Magnetic-aptamer targeting tumor tissue nano-drug delivery system as well as preparation method and application of magnetic-aptamer targeting tumor tissue nano-drug delivery system

Abstract

The invention discloses a magnetic-aptamer targeting tumor tissue nano-drug delivery system as well as a preparation method and application thereof, and belongs to the field of medicines. The system comprises a mesoporous ferroferric oxide magnetic core, a polyethyleneimine (PEI) layer coated outside the mesoporous ferroferric oxide magnetic core and an aptamer adsorbed outside the PEI layer, and an autophagy activator can be selectively loaded in the mesoporous core. The preparation method comprises the following steps: sequentially carrying out drug loading, PEI coating and aptamer modification on the mesoporous Fe3O4 nanoparticles. According to the invention, efficient enrichment and endocytosis of tumor tissues are realized through dual effects of magnetic targeting and active targeting of the aptamer; lysosome escape is promoted by using the proton sponge effect of PEI; finally, by virtue of the synergistic effect of iron ions released by degradation of the Fe3O4 nanoparticles and endogenous iron ions released by ferritin autophagy induced by an autophagy activator, ferroptosis of tumor cells is induced through enhanced Fenton-like reaction, so that high-efficiency and low-toxicity targeted therapy is realized.

Description

Technical Field

[0001] This invention relates to the field of drug development, specifically to a magnetic-aptamer-targeted nanomedicine delivery system for tumor tissues, its preparation method, and its application. Background Technology

[0002] Malignant tumors are a major disease that seriously threatens human health, and current chemotherapy methods still face many challenges in clinical application. Systemic chemotherapy drugs generally suffer from poor tumor tissue selectivity, making it difficult for effective therapeutic doses to accumulate sufficiently at the lesion site, while also potentially causing significant toxic side effects on normal tissues and organs. In addition, the heterogeneity of tumor cells and the complexity of the microenvironment often lead to low drug delivery efficiency, affecting treatment efficacy and potentially inducing drug resistance.

[0003] To improve the targeting of treatments, nanomedicine delivery systems have become a research hotspot. Loading drugs onto nanocarriers can enhance drug accumulation in tumor tissues to some extent by improving the penetration-retention (EPR) effect. To further achieve active targeting, researchers often modify the surface of nanocarriers with ligands that specifically recognize tumor cell surface markers, such as antibodies, peptides, or oligonucleotide aptamers, to enhance the carrier's binding and internalization of tumor cells. On the other hand, physical response-based targeting strategies, such as magnetic targeting technology that uses an external magnetic field to guide the aggregation of magnetic nanoparticles in a specific region, have also attracted attention due to their controllability. How to effectively integrate different targeting strategies to overcome the limitations of a single targeting mode is an important research direction for improving the accuracy of delivery systems.

[0004] In terms of tumor treatment mechanisms, in addition to traditional apoptosis induction, exploring other cell death pathways (such as iron-dependent cell death) provides new insights into overcoming drug resistance. These mechanisms typically involve alterations in specific intracellular metabolic states; for example, the abnormal accumulation of certain metal ions may catalyze the production of reactive oxygen species, thereby triggering cellular damage processes such as lipid peroxidation. Regulating related intracellular metabolic balance through external intervention holds promise for synergistically enhancing the killing effect on tumor cells.

[0005] While nanocarriers have shown promise in targeted cancer therapy, most existing systems have relatively limited functionality. For example, some carriers may focus on achieving only one aspect of physical or biological targeting, making efficient dual targeting difficult in complex physiological environments; others may focus on drug loading and controlled release, but fail to adequately consider how to synergistically regulate the intracellular microenvironment to enhance therapeutic effects. Furthermore, ensuring that carriers can effectively deliver drugs to the intended subcellular sites and function effectively after entering cells remains an area requiring optimization. Summary of the Invention

[0006] To address the aforementioned technical issues, this application provides a magnetic-aptamer-targeted tumor tissue nanomedicine delivery system, its preparation method, and its application.

[0007] The technical solution adopted in this application is as follows: In a first aspect, this application provides a magnetic-aptamer-targeted tumor tissue nanomedicine delivery system, comprising: The magnetic core formed by mesoporous iron oxide; A polyethyleneimine layer covering the magnetic core; An aptamer adsorbed on the outer surface of the polyethyleneimine layer and capable of specifically binding to tumor cells; And, optionally, an autophagy activator loaded in the mesopores of the magnetic core.

