Tumor-inhibiting programmed drug permeation biomimetic mineralized exosomes, their preparation methods and applications

Abstract

This invention relates to biomimetic mineralized exosomes for programmed drug penetration in tumors that inhibit cell burial, along with their preparation method and applications. Belonging to the field of novel excipients and dosage forms for pharmaceutical formulations, this invention co-loads the small-molecule chemotherapeutic drugs 10-hydroxycamptothecin and banoanthraquinone into exosomes. Then, through a biomimetic mineralization strategy, aTIM-4 is organically combined with mineralized calcium phosphate particles to obtain biomimetic mineralized exosomes. Under the acidic response of the tumor microenvironment, the calcium phosphate shell dissolves, and the exosome nucleus enters the tumor cells, inducing cell death and generating apoptotic bodies. This programmatically delivers the drug, delivering banoanthraquinone deep into the tumor. Simultaneously, the released aTIM-4 inhibits the cell burial of tumor-associated macrophages, amplifying the programmed drug penetration based on apoptotic bodies. This invention provides a new strategy and more options for overcoming the bottleneck of nanomedicine penetration in tumors, meeting the urgent clinical need for highly effective chemotherapeutic agents.

Description

Technical Field

[0001] This invention belongs to the field of new excipients and dosage forms for pharmaceutical preparations, and relates to biomimetic mineralized exosomes for tumor programmed drug permeation that inhibit cell burial, their preparation methods and applications. Specifically, it relates to a biomimetic mineralized exosome that simultaneously carries 10-hydroxycamptothecin, the hypoxia-activated prodrug banoanthraquinone, and the monoclonal antibody aTIM-4 that inhibits cell burial, its construction method, and its application in drug delivery. Background Technology

[0002] The incidence and mortality rates of cancer are increasing year by year, seriously endangering the health and lives of the Chinese people. Various novel nanoparticles for tumor treatment have become a research hotspot, such as biomimetic drug delivery systems constructed using exosomes as drug carriers, or functional mineralized nanoplatforms with good biocompatibility and long blood circulation time modified by inorganic mineralization. However, due to the dense extracellular matrix inside solid tumors, the continuously increasing interstitial hydrostatic pressure from the outside in, and the tightly packed tumor cells, nanomedicines that enter tumor tissue via blood vessels are often confined to the perivascular area, making it difficult to comprehensively kill the tumor and reducing their clinical therapeutic effect. Therefore, how to improve the penetration ability of nanomedicines into tumors remains a pressing clinical challenge.

[0003] Following apoptosis, the cell membrane shrinks and invaginates, cutting the cytoplasm to form apoptotic bodies containing nuclear fragments, organelles, and large protein molecules. Previous studies have shown that after small-molecule chemotherapeutic drugs induce apoptosis in tumor cells, the remaining drugs can be stored in apoptotic bodies. These bodies are then taken up by adjacent tumor cells, exerting cytotoxic effects, like peeling an onion, layer by layer killing tumor cells and penetrating deep into the tumor. However, macrophages enriched in the tumor site can also perform cytotoxicity, rapidly recognizing and clearing the generated apoptotic bodies, limiting their transfer between cells and hindering effective anti-tumor efficacy. Furthermore, the drug penetration mechanism based on apoptotic bodies remains unclear, and the differences in delivery efficiency among different drug models within apoptotic bodies need further investigation. These issues severely restrict the development of apoptotic body-based drug deep-tumor penetration nanotechnology. Summary of the Invention

[0004] The technical problem solved by this invention is to co-load two small-molecule chemotherapeutic drugs that act at different spatial locations of tumors into biomimetic exosomes, and then coat the surface of the biomimetic exosomes with a mineralized shell carrying a monoclonal antibody that inhibits cell burial through biomineralization, thus preparing a tumor-inhibiting programmed drug-permeable biomimetic mineralized exosome. The prepared biomimetic mineralized exosome nanoparticles have good stability, long cycling time, significant inhibitory effect on cell burial, and strong programmed penetration ability into tumor tissues, verifying the drug penetration mechanism based on apoptosis bodies and demonstrating the superiority of the formulation of this invention in tumor treatment.

