CD24-targeted nanodrug delivery system and its application in the preparation of antitumor drugs

Abstract

This invention provides a CD24-targeted nanomedicine delivery system and its application in the preparation of antitumor drugs. The nanomedicine delivery system of this invention comprises (1) a core structure: mesoporous manganese dioxide (hMnO₂) for co-loading glucose oxidase (GOx) and cysteine ​​(Cys); and (2) targeted delivery: nanoparticles encapsulated in a genetically engineered membrane EM-CD24 modified with the anti-CD24 single-chain variable fragment scFv. This invention can improve tumor penetration, reduce off-target accumulation, and enhance pharmacokinetics, achieving therapeutic Gox and cysteine ​​concentrations in the tumor while maintaining low systemic exposure, thus minimizing off-target toxicity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a CD24-targeting nanodrug delivery system and its application in the preparation of antitumor drugs. Background Technology

[0002] Neuroblastoma (NB) is the most common extracranial solid tumor in children. Surgery combined with radiotherapy and chemotherapy is the main treatment for neuroblastoma. However, chemotherapy often leads to bone marrow suppression, liver and kidney damage, and high-risk patients are not sensitive to chemotherapy, with a 5-year survival rate of less than 50%. Therefore, there is an urgent need to find new treatment methods. To overcome systemic toxicity, targeted drug delivery systems can be used to improve drug efficacy and reduce drug side effects. Exosomes (Exos) are naturally secreted extracellular vesicles, small in size (30-150 nm), with functions of intercellular communication, biocompatibility, and macromolecular delivery. Due to their inherent homing ability, exosomes can accumulate and target their tissues or locations of origin. Exosomes can evade phagocytosis through CD47 signaling, thus achieving long-term circulation in the body. However, natural exosomes still lack active targeting, and active targeting through receptor binding has become a hot topic in recent years. Among these, antibody-antigen binding is the most specific.

[0003] Studies have shown that CD24 is a highly variable glycosylated protein, highly expressed in various tumors and cancer stem cells, and has been shown to be highly expressed in neonatocytes (NBs) and associated with poor clinical prognosis. The inventors transfected an anti-CD24 nanobody encoding vector into HEK-293T (293T) cells and fused it with a decay-accelerating factor (DAF)-derived glycosylphosphatidylinositol (GPI) anchoring signal peptide to achieve nanobody expression on the cell membrane. Subsequently, nanoscale EMs expressing anti-CD24 nanobodies were successfully obtained through sequential extrusion, and their targeting ability was verified in vivo and in vitro.

[0004] However, because neuroblastoma is insensitive to chemotherapy, the developed liposomal doxorubicin, although capable of targeting the tumor site, still has unsatisfactory efficacy and is rarely used clinically. Engineered exosomes carrying doxorubicin, developed earlier by the inventors, have shown limited efficacy. This means that not only are new delivery systems needed, but also new therapeutic drugs and treatment modalities.

[0005] Disulfide death is a promising avenue for precision oncology. This unique cell death pattern is caused by cystine metabolism disorder. Tumor cells frequently overexpress the cystine / glutamate antitransporter SLC7A11, promoting excessive cystine uptake to support glutathione (GSH) biosynthesis, a key antioxidant defense mechanism and a contributing factor to chemotherapy resistance. However, this process consumes significant amounts of NADPH to convert ingested cystine into cysteine. NADPH is primarily produced via the pentose phosphate pathway (PPP), which depends on glucose metabolism. When glucose deficiency leads to NADPH insufficiency, cystine reduction is impaired, causing it to accumulate and form abnormal disulfide bonds between cytoskeletal proteins, ultimately leading to the collapse of the f-actin network and cell death. This mechanism re-emphasizes the therapeutic value of cystine. The inventors unexpectedly discovered that cystine exhibits metabolic bimorphism: under normal conditions, cystine promotes cell growth, while in glucose-deficient conditions, cystine significantly promotes disulfide death. This characteristic highlights the safety profile of cystine, allowing for a dual guarantee of efficacy and safety when combined with targeted delivery strategies. Summary of the Invention

[0006] To address the problems in the prior art, this invention provides a CD24-targeted nanodrug delivery system, its preparation method, and its application.

[0007] The present invention adopts the following technical solution:

[0008] A nanomedicine delivery system targeting CD24, characterized in that nanoparticles Cys-hMnO2@GOx are encapsulated in a genetically engineered membrane EM-CD24 nanoexosome modified with an anti-CD24 single-stranded variable fragment (scFv).

[0009] According to one embodiment of the present invention, the method for constructing the nanomedicine delivery system is as follows: Cys-hMnO2@GOx is dispersed in deionized water and mixed with CD24 engineered exosomes EM-CD24. The mixture is extruded through a polycarbonate membrane 18-22 times, and then extruded through a liposome extruder 8-12 times to obtain the nanomedicine delivery system Cys-hMnO2@GOx@EM-CD24.

[0010] According to one embodiment of the present invention, the method for constructing the genetically engineered membrane EM-CD24 nanoexosomes modified with anti-CD24 single-stranded variable fragment (scFv) is as follows: HEK-293T cells are transduced with a lentiviral vector encoding a glycosylphosphatidylinositol (GPI)-anchored anti-CD24 single-stranded variable fragment (anti-CD24 scFv), and specific primers are designed (Forward: 5′-CTGAGCCTGAGCGTGACCATTG-3′; Reverse: 5′-GGTAGGTCTTTCCATCGCTGTGC-3′). RT-PCR analysis confirms that the construct successfully expresses anti-CD24 scFv in HEK293T cells. Then, HEK293T-CD24 cells are extruded using a liposome extruder to generate EM-CD24 nanoexosomes.

