustained-release properties, effectively prolonging its duration of action in vivo. It also passively targets tumor tissue through the EPR effect. This nanomedicine demonstrates good biocompatibility, minimal side effects, and significant antitumor activity both in vivo and in vitro. Attached Figure Description

[0015] Figure 1 MTO@DSPE-PEG prepared in Example 1 of this invention 2000 - Transmission electron microscope (TEM) image of TPP nanomedicine.

[0016] Figure 2 MTO@DSPE-PEG prepared in Example 1 of this invention 2000 -Hydrated particle size distribution of TPP nanomedicines.

[0017] Figure 3 MTO and DSPE-PEG in Embodiment 3 of the present invention 2000 -TPP, MTO@DSPE-PEG 2000 - Absorption spectrum of TPP.

[0018] Figure 4 MTO and DSPE-PEG in Embodiment 3 of the present invention 2000 -TPP, MTO@DSPE-PEG2000 - Fluorescence emission spectrum of TPP.

[0019] Figure 5 This is a standard curve of MTO concentration-absorbance value in Example 4 of the present invention.

[0020] Figure 6 MTO and MTO@DSPE-PEG in Embodiment 5 of the present invention 2000 -Graph showing the release behavior of TPP.

[0021] Figure 7 MTO@DSPE-PEG prepared in Example 1 of this invention 2000 -Photothermal conversion results of TPP nanomedicine.

[0022] Figure 8 MTO (left) and MTO@DSPE-PEG in Embodiment 7 of the present invention. 2000 - Cellular uptake of TPP (right) (bar: 25 µm).

[0023] Figure 9 MTO@DSPE-PEG prepared in Example 1 of this invention 2000 -Mitochondrial colocalization of TPP nanomedicine (scale bar: 100 µm).

[0024] Figure 10 The changes in mitochondrial membrane potential under different treatments in Example 9 of this invention are shown (scale bar: 100 µm).

[0025] Figure 11 The figure shows the results of detecting intracellular adenosine triphosphate (ATP) content under different treatments in Example 10 of the present invention.

[0026] Figure 12 The image shows the results of cell antitumor activity detection under different treatments in Example 11 of this invention.

[0027] Figure 13 MTO and DSPE-PEG in Embodiment 12 of the present invention 2000 -TPP, MTO@DSPE-PEG 2000 -Graph showing the in vivo photothermal conversion results of TPP.

[0028] Figure 14 The graph shows the in vivo antitumor treatment results under different treatments in Example 13 of the present invention (n=5). p<0.001, p < 0.0001).

[0029] Figure 15The images show the blood routine test results under different treatments in Example 14 of this invention.

[0030] Figure 16 The image shows the blood biochemistry test results under different treatments in Example 14 of this invention.

[0031] Figure 17 The images show stained tissue sections under different treatments in Example 14 of this invention (scale bar: 100 µm). Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention and through specific examples. Any other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0033] Example 1 Nanomedicine MTO@DSPE-PEG 2000 Preparation of TPP Accurately weigh 1.5 mg of MTO and dissolve it in 2 mL of methanol, then sonicate for 15 min until completely dissolved. Accurately weigh DSPE-PEG. 2000 10 mg of TPP was placed in a round-bottom flask and dissolved in 25 mL of a 1:1 mixture of methanol and dichloromethane. The solution was sonicated for 30 min until completely dissolved. The two solutions were then sonicated to obtain a homogeneous and clear solution. The solution was placed in a rotary evaporator at 35 °C until the organic solvent was completely evaporated, forming a thin and uniform blue film. 1 mL of ultrapure water was added for hydration, and the mixture was stirred at room temperature for 8 h. After 8 h, the film was sonicated in an ice bath for 30 min to obtain a micelle dispersion. The dispersion was then freeze-dried for 24 h to obtain a solid nanomedicine, which was stored at -20 °C for later use.

[0034] Example 2 Nanomedicine MTO@DSPE-PEG 2000 Preparation of TPP Accurately weigh 1.5 mg of MTO and dissolve it in 2 mL of methanol, then sonicate for 15 min until completely dissolved. Accurately weigh DSPE-PEG. 2000-15 mg of TPP was placed in a round-bottom flask and dissolved in 25 mL of a 1:1 mixture of methanol and dichloromethane. The solution was sonicated for 30 min until completely dissolved. The two solutions were then sonicated to obtain a homogeneous and clear solution. The solution was placed in a rotary evaporator at 35 °C until the organic solvent was completely evaporated, forming a thin and uniform blue film. 1 mL of ultrapure water was added for hydration, and the mixture was stirred at room temperature for 8 h. After 8 h, the film was sonicated in an ice bath for 30 min to obtain a micelle dispersion. The dispersion was then freeze-dried for 24 h to obtain a solid nanomedicine, which was stored at -20 °C for later use.

