Phototherapy nano-drug with mitochondrion / STAT3 protein double-site targeting as well as preparation method and application of phototherapy nano-drug

Abstract

The invention discloses a phototherapy nano-drug with mitochondrial / STAT3 protein double-site targeting. The phototherapy nano-drug is ATO / CR nano-particles formed by self-assembling a compound CR and atorvaquone under the action of distearoyl phosphatidyl ethanolamine-polyethylene glycol; the structure of the compound CR is shown as a formula I in the specification. The invention discloses an application of the phototherapy nano-drug with mitochondrial / STAT3 protein double-site targeting in preparation of drugs for treating tumors. The phototherapy nano-drug can target mitochondria and STAT3 protein in tumor cells through ATO, and can also passively target tumor sites through the high-permeability long-retention effect of nano-particles, so that more nano-therapeutic agents are enriched around tumors, and the curative effect is improved. The phototherapy nano-drug provided by the invention has good photothermal performance, can effectively enhance the PTT effect of gastric cancer, and exerts the tumor synergistic treatment ability by promoting cell apoptosis, inhibiting angiogenesis and hindering the cell cycle.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of materials and biological medicine, and particularly relates to a kind of light therapy nano-drug with mitochondrion / STAT3 protein double-site targeting and its preparation method, and application in preparation fluorescent, photothermal imaging mediated multi-target tumor treatment drug. BACKGROUND

[0002] Gastric cancer is one of the common digestive tract malignant tumor diseases in the world, and its morbidity and mortality rank the fifth among malignant tumors. At present, the clinical treatment methods of gastric cancer mainly include radiotherapy, chemotherapy and surgery, but these treatment methods have the disadvantages of large toxic and side effects and poor curative effect. Therefore, it is still a great challenge to find more targeted and effective cancer treatment methods. In recent years, near-infrared light mediated photothermal therapy (PTT) has become a new treatment method for gastric cancer due to its small toxic and side effects, but due to the enhanced heat resistance of cancer cells, the overexpression of intracellular related heat shock proteins (HSPs) activated by high heat will hinder the curative effect of photothermal therapy. Therefore, overcoming tumor heat resistance is a potential strategy to improve the sensitivity and effectiveness in photothermal therapy.

[0003] In recent years, due to the rapid development of nanotechnology, passive targeting nanodrug delivery systems that selectively accumulate nanodrugs in tumors by enhancing permeability and retention (EPR) effect have attracted widespread attention, but due to the biological obstacles such as large diffusion resistance of tumor deep penetration site, extracellular matrix (caused by interstitial pressure) and rapid circulation in blood, the accumulation of nanodrugs in tumor site is limited, and the active targeting drug delivery strategy using specific targeting of biological receptors can improve the accumulation and uptake of drugs in tumor tissues to maximize the treatment efficiency.

[0004] The synthesis of HSPs cannot be separated from ATP supply, and inhibiting the synthesis of ATP will fundamentally reverse the photothermal resistance of tumors. Mitochondrial oxidative phosphorylation (OXPHOS) is the core process of ATP synthesis, and inhibiting OXPHOS will lead to a decrease in mitochondrial membrane potential and mitochondrial morphology instability, thereby reducing ATP production and enhancing ROS generation, further leading to organelle damage and cell dysfunction. Atovaquone (ATO) as an OXPHOS inhibitor can inhibit the activity of mitochondrial complex III, leading to a decrease in ATP content in tumors and further inhibiting the expression of HSPs, thereby further improving the effect of photothermal therapy. At the same time, atovaquone is also an effective STAT3 inhibitor, and abnormal activation of STAT3 can induce the expression of a large number of genes related to the occurrence of gastric cancer, and inhibition of the activation of STAT3 protein will inhibit cell proliferation, promote apoptosis, and reduce the expression of vascular endothelial growth factor, thereby hindering tumor development through multiple pathways.