[0008] Furthermore, the autophagy activator is selected from any one of gambogeylic acid, rapamycin, and cisplatin.

[0009] Furthermore, the aptor is AS1411.

[0010] Secondly, this application provides a method for preparing the above-mentioned magnetic-aptamer-targeted tumor tissue nanomedicine delivery system, which includes: (1) Superparamagnetic mesoporous iron oxide nanoparticles were prepared as magnetic cores; (2) Nanoparticles loaded with autophagy activators were obtained by co-incubating autophagy activators with magnetic cores; (3) The nanoparticles loaded with the autophagy activator are mixed with a polyethyleneimine solution to coat the surface of the nanoparticles with polyethyleneimine. (4) The PEI-coated nanoparticles and aptamers are incubated together to obtain a drug delivery system.

[0011] Furthermore, the co-incubation mass ratio of the autophagy activator to the mesoporous iron oxide nanoparticles is 0.8~1:1, and the incubation time is 4~8h.

[0012] Furthermore, the mass ratio of the nanoparticles loaded with the autophagy activator to the polyethyleneimine is 1:50~100, and the coating reaction is carried out under stirring for 1~3 hours.

[0013] Furthermore, the mass ratio of the PEI-coated nanoparticles to the aptamers was 1:120~200, and the mixture was incubated at 2~8℃ for 6~10h.

[0014] Furthermore, the above-mentioned mesoporous iron oxide nanoparticles have a particle size of 30~80nm and a charge of -15~-13mV.

[0015] Furthermore, the above-mentioned mesoporous iron oxide nanoparticles were prepared by a solvothermal method, with a reaction temperature of 120℃ to 200℃ and a reaction time of 12 to 20 h.

[0016] Thirdly, this application also provides the application of the above-mentioned nanomedicine delivery system in the preparation of drugs for treating tumors.

[0017] In summary, this application has the following beneficial effects: This invention utilizes a mesoporous Fe3O4 (MFe3O4) core with superparamagnetic properties to load autophagy activators (such as gambogeylic acid, abbreviated as GA), and coats it with polyethyleneimine (PEI), which can achieve lysosomal escape by utilizing the "proton sponge effect"; and modifies the surface of nanoparticles with aptamers that target nucleolins on the surface of tumor cells (such as AS1411) through electrostatic adsorption, thus synthesizing a drug delivery system that targets tumor tissues with both magnetic targeting and aptamer targeting.

[0018] The nanoparticles synthesized in this invention, after being injected into the tail vein of mice, aggregated in tumor tissue under the influence of an external magnetic field. They were then specifically endocytosed into tumor cells by recognizing nucleolin on the cell surface. Iron ions from the degradation of MFe3O4 under acidic conditions and autophagy activators, through inducing the release of iron ions from autophagy ferritin, jointly exert a Fenton-like effect, generating hydroxyl radicals that kill tumor tissue. GA inhibits GSH levels, reducing the scavenging of hydroxyl radicals and thus enhancing ferroptosis.

[0019] The nanomedicine delivery system provided by this invention has no toxic side effects on normal cells, is easily metabolized in vivo, and has good biocompatibility. The delivery system enables the effective release of drugs, which act precisely on different sites, causing ferritin autophagy in cells to cause a large accumulation of iron ions in the cytoplasm, and killing tumor cells by generating a large number of hydroxyl radicals through a Fenton-like reaction. Attached Figure Description

[0020] Figure 1 The N2 adsorption-desorption isotherm and pore size distribution of MFe3O4 prepared in Example 1 of this application are shown. Figure 2 Transmission electron microscopy image of MFe3O4@GA-PEI-AS1411 prepared in Example 1 of this application; Figure 3 The survival rate of tumor cells after treatment with different materials according to this application; Figure 4 The generation of reactive oxygen species after treating tumor cells with different materials according to this application; Figure 5 This shows the in vivo distribution of different materials in mice at different times after tail vein injection. Detailed Implementation

[0021] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] The inventive concept of this application stems from a profound understanding and integrated solution to the two core problems in existing tumor treatments: insufficient drug targeting and the reliance on a single cell death mechanism.

[0023] Existing nanodelivery systems either focus on single physical (e.g., magnetic targeting) or biological (e.g., ligand targeting) targeting strategies, and their positioning accuracy and cellular uptake efficiency in complex in vivo environments remain limited. Meanwhile, traditional chemotherapy mainly relies on inducing apoptosis, leading to drug resistance in tumor cells. Therefore, developing a novel synergistic therapeutic platform that can achieve highly efficient dual targeting and activate non-apoptotic cell death pathways is urgently needed and of great significance.