[0035] Example 3 Characterization of MTO@DSPE-PEG in Example 1 2000 - Physicochemical properties of TPP.

[0036] (1) Use TEM to characterize the morphology of nanomedicines. Figure 1 MTO@DSPE-PEG in Example 1 2000 The TEM image of TPP is shown in the figure. The aqueous dispersion of nanomedicine has good dispersibility and uniformity.

[0037] (2) The hydrodynamic size and distribution of the nanomedicine aqueous dispersion were detected using a Malvern particle size analyzer. Figure 2 MTO@DSPE-PEG in Example 1 2000 The particle size distribution of -TPP was analyzed, and the results showed that the average particle size was 126 nm and the polydispersity index was 0.319.

[0038] (3) Use a visible-ultraviolet absorption spectrometer to detect the absorption spectra of different drugs. Figure 3 The results showed that in Example 1, MTO@DSPE-PEG 2000 -TPP's absorption spectrum is similar to that of MTO, DSPE-PEG 2000 - The absence of characteristic absorption peaks in TPP indicates that MTO was successfully loaded.

[0039] (4) Use a transient steady-state fluorescence spectrometer to detect fluorescence emission spectra. Figure 4 The results showed that in Example 1, MTO@DSPE-PEG 2000 The emission spectrum of TPP is similar to that of MTO, and when the excitation wavelength is 600 nm, the emission wavelength of the nanomedicine is 714 nm, which is in the near-infrared band, proving that it can effectively penetrate biological tissues.

[0040] Example 4 Investigating MTO@DSPE-PEG in Example 1 2000 -TPP loading rate. The specific method is as follows: Prepare 0.2 mg / mL MTO@DSPE-PEG 2000 -TPP aqueous dispersion was analyzed, and its absorbance was measured (wavelength set to the characteristic absorption peak of MTO). Absorbance values ​​at the same wavelength were measured at different MTO concentrations (0, 10, 20, 30, 40, 50, 60, 70, 80 µM), and a concentration-absorbance standard curve was plotted. The content of free MTO in the nanomedicine was calculated using the MTO standard curve, with the formula: Drug loading rate (%) = m MTO / m 纳米药物 ×100%.

[0041] Figure 5 The concentration-absorbance standard curve of MTO shows that the drug loading rate of MTO in the nanomedicine is 12.05%.

[0042] Example 5 Investigating MTO@DSPE-PEG in Example 1 2000 -TPP release behavior. The specific method is as follows: Using dialysis to investigate MTO@DSPE-PEG in Example 1 2000 -TPP release behavior. Take 1 mL of MTO@DSPE-PEG. 2000 - TPP aqueous dispersion (6 mg / mL) was placed in a dialysis bag, with MTO of the same concentration serving as a control. Each sample was placed in a beaker containing 150 mL of phosphate-buffered saline (PBS) and shaken on a constant-temperature shaker (37°C, 100 rpm). At different time points (0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 20, 24, 36, 48, 60, 72, 80 h), 100 µL samples were taken and replenished with an equal volume of the corresponding drug solution. The absorbance values ​​were measured. Figure 5 The cumulative release rate is calculated using the standard curve, and the time-cumulative release rate curve is plotted.

[0043] The results are as follows Figure 6 As shown, after 24 hours, the cumulative release rate of MTO was 82.9%, and the cumulative release rate of nanomedicine was 41.98%, which is about half that of free drug, proving that nanomedicine has a sustained-release effect and effectively reduces toxic side effects.

[0044] Example 6 Investigating MTO@DSPE-PEG in Example 1 2000 - The photothermal conversion effect of TPP. The specific method is as follows: (1) Prepare 1 mL of 100 µM MTO solution and MTO@DSPE-PEG 2000-TPP aqueous dispersion (equivalent MTO concentration), using 660 nm and 0.6 W / cm², respectively. 2 Laser irradiation was used to record temperature changes every 30 seconds using an infrared thermal imager, and a time-temperature change curve was plotted. Pure water was used as a control group.

