Core-shell sequential drug delivery system for cascaded deep tumor penetration and differentiation induction therapy, its construction method and application

Abstract

This invention belongs to the field of novel excipients and dosage forms for pharmaceutical preparations, specifically relating to a core-shell structured sequential drug delivery system for cascaded deep penetration and differentiation-inducing therapy, its construction, and its application in drug delivery systems. The system has a core-shell structure, with the core consisting of hypoxia-responsive nanoparticles encapsulating differentiation-inducing agents, and the shell being an acid-responsive reversible polymer loaded with chemotherapeutic drugs. This invention overcomes the penetration barrier of solid tumors using a pulsed drug delivery strategy and performs programmed drug delivery targeting the heterogeneous distribution of different cells within the tumor, effectively reshaping the tumor microenvironment characterized by chemotherapy resistance and immune escape, thus achieving comprehensive tumor inhibition.

Description

Technical Field

[0001] This invention belongs to the field of new excipients and dosage forms for pharmaceutical preparations, specifically relating to a core-shell structured sequential drug delivery system for cascaded deep penetration and differentiation-inducing therapy, its construction, and its application in drug delivery systems. Background Technology

[0002] Currently, cancer remains one of the leading causes of death and death among humans, and various treatment methods have been developed to overcome this threat. As an emerging treatment strategy, chemoimmunotherapy leverages the synergistic advantages of chemotherapy and immunotherapy, reducing tumor burden while simultaneously enhancing the body's anti-tumor immune response, thereby improving treatment success rates and significantly improving prognosis. However, the complex microenvironment of solid tumors presents numerous factors detrimental to treatment. Among these, chemotherapy drug resistance caused by tumor stem cells and immunosuppression mediated by bone marrow-derived suppressor cells pose significant challenges to chemoimmunotherapy. Therefore, effectively targeting and eliminating tumor stem cells and bone marrow-derived suppressor cells, and breaking down the tumor's resistance to treatment, is a pressing clinical challenge.

[0003] Differentiation inducers (such as all-trans retinoic acid) have emerged as promising agents for eliminating tumor stem cells and bone marrow-derived suppressor cells due to their ability to promote cell differentiation and maturation. However, these cell populations are mostly distributed in the deep hypoxic regions of solid tumors. The dense extracellular matrix and high interstitial pressure make it difficult for differentiation inducers to penetrate deep into the tumor, severely limiting the efficacy and application of differentiation-inducing therapy. Previous studies have shown that during apoptosis, the cell membrane shrinks, simultaneously dividing and encapsulating the cytoplasm to form apoptotic bodies. During this process, drugs remaining in the cytoplasm enter the apoptotic bodies and are taken up by neighboring tumor cells as a drug reservoir, achieving layer-by-layer penetration into the deep tumor. However, the active drug released from the carrier rapidly and irreversibly binds to the intracellular target and becomes inactive. Therefore, uncontrolled burst release of the drug leads to a significant reduction in the proportion of intracellular active drug during apoptotic body-mediated penetration, ultimately resulting in unsatisfactory accessibility to deep tumors and apoptosis induction rates.

[0004] In conclusion, developing and constructing an efficient and safe drug delivery system that integrates deep tumor penetration and targeted differentiation induction therapy is a powerful means to overcome tumor chemoresistance and immune escape and enhance the efficacy of chemoimmunotherapy. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a core-shell structured sequential drug delivery system for cascaded deep penetration and induced differentiation therapy, its construction, and its application in drug delivery systems.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A core-shell structured sequential drug delivery system for cascaded deep tumor penetration and differentiation induction therapy, wherein the system has a core-shell structure, the core being a hypoxia-responsive nanoparticle encapsulating a differentiation inducing agent, and the shell being an acid-responsive reversible polymer loaded with chemotherapeutic drugs.

[0008] The acid-responsive reversible polymer loaded with the chemotherapeutic drug is formed by loading the chemotherapeutic drug onto the surface of the acid-responsive reversible polymer through stirring to form a corresponding shell layer; wherein, the loading amount of the chemotherapeutic drug accounts for 10%-40% of the shell layer mass; the chemotherapeutic drug is a chemotherapeutic drug containing conjugated anthracene rings or benzene rings; the acid-responsive reversible polymer is polymerized from tannic acid and tetraethylenepentamine.

[0009] The chemotherapy drugs are doxorubicin and mitoxantrone.

[0010] The hypoxia-responsive nanoparticles encapsulating the differentiation inducer are formed by encapsulating the differentiation inducer within the hypoxia-responsive nanoparticles using a single emulsion method; wherein the differentiation inducer accounts for 5%-10% of the mass of the hypoxia-responsive nanoparticles; the hypoxia-responsive nanoparticles are assembled from nitroimidazole-grafted hyaluronic acid polymers; the grafting degree of the nitroimidazole-grafted hyaluronic acid polymers is 10%; the differentiation inducer is all-trans retinoic acid.

[0011] The system achieves the sequential release of chemotherapeutic drugs and differentiation inducers by responding to changes in intracellular pH and the hypoxic characteristics of the tumor microenvironment, and synergistically addresses the problem of spatial heterogeneity in the distribution of different cells within the tumor.

[0012] The polytannic acid shell, by modulating the drug release characteristics, endows the drug with pulsed drug release capability and promotes apoptotic body-mediated permeation. Specifically, the polytannic acid shell reversibly responds to the acidic environment of the lysosome, undergoing protonation and swelling to release chemotherapeutic drugs and mediate lysosomal escape. In the cytoplasm, neutral conditions cause the shell to deprotonate and contract, thereby stopping drug release.

[0013] The chemotherapeutic drug is loaded in a polytannic acid shell via hydrogen bonds or π-π interactions.

[0014] The hyaluronic acid polymer grafted with a hydrophobic nitroimidazole group forms nanoparticles, which can responsively transform into hydrophilic aminoimidazole under hypoxic conditions, causing the nanoparticles to disintegrate and release the encapsulated cargo; the nitroimidazole-grafted hyaluronic acid polymer is obtained by reacting hyaluronic acid with 6-(2-nitroimidazole)hexylamine.

[0015] The preferred differentiation-inducing agent is all-trans retinoic acid, which can promote the differentiation of tumor stem cells and reduce their stemness and malignancy. Simultaneously, all-trans retinoic acid can also induce bone marrow-derived suppressor cells to differentiate into mature myeloid cells, such as dendritic cells and macrophages, thereby enhancing the immune response.

