ZIF-8-based intelligent nano bionic drug delivery system, preparation method and application

Abstract

The invention belongs to the technical field of tumor drugs, and particularly relates to an intelligent nano bionic drug delivery system based on ZIF-8, a preparation method and application, adriamycin, DNAzyme and an aptamer are loaded on ZIF-8, and the preparation method comprises the steps that after adriamycin is loaded to a ZIF-8 nano carrier, DNAzyme is further adsorbed by means of electrostatic interaction, and the ZIF-8 nano carrier is loaded with the DNAzyme; the intelligent nano bionic drug delivery system based on ZIF-8 is characterized in that a DOX / Dz-coated ZIF-8-RM / Apt nano drug delivery system is prepared by using a DOX / Dz-coated ZIF-8-RM / Apt nano drug delivery system, then using an erythrocyte membrane to perform surface modification, and finally inserting an aptamer drug into the surface of the cytomembrane through a lipid insertion method, and the intelligent nano bionic drug delivery system based on ZIF-8 is used for relieving chemotherapy drug resistance and improving the anti-tumor immunity efficiency; and the effect of the medicine in the tumor is improved to the greatest extent.

Description

Technical Field

[0001] This invention belongs to the field of tumor drug technology, specifically relating to a ZIF-8-based intelligent nanobiomimetic drug delivery system, its preparation method, and its application. Background Technology

[0002] Immunotherapy is a novel cancer treatment method following surgery, radiotherapy, and chemotherapy. It controls and destroys tumor cells by activating or enhancing the immune response, thereby strengthening or normalizing the body's immune system. Cancer immunotherapy has revolutionized the treatment of many solid tumors and hematologic malignancies, bringing benefits to cancer patients, especially those with advanced malignancies and multidrug-resistant cancers, fundamentally transforming cancer treatment.

[0003] Currently, chimeric antigen receptor T-cell immunotherapy and immune checkpoint blockade (ICB) are the most effective tumor immunotherapies in clinical practice. In recent years, an increasing number of cancer patients have benefited from ICB therapy. ICBs have shown significant promise in the clinical treatment of various cancers, working by repairing deficient immune responses. Chen Liping et al. discovered a novel immune checkpoint—Siglec-15 (sialic acid-binding immunoglobulin-like lectin-15)—that can inhibit antigen-specific T-cell responses. This checkpoint can directly reverse T-cell suppression and can be used for immunotherapy. However, due to the high heterogeneity of tumors and the redundancy of immune escape mechanisms, the overall response rate remains below ideal levels. Furthermore, strong clinical evidence suggests that patients with poor responses to ICB treatment are closely associated with insufficient T-cell infiltration and low immunogenicity.

[0004] Inducing immunogenic cell death (ICD) in tumor cells is an effective cancer treatment method that can enhance the efficacy of immune checkpoint blockade therapy by remodeling the immunosuppressive tumor microenvironment, making it possible to combine immunotherapy with many ICD-inducing modalities, such as chemotherapy. The chemotherapy drug doxorubicin (DOX) is an effective ICD inducer, promoting tumor cell death and the release of danger-associated molecular patterns (DAMPs) and tumor-associated antigens (TAAs), thereby leading to in situ activation of anti-tumor immunity. However, systemic administration of chemotherapy drugs can cause severe toxic side effects and drug resistance. Therefore, reducing multidrug resistance to chemotherapy drugs is crucial for effective cancer treatment. DNAzymes, due to their high specificity, high catalytic efficiency, good stability, and low immunogenicity, have great potential in reversing drug resistance. However, gene therapy is limited by poor stability and retention in the tumor microenvironment.

[0005] To this end, a ZIF-8-based nanobiomimetic drug delivery system was developed, which encapsulates chemotherapy drugs, gene therapy drugs, and ICB drugs in ZIF-8, named D / Dz@Z-RM / A, to alleviate chemotherapy resistance and improve the efficiency of anti-tumor immunotherapy, thereby maximizing the therapeutic effect of intratumoral therapy drugs. Summary of the Invention

[0006] To address the problems in the prior art, this invention provides a ZIF-8-based intelligent nanobiomimetic drug delivery system, its preparation method, and its application, aiming to alleviate chemotherapy resistance and improve anti-tumor immune efficiency, thereby maximizing the effect of drugs within tumors.

[0007] The technical problem solved by this invention is achieved by the following technical solution:

[0008] The present invention aims to provide a smart nanobiomimetic drug delivery system based on ZIF-8, comprising loading doxorubicin, DNAzyme and aptamer onto ZIF-8.

[0009] A method for preparing a ZIF-8-based intelligent nanobiomimetic drug delivery system involves loading doxorubicin onto a ZIF-8 nanocarrier, further adsorbing DNAzyme using electrostatic interactions, then modifying the surface with a erythrocyte membrane, and finally inserting an aptamer drug onto the cell membrane surface via lipid intercalation to prepare the DOX / Dz@ZIF-8-RM / Apt (D / Dz@Z-RM / A) nanodrug delivery system.