[0045] (2) Prepare 1 mL of nano-drug dispersions with concentrations of 20, 40, 80, and 100 µM, using 660 nm and 0.6 W / cm². 2 Laser irradiation was performed, and temperature changes were recorded using an infrared thermal imager. Concentration-temperature curves were plotted. 1 mL of the nano-drug dispersion (100 µM) was used with different power densities (0.2, 0.4, 0.6, 0.8 W / cm²). 2 Irradiate the sample with a 660 nm laser and record the temperature change using an infrared thermal imager. Plot the power density-temperature change curve.

[0046] (3) Investigate the photothermal stability of nanomedicines. Take 1 mL of nanomedicine dispersion (100 μM equivalent MTO concentration). Use 660 nm, 0.6 W / cm 2 Laser irradiation was performed for 10 minutes, followed by laser shutdown and a 10-minute natural cooling period. This cycle was repeated four times, with temperature changes recorded every 30 seconds using an infrared thermal imager. Finally, the photothermal conversion efficiency of the MTO was calculated.

[0047] Figure 7 MTO@DSPE-PEG is shown in Example 1. 2000 - The photothermal conversion effect of TPP. At 660 nm, 0.6 W / cm² 2 When 100 µM MTO was irradiated with a laser for 20 min, the temperature increased by 16.5 °C, and the photothermal conversion efficiency was 15.6%. Simultaneously, the nanomedicine maintained the photothermal conversion efficiency of MTO. Furthermore, the temperature change increased in a dependent manner with increasing nanomedicine concentration or laser power. Moreover, after cyclic laser irradiation and shutdown, the temperature of the nanomedicine exhibited periodic changes, demonstrating the excellent photothermal stability of the nanomedicine.

[0048] Example 7 Investigating MTO@DSPE-PEG in Example 1 2000 - TPP cellular uptake. The specific methods are as follows: (1) 4T1 cells were placed in a 37 ℃, 5% CO2 incubator for 24 h. The cells were completely adhered to the wall, had good morphology and reached a density of 80%. Cell passage culture was then carried out.

[0049] (2) 4T1 cells were fed at a dose of 2×10 4Cells were seeded per well in 24-well plates containing cell spreaders and incubated for 24 h until fully adhered. The original culture medium was then discarded, and 750 µL of a mixture containing MTO and MTO@DSPE-PEG was added. 2000 - New TPP culture medium (concentration of 1 μM MTO equivalent) was used for incubation at four time points: 1, 2, 4, and 8 h. The original culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 20 min, incubated with DAPI reagent for 20 min, and the cell nuclei were stained. Cell slides were removed, mounted, and observed under a laser confocal fluorescence microscope to observe the drug uptake by 4T1 cells.

[0050] Figure 8 For MTO, MTO@DSPE-PEG 2000 Cellular uptake of TPP. DAPI binds to the cell nucleus and emits blue fluorescence, while MTO emits red fluorescence. Quantitative results show that after 2 hours of cellular uptake, the autofluorescence intensity of the nanodrug is lower than that of free MTO; after 4 hours of cellular uptake, the average fluorescence intensity of both MTO and nanodrug reaches its maximum, with MTO at 37.62 and nanodrug at 48.91. The average fluorescence intensity of the nanodrug is greater than that of free MTO. This verifies that the nanodrug possesses a sustained-release effect and effectively enhances drug accumulation in tumor cells, which may be attributed to the nanodrug entering cells via endocytosis and targeting mitochondria.

[0051] Example 8 Investigating MTO@DSPE-PEG in Example 1 2000 -Mitochondrial colocalization of TPP. The specific method is as follows: 4T1 cells were fed at a rate of 2 × 10 4 Cells / well were seeded into 24-well plates containing cell spreaders. The plates were incubated for 24 h, and then MTO@DSPE-PEG containing different concentrations (0, 1, 2, 4 μM (MTO equivalent concentration)) was added. 2000 The complete culture medium of TPP was placed in an incubator and incubated for another 4 h. Mito-Tracker Green staining working solution was prepared according to the kit instructions. After the drug incubation was completed, 750 µL of staining working solution was added and incubated for 30 min. The co-localization of drug lines and particles was observed using a laser confocal fluorescence microscope.

