Cascaded core-shell structure sequential drug release system for deep penetration and induced differentiation treatment of tumors as well as construction method and application of core-shell structure sequential drug release system

Abstract

The invention belongs to the field of new auxiliary materials and new dosage forms of pharmaceutical preparations, and particularly relates to a core-shell structure sequential drug release system for cascade deep penetration and induced differentiation treatment, construction of the core-shell structure sequential drug release system and application of the core-shell structure sequential drug release system in a drug delivery system. The system is of a shell-core structure, an inner core is nanoparticles which encapsulate an induced differentiation agent and have hypoxia response capability, and a shell is an acid response reversible polymer loaded with chemotherapeutic drugs. According to the invention, a pulse drug release strategy is utilized to overcome penetration obstacles of solid tumors, programmed drug release is carried out aiming at heterogeneity distribution of different cells in the tumors, a tumor microenvironment characterized by chemotherapy drug resistance and immune escape is effectively remodeled, and comprehensive inhibition of the tumors is realized.

Description

Technical Field

[0001] The invention belongs to the field of new excipients and new dosage forms of pharmaceutical preparations, and specifically relates to a core-shell structure sequential drug release system for cascade deep penetration and induced differentiation therapy and its construction, as well as its application in a drug delivery system. Background Art

[0002] At present, cancer is still one of the main threats to human life and health, and people have developed different treatment methods to overcome the threat of cancer. As an emerging treatment strategy, chemoimmunotherapy takes advantage of the synergistic advantages of chemotherapy and immunotherapy, while reducing tumor burden and improving the body's anti-tumor immunity, thereby improving the success rate of treatment and significantly improving the prognosis. However, there are many factors that are unfavorable to treatment in the complex microenvironment of solid tumors. Among them, chemotherapy drug resistance caused by tumor stem cells and immunosuppression mediated by bone marrow-derived suppressor cells pose a huge challenge to chemoimmunotherapy. Therefore, how to effectively target and eliminate tumor stem cells and bone marrow-derived suppressor cells and break down the tumor's anti-treatment barrier is a difficult problem that needs to be solved clinically.

[0003] Differentiation inducers (such as all-trans retinoic acid) have emerged as a promising candidate for eliminating tumor stem cells and myeloid-derived suppressor cells by virtue of their ability to promote cell differentiation and maturation. However, most of these cell populations are distributed in the deep hypoxic areas of solid tumors. The dense extracellular matrix and high interstitial pressure make it difficult for differentiation inducers to penetrate deep into the tumor, which seriously restricts the efficacy and application of differentiation-induced therapy. Previous studies have shown that during cell apoptosis, the cell membrane shrinks, while dividing and encapsulating the cytoplasm to form apoptotic bodies. In this process, the drugs remaining in the cytoplasm will enter the apoptotic bodies together and be taken up by neighboring tumor cells in the form of drug reservoirs, achieving layer-by-layer penetration deep into the tumor. However, the active drugs released from the carrier will quickly and irreversibly bind to the intracellular target and then become inactivated. Therefore, the uncontrolled burst release of the drug will lead to a significant reduction in the proportion of intracellular active drugs during the apoptotic body-mediated penetration process, which ultimately makes the accessibility to deep tumors and the apoptosis induction rate unsatisfactory.

[0004] In summary, developing and constructing an efficient and safe drug delivery system that combines deep tumor penetration and targeted induced differentiation 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 the present invention is to overcome the deficiencies of the above-mentioned prior art and to provide a core-shell structure sequential drug release system for cascade deep penetration and induced differentiation therapy and its construction, as well as its application in a drug delivery system.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A core-shell sequential drug release system for cascade deep tumor penetration and differentiation induction therapy. The system is a core-shell structure, wherein the core is a hypoxia-responsive nanoparticle encapsulating a differentiation inducing agent, and the shell is an acid-responsive reversible polymer loaded with a chemotherapeutic drug.

[0008] The acid-responsive reversible polymer loaded with chemotherapeutic drugs is formed by loading the chemotherapeutic drugs on the surface of the acid-responsive reversible polymer by stirring, wherein the loading amount of the chemotherapeutic drugs accounts for 10%-40% of the mass of the shell layer; the chemotherapeutic drugs are chemotherapeutic drugs containing conjugated anthracene rings or benzene rings; and the acid-responsive reversible polymer is formed by polymerization of tannic acid and tetraethylenepentamine.

[0009] The chemotherapy drugs are doxorubicin and mitoxantrone.

[0010] The nanoparticles with hypoxia responsiveness that encapsulate the differentiation inducing agent are prepared by encapsulating the differentiation inducing agent in the nanoparticles with hypoxia responsiveness by a single emulsion method; wherein the differentiation inducing agent accounts for 5%-10% of the mass of the nanoparticles with hypoxia responsiveness; the nanoparticles with hypoxia responsiveness are assembled from hyaluronic acid polymers grafted with nitroimidazole; the grafting degree of the hyaluronic acid polymers grafted with nitroimidazole is 10%; and the differentiation inducing agent is all-trans retinoic acid.