[0005] Most inorganic nanomaterials suffer from poor biocompatibility and difficulty in metabolism. However, ketoacid dyes exhibit strong but narrow absorption in the near-infrared region, possessing high molar extinction coefficients, ideal fluorescence imaging properties, and excellent photothermal performance, making them widely used in bioimaging and therapeutics. Nevertheless, in the biomedical field, the water solubility and targeting properties of ketoacid dyes remain significant challenges, leading to low bioavailability, poor biocompatibility, and severe toxic side effects when administered alone. Furthermore, ketoacid dye-mediated photothermal therapy may more easily induce thermotherapy tolerance in tumor cells. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing tumor treatment agents, such as poor biocompatibility, lack of specific targeting, high heat resistance of tumor cells, and incomplete treatment. This invention utilizes the amphiphilic polymer distearate-phosphatidylethanolamine-polyethylene glycol (DSPE-PEG). 2000 Under the influence of [unspecified], compound CR and atovaquinone (ATO) self-assemble to form a phototherapy nanomedicine with dual-site targeting of mitochondria and STAT3 protein, passively accumulating around tumor cells through the EPR effect. Furthermore, it can target STAT3 protein, inhibiting STAT3 protein phosphorylation, thereby inhibiting downstream signaling pathways to promote apoptosis, inhibit angiogenesis, and inhibit cell cycle. On the other hand, ATO, as an OXPHOS inhibitor, is located in mitochondria, inhibiting ATP production by disrupting mitochondrial structure, thereby reducing HSP expression and enhancing PTT efficacy. The phototherapy nanomedicine with dual-site targeting of mitochondria and STAT3 protein described in this invention can serve as a novel multifunctional nanomedicine to enhance the PTT effect in gastric cancer.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] Another objective of this invention is to provide a phototherapy nanomedicine with dual-site targeting of mitochondria / STAT3 protein, which is an ATO / CR nanoparticle (ATO / CR NPs) formed by the self-assembly of compound CR and the OXPHOS / STAT3 protein inhibitor atovaquinone (ATO) under the action of distearate phosphatidylethanolamine-polyethylene glycol (DSPE-mPEG2000).

[0009] The structure of compound CR is shown in Formula I:

[0010]

[0011] Another object of the present application is to provide a preparation method of the light therapy nano drug with mitochondrial / STAT3 protein double site targeting, comprising: dissolving the compound CR and atovaquone in tetrahydrofuran to obtain a mixed solution; under stirring, the mixed solution is added dropwise into a di-stearoyl phosphatidyl ethanolamine-polyethylene glycol aqueous solution, after the dropwise addition is completed, the stirring is continued for 5-10 minutes, nitrogen is fully blown to remove tetrahydrofuran, after sedimentation, centrifugation is performed, the supernatant is taken, and an ATO / CR nanoparticle aqueous dispersion is obtained.

[0012] The mass ratio of the compound CR and atovaquone is 1:0.1-1:0.5, preferably 1:0.2.

[0013] The mass ratio of the compound CR and di-stearoyl phosphatidyl ethanolamine-polyethylene glycol is 1:1-1:2, preferably 1:1.5.

[0014] Specifically, the di-stearoyl phosphatidyl ethanolamine-polyethylene glycol can be DSPE-mPEG2000.

[0015] The mass-volume ratio of the compound CR and tetrahydrofuran is 1:1-1:2 mg / mL, preferably 1:1 mg / mL.

[0016] The di-stearoyl phosphatidyl ethanolamine-polyethylene glycol aqueous solution is prepared by using deionized water.

[0017] The concentration of the di-stearoyl phosphatidyl ethanolamine-polyethylene glycol aqueous solution is 0.2-0.5 mg / mL, preferably 0.3 mg / mL.

[0018] The dropwise addition speed of the mixed solution is 1-30 drops / min, and the stirring speed is 200-500 rpm.

[0019] Preferably, the mixed solution is slowly added dropwise into the di-stearoyl phosphatidyl ethanolamine-polyethylene glycol aqueous solution at room temperature; after the dropwise addition is completed, the stirring is continued at room temperature for 5-10 minutes.

[0020] The sedimentation is overnight sedimentation.

[0021] In the ATO / CR nanoparticle aqueous dispersion, the final concentration of the ATP / CR nano drug is 100-200 μM.

[0022] As a preferred technical solution of the preparation method of the light therapy nano drug with mitochondrial / STAT3 protein double site targeting, the ATO / CR nanoparticle aqueous dispersion is dried.

[0023] Another object of the present application is to provide the use of the phototherapy nano-drug with mitochondrial / STAT3 protein double site targeting in the preparation of a tumor treatment drug.

[0024] Another object of the present application is to provide the use of the phototherapy nano-drug with mitochondrial / STAT3 protein double site targeting in the preparation of a tumor treatment drug.

[0025] Preferably, the use is the use of the phototherapy nano-drug with mitochondrial / STAT3 protein double site targeting in the preparation of a tumor treatment drug mediated by photothermal treatment through multiple pathways such as promoting apoptosis, inhibiting angiogenesis, and hindering cell cycle.

[0026] The hindering of the cell cycle is that the levels of target proteins P-STAT3 / STAT3 and cell cycle proteins Cyclin D1, CDK4, and CDK6 are significantly reduced in a dose-dependent manner, which indicates that the ATO / CR nanoparticles can achieve tumor treatment by inhibiting STAT3 protein phosphorylation and hindering the cell cycle.