[0005] This invention co-loads a monoclonal antibody that inhibits cell burial and two small-molecule chemotherapeutic drugs that act at different spatial locations in the tumor into a biomimetic mineralized exosome. The study investigates the effects of the combined use of the three on drug penetration and pharmacodynamics in tumor tissue, and explores the drug penetration mechanism based on apoptotic bodies. This provides a new strategy for improving drug tumor penetration and guides the design of drugs that achieve deep tumor penetration based on apoptotic bodies, thus accelerating the clinical translation of nanomedicines.

[0006] The monoclonal antibody that inhibits cytotoxicity described in this invention is aTIM-4, a monoclonal antibody that binds to the TIM-4 receptor on the surface of macrophages, and can significantly inhibit the cytotoxicity of macrophages. The two small molecule chemotherapeutic drugs that act at different spatial locations of the tumor refer to hydrophobic small molecule drugs that are cytotoxic to all tumor regions, such as 10-hydroxycamptothecin (HCPT); and hydrophilic hypoxia-activating prodrugs that are cytotoxic only to hypoxic sites inside the tumor, such as banoanthraquinone (AQ4N). The biomimetic exosomes refer to exosomes homologous to tumor cells, such as exosomes secreted by 4T1 mouse breast cancer cells. The mineralized shell refers to a hard shell composed of inorganic ions that degrades in the acidic microenvironment of the tumor, such as a calcium phosphate shell. The components of the tumor-inhibiting programmed drug permeating biomimetic mineralized exosomes, by weight percentage, are as follows: mineralized shell 44%-56%, biomimetic exosomes 24%-38%, hydrophilic hypoxia-activating prodrug that is cytotoxic only to hypoxic sites within the tumor 7%-10%, hydrophobic small molecule drug that is cytotoxic to all tumor regions 1%-3%, and aTIM-4 3%-5%. The preferred weight ratio of each component is: mineralized shell : biomimetic exosomes : hydrophilic hypoxia-activating prodrug that is cytotoxic only to hypoxic sites within the tumor : hydrophobic small molecule drug that is cytotoxic to all tumor regions: aTIM-4 = 2.28 mg : 1 mg : 0.4 mg : 0.14 mg : 0.2 mg.

[0007] The calcium phosphate is prepared from calcium chloride (CaCl2) and disodium hydrogen phosphate (Na2HPO4). The weight ratio is calcium chloride:disodium hydrogen phosphate = (1-4):(2-6), preferably 1 mg:1.28 mg.

[0008] This invention also provides a method for preparing biomimetic mineralized exosomes that infiltrate tumor-inhibiting programmed drugs: Serum-free cell culture medium from mouse cancer cells is collected, and tumor cells, dead cells, and cell debris are removed by repeated centrifugation. After resuspending the cells in PBS containing a protease inhibitor, the supernatant is removed by ultracentrifugation to obtain blank exosomes. A hydrophobic small molecule drug cytotoxic to all tumor regions and a hydrophilic hypoxia-activating prodrug cytotoxic only to hypoxic sites within the tumor are dissolved in an organic solvent and deionized water, respectively, and mixed with the prepared blank exosomes. The mixture is then sonicated in an ice bath. After sonication, the resulting exosomes, co-loaded with the hydrophobic small molecule drug cytotoxic to all tumor regions and the hydrophilic hypoxia-activating prodrug cytotoxic only to hypoxic sites within the tumor, are incubated in a constant temperature incubator to restore the integrity of the exosome membrane. Then, the secretions were dispersed by stirring, and an aqueous solution containing the monoclonal antibody aTIM-4 that inhibits cell burial and CaCl2 was added dropwise. After stirring evenly, an aqueous solution of Na2HPO4 was added, and stirring was continued to obtain biomimetic mineralized exosomes.

[0009] The above-mentioned method for preparing tumor-infiltrating biomimetic mineralized exosomes by inhibiting cell burial includes:

[0010] The hydrophobic small molecule drug that is cytotoxic to all tumor regions is preferably HCPT; the hydrophilic hypoxia-activating prodrug that is cytotoxic only to hypoxic sites within the tumor is preferably AQ4N.