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

[0012] The polytannic acid shell modulates the release characteristics of the drug, giving it a pulsed drug release capability and promoting apoptotic body-mediated penetration. Specifically, the polytannic acid shell can reversibly respond to the acidic environment of the lysosome, become protonated and swell, thereby releasing the chemotherapeutic drug and mediating lysosomal escape. In the cytoplasm, neutral conditions deprotonate and shrink the shell, thereby stopping the release of the drug.

[0013] The chemotherapy drug is loaded in the polytannic acid shell through hydrogen bonds or π-π interactions.

[0014] The hyaluronic acid polymer grafted with hydrophobic nitroimidazole groups forms nanoparticles, which can be responsively converted into hydrophilic aminoimidazole under hypoxic conditions, resulting in the disintegration of the nanoparticles and the release of the encapsulated cargo; the nitroimidazole-grafted hyaluronic acid polymer is obtained by reacting hyaluronic acid with 6-(2-nitroimidazole)hexylamine.

[0015] The differentiation inducing agent is preferably all-trans retinoic acid, which can promote the differentiation of tumor stem cells and reduce their stemness and malignancy. At the same time, all-trans retinoic acid can also differentiate myeloid-derived suppressor cells into mature myeloid cells, such as dendritic cells and macrophages, to enhance immune response.

[0016] The core-shell sequential drug release system can respond to the pH gradient of lysosome-cytoplasm, thereby triggering the intracellular pulse release of doxorubicin, causing cell apoptosis and generating apoptotic bodies. The remaining doxorubicin is stored in the polymer shell of the sequential pulse drug release system and delivered to the deep part via the osmotic effect mediated by the apoptotic body. In response to the hypoxic characteristics of the deep area of ​​the tumor, the nanocore cleaves and releases all-trans retinoic acid, targeting the differentiation of tumor stem cells and myeloid-derived suppressor cells accumulated in the deep part.

[0017] A method for preparing a core-shell structure sequential drug release system for cascade tumor deep penetration and differentiation induction therapy:

[0018] Step (1): Preparation of the inner core: dissolve the hypoxia-responsive nanoparticles in the aqueous phase and set aside; dissolve the differentiation inducer in chloroform and slowly and uniformly drip it into the aqueous phase under ultrasonic treatment; remove the organic solvent in the mixture by suspension evaporation and then freeze-dry to obtain a solid inner core.

[0019] Step (2): Assembling the core and the shell: dissolving the core obtained in step (1) in Tris-HCl at pH 8.5 and a concentration of 10 mM, adding an acid-responsive reversible polymer to obtain a mixed solution, and centrifuging to remove impurities to obtain a solution in which the core-shell structure is dispersed;

[0020] Step (3): Loading of chemotherapy drugs: adding chemotherapy drugs to the solution dispersed with the core-shell structure obtained in step (2), and removing impurities by ultrafiltration after reaction, thereby obtaining a sequential drug release 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 as follows: ultrasonic power 100-150w, and ultrasonic time 10-20 minutes.

[0023] In the 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, stirred, 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 the chemotherapy drug to the system is 0.05:1-0.2:1, the reaction conditions are 15-25° C., protected from light, stirred, and the reaction time is 2-2-8 hours.

[0025] An application of the core-shell structure sequential drug release system for cascade tumor deep penetration and induced differentiation treatment, and the application of the core-shell structure sequential drug release system in promoting deep delivery of anti-tumor drugs and differentiation treatment of solid tumors.

[0026] The core-shell structure sequential drug release system is used in the preparation of breast cancer drugs, and the breast cancer drugs target 4T1 cells.

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

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

[0029] (2) The core-shell sequential drug release system of the present invention can respond to endogenous cell stimulation and release, uptake and lysosomal escape behavior, penetration performance in tumor tissue, differentiation of tumor stem cells and bone marrow-derived suppressor cells, and in vivo anti-tumor effect. Specifically, the core-shell structure characterization, response and drug release under weak acid and hypoxic conditions, tumor cell uptake experiments, lysosomal escape experiments, cytotoxicity experiments, tumor sphere penetration experiments, tumor stem cell differentiation, in vivo anti-tumor efficacy and in vivo anti-tumor immune evaluation were carried out. The results showed that the core-shell sequential drug release system responded to the pH gradient change of the intracellular lysosome-cytoplasm, and its shell reversibly swelled and contracted, accompanied by protonation and deprotonation processes, thereby achieving the pulsed release of doxorubicin. By adjusting the release performance of the drug, the core-shell sequential drug release system increased the proportion of intracellular active drugs, promoted the deep delivery mediated by apoptotic bodies, and thus achieved overall tumor killing. At the same time, the hypoxia-responsive hyaluronic acid core does not release drugs in the normoxic area, but after entering the hypoxic area of ​​the tumor with the help of apoptotic bodies, its nitroimidazole group is reduced to aminoimidazole, thereby releasing all-trans retinoic acid to differentiate tumor stem cells and myeloid-derived suppressor cells. Therefore, the hypoxia-responsive core can prevent all-trans retinoic acid from leaking prematurely in the normoxic area of ​​the tumor, and improve its targeted differentiation efficacy deep in the tumor. The sequential release drug delivery system of the present invention solves the problem of heterogeneous distribution of different cells in the tumor, provides new strategies and more options for the application of chemoimmunotherapy in the treatment of solid tumors, and meets the urgent clinical demand for efficient anti-tumor preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1The preparation and characterization experiment diagram of the core-shell structure sequential drug release system in Example 1 of the present invention

[0031] a: Transmission electron microscopy images 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: FTIR spectra of HNI, HNI@TT, DOX, and HNI@D-TT.

