A mitochondria-targeting autophagy-inhibiting polymer and a preparation method thereof, a nano-delivery particle and a preparation method and application thereof

Abstract

The present application relates to the technical field of targeted nanoparticles, and particularly relates to a mitochondria-targeting autophagy-inhibiting polymer and a preparation method thereof, a nano delivery particle and a preparation method and application thereof. A high molecule TPP-PEG-SH (TPS) containing a triphenylphosphonium group (triphenylphosphonium, TPP) is coupled with a mitochondria autophagy inhibitor Mdivi-1 through a disulfide bond to construct a polymer TPSM with mitochondria targeting ability and GSH responsiveness, and further co-assembled with a photosensitizer IT-4Cl to form a nano delivery system TPSM@IT-4Cl. The nano delivery system has mitochondria targeting ability, promotes continuous accumulation of oxidative stress at a tumor site, and can realize responsive drug release in a high-concentration GSH tumor microenvironment.

Description

Technical Field

[0001] This invention relates to the field of targeted nanoparticle technology, and in particular to a mitochondrial-targeted autophagy-inhibiting polymer and its preparation method, nanoparticles and their preparation method and applications. Background Technology

[0002] Osteosarcoma is a highly aggressive malignant tumor originating from bone tissue. Its pathogenesis stems from transformed primitive cells derived from mesenchyme, which abnormally differentiate into osteoblastic phenotypes and drive abnormal development of malignant bone tissue. This tumor is particularly prevalent in children and adolescents, and is one of the most common primary malignant bone tumors in this age group. Currently, conventional treatment strategies for osteosarcoma are still limited to chemotherapy, radiotherapy, and surgical resection. However, these treatments unfortunately have significant limitations: on the one hand, chemotherapy drugs have significant cytotoxicity to normal tissues, often causing severe adverse reactions; on the other hand, tumor cells are prone to developing multidrug resistance, leading to an increased risk of recurrence and metastasis, which greatly limits clinical efficacy and long-term survival rates for patients.

[0003] In recent years, numerous studies have shown that photosensitive drug delivery (PDT) is an effective means of enhancing anti-tumor immune responses, possessing advantages such as spatiotemporal controllability, low local toxicity, and low likelihood of inducing systemic drug resistance. Its principle involves photosensitizers generating reactive oxygen species (ROS) upon excitation by specific wavelengths of light, directly killing tumor cells and inducing immunogenic death (ICD). However, the immune activation induced by PDT in osteosarcoma is usually insufficient to establish durable systemic protection because: ROS have short lifespans, small diffusion radii, and limited effects; and tumor cells activate adaptive mechanisms such as antioxidant and stress repair mechanisms, weakening the therapeutic effect.

[0004] Mitochondria, as the core of cellular energy and redox regulation, are both a major source of reactive oxygen species (ROS) and a vulnerable target. PDT-induced ROS bursts can damage mitochondria, but tumor cells can evade oxidative damage by clearing damaged mitochondria through mitophagy. Mitophagy, through the PINK1 / Parkin pathway, marks and degrades depolarized mitochondria, aiding in ROS clearance. Currently, in the treatment of osteosarcoma, drug research targeting mitophagy is in the early stages of translating theoretical exploration into clinical application. Therefore, inhibiting mitophagy could be a reasonable strategy to improve the efficacy of PDT in osteosarcoma.

[0005] Nanodelivery systems can precisely deliver therapeutic drugs to tumor tissues and achieve controlled release at the subcellular level, while also being able to combine with multiple therapeutic modules (such as photosensitizers and autophagy inhibitors). Compared to traditional drugs, nanomedicines can achieve targeted enrichment of tumors and reduce off-target toxicity. Therefore, the synthesis of nanodelivery particle systems with mitochondrial targeting effects has become an important research topic in this field. Summary of the Invention

[0006] This invention discloses a mitochondrial-targeted autophagy-inhibiting polymer and its preparation method, nanoparticles and their preparation method and applications. The purpose is to provide polymers and nanoparticles that can precisely target mitochondria, thereby solving the problem of constructing PDT systems caused by mitochondrial autophagy.

[0007] To achieve the above objectives, the present invention provides a mitochondrial-targeted autophagy-inhibiting polymer, the chemical formula of which is:

[0008] Preferably, the molecular weight of the PEG polymer is 1500-2500.

