Preparation method and application of ROS-responsive nanodrugs crossing blood-brain barrier and loaded with OPN expression inhibitors

Abstract

This invention relates to a method for preparing and applying a ROS-responsive nanomedicine that crosses the blood-brain barrier and is loaded with an OPN expression inhibitor, belonging to the field of pharmaceutical technology. The invention designs and constructs an Angiopep-2 modified ROS-responsive nanomedicine delivery system. By loading an OPN expression inhibitor, it successfully solves key challenges of traditional OPN inhibitors, such as difficulty in penetrating the blood-brain barrier, poor targeting, easy in vivo clearance, inability to regulate the glioma microenvironment, and resistance to temozolomide chemotherapy. It achieves integrated synergistic therapy involving active targeting across the blood-brain barrier, tumor microenvironment-responsive drug release, SPP1⁺ tumor-associated macrophage regulation, tumor-promoting signaling pathway blockade, and chemotherapy sensitization, providing a more efficient, safer, and clinically promising novel treatment strategy for glioblastoma.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology, specifically relating to a nanomedicine delivery system that can efficiently cross the blood-brain barrier, achieve reactive oxygen species-responsive drug release in the tumor microenvironment, and target and regulate the function of glioma-related tumor-associated macrophages. In particular, it relates to the preparation method of the nanomedicine and its use in combination with temozolomide in the treatment of glioblastoma. Background Technology

[0002] Glioblastoma is the most malignant, aggressive, and poorly prognostic primary intracranial tumor of the central nervous system, characterized by high heterogeneity, strong invasiveness and metastasis, and a high recurrence rate. Current standard clinical treatment focuses on maximally safe tumor resection, combined with postoperative radiotherapy and concurrent temozolomide chemotherapy, followed by adjuvant temozolomide chemotherapy. However, overall efficacy remains limited, with a median overall survival of only 14-16 months, a 5-year survival rate of less than 10%, and a tumor recurrence rate approaching 100%. Chemotherapy resistance and the immunosuppressive microenvironment have become core scientific issues restricting treatment effectiveness.

[0003] Currently, the key bottlenecks in the clinical treatment of gliomas are mainly reflected in three aspects: First, the physiological barrier function of the blood-brain barrier. The blood-brain barrier is composed of brain microvascular endothelial cells, tight junctions, the basement membrane, and astrocyte foot processes. It has high selective permeability, blocking more than 98% of small molecule drugs and almost all large molecule preparations from entering the brain parenchyma. This results in most therapeutic drugs, including OPN inhibitors, failing to achieve effective therapeutic concentrations in the tumor area, and exhibiting extremely low drug utilization after systemic administration. Second, the dominant role of the tumor immunosuppressive microenvironment. The glioma microenvironment contains a large number of infiltrating SPP1 molecules. + Tumor-associated macrophages are a core cell type mediating immune escape, angiogenesis, and treatment resistance. They continuously activate downstream signaling pathways by highly expressing and secreting osteopontin (OPN / SPP1), remodeling the extracellular matrix, recruiting regulatory T cells, and inhibiting CD8. + T cell function is compromised, creating a highly immunosuppressive tumor microenvironment that significantly reduces the sensitivity to temozolomide chemotherapy. Thirdly, there are delivery defects in OPN targeted therapy drugs. Traditional OPN small molecule inhibitors often suffer from poor water solubility, short in vivo half-life, poor systemic stability, and a lack of active targeting capabilities. They cannot effectively cross the blood-brain barrier, are difficult to precisely accumulate in glioma regions, and cannot achieve specific regulation of SPP1⁺ tumor-associated macrophages. Single-drug therapy is insufficient to reverse immunosuppression and chemotherapy resistance.

[0004] Existing research has confirmed that SPP1 +OPN secreted by tumor-associated macrophages can specifically bind to integrin β3 on the cell membrane surface, activating the downstream NF-κB / p65 signaling pathway, further upregulating the expression and secretion of VEGFA, forming the OPN / integrin β3 / p65 / VEGFA tumor-promoting signaling axis. This significantly accelerates glioma angiogenesis, enhances tumor invasion and migration, and mediates temozolomide resistance. Therefore, targeting OPN as a key target and using nanodelivery systems to achieve brain-targeted delivery of inhibitors, combined with synergistic chemotherapy, has become an important strategy for overcoming the treatment challenges of glioma, reshaping the tumor microenvironment, and restoring chemosensitivity.

[0005] In recent years, microenvironment-responsive nanomedicines and active targeted delivery technologies have provided new insights for the treatment of brain tumors. Reactive oxygen species (ROS) are specifically highly expressed in the glioma microenvironment, with concentrations far exceeding those in normal brain tissue. These ROS can serve as ideal endogenous stimuli, enabling precise and controllable drug release at the lesion site. Angiopep-2 peptides can specifically recognize low-density lipoprotein receptor-related proteins on the blood-brain barrier, efficiently and safely crossing the blood-brain barrier and achieving active targeted enrichment in glioma tissue through receptor-mediated transcytosis. However, current technologies still lack an integrated nanomedicine delivery system that can simultaneously integrate multiple functions such as active targeting across the blood-brain barrier, ROS-responsive controlled-release drugs, precise inhibition of OPN expression, tumor-associated macrophage functional reprogramming, and temozolomide chemosensitization. This makes it difficult to achieve efficient, safe, and synergistic treatment of gliomas, and falls far short of meeting the practical clinical needs for precise treatment and long-term prognosis improvement in glioblastoma.