[0011] According to one embodiment of the present invention, the method for constructing Cys-hMnO2@GOx is as follows: Cys-hMnO2-PEI dispersion and glucose oxidase (GOx) are incubated with shaking at 400-600 rpm for 50-70 minutes, and then purified by centrifugation and washing to obtain Cys-hMnO2@GOx.

[0012] According to one embodiment of the present invention, the method for constructing the Cys-hMnO2-PEI dispersion is as follows: wash Cys-hMnO2 with deionized water until the pH value of the supernatant reaches 6.8-7.2, resuspend the precipitate in deionized water, add polyethyleneimine, incubate at 400-600 rpm for 1-3 hours under magnetic stirring, then centrifuge to obtain nanoparticles, wash with deionized water, and resuspend in deionized water to obtain the Cys-hMnO2-PEI dispersion.

[0013] According to one embodiment of the present invention, the method for constructing Cys-hMnO2 is as follows: hexadecyltrimethylammonium bromide (CTAB) and L-cysteine ​​are dissolved in deionized water, and the pH value is adjusted to 9-10. Then, triethylamine is added and the mixture is magnetically stirred at 90-100°C. Tetraethyl orthosilicate (TEOS) is then added, and the mixture is stirred for 3-5 hours. The solid is collected by centrifugation to obtain Cys-hSiO2 nanoparticles. After purification, the nanoparticles are dissolved in deionized water, potassium permanganate (KMnO4) is added, and the mixture is ultrasonically treated. The mixture is then continuously magnetically stirred at 1000-1500 rpm for 5-7 hours. The product is refluxed with sodium carbonate solution for 2-4 hours, and then refluxed with sodium bicarbonate solution for 9-11 hours to obtain Cys-hMnO2 hollow manganese dioxide nanostructures loaded with cysteine.

[0014] The aforementioned CD24-targeted nanodrug delivery system Cys-hMnO2@GOx@EM-CD24 is characterized by comprising the following steps:

[0015] (1) The construction method of Cys-hMnO2 is as follows:

[0016] Hexadecyltrimethylammonium bromide (CTAB) and L-cysteine ​​were dissolved in deionized water, and the pH was adjusted to 9-10. Triethylamine was then added, and the mixture was magnetically stirred at 90-100°C for 20-40 minutes. Tetraethyl orthosilicate (TEOS) was then added while continuously stirring, and the mixture was stirred for another 3-5 hours. The solid was collected by centrifugation to obtain Cys-hSiO2 nanoparticles, which were washed three times with ethanol, followed by washing with deionized water. The purified Cys-hSiO2 nanoparticles were then resuspended and dissolved in deionized water. Potassium permanganate (KMnO4) was then added, and the mixture was sonicated (40 kHz, 120 °C). W) 0.5-1.5 hours, followed by continuous magnetic stirring at 1000-1500 rpm for 5-7 hours, the product was refluxed in sodium carbonate solution at 50-70°C for 2-4 hours, followed by a second reflux treatment in sodium bicarbonate solution for 9-11 hours to etch the silica template, to obtain cystine-loaded hollow manganese dioxide nanostructure Cys-hMnO2;

[0017] (2) The method for constructing the Cys-hMnO2-PEI dispersion is as follows;

[0018] Cys-hMnO2 was washed with deionized water until the pH of the supernatant reached 6.8-7.2. The precipitate was resuspended in deionized water, polyethyleneimine was added, and the mixture was incubated at 400-600 rpm for 1-3 hours with magnetic stirring. The nanoparticles were then obtained by centrifugation, washed with deionized water, and resuspended in deionized water to obtain a PEI-functionalized Cys-hMnO2 (Cys-hMnO2-PEI) dispersion.

[0019] (3) The construction method of Cys-hMnO2@GOx is as follows:

[0020] Cys-hMnO2-PEI dispersion was incubated with glucose oxidase (GOx) at 400-600 rpm for 50-70 minutes, and then purified by centrifugation and washing to obtain Cys-hMnO2@GOx;

[0021] (4) The method for constructing the genetically engineered membrane EM-CD24 nanoexosomes modified with anti-CD24 single-stranded variable fragment (scFv) is as follows: HEK-293T cells are transduced with an anti-CD24 single-stranded variable fragment (anti-CD24 scFv) encoded by a lentiviral vector, specific primers are designed, and RT-PCR analysis confirms that the construct successfully expresses HEK293T-CD24 in HEK293T cells. Then, HEK293T-CD24 cells are squeezed with a liposome extruder to generate EM-CD24 nanoexosomes.

[0022] (5) The construction method of Cys-hMnO2@GOx@EM-CD24 is as follows:

[0023] Cys-hMnO2@GOx was dispersed in deionized water and mixed with EM-CD24 nano-exosomes. The mixture was extruded through a polycarbonate membrane 18-22 times, and then extruded through a liposome extruder 8-12 times to obtain Cys-hMnO2@GOx@EM-CD24.

[0024] According to one embodiment of the present invention, the polycarbonate membrane has a pore size of 400 nm; the liposome extruder has a pore size of 200 nm.

[0025] According to one embodiment of the present invention, the application of the CD24-targeted nanodrug delivery system Cys-hMnO2@GOx@EM-CD24 in the preparation of antitumor drugs.

[0026] According to one embodiment of the present invention, the application of the CD24-targeted nanodrug delivery system Cys-hMnO2@GOx@EM-CD24 in the preparation of drugs for the prevention or treatment of neuroblastoma.