[0010] Furthermore, the method for loading doxorubicin into the ZIF-8 nanocarrier includes: adding doxorubicin to a 2-methylimidazole solution at room temperature, stirring at 1300 rpm for 5 min, then adding zinc nitrate hexahydrate solution dropwise, stirring at room temperature to obtain a red suspension, centrifuging to obtain a red precipitate, and finally washing with water three times to obtain DOX@ZIF-8 (D@Z).

[0011] Furthermore, the method for adsorbing DNAzyme onto the ZIF-8 nanocarrier loaded with doxorubicin includes: adding DNAzyme to DOX@ZIF-8, stirring at 37 °C for 30 min, centrifuging at 10000 rpm for 10 min, collecting the precipitate, and redispersing the precipitate in deionized water to obtain DOX / Dz@ZIF-8 (D / Dz@Z).

[0012] Furthermore, the method for surface modification of erythrocyte membranes includes: mixing D / Dz@Z and erythrocyte membranes at a mass ratio of 1:5, then sonicating the mixture, and then extruding the mixture sequentially through 400 nm and 200 nm polycarbonate membranes 10 times to obtain DOX / Dz@ZIF-8-RM (D / Dz@Z-RM).

[0013] Furthermore, the method for inserting aptamer drugs onto the cell membrane surface includes: mixing equimolar amounts of pH-Apt and D / Dz@Z-RM by vortexing and incubating at 37 °C for 40 min to obtain D / Dz@Z-RM / A.

[0014] Application of a ZIF-8-based intelligent nanobiomimetic drug delivery system or a method for preparing a ZIF-8-based intelligent nanobiomimetic drug delivery system in the preparation of tumor drugs.

[0015] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0016] 1. The present invention discloses a ZIF-8-based intelligent nanobiomimetic drug delivery system, which loads the chemotherapy drug DOX, the gene therapy drug DNAzyme, and the ICB drug Apt onto ZIF-8, named D / Dz@Z-RM / A, to alleviate chemotherapy resistance and improve the efficiency of anti-tumor immunity, so as to maximize the effect of drugs within the tumor.

[0017] 2. This invention utilizes a one-pot method to load DOX onto a ZIF-8 nanocarrier, further adsorbing DNAzyme via electrostatic interactions. Subsequently, the surface is modified with erythrocyte membranes, and finally, aptamer drugs are inserted into the cell membrane surface via lipid intercalation, thus preparing the D / Dz@Z-RM / A nanodrug delivery system. In tumor tissues, this pH-responsive drug delivery system releases ICB drugs in the acidic tumor microenvironment, thereby relieving T-cell suppression and killing tumor cells. Furthermore, as the nanodrug further internalizes, it releases DOX, DNAzyme, and Zn. 2+ DNAzyme in Zn 2+ Under the action of DOX, P-gp mRNA is cleaved, reducing the expression of efflux proteins, reversing DOX-induced drug resistance, and further enhancing the effect of chemotherapy. At the same time, the DOX-induced ICD effect can work with ICB drugs to improve the immunosuppressive tumor microenvironment, so as to achieve effective tumor chemotherapy-immunotherapy synergy.

[0018] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the specification. Furthermore, in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0019] Figure 1 (a) is a flowchart of the preparation of D / Dz@Z; (b) is a scanning electron microscope image of ZIF-8; (c) is a particle size distribution diagram of ZIF-8; (d) is an infrared absorption spectrum; (e) is an ultraviolet absorption spectrum; scale bar: 100 nm.

[0020] Figure 2 (a) is a flowchart of the preparation process of D / Dz@ZR / A; (b) is the SDS-PAGE analysis of the nanoparticles (I: Marker, II: RM, III: D / Dz@Z, IV: D / Dz@ZR / A) and the transmission electron microscopy images of D / Dz@Z before (c) and after (d) coating; (e) is the particle size distribution map; (f) is the zeta potential map; scale bar: 200 nm.

[0021] Figure 3 (a) shows the XRD patterns of different nanomedicines; (b) shows the stability of D / Dz@ZR / A; (cd) shows the release curves of DOX and DNAzyme under different pH conditions.

[0022] Figure 4 This is a laser confocal image of 4T1 / ADR cells after incubation with D / Dz@ZR / A for 2 h in this invention; scale bar: 10 μm.

[0023] Figure 5 The cell survival rate of 4T1 / ADR cells after incubation with different nanomedicines for 24 h is shown in this invention.

[0024] Figure 6 (a) shows qRT-PCR and (b) shows Western blotting to assess the expression level of P-gp mRNA in 4T1 / ADR cells after different treatments; (c) shows normalized quantitative analysis of P-gp protein.