[0052] Figure 9 MTO@DSPE-PEG in Example 1 2000 Mitochondrial colocalization of TPP. Figure 9The results showed that the intensity of red fluorescence increased in a dependent manner with increasing nanodrug concentration. At concentrations of 2 and 4 µM, some red fluorescence overlapped with green fluorescence to form yellow fluorescence, and some red fluorescence overlapped with the blue fluorescence of the cell nucleus, indicating that the nanodrug can target mitochondria and the cell nucleus. This may be attributed to MTO inducing apoptosis by embedding in DNA, thereby achieving the effect of dual sub-organelle action.

[0053] Example 9 To investigate mitochondrial damage, the MTO@DSPE-PEG in Example 1 was tested. 2000 - Changes in mitochondrial membrane potential of TPP. The specific method is as follows: (1) 4T1 cells were fed at a rate of 2 × 10⁻⁶ 4 Cells were seeded per well in 24-well plates containing cell spreaders. There were 7 groups: I: Control group; II: DSPE-PEG. 2000 -TPP group; III:DSPE-PEG 2000 -TPP+Illumination Group; IV: MTO Group; V: MTO+Illumination Group; VI: MTO@DSPE-PEG 2000 -TPP group; VII: MTO@DSPE-PEG 2000 -TPP+ Illumination Group.

[0054] (2) Control group: The cells were incubated in an incubator for 48 h. JC-1 staining working solution was prepared according to the instructions of the enhanced mitochondrial membrane potential detection kit (JC-1). 750 µL of staining working solution was added and the cells were incubated in an incubator for 20 min to stain the nuclei and mitochondria. After washing, the slides were taken out and observed using a laser confocal fluorescence microscope.

[0055] (3) Non-light group: Incubated in an incubator for 24 h, each group was incubated with the corresponding drug solution (1 µM equivalent MTO concentration) for another 24 h, JC-1 staining working solution was prepared, 750 µL of staining working solution was added, and the incubator was incubated for 20 min to stain the cell nucleus and mitochondria. After washing, the slides were taken out and observed using a laser confocal fluorescence microscope.

[0056] (4) Light group: After culturing cells for 24 h, each group was incubated with the corresponding drug solution (1 µM equivalent MTO concentration) for 4 h, and then treated with 660 nm, 0.6 W / cm 2 Irradiate with laser for 2 min, continue culturing for 20 h, prepare JC-1 staining working solution, add 750 µL of staining working solution, incubate in an incubator for 20 min, stain the cell nucleus and mitochondria, wash and remove the slide, and observe using a laser confocal fluorescence microscope.

[0057] Figure 10 MTO@DSPE-PEG in Example 1 2000 The changes in mitochondrial membrane potential of TPP were observed. The merge plot shows that the control group and the blank vector DSPE-PEG... 2000 -TPP and its illuminated group mainly emit red fluorescence. MTO, MTO+illuminated group, and MTO@DSPE-PEG, on the other hand, emit red fluorescence. 2000 -TPP, MTO@DSPE-PEG 2000 -TPP+ illumination group showed a sequential increase in green fluorescence. Quantitative analysis (right figure) revealed that MTO@DSPE-PEG 2000 -TPP+ illumination group showed the highest proportion of green fluorescence, MTO@DSPE-PEG 2000 -TPP was the second most significant. This indicates that the decrease in mitochondrial membrane potential was most pronounced under 660 nm laser irradiation, which may be attributed to the nanomedicine targeting mitochondria and the photothermal conversion effect of MTO further exacerbating mitochondrial damage.

[0058] Example 10 To further investigate the MTO@DSPE-PEG in Example 1 2000 - TPP mitochondrial damage was assessed by detecting changes in intracellular ATP levels. The specific method is as follows: (1) 4T1 cells were fed at a rate of 4 × 10⁻⁶ 3 Cells / well were seeded into 96-well plates containing cell spreaders. Grouping was the same as in Example 8.

[0059] (2) Control group: Incubated in an incubator for 48 h, according to CellTiter-Glo ® Prepare the detection working solution according to the instructions of the chemiluminescence cell viability assay kit. Add 100 µL of the detection working solution, place it on a fixed-track shaker and shake for 2 min. Incubate at room temperature for 10 min, and use an ELISA reader to detect its chemiluminescence signal.

[0060] (3) Non-light group: Incubate in an incubator for 24 h, add the corresponding drug solution (1 µM equivalent MTO concentration) to each group and continue incubation for 24 h, prepare detection working solution, add 100 µL of detection working solution, place on a fixed-track shaker and shake for 2 min, incubate at room temperature for 10 min, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect its chemiluminescence signal.

