A bone-targeting, metabolic drive-relieving, chemotherapy-resistant polymer, dual-drug inhibitor nano-delivery particle, and preparation method and application thereof

Abstract

The present application relates to the technical field of targeted nanoparticles, in particular to a bone-targeting metabolic-driven chemotherapy resistance relieving polymer, a dual-drug inhibitor nano delivery particle and a preparation method and application thereof. The nano delivery particle comprises the bone-targeting metabolic-driven chemotherapy resistance relieving polymer MALss ALN , the polymer MALss Gi , and an MCT1 inhibitor. The present application discloses a metabolic reprogramming nano therapeutic system targeting osteosarcoma and a preparation and application method thereof, aiming to solve the problem of poor sensitivity of cisplatin, cope with the complex immune microenvironment of osteosarcoma, and propose a new nano therapeutic strategy for overcoming chemotherapy resistance and immune inactivation by precisely regulating the metabolic-immune axis.

Description

Technical Field

[0001] This invention relates to the field of targeted nanoparticle technology, and in particular to a bone-targeted polymer for alleviating metabolism-driven chemotherapy resistance, dual-drug inhibitor nanoparticles, their preparation methods, and applications. Background Technology

[0002] Osteosarcoma, one of the most common primary malignant bone tumors, predominantly affects adolescents and young adults. Its aggressive nature and tendency to metastasize early place a heavy burden on patients and their families. While comprehensive treatment strategies centered on surgical resection combined with multi-drug chemotherapy have improved patient prognosis to some extent, chemotherapy resistance remains a key bottleneck limiting further improvements in long-term survival rates. Simultaneously, osteosarcoma generally exhibits low immunogenicity and a significant immunosuppressive microenvironment, leading to significantly lower-than-expected efficacy of immunotherapy, particularly immune checkpoint inhibitors. Therefore, effectively reshaping the tumor immune microenvironment while reversing chemotherapy resistance, achieving a synergistic effect of "chemosensitizing" and "immune activation," has become a core clinical challenge urgently needing to be overcome in the treatment of osteosarcoma.

[0003] Recent studies have revealed that tumor metabolic reprogramming is a key link between chemotherapy resistance and immunosuppression. Osteosarcoma cells are highly dependent on glycolysis, taking up large amounts of glucose and producing lactate, leading to a continuous accumulation of lactate in the microenvironment. A high lactate state can both promote chemotherapy resistance and acidify the microenvironment, inhibiting immune cell function. More importantly, lactate, as a signaling molecule, mediates protein lactation and other modifications, establishing an intrinsic link between metabolic adaptation, drug resistance, and immunosuppression. Specifically, lactate can induce lactation modification of cGAS, inhibiting its activity and blocking the downstream STING signaling pathway, which is the core of innate immune activation. Therefore, cGAS lactation becomes a key molecular node connecting abnormal tumor metabolism and immunosuppression, providing a new entry point for simultaneously intervening in drug resistance and immune escape. Among related metabolic pathways, the glucose transporter GLUT1 (SLC2A1) and the monocarboxylic acid transporter MCT1 (SLC16A1), as key nodes regulating glucose uptake and lactate transport, exhibit abnormal activation in drug-resistant osteosarcoma cells and are closely related to chemotherapy response. This phenomenon suggests that inhibiting lactate production from the metabolic input end as a whole, while precisely intervening in lactate action at key signaling nodes, may be an effective strategy to break the vicious cycle of "metabolism-immunity" in osteosarcoma. Summary of the Invention

[0004] This invention discloses a metabolic reprogramming nanotherapy system for osteosarcoma and its preparation and application methods. The aim is to address the poor sensitivity of cisplatin, cope with the complex immune microenvironment of osteosarcoma, and propose a new nanotherapy strategy to overcome chemotherapy resistance and immune inactivation by precisely regulating the metabolic-immune axis.

[0005] To achieve the above objectives, the present invention provides a bone-targeted polymer for alleviating metabolism-driven chemotherapy resistance, the polymer having the following chemical formula: .

[0006] Preferably, the polymer has a molecular weight of 3000-5000.

[0007] Under the same technical concept, the present invention also provides a bone-targeted, metabolism-driven dual-drug inhibitor nanoparticle for resolving bone-related issues, wherein the nanoparticle comprises the aforementioned bone-targeted, metabolism-driven chemotherapy-resistant polymer MALss. ALN Polymer MALss Gi and MCT1 inhibitors; the polymer MALss Gi Including GLUT1 inhibitors, whose chemical formula is: .

[0008] Preferably, the polymer MALss ALN Polymer MALss Gi The mass ratio of the MCT1 inhibitor to the GLUT1 inhibitor is 3-5:3-5:1; the nanodelivery particles use a bone-targeted, metabolism-driven chemotherapy-resistant polymer as a backbone, and the MCT1 inhibitor and GLUT1 inhibitor are connected to the backbone by chemical bonds.