[0016] The aforementioned core-shell sequential drug delivery system responds to the lysosomal-cytoplasmic pH gradient, triggering intracellular pulsed release of doxorubicin, inducing apoptosis and the generation of apoptotic bodies. The remaining doxorubicin is preserved within the polymer shell of the sequential pulsed drug delivery system and delivered deeper into the tumor via apoptotic body-mediated permeation. In response to the hypoxic characteristics of deep tumor regions, the nanocore cleaves and releases all-trans retinoic acid, targeting and differentiating tumor stem cells and bone marrow-derived suppressor cells that have accumulated in the deep tumor.

[0017] A method for preparing the aforementioned cascaded tumor deep penetration and differentiation-inducing therapy core-shell structured sequential drug delivery system:

[0018] Step (1): Preparation of the core: Dissolve nanoparticles with hypoxia response capability in an aqueous phase for later use; dissolve the differentiation inducer in chloroform and slowly and uniformly drop it into the aqueous phase under ultrasonic treatment; remove the organic solvent in the mixture by evaporation and freeze-dry to obtain a solid core.

[0019] Step (2): Assembly of core and shell: Dissolve the core obtained in step (1) in Tris-HCl with pH 8.5 and a concentration of 10mM, add an acid-responsive reversible polymer to obtain a mixed solution, centrifuge to remove impurities, and obtain a solution with the core-shell structure dispersed therein.

[0020] Step (3): Loading of chemotherapy drugs: Add chemotherapy drugs to the solution containing the core-shell structure obtained in step (2), and remove impurities by ultrafiltration after reaction to obtain a sequential drug delivery system with a core-shell structure.

[0021] The mass ratio of the hypoxia-responsive nanoparticles to the differentiation inducer is (1-3):(10-16).

[0022] The reaction conditions in step (1) are an ultrasonic power of 100-150 W and an ultrasonic time of 10-20 minutes.

[0023] In step (2), the mass ratio of the core, tannic acid and tetraethylenepentamine is (10-30):(3-8):(1-3), the reaction conditions are 15-25°C, protected from light, and stirring, and the reaction time is 4-8 hours; the molar ratio of the chemotherapeutic drug and the differentiation inducer in the core-shell structure is 1:1-1:5.

[0024] In step (3), the mass ratio of chemotherapy drugs to the system is 0.05:1-0.2:1, the reaction conditions are 15-25°C, protected from light, and stirring, and the reaction time is 2-2-8 hours.

[0025] The application of a core-shell structured sequential drug delivery system for cascaded deep tumor penetration and differentiation induction therapy, wherein the core-shell structured sequential drug delivery system promotes deep delivery of antitumor drugs and differentiation therapy for solid tumors.

[0026] The application of the described core-shell structured sequential drug delivery system in the preparation of breast cancer drugs, wherein the breast cancer drugs target 4T1 cells.

[0027] The present invention has the following beneficial effects:

[0028] (1) The core-shell structured sequential drug delivery system with uniform particle size prepared by the present invention is used for cascade deep penetration and differentiation induction therapy. The preparation method is simple and efficient, with good stability, and achieves efficient dual-drug loading of chemotherapy drugs and all-trans retinoic acid.

[0029] (2) The core-shell sequential drug delivery system of this invention can respond to endogenous cellular stimuli and release drugs, exhibit uptake and lysosomal escape behavior, penetrate tumor tissues, differentiate tumor stem cells and bone marrow-derived suppressor cells, and demonstrate in vivo antitumor effects. Specifically, the core-shell structure was characterized, and the response and drug release under weakly acidic and hypoxic conditions were studied. Tumor cell uptake experiments, lysosomal escape experiments, cytotoxicity experiments, tumor spheroid penetration experiments, tumor stem cell differentiation, in vivo antitumor efficacy, and in vivo antitumor immune evaluation were conducted. The results showed that the core-shell sequential drug delivery system responds to changes in the pH gradient between intracellular lysosomes and cytoplasm, with its shell undergoing reversible swelling-contraction accompanied by protonation and deprotonation processes, thereby achieving pulsed release of doxorubicin. By regulating the drug release performance, the core-shell sequential drug delivery system increases the proportion of intracellular active drugs, promotes apoptotic body-mediated deep delivery, and thus achieves overall tumor killing. Simultaneously, the hypoxia-responsive hyaluronic acid core does not release drugs in normoxic regions. However, with the help of apoptotic bodies, it enters the hypoxic region of the tumor, where its nitroimidazole group is reduced to aminoimidazole, thereby releasing all-trans retinoic acid, which differentiates into tumor stem cells and bone marrow-derived suppressor cells. Therefore, the hypoxia-responsive core can prevent premature leakage of all-trans retinoic acid in normoxic tumor regions, improving its targeted differentiation efficacy deep within the tumor. This sequential drug delivery system solves the problem of heterogeneous distribution of different cells within the tumor, providing new strategies and more options for the application of chemoimmunotherapy in the treatment of solid tumors, and meeting the urgent clinical need for highly effective antitumor agents. Attached Figure Description

[0030] Figure 1This is an experimental diagram illustrating the preparation and characterization of the core-shell structured sequential drug delivery system in Example 1 of the present invention.

[0031] a: Transmission electron micrographs of HRA and HRA@D-TT.

[0032] b: Particle size distribution of HRA and HRA@D-TT.

[0033] c: Surface potential of HRA and HRA@D-TT.

[0034] d: Fluorescence spectra of HNI@D-TT, HNI@TT, and DOX.

[0035] e: Infrared spectra of HNI, HNI@TT, DOX, and HNI@D-TT.

[0036] Figure 2 The figure shows the stability study results of the core-shell structured sequential drug delivery system in Example 2 of this invention.

[0037] a: Colloidal stability of HRA@D-TT and HRA@D-TA.

[0038] b: Storage stability of HRA@D-TT and HRA@D-TA at 4°C.

[0039] Figure 3 This diagram illustrates the verification of the reversible acid response and oxygenation response capabilities of the core-shell structured sequential drug delivery system in Embodiment 3 of the present invention.

[0040] a: Particle size variation of HRA@D-TT and HRA@D-TA.

[0041] b: Potential changes of HRA@D-TT and HRA@D-TA.

[0042] c: UV spectra of HNI micelles under normal and hypoxic conditions.

[0043] d: Verification diagram of the Fret effect under normoxic and hypoxic conditions.

[0044] e: Change in Fret ratio.

[0045] Figure 4 This is a diagram showing the in vitro release results of the formulation in Example 4 of the present invention.

[0046] a: DOX release curves of HRA@D-TT under different pH conditions.

[0047] b: Release curves of ATRA from HRA@D-TT in different pH buffers under hypoxic or normoxic conditions.

[0048] Figure 5 This is a diagram showing the results of cell uptake in Example 5 of the present invention.

[0049] a: Confocal plot of cellular uptake at different time points in a pulsed sequential drug delivery system.