[0036] Figure 2 This is a graph showing the stability study results of the core-shell structure sequential drug release system of Example 2 of the present 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 is a diagram showing the reversible acid response and oxygen response capabilities of the core-shell structure sequential drug release system of Example 3 of the present invention.

[0040] a: Particle size changes 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 normoxic and hypoxic conditions.

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

[0044] e: Fret ratio change.

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

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

[0047] b: ATRA release curves of HRA@D-TT in different pH buffers under hypoxia or normoxia.

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

[0049] a: Confocal images of cellular uptake of the pulse sequential drug release system at different time points.

[0050] b: Flow cytometric results of cellular uptake of the pulse sequential drug release system at different time points.

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

[0052] a: Confocal image of lysosomal escape of the pulse sequential drug release system for 8 hours.

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

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

[0055] Figure 8 This is the extraction and characterization result of apoptotic bodies in Example 8 of the present invention.

[0056] a: Transmission electron microscopy image of apoptotic bodies.

[0057] b: Annexin V positive ratio of apoptotic bodies.

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

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

[0060] Fig. 9 This is a diagram showing the in vitro penetration verification results based on apoptotic bodies according to Example 9 of the present invention.

[0061] a: Confocal image of apoptotic body-mediated cell-to-cell drug delivery.

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

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

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

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

[0066] Fig.10 This is a graph showing the results of the tumor sphere penetration experiment in Example 10 of the present invention.

[0067] a: Confocal image of in vitro tumor sphere penetration by pulse sequential drug release system.

[0068] b: The corresponding quantitative results of tumor sphere permeation fluorescence by ImageJ.

[0069] Fig.11 This is a graph showing the apoptosis ratio of tumor stem cells after being treated with different preparations in Example 11 of the present invention.

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

[0071] a: CD44 after treatment with different preparations + / CD24 - Representative flow cytometry results of cell populations.

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

[0073] c: Yes Fig.12 Flow cytometry results of a.

[0074] d: Yes Fig.12 b Flow cytometry results.

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

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

[0077] Fig.13 This is a graph showing the in vivo distribution and permeation test results of the preparation of Example 13 of the present invention.

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

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

[0080] c: Fluorescence semi-quantitative images of major organs and tumors of mice 24 hours after injection of different DiR-labeled preparations.

[0081] d: Fluorescence quantification 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 after 24 h of treatment with DOX, HNI@D-TT, and HNI@D-TA.

[0083] Fig.14 This is a pharmacodynamic study of the core-shell structure sequential drug release system of Example 14 of the present invention.

[0084] a: Volume growth curve of mouse tumors after intravenous injection of different preparations.

[0085] b: Weight of excised 4T1 tumors after treatment with different preparations.

[0086] c: Photographs of exfoliated 4T1 tumors after treatment with different preparations.

[0087] d: Body weight changes of mice during the treatment period.

[0088] e: CD44 in 4T1 tumors after treatment with different agents + / CD24 - Flow cytometric quantification of cell populations.

[0089] f: CD133 in 4T1 tumors after treatment with different agents + Flow cytometric quantification of cell populations.

[0090] g: CD44 in 4T1 tumors after treatment with different agents + / CD24 - Representative flow cytometry analysis of cell populations.

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

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

[0093] Fig.15 This is an H&E staining image of the main organs of mice after intravenous injection of different preparations according to Example 14 of the present invention.

[0094] Fig.16 This is a graph of the in vivo anti-tumor immunity evaluation of the core-shell structure sequential drug release system of Example 15 of the present invention.

[0095] a: Volume growth curve of mouse tumors after intravenous injection of different preparations.

[0096] b: Weight of dissected 4T1 tumors after treatment with different preparations.

[0097] c: Photographs of dissected 4T1 tumors after treatment with different agents.

[0098] d: Body weight changes of mice during the treatment period.

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

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

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

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

[0103] i: CD8 in 4T1 tumors of mice after receiving different treatments + Representative flow cytometry analysis and quantitative results of T. DETAILED DESCRIPTION

[0104] The specific implementation modes of the present invention are further described below in conjunction with examples. It should be noted that the specific implementation modes described here are only for illustrating and explaining the present invention, and are not limited to the present invention.

[0105] The core-shell structure sequential drug release system of the present invention is used to deeply deliver differentiation inducers and overcome tumor anti-treatment barriers. The differentiation inducer is encapsulated in hypoxia-responsive polymer nano-micelles, and a reversible acid-responsive polymer shell layer loaded with chemotherapeutic drugs is wrapped on the outside to form a pulse sequential drug release system. The prepared sequential pulse drug release system has high drug loading, good stability, can achieve deep penetration of tumor tissues, effectively promote the differentiation of tumor stem cells and bone marrow-derived suppressor cells, and thus achieve enhanced tumor killing and anti-tumor immune activity.