[0006] Therefore, this invention designs and constructs an Angiopep-2 modified ROS-responsive nanomedicine delivery system. By loading an OPN expression inhibitor, it successfully solves key problems of traditional OPN inhibitors, such as difficulty in penetrating the blood-brain barrier, poor targeting, easy clearance in vivo, inability to regulate the glioma microenvironment, and resistance to temozolomide chemotherapy. It achieves active targeting across the blood-brain barrier, tumor microenvironment-responsive drug release, and SPP1. + The integrated synergistic therapy of tumor-associated macrophage regulation, tumor-promoting signaling pathway blockade, and chemotherapy sensitization provides a new, more efficient, safer, and clinically promising treatment strategy for glioblastoma. Summary of the Invention

[0007] Based on the above research background, this invention aims to provide a drug that can actively cross the blood-brain barrier, respond to ROS in the tumor microenvironment, and precisely target and regulate SPP1. + A nanomedicine delivery system for tumor-associated macrophages and synergistic effects with temozolomide is presented, along with the preparation method, structural characterization, mechanism of action, and application in the treatment of glioma.

[0008] This invention solves the technical problem through molecular design and self-assembly techniques, constructing an Angiopep-2-modified ROS-responsive polymeric nanocarrier, and using an OPN expression inhibitor as a regulator of SPP1. + A core drug for tumor-associated macrophages was developed, resulting in a nanomedicine (Ang-2-OPNi-1 NP) that integrates multiple functions, including brain-targeted delivery, microenvironment-responsive drug release, tumor microenvironment remodeling, chemotherapy sensitization, and angiogenesis inhibition.

[0009] This invention further provides a method for preparing the material, specifically a method for preparing ROS-responsive nanomedicines that cross the blood-brain barrier and are loaded with OPN expression inhibitors, comprising the following steps:

[0010] (I) Preparation of ROS-responsive polymers:

[0011] (1) First, HO-TK-OH and hydrogenated pyromellitic dianhydride are dissolved in a suitable organic solvent under anhydrous conditions, and stirred continuously at room temperature to ensure thorough mixing. Then, anhydrous triethylamine is added to the reaction solution as a catalyst, the temperature is raised to 50°C, and the reaction is carried out under nitrogen atmosphere for 48 hours to obtain a prepolymer with carboxyl groups at both ends;

[0012] (2) Polyethylene glycol mPEG-OH was added to the reaction system and the reaction was continued at the same temperature for 24 h. The hydrophilic polyethylene glycol was used for end capping, and finally a polymer with an amphiphilic structure was formed.

[0013] (3) After the reaction is complete, the crude product solution is transferred to a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed with deionized water for 48 h. The water is changed every once in a while to remove small molecule byproducts and monomers that have not participated in the reaction. Finally, after freeze drying, a block copolymer with reactive oxygen species (ROS) responsiveness (named ROS responsive polymer) is obtained.

[0014] In further step (1), each 100 mg HO-TK-OH corresponds to 114-115 mg of hydrogenated pyromellitic dianhydride and 61-62 mg of anhydrous triethylamine as catalysts.

[0015] Further step (2): Polyethylene glycol mPEG-OH is mPEG 2000 -OH, mPEG 3400 -OH, mPEG 5000 One or more of -OH groups; preferably, the selected compound is mPEG. 5000 -OH.

[0016] (II) Preparation of Ang-1-OPNi-1 NP nanoparticles:

[0017] (1) Weigh DSPE-PEG, ROS-responsive polymer, a polymer with brain-targeting activity and OPN expression inhibitor (OPNi-1), dissolve them together in tetrahydrofuran, and use ultrasound to assist dissolution to form a transparent mixed solution;

[0018] (2) Transfer the mixed solution to a round-bottom flask and slowly remove the organic solvent in a rotary evaporator, controlling the appropriate water bath temperature and rotation speed to form a uniform thin film on the flask wall.

[0019] (3) Slowly add the mixed solvent to the round-bottom flask in step (2). The mixed solvent consists of ultrapure water and tetrahydrofuran ((v:v) = 9:1). Then, perform oscillation and sonication treatment. The film is rapidly and uniformly dispersed. Transfer the resulting mixture into an 8000Da dialysis bag and dialyze it in the dark to remove the organic solvent THF and the remaining unreacted OPN expression inhibitor (OPNi-1) from the system. Finally, core-shell structured nanoparticles (named Ang-2-OPNi-1 NP) are obtained.

[0020] Further step (1) The DSPE-PEG is DSPE-PEG 1000 DSPE-PEG 2000 DSPE-PEG 3400 DSPE-PEG 5000 DSPE-PEG 6000 One or more of the following; preferably, the selected DSPE-PEG is DSPE-PEG. 2000 .

[0021] Further, in step (1), the polymer with brain-targeting effect is one or more of DSPE-PEG-RVG29, DSPE-PEG-T7, DSPE-PEG-TGN, and DSPE-PEG-Angiopep-2; preferably, the selected polymer is DSPE-PEG-Angiopep-2.

[0022] In a further step (1), the mass ratio of DSPE-PEG, ROS-responsive polymer, brain-targeting polymer and OPN expression inhibitor (OPNi-1) is 10:5:5:2.