[0050] b: Flow cytometry results of cellular uptake quantification of the pulsed sequential drug delivery system at different time points.

[0051] Figure 6 This is a diagram showing the lysosomal escape results in Example 6 of the present invention.

[0052] a: Confocal image of lysosomal escape after 8 hours in a pulsed sequential drug delivery system.

[0053] b: The corresponding Pearson coefficient quantified by ImageJ.

[0054] Figure 7 This is a graph showing the cell viability results after 48 hours of treatment with different formulations in Example 7 of the present invention.

[0055] Figure 8 The results of extraction and characterization of apoptotic bodies are shown in Example 8 of this invention.

[0056] a: Transmission electron micrograph of apoptotic bodies.

[0057] b: The percentage of Annexin V positive in apoptotic bodies.

[0058] c: Expression of characteristic proteins in apoptotic bodies.

[0059] d: Confocal image of cellular uptake by apoptotic bodies.

[0060] Figure 9 This is a diagram showing the in vitro permeation verification results based on apoptotic bodies in Example 9 of the present invention.

[0061] a: Confocal image of intercellular drug delivery mediated by apoptosis bodies.

[0062] b: The proportion of apoptotic cells during the apoptotic body-mediated infiltration process.

[0063] c: Quantitative analysis results of free drugs in cells.

[0064] d: Quantitative analysis results of free drug in apoptotic bodies.

[0065] f: Confocal image of apoptotic bodies extracted from HNI@D-TT cells labeled with coumarin 6 after incubation.

[0066] Figure 10 This is a diagram showing the results of a tumor sphere permeation experiment in Example 10 of the present invention.

[0067] a: In vitro tumor sphere permeation confocal image of a pulsed sequential drug delivery system.

[0068] b: Quantitative results of tumor sphere permeation fluorescence obtained using ImageJ.

[0069] Figure 11 This is a graph showing the apoptosis rate of tumor stem cells after treatment with different formulations in Example 11 of the present invention.

[0070] Figure 12 This is a graph showing the characterization results of changes in stemness-related characteristics of tumor stem cells in Example 12 of the present invention.

[0071] a: CD44 after treatment with different formulations + / CD24 - A representative flow cytometry result of the cell population.

[0072] b: Representative flow cytometry results showing changes in the proportion of side population cells after treatment with different formulations.

[0073] c: Yes Figure 12 The flow cytometry results of a.

[0074] d: Yes Figure 12 The flow cytometry results for b.

[0075] e: The number of tumor spheres formed by 4T1 cells after treatment with different agents.

[0076] f: Western blot analysis of the protein expression of SOX2, Nanog and Oct4 after treatment with different formulations.

[0077] Figure 13 This is a diagram showing the in vivo distribution and permeation test results of the formulation in Example 13 of this invention.

[0078] a: In vivo fluorescence images of 4T1 tumor-bearing mice at different time points after treatment with different DiR-labeled formulations.

[0079] b: In vitro fluorescence distribution of major organs (heart, liver, spleen, lung, kidney) and tumors in mice 24 hours after injection of different DiR-labeled formulations.

[0080] c: Semi-quantitative fluorescence images of major organs and tumors in mice 24 hours after injection of different DiR-labeled formulations.

[0081] d: Quantitative fluorescence results of mouse tumor sections 24 hours after intravenous injection of DOX, HNI@D-TT and HNI@D-TA.

[0082] e: Fluorescence images of mouse tumor sections 24 hours after treatment with DOX, HNI@D-TT, and HNI@D-TA.

[0083] Figure 14 This is a pharmacodynamic study diagram of the core-shell structure sequential drug delivery system of Embodiment 14 of the present invention.

[0084] a: Tumor volume growth curves in mice after intravenous injection of different formulations.

[0085] b: Weight of the 4T1 tumors after treatment with different formulations.

[0086] c: Photographs of the detached 4T1 tumor after treatment with different formulations.

[0087] d: Changes in mouse body weight during treatment.

[0088] e: CD44 levels in 4T1 tumors after treatment with different agents + / CD24 - Flow cytometry results of cell population.

[0089] f: CD133 in 4T1 tumors after treatment with different agents + Flow cytometry results of cell population.

[0090] g: CD44 levels in 4T1 tumors after treatment with different formulations + / CD24 - Representative flow cytometry plot of the cell population.

[0091] h: CD133 in 4T1 tumors after treatment with different agents + Representative flow cytometry plot of the cell population.

[0092] i: Images of H&E staining, Ki67 and TUNEL staining of mouse tumors after different treatments.

[0093] Figure 15 This is an H&E staining image of the major organs of mice after intravenous injection of different formulations in Example 14 of the present invention.

[0094] Figure 16 This is an in vivo antitumor immune evaluation diagram of the core-shell structure sequential drug delivery system of Embodiment 15 of the present invention.

[0095] a: Tumor volume growth curves in mice after intravenous injection of different formulations.

[0096] b: Weight of 4T1 tumors after dissection following treatment with different formulations.

[0097] c: Photographs of 4T1 tumors after dissection following treatment with different formulations.

[0098] d: Changes in mouse body weight during treatment.

[0099] e: H&E staining images of mouse lung tissue after treatment with different formulations.

[0100] f: In vitro fluorescence images of mouse lungs after treatment with different formulations.

[0101] g: Representative flow cytometry analysis and quantitative results of bone marrow-derived suppressor cells in 4T1 tumors after treatment with different formulations.

[0102] h: Representative flow cytometry analysis and quantitative results of dendritic cells in 4T1 tumors after treatment with different formulations.

[0103] i: CD8+ in 4T1 tumors of mice after receiving different treatments + Representative flow cytometry plots and quantitative results for T. Detailed Implementation

[0104] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0105] This invention relates to a core-shell structured sequential drug delivery system for deep delivery of differentiation inducers and overcoming the tumor anti-therapeutic barrier. The differentiation inducer is encapsulated in hypoxia-responsive polymer nanomicelles, and then surrounded by a reversible acid-responsive polymer shell loaded with chemotherapeutic drugs, forming a pulsed sequential drug delivery system. The prepared sequential pulsed drug delivery system exhibits high drug loading capacity, good stability, and the ability to penetrate deep into tumor tissues, effectively promoting the differentiation of tumor stem cells and bone marrow-derived suppressor cells, thereby achieving enhanced tumor killing and anti-tumor immune activity.