[0025] Figure 7 Immunofluorescence images of (a) CRT and (b) HMGB1 cells after different treatments according to the present invention; scale bar 10 μm.

[0026] Figure 8 This is a confocal microscope image of 4T1 / ADR or CT26 cells stained with TAMRA-labeled aptamers or random sequence nanomedicines according to the present invention; scale bar 10 μm.

[0027] Figure 9 The hemolysis rate is calculated by incubating the red blood cell suspension of the present invention with D / Dz@ZR / A at different concentrations.

[0028] Figure 10This invention relates to the construction of a breast cancer model and its treatment regimen by subcutaneous injection of 4T1 / ADR cells.

[0029] Figure 11 This invention demonstrates the in vivo tumor suppression effect. (a) Images of tumors removed from mice after different treatments; (b) tumor growth curves; (c) weight of the removed tumor.

[0030] Figure 12 Images of major organs (heart, liver, spleen, lung, and kidney) collected from mice treated with different methods in this invention, showing H&E staining; scale bar: 50 μm.

[0031] Figure 13 This invention utilizes flow cytometry to detect tumor-infiltrating CD8+ in mice treated with different methods. + T cells. Detailed Implementation

[0032] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0033] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing from the market or prepared by existing methods.

[0034] Example 1

[0035] A method for preparing a ZIF-8-based intelligent nanobiomimetic drug delivery system includes the following steps:

[0036] Preparation of D / Dz@Z

[0037] 1.549 g of 2-methylimidazole was weighed and dissolved in 4 mL of water, and 0.0585 g of zinc nitrate hexahydrate was weighed and dissolved in 0.4 mL of water. At room temperature, 8 mg of DOX was added to the 2-methylimidazole solution and stirred at 1200 rpm for 5 min. Then, the zinc nitrate hexahydrate solution was added dropwise to the above mixture and stirred at room temperature. The drug-loaded nanoparticles spontaneously formed, resulting in a red suspension. Centrifugation yielded a red precipitate, which was washed three times with water to remove unreacted reactants and drug adsorbed weakly on the ZIF-8 surface, yielding D@Z.

[0038] Take an appropriate amount of D@Z solution, add 200 μL of DNAzyme, and stir at 37℃ for 30 min. Then centrifuge at 12000 rpm for 10 min, collect the precipitate, and redisperse it in deionized water to obtain D / Dz@Z.

[0039] Extraction of red blood cell membranes

[0040] BALB / c mice were anesthetized and placed head down. The eyeballs were removed with forceps, and blood was collected using EP tubes containing sodium heparin. Whole blood was centrifuged at 2000 rpm for 10 min at 4°C to carefully remove serum and leukocytes. Red blood cells were washed with 1×PBS solution, repeated three times. Then, 0.25×PBS hypotonic solution was added to the red blood cell pellet, mixed, and incubated at 4°C. The hypotonic red blood cell suspension was centrifuged at 12000 rpm for 30 min at 4°C. The supernatant was discarded, and the mixture was washed repeatedly until the supernatant was nearly colorless, yielding the red blood cell membrane. This membrane was dispersed in 1×PBS solution to obtain purified red blood cell membranes (RM). Red blood cell membrane protein concentration was determined using the BCA method. The final concentration of the purified cell membrane protein was adjusted to 1.5 mg / mL.

[0041] Preparation of D / Dz@ZR / A

[0042] D / Dz@Z and RM were mixed at a mass ratio of 1:5 and briefly sonicated. The mixture was then extruded sequentially through 400 nm and 200 nm polycarbonate films 10 times to prepare D / Dz@ZR.

[0043] D / Dz@ZR / A was finally obtained by vortexing equal molar amounts of pH-Apt and D / Dz@ZR and incubating at 37 °C for 40 min.

[0044] Experimental Example 1: Particle size potential and morphology of nanomedicines

[0045] ZIF-8, D@Z, D / Dz@Z, D / Dz@ZR, and D / Dz@ZR / A were diluted to a certain concentration. The nanoparticle size and surface potential were measured using a NanoBrook 90Plus PALS microscope, and the morphological characteristics were observed using a Zeiss Sigma 300 scanning electron microscope and a 200kV JEM-2100 transmission electron microscope.

[0046] Conclusion: Scanning electron microscopy (SEM) revealed that ZIF-8 exhibits a uniform hexagonal-like structure. Figure 1 b), with a particle size of 122 nm ( Figure 1 c). The infrared spectrum shows that D@Z is at 3326 cm⁻¹. -1 and 1730cm -1 The stretching vibration peaks of -OH and -NH2 of DOX are present. Figure 1 d), Ultraviolet absorption spectrum ( Figure 1e) The absorption peaks observed at 480 and 260 nm demonstrate the successful encapsulation of DOX and DNAzyme in ZIF-8, and these results prove the successful preparation of D / Dz@Z.