[0009] Under the same technical concept, the present invention also provides a method for preparing a bone-targeted, metabolism-driven chemotherapy-resistant polymer, comprising the following steps: (1) Dissolve 2-mercaptoethanol and sodium acetate in methanol, add dibromomaleimide while stirring, and continue stirring for 2-6 h to carry out the reaction; add deionized water, extract the mixture with ethyl acetate, dry the organic phase, filter, concentrate under reduced pressure to obtain crude product; elute and purify by silica gel column chromatography, and then further purify by a second column chromatography, and remove under reduced pressure to obtain 3,4-bis[(2-hydroxyethyl)thio]-2,5-dihydro-1H-pyrrole-2,5-dione sensitive monomer; (2) Take the 3,4-bis[(2-hydroxyethyl)thio]-2,5-dihydro-1H-pyrrole-2,5-dione sensitive monomer and 1,2,4,5-cyclohexanetetracarboxylic acid dihydrogenate obtained in step (1) and stir for 36-60 h under light protection at 45-55℃; add polyethylene glycol and stir for 12-30 h at 45-55℃. After the reaction is completed, dialyze the mixture under deionized water for 60-84 h through a MWCO: 3000-5000 Da dialysis bag, purify and freeze dry to obtain polymer MALss; (3) Dissolve the polymer MALss obtained in step (2) in DMF, add EDCl and NHS, stir, add alendronate and stir at 45-55℃ for 12-30 h. After the reaction is completed, dialyze in deionized water for 60-84 h through a MWCO: 3000-5000 Da dialysis bag, purify and freeze dry to obtain bone-targeted metabolism-driven chemotherapy-resistant polymer MALss. ALN .

[0010] Preferably, in step (1), the molar ratio of 2-mercaptoethanol to sodium acetate is 1:1-1.2, and the molar ratio of dibromomaleimide to 2-mercaptoethanol is 1.96-3.92:4.3; the drying agent for the dried organic phase is sodium sulfate, and the eluent used in the silica gel column chromatography is petroleum ether and ethyl acetate, with a volume ratio of 2-4:1; the eluent used in the second column chromatography is dichloromethane and methanol, with a volume ratio of 80-120:1. The molar ratio of the 3,4-bis[(2-hydroxyethyl)thio]-2,5-dihydro-1H-pyrrole-2,5-dione sensitive monomer, 1,2,4,5-cyclohexanetetracarboxylic acid dihydrogenate and polyethylene glycol in step (2) is 50-75:55-80:10. In step (3), the mass ratio of polymer MALss, EDCl, NHS and alendronate is 200-300:17.3-34.6:10.4-20.8:20; the stirring time is 20-40 min.

[0011] Under the same technical concept, the present invention also provides a method for preparing bone-targeted, metabolism-driven dual-drug inhibitor nanoparticles, comprising the following steps: S1. Preparation of bone-targeted, metabolism-driven chemotherapy-resistant polymer MALss ALN ; S2, Preparation of polymer MALss Gi ; S3. Continuously stir the deionized water to remove the polymer MALss. ALN Polymer MALss GiA DMSO solution containing MCT1 inhibitor at a mass ratio of 3-5:3-5:1 was added dropwise to the aqueous phase. The resulting mixture was then transferred to a MWCO: 3000-5000 Da dialysis bag and dialyzed under deionized water for 12-30 hours to obtain bone-targeted metabolic-driven dual-drug inhibitor nanoparticles.

[0012] Preferably, the preparation of polymer MALss in step S2 is... Gi Specifically, it includes: The polymer MALss was dissolved in DMF, EDCl and DMAP were added, and the mixture was stirred. A GLUT1 inhibitor was then added and the mixture was stirred at 45-55°C for 12-30 hours. After the reaction was complete, the mixture was dialyzed in deionized water for 60-84 hours using a MWCO: 3000-5000 Da dialysis bag. The resulting purified product was then lyophilized to obtain the bone-targeted, metabolism-driven chemotherapy-resistant polymer MALss. ALN ; The molar ratio of the polymer MALss, EDCl, DMAP and GLUT1 inhibitor is 200-300:9.6-19.2:6.1-12.2:15.

[0013] Under the same technical concept, the present invention also provides an application of bone-targeted metabolic-driven dual drug inhibitor nanoparticles for preparing bone tumor-targeted delivery systems.

[0014] Preferably, the bone tumor targeted delivery system is used to prepare injectable formulations for targeted therapy.

[0015] The above-described solution of the present invention has the following beneficial effects: (1) The nanomaterials of the present invention can achieve active targeting and microenvironment-responsive release of bone tumors. Alendronic acid (ALN) modification enables the nanoparticles to have a high affinity for hydroxyapatite in bone tissue, preferentially accumulating in the bone tumor area after intravenous injection. Simultaneously, the GSH-responsive monomer synthesized using dibromomaleimide specifically depolymerizes the polymer in the high-concentration GSH microenvironment of osteosarcoma, achieving synergistic drug release and reducing systemic toxicity.