[0023] Further, in step (2), the temperature range for rotary evaporation to remove the organic solvent is 35-40°C and the rotation speed is 90-110 rpm; preferably, the temperature is 35°C and the rotation speed is 100 rpm.

[0024] The nanomedicine obtained in this invention is used to prepare drugs for treating glioblastoma. It can also be used to prepare combination drugs that synergistically enhance sensitization when combined with temozolomide.

[0025] This invention further provides the application of this nanomaterial in tumor treatment, including the nanomaterial's ability to penetrate the blood-brain barrier and regulate SPP1. + Application of glioma-associated macrophages and sensitization to temozolomide chemotherapy. This nanomedicine uses an amphiphilic ROS-responsive polymer as the drug carrier framework. The amphiphilic block copolymer can self-assemble in the aqueous phase through intermolecular hydrophobic interactions to encapsulate an OPN expression inhibitor. The surface of the nanoparticles is modified using polymers with brain-targeting activity, especially DSPE-PEG-Angiopep-2, to endow them with receptor-mediated trans-blood-brain barrier transport capabilities. Thioketal (TK) sensitive groups are introduced into the carrier structure, causing chemical bond breakage under high concentrations of reactive oxygen species in the glioma region, triggering carrier depolymerization and rapid drug release. The released OPNi-1 can specifically target SPP1⁺ tumor-associated macrophages, significantly downregulate OPN expression, block the OPN / integrin β3 / p65 / VEGFA tumor-promoting signaling axis, inhibit tumor angiogenesis, and reduce tumor invasiveness. At the same time, the combined action of Ang-2-OPNi-1 NP and temozolomide achieves synergistic killing, significantly increases the level of glioma cell apoptosis, inhibits in situ tumor growth, and effectively prolongs the survival of tumor-bearing animals.

[0026] Compared with the prior art, the present invention has the following outstanding advantages:

[0027] (1) Strong ability to cross the blood-brain barrier: The high molecular weight polymer with brain-targeting effect in this invention, especially DSPE-PEG-Angiopep-2, can specifically recognize and bind to low-density lipoprotein receptor-associated protein (LRP) highly expressed on the endothelial cells of the blood-brain barrier. Furthermore, when DSPE-PEG-Angiopep-2 accounts for 25% of the nanocarrier shell material, it can ensure the ideal drug encapsulation rate and endow the nanoparticles with excellent blood-brain barrier penetration ability. Then, through receptor-mediated transcytosis, it can efficiently penetrate the blood-brain barrier, significantly increase the enrichment concentration of the drug in the brain parenchyma and glioma area, and greatly improve the problem of traditional OPN inhibitors being difficult to enter the brain and having extremely low bioavailability, so as to achieve precise and efficient delivery from systemic administration to brain lesions.

[0028] (2) High specificity of microenvironment response: The nanocarrier framework constructed in this invention introduces ROS-sensitive thioacetate (TK) groups, which can accurately identify the highly expressed reactive oxygen species in the microenvironment of glioma. Under the stimulation of high concentration of ROS at the lesion site, specific chemical bond breaking occurs, triggering rapid depolymerization of the carrier and achieving drug burst release. In the physiological low ROS environment of normal brain tissue and peripheral blood, the nanoparticle structure remains highly stable and the drug leakage rate is extremely low, thereby greatly improving the selectivity of treatment, prolonging the drug action window, reducing non-specific tissue exposure, and significantly improving the safety of in vivo treatment.

[0029] (3) Clear target and mechanism: This invention targets SPP1⁺ tumor-associated macrophages, the core tumor-promoting cell subset in the glioma microenvironment, and effectively blocks the OPN / integrin β3 / p65 / VEGFA malignant signaling axis by inhibiting the expression of its key effector molecule osteopontin (OPN / SPP1), thereby inhibiting tumor angiogenesis and reducing tumor invasion ability from the root. At the same time, it intervenes in the core driving link of temozolomide resistance, solving the problem of chemotherapy resistance from the dual levels of tumor microenvironment remodeling and signaling pathway blocking. The action path is clear and the regulatory mechanism is clear, providing a reliable molecular target and action basis for the precision treatment of glioma.

[0030] (4) Significant synergistic therapeutic effect: This invention adopts an integrated synergistic therapeutic mode of tumor microenvironment remodeling combined with chemotherapy. Through the regulation of SPP1⁺ tumor-associated macrophages by nanomedicine, it reverses tumor immunosuppression and abnormal angiogenesis, while enhancing the chemosensitivity of glioma cells to temozolomide. In vitro experiments show that it can significantly increase the apoptosis level of tumor cells, and the apoptosis rate is much higher than that of the single drug group. In vivo experiments show that it can inhibit the proliferation and invasion of glioma in situ, significantly slow down the tumor growth rate, and effectively prolong the median survival of tumor-bearing animals. The overall therapeutic effect is significantly better than that of a single chemotherapy regimen, and the synergistic sensitization effect is outstanding.

[0031] (5) Good biocompatibility in vivo: The polymer carrier and components used in this invention have excellent biocompatibility. The in vitro and in vivo safety evaluation of the system has confirmed that the nanomedicine does not cause significant pathological damage or functional abnormalities to important organs such as heart, liver, spleen, lungs and kidneys in mice at effective therapeutic doses. During the treatment, the animals are in a stable overall condition and do not experience significant weight loss. No obvious systemic inflammatory response or toxic side effects are triggered. The safety window is wide and the biocompatibility is high, which has reliable potential for further clinical translation and practical application.