[0027] Beneficial Effects: This invention provides a CD24-targeted nanodrug delivery system and its application in the preparation of antitumor drugs. A multifunctional cascaded nanoplatform (Cys-hMnO2@GOx@EM-CD24) is designed, comprising (1) a core structure: mesoporous manganese dioxide (hMnO2) for co-loading GOx and cystine. GOx consumes glucose to produce hydrogen peroxide, and its inherent catalase-mimicking activity decomposes H2O2 into O2, alleviating local hypoxia and maintaining GOx activity. Exogenous cystine delivery bypasses the expression limit of SLC7A11 and directly induces disulfide death; (2) targeted delivery: nanoparticles are encapsulated in a genetically engineered membrane modified with anti-CD24 single-chain variable fragment (scFv). Compared with traditional monoclonal antibodies, scFv, due to its smaller size and flexible linker design, can improve tumor penetration, reduce off-target accumulation, and enhance pharmacokinetics. This achieves therapeutic cystine concentrations in tumors while maintaining low systemic exposure, minimizing off-target toxicity. Attached Figure Description

[0028] Figure 1 This is a diagram showing the high expression of CD24 in neuroblastoma cells;

[0029] Figure 2 This is a production process and results diagram of an engineered exosome mimic targeting CD24 (EM-CD24);

[0030] Figure 3 The figure shows the synthesis and characterization results of Cys-hMnO2@GOx@EM-CD24;

[0031] Figure 4 This is a graph showing the functional identification results of Cys-hMnO2@GOx@EM-CD24;

[0032] Figure 5 This is a diagram showing the results of Cys-hMnO2@GOx@EM-CD24 inducing neuroblastoma cell death in vitro;

[0033] Figure 6 This is a graph showing the identification results of Cys-hMnO2@GOx@EM-CD24 induced disulfide death in vitro;

[0034] Figure 7 This is a diagram showing the results of Cys-hMnO2@GOx@EM-CD24 targeting orthotopic tumors in mice;

[0035] Figure 8 This is a flowchart and protocol design for Cys-hMnO2@GOx@EM-CD24 treatment of neuroblastoma;

[0036] Figure 9This is a graph showing the efficacy evaluation results of Cys-hMnO2@GOx@EM-CD24 in the treatment of neuroblastoma;

[0037] Figure 10 This is a diagram showing the mechanistic results of Cys-hMnO2@GOx@EM-CD24 in an orthotopic neuroblastoma model. Detailed Implementation

[0038] To further illustrate the present invention and its advantages, the technical solution of the present invention will be further described below through specific embodiments. It should be understood that these embodiments are only helpful in understanding the present invention and should not be regarded as specific limitations of the present invention.

[0039] Unless otherwise specified, all parts mentioned in this invention are parts by weight, and all percentages are mass percentages.

[0040] Example 1

[0041] Engineering and functional validation of CD24-targeted nanoexosomes

[0042] CD24 has become a key target in cancer therapy. The inventors' previously designed CD24-targeted antibody-drug conjugates (ADCs) exhibited good tumor selectivity, but the limited payload capacity of monoclonal antibodies restricted their therapeutic versatility. To enable precise co-delivery of multiple disulfide death inducers, the inventors developed extracellular exosomes functionalized with an anti-CD24 single-chain variable fragment (anti-CD24 scFv).

[0043] To confirm the targetability of CD24 in neuroblastoma, single-cell RNA sequencing data from 16 primary neuroblastoma cases (GSE137804) were rigorously analyzed. After strict quality control, 171,636 cells were subjected to UMAP-based clustering and marker-guided annotation, identifying eight distinct subpopulations, predominantly of neurogenic origin. Figure 1 A). Analysis showed that CD24 expression was significantly increased in tumor cells compared to stromal / immune subsets. Figure 1 B). Immunohistochemical analysis of 32 clinical specimens confirmed that 87.5% (28 / 32) of the cases were positive for CD24 in the membrane / cytoplasm, indicating broad clinical applicability. Figure 1 C and D). Parallel validation in the neuroblastoma cell line (SK-N-DZ / SH-SY5Y) showed that CD24 is mainly expressed at membrane localization (C and D). Figure 1 E and Figure 1 F).

[0044] CD24 is highly expressed in neuroblastoma cells. The results are as follows: Figure 1 As shown; (A) Single-cell RNA sequencing analysis of neuroblastoma (GSE137804 dataset) and visualization of cell clusters using UMAP; (B) Expression of CD24 in different cell clusters; (C, D) Immunohistochemical verification of CD24 expression in clinical neuroblastoma specimens; (EF) Western blot (E) and immunofluorescence (F) confirming CD24 expression in neuroblastoma cell lines.