[0027] Another object of the present application is to provide the use of the phototherapy nano-drug with mitochondrial / STAT3 protein double site targeting in the preparation of a tumor treatment drug.

[0028] Another object of the present application is to provide the use of the compound CR or nanoparticles thereof in the preparation of a tumor treatment drug.

[0029] Another object of the present application is to provide a preparation method of the compound CR nanoparticles, which comprises the following steps: dissolving the compound CR in tetrahydrofuran to obtain a compound CR solution; adding the compound CR solution dropwise into a di-stearoyl phosphatidyl ethanolamine-polyethylene glycol aqueous solution under stirring, continuing to stir for 5-10 minutes after the dropwise addition is completed, removing the tetrahydrofuran by blowing nitrogen, centrifuging after overnight sedimentation, and taking the supernatant to obtain a compound CR nanoparticle aqueous dispersion.

[0030] The mass ratio of the compound CR to the di-stearoyl phosphatidyl ethanolamine-polyethylene glycol is 1:1-1:2, and preferably 1:1.

[0031] The mass-volume ratio of the compound CR to tetrahydrofuran is 1:1-1:2 mg / mL, and preferably 1:1 mg / mL.

[0032] The di-stearoyl phosphatidyl ethanolamine-polyethylene glycol aqueous solution is prepared by using deionized water.

[0033] The concentration of the di-stearoyl phosphatidyl ethanolamine-polyethylene glycol aqueous solution is 0.2-0.5 mg / mL, and preferably 0.2 mg / mL.

[0034] The concentration of compound CR nanoparticles in the aqueous dispersion of compound CR nanoparticles is 100-200 μM.

[0035] The tumor is gastric cancer.

[0036] Another object of the present application is to provide a preparation method of the compound CR, and the synthetic route is as follows:

[0037]

[0038] The preparation method comprises the following steps:

[0039] In step (1), the molar ratio of 4-iodobenzene hydrazine hydrochloride to methyl isopropyl ketone is 1:1.

[0040] In step (2), the molar ratio of 2,3,3-trimethyl-5-iodo-3H-indole to croconic acid is 1:2.0-1:2.5.

[0041] In step (1), the molar ratio of 4-iodobenzene hydrazine hydrochloride to methyl isopropyl ketone is 1:1.

[0042] The temperature of the heating and stirring is 25-100 ℃, and the time of the heating and stirring is 12-36 hours.

[0043] Preferably, the temperature of the heating and stirring is 80-90 ℃, and the time of the heating and stirring is 18-30 hours.

[0044] In step (2), the molar ratio of 2,3,3-trimethyl-5-iodo-3H-indole to croconic acid is 1:2.0-1:2.5.

[0045] The volume ratio of toluene to n-butanol is 1:1-1:4, preferably 1:1-1:2.

[0046] The temperature of the heating reaction is 25-110 ℃, and the time of the heating reaction is 8-24 hours.

[0047] Preferably, the temperature of the heating reaction is 100-110 ℃, and the time of the heating reaction is 10-16 hours.

[0048] The molecular sieve is 4A molecular sieve.

[0049] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0050] (1) The present invention has a simple and high-yield phototherapy nanomedicine with dual-site targeting of mitochondria / STAT3 protein.

[0051] (2) The phototherapy nanomedicine of the present invention has dual-site targeting of mitochondria / STAT3 protein, which has precise active and passive tumor targeting. It can target mitochondria and STAT3 protein in tumor cells through ATO, and can also passively target tumor sites through the high-penetration and long-retention effect of nanoparticles, so that the nanotherapeutic agent is more enriched around the tumor and improves the efficacy.

[0052] (3) The phototherapy nanomedicine targeting the mitochondrial / STAT3 protein dual site of the present invention has good photothermal properties, can effectively enhance the PTT effect of gastric cancer, and exert the ability of tumor synergistic treatment by promoting cell apoptosis, inhibiting angiogenesis and hindering cell cycle, thus realizing multimodal tumor treatment and has broad application prospects.

[0053] (4) The phototherapy nanomedicine of the present invention, which targets mitochondria / STAT3 protein dual sites, has ideal metabolizable properties and biocompatibility. Attached Figure Description

[0054] Figure 1 For compound CR 1 H-NMR spectrum (400MHz, CDCl3), with the horizontal axis representing chemical shift and the vertical axis representing intensity.

[0055] Figure 2 This is a scanning electron microscope image of the phototherapy nanomedicine ATO / CR NPs with dual mitochondrial / STAT3 protein targeting of the present invention.