[0009] Under the same technical concept, the present invention also provides a mitochondrial-targeted autophagy inhibitory polymer nanoparticle, wherein the nanoparticle comprises the mitochondrial-targeted autophagy inhibitory polymer TPSM and photosensitizer IT-4Cl; the mass ratio of the polymer TPSM to the photosensitizer IT-4Cl is 5-10:1.

[0010] Preferably, the nanoparticles use a mitochondrial-targeted autophagy inhibitor polymer as a self-assembly carrier, the mitochondrial-targeted autophagy inhibitor polymer containing the mitochondrial autophagy inhibitor Mdivi-1 and encapsulating the photosensitizer IT-4Cl.

[0011] Under the same technical concept, the present invention also provides a method for preparing the aforementioned mitochondrial-targeted autophagy-inhibiting polymer, comprising the following steps: (1) Under magnetic stirring, TPP-PEG-SH solution was slowly added dropwise to 2,2'-dipyridine disulfide solution; after the addition was completed, the reaction was stirred for 4-8 hours, followed by rotary evaporation, dialyzing with deionized water, and freeze-drying to obtain TPP-PEG-SS-Py. The relevant molecular formula and synthesis process are as follows:

[0012] (2) TPP-PEG-SS-Py was dissolved in dimethylformamide, and then Mdivi-1 solution was added. The mixture was stirred at room temperature for 12-48 h. The reaction solution was rotary evaporated, dialyzed with deionized water, and freeze-dried to obtain the final product, mitochondrial-targeted autophagy inhibitory polymer TPSM.

[0013] The relevant molecular formula and synthesis process are as follows:

[0014] Preferably, the solvent for the TPP-PEG-SH solution and the 2,2'-dipyridine disulfide solution in step (1) is CH2Cl2, and the mass ratio of the TPP-PEG-SH solution to the 2,2'-dipyridine disulfide solution is 1-5:1; In step (2), the mass ratio of TPP-PEG-SS-Py to Mdivi-1 solution is 3.5-15:1; the pyridine disulfide in TPP-PEG-SS-Py is connected to the -SH group in Mdivi-1 solution as a -ss- bond. To ensure bond connection, the molar mass ratio of pyridine disulfide in the synthesized TPP-PEG-SS-Py to the -SH group in Mdivi-1 solution is 1:2-5.

[0015] Under the same technical concept, the present invention also provides a method for preparing the aforementioned mitochondrial-targeted autophagy-inhibiting polymer nanoparticles, comprising the following steps: S1. Preparation of mitochondrial-targeted autophagy-inhibiting polymer TPSM; S2. Dissolve IT-4Cl in tetrahydrofuran. Under ultrasonic conditions, slowly add the IT-4Cl solution dropwise to deionized water containing polymer TPSM, and then continue ultrasonic stirring to promote its self-assembly into nanoparticles. Transfer the resulting dispersion to a dialysis bag and dialyze with deionized water to obtain the mitochondrial-targeted autophagy-inhibiting polymer nanoparticle dispersion.

[0016] Preferably, in step S2, the mass ratio of IT-4Cl to polymer TPSM is 1:2-10; the dialysis bag used for deionized water dialysis is MWCO 1000-2000 Da, and the dialysis time is 12-48h.

[0017] Under the same technical concept, the present invention also provides an application of the aforementioned mitochondrial-targeted autophagy-inhibiting polymer nanoparticles, wherein the nanoparticles are used in a high-concentration GSH tumor microenvironment.

[0018] Preferably, the mitochondrial-targeted autophagy-inhibiting polymer nanoparticles are used to prepare injectable formulations for mitochondrial-targeted therapy.