[0106] Example 1: Preparation and Characterization of Core-Shell Sequential Drug Delivery System

[0107] Hypoxia-responsive nanocores were prepared using a single emulsion method. Specifically, 13 mg of nitroimidazole-grafted hyaluronic acid polymer (HNI, prepared according to the method described in "Glucose-Responsive Insulin Delivery by Microneedle-Array Patches Loaded with Hypoxia-Sensitive Vesicles" to achieve a grafting rate of 7%-12%) was dissolved in deionized water as the aqueous phase. 2 mg of all-trans retinoic acid (ATRA) was dissolved in chloroform and slowly and uniformly added dropwise to the aqueous phase under ultrasonic treatment. The organic solvent in the emulsion was removed by rotary evaporation, and the resulting liquid was filtered through a 0.45 μm filter to remove unencapsulated drug, yielding a homogeneous and clear ATRA-loaded micelle solution (HRA).

[0108] Next, a reversible acid-responsive tannic acid-tetraethylenepentamine (TA-TEPA) shell loaded with doxorubicin (DOX) was coated onto the surface of the nanocore. Specifically, 20 mg of the nanocore was dispersed in a Tris-HCl buffer at pH 8.5, then 2 mg of TA and 5 mg of TEPA were added to the system. The mixture was stirred overnight in the dark, and unreacted compounds were removed by repeated centrifugation and washing. The resulting product was freeze-dried to form a solid. To load DOX, 20 mg of the solid was dissolved in water, and 1 mL of a DMSO solution containing 2 mg DOX was added dropwise, with stirring at room temperature for 4 h. Finally, the free drug was removed by repeated ultrafiltration and washing to obtain a core-shell structured pulsed sequential drug delivery system (HRA@D-TT).

[0109] Meanwhile, in the above preparation process, a pure tannic acid shell formed by the oxidative polymerization of tannic acid monomers under alkaline conditions was used as a control for irreversible acid response (HRA@D-TA). Furthermore, to facilitate observation of the shell's function, TA-TEPA binary core-shell nanoparticles loaded only with DOX (HNI@D-TT) were prepared. Specifically, 13 mg of HNI was dissolved in deionized water, then 2 mg of TA and 5 mg of TEPA were added to the system, stirred overnight in the dark, and unreacted compounds were removed by repeated centrifugation and washing. Finally, TA mono-core-shell nanoparticles loaded only with DOX (HNI@D-TA) were prepared by self-polymerization under alkaline conditions.

[0110] The core-shell structure of the drug delivery system was verified by transmission electron microscopy, and the results are as follows: Figure 1 As shown in Figure a, all nanoparticles are uniformly spherical with an outer shell, demonstrating the successful synthesis of the core-shell structure. The particle size changes before and after the shell coating were analyzed using a Malvern particle size analyzer, and the results are as follows. Figure 1 As shown in b, the particle size of HRA is approximately 130 nm. After coating, the particle size increases to approximately 160 nm, accompanied by an increase in surface potential. The loading of doxorubicin was verified by fluorescence spectroscopy, and the results are shown in Figure b. Figure 1 As shown in c. Compared to doxorubicin solutions of the same concentration, doxorubicin in HNI@D-TT exhibited a significant fluorescence quenching effect, suggesting that doxorubicin may be embedded in the shell of polytannic acid through π-π interactions. Furthermore, as... Figure 1 As shown in d, the infrared spectrum also shows the successful coating of the polymer shell and the successful loading of doxorubicin.

[0111] Example 2: In vitro stability study of a core-shell structured sequential drug delivery system

[0112] The obtained 1 mg / mL HRA@D-TT and HRA@D-TA were placed in PBS supplemented with 10% FBS and incubated at room temperature for 48 hours. The colloidal stability of the drug delivery system was studied by measuring particle size changes. Furthermore, the long-term stability of the formulation was assessed by storing the drug delivery system at 4°C for 2 weeks. Figure 2 a and Figure 2 As shown in b, both HRA@D-TA and HRA@D-TT exhibit high stability under storage conditions.

[0113] Example 3: In vitro reversible acid and hypoxia response performance study of a core-shell structured sequential drug delivery system

[0114] To verify the reversible acid-response characteristics of the formulations, the 1 mg / mL HRA@D-TT and HRA@D-TA prepared in Example 1 were repeatedly cyclically placed in PBS at pH 7.4 and pH 5.0, incubated for 8 hours each time. The particle size changes and potential changes of both were measured using a Malvern particle size analyzer, and the results are as follows: Figure 3 a and Figure 3 As shown in b, compared to a single tannic acid coating, HRA@D-TT exhibits a reversible size change from 160 nm at pH 7.4 to nearly 300 nm at pH 5.0, with its surface potential showing greater sensitivity to increased acidity. This indicates that HRA@D-TT possesses excellent reversible acid-response characteristics under different pH conditions, further demonstrating its ability to effectively respond to environmental pH changes during lysosomal-cytoplasmic transport, achieving pulsed drug release.

[0115] To verify the hypoxia-responsiveness of the nanocore, the HNI polymer was dissolved in PBS, and 10 mM sodium dithionite was added to create a hypoxia environment. After incubation for 8 hours, UV spectroscopy analysis was performed. Figure 3As shown in Figure c, after incubation under hypoxic conditions, the nitro absorption peak at 325 nm disappeared, while an amino absorption peak appeared at 278 nm, indicating that hypoxia induced a hydrophilic transition in the micelle structure, leading to its cleavage. The hypoxia-responsiveness of the formulation was verified by the FRET experiment. To prepare FRET nanoparticles, DiI, DiO, and HNI polymers were co-encapsulated in HNI polymer at a feed ratio of 1:1:10 (DiI:DiO:HNI, w / w / w). Specifically, HNI was dissolved in deionized water for later use. DiI and DiO were dissolved in ethanol and added dropwise to the HNI aqueous solution. After stirring for 24 h, the mixture was sonicated for 10 min. Then, 20 mg of the resulting mixture was lyophilized and resuspended in Tris-HCl buffer at pH 8.5, and 2 mg TA and 5 mg TEPA were added to obtain HNI-(DiI+DiO)@TT. Under normoxic or simulated hypoxic conditions, 4T1 cells were cultured in 24-well plates (2 × 10⁶ cells per well). 4 Cells were cultured overnight in HNI-(DiI+DiO)@TT at a dye concentration of 2 μg / mL for 8 hours. Excess micelles were removed by washing three times with cold PBS, followed by fixation with 4% formaldehyde and staining with Hoechst 33342. Observation was performed using confocal microscopy: fluorescence was excited at 488 nm, with a 505–550 nm filter for green emission and a 560–615 nm filter for red (FRET) emission. Cell fluorescence was quantitatively assessed using ImageJ. Figure 3 As shown in 3d and 3e, a significant decrease in FRET efficiency can be observed under hypoxic conditions, indicating an increased distance between DiI and DiO, thus verifying the responsiveness of HNI-(DiI+DiO)@TT nanoparticles in hypoxic environments. This further demonstrates that the system can effectively release encapsulated drugs under hypoxic conditions, providing strong support for its application in the tumor microenvironment.