[0032] This invention is the first to integrate cross-blood-brain barrier targeting, ROS-responsive drug release, SPP1⁺ macrophage regulation, and temozolomide chemosensitization, providing a novel, effective, and safe synergistic treatment strategy for glioblastoma. It has significant value and broad prospects in the field of targeted therapy and clinical translation of glioma. Attached Figure Description

[0033] Figure 1 This invention relates to a ROS-responsive polymer. 1 H-NMR spectrum.

[0034] Figure 2 This is a measurement diagram of the critical micelle concentration of the ROS-responsive polymer and the Ang-2-targeting polymer of this invention.

[0035] Figure 3 These are DLS and TEM images of the Ang-2-OPNi-1 NP nanoparticles of this invention.

[0036] Figure 4 This is a stability test diagram of the Ang-2-OPNi-1 NP nanoparticles of the present invention.

[0037] Figure 5 This is a drug release curve of the Ang-2-OPNi-1 NP nanoparticles of the present invention under different conditions.

[0038] Figure 6 This invention relates to the CCK-8 method for determining the cell proliferation level under the action of Ang-2-OPNi-1 NP nanoparticles.

[0039] Figure 7 This is the experimental result of the anti-angiogenic effect of the nanoparticles Ang-2-OPNi-1 NP of the present invention on HUVEC cells.

[0040] Figure 8 This invention presents the results of immunofluorescence assays to detect the co-localization of OPN and Integrinβ3 under the action of Ang-2-OPNi-1 NP nanoparticles.

[0041] Figure 9 These are the flow cytometry results of cell apoptosis after administration of the Ang-2-OPNi-1 NP nanoparticles of this invention in combination with TMZ;

[0042] Figure 10 This invention provides imaging of the dynamic distribution of Cy5.5-labeled nanoparticles in mice and imaging of ex vivo organs.

[0043] Figure 11 This is an in vivo imaging image showing the effect of the Ang-2-OPNi-1 NP nanoparticles of the present invention combined with TMZ treatment on the growth of orthotopic gliomas in mice.

[0044] Figure 12 This is the result of the effect of the present invention's nanoparticles Ang-2-OPNi-1 NP combined with TMZ treatment on the survival of mice with orthotopic gliomas.

[0045] Figure 13 This study investigates the effects of the Ang-2-OPNi-1 NP nanoparticles combined with TMZ treatment on the expression of Ki67 and SPP1 in mouse glioma tissues.

[0046] Figure 14 HE staining results of major organs (heart, liver, spleen, lung, and kidney) in mice after treatment with Ang-2-OPNi-1 NP nanoparticles of this invention. Detailed Implementation

[0047] The following description, in conjunction with specific implementation examples and accompanying drawings, further illustrates the present invention, but it is not limited to the following embodiments.

[0048] Example 1: A method for preparing a ROS-responsive nanomedicine that crosses the blood-brain barrier and is loaded with an OPN expression inhibitor.

[0049] This embodiment provides a method for preparing nanomedicines modified with Angiopep-2, ROS-responsive, and loaded with OPNi-1. The specific steps are as follows:

[0050] (1) Synthesis of ROS-responsive polymeric carrier: 100 mg of HO-TK-OH and 114.3 mg of hydrogenated pyromellitic dianhydride were weighed and dissolved in anhydrous N,N-dimethylformamide. The mixture was magnetically stirred at room temperature until completely clear and transparent. Then, 61.8 mg of anhydrous triethylamine was added as a catalyst. After thorough mixing, the reaction system was placed in a nitrogen-protected, 50°C constant-temperature oil bath and stirred continuously for 48 h to obtain a ROS-responsive prepolymer with carboxyl active groups at both ends. After the prepolymer reaction was completed, methoxy polyethylene glycol monohydroxy ether (mPEG5000-OH) was added to the system for hydrophilic segment end capping. The reaction was continued at a constant temperature of 50°C for 24 h to ensure complete formation of the amphiphilic block structure. After the reaction, the crude product was transferred to a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed with ultrapure water in the dark for 48 h, with the dialysate changed every 8 h to thoroughly remove unreacted monomers, organic solvents, and small molecule byproducts. After dialysis, the product was rapidly frozen and thoroughly dried in a freeze dryer to obtain a pale yellow, loose, ROS-responsive amphiphilic polymer solid, which was then stored in a desiccator at low temperature and sealed for later use. The 1H NMR spectrum showed successful synthesis. Figure 1 ).