[0016] (2) The nanomaterials of this invention can inhibit glycolysis and lactate production, thus alleviating chemotherapy resistance. The nanoparticles co-deliver GLUT1 inhibitor (inhibiting glucose uptake) and MCT1 inhibitor (blocking lactate efflux). The two drugs are encapsulated in the same nanoparticle, ensuring that they reach the same tumor cells simultaneously, achieving precise drug efficacy coordination in time and space, and avoiding the problem of inconsistent distribution of the two free drugs in vivo. After the nanomaterials reach the tumor cells, they can significantly reduce the lactate levels inside and outside the drug-resistant osteosarcoma cells. This metabolic remodeling directly alleviates lactate-driven chemotherapy resistance and enhances the killing effect of drugs such as cisplatin. Furthermore, by reducing intracellular lactate accumulation, it effectively reverses the lactation modification of cGAS protein, restoring its ability to bind to cytoplasmic dsDNA and form a signaling complex. This process does not directly activate the immune pathway, but rather repairs the intrinsic function of the DNA sensing system through metabolic regulation, creating a prerequisite for subsequent immune responses.

[0017] (3) The nanomaterials of the present invention can amplify the activation of the cGAS–STING pathway and enhance anti-tumor immunity. In the context of cisplatin-induced DNA damage, the cGAS–STING pathway signal after metabolic remodeling is significantly enhanced: the phosphorylation level of STING increases, and the secretion of downstream type I interferon and various pro-inflammatory cytokines (such as IFN, IL-6, etc.) increases, thereby driving the cascade reaction of innate immunity and adaptive immunity.

[0018] (4) The preparation method of this invention is mild and simple to operate, requiring no strict reaction conditions or complex separation and purification steps, and is easy to prepare. The obtained nanoparticles have uniform particle size and controllable distribution, and exhibit good stability and storability in aqueous systems, which is beneficial for targeted delivery in vivo. At the same time, the nanosystem has excellent biosafety, and has not caused obvious systemic toxicity or local adverse reactions in in vitro and in vivo experiments, demonstrating good biocompatibility. Attached Figure Description

[0019] Figure 1 For bone-targeted MALss Gi / A Transmission electron microscopy image of @Mi nanoparticles. Scale bar: 100 nm.

[0020] Figure 2 To measure MALss via DLS Gi / A The average particle size of @Mi and the results of PDI are shown in the figure.

[0021] Figure 3 To measure MALss within 7 days Gi / A The results are shown in the figure, which illustrates the changes in the average diameter of @Mi and PDI in PBS buffer to reflect its activity.

[0022] Figure 4MALss in the presence of 10 mM GSH or PBS Gi / A Cumulative release curve of @Mi's MCT1i.

[0023] Figure 5 MALss labeled with Cy5.5 fluorescence Gi / A @Mi nanoparticles co-incubated with HOS-R cells for 1, 4, and 7 h using confocal laser scanning microscopy (CLSM). Scale bar: 20 μm.

[0024] Figure 6 Representative images of cisplatin-resistant osteosarcoma cells under different treatment conditions were obtained by flow cytometry, and statistical analysis of the apoptosis ratio was performed based on flow cytometry.

[0025] Figure 7 For observation of PBS or MALss using confocal laser scanning microscopy (CLSM) Gi / A @Mi Fluorescence distribution of GLUT1 (red) and MCT1 (green) in cisplatin-resistant osteosarcoma cells after nanoparticle treatment, and quantitative analysis of fluorescence intensity based on CLSM images. Scale bar: 20 μm.

[0026] Figure 8 To observe the subcellular distribution and co-localization of cGAS (red) and cytoplasmic dsDNA (green) in HOS-P and HOS-R cells under different treatment conditions using confocal laser scanning microscopy, cell nuclei were stained with DAPI (blue). Scale bar: 20 μm.

[0027] Figure 9 The figure shows the results of Western blot analysis of p-STING expression levels in HOS-R cells under different treatment conditions.

[0028] Figure 10 To utilize IVIS technology, MALss Gi / A @Cy7.5 and MALss Gi @Cy7.5 fluorescence biodistribution at different time points in K7M2-R tumor-bearing mice.

[0029] Figure 11 To record tumor growth inhibition curves and collect tumor weights from mice in different treatment groups on day 24, as well as representative gross photographs of tumor resection. Scale bar: 1 cm.

[0030] Figure 12 For MALss Gi / A Physical images and quantitative diagrams of @Mi nanoparticles in hemolysis experiments.