[0011] The organic solvent is one of methanol, anhydrous ethanol, and dimethyl sulfoxide, preferably dimethyl sulfoxide. The organic solvent dissolves hydrophobic small molecule drugs that are cytotoxic to all tumor regions; deionized water dissolves hydrophilic hypoxia-activating prodrugs, aTIM-4, CaCl2, and Na2HPO4 that are cytotoxic only to hypoxic sites within the tumor.

[0012] The ultrasound is performed in an ice bath with an ultrasonic power of 100W-200W, preferably 180W.

[0013] The present invention also provides the application of the above-mentioned tumor-inhibiting programmed drug permeation biomimetic mineralized exosomes in injection, oral or local drug delivery systems.

[0014] The present invention has the following beneficial effects:

[0015] (1) A biomimetic mineralized exosome preparation with a calcium phosphate shell carrying aTIM-4, HCPT and AQ4N with uniform particle size was prepared. The preparation method is simple, efficient and stable, and achieves efficient co-delivery of small molecule drugs and monoclonal antibodies.

[0016] (2) The study investigated the response and disintegration of calcium phosphate shells in the tumor microenvironment, the inhibitory effect of aTIM-4 on the burial of tumor-associated macrophages, the intercellular delivery efficiency of two different drug models, HCPT and AQ4N, based on apoptotic bodies, the penetration of biomimetic mineralized exosomes into tumor tissue, and their inhibitory effect on tumor growth. The formulation characterization of biomimetic mineralized exosomes, detection of acid-responsive drug release behavior, extraction of apoptotic bodies and their adjacent effect experiments, analysis of drug content changes during drug delivery by apoptotic bodies, investigation of the burial inhibition effect of monoclonal antibodies, tumor spheroid experiments, and pharmacodynamic studies in animals were conducted. The results showed that the biomimetic mineralized exosomes have uniform particle size and good stability, and can disintegrate in an acidic environment. By inhibiting burial, they amplify the sorting behavior during drug delivery by apoptotic bodies, achieving programmed penetration deep into the tumor and complete inhibition of tumor growth. This provides a new strategy and more options for overcoming the bottleneck of nanomedicine penetration in tumors, meeting the urgent clinical need for highly efficient chemotherapy agents. Attached Figure Description

[0017] Figure 1 This is an experimental diagram of the preparation of small molecule drug co-loaded exosomes in Example 1 of the present invention.

[0018] A: Drug ratio screening.

[0019] B: Transmission electron microscope image of AH-Exos.

[0020] Figure 2 This is an experimental diagram illustrating the characteristics of biomimetic mineralized exosomes in Embodiment 2 of the present invention.

[0021] A: Particle size and potential diagram of AH-Exos@CPM.

[0022] B: Transmission electron micrograph of AH-Exos@CPM.

[0023] C: Elemental energy spectrum analysis of AH-Exos@CPM.

[0024] D: SDS-PAGE images of AH-Exos@CPM, AH-Exos, and Exosomes.

[0025] E: Scans of AH-Exos@CPM and AH-Exos using UV-Vis spectrophotometry and fluorescence spectroscopy.

[0026] Figure 3 This is an in vitro release detection diagram of biomimetic mineralized exosomes in Example 3 of the present invention.

[0027] A: Graph showing the release kinetics of calcium ions under different pH conditions, ***p<0.001.

[0028] B: Release kinetics of aTIM-4 under different pH conditions, ***p<0.001.

[0029] C: Release kinetics of HCPT under different pH conditions.

[0030] D: Release kinetics of AQ4N under different pH conditions.

[0031] Figure 4 This is a diagram showing the colloidal stability and placement stability of the biomimetic mineralized exosomes in Example 4 of the present invention.

[0032] Figure 5 This is a diagram of the in vitro cellular uptake experiment of biomimetic mineralized exosomes in Example 5 of the present invention.