[0106] Example 1: Preparation and characterization of core-shell sequential drug release system

[0107] The hypoxia-responsive nanocore was prepared by a single emulsion method. Specifically, 13 mg of nitroimidazole-grafted hyaluronic acid polymer (HNI, which was prepared according to the method mentioned in Glucose-Responsive Insulin Delivery by Microneedle-Array Patches Loaded with Hypoxia-Sensitive Vesicles to obtain a grafting amount of 7%-12%) was dissolved in deionized water and used as the aqueous phase. 2 mg of all-trans retinoic acid (ATRA) was dissolved in chloroform and slowly and uniformly dripped into 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 membrane to remove the unencapsulated drug, obtaining a uniform and clear ATRA-loaded nanomicelle solution (HRA).

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

[0109] At the same time, in the above preparation process, the pure tannic acid shell formed by oxidative polymerization of tannic acid monomers under alkaline conditions was used as a control for irreversible acid response (HRA@D-TA). In addition, in order to facilitate the observation of the role of the shell, TA-TEPA binary core-shell nanoparticles loaded only with DOX (HNI@D-TT) were prepared. The specific method is to dissolve 13 mg of HNI in deionized water, then add 2 mg of TA and 5 mg of TEPA to the system, stir overnight in the dark, and remove the unreacted compounds by multiple centrifugation washings. 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. Figure 1 All nanoparticles are uniformly spherical and covered with a shell layer on the outside, proving the successful synthesis of the core-shell structure. The particle size change before and after the shell layer was analyzed by Malvern particle size analyzer. The results are shown in Figure 1 b. The particle size of HRA is about 130 nanometers. After being coated with the shell, the particle size increases to about 160 nanometers, accompanied by an increase in surface potential. The loading of doxorubicin was verified by fluorescence spectroscopy. The results are shown in Figure 1 c. Compared with the doxorubicin solution of the same concentration, doxorubicin in HNI@D-TT showed a significant fluorescence quenching effect, indicating that doxorubicin may be embedded in the shell of polytannic acid through π-π interaction. In addition, 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 core-shell sequential drug release system

[0112] The 1 mg / mL HRA@D-TT and HRA@D-TA obtained above 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 the change in particle size. In addition, the long-term stability of the formulation was evaluated 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 ambient conditions.

[0113] Example 3: Study on the in vitro reversible acid response and hypoxia response performance of the core-shell sequential drug release system

[0114] In order to verify the reversible acid response characteristics of the preparation, 1 mg / mL HRA@D-TT and HRA@D-TA prepared in Example 1 were placed in PBS at pH 7.4 and pH 5.0 repeatedly, and incubated for 8 hours each time. The particle size change and potential change of the two were measured by Malvern particle size analyzer, and the results were as follows: Figure 3 a and Figure 3 As shown in Figure 2b. Compared with the tannic acid coating alone, HRA@D-TT showed a reversible size change from 160 nm at pH 7.4 to nearly 300 nm at pH 5.0, and the surface potential showed a higher sensitivity to increased acidity. This indicates that HRA@D-TT has good reversible acid response properties under different pH conditions, further proving that it can effectively respond to environmental pH changes and achieve pulsed drug release during lysosome-cytoplasm transport.

[0115] To verify the hypoxia responsiveness of the nanocore, HNI polymer was dissolved in PBS and 10 mM sodium dithionite was added to create a hypoxic environment. After incubation for 8 h, UV spectroscopy analysis was performed. Figure 3 As shown in c, after incubation under hypoxic conditions, the nitro absorption peak at 325nm disappeared, while the amino absorption peak appeared at 278nm, indicating that hypoxia induced a hydrophilic transformation of the micelle structure, resulting in its cleavage, and the hypoxia response ability of the preparation was verified by FRET experiments. In order 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). The specific method is to dissolve HNI in deionized water for use. DiI and DiO were dissolved in ethanol and added dropwise to the HNI aqueous solution, stirred for 24h and then sonicated for 10min. The resulting 20mg mixture was then lyophilized and resuspended in Tris-HCl buffer at pH 8.5, and 2mg TA and 5mg TEPA were added to obtain HNI-(DiI+DiO)@TT. Under normoxic or simulated hypoxic conditions, 4T1 cells were plated in 24-well plates (2×104 Cells were cultured overnight in 4% paraformaldehyde (400 μg / mL) and then further treated with HNI-(DiI+DiO)@TT at a dye concentration of 2 μg / mL for 8 hours. Excess micelles were removed by washing the cells three times with cold PBS, followed by fixation with 4% formaldehyde and staining with Hoechst 33342. Observation was performed by confocal microscopy: fluorescence was excited with an excitation wavelength of 488 nm, a 505-550 nm filter was used to receive green emission light, and a 560-615 nm filter was used to receive red (FRET) emission light. In addition, cell fluorescence was quantitatively evaluated by ImageJ. Figure 3 As shown in Figures 3d and 3e, it can be observed that under hypoxic conditions, the FRET efficiency is significantly reduced, indicating that the distance between DiI and DiO increases, thus verifying the responsiveness of HNI-(DiI+DiO)@TT nanoparticles under hypoxic conditions. This further proves that the system can effectively release encapsulated drugs under hypoxic conditions, providing strong support for its application in tumor microenvironment.