[0031] Figure 13Hematoxylin-eosin (he) staining of major organs in mice for different drugs. Scale bar: 50 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] The complete technical solution of this application's embodiments is as follows: (1) Synthesis of 3,4-bis[(2-hydroxyethyl)thio]-2,5-dihydro-1H-pyrrole-2,5-dione GSH-sensitive monomer: 2-Mercaptoethanol (335.9 mg, 4.3 mmol) and sodium acetate (352.7 mg, 4.3 mmol) (molar ratio 1:1) were added to a dry round-bottom flask and dissolved in methanol. Dibromomaleimide (500.0 mg, 1.96 mmol) was then slowly added with stirring at room temperature. The reaction mixture was continuously stirred at room temperature for 2–6 h (4 h optimal). After the reaction was complete, deionized water was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The residue was purified by silica gel column chromatography using petroleum ether / ethyl acetate (2:1, v / v) as the eluent. The collected fraction containing the target compound was concentrated and further purified by a second column chromatography (dichloromethane / methanol = 99:1, v / v). After solvent removal under reduced pressure, 3,4-bis[(2-hydroxyethyl)thio]-2,5-dihydro-1H-pyrrole-2,5-dione was obtained as a pale solid. The relevant molecular formula and synthetic process are as follows: (2) Synthesis of polymer MALss 3,4-bis[(2-hydroxyethyl)thio]-2,5-dihydro-1H-pyrrole-2,5-dione (136.0 mg, 0.50 mmol) and 1,2,4,5-cyclohexanetetracarboxylic acid dihydrogenate (123.3 mg, 0.55 mmol) were added to a round-bottom flask and stirred under light protection at 45-55 °C (50 °C optimal) for 36-60 h (48 h optimal). Subsequently, polyethylene glycol (PEG) was added... 5k 500.0 mg (0.1 mmol) was introduced into the reaction mixture, and then stirred further at 45-55 °C (50 °C optimal) for 12-30 h (24 h optimal). After the reaction, the resulting solution was transferred to a dialysis bag (MWCO: 3000-5000 Da, 3500 Da optimal) and dialyzed under deionized water for 60-84 h (72 h optimal) to remove unreacted small molecules and impurities. The purified solution was then lyophilized to obtain the polymer MALss (P1). The relevant molecular formula and synthesis process are as follows: (3) Polymer MALss ALN Synthesis Polymer MALss (200 mg) was dissolved in 5 mL of DMF, followed by the addition of EDCI (17.3 mg, 0.09 mmol) and NHS (10.4 mg, 0.09 mmol). The mixture was stirred at room temperature for 30 min to activate the carboxyl groups. Alendronic acid (20.0 mg, 0.08 mmol) was then added, and the reaction was carried out at 45–55 °C (50 °C optimal) for 12–30 h (24 h optimal). After the reaction was complete, the reaction mixture was transferred to a dialysis bag (MWCO: 3000–5000 Da, 3500 Da optimal) and dialyzed against deionized water for 60–84 h (72 h optimal) to remove unreacted small molecules and byproducts. The purified solution was then lyophilized to obtain polymer MALss. ALN (P2). The relevant molecular formula and synthesis process are as follows: (4) Polymer MALss Gi Synthesis Polymer MALss (200 mg) was dissolved in 5 mL of DMF, followed by the addition of EDCI (9.6 mg, 0.05 mmol) and DMAP (6.1 mg, 0.05 mmol). The mixture was stirred at room temperature for 30 min to allow activation. Subsequently, a GLUT1 inhibitor (GLUT1i, WZB117) (15 mg, 0.04 mmol) was added, and the reaction was carried out at 45–55 °C (50 °C optimal) for 12–30 h (24 h optimal). After the reaction was complete, the reaction mixture was transferred to a dialysis bag (MWCO: 3000–5000 Da, 3500 Da optimal) and dialyzed under deionized water for 60–84 h (72 h optimal) to remove unreacted small molecules and byproducts. The purified solution was then lyophilized to obtain polymer MALss. Gi (P3) (5) MALss Gi / A Preparation of @Mi Simply put, vigorously stir 9 mL of deionized water. With continuous stirring, the solution containing MALss will be removed. ALN (P2, 50mg), MALss GiA DMSO solution (1 mL) of P3 (50 mg) and an MCT1 inhibitor (MCT1i, BAY8002, 10 mg) was added dropwise to the aqueous phase. The resulting mixture was then transferred to a dialysis membrane (MWCO: 3000-5000 Da, 3500 Da optimal) and dialyzed under deionized water for 12-30 h (24 h optimal) to remove residual solvent and free drug. A stable aqueous dispersion, MALss, was finally obtained. Gi / A @MiNanoParticles Example 1: Related MALss Gi / A Preparation of @Mi nanoparticles Synthesis of GSH-sensitive monomer: 2-mercaptoethanol (335.9 mg, 4.3 mmol) and sodium acetate (352.7 mg, 4.3 mmol) were dissolved in methanol, and dibromomaleimide (500.0 mg, 1.96 mmol) was slowly added under stirring at room temperature for 4 hours. The reaction solution was extracted with deionized water and ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then purified by column chromatography successively with petroleum ether / ethyl acetate (2:1, v / v) and dichloromethane / methanol (99:1, v / v). The solvent was removed under reduced pressure to obtain the target product as a light-colored solid.