[0056] Figure 3 The photothermal conversion efficiency of the phototherapy nanomedicine ATO / CR NPs with dual mitochondrial / STAT3 protein targeting in aqueous solution was detected. Deionized water blank detection was used as a comparison. The horizontal axis represents time and the vertical axis represents temperature difference.

[0057] Figure 4 The apoptosis mechanism of the phototherapy nanomedicine ATO / CR NPs with dual mitochondrial / STAT3 protein targeting of the present invention was analyzed by flow cytometry at different concentrations (0 μM, 6 μM, 12 μM, 18 μM).

[0058] Figure 5 The subcellular localization of the phototherapy nanomedicine ATO / CR NPs with dual mitochondrial / STAT3 protein targeting of this invention was observed using fluorescence confocal microscopy.

[0059] Figure 6 The changes in mitochondrial membrane potential were observed using fluorescence confocal microscopy to detect the phototherapy nanomedicine ATO / CR NPs, which have dual-site targeting of mitochondrial / STAT3 proteins, according to the present invention.

[0060] Figure 7 This invention relates to an experiment demonstrating the inhibitory effect of the phototherapy nanomedicine ATO / CR NPs, which targets both mitochondrial and STAT3 proteins, on angiogenesis using human umbilical vein endothelial cells.

[0061] Figure 8 The phototherapy nanomedicine ATO / CR NPs of this invention, which targets both mitochondria and STAT3 proteins, inhibits STAT3 protein phosphorylation (a), inhibits cell cycle (b), and downregulates HSP70 expression (c).

[0062] Figure 9 This invention utilizes the phototherapy nanomedicine ATO / CR NPs, which targets both mitochondria and STAT3 proteins, to monitor the fluorescence changes in various organs of nude mice after intravenous injection of the drug via a live in vivo imaging system.

[0063] Figure 10 This invention utilizes the phototherapy nanomedicine ATO / CR NPs, which targets both mitochondria and STAT3 proteins, to observe in real-time temperature changes at the tumor site after intravenous injection in nude mice using a thermal imager.

[0064] Figure 11 This study describes the tumor volume change process in nude mice treated with ATO / CR NPs, a phototherapy nanomedicine with dual mitochondrial / STAT3 protein targeting sites. Detailed Implementation

[0065] The technical solution of the present invention will be further illustrated below through embodiments, so as to better understand the content of the present invention.

[0066] Example 1

[0067] Synthesis of compound CR

[0068]

[0069] Step (1): 4-Iodophenylhydrazine hydrochloride (2.00 g, 7.4 mmol) and methyl isopropyl ketone (0.690 g, 8.0 mmol) were added to acetic acid (15 mL), refluxed at 80 °C for 6 h, cooled to room temperature, and stirred for 18 h. Acetic acid was removed under vacuum at 80 °C. The residue was adjusted to pH 9 with 10% potassium hydroxide aqueous solution and then extracted with DCM (dichloromethane). The organic phase was collected and DCM was removed under vacuum at 40 °C to obtain the product 2,3,3-trimethyl-5-iodo-3H-indole, with a yield of 94%.

[0070] Step (2): 2,3,3-trimethyl-5-iodo-3H-indole (0.407 g, 1.43 mmol) and ketone acid (0.102 g, 0.7 mmol) were added to a mixed solvent of toluene (5 mL) and n-butanol (5 mL), and then 0.8 g of 4A molecular sieve was added. The mixture was refluxed at 110 °C for 8 h, cooled to -20 °C, and allowed to stand for 24 h. The mixture was then filtered with diethyl ether to obtain compound CR with a yield of 58%.

[0071] Compound CR 1 See H-NMR image Figure 1 . 1 H NMR (400MHz, CDCl3): δ15.93-14.43(m,2H),7.74-7.64(m,4H),7.05-6.97(m,2H),6.12-5.93(m,2H),1.52(s,12H).

[0072] Preparation of ATO / CR nanoparticles (ATO / CR NPs)

[0073] 1.5 mg DSPE-mPEG 2000 Sonication dissolved in 5 mL of deionized water yielded DSPE-mPEG. 2000 Solution; dissolve 1 mg of compound CR and 0.2 mg of ATO in 1 mL of tetrahydrofuran (THF) to obtain a mixed solution; maintain DSPE-mPEG 2000 The solution was stirred rapidly (500 rpm) at room temperature, and the mixture was slowly added dropwise to DSPE-mPEG at a rate of 20 drops / minute. 2000 After the addition of the solution was complete, the mixture was stirred for 5 minutes at room temperature. Nitrogen gas was used to purge the tetrahydrofuran from the solution. After overnight sedimentation, the supernatant was collected to obtain an aqueous dispersion of the multifunctional nanotherapeutic agent ATO / CR nanoparticles (ATO / CR NPs). The concentration of the ATO / CR nanoparticles was 150 μM.