[0019] The above-described solution of the present invention has the following beneficial effects: (1) The TPSM@IT-4Cl nanosystem successfully constructed in this invention integrates mitochondrial targeting and GSH responsiveness into a polymer delivery system, overcoming the limitations of single-therapy and achieving multiple therapeutic goals of "intelligent, precise, and efficient". PEG linkage imparts high dispersibility, uniform particle size distribution, good physicochemical stability, and excellent blood compatibility, meeting the basic requirements for material safety in both in vivo and in vitro applications. Under 735 nm laser irradiation, these nanoparticles can efficiently generate ROS, providing a reliable photodynamic activity basis for subsequent anti-tumor research; (2) This invention has significant in vitro anti-osteosarcoma activity and immune activation potential. In vitro experiments show that TPSM@IT-4Cl has excellent inhibitory ability against osteosarcoma cells. Mechanistic studies have confirmed that PDT-induced mitochondrial damage can activate protective mitophagy; while this invention, by introducing a mitochondrial division inhibitor (Mdivi-1), effectively inhibits mitochondrial damage, thereby inhibiting the autophagy process, preventing the timely clearance of damaged mitochondria, leading to the continuous accumulation of oxidative damage and significantly enhancing cytotoxicity. In addition, combined with light-exposed TPSM@IT-4Cl, it can improve the immunogenicity of tumor cells, promote BMDC maturation, and has the ability to induce immunogenic cell death (ICD) and initiate anti-tumor immune responses. Its mechanism of action has both direct tumor killing and immune regulation functions. (3) This invention has excellent in vivo tumor enrichment, biosafety, and anti-tumor immune enhancement effects. In in vivo experiments, TPSM@IT-4Cl showed good tumor-targeting enrichment characteristics and biosafety. After phototherapy, the delivery system significantly inhibited tumor growth and showed characteristics of inhibited mitophagy and enhanced ICD in tumor tissue. At the same time, the proportion of mature DCs in tumor draining lymph nodes increased, the number of activated CD8+ T cells in spleen increased, CD8+ T cell infiltration in tumor tissue was enhanced, and TAM polarized to M1-like phenotype, proving that this invention can effectively improve the immunosuppressive microenvironment of osteosarcoma and stimulate a strong anti-tumor immune response; (4) This invention has translational potential in preclinical models. In a human PDX tumor model that is closer to clinical reality, TPSM@IT-4Cl combined with light irradiation still maintains excellent anti-tumor effects, suggesting that this invention has good clinical translational potential and is worth further in-depth research. The preparation method used in this invention is mild and simple to operate, does not rely on strict reaction conditions, and does not require complex separation and purification steps, thus having good operability and reproducibility. The obtained nanoparticles have uniform particle size, controllable distribution, and good biocompatibility, providing favorable conditions for their targeted delivery in vivo. Attached Figure Description

[0020] Figure 1The 1H NMR spectra of Mdivi-1, TPS, TPP-PEG-SS-Py and TPSM are shown.

[0021] Figure 2 SEM image of TPSM@IT-4Cl, scale bar = 40 nm.

[0022] Figure 3 The UV-Vis absorption spectra of TPS, Mdivi-1, TPSM, IT-4Cl, and TPSM@IT-4Cl are shown.

[0023] Figure 4 For 735 nm laser (0.5 W / cm) 2 Under irradiation, the time-dependent fluorescence change curve of ROS generated by TPSM@IT-4Cl was measured using SOSG and DHR123 probes.

[0024] Figure 5 This is a confocal fluorescence image of nanoparticles co-localizing with mitochondria in HOS cells, scale bar = 20 μm.

[0025] Figure 6 The figure shows the CCK-8 experimental results of TPSM@IT-4Cl nanomaterials against HOS, MG63, and K7M2.

[0026] Figure 7 This image shows the confocal fluorescence imaging and quantitative analysis results of intracellular ROS. Scale bar = 10 μm.

[0027] Figure 8 The results of Western blot and immunofluorescence co-localization detection of mitochondrial autophagy levels in HOS cells are shown in the figures. (A) Western blot images of GAPDH, PINK1, Parkin, p62, and LC3B proteins in HOS cells after different treatments. (B) Quantitative analysis of the relative expression levels of PINK1, Parkin, p62, and LC3B in Western blot. (C) Confocal fluorescence images of mitochondrial autophagy in HOS cells after different treatments detected by immunofluorescence co-localization, scale bar = 30 μm. (D) Quantitative analysis of LC3B fluorescence images.

[0028] Figure 9 The images show the in vivo drug fluorescence imaging results at different time points after intravenous administration to osteosarcoma model mice.

[0029] Figure 10 The image shows the tumor volume change curve, endpoint tumor weight, and gross photograph of the orthotopic osteosarcoma in mice after TPSM@IT-4Cl was applied. Scale bar = 1 cm.

[0030] Figure 11 For the blood compatibility study of TPSM@IT-4Cl. (A) Hemolysis test of nanoparticles at different concentrations. (B) Background color of nanoparticles at different concentrations. (C) Hemolysis rate of nanoparticles at different concentrations.