[0047] SDS-PAGE results showed that ( Figure 2 b), D / Dz@ZR / A exhibits banding consistent with RM, providing evidence of the successful coating, as shown by transmission electron microscopy images ( Figure 2 (cd) It can also be seen that the surface of D / Dz@ZR / A has a distinct membrane structure. The thickness of the erythrocyte membrane is approximately 10-16 nm. Dynamic light scattering (DLS) measurements show that the hydrodynamic size of D / Dz@Z is 163.55 ± 1.55 nm. After coating D / Dz@ZR with cell membrane and aptamer drugs, the DLS size increases to 202.88 ± 2.12 nm. Figure 2 e). Furthermore, the surface charge changed from 18.81 ± 1.2 mV to -25.68 ± 0.54 mV ( Figure 2 f), which indicates the successful preparation of D / Dz@ZR / A.

[0048] Experimental Example 2: Stability Experiment and Nanodrug Release Experiment of D / Dz@ZR / A

[0049] Stability experiment of D / Dz@ZR / A: D / Dz@ZR / A was dispersed in PBS, and samples were taken on days 1, 2, 3, 4, 5, 6, and 7, and the changes in nanoparticle size were measured using a laser particle size analyzer.

[0050] Nanodrug release assay: PBS solutions with pH 7.4 and 5.0 were prepared. The nanodrugs were resuspended in the corresponding pH PBS solutions and incubated at 37 °C for different times (0, 0.5, 1, 2, 4, 6, 12, 24 h). After centrifugation, 100 μL of the supernatant was collected, and the absorbance values ​​of DOX and DNAzyme were measured using an ELISA reader and a fluorescence spectrophotometer, respectively, to obtain the relative DOX and DNAzyme contents. After measurement, 100 μL of supernatant was aspirated back into the corresponding EP tube, the nanoparticles were resuspended, and the tubes were allowed to stand in the dark until the next measurement time point.

[0051] Cell membrane encapsulation of nanomedicines: RM and D / Dz@ZR / A were lysed separately using protein lysis buffer, followed by centrifugation at 4 °C and 10,000 rpm for 5 min. The supernatant was thoroughly mixed with loading buffer and heated in a 100 °C metal bath for 5 min to obtain protein samples. Next, a stacking gel and a separating gel were prepared in the correct proportions, and 10 μL of protein sample was added to each well for electrophoresis to separate the proteins. The electrophoresis conditions were: first, electrophoresis at 80 V for 30 min, then adjusted to 120 V for 100 min. After electrophoresis, the protein gel was stained with Coomassie Brilliant Blue for 30 min, followed by destaining with destaining solution. The protein bands were then observed using a gel imaging system.

[0052] Conclusion: Powder X-ray diffraction (XRD) patterns ( Figure 3 a) The diffraction patterns of D@Z and D / Dz@Z are basically the same as those of the blank ZIF-8 nanoparticles, indicating that the drug loading did not damage the crystal structure of ZIF-8. However, after RM modification, the intensity of the diffraction peaks of D / Dz@ZR / A at low angles decreased, which may be due to the amorphous RM coating on the surface of the nanoparticles having a certain shielding effect on the crystal structure.

[0053] Furthermore, in PBS solution, the fluid size distribution and polydispersity index (PDI) of D / Dz@ZR / A showed minimal changes over 7 days. Figure 3 (b) indicates that the nanoparticles have good stability. ZIF-8 is a natural pH-responsive nanocarrier that degrades at acidic pH values.

[0054] In vitro release curves of DOX and DNAzyme in D / Dz@ZR / A nanoparticles ( Figure 3 (cd) confirmed that ZIF-8 is responsive at low pH. The amount of DOX and DNAzyme released by D / Dz@ZR / A in pH 5.0 buffer was approximately 6.84 and 5.15 times that released in pH 7.4 buffer, respectively.

[0055] Experimental Example 3

[0056] I. Cell Culture

[0057] 4T1 cells (mouse breast cancer cells), 4T1 / ADR cells (mouse drug-resistant breast cancer cells), CT26 (mouse colon cancer cells), RAW 264.7 (mouse monocytes / macrophages), CD8 +T cells (mouse spleen cytotoxic T lymphocytes) were all obtained from the ATCC cell bank (American Type Culture Collection, Manassas, USA). They were incubated at 37°C in a cell culture incubator containing 95% relative humidity and 5% CO2. 4T1 cells, 4T1 / ADR cells, and CT26 cells were cultured in 1640 medium containing 10% FBS and 1% penicillin-dextrose antibody, while RAW 264.7 cells were cultured in DMEM medium containing 10% FBS and 1% P / S. CD8+... + T cells were cultured in DMEM medium containing 10% FBS, 1% triple antibiotics, high glucose, and no sodium pyruvate.