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

[0117] To verify 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 PBS containing 2% Tween 80. To evaluate the pH-induced DOX release, the release medium was adjusted to a series of acidity gradients (pH 7.4, 6.8 and 5.0). In addition, a hypoxic system was established by adding 10 mM sodium dithionite to the above-mentioned buffers with different acidity gradients to evaluate the hypoxia-responsive release of ATRA. 200 μL samples were taken out at predetermined time intervals (i.e. 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h) and immediately replenished with the same volume of fresh release medium. The release of DOX and ATRA from the nanoplatform was monitored by fluorescence spectrometer and HPLC, respectively. As Figure 4 a and Figure 4 As shown in (b), at pH 7.4, less than 20% of DOX leaked from HRA@D-TT, minimizing the systemic toxicity of burst release. However, when the pH of the medium was lowered to 5.0, the drug release increased significantly by about 3 times. In addition, HRA@D-TT showed excellent hypoxia sensitivity in a hypoxic environment, promoting the rapid release of ATRA. In particular, under the combined stimulation of hypoxia and high acidity, the release of ATRA significantly exceeded the release triggered by hypoxia alone, indicating that the structure of the polymer shell limits the release of ATRA from the core. The enhanced release can be attributed to the porosity of the surface at low pH, which gives the nanoplatform excellent ability for rapid drug release under specific conditions.

[0118] Example 5: Cellular uptake experiment

[0119] In order to visualize the intracellular process of the preparation, nanoparticles loaded with DOX only (HNI@D-TT and HNI@D-TA) were prepared. 4T1 cells were seeded in 24-well plates (2×10 4 cells) and cultured for 12 hours to allow cells to adhere. Subsequently, the 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 hours and 8 hours, respectively. After incubation, the drug-containing culture medium was discarded and washed three times with ice-cold PBS (pH 7.4) to terminate the cell uptake process. The cells were fixed with 4% paraformaldehyde for 10 minutes, and the cell nuclei were stained with Hoechst 33342 at 37°C for 10 minutes and observed by confocal microscopy. For flow cytometric quantitative analysis of cell uptake, 24-well plates were used for culture. After drug administration, the cells were digested with 0.05% trypsin, the cells were collected by centrifugation, and the cells were evenly dispersed in PBS (pH 7.4). The cell uptake was quantitatively analyzed by flow cytometry. Figure 5 As shown in Figures 5a and 5b, the group treated with DOX solution showed rapid fluorescence colocalization and higher fluorescence intensity in the cell nucleus due to unregulated drug release. In addition, the internalization efficiency of HNI@D-TT was 1.5 times that of HNI@D-TA, which may be 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 × 10 4 cells) and cultured for 12 hours to allow the cells to adhere. Subsequently, the cells were treated with fresh culture medium containing 5 μg / mL DOX equivalent doses of HNI@D-TT and HNI@D-TA for 4 hours and 8 hours, respectively. After the incubation, the drug solution was discarded and washed three times with ice-cold PBS (pH 7.4) to terminate the cell uptake process. After staining the lysosomes with Lysotracker-red, wash three times with ice-cold PBS (pH 7.4), add cells and fix with 4% paraformaldehyde for 10 minutes, and stain the cell nuclei with Hoechst 33342 for 10 minutes at 37°C, and observe by confocal microscopy. Figure 6 As shown in Figures 6a and 6b, after treatment with HNI@D-TT, the colocalization coefficient of red fluorescence and green fluorescence decreased, indicating that doxorubicin successfully escaped into the cytoplasm. However, when treated with HNI@D-TA, most of the red fluorescence overlapped with the green fluorescence, indicating that the simple tannic acid coating could not achieve the ideal lysosomal escape effect.

[0122] Example 7: Cytotoxicity Experiment

[0123] To evaluate in vitro cytotoxicity against tumor cells, 4T1 cells were seeded in 96-well plates at a density of 2000 cells per well. After 12 h, the culture medium was replaced with gradient dilutions containing DOX, HNI@D-TT, HNI@D-TA, and HRA@D-TT. After incubation in normoxia or hypoxia for 48 h, MTT solution was added and incubated at 37 °C for 4 h. To dissolve the produced formazan crystals, 200 μL DMSO was added to each well, and the absorption at 570 nm was detected using a microplate reader. IC was determined using GraphPad Prism 8 using molar concentration and cell viability ratio as reference values. 50 Value. Figure 7 As shown in Figure 2, the inhibitory ability of the agents on tumor cell viability follows the order of DOX>HNI@D-TT>HNI@D-TA. In addition, when cultured under normoxic conditions, cells treated with HRA@D-TT showed similar toxicity differences compared with cells treated with HNI@D-TT, with an IC 50 The values ​​were 277.9 nM and 253.6 nM, respectively. In contrast, incubation under hypoxia significantly enhanced the cytotoxicity of HRA@D-TT, with an IC 50 The difference in toxicity caused by different incubation environments further confirmed that HNI micelles could disintegrate under hypoxic conditions, release ATRA and exert synergistic anti-tumor advantages.