[0024] Based on this, this application creatively designs and constructs an integrated nanomedicine delivery system for "magnetic-aptamer dual-targeted ferroptosis-autophagy synergistic therapy".

[0025] This application provides a magnetic-aptamer-targeted tumor tissue nanomedicine delivery system, comprising: A magnetic core formed by mesoporous iron oxide.

[0026] A polyethyleneimine layer covering the magnetic core.

[0027] An aptamer adsorbed on the outer surface of the polyethyleneimine layer and capable of specifically binding to tumor cells.

[0028] And, optionally, an autophagy activator loaded in the mesopores of the magnetic core.

[0029] The aptamer is AS1411. AS1411 is a DNA oligonucleotide that specifically recognizes and binds to nucleolin on the surface of tumor cells. Nucleolin is abnormally highly expressed on the surface of various cancer cells, but almost not expressed on the surface of normal somatic cells. This characteristic makes AS1411 a highly promising tumor-targeting molecule.

[0030] The autophagy activator is selected from any one of gambogeylic acid, rapamycin, and cisplatin, preferably gambogeylic acid.

[0031] The technical solution of this application has the following characteristics: On the one hand, at the level of targeted delivery, this invention achieves simultaneous synergy between physical and biological targeting. The inventors selected superparamagnetic mesoporous iron(III) oxide (MFe3O4) nanoparticles as the core carrier. Their inherent magnetic responsiveness allows the system to actively accumulate in the tumor region under the guidance of an external magnetic field, achieving first-level physical targeting. Furthermore, by modifying the particle surface with the aptamer AS1411, the system is endowed with second-level active molecular recognition targeting capabilities. This dual-targeting strategy of "magnetic accumulation + aptamer-specific binding" is not a simple superposition, but rather aims to overcome the limitations of each component, progressively enhancing the drug's accumulation concentration and cellular uptake efficiency at the tumor site from the tissue to the cell level, laying a foundation for subsequent highly effective treatment.

[0032] On the other hand, at the intracellular therapeutic mechanism level, this invention conceives of a cascade reaction of "lysosomal escape - synergistic amplification of iron supply - ferroptosis-dominant". The drug delivery system, through a PEI coating layer, utilizes its "proton sponge effect" to promote escape from lysosomes after endocytosis, precisely delivering the active ingredient to the cytoplasm. More importantly, this invention transforms the carrier itself (MFe3O4) into a therapeutic component: it degrades in the tumor's slightly acidic environment, releasing iron ions, which directly participate in the reaction as an exogenous iron source. Simultaneously, the loaded autophagy activator (such as gambogeylic acid) exerts a dual pharmacological effect: firstly, by inhibiting pathways such as Nrf2, it enhances NCOA4-mediated ferritin autophagy, prompting the release of large amounts of stored iron within the cell, providing an endogenous iron source; secondly, it consumes antioxidants such as glutathione (GSH), weakening the cell's defense against oxidative damage. The convergence of endogenous and exogenous iron leads to a sharp increase in the level of unstable iron pools within cells, generating excess reactive oxygen species (ROS) through an enhanced Fenton / Fenton-like reaction, triggering irreversible lipid peroxidation, and thus specifically and effectively inducing ferroptosis in tumor cells. This synergistic design of "carrier iron supply + drug iron release and weakening defense" achieves multi-site and potent activation of the ferroptosis pathway.

[0033] In summary, the essence of this application lies in the innovative integration of a dual-targeted delivery system, lysosomal escape function, and ferroptosis-autophagy synergistic therapeutic mechanism into a single nanostructure, providing a novel and promising solution for developing highly efficient and low-toxicity new strategies for tumor treatment.

[0034] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0035] Example 1 This embodiment provides a magnetic-aptamer-targeted tumor tissue nanomedicine delivery system, the preparation method of which includes: (1) Preparation of superparamagnetic MFe3O4 nanoparticles: 1 g of FeCl3·6H2O and 3 g of CH3COONa were added to 10 mL of ethylene glycol solution under stirring. After dissolution, 20 mL of ethylenediamine was added and stirred until the mixture became clear. The solution was then transferred to a reaction vessel and reacted at 150 °C for 18 h. After the reaction, the solution was washed alternately with ethanol and ultrapure water and then lyophilized. The specific surface area and pore size of MFe3O4 were determined by BET analysis. The results are as follows: Figure 1 As shown, the specific surface area and average pore size of MFe3O4 are 98.8762 m². 3 g −1 The wavelength of 10.9583 nm indicates that MFe3O4 has a mesoporous structure and a high drug loading rate.