[0058] II. Cell Uptake Experiment

[0059] 4T1 / ADR cells were loaded at 3 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells / day in confocal culture dishes (35 mm) and incubated at 37 °C for 24 h in a cell culture incubator containing 5% CO2. After washing the cells with 1×PBS, fresh medium containing D / Dz@ZR / A was added, and the cells were incubated for 2 h. The cells were then washed three times with 1×PBS buffer to remove uninternalized drug. The cells were fixed with 4% paraformaldehyde for 10 min, the fixative was discarded, and the cells were washed three times with 1×PBS buffer. DAPI was added to stain the cell nuclei for 10 min. After washing three times with 1×PBS buffer, endocytosis was immediately observed using a confocal laser scanning microscopy (CLSM). For flow cytometry analysis, 4T1 / ADR cells were incubated with D / Dz@ZR / A for different time periods, and the intracellular DOX fluorescence intensity was measured by flow cytometry.

[0060] Conclusion: Cellular distribution of nanomedicines was analyzed using laser confocal imaging. DNAzymes in D / Dz@ZR / A cells were labeled with the fluorescent dye FAM and incubated with 4T1 / ADR cells for 2 h. Cell distribution was then observed using laser confocal imaging. Results are as follows: Figure 4 As shown, green and red fluorescence were clearly observed around the cells, indicating that the ZIF-8 vector can effectively deliver therapeutic drugs into the cells.

[0061] III. Cytotoxicity Experiment

[0062] The viability of 4T1 / ADR cells was detected using a CCK-8 cell proliferation assay kit. 4T1 cells were cultured at 2 × 10⁶ cells per well. 4Cells were seeded at a density of [number] cells / well in 96-well plates and incubated at 37 °C for 12 h in a cell culture incubator containing 5% CO2. The original cell culture medium was aspirated, and culture medium containing DOX, D@ZR, D / Dz@ZR, and D / Dz@ZR / A (DOX equivalent dose 20 μg / mL) was added to each treatment well. After incubation for 24 h, 10% CCK8 diluted with 1640 medium was added to the wells. After incubation at 37 °C for 30 min, the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula:

[0063] .

[0064] Conclusion: The synergistic effect of gene silencing and chemotherapy was further demonstrated by the CCK-8 assay, indicating that D / Dz@ZR and D / Dz@ZR / A can effectively reverse multidrug resistance in 4T1 / ADR cells, thereby effectively killing tumor cells. Figure 5 ).

[0065] IV. Protein Immunoblot Experiment

[0066] Extraction of total protein from cells: After culturing for 48 h, the culture medium in the well plate was discarded, and the cells were washed three times with pre-cooled PBS buffer. Then, 200 μL of cell lysis buffer prepared with RIPA:protease inhibitor = 1:100 was added to the cell well plate, and the plate was placed on ice and shaken for 30 min to lyse. Cells were collected using a cell scraper, and the cell lysis buffer was collected into a 1.5 mL centrifuge tube and centrifuged at 4 ℃ for 10 min (12000 rpm). The supernatant was then collected into a new centrifuge tube, which is the total protein in the cells.

[0067] BCA method for protein concentration determination: Using the BCA protein quantification kit, dilute the BCA standard to create a concentration gradient. Add the diluted protein standard and sample to a 96-well plate, with three replicates per group. Add the prepared BCA working solution to the corresponding wells, then incubate at 37 ℃ for 30 min. Measure the absorbance at 562 nm in each well using a microplate reader and plot a standard concentration curve. Substitute the OD value of the sample into the standard curve to obtain the protein concentration of the sample. Adjust the final concentration of the sample based on the measurement results, and adjust the solution used to be cell lysis buffer. Then add 5× Loading buffer proportionally, incubate at 100 ℃ for 5 min in a metal bath to fully denature the protein. After cooling to room temperature, aliquot the samples and store at -80 ℃ for later use.

[0068] Preparation of SDS-PAGE separating gel and stacking gel:

[0069] ① Prepare 10% SDS-PAGE gel according to the instructions.

[0070] ② Sample loading: The sample protein loading amount for each group is 20 μg, and 5 μL of protein marker is added to the sample loading wells at both ends.

[0071] ③ Electrophoresis: Add electrophoresis solution to the electrophoresis tank, first fix the voltage at 80 V, and after it runs out of the stacking gel, increase the voltage to 120 V. Stop electrophoresis when the blue indicator band runs to the bottom of the separating gel.

[0072] ④ Transfer: Activate the PVDF membrane in anhydrous methanol for 5 min beforehand. Soak the sandwich clamp, filter paper, and sponge in transfer buffer. Cut off any excess gel after electrophoresis. Open the transfer clamp and place the sponge, four layers of filter paper, gel, PVDF membrane, four layers of filter paper, and sponge in sequence, avoiding air bubble formation during this process. Then, place the transfer clamp into the transfer tank containing 1× transfer buffer and transfer at a constant current of 200 mA for 90 min. After transfer, remove the PVDF membrane and wash it three times in 1× TBST buffer, 5 min each time.