[0045] HEK293T-CD24 cells were generated by lentiviral transduction of 293T cells, whose structure encodes the anti-CD24 antibody display sequence. This sequence was cloned into a vector co-expressing Myc and HA tags fused with a DAF peptide (HA-scFv(CD24)-Myc-GPI), thereby achieving membrane anchoring and subsequent detection. Figure 2 A and Figure 2B). RT-PCR using specific primers designed for the CD24 scFv sequence confirmed successful expression in engineered HEK-293T cells (HEK293T-CD24). Figure 2 C). Western blot analysis further validated the expression of the HA-tagged fusion protein, confirming effective transduction (C). Figure 2 D). HEK293T-CD24 cells were then extruded using a liposome extruder to generate engineered exosomes (EM-CD24). Western blot analysis confirmed that EM-CD24 carried classic exosome markers such as CD63, TSG101, and Alix, consistent with the proteomic characteristics of native exosomes. Figure 2 E). Importantly, the presence of the HA marker protein in EM-CD24 validates the successful inheritance of anti-CD24 scFv from donor cells. Figure 2 F). The average diameter of EM-CD24 exosomes was approximately 160 nm, slightly larger than that of natural exosomes (134 nm). Scanning electron microscopy further confirmed the structural integrity of the exosomes, showing the typical spherical, bilayer membrane morphology of the EM-CD24 formulation. Figure 2 G). Zeta potential measurements showed that both EM-CD24 and native exosomes possess negative surface charges, ranging from -10 mV to -15 mV. Figure 2 H). Functional validation demonstrated superior targeting specificity: when incubated with SK-N-DZ cell lysates, EM-CD24 exhibited more than 10-fold higher CD24 capture capacity than non-targeted exosomes, confirming precise antibody-mediated targeting. Figure 2 I and Figure 2 J).

[0046] Production of engineered exosome mimics targeting CD24 (EM-CD24) Figure 2As shown; (A) The nanobody-DAF sequence was cloned into the pLNCX vector under the CMV promoter, encoding Igκleader (LS), HA tag, nanobody, Myc tag, GGGGS 2 linker and C-terminal gpi anchored signal peptide (DAF); (B) After expression, the gpi anchored nanobody is expected to localize to the lipid raft of the cell membrane; (C) RT-PCR confirming the expression of scFv transcript; (D) Western blot detection of the expression of the marker protein (HA); EM-CD24 characteristics: Western blot detection of classic exosome markers (CD63 / TSG101 / Alix) (E) and HA (F); (G) Transmission electron microscopy (TEM) showing the well-preserved ultrastructure of EM-CD24; (H) Zeta potential analysis; SK-N-DZ cell lysate was incubated with EM-CD24, and the ability of EM-CD24 to bind CD24 protein was detected by Western blot (I), and the corresponding statistical bar graph (J). Example 2

[0047] Construction and characterization of Cys-hMnO2@GOx@EM-CD24

[0048] Synthesis of Cys-hMnO2@GOx@EM-CD24 nanocomposite materials

[0049] First, 0.4 g of cetyltrimethylammonium bromide (CTAB) and 1000 mg of L-cysteine ​​were dissolved in 10 mL of deionized water, and the pH was adjusted to 9.5. After adding 100 μL of triethylamine, the mixture was magnetically stirred at 95°C for 30 minutes. Then, 1.2 mL of tetraethyl orthosilicate (TEOS) was added dropwise with continuous stirring, and stirring was continued for 4 hours. After collecting the product by centrifugation, the Cys-hSiO2 nanoparticles were washed three times with ethanol, followed by washing with deionized water. Finally, the purified product was resuspended and brought to a final volume of 50 mL with deionized water. Then, 50 mg of potassium permanganate (KMnO4) was added to the suspension. The mixture was sonicated (40 kHz, 120 W) for 1 hour, followed by continuous magnetic stirring at 1200 rpm for 6 hours. Next, the product was refluxed at 60°C with 24 mL of 2 mol / L sodium carbonate solution for 3 hours, followed by a second reflux in 3% sodium bicarbonate solution for 10 hours to etch the silica template, yielding cystine-loaded hollow manganese dioxide nanostructures (Cys-hMnO2). The particles were washed with deionized water until the pH of the supernatant reached 7 (tested with pH paper). After the final wash, the precipitate was resuspended in deionized water and the volume was adjusted to 5 mL. This nanoparticle suspension was incubated with 5.0 mL of polyethyleneimine solution (PEI, molecular weight 10000, 1 mg / mL) under magnetic stirring (500 rpm) for 2 hours. The nanoparticles were purified by centrifugation and resuspended in deionized water, adjusted to a final volume of 10 mL, yielding PEI-functionalized Cys-hMnO2 (Cys-hMnO2-PEI). The dispersion was incubated with 20 mg of glucose oxidase (GOx) under shaking (500 rpm, 60 min), purified again by centrifugation, and brought to a final volume of 10 mL to obtain Cys-hMnO2@GOx. For EM or EM-CD24 coating: 0.3 mL of Cys-hMnO2@GOx was mixed with 0.3 mL of CD24-engineered exosomes (EM-CD24) or ordinary exosomes (EM) solution (4 mg / mL). The mixture was extruded 20 times through a polycarbonate membrane (400 nm pore size) and then 10 times through a liposome extruder (200 nm pore size). The final product was centrifuged and resuspended in 0.6 mL of phosphate-buffered saline (PBS). The control formulations (hMnO2@GOx@EM-CD24 without cystine, and Cys-hMnO2@EM-CD24 without GOx) were prepared using the same method, only omitting the corresponding components.