[0037] Synthesis of polymer MALss: The above product (136.0 mg, 0.50 mmol) was reacted with 1,2,4,5-cyclohexanetetracarboxylic acid dihydrogenate (123.3 mg, 0.55 mmol) at 50 °C in the dark for 48 hours. Polyethylene glycol (PEG5k, 500.0 mg, 0.1 mmol) was added, and the reaction was continued for another 24 hours. The reaction solution was dialyzed against deionized water for 72 hours using a dialysis bag with a molecular weight cutoff of 3000-5000 Da, and then lyophilized to obtain polymer MALss.

[0038] Polymer MALss ALN Synthesis: MALss (200 mg) was dissolved in 5 mL DMF, and EDCI (17.3 mg, 0.09 mmol) and NHS (10.4 mg, 0.09 mmol) were added. The mixture was activated at room temperature for 30 minutes, and alendronic acid (20.0 mg, 0.08 mmol) was added. The reaction mixture was then reacted at 50 °C for 24 hours. The reaction solution was dialyzed against deionized water for 72 hours using a dialysis bag with a molecular weight cutoff of 3000-5000 Da, and then lyophilized to obtain the polymer MALss. ALN The polymer has a molecular weight of 5000.

[0039] Polymer MALss GiSynthesis: MALss (200 mg) was dissolved in 5 mL DMF, and EDCI (9.6 mg, 0.05 mmol) and DMAP (6.1 mg, 0.05 mmol) were added. The mixture was activated at room temperature for 30 minutes, and the GLUT1 inhibitor WZB117 (15 mg, 0.04 mmol) was added. The reaction mixture was then reacted at 50 °C for 24 hours. The reaction solution was dialyzed against deionized water for 72 hours using a dialysis bag with a molecular weight cutoff of 3000-5000 Da, and then lyophilized to obtain the polymer MALss. Gi .

[0040] MALss Gi / A Preparation of @Mi nanoparticles: MALss ALN (50 mg), MALss Gi MALss (50 mg) and the MCT1 inhibitor BAY8002 (10 mg) were co-dissolved in 1 mL DMSO and added dropwise to 9 mL deionized water under vigorous stirring. After emulsification, the solution was transferred to a dialysis membrane with a molecular weight cutoff of 3500 Da and dialyzed in deionized water for 24 hours to obtain MALss. Gi / A @Mi nanoparticle aqueous dispersion.

[0041] Example 2: MALss Gi / A Characterization of @Mi nanoparticles Transmission electron microscopy (TEM): MALss was characterized by TEM (HT-7700, Hitachi, Japan). Gi / A @Mi's appearance. For example... Figure 1 As shown, MALss can be detected using transmission electron microscopy (TEM). Gi / A @Mi has a particle size of approximately 100nm and consists of homogeneous spheres.

[0042] DLS (Dielectric Light Scattering): The hydrated particle size was further determined using DLS, and the results are as follows. Figure 2 Display nano MALss Gi / A The average particle size of @Mi is 121.8 nm, and the corresponding polydispersity index (PDI) is 0.12, indicating that the nanoparticles have good size uniformity.

[0043] Structural stability assessment: To assess MALss Gi / A To assess the stability of @Mi in a fluid-like environment, we used PBS buffer to simulate physiological conditions and dynamically monitored changes in particle size and PDI. Figure 3 The results showed that during the 7-day continuous incubation period, MALss Gi / AThe particle size distribution and PDI of @Mi did not show significant changes, suggesting that the nanosystem has good colloidal stability in physiological environments.

[0044] GSH in vitro response characteristics assay: We investigated MALss in PBS and buffer containing 10 mM GSH, respectively. Gi / A The in vitro drug release behavior of @Mi. Results are as follows: Figure 4 The results show that, in the presence of GSH, MALss Gi / A @Mi released approximately 87.42% of the MCT1 inhibitor within 48 hours; in contrast, the cumulative release under PBS conditions was only 19.73% within 48 hours. These results indicate that the nanoparticles exhibit significant GSH-triggered drug release characteristics, enabling selective responses to the reducing environment within tumor cells.