[0073] ⑤ Sealing: Use tweezers to transfer the PVDF membrane into 5% skim milk powder and seal it by gently shaking it on a shaker at room temperature for 1 hour.

[0074] ⑥ Primary antibody incubation: After blocking, transfer the PVDF membrane to 1×TBST buffer and wash three times for 5 min each time. Then place it in pre-diluted primary antibody and incubate overnight at 4 ℃. After incubation, wash three times with 1×TBST buffer for 5 min each time.

[0075] ⑦ Incubate with secondary antibody: Place the membrane in the pre-diluted secondary antibody and incubate at room temperature for 1 h. After incubation, wash the membrane 3 times with 1×TBST buffer for 5 min each time.

[0076] ⑧ Development: Immerse the PVDF membrane in ECL colorimetric solution, and observe and photograph it using a chemiluminescent gel imaging system.

[0077] Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis

[0078] 4T1 / ADR cells were loaded at 3 × 10⁻⁶ cells per well. 5Cells were seeded at a density of [number] cells / well in 6-well plates and incubated at 37 °C for 12 h in a cell culture incubator containing 5% CO2. The original cell culture medium was aspirated, and the cells were treated with the drug for 24 h. Total RNA was isolated from cells in each treatment group using a total RNA extraction kit, and then the extracted total RNA was reverse transcribed into cDNA using a rapid reverse transcription kit. The concentration of cDNA was measured using a Nanodrop 2000 spectrophotometer, and qRT-PCR analysis was performed using the SYBRPRIME qPCR Kit. The primer sequences used for PCR analysis are as follows:

[0079] P-gp forward primer: 5'-AGTGGCTCTTGAAGCCGTAA-3';

[0080] P-gp reverse primer: 5'-AAACTCCATCACCACCTCACG-3';

[0081] β-actin forward primer: 5'-GTCCCTGACCCTCCCAAAAG-3';

[0082] β-actin reverse primer: 5'-GCTGCCTCAACACCTCAACCC-3'.

[0083] In vitro T cell activation evaluation

[0084] An appropriate amount of tumor cells were seeded into 6-well plates. After 24 hours, they were co-cultured with activated T cells at a ratio of 1:5 (tumor cells: activated T cells). After incubation for 48 hours with different drugs, tumor cells were collected, and the apoptosis rate was determined using the Annexin V-APC / PI apoptosis detection kit. The collected tumor cells were resuspended in PBS solution, counted using a cell counter, and diluted to 1×10⁶ cells with PBS solution. 7 After mixing by pipetting, 100 µL of tumor cells were added to a sample tube, along with 5 µL of Annexin V-APC reagent. After staining in the dark for 30 min, 5 µL of PI solution was added to each sample, and the reaction was carried out in the dark for 30 min. The apoptosis rate was then detected by flow cytometry.

[0085] Conclusion: A nanomedicine, named D / cDz@ZR, was synthesized by replacing DNAzyme with cDNA. 4T1 / ADR cells were treated with PBS, D / cDz@ZR, D@ZR, and D / Dz@ZR / A, respectively, and the expression level of intracellular P-gpmRNA after different treatments was quantitatively analyzed by qRT-PCR. Figure 6As shown in Figure a, compared with the PBS control group, the expression level of P-gp mRNA in 4T1 / ADR cells was significantly reduced after treatment with D / Dz@ZR / A, exhibiting a significant gene silencing effect. However, no significant decrease in P-gp mRNA expression was observed in the D / cDz@ZR treatment group, because cDNA has no cleavage activity against P-gp mRNA. WB ( Figure 6 b) The bands exhibit a similar phenomenon.

[0086] Studies have shown that anthracycline drugs like DOX can induce immune cell death (ICD) effects, stimulating adaptive antitumor immunity by triggering the release of various damage-associated molecular patterns (DAMPs), including the release of calreticulin (CRT) and high-mobility group box 1 (HMGB1) from dying tumor cells. This process is crucial for evoking an immune response. Therefore, CLSM was used to detect the expression levels of CRT and HMGB1 to assess the in vitro ICD effect. After immunofluorescence staining, as shown... Figure 7 As shown, intracellular ROS synergistically induces significant ICD with DOX, which can significantly increase the expression of CRT and HMGB1 in tumor cells. Treatment with D / Dz@ZR / A results in the greatest CRT exposure and HMGB1 migration.

[0087] The ability of D / Dz@ZR / A to target siglec-15 on the cell surface was verified. D / Dz@ZR / A or D / Dz@ZR / RS was co-incubated with 4T1 / ADR or CT26 cells for 1 h. Figure 8 A distinct red fluorescence was observed on the surface of 4T1 / ADR cells, while no fluorescence was observed in CT-26 cells. This indicates that D / Dz@ZR / A can release Apt under acidic conditions and target the surface of 4T1 / ADR cells.