[0050] High-resolution transmission electron microscopy revealed that Cys-hMnO2-PEI nanoparticles possess a hollow structure with a crystalline core and a porous shell. After successive functionalization with GOx and EM, they transformed into a solid core-shell morphology, confirming the success of the surface modification. Figure 3 A). Dynamic light scattering analysis showed that the diameter of Cys-hMnO2@GOx@EM-CD24 was between 150 and 300 nm, slightly larger than that of drug-free EM-CD24. Figure 3 B). Zeta potential measurements showed that after the addition of EM-CD24, the particle surface carried a negative charge (-18.06 ± 2.18 mV). Figure 3 C). Energy-dispersive X-ray spectroscopy (EDS) elemental mapping confirmed a uniform distribution of Mn (14.27 wt%), O (19.12 wt%), C (31.11 wt%), N (1.5 wt%), and S (0.06 wt%). X-ray photoelectron spectroscopy (XPS) analysis showed that the surface composition of Cys-hMnO2-PEI nanoparticles consisted of Mn, O, C, N, Si, and S. High-resolution Mn 2p spectra (peaks at 642.5 eV and 654.1 eV) confirmed the uniform distribution of Mn. 4+ The dominant oxidation state is σ, while the 0.6 eV shift at Mn 2p indicates coordination of Mn-s with cysteine. The S2p spectrum shows 73.5% of the sulfhydryl groups are surface-bound, with the remainder forming disulfide bonds. Deconvolutional O1s spectroscopy identifies lattice oxygen, hydroxyl groups (34.8%), and Mn-OC bonds; the hydroxyl groups are linked to catalytic activity via oxygen vacancies. The N1s peak confirms the PEI coating through signals from primary, secondary, and protonated amines. High-performance liquid chromatography (HPLC) analysis shows the presence of cysteine ​​(…). Figure 3 D).

[0051] The synthesis and characterization results of Cys-hMnO2@GOx@EM-CD24 are as follows: Figure 3 As shown; (A) Transmission electron microscopy (TEM) images reveal a uniform spherical morphology and nanoscale structure; (B) Dynamic light scattering (DLS) analysis shows the hydrodynamic diameter of the nanosystem; (C) Zeta potential measurements confirm the surface charge properties; (D) X-ray photoelectron spectroscopy (XPS) provides atomic-level insights into the surface chemical state and functional bond configuration, supporting successful loading (Cys-hMnO2-PEI); (D) Representative images of cystine quantification using high-performance liquid chromatography (HPLC).

[0052] To further evaluate the functional performance of the nanocarrier, the inventors quantified its drug loading and release behavior. High-performance liquid chromatography (HPLC) was used to determine the content; Cys-hMnO2@EM-CD24 showed higher cysteine ​​loading, with an encapsulation efficiency of 82.73% and a drug loading of 63.73%, which was superior to Cys-hMnO2@GOx@EM-CD24 (77.85%, 56.58%) and Cys-hMnO2@GOx@EM (76.19%, 54.63%). Figure 4 A and Figure 4 B). Cystine release kinetics exhibited similar initial burst rates (0–10 min), followed by accelerated release (10–30 min), with GOx-modified anoparticles (Cys-hMnO2@GOx@EM, Cys-hMnO2@GOx@EM-CD24) showing 37–46% higher release rates than Cys-hMnO2@EM-CD24. The sustained release rates (0.5–24 h) of the GOx-modified systems reached 51.78% and 48.28%, respectively, which were 23.6–32.5% higher than the unmodified system (39.07%). GOx may promote release through enzymatic hydrogen peroxide generation or local pH regulation, disrupting the stability of the nanoparticle matrix and promoting cystine desorption. Figure 4 C). Dissolved oxygen assays showed that hMnO2@GOx@EM-CD24 rapidly released O2 (12.80 mg / L) at 0.5 h, but rapidly decayed at 6 h (7.13 mg / L). In contrast, Cys-hMnO2@GOx@EM-CD24 maintained stable O2 levels (≥9.5 mg / L over 6 h, decay rate <4.7%) through a confined catalytic microenvironment and dynamic regulation of cysteine-mediated active site exposure, effectively balancing H2O2 decomposition and O2 generation. Figure 4 D). This invention achieves sustained tumor microenvironment remodeling through spatiotemporal control of metabolic intervention. Cellular uptake experiments demonstrated the targeted drug delivery capability of the nanosystem. The system was tracked using dual fluorescent labeling: FITC-coupled glucose oxidase (GOx) was coated within the intermediate exosome layer, and a PKH26-labeled engineered membrane (EM) was anchored on the outer surface. After 6 hours of incubation, Cys-hMnO2@GOx@EM-CD24 and non-targeted Cys-hMnO2@GOx@EM exhibited effective cell internalization, with co-localization between FITC-GOx and PKH26 signals. The simultaneous presence of the outer EM and intermediate GOx layers within the cell indirectly confirmed the successful delivery of the innermost Cys-hMnO2 payload. Compared to non-targeted exosomes, Cys-hMnO2@GOx@EM-CD24 showed significantly higher cellular uptake due to antibody-antigen interaction-mediated endocytosis. Figure 4 E).

[0053] Functional identification of Cys-hMnO2@GOx@EM-CD24, such as Figure 4 As shown; (A and B) Quantitative determination of cystine loading and encapsulation efficiency by high performance liquid chromatography (HPLC); (C) In vitro cystine release kinetics; (D) Evaluation of oxygen production to confirm nitrogen dioxide-mediated H2O2 catalytic decomposition, verifying the ability of the nanoplatform to regulate hypoxia; (E) Fluorescence imaging showing effective uptake of Cys-hMnO2@GOx@EM-CD24 by neuroblastoma cells. Example 3

[0054] Cys-hMnO2@GOx@EM-CD24 induces disulfide death in neuroblastoma cells in vitro.

[0055] To evaluate the therapeutic potential of the delivery system of the present invention, the inventors first conducted a CCK-8 assay to determine the half-maximal inhibitory concentration (IC50) of the treatment group (Cys-hMnO2@GOx@EM-CD24). 50 For comparison, the IC50 of the pure GOx formulation (hMnO2@GOx@EM-CD24) was also measured. 50 .like Figure 5 As shown in Figure A, the IC of Cys-hMnO2@GOx@EM-CD24 50 The values ​​were significantly lower (SK-N-DZ: 9.93 μg / mL; SH-SY5Y: 7.71 μg / mL) than hMnO2@GOx@EM-CD24 (SK-N-DZ: 14.03 μg / mL; SH-SY5Y: 11.88 μg / mL), indicating that its cytotoxic effect was significantly enhanced.