[0031] Figure 12 H&E staining results of heart, liver, spleen, lung and kidney of mice in different treatment groups. Scale bar = 100 μm. Detailed Implementation

[0032] To make the technical problems, solutions, and advantages of this invention clearer, a detailed description will be provided below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0033] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0034] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a locking connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0035] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0036] This invention discloses a polymer TPP-PEG-SH (TPS) containing a triphenylphosphonium (TPP) group, coupled with the mitochondrial autophagy inhibitor Mdivi-1 via disulfide bonds, to construct a polymer TPSM with mitochondrial targeting capability and GSH responsiveness. This polymer is further co-assembled with the photosensitizer IT-4Cl to form a nanodelivery system TPSM@IT-4Cl. This nanodelivery system exhibits mitochondrial targeting capability, promoting the continuous accumulation of oxidative stress at the tumor site, and enabling responsive drug release in a high-concentration GSH tumor microenvironment.

[0037] Complete technical solution of this invention: (1) Synthesis of TPP-PEG-SS-Py: Under magnetic stirring, a TPP-PEG-SH (TPS) solution (80-120 mg, dissolved in 5-10 mL CH2Cl2) was slowly added dropwise over 1 hour to a 2,2'-dipyridine disulfide solution (0.024-0.048 g, dissolved in 5-10 mL CH2Cl2). After the addition was complete, the reaction was stirred for 4-8 hours (6 hours being optimal). Subsequently, CH2Cl2 was removed by rotary evaporation, and unreacted matter was removed by dialysis with deionized water. After freeze-drying, TPP-PEG-SS-Py was obtained.

[0038] The relevant molecular formula and synthesis process are as follows:

[0039] (2) Synthesis of TPSM TPP-PEG-SS-Py (20-40 mg, containing 8-16 μmol pyridine disulfide groups) was dissolved in dimethylformamide (N,N-Dimethylformamide, DMF, 2.0-4.0 mL). Then, Mdivi-1 solution (2.8-5.6 mg, containing 8-16 μmol -SH groups, dissolved in 1.2-2.4 mL DMF) was added, and the reaction was stirred at room temperature for 12-48 h (24 h optimal). The reaction solution was purified by dialysis and lyophilized using the same method to obtain the final product TPP-PEG-SS-Mdivi-1 (TPSM). The relevant molecular formula and synthesis process are as follows:

[0040] (3) Synthesis of TPSM@IT-4Cl nanoparticles IT-4Cl (1-5 mg) was dissolved in tetrahydrofuran (THF, 1-5 mL). Under sonication, the IT-4Cl solution was slowly added dropwise to deionized water containing TPSM (10-50 mL), followed by continued sonication / stirring to promote self-assembly into nanoparticles. The resulting dispersion was transferred to a dialysis bag (MWCO 1000 Da) and dialyzed with deionized water to remove free or unencapsulated IT-4Cl. Dialysis was performed at room temperature with gentle stirring for 12-48 hours (24 h optimal) to obtain a TPSM@IT-4Cl nanoparticle dispersion.

[0041] Example 1: Preparation of related TPSM@IT-4Cl nanoparticles.

[0042] The nanoparticles TPSM@IT-4Cl of the present invention are prepared by the following three-step method: (1) Synthesis of intermediate TPP-PEG-SS-Py First, under magnetic stirring, a TPP-PEG-SH (TPS) solution (100 mg, dissolved in 5 mL of dichloromethane CH2Cl2) was slowly added dropwise to a 2,2'-dipyridine disulfide solution (0.036 g, 0.165 mmol, also dissolved in 5 mL of CH2Cl2). The addition was slow to ensure the homogeneity and controllability of the reaction. After the addition was complete, the reaction was stirred at room temperature for 4 hours to allow the thiol and dipyridine disulfide to undergo a complete exchange reaction. After the reaction was complete, the organic solvent CH2Cl2 was removed by rotary evaporation, followed by dialysis with deionized water to remove unreacted small molecule impurities. Finally, the target intermediate TPP-PEG-SS-Py was obtained by freeze-drying. (2) Preparation of functionalized polymer TPSM (TPP-PEG-SS-Mdivi-1) The intermediate TPP-PEG-SS-Py (20 mg, containing approximately 8 μmol of pyridine disulfide active groups) was dissolved in 2.0 mL of N,N-dimethylformamide (DMF). Mdivi-1 (2.8 mg, containing an equimolar amount of 8 μmol of thiol-SH groups) was dissolved in 1.2 mL of DMF. The Mdivi-1 solution was then added to the polymer solution, and the reaction was stirred at room temperature for 24 hours. Mdivi-1 was covalently linked to the polymer chain via a thiol-disulfide exchange reaction. After the reaction, the final functionalized polymer product, denoted as TPP-PEG-SS-Mdivi-1 (TPSM), was obtained using the same dialysis purification method (dialysis with deionized water) and lyophilization process as in step one. (3) Encapsulation of photosensitizer IT-4Cl and self-assembly of nanoparticles to synthesize TPSM@IT-4Cl nanoparticles First, the photosensitizer IT-4Cl (1 mg) was dissolved in 1 mL of tetrahydrofuran (THF) to form a homogeneous organic solution. Under ultrasonic assistance, this IT-4Cl solution was slowly added dropwise to 10 mL of deionized water containing TPSM polymer. After the addition was complete, ultrasonic / stirring treatment was continued to promote the self-assembly of the amphiphilic polymer and photosensitizer into nanoparticles through hydrophobic interactions. The resulting nanoparticle dispersion was transferred to a dialysis bag (molecular weight cutoff MWCO 1000 Da) and dialyzed gently with stirring at room temperature for 24 hours to effectively remove the organic solvent THF and unencapsulated free IT-4Cl. Finally, a pure TPSM@IT-4Cl nanoparticle dispersion was obtained.