[0088] V. Hemolysis Test

[0089] To preliminarily evaluate the feasibility of intravenous administration of D / Dz@ZR / A, a hemolytic toxicity study was conducted. One mL of blood was collected from the orbital sinus of mice and placed in a 1.5 mL EP tube containing sodium heparin. The tube was centrifuged at 3000 rpm for 15 min at 4 °C. The supernatant plasma and white interfacial material were carefully removed. The supernatant was then washed three times with pre-cooled PBS buffer at 4 °C until clear. A 2% red blood cell suspension (v / v) was prepared using physiological saline. The 2% red blood cell suspension and different concentrations of D / Dz@ZR / A (5, 10, 50, 100, 500, 1000 μg / mL) were added to centrifuge tubes. Physiological saline was used as the negative control, and deionized water as the positive control. Three replicates were prepared for each group. After mixing, the mixture was incubated at 37 °C for 3 h. After incubation, the tube was centrifuged at 1500 rpm for 15 min, and the supernatant was collected. The absorbance was measured at 545 nm using a microplate reader. The corresponding hemolysis rate was calculated.

[0090] .

[0091] Conclusion: The biocompatibility and safety of D / Dz@ZR / A as an intravenous administration formulation were preliminarily assessed through a hemolysis test. Figure 9 At therapeutic concentrations, the hemolysis rate of all groups of nanoparticle drugs was less than 5%, meeting the requirements for intravenous administration.

[0092] Test Example 4

[0093] I. Construction of a 4T1 / ADR tumor-bearing mouse model

[0094] A 4T1 / ADR tumor-bearing mouse model was established using six-week-old female BALB / c mice. Hair was removed from the right posterior back of the mice using depilatory cream. 100 μL of cell suspension (5 × 10⁶ cells) was then subcutaneously injected into the right posterior back of the mice using a 1 mL insulin syringe. 6 Mice were housed in an SPF-protected animal facility, and tumor growth was monitored daily. When the tumor volume reached 100 mm², [the tumor was considered closed]. 3 Mice were treated differently according to their experimental groups. Tumor volume (V) was calculated using the following formula:

[0095] V=A×B 2 / 2;

[0096] Where A and B represent the longest and shortest diameters of the tumor, respectively.

[0097] II. Determination of the biodistribution of tumor-bearing mice

[0098] The distribution of nanomedicines in mice was evaluated using a 4T1 / ADR tumor-bearing mouse model, with tumor volume reaching approximately 100 mm. 3 The drug was administered via tail vein injection (at IR 7834 mg / kg). In vivo drug distribution was observed at 1, 2, 4, 8, 12, and 24 hours after tail vein injection. Mice were sacrificed 24 hours later, and the fluorescence intensity of isolated organs (heart, liver, spleen, lung, and kidney) was observed.

[0099] III. In vivo anti-tumor effects

[0100] The inhibitory effect of nanomedicine on tumors was evaluated using a 4T1 / ADR tumor-bearing mouse model until the tumor volume grew to approximately 100 mm. 3 Mice were randomly divided into 5 groups of 5 mice each. The first administration was recorded as day 0. 100 μL of the nanomedicine was administered via tail vein injection every 2 days for a total of 6 administrations. The mice were treated according to the following groups: PBS, DOX, D@ZR, D / Dz@ZR, and D / Dz@ZR / A (the PBS treatment group served as the control group; the nanomedicine dosage was the equivalent dose of DOX, 4 mg / kg). During treatment, mouse weight and tumor length and width were measured every 2 days. Tumor volume was calculated using the following formula: After the treatment cycle, all mice were sacrificed, and tumors and major organs (heart, liver, spleen, lungs, and kidneys) were dissected and preserved in 4% paraformaldehyde solution. One mouse from each group was randomly selected for hematoxylin-eosin (H&E) staining analysis.

[0101] Conclusion: The antitumor effect of D / Dz@ZR / A in 4T1 / ADR tumor-bearing mice was studied. The mice were randomly divided into five treatment groups. The relative tumor volume after treatment was as follows: Figure 11 a. In the PBS group, the average tumor volume reached approximately 1200 mm after 14 days. 3 In contrast, D@ZR treatment showed some tumor-suppressive effects, which is attributed to the chemotherapeutic effect of DOX. Different degrees of tumor growth inhibition were observed in the D / Dz@ZR and D / Dz@ZR / A groups, with the D / Dz@ZR / A group showing the most significant tumor-suppressive effect, maintaining the tumor volume at approximately 200 mm. 3 This is due to DOX, DNAzyme, Zn 2+ The study explored the synergistic therapeutic effects of Apt-mediated drug resistance reversal and immune activation. Subsequently, tumor-bearing mice were sacrificed after treatment, and all tumor samples were collected, photographed, and weighed for further analysis of their anti-tumor efficacy. Figure 11(b and 11c). Tumor weight was consistent with relative volume, with the average tumor weight in the PBS group being close to 0.95 g, while it was approximately 0.3 g in the D / Dz@ZR / A group, which confirms the significant advantage of D / Dz@ZR / A in inhibiting tumor growth.