[0033] A: Confocal fluorescence images of 4T1 cells treated with AQ4N solution, HCPT solution, AH-Exos, AH-Exos@CPM, and AH-Exos@CPM (pH 6.5) for 12 h.

[0034] B: Flow cytometry measurements of 4T1 cells treated with each formulation for 12 hours, ***p<0.001, ****p<0.0001.

[0035] C: Confocal fluorescence images of 4T1 cells treated with each formulation for 6 hours.

[0036] D: Flow cytometry measurements of 4T1 cells treated with each formulation for 6 hours.

[0037] Figure 6 This is a diagram of the 4T1 cytotoxicity experiment of biomimetic mineralized exosomes in Example 6 of the present invention.

[0038] A: Survival rates of 4T1 cells treated with AQ4N solution, HCPT solution, a mixture of AQ4N and HCPT, AH-Exos, AH-Exos@CPM, and AH-Exos@CPM (pH 6.5) for 48 h under normoxic conditions.

[0039] B: Survival rate of 4T1 cells after 48 hours of treatment with various agents under hypoxic conditions.

[0040] Figure 7 This is a diagram showing the extraction and characterization of apoptotic bodies in Example 7 of the present invention.

[0041] A: Transmission electron micrograph of apoptotic bodies extracted from cells induced by AH-Exos@CPM (pH 6.5).

[0042] B: Western blot analysis of characteristic proteins of apoptotic bodies.

[0043] C: Confocal fluorescence image of apoptotic bodies and uptake fluorescence image of 4T1 tumors.

[0044] D: Cytotoxicity of apoptotic bodies on 4T1 cells.

[0045] Figure 8 This is a diagram illustrating the neighbor effect based on apoptotic bodies in Example 8 of the present invention.

[0046] A: Schematic diagram of the neighbor effect investigation method.

[0047] B: Confocal fluorescence images of 4T1 cells on each plate.

[0048] C: Confocal fluorescence images of the drug in apoptotic bodies (i-ii) and (ii-iii) in the AH-Exos@CPM (pH 6.5) treatment group.

[0049] D: A graph showing the changes in AQ4N content in cells (iI), apoptotic bodies (i-ii), and cells (ii-I) in the AH-Exos@CPM (pH 6.5) treatment group.

[0050] E: Graph showing changes in HCPT levels in cells (iI), apoptotic bodies (i-ii), and cells (ii-I).

[0051] F: The total molar ratio of AQ4N to HCPT in cells (iI), apoptotic bodies (i-ii), and cells (ii-I), ****p<0.0001.

[0052] G: The graph showing the change in AQ4N content relative to AQ4 in iI and iA cells in the AH-Exos@CPM (pH 6.5) treatment group.

[0053] Western blot image of apoptosis marker proteins in H:iI and iA cells.

[0054] Figure 9 This is a diagram illustrating the effect of inhibiting cell burial and enhancing the permeability of apoptotic bodies in Example 9 of the present invention.

[0055] A: Flow cytometry measurements of aTIM-4's inhibition of macrophage clearance of apoptotic bodies.

[0056] B: In the tumor sphere experiment, the permeation of AQ4N solution, HCPT solution, AQ4N and HCPT mixed solution, AH-Exos and AH-Exos@CPM was observed by confocal microscopy.

[0057] C: Quantitative fluorescence intensity diagram of AQ4N and HCPT in tumor spheres.

[0058] Figure 10 This is a plasma drug concentration-time curve of biomimetic mineralized exosomes in Example 10 of the present invention.

[0059] Figure 11 This is an in vivo tissue distribution diagram of the biomimetic mineralized exosomes in Example 11 of the present invention.

[0060] A: Fluorescence distribution of AQ4N solution, AH-Exos, and AH-Exos@CPM in mice at different time points.

[0061] B: In vitro fluorescence distribution of major organs (heart, liver, spleen, lung, kidney, and tumor) in mice 24 hours after injection of different formulations.

[0062] C: Semi-quantitative in vitro fluorescence intensity of major organs in mice 24 hours after injection of different formulations, ***p<0.001, ****p<0.0001.