[0045] Example 3: Cellular uptake of nanoparticles and in vitro drug resistance assay Cellular uptake assay: We first prepared MALss containing Cy5.5 fluorescent probes. Gi / A @Mi nanoparticles were prepared by dissolving nanoparticles and Cy5.5 dye (1 mg) in DMSO (1 mL). This solution was added dropwise to deionized water (10 mL) with continuous stirring. The resulting suspension (MWCO: 3500 Da) was dialyzed against deionized water for 24 h to remove DMSO. The supernatant was collected by centrifugation and named MALss. Gi / A @Mi-Cy5.5. It was then co-incubated with HOS-R cells for 1, 4, and 7 hours. The results of confocal laser scanning microscopy are as follows: Figure 5 As shown, a weak intracellular red fluorescence signal could be detected after 1 hour of co-incubation. With prolonged incubation, the intracellular red fluorescence signal gradually increased, suggesting that MALss... Gi / A @Mi can be effectively internalized by HOS-R cells and accumulate intracellularly.

[0046] Assay for drug resistance: Experiments were conducted using cisplatin-resistant osteosarcoma cells. Flow cytometry was used to detect the level of apoptosis in each treatment group under cisplatin (cisPt) treatment. Figure 6 The results showed that, compared with the 27.02% apoptosis rate of cisPt single treatment, cisPt+GM and cisPt+MALss... Gi / A @Mi treatment significantly increased the apoptosis rate of tumor cells, with MALss Gi / A The group treated with @Mi showed the highest level of apoptosis-induced apoptosis, reaching 46.23%. This indicates that MALss... Gi / A@Mi, when used in combination with cisplatin, not only promoted cell death but also significantly inhibited the generation of cisplatin-resistant osteosarcoma cells.

[0047] Example 4: MALss Gi / A Exploring the Mechanism of Action of @Mi Assay for inhibition of related gene expression: HOS-R cells were subjected to a 5×10⁻⁶ m² / h ... 4 Seeds were planted per well in 24-well plates and incubated for 12 h to allow adhesion. After medium change, the plates were treated with PBS, Gi, Mi, GM, or MALss. Gi / A Cells were treated with @Mi for 24 h (Gi and Mi concentrations were both 60 μM). After treatment, cells were washed with PBS, fixed with 4% paraformaldehyde, blocked with 1% BSA, and permeabilized with 0.1% Triton X-100. GLUT1 staining: rabbit monoclonal antibody was added and incubated at 37°C for 2 h, followed by incubation with Alexa Fluor® 555-labeled goat anti-rabbit IgG for 2 h. MCT1 staining: rabbit polyclonal antibody was added and incubated at 37°C for 2 h, followed by incubation with Alexa Fluor® 488-labeled goat anti-rabbit IgG for 2 h. After nuclear staining with DAPI, the cells were mounted, and GLUT1 expression and distribution were observed using CLSM. The MCT1 staining procedure was the same. We verified MALss using confocal laser scanning microscopy. Gi / A @Mi, after being efficiently internalized by cisplatin-resistant osteosarcoma cells, significantly inhibited the functional activity of GLUT1 and MCT1 within the cells. For example... Figure 7 As shown, compared with the control group, MALss Gi / A @Mi treatment significantly reduced the fluorescence signals of GLUT1 (red) and MCT1 (green) in drug-resistant cells, with intensities 0.42 and 0.32 times that of the control group, respectively. These results indicate that this nanodelivery system can simultaneously intervene in two key metabolic processes within cells: glucose uptake and lactate transmembrane transport.

[0048] cGAS-STING pathway assay: The staining process was the same as described above. We further analyzed the subcellular distribution characteristics of cGAS and its co-localization with cytoplasmic dsDNA using confocal laser scanning microscopy (cGAS, red; dsDNA, green; DAPI, blue). Figure 8As shown, in the untreated HOS-R control group, the overall cGAS signal was weak. After cisplatin treatment, cGAS in HOS-R cells exhibited a diffuse, hazy distribution, indicating a significantly impaired binding ability to cytoplasmic dsDNA and failure to effectively form the cGAS–dsDNA complex. In contrast, in cisplatin-treated HOS-P cells, cGAS showed a typical punctate aggregation distribution, indicating its ability to effectively sense and bind to cytoplasmic dsDNA. Notably, in cisPt+GM and cisPt+MALss cells... Gi / A In HOS-R cells treated with @Mi, punctate aggregation of cGAS was significantly restored, with the latter showing a more pronounced effect. This result indicates that reducing intracellular lactate levels and inhibiting cGAS lactation modification can effectively restore its ability to sense and bind to cytoplasmic DNA.

[0049] Western blot (WB) experiment: The activation status of the STING pathway is verified using a WB experiment. For example... Figure 9 Experimental results showed that HOS-R cells cisPt+MALss Gi / A The relative expression levels of p-STING in the @Mi and cisPt+GM treatment groups were significantly higher than those in the cisplatin-only treatment group, at 2.49 and 2.20 times higher, respectively, confirming the activation of STING-related pathways.