[0051] (2) Critical micelle concentration test of ROS-responsive polymer and Ang-2 targeted polymer carrier: A small amount of Nile Red dye was mixed with 10 mg of ROS-responsive polymer and 10 mg of DSPE-PEG-Angiopep-2 to prepare nanoparticles by mixing and stirring. The polymer concentration of the nanoparticles was diluted to 1 mg / mL as the mother liquor. By continuously diluting the concentration of the mother liquor, the fluorescence emission spectra of polymer solutions of different concentrations at an excitation wavelength of 578 nm were measured at 590-750 nm using a fluorescence spectrophotometer. By observing the change in fluorescence intensity with polymer concentration, a curve of fluorescence intensity versus concentration can be plotted. The concentration corresponding to the inflection point of the sudden change in fluorescence intensity on the curve is the critical micelle concentration (the critical micelle concentration, or CMC, is an important characteristic parameter of the self-assembly behavior of amphiphilic polymers, referring to the minimum critical concentration at which amphiphilic molecules spontaneously aggregate in the aqueous phase to form a stable micelle structure. The lower the CMC value, the stronger the hydrophobic association ability of the polymer, and the less likely the formed nanomicelles are to dissociate in the diluted environment of body fluid). Driven by hydrophobic interactions, the hydrophobic segments curl inward and the hydrophilic segments face outward, leading to the spontaneous assembly of numerous molecules and the formation of stable core-shell micelles. Therefore, this invention tested their critical micelle concentration (CMC) to evaluate the minimum concentration required for ROS-responsive polymers and DSPE-PEG-Angiopep-2 to form micelles. Analysis using the Nile Red dye method revealed low CMC values: 0.0835 mg / mL for the ROS-responsive polymer and 0.0425 mg / mL for DSPE-PEG-Angiopep-2. Figure 2 ).

[0052] (3) Preparation of Ang-2-OPNi-1 NP nanoparticles by ultrasonic thin film dispersion method: Weigh 25 mg of ROS-responsive polymer, 50 mg of DSPE-PEG2000, 25 mg of DSPE-PEG-Angiopep-2, and 10 mg of OPNi-1, and dissolve them together in 3 mL of tetrahydrofuran. Use ultrasound to assist complete dissolution. Transfer the solution to a round-bottom flask and remove the organic solvent by rotary evaporation to form a uniform and transparent film on the flask wall. Slowly add 10 mL of mixed solvent to the flask. The mixed solution consists of ultrapure water and tetrahydrofuran (v:v = 9:1). Use ultrasound to completely disperse the film. Transfer the mixture to an 8000 Da dialysis bag and dialyze for 48 h in the dark to remove unreacted free drugs and organic solvents, thus obtaining Ang-2-OPNi-1 NP nanoparticles.

[0053] (4) Characterization of Ang-2-OPNi-1 NP nanoparticles: For particle size and potential testing, the nanoparticles were diluted with ultrapure water to a suitable concentration of 0.1-1 mg / mL to ensure that the detection signal was within the sensitive range of the instrument. Dynamic light scattering was used to measure the nanoparticles at 25℃. Each sample was tested more than 5 times, and the average value was taken as the final result. The results showed that the average particle size of the obtained nanoparticles was 119 nm, the polydispersity index (PDI) was 0.122, the particle size distribution was uniform, and the dispersion was good. The zeta potential was -14.9 mV, and the surface was electronegative, which is beneficial for long-term cycling stability and reduces non-specific adsorption. For transmission electron microscopy (TEM) morphology characterization, a clean 200-mesh carbon-supported copper mesh was placed on filter paper. 10 μL of a suspension of Ang-2-OPNi-1NP nanoparticles (approximately 1 mg / mL) was gently added to the copper mesh surface and allowed to stand at room temperature for 3 min for adsorption. Excess liquid was gently blotted away with filter paper, followed by staining with 1% phosphotungstic acid solution for 50 s. The sample was then blotted dry again and allowed to air dry naturally. TEM observation showed that the nanoparticles were regularly spherical, with clear outlines, uniform size, and no obvious aggregation, consistent with the dynamic light scattering particle size results, further confirming the intact structure and regular morphology of the nanomedicine. Figure 3 ).

[0054] (5) In vitro stability experiment of Ang-2-OPNi-1 NP nanoparticles: To evaluate the structural stability of the nanomedicine under physiological conditions, a PBS buffer group (pH 7.4) and a 10% fetal bovine serum (FBS) simulated physiological fluid group were set up for comparison. Equal amounts of nanomedicine samples were taken and thoroughly mixed with equal volumes of PBS and PBS solution containing 10% FBS, respectively, and placed in a constant temperature shaker at 37℃ in the dark to simulate the in vivo blood circulation environment. Samples were taken at 0, 1, 2, 4, 7, and 14 days, and the average particle size and polydispersity index (PDI) of each group of samples were measured using a dynamic light scattering instrument. The results showed that after 14 days of incubation in PBS and 10% serum systems, the average particle size of the nanomedicine did not increase significantly, and the PDI remained at a low level. No obvious aggregation, sedimentation, or structural dissociation was observed, proving that the prepared nanomedicine has excellent colloidal stability and structural integrity under physiological conditions and can meet the requirements of long-term in vivo delivery. Figure 4 ).

[0055] (6) Drug release experiment of Ang-2-OPNi-1 NP nanoparticles: Equal amounts of drug-loaded nanoparticles were added to 8000 Da dialysis bags, and the dialysis bags were placed in two different release media: one group was PBS buffer (pH 7.4) to simulate the normal physiological environment; the other group was PBS solution containing 10 mM H2O2 to simulate the highly reactive oxygen species tumor microenvironment of glioma. Both groups were subjected to drug release experiments at 37℃ and 100 rpm in the dark with stirring. Samples were taken from the release media at predetermined time points, and fresh media of the same temperature and volume were replenished in a timely manner. The drug concentration was determined by high performance liquid chromatography, and the cumulative drug release curve was calculated and plotted. The results showed that the release was slow under the physiological conditions of PBS, and the cumulative release amount was less than 30% after 72 h; while under the ROS stimulation induced by 10 mM H2O2, the carrier rapidly responded and disintegrated, and the drug release rate was significantly accelerated, with a cumulative release amount of up to 78% after 72 h, showing excellent ROS-specific response drug release performance, which can realize the controlled release of drugs at the tumor site. Figure 5 ).