[0061] (4) Light group: Incubated in an incubator for 24 h, each group was incubated with the corresponding drug solution (1 µM equivalent MTO concentration) for 4 h, and then treated with 660 nm, 0.6 W / cm 2Irradiate with laser for 90 s, continue culturing for 20 h, prepare detection working solution, add 100 µL of detection working solution, place on a fixed-track shaker and shake for 2 min, incubate at room temperature for 10 min, and use an ELISA reader to detect its chemiluminescence signal.

[0062] Figure 11 The image shows the results of intracellular ATP content detection under different treatments. As shown in the figure, DSPE-PEG... 2000 Compared with the control group, there was no significant difference in intracellular ATP content in the TPP and its light-illuminated group. The intracellular ATP content from high to low was as follows: MTO group, MTO+ light-illuminated group, MTO@DSPE-PEG. 2000 -TPP group, MTO@DSPE-PEG 2000 -TPP + light irradiation group. The results were consistent with the JC-1 experiment, suggesting that TPP's mitochondrial targeting effect led to drug accumulation in tumor tissue, and light irradiation further induced mitochondrial damage, disrupted mitochondrial energy metabolism, and promoted tumor cell death.

[0063] Example 11 To investigate the MTO@DSPE-PEG in Example 1 2000 The in vitro tumor-killing effect of TPP was assessed using a CCK-8 assay kit (cell counting kit-8, CCK-8). The specific method is as follows: (1) 4T1 cells were fed at a rate of 4 × 10⁻⁶ 3 Cells were seeded per well in 96-well plates and divided into 6 groups: MTO group, MTO + light group, MTO@DSPE-PEG group, etc. 2000 -TPP group, MTO@DSPE-PEG 2000 -TPP+ Illumination Group, DSPE-PEG 2000 -TPP group, DSPE-PEG 2000 -TPP+ Illumination Group.

[0064] (2) Control group: Cells were cultured in an incubator for 48 h. 100 μL of complete culture medium containing 10% CCK-8 reagent was added to each well (a blank well containing only CCK-8 medium and no cells was also set up). The cells were incubated for another 20 min. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. Cell viability (%) = (As-Ab) / (Ac-Ab) × 100%. Where As is the absorbance value of the experimental group, Ac is the absorbance value of the control group, and Ab is the absorbance value of the blank group.

[0065] (3) Non-light group: placed in an incubator and cultured for 24 h. Each group was added with the corresponding drug solution (1 µM equivalent MTO concentration) and incubated for another 24 h. 100 μL of complete culture medium containing 10% CCK-8 reagent was added to each well (and blank wells containing only CCK-8 culture medium without cells were set up at the same time). The cells were placed in an incubator and incubated for another 20 min. The absorbance value at 450 nm was detected using an ELISA reader. The cell viability was calculated according to the method in (2) of this example.

[0066] (4) For the light-illuminated group, the group was incubated in an incubator for 24 h. Each group was then incubated with the corresponding drug solution (1 µM equivalent MTO concentration) for 4 h, using 660 nm and 0.6 W / cm 2 Irradiate each well with laser for 90 s, continue culturing for 20 h, add 100 μL of complete culture medium containing 10% CCK-8 reagent to each well (and set up blank wells containing only CCK-8 culture medium without cells), place in an incubator and continue incubating for 20 min, use an ELISA reader to detect the absorbance value at 450 nm wavelength, and calculate the cell viability according to the method in (2) of this example.

[0067] Figure 12 The figure shows the cytotoxicity test results under different treatments. As can be seen from the figure, DSPE-PEG... 2000 -TPP and its light-irradiated group showed near 100% cell survival and good cell compatibility. The cytotoxicity of both MTO and nanomedicines increased in a concentration-dependent manner; furthermore, light irradiation exacerbated their cytotoxicity. At a MTO concentration of 2 μM, the survival rate of free MTO cells was 73.36%, while the nanomedicine resulted in a survival rate of 48.15% (p<0.05). After laser irradiation, the cell survival rate of the nanomedicine group was 39.52%, significantly better than the control group (p<0.05). This indicates that nanomedicines achieve synergistic effects of chemotherapy-photothermal combined therapy at the cellular level through mitochondrial targeting.