[0050] Example 5: MALss Gi / A In vivo targeting and efficacy study of @Mi MALss Gi / A @Mi's in vivo targeted investigation: Using BALB / c mice (female, SPF grade, purchased from Spiford (Beijing) Biotechnology Co., Ltd.), we established a K7M2 cell orthotopic osteosarcoma mouse model of the lower limb to study MALss. Gi / A The in vivo behavior of @Mi. To clarify the effect of the bone-targeting group ALN on the in vivo distribution of nanoparticles, we used the polymer MALss Gi Cy7.5 fluorescent probe-labeled nanoparticles MALss were prepared. Gi @Cy7.5 and using polymer MALss Gi and MALss ALN Nanoparticles labeled with the Cy7.5 fluorescent probe ALN, MALss, were prepared. Gi / A @Cy7.5, the labeling method is similar to Cy5.5. The mouse orthotopic osteosarcoma was allowed to grow to 100 mm in volume. 3Around 1000 mg / L, an equal dose of Cy7.5-labeled nanoparticles was injected into tumor-bearing mice via the tail vein. The fluorescence signal in vivo was then dynamically monitored using a small animal in vivo imaging system (IVIS). The in vivo imaging results are shown below. Figure 10 As shown, 2 hours after injection. MALss Gi / A @Cy7.5 The fluorescence signal in the tumor region gradually increased and peaked at about 24 h, and remained at a high level for 48 h, which confirmed the in vivo targeting ability of the nanoparticles.

[0051] MALss Gi / A @Mi's in vivo efficacy study: A mouse model of lower limb orthotopic osteosarcoma was constructed using drug-resistant K7M2 cells, with the tumor volume growing to approximately 100 mm. 3 Subsequently, female BALB / c mice were randomly divided into 5 groups (PBS, CisPt, CisPt+GM, CisPt+MALss). Gi @Mi、CisPt+MALss Gi / A @Mi, n = 5, cisplatin dose 3 mg / kg, Gi and Mi doses both 10 mg / kg). Mice in each group were injected with the corresponding drugs via tail vein every 3 days, and tumor volume growth was monitored simultaneously. Figure 11 As shown, the tumor inhibition rate of CisPt alone was only 21.62%, while the tumor inhibition rate of the small molecule inhibitor GM was 50.59%, both significantly lower than the nanodelivery combined therapy group. Among them, CisPt+MALss Gi The tumor inhibition rate in the @Mi group was 58.24%, while that of CisPt+MALss Gi / A The @Mi group showed a further increase to 68.37%, demonstrating the most significant anti-tumor effect. Mice were sacrificed 24 days after treatment, and tumor tissue was collected for weighing and analysis. The results indicate that CisPt+MALss... Gi / A The average tumor weight of mice in the @Mi group was 0.63 ± 0.05 g, significantly lower than that in the PBS group (2.03 ± 0.17 g) and the CisPt group (1.51 ± 0.16 g), and also significantly lower than that in the CisPt+MALss group. Gi The @Mi group (0.86 ± 0.05 g) further validated MALss. Gi / A @Mi's superior tumor-suppressing ability in the body.

[0052] Example 6: MALss Gi / A @Mi's biosafety research In vitro hemolysis assay: Different concentrations of MALss were used to determine the hemolysis assay. Gi / A@Mi The saline solution was mixed with mouse whole blood at an equal volume ratio and incubated for 4 h, after which the hemolysis rate was measured. The results are as Figure 12 shown. No obvious hemolysis was observed in each concentration MALss Gi / A @Mi treatment group; although the hemolysis rate showed a slight upward trend with the increase of concentration, it was all lower than 5%, and there was no statistical difference. It was confirmed that it had good blood biocompatibility.

[0053] Determination of important organs: Healthy 6-week-old BALB / c mice (female, SPF grade, purchased from Spf(Beijing) Biotechnology Co., Ltd.) were respectively treated with PBS, cisPt, cisPt+GM, cisPt+MALss Gi @Mi and cisPt+MALss Gi / A @Mi (the dose of cisplatin was 3 mg / kg, and the doses of Gi and Mi were both 10 mg / kg, injected once every 3 days) were injected via the tail vein. After continuous injection for 14 days, the main organs of the mice (heart, liver, spleen, lung, kidney) were taken for H&E staining analysis. The histological results are as Figure 13 , and no obvious tissue damage or pathological abnormalities were observed in the main organs of each treatment group, and their morphological characteristics were basically the same as those of the PBS control group.

Claims

1. A bone-targeted polymer for alleviating metabolism-driven chemotherapy resistance, 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 3000-5000.

3. A bone-targeted, metabolism-driven dual-drug inhibitor nanoparticle delivery system, characterized in that, The nanodelivered particles comprise the bone-targeting, metabolism-driven chemotherapy-resistant polymer MALss as described in any one of claims 1-2. ALN Polymer MALss Gi and MCT1 inhibitors; the polymer MALss Gi Including GLUT1 inhibitors, whose chemical formula is:

4. The nanoparticles as described in claim 3, characterized in that, The polymer MALss ALN Polymer MALss Gi The mass ratio of the MCT1 inhibitor to the GLUT1 inhibitor is 3-5:3-5:1; the nanodelivery particles use a bone-targeted, metabolism-driven chemotherapy-resistant polymer as a backbone, and the MCT1 and GLUT1 inhibitors are connected to the backbone by chemical bonds.