[0074] Transmission electron microscopy images of ATO / CR NPs are shown below. Figure 2The average size is 102.5 nm.

[0075] The preparation methods for ATO nanoparticles (ATO NPs) and CR nanoparticles (CR NPs) are the same as those for ATO / CR NPs.

[0076] Preparation of ATO nanoparticles (ATO NPs)

[0077] 1.0 mg DSPE-mPEG 2000 Sonication dissolved in 5 mL of deionized water yielded DSPE-mPEG. 2000 Solution: Dissolve 1 mg ATO in 1 mL of tetrahydrofuran (THF) to obtain an ATO solution, and slowly add it dropwise to DSPE-mPEG. 2000 After the addition of the solution is complete, continue stirring for 5 minutes. Use nitrogen gas to purge the tetrahydrofuran from the solution. Allow it to settle overnight, and take the supernatant to obtain an aqueous dispersion of ATO nanoparticles (ATO NPs). The concentration of ATO nanoparticles is 100 μM.

[0078] Preparation of CR nanoparticles (CR NPs)

[0079] 1.0 mg DSPE-mPEG 2000 Sonication dissolved in 5 mL of deionized water yielded DSPE-mPEG. 2000 Solution: Dissolve 1 mg of compound CR in 1 mL of tetrahydrofuran (THF) to obtain CR solution, and slowly add it dropwise to DSPE-mPEG. 2000 After the addition of the solution was complete, the mixture was stirred for 5 minutes. Nitrogen gas was then used to purge the tetrahydrofuran from the solution. The mixture was allowed to settle overnight, and the supernatant was collected to obtain an aqueous dispersion of CR nanoparticles (CR NPs). The concentration of the CR nanoparticles was 200 μM.

[0080] Example 2

[0081] Photothermal conversion efficiency of ATO / CR nanoparticles

[0082] ATO / CR nanoparticle aqueous dispersion under 808nm laser (1.2w / cm²) 2 Irradiated for 10 minutes, and deionized water (ddH2O) was measured under the same conditions as a blank control. The results are as follows. Figure 3 When the temperature reaches equilibrium, it cools to room temperature; the significant increase in temperature indicates that ATO / CR nanoparticles have extremely strong photothermal conversion capabilities.

[0083] Example 3

[0084] Detection of ATO / CR nanoparticles' quantitative in vitro toxicity to tumor cells, subcellular localization, changes in mitochondrial membrane potential, and in vitro anti-angiogenic effects.

[0085] Cytotoxicity assay: MKN45 tumor cells (purchased from Pronosei Biotechnology Co., Ltd.) were selected for in vitro quantitative toxicity experiments to test their laser toxicity. The specific experimental steps are as follows: ATO / CR nanoparticle aqueous dispersion (Example 1) was diluted with RPMI-1640 medium to different concentrations (0 μM, 6 μM, 12 μM, 18 μM). MKN45 cells were seeded in 12-well culture plates and cultured at 37°C for 24 hours to allow adherence. After adding 500 μL of the drug and culturing in the dark for 4 hours, the cells were irradiated with an 808 nm laser (power 1.2 W / cm²). 2 After 5 minutes, the culture medium was replaced with fresh medium, and the cells were cultured for another 12 hours. Then, staining was performed with Annexin V-FITC and propidium iodide (PI). Absolute cell viability was calculated using flow cytometry. Figure 4 As shown, cell survival rate is inversely concentration-dependent; the higher the drug concentration, the lower the cell survival rate.

[0086] ATO / CR nanoparticle aqueous dispersion (Example 1) was diluted to a final concentration of 18 μM using RPMI-1640 medium. MKN45 cells were seeded in confocal dishes and cultured at 37°C for 24 hours to allow adherence. After 4 hours of incubation in the dark, the cells were stained with Lyso Tracker and Mito Tracker, respectively, and the intracellular fluorescence intensity was observed using a laser scanning inverted fluorescence microscope. Figure 5 As shown, the red fluorescence distribution of the drug matched well with the green fluorescence of the mitochondria, indicating that the ATO / CR nanoparticles have mitochondrial localization capabilities.

[0087] ATO / CR nanoparticle aqueous dispersion (Example 1) was diluted with RPMI-1640 medium to a final concentration of 18 μM to obtain ATO / CR NPs culture medium; ATO NPs aqueous dispersion (Example 1) was diluted with RPMI-1640 medium to a final concentration of 18 μM to obtain ATO NPs culture medium; CR NPs aqueous dispersion (Example 1) was diluted with RPMI-1640 medium to a final concentration of 18 μM to obtain CR NPs culture medium.