[0036] (2) Preparation of drug-loaded nanoparticles MFe3O4@GA: The autophagy activator gambogeylic acid (GA) and MFe3O4 were dispersed in ultrapure water at a mass ratio of 1 mg:1 mg, incubated at room temperature by rotation for 7 h, and then subjected to magnetic separation, washing and freeze-drying to obtain MFe3O4@GA.

[0037] (3) Preparation of polyethyleneimine-coated nanoparticles MFe3O4@GA-PEI: The mass ratio of nanoparticles MFe3O4@GA to PEI was 1:70 mg; they were dissolved in ultrapure water and stirred with a stirrer at room temperature for 2 h at a speed of 400 r / min; MFe3O4@GA-PEI was obtained by magnetic separation, washing and freeze drying.

[0038] (4) Preparation of the nanomedicine delivery system MFe3O4@GA-PEI-AS1411: MFe3O4@GA-PEI and AS1411 were mixed at a mass ratio of 150:1; the rotational incubation time was 8 h; and the temperature was 5 °C. MFe3O4@GA-PEI-AS1411 was obtained by magnetic separation and washing.

[0039] The transmission electron microscope (TEM) images were obtained through examination, such as... Figure 2 As shown, MFe3O4@GA-PEI-AS1411 exhibits good dispersibility, and PEI adhesion to the nanoparticle surface can be observed, indicating the successful synthesis of MFe3O4@GA-PEI-AS1411.

[0040] Example 2 The difference between this embodiment and embodiment 1 is that rapamycin is used as an autophagy activator in step (2).

[0041] Example 3 The difference between this embodiment and embodiment 1 is that cisplatin is used as an autophagy activator in step (2).

[0042] Example 4 The difference between this embodiment and Example 1 is that the reaction temperature in step (1) when preparing MFe3O4@GA is 170℃ and the reaction time is 14h.

[0043] Example 5 The difference between this embodiment and embodiment 1 is that in step (2), when preparing drug-loaded nanoparticles MFe3O4@GA, gambogeylic acid (GA) and MFe3O4 are incubated at room temperature for 5 hours by rotation at a mass ratio of 0.8 mg: 1 mg.

[0044] Example 6 The difference between this embodiment and embodiment 1 is that: in step (3) when preparing MFe3O4@GA-PEI, the mass ratio of MFe3O4@GA to PEI is 1:80mg; it is dissolved in ultrapure water and stirred with a stirring paddle at room temperature for 2.5h; the rotation speed is 420r / min.

[0045] Example 7 The difference between this embodiment and Example 1 is that: in step (4) when preparing MFe3O4@GA-PEI-AS1411, the mass ratio of MFe3O4@GA-PEI to AS1411 is 160:1; the rotation incubation time is 9h; and the temperature is 4℃.

[0046] Performance testing 1. Cell viability assay: 4T1 cells were transplanted into 96-well plates, with 0.5 × 10⁶ cells per well. 4 Cells were collected. After 24 hours, different material treatment groups (provided in Example 1) were set up, including PEI, MFe3O4, GA, MFe3O4@GA, and MFe3O4@GA-PEI-AS1411 at concentrations of 0.1 mg / mL, with a PBS blank control group. The relative cell viability of each group was assessed using the CCK-8 assay.

[0047] Cell survival rate, such as Figure 3 As shown, the tumor cell killing ability of different materials was verified. After treatment of 4T1 cells with MFe3O4 and GA alone, the average cell viability was 73.7% and 65.2%, respectively, indicating that single treatment with MFe3O4 and GA had an inhibitory effect on the proliferation of 4T1 cells. Notably, compared with the PBS group, MFe3O4@GA and MFe3O4@GA-PEI-AS141 significantly inhibited the activity of 4T1 cells, and the cell viability was significantly reduced to 44.1% and 17.1%, respectively.

[0048] Intracellular reactive oxygen species assay: 4T1 cells were transplanted into 24-well plates, with 1×10⁶ cells per well. 5 Cells were collected. After 24 hours, different material treatment groups (provided in Example 1) were set up, including PEI, MFe3O4, GA, and MFe3O4@GA-PEI-AS1411 at a concentration of 0.1 mg / mL, and a PBS blank control group was set up. Intracellular reactive oxygen species production was assessed using DCFH-DA.