[0116] Example 4: In vitro drug release study of a core-shell structured sequential drug release system

[0117] To validate the release kinetics of the formulation, HRA@D-TT (1 mL) was placed in a dialysis bag (MWCO, 3.5 kDa) and immersed in 30 mL of PBS containing 2% Tween 80. To assess pH-induced DOX release, the release medium was adjusted to a series of acidity gradients (pH 7.4, 6.8, and 5.0). Furthermore, a hypoxia-responsive release of ATRA was assessed by adding 10 mM sodium dithionite to the buffers of the different acidity gradients. 200 μL samples were taken at predetermined time intervals (i.e., 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h) and immediately replenished with the same volume of fresh release medium. The release of DOX and ATRA from the nanoplatform was monitored by fluorescence spectroscopy and HPLC, respectively. Figure 4 a and Figure 4 As shown in b, less than 20% of DOX leaked from HRA@D-TT at pH 7.4, minimizing systemic toxicity from sudden release. However, when the pH of the medium was lowered to 5.0, drug release increased significantly by approximately 3-fold. Furthermore, HRA@D-TT exhibited excellent hypoxia sensitivity in low-oxygen environments, promoting rapid ATRA release. Particularly under the combined stimulation of hypoxia and high acidity, ATRA release significantly exceeded that induced by hypoxia alone, indicating that the polymer shell structure restricts ATRA release from the core. The enhanced release can be attributed to the porosity of the surface at low pH, which endows the nanoplatform with excellent capabilities for rapid drug release under specific conditions.

[0118] Example 5: Cell Uptake Experiment

[0119] To visualize the intracellular processes of the formulation, DOX-only nanoparticles (HNI@D-TT and HNI@D-TA) were prepared. 4T1 cells were seeded in 24-well plates (2 × 10⁶ cells per well). 4 Cells were cultured for 12 hours to allow cell adhesion. Subsequently, cells were treated with fresh 1640 medium containing 5 μg / mL DOX equivalent doses of free DOX, HNI@D-TT, and HNI@D-TA for 4 and 8 hours, respectively. After incubation, the drug-containing medium was discarded, and cells were washed three times with ice-cold PBS (pH 7.4) to terminate cell uptake. Cells were fixed with 4% paraformaldehyde for 10 minutes and the nuclei were stained with Hoechst 33342 at 37°C for 10 minutes for observation using a confocal microscope. For flow cytometry analysis of cell uptake, cells were cultured in 24-well plates. After drug administration, cells were digested with 0.05% trypsin, collected by centrifugation, and uniformly dispersed in PBS (pH 7.4). Cell uptake was then quantitatively analyzed by flow cytometry. Figure 5As shown in a and 5b, the group treated with DOX solution exhibited rapid fluorescent colocalization and higher fluorescence intensity in the cell nucleus due to unregulated drug release. Furthermore, the internalization efficiency of HNI@D-TT was 1.5 times that of HNI@D-TA, which is likely due to the higher surface potential of the nanoparticles.

[0120] Example 6: Lysosomal Escape Experiment

[0121] 4T1 cells were seeded in 24-well plates (2 × 10⁴ cells per well). 4 Cells were cultured for 12 hours to allow cell adhesion. Subsequently, cells were treated with fresh medium containing 5 μg / mL DOX equivalent doses of HNI@D-TT and HNI@D-TA for 4 and 8 hours, respectively. After incubation, the drug solution was discarded, and cells were washed three times with ice-cold PBS (pH 7.4) to terminate cell uptake. Lysosomes were stained with Lysotracker-red, washed three times with ice-cold PBS (pH 7.4), and cells were fixed with 4% paraformaldehyde for 10 min. Nuclei were stained with Hoechst 33342 at 37°C for 10 min and observed using a confocal microscope. Figure 6 As shown in a and b, after treatment with HNI@D-TT, the colocalization coefficient of red and green fluorescence decreased, indicating that doxorubicin successfully escaped into the cytoplasm. However, when treated with HNI@D-TA, most of the red and green fluorescence overlapped, indicating that the simple tannic acid coating could not achieve the desired lysosomal escape effect.

[0122] Example 7: Cytotoxicity Experiment

[0123] To evaluate in vitro cytotoxicity against tumor cells, 4T1 cells were seeded at a density of 2000 cells per well in 96-well plates. After 12 hours, the medium was replaced with a series of dilutions containing DOX, HNI@D-TT, HNI@D-TA, and HRA@D-TT. After incubation for 48 hours under normal or hypoxic conditions, MTT solution was added and incubation was carried out at 37°C for 4 hours. To dissolve the generated methylxanidine crystals, 200 μL of DMSO was added to each well, and the absorbance at 570 nm was detected using a microplate reader. The IC50 was determined using a GraphPad Prism 8 microplate reader, with molar concentration and cell viability ratio as reference values. 50 Value. For example... Figure 7 As shown, the inhibitory effect of the formulations on tumor cell viability followed the order DOX > HNI@D-TT > HNI@D-TA. Furthermore, when cultured under normoxic conditions, cells treated with HRA@D-TT showed similar toxicity differences compared to cells treated with HNI@D-TT, with IC50 values ​​of [missing value]. 50The values ​​were 277.9 nM and 253.6 nM, respectively. In contrast, incubation under hypoxic conditions significantly enhanced the cytotoxicity of HRA@D-TT, with an IC50 value of 277.9 nM and 253.6 nM, respectively. 50 The concentration decreased to 116.7 nM. This difference in toxicity caused by different incubation environments further confirms that HNI micelles can disintegrate under hypoxic conditions, releasing ATRA and exerting a synergistic antitumor effect.

[0124] Example 8: Extraction and characterization of apoptotic bodies

[0125] 4T1 cells were cultured in 6-well plates and treated with HRA@D-TT at a DOX equivalent dose of 5 mg / mL for 8 hours. After incubation with fresh culture medium for 12 hours, the culture was harvested and centrifuged at 100g for 10 minutes to remove cell debris. The resulting supernatant was then centrifuged at 1000g for 15 minutes to separate apoptotic bodies. The morphology of the apoptotic bodies was observed by transmission electron microscopy, and the results showed that the apoptotic bodies were spherical vesicles with a diameter of approximately 1.5 μm. Figure 8 a). Since annexin V specifically marks externalized phosphatidylserine residues in apoptotic cells, we used the annexin V-FITC / PI kit to determine the positive rate of annexin V in apoptotic bodies and analyzed it by flow cytometry. Figure 8 As shown in b, 90.9% of the isolated apoptotic bodies exhibited a high positive rate of annexin V, indicating high purity. Furthermore, we validated the expression of characteristic proteins using Western blotting. Similar to 4T1 cells, the apoptotic bodies also expressed similar levels of the membrane markers CD44 and CD326. However, due to the high degree of apoptosis, they showed high expression of cleaved caspase-3 (…). Figure 8 c).