[0088] MKN45 cells were seeded into confocal culture dishes and cultured at 37°C. After cell attachment, the original culture medium was discarded, and control, ATO NPs, ATO / CR NPs(-), and ATO / CR NPs(+) groups were set up. 1 mL of 18 μM ATO / CR NPs culture medium was added to each well of the ATO / CR NPs(+) and ATO / CR NPs(-) groups, and 1 mL of 18 μM ATO NPs culture medium was added to each well of the ATO NPs group. An equal volume of PBS was added to each well of the control group. Cells were co-cultured for 4 hours. The ATO / CR NP(+) group cells were then irradiated with an 808 nm laser (power 1.2 W / cm²). 2 The irradiation time was 5 minutes. After the irradiation, the culture medium was replaced with fresh medium, and the cells were cultured for another 12 hours. The control group, ATO NPs group, and ATO / CR NPs(-) group were not exposed to light. After 4 hours of co-culturing, the culture medium was replaced with fresh medium, and the cells were cultured for another 12 hours. Then, the cells were stained using a mitochondrial membrane potential detection kit (JC-1), and finally, the cells were photographed using a confocal microscope. Figure 6 As shown, in the control group, ATO NPs group, ATO / CRNPs(-) group and ATO / CR NPs(+) group, the red fluorescence in MNK45 cells gradually decreased while the green fluorescence gradually increased. The ATO / CR NPs(+) group showed the most significant change in red and green fluorescence compared to the other groups, indicating that ATO / CRNPs have a significant ability to damage mitochondrial function under laser irradiation.

[0089] ATO / CR nanoparticle aqueous dispersions (Example 1) were diluted with RPMI-1640 medium to prepare ATO / CR NPs culture media at different concentrations (15 μM, 30 μM, 45 μM). The matrix gel was thawed to 4°C beforehand, and 15 μL of matrix gel was added to each well of a 384-well plate, incubated at 37°C for 1 hour. Different concentration drug administration groups were set up: HUVEC cells (2 × 10⁻⁶ cells / well) were used. 4 Cells / well were seeded into wells with 40 μM ATO / CRNPs culture medium (15, 30, and 45 μM), and a blank control group (using an equal volume of PBS instead of ATO / CRNPs culture medium) was set up. The wells were incubated at 37°C, and the vascular structures were observed and photographed using an inverted fluorescence microscope. Figure 7 As shown, the blood vessels in the blank group (0 μM) gradually formed a dense vascular network over time, while the formation of the vascular network in the drug-treated group was significantly inhibited, and this inhibition was concentration-dependent.

[0090] Example 4

[0091] Western blot experiments confirmed the ability of ATO / CR NPs to inhibit STAT3 protein phosphorylation, inhibit cell cycle, and downregulate HSP70 expression.

[0092] The ATO / CR nanoparticle aqueous dispersion (Example 1) was diluted to different concentrations (6, 12, 18 μM) using RPMI-1640 medium. MKN45 cells were seeded in 6-well plates and cultured at 37°C until adherence. Afterward, the cells were treated with the drug (1 mL) in the dark. The control group received 1 mL of PBS. The cells were co-cultured for 4 hours. Each group of cells was then irradiated with an 808 nm laser (power 1.2 W / cm²). 2 The cells were cultured for 5 minutes, and after the light exposure, the culture medium was replaced with fresh medium, and the cells were cultured for another 12 hours. Cells were then lysed with 100 μL of RIPA lysis buffer containing protease inhibitors, centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was collected. Western blotting was used to detect protein expression in MKN45 cells under different treatments. Proteins were separated by SDS-PAGE gel electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane, and then blocked with the corresponding primary antibodies (anti-Cyclin D1, anti-CDK4, anti-CDK6, anti-STAT3, and anti-P-STAT3 antibodies) in 5% skim milk powder, and incubated overnight at 4°C. Finally, chemiluminescence imaging was performed using a horseradish peroxidase-conjugated secondary antibody. Figure 8 a and Figure 8 As shown in b, compared with the control group (ATO / CR NPs 0 μM), the levels of P-STAT3 / STAT3 and cyclin D1, CDK4, and CDK6 were significantly decreased in a dose-dependent manner. This indicates that ATO / CR nanoparticles can achieve tumor therapy by inhibiting STAT3 protein phosphorylation and inhibiting the cell cycle.