[0102] H&E staining of the heart, liver, spleen, lungs, and kidneys of mice revealed no pathological damage in the major organs of the free DOX group (except for slight inflammatory infiltration in the heart). Figure 12 This demonstrates that nanomedicines can avoid systemic toxicity caused by DOX and overcome multidrug resistance in tumors, reflecting the biocompatibility and safety of nanomedicines during use.

[0103] IV. In vivo antitumor immunological evaluation

[0104] Tumor tissue was ground, digested with a digestive solution (a mixture of 400 μg / mL type I collagen and 100 μg / mL type IV collagen), and filtered through a 70 μm cell sieve to obtain a single-cell suspension of the tumor tissue. The specific procedures were as follows: the extracted single-cell suspension was stained with PE Anti-Mouse CD3 antibody, APC Anti-Mouse CD8a antibody, and FITC Anti-Mouse CD4 antibody, incubated at room temperature in the dark for 1 h, washed with PBS, resuspended, and finally analyzed by flow cytometry.

[0105] Conclusion: To further explore the in vivo anti-tumor immune mechanism, flow cytometry analysis was used to identify tumor-infiltrating CTLs (i.e., CD8+) in mice treated with different drugs. + The percentage of T cells. Figure 13 In mice treated with PBS, CD8 + The proportion of T cells was the lowest, at 15.15 ± 3.31%. Mice treated with the aptamer had a higher proportion of tumor-infiltrating CTLs (cytotoxic T lymphocytes) than other groups, because the aptamer drug can activate T cells and produce a synergistic immune effect with ICD. CD8 was detected in free DOX, D@ZR, and D / Dz@ZR. + The percentages of T cells were 28.46±1.13%, 32.29±3.04%, and 40.55±2.24%, respectively. The D / Dz@ZR / A treatment group showed the highest CTL infiltration rate in the tumors of mice, at 51.21±2.65%, which was 2.0-fold, 1.6-fold, and 1.3-fold higher than that of the DOX, D@ZR, and D / Dz@ZR treatment groups, respectively. This indicates that this multimodal synergistic therapy has potential anti-tumor effects.

[0106] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0107] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A smart nanobiomimetic drug delivery system based on ZIF-8, characterized in that, This includes loading doxorubicin, DNAzyme, and aptamers onto ZIF-8.

2. A method for preparing a ZIF-8-based intelligent nanobiomimetic drug delivery system, characterized in that: After loading doxorubicin into the ZIF-8 nanocarrier, DNAzyme was further adsorbed using electrostatic interactions. Subsequently, the surface was modified with erythrocyte membranes, and finally, aptamer drugs were inserted into the cell membrane surface via lipid intercalation to prepare the D / Dz@Z-RM / A nanodrug delivery system.

3. The method for preparing a ZIF-8-based intelligent nanobiomimetic drug delivery system as described in claim 2, characterized in that: The method for loading doxorubicin into the ZIF-8 nanocarrier includes: adding doxorubicin to a 2-methylimidazole solution at room temperature, stirring at 1200 rpm for 5 min, then adding zinc nitrate hexahydrate solution dropwise, stirring at room temperature to obtain a red suspension, centrifuging to obtain a red precipitate, and finally washing with water 3 times to obtain D@Z.

4. The method for preparing a ZIF-8-based intelligent nanobiomimetic drug delivery system as described in claim 3, characterized in that: The method for adsorbing DNAzyme onto the ZIF-8 nanocarrier loaded with doxorubicin includes: adding DNAzyme to D@Z, stirring at 37°C for 30 min, centrifuging at 10,000 rpm for 10 min, collecting the precipitate, and redispersing the precipitate in deionized water to obtain D / Dz@Z.

5. The preparation method of the ZIF-8-based intelligent nanobiomimetic drug delivery system as described in claim 4, characterized in that: The method for surface modification of erythrocyte membranes includes: mixing D / Dz@Z and erythrocyte membranes at a mass ratio of 1:5, then sonicating the mixture, and then extruding the mixture sequentially through 400 nm and 200 nm polycarbonate membranes 10 times to obtain D / Dz@Z-RM.

6. The method for preparing a ZIF-8-based intelligent nanobiomimetic drug delivery system as described in claim 5, characterized in that: Methods for inserting aptamer drugs onto the cell membrane surface include: mixing equimolar amounts of pH-Apt and D / Dz@Z-RM by vortexing and incubating at 37°C for 40 min to obtain D / Dz@Z-RM / A.

7. The application of the ZIF-8-based intelligent nanobiomimetic drug delivery system as described in claim 1 or the preparation method of the ZIF-8-based intelligent nanobiomimetic drug delivery system as described in any one of claims 2-6 in the preparation of tumor drugs.

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