[0126] To study cellular uptake by apoptotic bodies, 4T1 cells were incubated with apoptotic bodies for 8 hours. After nuclear staining, the cells were observed using a confocal microscope. Figure 8 As shown in d, DOX fluorescence can be detected in the isolated apoptotic bodies, and it can be internalized by 4T1 cells, indicating that apoptotic bodies can serve as a reservoir of residual drugs in the cell and be taken up by surrounding cells.

[0127] Example 9: Apoptosis Body-Mediated In Vitro Permeation Experiment

[0128] 4T1 cells were seeded in 6-well plates and incubated overnight, designated as plate (i). Cells in plate (i) were treated for 8 hours under normoxic conditions with an equivalent dose of 5 μg / mL DOX, HNI@D-TT, and HNI@D-TA, followed by incubation in fresh culture medium for 12 hours to induce apoptosis. Apoptotic bodies (i-ii) were then collected from the supernatant and co-incubated with untreated fresh cells for 8 hours, designated as plate (ii). This process was repeated three times to obtain plate (iii). After washing the cells, they were stained with Hoechst 33342, and DOX internalization was assessed using a fluorescence confocal microscope. Figure 9 As shown in a, cells treated with DOX solution or HNI@D-TA showed weak red fluorescence in the second incubation cycle, which disappeared in the third cycle, indicating their limited delivery capacity to neighboring cells. Conversely, cells in plate (iii) treated with HNI@D-TT still showed a significant fluorescent signal despite the gradual decrease in fluorescence intensity, which is attributed to the controlled pulsed release of DOX promoting the retention of the remaining drug.

[0129] To assess apoptosis, treated cells were stained with an annexin V-FITC / PI kit, and the apoptosis rate was examined by flow cytometry. Figure 9 As shown in b, HNI@D-TT treated cells still exhibited an 11% apoptosis rate in the third incubation cycle, demonstrating satisfactory delivery. Furthermore, although the DOX solution initially induced extensive apoptosis, its excessive consumption significantly impaired the effectiveness of subsequent treatments.

[0130] Next, we quantified the ratio of free drug in cells and apoptotic bodies in each plate. Cells and apoptotic bodies from plates (i)-(iii) were collected, and proteins were precipitated with methanol under sonication in an ice bath. After centrifugation and ultrafiltration, the DOX content in the filtrate was measured using a microplate reader and normalized to its protein content. Figure 9 As shown in c, during each incubation cycle, approximately 15% to 20% of the DOX in HNI@D-TT entered the nucleus and bound tightly to topoisomerase II, while the remaining drug remained active. Furthermore, in the HNI@D-TT group, higher accumulations of unbound active drug were measured in apoptotic bodies (i-ii) and (ii-iii), indicating that by increasing the proportion of active drug in apoptotic bodies, drug penetration based on the orthoside effect was enhanced. Figure 9 d).

[0131] To determine drug retention in apoptotic bodies, coumarin 6 was encapsulated in the hypoxic core of HNI@D-TT cells and incubated with cells in well (i). This process was repeated three times. Apoptotic bodies (i-ii) and (ii-iii) were then extracted from the cell culture, dropped onto a glass slide, and observed using a confocal microscope. Figure 9 As shown in e, the overlapping fluorescence signals between red and green confirm the co-encapsulation of DOX and coumarin 6 in the apoptotic bodies. However, with continued intercellular transport, DOX consumption leads to a decrease in red fluorescence within the apoptotic bodies (ii-iii), while the fluorescence of coumarin 6 remains stable. This indicates that the HNI micelles maintain their intact structure under normoxic conditions.

[0132] Example 10: Tumor sphere permeation experiment

[0133] 4T1 cell suspension was mixed with 0.24% methylcellulose at a 1:1 volume ratio, and 15 μL of the mixture was pipetted onto the lid of a 96-well round-bottom plate. The lid containing the cell suspension droplet was inverted onto the 96-well round-bottom plate containing culture medium and incubated under standard culture conditions for 24 hours, followed by centrifugation at 2800 rpm for 3 minutes. After 6 days of culture, tumor spheroids were incubated for 24 hours with an equivalent dose of 5 μg / mL DOX, HNI@D-TT, and HNI@D-TA. DOX fluorescence in the spheroids was observed using confocal microscopy. In the HNI@D-TT group, red fluorescence was observed inside the tumor spheroids. In contrast, the red fluorescence after treatment with DOX and HNI@D-TA was concentrated only at the periphery of the tumor spheroids, further illustrating that the pulsed drug release of the polymer shell facilitated the delivery of the formulation deep into the tumor. Figure 10 a and 10 (b).

[0134] Example 11: Tumor stem cell apoptosis experiment

[0135] First, tumor stem cells were enriched using a serum-free culture method with a low adhesion system. 4T1 cells were seeded in ultra-low adhesion 6-well plates (2 × 10⁶ cells per well). 4 The cells were cultured in DMEM / F12 medium supplemented with 20 ng / mL epidermal growth factor, 20 ng / mL basic fibroblast growth factor and 1x B27 for 7 days, and the resulting spheroids were collected by low-speed centrifugation.

[0136] Next, to investigate whether a core-shell-based pulsed sequential drug delivery system would affect tumor cells deep within the tumor, 4T1 cells were incubated for 8 h with equivalent doses of DOX (5 μg / mL), HNI@D-TT (8 μg / mL), HNI@D-TT+ATRA (8 μg / mL), and HRA@D-TT (8 μg / mL), respectively. Apoptotic bodies were then extracted from each treatment group and recultured with enriched tumor stem cell spheres under hypoxic conditions for 48 h. The treated cells were stained using an annexin V-FITC / PI kit, and the apoptosis rate was examined by flow cytometry. Figure 11 As shown in a and b, the HRA@D-TT group exhibited superior anti-tumor stem cell efficacy across all treatments. Furthermore, HNI@D-TT also inhibited tumor stem cell survival due to the toxicity of residual DOX within it. Compared to HRA@D-TT, HNI@D-TT+ATRA treatment induced lower cytotoxicity, while HRA@D-TT alone showed high cytotoxicity, highlighting the effectiveness of the HNI nanocore in preventing ATRA depletion in the normoxic region.