[0043] Example 2: Material characterization of related TPSM@IT-4Cl nanoparticles 1H NMR (1H NMR) 1 1H NMR): 1H NMR results are as follows Figure 1 As shown, Mdivi-1 exhibits two characteristic peaks: 3.8 ppm for methoxy (-OCH3) and 13.2 ppm for thiol (-SH). Compared to TPS and TPP-PEG-SS-Py, the presence of the -OCH3 characteristic peak in TPSM indicates that Mdivi-1 has been successfully coupled to TPS. Simultaneously, the -SH characteristic peak of Mdivi-1 disappears in TPSM, suggesting that -SH has participated in disulfide bond formation. These findings collectively demonstrate the successful preparation of the polymer TPSM.

[0044] Scanning electron microscopy (SEM): SEM is used to characterize the morphology and microstructure of samples. For example... Figure 2 As shown in the figure, SEM revealed that the TPSM@IT-4Cl nanoparticles were well dispersed and exhibited a uniform spherical shape.

[0045] Ultraviolet-Vis absorption spectrum (UV-Vis): Figure 3 The UV-Vis absorption spectra of TPS, Mdivi-1, TPSM, IT-4Cl, and TPSM@IT-4Cl are shown. Successful drug loading was determined by the unique absorption peaks of each substance. Figure 3 UV-Vis results showed that TPSM had a characteristic absorption peak at 277 nm, while IT-4Cl had a characteristic absorption peak at 677 nm. Notably, TPSM@IT-4Cl exhibited absorption peaks at both 277 nm and 754 nm. The 277 nm peak corresponds to the characteristic absorption of TPSM, while the 754 nm peak shows a redshift compared to free IT-4Cl, suggesting a change in the aggregation state of IT-4Cl after encapsulation, further confirming its successful loading.

[0046] ROS generation capacity determination of TPSM@IT-4Cl: such as Figure 4 As shown, SOSG and DHR123 were used to detect its concentration in aqueous solution. 1 O2、O2• - The fluorescence intensity of SOSG significantly increased with irradiation time in the presence of TPSM@IT-4Cl under 735 nm laser irradiation. Similar results were obtained after replacing the probe with DHR123, indicating that TPSM@IT-4Cl possesses excellent O2•- generation capability. - Generation capability.

[0047] Example 3: Investigation of the in vitro antitumor effect of TPSM@IT-4Cl Cellular uptake and mitochondrial targeting experiment: Cells were incubated at 5 × 10⁻⁶ cells per cell. 4 Cells were seeded at a density of [number] cells / dish in confocal culture dishes. After cell adhesion, FITC-labeled TPSM@IT-4Cl was added to fresh complete culture medium with a final Mdivi-1 concentration of 10 μM, and incubated for 0, 1, 2, 4, and 8 hours, respectively. Mitochondria were then stained with Mito-Tracker Red, and nuclei were stained with Hoechst 33342. Finally, the distribution of the nanocomposite within the cells and its co-localization with mitochondria were observed using confocal laser scanning microscopy. Figure 5 As shown, with prolonged incubation time, the green fluorescence signal in the cells gradually increased and overlapped extensively with the red mitochondrial fluorescence signal, exhibiting yellow co-localization fluorescence, confirming that the nanoparticle has good mitochondrial targeting ability.