[0056] To further compare the efficacy of each group, hMnO2@GOx@EM-CD24 was used as a positive control. Its 2-fold IC50... 50 The value was used as a fixed total dose for subsequent in vitro experiments. To systematically evaluate the effects of each component, the inventors established six experimental groups: PBS (Group 1), hMnO2@EM-CD24 (Group 2), Cys-hMnO2@EM-CD24 (Group 3), hMnO2@GOx@EM-CD24 (Group 4), Cys-hMnO2@GOx@EM-CD24 (Group 5), and Cys-hMnO2@GOx@EM (Group 6). Figure 5 B). hMnO2@EM-CD24 alone showed no cytotoxicity, confirming its biosafety as a nanocarrier material.

[0057] Interestingly, treatment with Cys-hMnO2@EM-CD24 slightly inhibited cell proliferation; however, exogenous cystine alone may not be sufficient to induce disulfide death. Therefore, co-administration with an NADPH depletion strategy is necessary to achieve the desired effect. hMnO2@GOx@EM-CD24 exhibited moderate cytotoxicity. Notably, at the same dose, Cys-hMnO2@GOx@EM-CD24 showed the most pronounced cytotoxicity. The inventors also observed that nanomaterials encapsulated in unmodified EMs were less effective at inhibiting tumor growth than nanomaterials delivered via anti-CD24 modified EMs, highlighting the need for targeted surface modification to enhance therapeutic efficacy. Figure 5 C).

[0058] The degree of NADPH depletion was proportional to the observed level of cell death, providing preliminary evidence to support the occurrence of disulfide death. Figure 5 D). Non-reducing Western blot analysis confirmed the presence of disulfide-specific biomarkers, including high molecular weight aggregates of MYH9, Drebrin, and FLNA. Figure 6 A), thus validating the cell death pattern. Furthermore, phalloidin staining revealed extensive cytoskeleton disintegration (A). Figure 6 B). Interestingly, GOx-mediated glucose deprivation induces compensatory upregulation of SLC7A11 expression ( Figure 6 C and Figure 6 (D) This is a well-established resistance mechanism in the context of apoptosis. However, in this invention, this adaptive response paradoxically enhances the therapeutic effect: under NADPH depletion conditions, the upregulation of SLC7A11 promotes cystine input, further exacerbating disulfide accumulation, ultimately leading to cytoskeleton collapse and cell death.

[0059] Results of Cys-hMnO2@GOx@EM-CD24 inducing neuroblastoma cell death in vitro: Figure 5 As shown, (A) uses the CCK-8 method to determine the IC of nanomaterials. 50 (B) Six groups were established to evaluate the efficacy of combined therapy and targeted modification strategies; (C) Cell viability assays showed that neuroblastoma cell proliferation was significantly reduced after treatment with the nanoplatform; (D) Intracellular NADPH levels were significantly reduced after treatment.

[0060] Identification of in vitro induced disulfide death by Cys-hMnO2@GOx@EM-CD24 as follows: Figure 6As shown; (A) Non-reducing Western blot analysis showed disulfide-linked aggregation of cytoskeletal proteins FLNA, Drebrin, and MYH9; (B) f-actin immunofluorescence staining showed severe cytoskeletal disorder; (C and D) Western blot analysis showed upregulated expression of SLC7A11. Example 4

[0061] In vivo targeting effects of Cys-hMnO2@GOx@EM-CD24 nanoexosomes

[0062] Animal research

[0063] Male NOD / SCID mice (6 weeks old) were placed in a specific pathogen-free facility under controlled conditions. In an orthotopic neuroblastoma model, SK-N-DZ cells transfected with luciferase (2 * 10T) were used. 6 SK-N-DZ cells (50 μL culture medium) were surgically implanted into the left adrenal gland. Tumor implantation was confirmed by bioluminescence imaging (BLI) on day 10. Mice were randomly assigned to 6 groups: PBS (carrier control, group 1), hMnO2@EM-CD24 (vacuolated, group 2), Cys-hMnO2@EM-CD24 (cystine monotherapy, group 3), hMnO2@GOx@EM-CD24 (glucose oxidase monotherapy, group 4), Cys-hMnO2@GOx@EM-CD24 (combination therapy, group 5), and Cys-hMnO2@GOx@EM (non-targeted control, group 6). SK-N-DZ cells (1*10 cells, 50 μL culture medium) were implanted into the left adrenal gland. Tumor implantation was confirmed by bioluminescence imaging (BLI) on day 10. Mice were randomly assigned to 6 groups: PBS (carrier control, group 1), hMnO2@EM-CD24 (vacuolated, group 2), Cys-hMnO2@GOx@EM-CD24 (combination therapy, group 5), and Cys-hMnO2@GOx@EM (non-targeted control, group 6). SK-N-DZ cells were administered via tail vein every 48 hours (40 mg / kg) until the end of the study. Tumor progression was monitored using an in vivo imaging system, and mouse body weight was recorded. In a metastatic lung model, SK-N-DZ cells (1*10 cells, 50 μL culture medium) were implanted into the left adrenal gland. 6 100 μL of PBS (100 cells) was injected into the tail vein of NOD / SCID mice, and lung metastases were confirmed by BLI on day 14 before the start of treatment. Subsequent drug administration and continuous monitoring of changes in luminescence intensity were performed. At the study endpoint, mice were euthanized by CO2 asphyxiation. Tumors and major organs (heart, liver, spleen, kidney, lung) were harvested, weighed, and fixed in 10% neutral buffered formalin for histopathological analysis. Serum biochemical parameters (ALT, AST, creatinine, CK-MB, urea) were analyzed by the Department of Laboratory Medicine, Chongqing Children's Hospital. Paraffin-embedded tissue sections were stained with hematoxylin and eosin (H&E) to assess tumor morphology. Similarly, another group of mice was modeled and intervened, but not sacrificed, and their survival was observed.