[0049] The results are as follows Figure 4 As shown, a large amount of ROS (green fluorescence) was generated after treatment with MFe3O4@Cy5.5-GA-PEI-AS1411.

[0050] Drug distribution assay in vivo: Balb / c mice were subcutaneously injected with 4×10⁴ ppm of the drug under the left axilla. 6 4T1 cells were used to construct a mouse model carrying a solid tumor. When the tumor volume reached 1000 mm², the tumor was further investigated. 3 Mice were treated by intravenous injection of Cy5.5-GA (Cyanine 5.5-Gambogicacid, Xi'an Ruixi Biotechnology Co., Ltd.), MFe3O4@Cy5.5-GA-PEI-AS1411 (MFGPA), and MFe3O4@Cy5.5-GA-PEI-AS1411+magnetic field (MFGPA+magnetic field), respectively, via the tail vein. MFGPA was provided in Example 1. In vivo fluorescence imaging was performed in mice using the IVIS Spectrum imaging system, with real-time tracking at 1, 3, 6, 12, 24, and 48 hours. The spatiotemporal distribution of the drugs was monitored using Cy5.5 fluorescence (excitation wavelength: 680 nm; emission wavelength: 710 nm).

[0051] The results are as follows Figure 5 As shown, compared to free Cy5.5-GA, MFGPA nanoparticles exhibit stronger fluorescence signals at the tumor site due to the passive targeting of the nanoparticles and the active targeting of nucleolin on the surface of tumor cells by AS1411. After applying an external magnetic field for 1 hour, the fluorescence signal at the tumor site was significantly enhanced.

[0052] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A nano-drug delivery system of magnetic-aptamer targeting tumor tissue, characterized in that, It comprises: a magnetic core formed by mesoporous ferroferric oxide; a polyethyleneimine layer coated outside the magnetic core; aptamers adsorbed outside the polyethyleneimine layer and capable of specifically binding to tumor cells; and, optionally, an autophagy activator loaded in the mesopores of the magnetic core. 2.The magnetic-aptamer targeted tumor tissue nano-drug delivery system according to claim 1, characterized in that, The autophagy activator is selected from any one of gambogic acid, rapamycin and cisplatin. 3.The magnetic-aptamer targeted tumor tissue nano-drug delivery system of claim 1, wherein, The aptamer is AS1411.

4. A method for preparing the magnetic-aptamer targeted tumor tissue nano-drug delivery system according to any one of claims 1-3, characterized in that, It comprises: superparamagnetic mesoporous ferroferric oxide nanoparticles are prepared as a magnetic core; autophagy activators are co-incubated with the magnetic core to obtain nanoparticles loaded with autophagy activators; nanoparticles loaded with the autophagy activators are mixed with a polyethyleneimine solution to coat the nanoparticles with polyethyleneimine on the surface; and, the PEI-coated nanoparticles and aptamers are co-incubated to obtain a drug delivery system.

5. The method for preparing the magnetic-aptamer-targeted tumor tissue nanomedicine delivery system according to claim 4, characterized in that, The mass ratio of the autophagy activator to the mesoporous ferroferric oxide nanoparticles in the co-incubation is 0.8-1:1, and the incubation time is 4-8h.

6. The method for preparing the magnetic-aptamer-targeted tumor tissue nanomedicine delivery system according to claim 4, characterized in that, The mass ratio of the nanoparticles loaded with the autophagy activators to the polyethyleneimine is 1:50-100, and the coating reaction is carried out under stirring for 1-3h.

7. The method for preparing the magnetic-aptamer-targeted tumor tissue nanomedicine delivery system according to claim 4, characterized in that, The mass ratio of the PEI-coated nanoparticles to the aptamers is 1:120-200, and the incubation is carried out at 2-8℃ for 6-10h.

8. The method for preparing the magnetic-aptamer-targeted tumor tissue nanomedicine delivery system according to claim 4, characterized in that, The particle size of the mesoporous ferroferric oxide nanoparticles is 30-80nm, and the charge is -15--13mV.

9. The method for preparing the magnetic-aptamer-targeted tumor tissue nanomedicine delivery system according to claim 8, characterized in that, The mesoporous ferroferric oxide nanoparticles are prepared by a solvothermal method, the reaction temperature is 120-200℃, and the reaction time is 12-20h.

10. Use of the magnetic-aptamer targeted tumor tissue nanodrug delivery system of any one of claims 1-3 in the preparation of a medicament for treating tumors.

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