[0048] CCK-8 cell viability assay: The in vitro antitumor activity of TPSM@IT-4C was evaluated using the CCK-8 assay, and the effect of TPSM@IT-4C on the 24-hour proliferation of three osteosarcoma cell lines was investigated. HOS, MG63, and K7M2 cells were purchased from Wuhan Shangen Biotechnology Co., Ltd. HOS, MG63, and K7M2 cells were cultured at 1×10⁻⁶ cells / cells. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and incubated overnight at 37°C with 5% CO2. After cell adhesion, fresh complete culture medium containing different treatment conditions was added. The treatment conditions were: PBS control group (Ⅰ), free Mdivi-1 group (Ⅱ), TPSM group (Ⅲ), TPS@IT-4Cl+L group (Ⅳ), TPSM@IT-4Cl group (Ⅴ), and TPSM@IT-4Cl+L group (Ⅵ), with the final Mdivi-1 concentration remaining consistent across all groups at 1, 2, 4, 5, 10, and 20 μM. After 8 hours of incubation, the irradiation group (L) was irradiated with a 735 nm laser at a power density of 0.1 W / cm². 2Irradiation time was 10 minutes, followed by 24 hours of incubation. Fresh culture medium containing 10% CCK-8 reagent (C0038, Beyotime, China) was added, and incubation continued for 2 hours. Finally, the absorbance was measured at 450 nm using a microplate reader. Figure 6 The results showed that 1, 2, and 5 μM Mdivi-1 had no significant effect on the proliferation of the three osteosarcoma cell types. At 10 μM, free Mdivi-1, TPSM, and TPSM@IT-4Cl only mildly inhibited HOS cell viability. Cell viability in the PDT-only group (TPS@IT-4Cl+L) was approximately 65%, while in the combination therapy group (TPSM@IT-4Cl+L) it decreased to 30%, indicating that inhibition of mitophagy enhances the cytotoxic effect of PDT. Although 20 μM Mdivi-1 produced stronger inhibition, previous studies suggested that this dose might interfere with mitochondrial oxidative metabolism, thus introducing additional metabolic confounding factors; therefore, 10 μM was chosen for this experiment.

[0049] DCFH-DA staining: HOS cells were cultured and adhered to the culture medium according to the pretreatment method described in the cck-8 experiment. Serum-free medium containing 10 μM 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was added, and the cells were incubated at 37°C in the dark for 30 minutes. After staining, the cells were washed twice with PBS to remove excess probe. Subsequently, Hoechst 33342 was added to counterstain the cell nuclei, and fluorescence images were acquired using a confocal laser scanning microscope to determine intracellular ROS levels. Results are as follows: Figure 7 As shown, compared with the PBS group, both TPS@IT-4Cl+L and TPSM@IT-4Cl+L produced significantly enhanced green fluorescence after light irradiation, indicating that the photosensitizer can efficiently generate ROS values ​​under irradiation. The fluorescence intensity of the TPSM@IT-4Cl+L group further increased "explosively", reaching 2.39 times that of the TPS@IT-4Cl+L group, suggesting that inhibiting mitophagy can increase intracellular ROS levels.

[0050] Example 4: Investigation into the antitumor mechanism of TPSM@IT-4Cl Mitophagy is an important protective mechanism for cells to clear damaged mitochondria after oxidative damage, reducing ROS burden and limiting the accumulation of oxidative damage. Therefore, it may weaken the efficacy of anti-tumor therapies such as phototherapy (PDT). Mitophagy is a type of selective autophagy that can be regulated by multiple signaling axes, among which the PINK1 / Parkin pathway is the classic mechanism for clearing damaged mitochondria.

[0051] We used Western blot to detect the expression of key mitophagy proteins such as PINK1, Parkin, p62, and LC3B after different treatment conditions. The treatment conditions were: PBS control group (Ⅰ), free Mdivi-1 group (Ⅱ), TPSM group (Ⅲ), TPS@IT-4Cl+L group (Ⅳ), TPSM@IT-4Cl group (Ⅴ), and TPSM@IT-4Cl+L group (Ⅵ). Western blot results showed ( Figure 8 A) In the PDT-related treatment groups (TPS@IT-4Cl+L group and TPSM@IT-4Cl+L group), the expression of PINK1 and Parkin was significantly increased, suggesting that PDT-induced mitochondrial damage activated PINK1 / Parkin-dependent mitophagy. An increased LC3B-II / I ratio is a classic molecular indicator of enhanced autophagosome formation, while p62, as a selective autophagy receptor, is negatively correlated with mitophagy activity. Quantitative analysis showed that ( Figure 8 (B) Compared with the TPS@IT-4Cl+L group, the TPSM@IT-4Cl+L group showed significantly increased p62 expression and a significantly decreased LC3B-II / I ratio (p < 0.0001). These results suggest that, in the context of PDT-induced mitochondrial damage, TPSM@IT-4Cl+L can significantly inhibit mitophagy, thereby amplifying oxidative damage and enhancing the therapeutic effect.