[0063] Figure 12 In Example 11 of this invention, confocal microscopy was used to observe the penetration of AQ4N solution, HCPT solution, AQ4N and HCPT mixed solution, AH-Exos and AH-Exos@CPM in a mouse tumor model.

[0064] Figure 13 This is a pharmacodynamic study diagram of biomimetic mineralized exosomes in Example 13 of the present invention.

[0065] A: Changes in tumor volume at 4T1 levels after injection of physiological saline, HCPT solution, AQ4N solution, a mixture of HCPT and AQ4N, AH-Exos, and AH-Exos@CPM via tail vein. **p<0.01,***p<0.001.

[0066] B: Figure showing the weight changes of 4T1 tumor-bearing mice after injection of physiological saline, HCPT solution, AQ4N solution, HCPT and AQ4N mixed solution, AH-Exos, and AH-Exos@CPM via tail vein.

[0067] C: Anatomical images of detached 4T1 tumors after treatment with different types of agents.

[0068] D: Weight of 4T1 tumors after anatomical dissection following treatment with different types of agents, **p<0.01, ****p<0.0001.

[0069] E: Liver function indicators in 4T1 tumor-bearing mice after treatment with different types of formulations.

[0070] F: Renal function indicators in 4T1 tumor-bearing mice after treatment with different types of formulations.

[0071] G: H&E staining, TUNEL staining, and Ki67 staining images of anatomically excised 4T1 tumors after treatment with different types of agents.

[0072] H: H&E staining images of the heart, liver, spleen, lungs, and kidneys of 4T1 tumor-bearing mice after treatment with different types of formulations. Detailed Implementation

[0073] Example 1: Preparation of small molecule drug co-loaded exosomes

[0074] Exosomes were extracted using ultracentrifugation. Cells were first cultured in cell culture medium containing 10% fetal bovine serum (FBS) containing exosomes. The cell supernatant was collected and centrifuged at 300g for 10 min to remove floating cells. Then, the cells were centrifuged at 15,000g for 30 min to remove cell debris. Finally, the cells were centrifuged at 100,000g for 120 min to obtain purified exosomes. The obtained exosomes were dissolved in PBS, filtered through a 0.22 μm syringe filter for sterilization, and quantified and aliquoted using a BCA protein quantification kit. The exosomes were then stored at -80℃ for later use. All operations were performed at 4℃. Different ratios of HCPT and AQ4N were dissolved in 14 μl of dimethyl sulfoxide and 100 μl of deionized water, respectively, and mixed with exosomes containing 1 mg of membrane protein. The mixture was sonicated under ice bath conditions at 180W for 2 seconds with a 4-second interval, for a total sonication time of 2 min. After sonication, the prepared exosomes co-loaded with HCPT and AQ4N (AH-Exos) were incubated at 37°C for 1 hour to restore the integrity of the exosome membrane. The particle size and potential of the exosomes were measured using a Malvern laser particle size analyzer. 1 ml of the formulation was added to an ultrafiltration centrifuge tube with a pore size of 10 kDa and centrifuged. The encapsulation efficiency of AQ4N and HCPT was determined using a UV-Vis spectrophotometer and a fluorescence spectrometer. The calculation formula is as follows:

[0075] Encapsulation efficiency (%) = (mass of encapsulated drug) / (total drug mass) * 100%

[0076] Experimental results are as follows Figure 1 As shown, when the molar ratio of AQ4N to HCPT is 2:1, it has the optimal particle size and moderate encapsulation efficiency, and appears as a disc-shaped vesicle under transmission electron microscopy.

[0077] Example 2: Experiment on the characteristics of biomimetic mineralized exosomes

[0078] The exosomes (AH-Exos) co-loaded with HCPT and AQ4N prepared in Example 1 were dispersed at 4°C using a magnetic stirrer. An aqueous solution containing 0.2 mg aTIM-4 and 1 mg CaCl2 was added dropwise. After stirring until homogeneous, an equal volume of an aqueous solution containing 1.28 mg Na2HPO4 was added, and stirring continued for 2 hours to obtain biomimetic mineralized exosomes (AH-Exos@CPM). The particle size and potential were measured using a Malvern laser particle size analyzer, and the morphological characteristics of AH-Exos@CPM were observed using a transmission electron microscope (TEM). Figure 2 As shown in A and 2B, the morphology of the nanoparticles changed from a disk-like shape to a core-shell shape, and the particle size and potential also changed, which initially proved the successful preparation of biomimetic mineralized exosomes.