[0137] Example 12: Tumor stem cell differentiation experiment

[0138] Tumor stem cells were enriched using the method described above and cultured with extracted apoptotic bodies under hypoxic conditions for 48 h. After incubation, cells were collected and stained with CD44-PE and CD24-FITC. CD44 levels were detected by flow cytometry. + / CD24 - The proportion of the cell population. Furthermore, to determine the proportion of side population cells, 4T1 cells were incubated with the aforementioned apoptotic bodies under hypoxic conditions for 48 h, then the cells were collected and incubated with 5 μg / mL Hoechst 33342 at 37°C for 1 h, followed by washing with PBS. Verapamil hydrochloride (100 µM) was added as a control. The proportion of side population cells was detected by flow cytometry. Figure 12 As shown in Figure ae, the cell population with tumor stem cell phenotypes was significantly reduced in the HRA@D-TT treatment group, demonstrating the considerable potential of HRA@D-TT in inducing differentiation of hypoxic deep tumor cells and reducing the proportion of malignant cells.

[0139] Furthermore, Western blotting was used to assess the expression levels of SOX2, Nanog, and Oct4 proteins in 4T1 cells after treatment with different apoptotic bodies. Figure 12 As shown in f, the expression levels of these stemness-related proteins were significantly downregulated in the HNI@D-TT group, demonstrating the synergistic benefits of a cascaded nano-strategy for tumor penetration and differentiation therapy in eliminating tumor stem cells.

[0140] Example 13: In vivo distribution and tumor penetration experiment

[0141] First, core-shell nanoparticles internally loaded with DiR were prepared according to the method in Example 1 to indicate their in vivo process. Specifically, HNI was dissolved in deionized water for later use, DiR was dissolved in ethanol and added dropwise to the HNI aqueous solution. After stirring for 24 h, the mixture was sonicated for 10 min to obtain the DiR-loaded nanocore. Then, 20 mg of the resulting mixture was lyophilized and resuspended in a Tris-HCl buffer at pH 8.5, and 2 mg of TA and 5 mg of TEPA were added to obtain HDiR@TT. Next, 1 mL of DMSO solution containing 2 mg of DOX was added dropwise to the above system. After stirring for 4 h, free drug was removed by multiple washes and ultrafiltration to obtain HDiR@D-TT. Simultaneously, control group nanoparticles were prepared by the self-polymerization of tannic acid monomers under alkaline conditions and loaded with DOX using the same method.

[0142] To investigate the biodistribution of HRA@D-TT, 1×10⁻⁶ t / t was administered subcutaneously. 6 A mouse model of 4T1 tumor xenograft was established using 4T1 cells. The cells reached approximately 400 mm in size. 3 After tumor volume was determined, mice were divided into three groups and intravenously injected with free DiR, HDiR@D-TA, and HDiR@D-TT (1 mg / kg DiR equivalent dose). At predetermined time points, the DiR-labeled nanoparticles were imaged using an in vivo imaging system. Figure 13 As shown in Figure a, HDiR@D-TT exhibits higher fluorescence intensity at the tumor site compared to DiR solution. Fluorescence at the tumor site remained in the HDiR@D-TT group 24 h after injection, indicating that the formulation has good tumor targeting and accumulation effects. Furthermore, mice were euthanized, and major organs (heart, liver, spleen, lungs, and kidneys) and tumors were collected for fluorescence imaging. Figure 13 As shown in b and 13c, the nanoparticles mainly accumulate in the liver, spleen, and tumors.

[0143] Next, the penetration effect of the formulations on tumor tissue was evaluated by intravenous injection of free DOX, HNI@D-TA, and HNI@D-TT (2 mg / kg DOX equivalent dose) into tumor-bearing mice. Twenty-four hours later, the mice were euthanized, tumor tissue was collected, and tumor sections were stained with DAPI and CD31 and observed using a confocal microscope. Figure 13As shown in d and 13e, the DOX fluorescence distribution in the tumors of the HNI@D-TT treatment group was more extensive, indicating that HNI@D-TT overcame the penetration barrier and achieved optimal penetration efficiency. Although HNI@D-TA showed similar tumor accumulation to HNI@D-TT, its distribution was limited to the perivascular region. The DOX solution exhibited the worst tumor permeability, which is attributed to its poor tumor targeting and accelerated in vivo metabolism.

[0144] Example 14: Pharmacodynamic Study

[0145] 100 μL containing 1×10 6 A 4T1 tumor-bearing mouse model was constructed using PBS solution containing 4T1 tumor cells. When the tumor grew to approximately 100 mm³, the mice were randomly divided into six groups (G1: saline, G2: DOX, G3: HNI@D-TA, G4: HNI@D-TT, G5: HNI@D-TT+ATRA, G6: HRA@D-TT). During an 11-day treatment period, mice received intravenous injections on days 0, 3, 6, and 9, each dose containing an equivalent concentration of 2 mg / kg DOX and 3 mg / kg ATRA. Tumor volume and body weight were measured daily. After treatment, the tumor tissue was dissected and photographed. Figure 14 As shown in Figures bd, the HNI@D-TT treatment group exhibited the slowest tumor growth rate, indicating its effective inhibition of tumor growth. Throughout the treatment period, the doxorubicin solution group showed more significant weight loss, suggesting systemic toxicity, while HNI@D-TT demonstrated better biocompatibility. Figure 14 e). To analyze the stem cell population in tumors, tumor samples were minced and digested in 1640 medium containing collagenase IV, hyaluronidase, and DNase I. The resulting single-cell suspension was incubated with the fluorescent antibodies CD44-PE and CD24-FITC at 4°C for 40 minutes. Figure 14 As shown in eh, HRA@D-TT significantly reduced CD44 + / CD24 - and CD133 + The proportion of tumor stem cell populations was identified as a key characteristic. Furthermore, after treatment, tumor tissue was H&E stained and TUNEL and Ki67 immunofluorescence staining was used to assess tumor cell apoptosis and proliferation levels. Figure 14 As shown in i, HRA@D-TT induced maximal tumor tissue apoptosis. To assess the biocompatibility of the nano-drug delivery system, H&E staining was performed on major organs after treatment. Figure 15 As shown, HRA@D-TT has good safety profile, with no significant damage to major organs.