[0064] To assess the accumulation of tumor-specific nanoexosomes, the inventors orally implanted SK-N-DZ cells expressing luciferase to establish neuroblastoma xenografts closely associated with the native tumor microenvironment. Bioluminescence imaging confirmed successful tumor establishment on day 10 post-implantation. The targeting capabilities of the Cys-hMnO2@GOx@EM-CD24 group (dEM-CD24) and free DiR were evaluated by comparison with unmodified Cys-hMnO2@GOx@EM, dEM. Figure 7 A) Mice were intravenously injected with DiR-labeled dEM-CD24, dEM, or free DiR. Whole-body fluorescence imaging showed progressive accumulation in the liver of all three formulations between 6 and 24 hours post-injection, with the strongest signal in the liver and spleen. Fluorescence intensity began to decline after 24 hours. Although the fluorescence intensity of dEM-CD24 and dEM in the liver and spleen was comparable, the in vivo retention time of dEM-CD24 was significantly prolonged compared to free DiR.

[0065] The inventors then focused on fluorescence signals within the tumor region. However, due to the anatomical overlap between the in situ tumor and major visceral organs (liver / spleen), accurately quantifying tumor-specific in vivo uptake is challenging. For example, some researchers have mistakenly interpreted fluorescence observed in the dorsal region of the liver and spleen in mice as tumor-specific (or kidney) accumulation. However, this interpretation is inaccurate based on fluorescence intensity and anatomical location. Therefore, ex vivo fluorescence imaging of the resected organs was performed 24 hours after injection. The nanoparticles primarily accumulated in the liver and spleen, exhibiting a weak but identifiable signal in the tumor. Figure 7 B. Figure 7 C). Notably, tumor fluorescence was stronger than that of the kidney, lung, and heart. This was after tumor volume correction based on luciferase (LUC) bioluminescence. Figure 7 Quantitative comparison showed that, compared with the dEM treatment group, the fluorescence intensity of the in situ tumor in the dEM-CD24 treatment group increased by more than two times (D). Figure 7 E), confirming its tumor-selective delivery capability.

[0066] Results of Cys-hMnO2@GOx@EM-CD24 targeting mouse orthotopic tumors as follows: Figure 7 As shown, (A) targeting specificity was evaluated using DiR-labeled Cys-hMnO2@GOx@EM-CD24 (dEM-CD24), Cys-hMnO2@GOx@EM (dEM), and free DiR; biodistribution of DiR-labeled dEM-CD24, dEM, and free DiR in mice; (B, C) fluorescence distribution and intensity quantification of isolated organs (24h); (D) bioluminescence of luciferase (LUC) in an orthotopic tumor model; and (E) fluorescence distribution and intensity analysis of orthotopic tumors.

[0067] Cys-hMnO2@GOx@EM-CD24 inhibits tumor growth by inducing disulfide death.

[0068] To evaluate the anti-tumor efficacy of nanomedicines, the inventors established a mouse model of tumors, such as... Figure 8 As shown in Figure A, patients were treated accordingly. Tumor progression was monitored using in vivo bioluminescence imaging, and body weight was recorded to assess systemic toxicity. No significant weight loss was observed in either treatment group, indicating good biocompatibility and low systemic toxicity. Figure 8 B). Bioluminescence imaging showed continued tumor growth in both the PBS control group and the hMnO2@EM-CD24 treatment group, reflecting the natural progression of the tumor. Cystine alone had no inhibitory effect on tumor growth and no promoting effect on tumor development. In contrast, glucose oxidase (GOx) monotherapy (hMnO2@GOx@EM-CD24, group 4) showed moderate antitumor activity, which was significantly enhanced when co-administered with cystine, confirming their synergistic therapeutic effect. Notably, CD24-targeted exosomes showed better tumor suppression compared to non-targeted agents, which can be attributed to enhanced tumor accumulation mediated by CD24 antibody-directed targeting (…). Figure 8 C and Figure 8 D). In vitro tumor measurements visually confirmed the significant tumor regression in the combined treatment group ( Figure 9 A) Tumor weight data provided consistent evidence of efficacy. Figure 9 B). Hematoxylin and eosin (H&E) staining confirmed the successful establishment of the in situ tumor model and the therapeutic effect, with clear boundaries between the tumor and adjacent kidney tissue. After treatment, another group of tumor-bearing mice were allowed to grow naturally to assess long-term survival. Mice treated with Cys-hMnO2@GOx@EM-CD24 had the longest survival time, while all mice in the PBS, hMnO2@EM-CD24, and Cys-hMnO2@EM-CD24 groups died within 60 days. Postmortem analysis showed that death was mainly due to tumor invasion outside the renal capsule, leading to extensive peritoneal metastasis and severe deterioration of nutritional status.