[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 h. After incubation with fresh medium for 12 h, the culture was harvested and centrifuged at 100 g for 10 min to eliminate cell debris. The resulting supernatant was then centrifuged at 1000 g for 15 min to isolate apoptotic bodies. The morphology of apoptotic bodies was observed using transmission electron microscopy, and the results showed that apoptotic bodies were spherical vesicles with a diameter of approximately 1.5 μm ( Figure 8 a). Since Annexin V can specifically label externalized phosphatidylserine in apoptotic cells, we used the Annexin V-FITC / PI kit to determine the positive ratio 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 showed a high annexin V positive ratio, with high purity. In addition, we performed Western blotting experiments to verify the expression of characteristic proteins. Similar to 4T1 cells, apoptotic bodies also expressed similar levels of membrane markers CD44 and CD326. However, due to the high degree of apoptosis, it showed high expression of cleaved caspase-3 ( Figure 8c).

[0126] To study the cellular uptake of apoptotic bodies, 4T1 cells were incubated with apoptotic bodies for 8 h. 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 can be internalized by 4T1 cells, indicating that the apoptotic bodies can serve as a reservoir for the remaining drug in the cells and be taken up by surrounding cells.

[0127] Example 9: In vitro permeation assay mediated by apoptotic bodies

[0128] 4T1 cells were seeded in a 6-well plate and incubated overnight, denoted as plate (i). The cells in plate (i) were treated with 5 μg / mL DOX equivalent doses of free DOX, HNI@D-TT and HNI@D-TA under normoxic conditions for 8 hours, and then continued to incubate in fresh culture medium for 12 hours to induce cell apoptosis. Then, apoptotic bodies (i-ii) were collected from the supernatant and incubated with untreated fresh cells for 8 hours, denoted as plate (ii). This process was repeated three times to obtain plate (iii). After washing the cells, they were stained with Hoechst33342 and observed by fluorescence confocal microscopy to evaluate the internalization of DOX. Fig. 9 As shown in (a), cells treated with DOX solution or HNI@D-TA showed weak red fluorescence in the second incubation cycle and the fluorescence disappeared in the third cycle, indicating their limited delivery capacity to neighboring cells. In contrast, cells in plate (iii) treated with HNI@D-TT still showed obvious fluorescence signals, although the fluorescence intensity gradually decreased, which was attributed to the controlled pulse release of DOX promoting the retention of the remaining drug.

[0129] To evaluate cell apoptosis, the treated cells were stained with Annexin V-FITC / PI kit, and the apoptotic rate was examined by flow cytometry. Fig. 9 As shown in (b), HNI@D-TT-treated cells still exhibited an 11% apoptosis rate in the third incubation cycle, indicating a satisfactory delivery effect. In addition, although the DOX solution initially induced extensive apoptosis, its excessive consumption greatly impaired the effectiveness of subsequent treatment.

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

[0131] To determine the drug retention in apoptotic bodies, coumarin 6 was encapsulated into the anoxic core of HNI@D-TT and incubated with the cells in well (i). After repeating the above steps three times, apoptotic bodies (i-ii) and (ii-iii) were extracted from the cell culture and observed by confocal microscopy after dropping onto a glass slide. Fig. 9 As shown in Figure e, the overlapping fluorescence signals between red and green confirmed the co-encapsulation of DOX and coumarin 6 in the apoptotic bodies. However, as the intercellular transport continued, the depletion of DOX led to a decrease in the red fluorescence within the apoptotic bodies (ii-iii), while the fluorescence of coumarin 6 remained stable. This indicates that the HNI micelles maintained their intact structure under normoxic conditions.

[0132] Example 10: Tumor sphere penetration experiment

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

[0134] Example 11: Tumor stem cell apoptosis experiment

[0135] First, tumor stem cells were enriched by low-adhesion serum-free culture. 4T1 cells were seeded in ultra-low-adhesion 6-well plates (2×10 4cells) and 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 formed spheres were collected by low-speed centrifugation.

[0136] Next, in order to explore whether the pulsed sequential drug release system based on the core-shell structure would have an effect on tumor cells deep in the tumor, 4T1 cells were incubated with DOX, HNI@D-TT, HNI@D-TT+ATRA, and HRA@D-TT at equivalent doses of 5μg / mL DOX and 8μg / mL ATRA for 8 hours, and then the apoptotic bodies produced in each treatment group were extracted and re-cultured with enriched tumor stem cell spheres under hypoxic conditions for 48 hours. The treated cells were stained with the Annexin V-FITC / PI kit, and the cell apoptosis rate was examined by flow cytometry. Fig.11 As shown in Figures 1a and 11c, the HRA@D-TT group showed excellent anti-tumor stem cell efficacy in all treatments. In addition, HNI@D-TT also inhibited the survival of tumor stem cells due to the toxicity of the remaining DOX in HNI@D-TT. Compared with HRA@D-TT, the cytotoxicity caused by HNI@D-TT+ATRA treatment was low, while HRA@D-TT had high cytotoxicity, which highlights the effectiveness of HNI nanocores in preventing ATRA depletion in the normoxic region.

[0137] Example 13: Tumor stem cell differentiation experiment

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

[0139] In addition, Western blotting was used to evaluate the expression levels of SOX2, Nanog, and Oct4 proteins in 4T1 cells after treatment with different apoptotic bodies. Fig.12As 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 the nano-strategy of cascading tumor penetration and differentiation therapy in eliminating cancer stem cells.