[0052] To confirm whether the observed autophagy was mitophagy, this study performed colocalization analysis on LC3B and mitochondria. The results showed that ( Figure 8 (C) Both the TPS@IT-4Cl+L and TPSM@IT-4Cl+L groups showed obvious yellow colocalization fluorescence, indicating that mitophagy was activated. Further comparison revealed that the colocalization signal in the TPSM@IT-4Cl+L group was significantly lower than that in the TPS@IT-4Cl+L group, and LC3B expression decreased synchronously (p < 0.0001). Figure 8 D).

[0053] Example 5: Investigation of the in vivo antitumor effect of TPSM@IT-4Cl Investigation into the mitochondrial targeting and biodistribution characteristics of TPSM@IT-4C nanoparticles: 4×10 6K7M2 cells were injected intramuscularly into the tibial bone marrow cavity of 4-6 week old BALB / c mice to establish an orthotopic osteosarcoma model. BALB / c mice (SPF grade, purchased from Hunan Silek Jingda Experimental Animal Co., Ltd.) were injected intravenously via the tail vein into the mice with TPSM@IT-4Cl (containing 2.5 mg / kg Mdivi-1, 100 μL PBS). Mice were anesthetized at 1, 2, 4, 12, 24, and 48 hours post-injection, and fluorescence images were acquired using an IVIS in vivo imaging system. Results are as follows: Figure 9 As shown, the fluorescence signal in the tumor area gradually increased after drug administration and reached a peak at 24 hours; the signal then weakened but was still detectable at 48 hours, indicating that the nanoparticles have a long retention time in the tumor site, demonstrating that TPSM@IT-4C nanoparticles have the ability to target and bioaccumulate at the tumor site.

[0054] Investigation of the tumor-suppressive effect of TPSM@IT-4C nanoparticles in a mouse orthotopic osteosarcoma model: The osteosarcoma model was established as described in Example 5 above. When the tumor volume reached 200 mm², the tumor was subjected to TPSM@IT-4C nanoparticles. 3 Mice were randomly divided into 6 groups (n = 5): PBS control group (Ⅰ), free Mdivi-1 group (Ⅱ), TPSM group (Ⅲ), TPS@IT-4Cl+L group (Ⅳ), TPSM@IT-4Cl group (Ⅴ), and TPSM@IT-4Cl+L group (Ⅵ). The nanomedicine (containing 2.5 mg / kg Mdivi-1, 100 μL PBS) was injected via the tail vein every 96 hours for a total of 4 injections. The irradiation group (+L) received a 735 nm laser (0.1 W / cm²) 24 hours after injection. 2 Irradiate the tumor site for 10 minutes. Measure body weight and tumor volume every 3 days. Results are as follows: Figure 10 As shown in the tumor growth curves, the tumor inhibition rate of the TPS@IT-4Cl+L group was 51.32%, while that of the TPSM@IT-4Cl+L group was as high as 75.03%, indicating that the combined treatment group had a better effect. On day 25, mice were sacrificed and tumors were harvested and weighed. The average tumor weight in the TPSM@IT-4Cl+L group was only 0.84 ± 0.07 g, significantly lower than that in the PBS group (2.41 ± 0.26 g) and the TPS@IT-4Cl+L group (1.40 ± 0.17 g). The endpoint tumor image showed a significant reduction in tumor burden in the TPSM@IT-4Cl+L group, suggesting that TPSM@IT-4Cl has a significant tumor-suppressive effect in vivo, and that the combined effect of PDT (proton pump inhibitory therapy) with mitophagy inhibition can further enhance the tumor-suppressive effect.

[0055] Example 6: Investigation of the biosafety of TPSM@IT-4Cl Blood compatibility study of TPSM@IT-4Cl: To evaluate the blood compatibility of this nanoparticle, a hemolysis experiment was conducted. The results are as follows: Figure 11 As shown in A and B, the negative control (PBS group) caused almost no hemolysis, while the positive control (deionized water) resulted in complete hemolysis. After treatment with TPSM@IT-4Cl at concentrations of 2-40 μg / mL, the supernatant remained largely clear; the slight color changes observed in the high-concentration groups were mainly due to the background color of the material, not hemolysis. Quantitative analysis showed that the hemolysis rate in each group increased only slightly with increasing concentration and remained consistently below 5%, indicating good blood compatibility. Figure 11 C).