[0079] Next, elemental energy dispersive spectroscopy (EDS) was used to perform semi-quantitative analysis of the surface elements of the biomimetic mineralized exosomes. It was clearly observed that calcium phosphate and the characteristic elements of aTIM-4, Ca, P, O, and S, were uniformly dispersed on the particle surface. Figure 2 C), and simultaneously, the characteristic proteins on the surface of the formulation were detected by SDS-PAGE. It was found that both AH-Exos and AH-Exos@CPM exhibited exosome characteristic proteins, and the mineralized exosomes also showed the characteristic protein aTIM-4 (C). Figure 2 D), AH-Exos@CPM was spectrally scanned using a multi-functional microplate reader, and characteristic peaks containing both AQ4N and HCPT were detected. Figure 2 E). The encapsulation efficiency of aTIM-4 formulation, measured using an ELISA kit, was 87.3 ± 7.3%.

[0080] Example 3: In vitro release experiment of AH-Exos@CPM

[0081] 1 ml of AH-Exos@CPM was placed in 20 ml of PBS at different pH values ​​(pH=7.4 and pH=6.5) and incubated. A simulated release was performed in a 37°C water bath with a shaker. The mixed release solution was collected at predetermined time points (0, 0.5, 1, 2, 4, 8, 12, 24 h), centrifuged at 13,000 g for 10 min, and the supernatant was collected. The concentrations of calcium ions and aTIM-4 were monitored using a calcium ion concentration analyzer and an ELISA kit, respectively. Results are as follows: Figure 3 As shown, within 12 hours, 63.7 ± 3.4% of calcium ions and 33.5 ± 0.9% of aTIM-4 were released from AH-Exos@CPM at pH 6.5, while less than 20% of calcium ions and aTIM-4 were released in a neutral environment at pH 7.4, demonstrating the tumor microenvironment responsiveness of calcium phosphate. Figure 3 A, 3B).

[0082] Another 1 ml of AH-Exos@CPM was placed in 20 ml of PBS at different pH values ​​(pH 7.4, pH 6.5, pH 5.0). At pre-set time points, the mixed release solution was removed, centrifuged, and the release levels of AQ4N and HCPT in the solution were measured using a multi-mode microplate reader. It was found that they also exhibited a response release under acidic conditions. Figure 3 C, 3D).

[0083] Example 4: Stability testing of AH-Exos@CPM

[0084] AH-Exos@CPM was mixed with PBS (pH 7.4) (1:20, v / v) and incubated in a 37°C shaker. Particle size was measured using dynamic light scattering at 0, 2, 4, 6, 8, 12, 18, and 24 h to evaluate its colloidal stability. Furthermore, it was stored at 4°C for 7 days to observe particle size changes. Results are as follows: Figure 4 As shown, the biomimetic mineralization exhibits good stability, with no significant change in particle size.

[0085] Example 5: In vitro uptake experiment of AH-Exos@CPM

[0086] 4T1 cells (1×10) 5 Cells (per well) were seeded into 12-well plates and incubated at 37°C for 24 hours. The old culture medium was discarded, and medium containing AQ4N solution, HCPT solution, AH-Exos nanoparticles, or AH-Exos@CPM nanoparticles (10 μM HCPT) at pH 7.4 or pH 6.5 was added. The plates were then incubated at 37°C for 6 hours and 12 hours, respectively. After incubation, the drug-containing medium was discarded, and the cells were washed three times with ice-cold PBS (pH 7.4) to terminate cell uptake. Cells were fixed with 4% paraformaldehyde for 10 minutes and stained with Hoechst 33342 at 37°C for 10 minutes. The smears were then placed on slides with anti-quenching mounting medium. The slides were then examined under a confocal microscope to observe the red and green drug fluorescence within the cells. For flow cytometry quantitative analysis of cell uptake, cells were cultured in 24-well plates. After drug administration, cells were digested with 0.05% trypsin, collected by centrifugation, and uniformly dispersed in PBS (pH 7.4). Cell uptake was then quantitatively analyzed using flow cytometry. Figure 5 As shown, exosomes are more easily taken up by tumor cells due to their homology, exhibiting the highest uptake efficiency. The calcium phosphate shell of AH-Exos@CPM dissolves at pH 6.5, exposing the exosome core, and has similar uptake characteristics to AH-Exos.