[0093] The ATO NPs aqueous dispersion (Example 1) was diluted to a final concentration of 18 μM using RPMI-1640 medium to obtain the ATO NPs culture medium; the CR NPs aqueous dispersion (Example 1) was diluted to a final concentration of 18 μM using RPMI-1640 medium to obtain the CR NPs culture medium; the ATO / CR nanoparticle aqueous dispersion (Example 1) was diluted to a final concentration of 18 μM using RPMI-1640 medium to obtain the ATO / CR NPs culture medium. MKN45 cells were seeded into 6-well plates and cultured at 37°C. After cell attachment, the original culture medium was discarded, and the following groups were set up: control group, ATO NPs group, CR NPs(+) group, ATO / CR NPs(+) group, and ATO / CR NPs(-) group. 1 mL of 18 μM ATO / CR NPs culture medium was added to each well of the ATO / CR NPs(+) and ATO / CR NPs(-) groups, 1 mL of 18 μM ATO NPs culture medium was added to each well of the ATO NPs group, and 1 mL of 18 μM CR NPs culture medium was added to each well of the CR NPs(+) group. An equal volume of PBS was added to each well of the control group. Cells were co-cultured for 4 hours. Cells in the CR NPs(+) and ATO / CR NPs(+) groups were then irradiated with an 808 nm laser (power 1.2 W / cm²). 2 The light exposure time was 5 minutes. After the light exposure, the medium was replaced with fresh medium, and the cells were cultured for another 12 hours. Cells in the control group, ATO NPs group, and ATO / CR NPs(-) group were not exposed to light. After 4 hours of co-culturing, the medium was replaced with fresh medium, and the cells were cultured for another 12 hours. Protein extraction was performed using the same procedure as Western blot, incubated with anti-HSP-70 antibody. Figure 8 As shown in Figure c, HSP70 expression in the CR NPs(+) group was higher than that in the control group after irradiation with 808nm laser, indicating that high heat leads to increased HSP expression. However, the HSP70 expression levels in the ATO NPs group, ATO / CR NPs(-) group, and ATO / CRNPs(+) group were all lower than those in the control group and CR(+) NPs group, respectively, indicating that ATO reduces ATP in mitochondria and inhibits HSP expression.

[0094] Example 5

[0095] In vivo fluorescence detection and tumor therapy performance of ATO / CR nanoparticles

[0096] The ATO / CR NPs aqueous dispersion (Example 1) was diluted with PBS to prepare an ATO / CR NPs dispersion (120 μM); the ATO nanoparticle aqueous dispersion (Example 1) was concentrated using a vacuum concentrator for 2 h, and then diluted with PBS to prepare ATO nanoparticles (120 μM); the CR nanoparticle aqueous dispersion (Example 1) was diluted with PBS to prepare CR nanoparticles (120 μM).

[0097] First, nude mice with MKN45 tumor cells injected into their axilla were selected as a tumor model. 200 μL of ATO / CR NPs dispersion was then injected into the tail vein of the mice. Next, major tissues and organs (tumor, heart, liver, spleen, lungs, and kidneys) were collected from the mice at 0, 1, 2, 3, 4, and 6 hours, and fluorescence signals were detected using an in vivo animal imaging system. Figure 9 As shown, after intravenous injection, due to the EPR target effect, the fluorescence intensity at the tumor site gradually increased, reaching a maximum at 2 hours. Subsequently, the fluorescence decreased as ATO / CRNPs were metabolized, indicating that 2 hours is the optimal time point for in vivo tumor treatment. The fluorescence phenomena in the liver and kidneys are due to their status as the most important detoxification and metabolic organs; the weakening of fluorescence within these organs indicates that ATO / CR NPs possess ideal metabolic properties and biocompatibility in vivo. This also demonstrates that ATO / CR NPs can be used for tumor diagnosis based on fluorescence imaging.

[0098] Nude mice with MKN45 tumor cells injected into the axilla were selected as a tumor model. Mice were then injected via tail vein with 200 μL of LATO / CR NPs dispersion (120 μM) or PBS. Two hours later, the mice were treated with an 808 nm laser (1.2 W / cm²). 2 The tumor site on the left upper limb of mice was irradiated for 6 minutes, and temperature changes were monitored using infrared thermal imaging. Figure 10 As shown, the temperature at the tumor site in the control group mice (injected with PBS, without injection of ATO / CR NPs nanomedicine) was measured by laser (808 nm, 1.2 W / cm²). 2 After 6 minutes of irradiation, the temperature of mice in the treatment group (injected with ATO / CR NPs) increased by only 4.3°C, while the temperature of mice in the treatment group (injected with ATO / CR NPs) increased by 20.1°C under the same conditions, indicating that ATO / CR NPs nanomedicines have good photothermal properties in vivo.