[0056] Measurement of vital organs: At the study endpoint, major organs such as the heart, liver, spleen, lungs, and kidneys were collected for pathological analysis. For example... Figure 12 As shown, H&E staining did not reveal any obvious structural damage, necrotic foci, or inflammatory infiltration, and its morphological characteristics were basically consistent with those of the PBS control group.

Claims

1. A mitochondrial-targeted autophagy-inhibiting polymer, characterized in that, The chemical formula of the polymer is: 。 2. The polymer according to claim 1, characterized in that, The polymer has a molecular weight of 1500-2500.

3. A mitochondrial-targeted autophagy-inhibiting polymer nanoparticle, characterized in that, The nanoparticles comprise the mitochondrial-targeted autophagy-inhibiting polymer TPSM as described in any one of claims 1-2 and the photosensitizer IT-4Cl; the mass ratio of the polymer TPSM to the photosensitizer IT-4Cl is 5-10:

1.

4. The nanoparticles as described in claim 3, characterized in that, The nanoparticles are self-assembled using a mitochondrial-targeted autophagy inhibitor polymer as a carrier. The mitochondrial-targeted autophagy inhibitor polymer contains the mitochondrial autophagy inhibitor Mdivi-1 and is encapsulated with the photosensitizer IT-4Cl.

5. A method for preparing the mitochondrial-targeted autophagy-inhibiting polymer as described in any one of claims 1-2, characterized in that, Includes the following steps: (1) Under magnetic stirring, TPP-PEG-SH solution was slowly added dropwise to 2,2'-dipyridine disulfide solution; after the addition was completed, the reaction was stirred for 4-8 hours, followed by rotary evaporation, dialyzing with deionized water, and freeze-drying to obtain TPP-PEG-SS-Py. (2) TPP-PEG-SS-Py was dissolved in dimethylformamide, and then Mdivi-1 solution was added. The mixture was stirred at room temperature for 12-48 h. The reaction solution was rotary evaporated, dialyzed with deionized water, and freeze-dried to obtain the final product, mitochondrial-targeted autophagy inhibitory polymer TPSM.

6. The preparation method according to claim 5, characterized in that, The solvent for the TPP-PEG-SH solution and the 2,2'-dipyridine disulfide solution in step (1) is CH2Cl2, and the mass ratio of the TPP-PEG-SH solution to the 2,2'-dipyridine disulfide solution is 1-5:1; In step (2), the mass ratio of TPP-PEG-SS-Py to Mdivi-1 solution is 3.5-15:1; the pyridine disulfide in TPP-PEG-SS-Py is connected to the -SH group in Mdivi-1 solution as a -ss- bond, and the molar mass ratio of pyridine disulfide in TPP-PEG-SS-Py to the -SH group in Mdivi-1 solution is 1:2-5.

7. A method for preparing mitochondrial-targeted autophagy-inhibiting polymer nanoparticles as described in any one of claims 3-4, characterized in that, Includes the following steps: S1. Preparation of mitochondrial-targeted autophagy-inhibiting polymer TPSM; S2. Dissolve IT-4Cl in tetrahydrofuran. Under ultrasonic conditions, slowly add the IT-4Cl solution dropwise to deionized water containing polymer TPSM, and then continue ultrasonic stirring to promote its self-assembly into nanoparticles. Transfer the resulting dispersion to a dialysis bag and dialyze with deionized water to obtain the mitochondrial-targeted autophagy-inhibiting polymer nanoparticle dispersion.

8. The preparation method according to claim 7, characterized in that, In step S2, the mass ratio of IT-4Cl to polymer TPSM is 1:2-10; the dialysis bag used for deionized water dialysis is MWCO 1000-2000 Da, and the dialysis time is 12-48h.

9. An application of the mitochondrial-targeted autophagy-inhibiting polymer nanoparticles as described in any one of claims 3-4, characterized in that, The nanoparticles are used to create a high-concentration GSH tumor microenvironment.

10. The application as described in claim 9, characterized in that, The mitochondrial-targeted autophagy-inhibiting polymer nanoparticles are used to prepare injectable formulations for mitochondrial-targeted therapy.

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