[0069] The procedure and protocol for Cys-hMnO2@GOx@EM-CD24 treatment of neuroblastoma are as follows: Figure 8 As shown, (A) a schematic diagram of the in situ tumor model establishment process and treatment intervention plan; (B) longitudinal monitoring of weight changes during treatment; (C) real-time tumor progression tracking using luciferase (LUC) bioluminescence imaging; and (D) dynamic quantitative analysis of tumor-related bioluminescence intensity.

[0070] Evaluation of the efficacy of Cys-hMnO2@GOx@EM-CD24 in the treatment of neuroblastoma, as follows: Figure 9As shown, (A) in situ tumor weight quantification; (B) gross morphological evaluation after in situ tumor resection; (C, D) Kaplan-Meier survival curves across treatment cohorts; (E) histopathological evaluation of tumor tissue by hematoxylin and eosin (H&E) staining.

[0071] To investigate whether disulfide death occurs in vivo, the inventors performed non-reducing Western blot analysis. The results showed that Cys-hMnO2@GOx@EM-CD24-induced disulfide death was the most prominent, consistent with the observed tumor growth inhibition, thus supporting the conclusion that disulfide death mediates antitumor activity. Figure 10 A and Figure 10 B). To further assess biocompatibility, the inventors measured serum biochemical parameters, creatine kinase (CK), urea, aspartate aminotransferase (AST), and alanine aminotransferase (ALT), all of which remained within the normal physiological range. Figure 10 C). Histological analysis of the major organs revealed no significant pathological changes or tissue damage. Figure 10 D). In summary, these findings confirm the low toxicity and good biocompatibility of the nanoplatform, highlighting its great potential for clinical translation.

[0072] Mechanistic evaluation results of Cys-hMnO2@GOx@EM-CD24 in an orthotopic neuroblastoma model are as follows: Figure 10 As shown, (A and B) non-reducing Western blot analysis confirmed successful induction of disulfide death after treatment; (C) blood biochemical parameters showed no signs of systemic toxicity associated with the nanosystem; and (D) H&E staining of major organs showed no significant histological damage among the treatment groups, supporting the safety of Cys-hMnO2@GOx@EM-CD24 in vivo.

Claims

1. A nanomedicine delivery system targeting CD24, characterized in that, The nanoparticles Cys-hMnO2@GOx were encapsulated in a genetically engineered membrane EM-CD24 nanoexosome modified with the CD24 single-stranded variable fragment scFv. The construction method was as follows: Cys-hMnO2@GOx was dispersed in deionized water and mixed with CD24-engineered exosomes EM-CD24. The mixture was extruded through a polycarbonate membrane 18-22 times, followed by extrusion through a liposome extruder 8-12 times to obtain the nanodrug delivery system Cys-hMnO2@GOx@EM-CD24. The method for constructing the genetically engineered membrane EM-CD24 nanoexosomes modified with the anti-CD24 single-stranded variable fragment scFv is as follows: HEK-293T cells are transduced with an anti-CD24 single-stranded variable fragment anti-CD24 scFv encoded by a lentiviral vector. Specific primers are designed, and RT-PCR analysis confirms that the construct successfully expresses HEK293T-CD24 in HEK293T cells. Then, HEK293T-CD24 cells are extruded using a liposome extruder to generate EM-CD24 nanoexosomes. The method for constructing Cys-hMnO2@GOx is as follows: Cys-hMnO2-PEI dispersion was incubated with glucose oxidase GOx at 400-600 rpm for 50-70 minutes, and then purified by centrifugation and washing to obtain Cys-hMnO2@GOx; The method for constructing the Cys-hMnO2-PEI dispersion is as follows; Wash Cys-hMnO2 with deionized water until the pH of the supernatant reaches 6.8-7.

2. Resuspend the precipitate in deionized water, add polyethyleneimine, and incubate at 400-600 rpm for 1-3 hours with magnetic stirring. Then centrifuge to obtain nanoparticles, wash with deionized water, and resuspend in deionized water to obtain Cys-hMnO2-PEI dispersion. The specific primers are: Forward: 5′-CTGAGCCTGAGCGTGACCATTG-3′; Reverse: 5′-GGTAGGTCTTTCCATCGCTGTGC-3′; The method for constructing Cys-hMnO2 is as follows: Hexadecyltrimethylammonium bromide (CTAB) and L-cysteine ​​were dissolved in deionized water, and the pH was adjusted to 9-10. Triethylamine was then added, and the mixture was magnetically stirred at 90-100°C for 20-40 minutes. Tetraethyl orthosilicate (TEOS) was then added under continuous stirring, and stirring was continued for 3-5 hours. The solid was collected by centrifugation to obtain Cys-hSiO2 nanoparticles, which were washed three times with ethanol, followed by washing with deionized water. The purified Cys-hSiO2 nanoparticles were then resuspended and dissolved in deionized water. Potassium permanganate (KMnO4) was added, and the mixture was sonicated for 0.5-1.5 hours. The mixture was then magnetically stirred continuously at 1000-1500 rpm for 5-7 hours. The product was refluxed in sodium carbonate solution at 50-70°C for 2-4 hours, followed by a second reflux in sodium bicarbonate solution for 9-11 hours to etch a silica template, thus obtaining Cys-hMnO2 hollow manganese dioxide nanostructures loaded with cysteine.

2. The CD24-targeting nanodrug delivery system as described in claim 1, characterized in that, The polycarbonate membrane has a pore size of 400 nm.

3. The CD24-targeting nanodrug delivery system according to any one of claims 1-2, characterized in that, The liposome extruder has an pore size of 200 nm.

4. The application of the CD24-targeting nanodrug delivery system according to any one of claims 1-3 in the preparation of an antitumor drug, wherein the tumor is neuroblastoma.

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