[0056] Example 2: In vitro antitumor and mechanism study of nanomedicines

[0057] The cells used in this embodiment included mouse glioma GL261 cells, human monocytes THP-1, and human umbilical vein endothelial cells (HUVECs), all cultured in a medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody at 37°C and 5% CO2. Within the GL261 tumor microenvironment, THP-1 cells were recruited and polarized into M2-like SPP1 cells. + (OPN-overexpressing) tumor-associated macrophages, HUVECs, under OPN stimulation promote angiogenesis and accelerate tumor malignant progression.

[0058] (1) Cell proliferation experiment: The effect of each group of drugs on cell proliferation was detected by CCK-8 method. The groups were set as follows: LVCtrl (lentivirus empty vector control group, negative control), LV SPP1 (lentivirus overexpression SPP1 / OPN group), LV SPP1+DMSO (SPP1 overexpression + solvent DMSO treatment, excluding the effect of DMSO itself on cells, ensuring that the difference in the inhibitor group is caused by the drug), LV SPP1+OPNi-1 (SPP1 overexpression + OPN expression inhibitor, directly inhibiting OPN function), LV SPP1+Ang-2-blank NP (SPP1 overexpression + Ang-2 blank vector, excluding the influence of the vector itself) and LV SPP1+Ang-2-OPNi-1NP (SPP1 overexpression + Ang-2 targeted OPN inhibitor, compared with OPNi-1 to verify whether targeted delivery enhances the inhibitory effect). HUVEC cells were co-cultured with THP-1 cells, and drugs were added according to the group. The concentration of OPNi-1 50 μM was used as the standard. After 24 h, 48 h, 72 h and 96 h of treatment, the cells were digested and seeded into 96-well plates. After adhesion, the old culture medium was discarded, and 100 μL of complete culture medium containing 10% CCK-8 was added to each well. The cells were incubated at 37℃ for 2 to 4 hours. The absorbance was read at 450 nm using a microplate reader. The blank control was an equal volume of mixed solution without cells. Cell proliferation was expressed by absorbance values. The results showed that the LV SPP1+Ang-2-OPNi-1 NP group had a more significant inhibitory effect on HUVEC cell proliferation, which was significantly stronger than the control group and the blank drug-loaded group. Figure 6 ).

[0059] (3) Angiogenesis experiment: The effect of drugs on HUVEC angiogenesis was evaluated using the matrix gelation tube experiment. Before the experiment, the matrix gel was melted at 4℃, and the 24-well plates were pre-cooled. 50-70 μL of matrix gel was added to each well, and the plates were incubated at 37℃ for 30-60 min until completely solidified. HUVEC and THP-1 cells in the logarithmic growth phase were selected. THP-1 cells were activated by induction with 10-50 ng / mL PMA for 24-48 h. After digestion, washing, and counting, HUVEC and THP-1 cells were mixed at a 3:1 ratio and cultured in EGM-2 complete medium (based on 500 mL EBM-2 basal medium, with the following added sequentially: 10.00 mL fetal bovine serum, 0.20 mL hydrocortisone, 2.00 mL hFGF-B, 0.50 mL VEGF, 0.50 mL R3-IGF-1, 0.50 mL ascorbic acid, 0.50 mL hEGF, and 0.50 mL...). Adjust cell density with GA-1000 and 0.50 mL heparin, and add 300 μL of cell suspension (3 × 10⁶ HUVEC cells) to each well. 4 cells / well, 1 × 10⁶ THP-1 cells 4 Cells / well were incubated at 37°C in a 5% CO2 incubator. After 48 h, cells were observed and images were taken using an inverted microscope. Results showed that SPP1 overexpression significantly promoted tubular structure formation; the addition of OPNi-1 significantly reduced tubular formation; Ang-2-OPNi-1 NP effectively reversed the SPP1-induced angiogenesis effect, significantly reducing total vessel length and branching point number. Figure 7 ).

[0060] (4) Immunofluorescence assay: Cells were seeded in 24-well plates, and cell smears with a diameter of 14 mm were placed at the bottom of each well beforehand. The smears were pretreated with poly-L-lysine to enhance cell adhesion. After treatment with the drug (concentration based on the corresponding OPNi-1 50 μM) for 24 h, the plates were fixed with 4% paraformaldehyde at room temperature for 10 min, and then blocked with QuickBlock blocking solution at room temperature for 15 min. After blocking, the smears were incubated overnight at 4°C with the corresponding OPN primary antibody and Integrin β3 primary antibody. The corresponding fluorescent secondary antibodies were selected according to the source of the primary antibody. The secondary antibodies were goat anti-rabbit IgG H&L (Alexa Fluor-488), goat anti-mouse IgG H&L (Alexa Fluor-488), and goat anti-rabbit IgG H&L (Alexa Fluor-594). All secondary antibodies were diluted at a ratio of 1:500 and added to the sample. The plates were incubated at room temperature in the dark for 1 h. After incubation, the plates were mounted with anti-fluorescence quenching mounting medium containing DAPI. Fluorescence microscopy was used to perform quantitative and statistical analysis of the results obtained using ImageJ software. The results confirmed that Ang-2-OPNi-1 NP specifically reverses SPP1-mediated enhanced OPN-Integrin β3 binding. OPN and Integrin β3 are key ligand-receptor pairs for communication between macrophages and endothelial cells. Figure 8 ).