[0140] Example 14: In vivo distribution and tumor penetration experiments

[0141] First, core-shell nanoparticles containing DiR were prepared according to the method in Example 1 to indicate its in vivo process. The specific method is to dissolve HNI in deionized water for standby use, dissolve DiR in ethanol, and add it dropwise to the HNI aqueous solution, stir for 24 hours, and then ultrasonicate for 10 minutes to obtain the nanocore containing DiR. Then, the obtained 20 mg 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 HDiR@TT. Next, 1 mL of DMSO solution containing 2 mg DOX was added dropwise to the above system, and after stirring for 4 hours, the free drug was removed by multiple washing and ultrafiltration to obtain HDiR@D-TT. At the same time, the control group nanoparticles were prepared by self-polymerization of tannic acid monomers under alkaline conditions, and DOX was loaded in the same way.

[0142] To study the biodistribution of HRA@D-TT, 1×10 6 4T1 cells were used to establish a 4T1 tumor xenograft mouse model. 3 After the tumor volume reached 100 μg / kg, the 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 the predetermined time points, the DiR-labeled nanoformulations were imaged using an in vivo imaging system. Fig.13 As shown in a, HDiR@D-TT has a higher fluorescence intensity at the tumor site compared with the DiR solution. After 24 h of injection, the fluorescence at the tumor site of the HDiR@D-TT group still existed, indicating that the preparation has good tumor targeting and accumulation effects. In addition, the mice were euthanized, and the main organs (heart, liver, spleen, lungs, and kidneys) as well as tumors were collected for fluorescence imaging. Fig.13 As shown in Figures 13b and 13c, the nanoparticles were mainly accumulated in the liver, spleen, and tumors.

[0143] Next, the penetration effect of the formulations into tumor tissues 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. After 24 hours, the mice were euthanized, and the tumor tissues were collected. The tumor sections were then stained with DAPI and CD31 and observed using a confocal microscope. Fig.13As shown in Figures 13d and 13e, the DOX fluorescence distribution in the tumor of the HNI@D-TT-treated group was wider, indicating that HNI@D-TT overcame the penetration barrier and achieved the best penetration efficiency. Although HNI@D-TA showed similar tumor accumulation as HNI@D-TT, its distribution was limited to the perivascular area. The DOX solution showed the worst tumor penetration, which was attributed to its poor tumor targeting and accelerated in vivo metabolism.

[0144] Example 15: Pharmacodynamics Study

[0145] Subcutaneously inject 100 μL containing 1 × 10 6 PBS solution of 4T1 tumor cells was used to construct a 4T1 tumor-bearing mouse model. When the tumor grew to about 100 mm 3 At the time of the study, 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 the 11-day treatment period, mice received intravenous injections on days 0, 3, 6, and 9, with each dose containing an equivalent concentration of 2 mg / kg of DOX and 3 mg / kg of ATRA. The tumor volume and body weight of the mice were measured every day. After the treatment, the tumor tissue was removed and photographed. Fig.14 As shown in bd, the tumor growth rate of the HNI@D-TT treatment group was the slowest, indicating that it can effectively inhibit tumor growth. During the entire treatment period, the weight loss of the doxorubicin solution group was more obvious, indicating that it has systemic toxicity, while HNI@D-TT has better biosafety ( Fig.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 fluorescent antibodies CD44-PE and CD24-FITC at 4°C for 40 minutes. Fig.14 As shown in eh, HRA@D-TT significantly reduced the expression of CD44 + / CD24 - and CD133 + In addition, after the treatment, the tumor tissues were stained with H&E and the apoptosis and proliferation levels of tumor cells were evaluated by TUNEL and Ki67 immunofluorescence staining. Fig.14 As shown in Fig. i, HRA@D-TT can induce the greatest degree of tumor tissue apoptosis. In order to evaluate the biocompatibility of the nano-drug delivery system, H&E staining was performed on the main organs after the treatment. Fig.15 As shown in the figure, HRA@D-TT has good safety and no obvious damage to its major organs.

[0146] Example 16: In vivo anti-tumor immunity assessment

[0147] We established a 4T1 tumor-bearing mouse model and injected 2×10 5 4T1-Luc cells were injected intravenously into mice to simulate the hematogenous metastasis of advanced tumors. The mice were randomly divided into six groups and treated as follows: G1 (normal 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 doses of 2 mg / kg and ATRA doses of 3 mg / kg were injected intravenously on days 0, 3, 6 and 9. aPD-1 antibody (100 μg per mouse per injection) was injected intraperitoneally on the day after HRA@D-TT administration (days 1, 4, 7 and 10). Tumor size and body weight were recorded every day. Fig.16 As shown in Figures ac, the combined treatment of HRA@D-TT and aPD-1 had the best therapeutic effect and achieved tumor regression. In addition, the weight of mice did not decrease during the entire treatment period, indicating that the combined therapy has good biosafety ( Fig.16 d)