[0068] Example 12 Investigating MTO@DSPE-PEG in Example 1 2000 - The in vivo photothermal conversion effect of TPP. The specific method is as follows: (1) 4T1 cells were fed at a rate of 2 × 10⁻⁶ 6 100 μL of 4T1 cells per mouse was injected subcutaneously on the right posterior side of the mouse to establish a subcutaneous 4T1 cell tumor model.

[0069] (2) When the tumor volume in mice reaches 100 mm 3 Mice were divided into three groups: PBS group, MTO group, and MTO@DSPE-PEG group. 2000-TPP group (all concentrations were 5 mg / kg MTO equivalent), administered via tail vein injection for 8 h, followed by treatment with 660 nm, 0.6 W / cm 2 The tumor area of ​​mice was irradiated with laser for 5 minutes, and the temperature change was recorded every 30 seconds using an infrared thermal imager. Photothermal images were acquired, and time-temperature change curves were plotted. The PBS group served as the control group.

[0070] Figure 13 The study demonstrated the in vivo photothermal conversion efficiency under different treatments. The nanomedicine group showed a temperature increase of 24.5 °C after 5 minutes. In contrast, the PBS and MTO groups showed slower temperature increases. Furthermore, when the tumor site of the nanomedicine group was irradiated with a 660 nm laser for 2 minutes, the temperature rose to 52.4 °C, sufficient to induce thermal ablation of tumor cells. This indicates that the nanomedicine maintains a more efficient photothermal conversion effect in vivo, validating the feasibility of its photothermal conversion (PTT) method.

[0071] Example 13 Investigating MTO@DSPE-PEG in Example 1 2000 - The in vivo anti-tumor effect of TPP. The specific method is as follows: (1) Mice were randomly divided into 5 groups (n=5): I: PBS group; II: MTO group; III: MTO + light group; IV: MTO@DSPE-PEG group. 2000 -TPP group; V: MTO@DSPE-PEG 2000 -TPP+light group (concentration of 5 mg / kg MTO equivalent).

[0072] (2) Control group: The tumor volume of mice reached 100 mm 3 Mice were injected with 100 μL of PBS solution via tail vein, once every 3 days, for a total of two injections. In the non-light-exposed group, the tumor volume of mice reached 100 mm. 3 100 μL of the corresponding drug solution was injected via the tail vein, once every 3 days, for a total of two injections. In the light-exposed group, the tumor volume of mice reached 100 mm. 3 100 μL of the corresponding drug solution was injected intravenously via the tail vein. Eight hours later, the drug was administered at 660 nm and 0.6 W / cm². 2 Laser irradiation for 2 minutes, with injection and laser irradiation once every 3 days, for a total of 2 times.

[0073] (3) After treatment began, the long and short diameters of the mouse tumor and the mouse's body weight were recorded every two days. The tumor volume was calculated, and curves showing the changes in tumor volume and body weight were plotted. Mouse volume V = (L × W²) / 2, where V is the tumor volume (mm). 3 L represents the long diameter of the tumor (mm), and W represents the short diameter of the tumor (mm). The treatment cycle is 16 days.

[0074] (4) After treatment, the mice were euthanized, their tumor tissue was dissected, and the tumor growth inhibition rate of the nanomedicine was calculated. Tumor growth inhibition rate (%) = (V C -V T ) / V C ×100%. Where V T V represents the average volume of the treatment group. C This represents the average volume of the control group.

[0075] Figure 14 The study demonstrated the in vivo antitumor effects under different treatments. At the end of treatment, tumor volume decreased to varying degrees in all treatment groups, and MTO@DSPE-PEG showed [significant improvement]. 2000 -TPP+ light irradiation group showed the most significant tumor inhibition. Calculations show that MTO@DSPE-PEG... 2000 The tumor growth inhibition rate in the TPP+ light irradiation group was 83.7%, validating its highly effective anti-tumor effect. Furthermore, the MTO+ light irradiation group and the MTO@DSPE-PEG group... 2000 The tumor volume changes were relatively small in the TPP group, which may be attributed to the combined effect of the photothermal properties of MTO and the inhibitory effect of the nano-drug delivery system on tumor tissue. There was no significant difference in mouse body weight between the treatment groups and the control group, suggesting that the drug had no significant toxic side effects on mice.