[0061] (5) Apoptosis assay: Apoptosis of GL261 cells was detected by Annexin V-FITC / PI double staining flow cytometry. Annexin V-FITC labeled early apoptotic cells (green fluorescence), and PI labeled necrotic or late apoptotic cells (red fluorescence). The degree of DNA fragmentation and apoptosis level of cells were reflected by flow cytometry. Experimental groups were set up as follows: LV SPP1 group, LV SPP1+OPNi-1 group, LV SPP1+Ang-2-OPNi-1NP group, LV SPP1+TMZ group, LV SPP1+OPNi-1+TMZ group and LV SPP1+Ang-2-OPNi-1 NP +TMZ group. GL261 cells and THP-1 cells in logarithmic growth phase were co-cultured and incubated for 24 h. Different drugs were added according to the group (the concentration was based on the corresponding OPNi-1 50 μM and TMZ 40 µM as the standard), and then cultured for another 48 h. Discard the original culture medium, digest with 100 μL of trypsin, collect the cell suspension, centrifuge at 1000 rpm for 5 min, and adjust the cell number per group to approximately 5 × 10⁶ cells / mL. 5Each tube. GL261 cell pellet was washed three times with 1×PBS buffer pre-cooled to 4°C, and the supernatant was discarded after each centrifugation to remove residual culture medium and free drugs. GL261 cells were resuspended in 1×Binding Buffer, Annexin V-FITC staining solution was added, and the cells were incubated in the dark for 15 min. PI staining solution was added, and the cells were incubated at room temperature in the dark for another 5 min. GL261 cell counts were analyzed using flow cytometry. The results showed that the apoptosis rate was only 1.74% in the LV SPP1 group, 37.93% in the LV SPP1+TMZ group, 62.67% in the LV SPP1+OPNi-1+TMZ group, and as high as 88.93% in the LV-SPP1+Ang-2-OPNi-1 NP+TMZ combined group, indicating that synergistic therapy can significantly induce tumor cell apoptosis. Figure 9 ).

[0062] Example 3: Evaluation of the in vivo antitumor efficacy and safety of nanomedicines

[0063] In this embodiment, SPF-grade C57BL / 6 male mice, aged 6-8 weeks, were used to construct a GL261-Luc orthotopic glioma model. The tumor-bearing mice were randomly divided into 4 groups (n=5): PBS control group, TMZ single drug group, Ang-2-OPNi-1 NP group, and Ang-2-OPNi-1 NP + TMZ combination group.

[0064] (1) Dosing regimen: blank control group, i.e. PBS group; positive control group, i.e. TMZ group, Ang-2-OPNi-1 NP group, Ang-2-OPNi-1 NP+TMZ group.

[0065] Ang-2-OPNi-1 NP is administered via tail vein injection at a dose of 0.5 mg / kg, for a total of 7 administrations, with a dosing frequency of once every 2 days. TMZ is administered via intraperitoneal injection at a dose of 50 mg / kg, with a dosing regimen of 5 consecutive days followed by a 2-day rest period, for a total dosing cycle of 2 weeks, for a total of 10 administrations.

[0066] Throughout the treatment process, on day 0 (baseline before administration), day 7, day 14, and day 21, tumor fluorescence imaging was performed on mice in each group using a small animal in vivo imaging system. By analyzing the fluorescence intensity of the tumor region in the imaging results, the growth size and progression of the mouse tumors were dynamically monitored.

[0067] (2) In vivo distribution and brain targeting experiment: Cy5.5-labeled nanomedicines were used, and their in vivo distribution was observed using in vivo imaging in small animals. The results showed that the fluorescence signal in the brain tumor region of the Ang-2 modified targeting group was significantly stronger than that of the non-targeting group; 48 h after administration, the fluorescence intensity at the brain tumor site was 24.77 times that of the non-targeting group, proving that the nanomedicines can efficiently cross the blood-brain barrier and target and accumulate in the glioma site. Figure 10 ).

[0068] (3) Tumor growth inhibition effect: Tumor growth was dynamically monitored by in vivo bioluminescence imaging. The results showed that the PBS group had rapid tumor proliferation and continuous enhancement of fluorescence signal; the TMZ single drug group showed only limited inhibitory effect; the Ang-2-OPNi-1 NP+TMZ combination group showed significant inhibition of tumor growth, and the fluorescence signal was much lower than that of other groups ( Figure 11 ).

[0069] (4) Mouse survival analysis: Survival statistics showed that the median survival in the PBS group was only 31 days; the median survival in the TMZ monotherapy group was 35 days; and the median survival in the Ang-2-OPNi-1 NP+TMZ combination group was significantly prolonged to more than 50 days, with extremely statistically significant differences. Figure 12 ).