[0146] Example 15: In vivo anti-tumor immune assessment

[0147] We established a 4T1 tumor-bearing mouse model and administered 2×10⁶ mice on day 10 post-inoculation. 5 4T1-Luc cells were intravenously injected into mice to mimic hematogenous metastasis of advanced tumors. Mice were randomly divided into six groups and treated as follows: G1 (saline), G2 (HRA@D-TT+aPD-1), G3 (HNI@D-TT+aPD-1), G4 (HNI@D-TT+aPD-1+ATRA), G5 (HRA@D-TT+aPD-1), and G6 (HRA@D-TT). DOX was administered intravenously at a dose of 2 mg / kg and ATRA at a dose of 3 mg / kg on days 0, 3, 6, and 9. APD-1 antibody (100 μg per mouse) was administered intraperitoneally on days 1, 4, 7, and 10 following HRA@D-TT administration. Tumor size and body weight were recorded daily. Figure 16 As shown in the diagram, the combination therapy of HRA@D-TT and aPD-1 had the best therapeutic effect, achieving tumor regression. Furthermore, no weight loss was observed in mice throughout the treatment period, indicating that the combination therapy had good biocompatibility. Figure 16 d)

[0148] After treatment, all mice were euthanized, and samples were taken from the lungs, tumor-draining lymph nodes, and tumor tissue. The harvested lungs were immersed in D-fluorescein solution (15 mg / mL) for 10 minutes and then subjected to bioluminescence imaging to observe the invasion of malignant cells into the lungs. Simultaneously, H&E staining was performed on the lung tissue to assess pathological changes. Figure 16 As shown in the figure, HNI@D-TT+aPD-1 significantly reduced the number of metastatic nodules in lung tissue and reduced the shadow area in H&E images, indicating that it can significantly inhibit tumor metastasis.

[0149] To assess changes in immune response levels, tumor cells collected from mice were isolated into single cells using digestive enzymes. Furthermore, cell suspensions from lymph nodes were prepared by grinding. After filtration through a 70 μm filter membrane, the cells were stained with various fluorescent antibodies, and the immune cell populations in tumors and lymph nodes were determined by flow cytometry and analyzed using FlowJo. Figure 16 As shown in GI, HNI@D-TT+aPD-1 significantly reduced the proportion of myelosuppressive cells in tumors and increased the number of dendritic cells. Furthermore, treatment increased tumor-invasive CD8+ cells. + The number of T cells indicates that HRA@D-TT can promote enhanced anti-tumor immunity by reversing the intratumoral immunosuppressive microenvironment.

[0150] In summary, the core-shell structured sequential drug delivery system for cascaded deep tumor penetration and differentiation induction therapy of this invention possesses an acid-responsive reversible polymer shell loaded with cytotoxic drugs. This allows the sequential drug delivery system to pulse-release drugs in response to pH changes during lysosomal-cytoplasmic transport, promoting deep drug penetration. Simultaneously, the internal encapsulation of differentiation-inducing agents in hypoxia-responsive polymer nanoparticles enables specific deep tumor release of the differentiation-inducing agents.

Claims

1. A core-shell structured sequential drug delivery system for cascaded deep tumor penetration and differentiation induction therapy, characterized in that: The system has a core-shell structure, with the core being nanoparticles encapsulating differentiation-inducing agents and exhibiting hypoxia-responsive capabilities, and the shell being an acid-responsive reversible polymer loaded with chemotherapeutic drugs. The acid-responsive reversible polymer is formed by the polymerization of tannic acid and tetraethylenepentamine. The hypoxia-responsive nanoparticles are assembled from nitroimidazole-grafted hyaluronic acid polymers; the grafting degree of the nitroimidazole-grafted hyaluronic acid polymers is 7%-12%.

2. The core-shell structured sequential drug delivery system for cascaded deep tumor penetration and differentiation induction therapy according to claim 1, characterized in that: The acid-responsive reversible polymer loaded with the chemotherapeutic drug is formed by loading the chemotherapeutic drug onto the surface of the acid-responsive reversible polymer through stirring to form a corresponding shell layer; wherein, the loading amount of the chemotherapeutic drug accounts for 10%-40% of the shell layer mass; the chemotherapeutic drug is a chemotherapeutic drug containing conjugated anthracene rings or benzene rings.

3. The core-shell sequential drug delivery system for cascaded deep tumor penetration and differentiation induction therapy according to claim 1 or 2, characterized in that: The chemotherapy drugs are doxorubicin and mitoxantrone.

4. The core-shell sequential drug delivery system for cascaded deep tumor penetration and differentiation induction therapy according to claim 1, characterized in that: The hypoxia-responsive nanoparticles encapsulating the differentiation inducer are formed by encapsulating the differentiation inducer within the hypoxia-responsive nanoparticles using a single emulsion method; wherein the differentiation inducer accounts for 5-10% of the mass of the hypoxia-responsive nanoparticles; and the differentiation inducer is all-trans retinoic acid.

5. A method for preparing a core-shell structured sequential drug delivery system for cascaded deep tumor penetration and differentiation induction therapy as described in claim 1, characterized in that... Step (1): Core preparation: Dissolve nanoparticles with hypoxia response capability in an aqueous phase for later use; dissolve differentiation inducer in chloroform and slowly and uniformly drop it into the aqueous phase under ultrasonic treatment; remove organic solvent from the mixture by evaporation and freeze-dry to obtain solid core; Step (2): Assembly of core and shell: Dissolve the core obtained in step (1) in Tris-HCl with pH 8.5 and a concentration of 10 mM, add an acid-responsive reversible polymer to obtain a mixed solution, centrifuge to remove impurities, and obtain a solution with the core-shell structure dispersed therein. Step (3): Loading of chemotherapy drugs: Add chemotherapy drugs to the solution containing the core-shell structure obtained in step (2), and remove impurities by ultrafiltration after reaction to obtain a sequential drug delivery system with a core-shell structure.

6. The method for preparing the core-shell structured sequential drug delivery system for cascaded deep tumor penetration and differentiation induction therapy according to claim 5, characterized in that: The mass ratio of the hypoxia-responsive nanoparticles to the differentiation inducer is (1-3):(10-16).

7. The method for preparing the core-shell structured sequential drug delivery system for cascaded deep tumor penetration and differentiation induction therapy according to claim 5, characterized in that: The reaction conditions in step (1) are an ultrasonic power of 100-150 W and an ultrasonic time of 10-20 minutes.

8. The method for preparing the core-shell structured sequential drug delivery system for cascaded deep tumor penetration and differentiation induction therapy according to claim 5, characterized in that: In step (2), the mass ratio of the core, tannic acid and tetraethylenepentamine is (10-30):(3-8):(1-3), the reaction conditions are 15-25°C, protected from light, and stirring, and the reaction time is 4-8 hours; the molar ratio of the chemotherapeutic drug and the differentiation inducer in the core-shell structure is 1:1-1:

5.

9. The application of the core-shell structured sequential drug delivery system for cascaded deep tumor penetration and differentiation induction therapy as described in claim 1, characterized in that: The application of the core-shell structured sequential drug delivery system in the preparation of drugs that promote deep delivery of anti-tumor drugs and differentiation therapy for solid tumors.

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