[0148] After treatment, all mice were killed and the lungs, tumor-draining lymph nodes, and tumor tissues were sampled. The harvested lungs were immersed in D-luciferin solution (15 mg / mL) for 10 minutes and then subjected to bioluminescence imaging to observe the invasion of malignant cells into the lungs. At the same time, H&E staining was also performed on the lung tissues to evaluate pathological changes. Fig.16 As shown in Figures ef, 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 evaluate changes in the level of immune response, tumors collected from mice were separated into single cells using digestive enzymes. In addition, cell suspensions from lymph nodes were prepared by grinding. After filtering through a 70 μm filter membrane, the cells were stained with various fluorescent antibodies, and immune cell populations in tumors and lymph nodes were determined by flow cytometry and analyzed with Flow Jo. Fig.16 As shown in gi, HNI@D-TT+aPD-1 significantly reduced the proportion of myeloid-derived suppressor cells in tumors and increased the number of dendritic cells. In addition, treatment increased tumor-infiltrating CD8 + The number of T cells indicated that HRA@D-TT could promote the improvement of anti-tumor immunity by reversing the intratumor immunosuppressive microenvironment.

[0150] In summary, the core-shell sequential drug release system for cascade deep tumor penetration and differentiation induction therapy of the present invention has an acid-responsive reversible polymer shell layer and is loaded with cytotoxic drugs, so that the sequential drug release system can respond to pH changes during the lysosome-cytoplasm transport process and pulse release the drug to promote the deep penetration of the drug. At the same time, the hypoxia-responsive polymer nanoparticles encapsulating the differentiation inducing agent are internally wrapped to achieve deep tumor-specific release of the differentiation inducing agent.

Claims

1. A core-shell sequential drug release system for cascade tumor deep penetration and differentiation induction therapy, characterized in that: The system is a core-shell structure, wherein the core is a hypoxia-responsive nanoparticle encapsulating a differentiation-inducing agent, and the shell is an acid-responsive reversible polymer loaded with a chemotherapeutic drug.

2. The core-shell sequential drug release system for cascade tumor deep penetration and differentiation induction therapy according to claim 1, characterized in that: The acid-responsive reversible polymer loaded with chemotherapeutic drugs is formed by loading the chemotherapeutic drugs on the surface of the acid-responsive reversible polymer by stirring, wherein the loading amount of the chemotherapeutic drugs accounts for 10%-40% of the mass of the shell layer; the chemotherapeutic drugs are chemotherapeutic drugs containing conjugated anthracene rings or benzene rings; and the acid-responsive reversible polymer is formed by polymerization of tannic acid and tetraethylenepentamine.

3. The core-shell structure sequential drug release system for cascade tumor deep 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 release system for cascade tumor deep penetration and differentiation induction therapy according to claim 1, characterized in that: The nanoparticles with hypoxia responsiveness that encapsulate the differentiation inducing agent are prepared by encapsulating the differentiation inducing agent in the nanoparticles with hypoxia responsiveness through a single emulsion method; wherein the differentiation inducing agent accounts for 5-10% of the mass of the nanoparticles with hypoxia responsiveness; the nanoparticles with hypoxia responsiveness are assembled from hyaluronic acid polymers grafted with nitroimidazole; the grafting degree of the hyaluronic acid polymers grafted with nitroimidazole is 7%-12%; and the differentiation inducing agent is all-trans retinoic acid.

5. A method for preparing the core-shell sequential drug release system for cascade tumor deep penetration and differentiation induction therapy according to claim 1, characterized in that Step (1): Preparation of the inner core: dissolving the nanoparticles with hypoxia responsiveness in the aqueous phase for later use; dissolving the differentiation inducer in chloroform and slowly and uniformly dripping it into the aqueous phase under ultrasonic treatment; removing the organic solvent in the mixture by suspension evaporation and then freeze-drying to obtain a solid inner core; Step (2): Assembling the core and the shell: dissolving the core obtained in step (1) in Tris-HCl at a pH of 8.5 and a concentration of 10 mM, adding an acid-responsive reversible polymer to obtain a mixed solution, and centrifuging to remove impurities to obtain a solution in which the core-shell structure is dispersed; Step (3): Loading of chemotherapy drugs: adding chemotherapy drugs to the solution dispersed with the core-shell structure obtained in step (2), and removing impurities by ultrafiltration after reaction to obtain a sequential drug release system of the core-shell structure.

6. The method for preparing the core-shell structure sequential drug release system for cascade tumor deep 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 sequential drug release system for cascade tumor deep penetration and differentiation induction therapy according to claim 5, characterized in that: The reaction conditions in step (1) are as follows: ultrasonic power 100-150w, and ultrasonic time 10-20 minutes.

8. The method for preparing the core-shell sequential drug release system for cascade tumor deep penetration and differentiation induction therapy according to claim 5, characterized in that: In the 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, stirred, 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 method for preparing the core-shell sequential drug release system for cascade tumor deep penetration and differentiation induction therapy according to claim 5, characterized in that: In step (3), the mass ratio of the chemotherapy drug to the system is 0.05:1-0.2:1, the reaction conditions are 15-25° C., protected from light, stirred, and the reaction time is 2-2-8 hours.

10. An application of the core-shell structure sequential drug release system for cascade tumor deep penetration and differentiation induction therapy according to claim 1, characterized in that: The core-shell structure sequential drug release system is used to promote the deep delivery of anti-tumor drugs and the differentiation treatment of solid tumors.

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