[0070] (5) Tumor tissue pathological examination: Immunohistochemical staining (Ki-67, SPP1) was performed on the tumor tissue. The results showed that the SPP1 expression level was significantly downregulated in the Ang-2-OPNi-1 NP+TMZ group, confirming that OPNi-1 can effectively inhibit SPP1 expression, and that the combination of OPN expression inhibitor nanomedicine and TMZ can synergistically exert antitumor effects and inhibit tumor proliferation. Figure 13 ).

[0071] (6) In vivo safety evaluation: After treatment, mouse heart, liver, spleen, lung, and kidney were stained with hematoxylin and eosin (HE). The results showed that the structures and morphologies of the major organs were intact and normal, with no obvious inflammatory infiltration, necrosis, or damage. There was no significant difference compared with the PBS group, indicating that Ang-2-OPNi-1 NP has good in vivo biosafety and can meet the needs of clinical application. Figure 14 ).

Claims

1. A method for preparing a ROS-responsive nanomedicine that crosses the blood-brain barrier and is loaded with an OPN expression inhibitor, characterized in that, Includes the following steps: (I) Preparation of ROS-responsive polymers: (1) First, HO-TK-OH and hydrogenated pyromellitic dianhydride are dissolved in a suitable organic solvent under anhydrous conditions, and stirred continuously at room temperature to ensure thorough mixing. Then, anhydrous triethylamine is added to the reaction solution as a catalyst, the temperature is raised to 50°C, and the reaction is carried out under nitrogen atmosphere for 48 hours to obtain a prepolymer with carboxyl groups at both ends; (2) Polyethylene glycol mPEG-OH was added to the reaction system and the reaction was continued at the same temperature for 24 h. The hydrophilic polyethylene glycol was used for end capping, and finally a polymer with an amphiphilic structure was formed. (3) After the reaction was completed, the crude product solution was transferred to a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed with deionized water for 48 h. The water was changed every once in a while to remove small molecule byproducts and monomers that did not participate in the reaction. Finally, after freeze drying, a block copolymer with reactive oxygen species (ROS) responsiveness was obtained and named ROS responsive polymer. (II) Preparation of Ang-1-OPNi-1 NP nanoparticles: (1) Weigh DSPE-PEG, ROS-responsive polymer, a polymer with brain-targeting activity and OPN expression inhibitor (OPNi-1), dissolve them together in tetrahydrofuran, and use ultrasound to assist dissolution to form a transparent mixed solution; (2) Transfer the mixed solution to a round-bottom flask and slowly remove the organic solvent in a rotary evaporator, controlling the appropriate water bath temperature and rotation speed to form a uniform thin film on the flask wall. (3) Slowly add the mixed solvent to the round-bottom flask of step (2). The mixed solvent consists of ultrapure water and tetrahydrofuran in a volume ratio of 9:

1. Then, perform oscillation and sonication treatment. The film is quickly and uniformly dispersed. Transfer the resulting mixture to an 8000Da dialysis bag and dialyze it in the dark to remove the organic solvent THF and the remaining unreacted OPN expression inhibitor in the system. Finally, the core-shell structured nanoparticles are obtained and named Ang-2-OPNi-1 NP.

2. The method according to claim 1, characterized in that, (I) In step (1) of the preparation of ROS responsive polymer, every 100 mg of HO-TK-OH corresponds to 114-115 mg of hydrogenated pyromellitic dianhydride and 61-62 mg of anhydrous triethylamine as catalyst.

3. The method according to claim 1, characterized in that, (I) In the preparation of ROS-responsive polymers, step (2) involves polyethylene glycol mPEG-OH as mPEG. 2000 -OH, mPEG 3400 -OH, mPEG 5000 One or more of -OH groups; preferably, the selected compound is mPEG. 5000 -OH.

4. The method according to claim 1, characterized in that, (II) Steps in the preparation of Ang-1-OPNi-1 NP nanoparticles (1) The DSPE-PEG is DSPE-PEG 1000 DSPE-PEG 2000 DSPE-PEG 34000 DSPE-PEG 5000 DSPE-PEG 6000 One or more of the following; preferably, the selected DSPE-PEG is DSPE-PEG. 2000 .

5. The method according to claim 1, characterized in that, (II) Steps in the preparation of nanoparticles Ang-1-OPNi-1 NP (1) The polymer with brain-targeting effect is one or more of DSPE-PEG-RVG29, DSPE-PEG-T7, DSPE-PEG-TGN, and DSPE-PEG-Angiopep-2; preferably, the selected polymer is DSPE-PEG-Angiopep-2.

6. The method according to claim 1, characterized in that, (II) In step (1) of the preparation of Ang-1-OPNi-1 NP nanoparticles, the mass ratio of DSPE-PEG, ROS-responsive polymer, brain-targeting polymer and OPN expression inhibitor (OPNi-1) is 10:5:5:

2.

7. The method according to claim 1, characterized in that, (II) In the preparation of nanoparticles Ang-1-OPNi-1 NP, step (2) describes the temperature range for removing organic solvent by rotary evaporation as 35-40℃ and the rotation speed as 90-110 rpm; preferably, the temperature is 35℃ and the rotation speed is 100 rpm.

8. Nanomedicine prepared according to any one of claims 1-7.

9. The application of the nanomedicine prepared according to any one of claims 1-7, for the preparation of a drug for treating glioblastoma.

10. The application according to claim 9, used to prepare a combination drug for synergistic sensitization with temozolomide.

Images