[0076] Example 14 To investigate the MTO@DSPE-PEG in Example 1 2000 - To assess the biocompatibility of TPP, routine blood tests, blood biochemistry tests, histological analysis, and H&E staining were performed. The specific methods are as follows: (1) After the mice were treated, they were anesthetized with isoflurane. Whole blood samples were collected by blood collection from the eyeballs. The samples were divided into two parts. One part was used for routine blood tests, including hemoglobin (HGB), lymphocyte percentage (Lymph%), platelet count (PLT), red blood cell count (RBC), and white blood cell count (WBC). The other part was used for blood biochemistry tests, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), total protein (TP), albumin (ALB), creatinine (CR), and blood urea nitrogen (BUN).

[0077] (2) After collecting blood samples, the mice were euthanized and the heart, liver, spleen, lung, kidney and tumor tissues of each component mouse were collected, fixed with 4% paraformaldehyde, routinely embedded in paraffin, sectioned (thickness 4-5 µm), and hematoxylin and eosin (H&E) staining and histopathological analysis were performed.

[0078] Figure 15 The graph shows the results of routine blood tests under different treatments. As can be seen from the graph, the white blood cell count was elevated in the control group and all treatment groups, while other indicators fluctuated within the normal range. This suggests that the mice had mild inflammation.

[0079] Figure 16 The figures show the results of routine blood tests under different treatments. As can be seen from the figures, compared with the control group, the mice in the treatment group had higher levels of ALT and AST in their blood, indicating that the drug caused mild damage to the liver of the mice. Other indicators in the treatment group, including TBIL, ALB, TP, ALP, UREA, and CREA, showed no significant differences compared with the control group, indicating that the drug caused limited and reversible damage to liver and kidney function, which may be attributed to the fact that MTO is metabolized by the liver.

[0080] Figure 17 The images show the H&E staining results of heart, liver, spleen, lung, kidney, and tumor tissues from different groups under different treatments. As can be seen from the images, the structures of the heart, liver, spleen, lung, and kidneys in each group of mice were normal and no obvious damage was observed, demonstrating the excellent tissue compatibility of the drug. Furthermore, the H&E staining results of the tumor tissues showed that, compared to the control group, the treatment group had decreased cell density and exhibited nuclear pyknosis and karyorrhagia. MTO@DSPE-PEG 2000 - Tissue necrosis was observed in the TPP+ phototherapy group, further validating the significant anti-tumor effect of nanomedicine chemotherapy-photothermal combined therapy.

[0081] Finally, the embodiments of the present invention are not limited to those described above. Any other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for preparing mitochondrial-targeted drug-loaded micelles for chemotherapy-photothermal combined therapy of tumors, characterized in that: Includes the following steps: S1. Weigh out MTO and dissolve it in methanol. Sonicate for 15 min until completely dissolved to obtain solution one. S2, Weigh out DSPE-PEG 2000 - TPP was placed in a round-bottom flask and dissolved in a mixed solvent of methanol and dichloromethane in a volume ratio of 1:

1. The solution was sonicated for 30 min until the solute was completely dissolved, resulting in solution two. S3. Mix solution one and solution two by ultrasonication to obtain a homogeneous and clear solution. Place the clear solution in a rotary evaporator and set the temperature to 35°C. Wait for the organic solvent to evaporate completely to form a thin and uniform blue film. S4. Add the membrane to ultrapure water for hydration, stir at room temperature for 8 h, and then place it in an ice bath for sonication for 30 min to obtain a micelle dispersion. S5. The micelle dispersion was freeze-dried in a freeze dryer for 24 h to obtain a solid nanomedicine, denoted as MTO@DSPE-PEG. 2000 -TPP nanomedicines should be stored at -20°C for later use.

2. The method for preparing mitochondrial-targeted drug-loaded micelles for chemotherapy-photothermal combined therapy of tumors according to claim 1, characterized in that: In S1, the amount of MTO used is 1.5 mg, and the amount of methanol used is 2 mL.

3. The method for preparing mitochondrial-targeted drug-loaded micelles for chemotherapy-photothermal combined therapy of tumors according to claim 1, characterized in that: In S2, the DSPE-PEG 2000 The dosage of TPP is 10-15 mg, and the dosage of the mixed solvent is 25 mL.

4. The method for preparing mitochondrial-targeted drug-loaded micelles for chemotherapy-photothermal combined therapy of tumors according to claim 1, characterized in that: In S3, the amount of ultrapure water used is 1 mL.

5. The application of mitochondrial-targeted drug-loaded micelles prepared by the preparation method according to any one of claims 1 to 4 in the preparation of antitumor drugs.

Images