5. A method for preparing a bone-targeted, metabolism-driven chemotherapy-resistant polymer as described in any one of claims 1-2, characterized in that, Includes the following steps: (1) Dissolve 2-mercaptoethanol and sodium acetate in methanol, add dibromomaleimide while stirring, and continue stirring for 2-6 h to carry out the reaction; add deionized water, extract the mixture with ethyl acetate, dry the organic phase, filter, concentrate under reduced pressure to obtain crude product; elute and purify by silica gel column chromatography, and then further purify by a second column chromatography, and remove under reduced pressure to obtain 3,4-bis[(2-hydroxyethyl)thio]-2,5-dihydro-1H-pyrrole-2,5-dione sensitive monomer; (2) Take the 3,4-bis[(2-hydroxyethyl)thio]-2,5-dihydro-1H-pyrrole-2,5-dione sensitive monomer and 1,2,4,5-cyclohexanetetracarboxylic acid dihydrogenate obtained in step (1) and stir for 36-60 h under light protection at 45-55℃; add polyethylene glycol and stir for 12-30 h at 45-55℃. After the reaction is completed, dialyze the mixture under deionized water for 60-84 h through a MWCO: 3000-5000 Da dialysis bag, purify and freeze dry to obtain polymer MALss; (3) Dissolve the polymer MALss obtained in step (2) in DMF, add EDCl and NHS, stir, add alendronate and stir at 45-55℃ for 12-30 h. After the reaction is completed, dialyze in deionized water for 60-84 h through a MWCO: 3000-5000 Da dialysis bag, purify and freeze dry to obtain bone-targeted metabolism-driven chemotherapy-resistant polymer MALss. ALN .

6. The preparation method according to claim 5, characterized in that, In step (1), the molar ratio of 2-mercaptoethanol to sodium acetate is 1:1-1.2, and the molar ratio of dibromomaleimide to 2-mercaptoethanol is 1.96-3.92:4.3; the drying agent for the organic phase is sodium sulfate, and the eluent used in the silica gel column chromatography is petroleum ether and ethyl acetate, with a volume ratio of 2-4:1; the eluent used in the second column chromatography is dichloromethane and methanol, with a volume ratio of 80-120:

1. The molar ratio of the 3,4-bis[(2-hydroxyethyl)thio]-2,5-dihydro-1H-pyrrole-2,5-dione sensitive monomer, 1,2,4,5-cyclohexanetetracarboxylic acid dihydrogenate and polyethylene glycol in step (2) is 50-75:55-80:

10. In step (3), the mass ratio of polymer MALss, EDCl, NHS and alendronate is 200-300:17.3-34.6:10.4-20.8:20; the stirring time is 20-40 min.

7. A method for preparing bone-targeted, metabolism-driven dual-drug inhibitor nanoparticles as described in any one of claims 3-4, characterized in that, Includes the following steps: S1. Preparation of bone-targeted, metabolism-driven chemotherapy-resistant polymer MALss ALN ; S2, Preparation of polymer MALss Gi ; S3. Continuously stir the deionized water to remove the polymer MALss. ALN Polymer MALss Gi A DMSO solution containing MCT1 inhibitor at a mass ratio of 3-5:3-5:1 was added dropwise to the aqueous phase. The resulting mixture was then transferred to a MWCO: 3000-5000 Da dialysis bag and dialyzed under deionized water for 12-30 hours to obtain bone-targeted metabolic-driven dual-drug inhibitor nanoparticles.

8. The preparation method according to claim 7, characterized in that, The preparation of polymer MALss in step S2 Gi Specifically, it includes: The polymer MALss was dissolved in DMF, EDCl and DMAP were added, and the mixture was stirred. A GLUT1 inhibitor was then added and the mixture was stirred at 45-55°C for 12-30 hours. After the reaction was complete, the mixture was dialyzed in deionized water for 60-84 hours using a MWCO: 3000-5000 Da dialysis bag. The resulting purified product was then lyophilized to obtain the bone-targeted, metabolism-driven chemotherapy-resistant polymer MALss. ALN ; The mass ratio of the polymer MALss, EDCl, DMAP and GLUT1 inhibitor is 200-300:9.6-19.2:6.1-12.2:

15.

9. The application of a bone-targeting, metabolism-driven dual-drug inhibitor nanoparticle as described in any one of claims 3-4, characterized in that, The nanoparticles are used to prepare a bone tumor targeted delivery system.

10. The application as described in claim 9, characterized in that, The bone tumor targeted delivery system is used to prepare injectable formulations for targeted therapy.

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