[0099] Nude mice were selected as tumor models by injecting MKN45 tumor cells into the axilla. Thirty nude mice were randomly divided into six groups: Group 1 (Control), Group 2 (ATO NPs), Group 3 (CR NPs(-)), Group 4 (ATO / CR NPs(-)), Group 5 (CR NPs(+)), and Group 6 (ATO / CR NPs(+)). When the tumor volume was approximately 100 mm², the tumor cells were injected into the axilla of the mice. 3At the time of administration, nude mice in the first group (Control) were injected with PBS (200 μL) via the tail vein; nude mice in the second group (ATO NPs) were injected with ATO nanoparticles (120 μM, 200 μL) via the tail vein; nude mice in the third group (CR NPs) were injected with CR nanoparticles (120 μM, 200 μL) via the tail vein; nude mice in the fourth group (ATO / CR NPs) were injected with ATO / CR nanoparticles (120 μM, 200 μL) via the tail vein; nude mice in the fifth group (CR NPs(+)) were injected with CR nanoparticles (120 μM, 200 μL) via the tail vein; and nude mice in the sixth group (ATO / CR NPs(+)) were injected with ATO / CR nanoparticles (120 μM, 200 μL) via the tail vein. Two hours later, the tumors of the nude mice in the fifth and sixth groups were treated with 808 nm (1.2 W / cm²) PBS. 2 The group was irradiated with laser for 6 minutes, while other groups did not receive light irradiation. Tumor size was measured every 3 days, and the results were as follows: Figure 11 As shown, the tumor tissue in the sixth group of mice was basically eliminated by day 7, while the other groups all had varying degrees of residual tumor tissue. The tumor tissue in the fifth group of mice was also basically eliminated by day 10.

Claims

1. A phototherapy nanodrug with mitochondrial / STAT3 protein dual-site targeting, characterized in that: The light therapy nano-drug is ATO / CR nanoparticles formed by self-assembly of compound CR and atovaquone under the action of distearoyl phosphatidyl ethanolamine-polyethylene glycol; The structure of the compound CR is shown in formula I:

2. The preparation method of the phototherapy nanomedicine with mitochondrial / STAT3 protein dual-site targeting according to claim 1, characterized in that: The method comprises the following steps: The compound CR and atovaquone are dissolved in tetrahydrofuran to obtain a mixed solution; The mixed solution is added dropwise into an aqueous solution of distearoyl phosphatidyl ethanolamine-polyethylene glycol under stirring, and after the dropwise addition is completed, stirring is continued for 5-10 minutes, and tetrahydrofuran is removed by blowing nitrogen; after sedimentation, centrifugation is performed, and the supernatant is taken to obtain an aqueous dispersion of ATO / CR nanoparticles.

3. The preparation method of the phototherapy nanodrug with mitochondrial / STAT3 protein dual-site targeting according to claim 2, characterized in that: The mass ratio of the compound CR to atovaquone is 1:0.1-1:0.5, and the mass ratio of the compound CR to distearoyl phosphatidyl ethanolamine-polyethylene glycol is 1:1-1:

2.

4. The preparation method of the phototherapy nanodrug with mitochondrial / STAT3 protein dual-site targeting according to claim 2 or 3, characterized in that: The mass ratio of the compound CR to atovaquone is 1:0.2, and the mass ratio of the compound CR to distearoyl phosphatidyl ethanolamine-polyethylene glycol is 1:1.

5.

5. The preparation method of the phototherapy nanodrug with mitochondrial / STAT3 protein dual-site targeting according to claim 2, characterized in that: The mass-volume ratio of the compound CR to tetrahydrofuran is 1:1-1:2 mg / mL.

6. The preparation method of the phototherapy nanodrug with mitochondrial / STAT3 protein dual-site targeting according to claim 2, characterized in that: The concentration of the aqueous solution of distearoyl phosphatidyl ethanolamine-polyethylene glycol is 0.2-0.5 mg / mL, and preferably 0.3 mg / mL.

7. The light therapy nano-drug with mitochondrial / STAT3 protein dual-site targeting according to claim 1 is used for preparing a tumor treatment drug.

8. The light therapy nano-drug with mitochondrial / STAT3 protein dual-site targeting according to claim 1 is used for preparing a photothermal tumor treatment drug, and preferably a photothermal-mediated tumor treatment drug through promoting cell apoptosis, inhibiting angiogenesis, and hindering cell cycle.

9. The light therapy nano-drug with mitochondrial / STAT3 protein dual-site targeting according to claim 1 is used for preparing a tumor diagnosis reagent.

10. The compound CR or nanoparticles thereof are used for preparing a photothermal tumor treatment drug; the structure of the compound CR is shown in formula I:

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