[0087] Example 6: Cytotoxicity assay of AH-Exos

[0088] 4T1 cells (2000 cells / well) were seeded in 96-well plates and placed in a 37°C CO2 cell culture incubator for 12 h under normoxic conditions. Then, the cells were cultured for another 12 h under normoxic conditions or in a hypoxic culture chamber with a hypoxic gas-generating bag. The old culture medium was discarded, and a series of gradient concentrations (HCPT: 20 nM, 50 nM, 100 nM, 200 nM, 500 nM; AQ4N: 40 nM, 100 nM, 200 nM, 400 nM, 1000 nM) of AQ4N solution, HCPT solution, AH-Exos nanoparticles, or AH-Exos@CPM nanoparticles were added to pH 7.4 or pH 6.5 medium. The cells were then incubated for another 48 h under normoxic conditions or in a hypoxic culture chamber with a hypoxic gas-generating bag. Add 0.5 mg / ml MTT to each well and incubate for 4 hours. Discard the culture medium, add 200 μl of dimethyl sulfoxide, shake for 10 minutes, and measure the absorbance at 490 nm using a multi-mode microplate reader. Figure 6 As shown, AQ4N has almost no cytotoxicity under normoxic conditions but can effectively kill cells under hypoxic conditions. HCPT has significant cytotoxicity under both normoxic and hypoxic conditions. Therefore, the combined use of HCPT and AQ4N can comprehensively kill tumor cells.

[0089] Example 7: Extraction and Characterization of Apoptotic Bodies

[0090] After incubating 4T1 cells with AH-Exos@CPM (20 μM HCPT) at pH 6.5 for 12 h, the old culture medium was discarded, the cells were washed, and fresh blank culture medium was added. The cells were then cultured for another 24 h. The culture medium was collected and apoptotic bodies (ApoBDs) were collected by centrifugation at 1500 g for 20 min using a low-temperature centrifuge. The particle size and zeta potential of the apoptotic bodies were analyzed using a particle size analyzer, and the morphology of the apoptotic bodies was observed using transmission electron microscopy. Drug fluorescence within the apoptotic bodies was observed using a confocal microscope. Figure 7 As shown, the red and green fluorescence overlaps in the apoptotic bodies, indicating that the apoptotic bodies can serve as drug delivery carriers, loading residual drugs within tumor cells after apoptosis. The collected apoptotic bodies were added to 20 mm glass-bottomed culture dishes containing 4T1 cells and incubated for 24 h. After washing three times with ice-cold PBS (pH 7.4) and fixing with 4% paraformaldehyde for 10 min, the cell nuclei were stained with Hoechst 33342 for 10 min. The slides were then removed and placed on slides with anti-quenching mounting medium. Microscopic observation revealed that tumor cells clearly took up the apoptotic bodies. The toxicity of the apoptotic bodies to tumor cells was determined by the MTT assay, showing that the apoptotic bodies still had a significant killing effect on tumor cells.

[0091] Example 8: Investigation of the Neighbor Effect Mechanism Based on Apoptotic Bodies

[0092] like Figure 8 As shown, 4T1 cells (4×10⁻⁶) were used. 5 Cells (10 cells / well) were seeded into 6-well plates (i) and incubated in a 37°C CO2 cell culture incubator for 24 h. The old culture medium was discarded, and medium containing AQ4N solution, HCPT solution, a mixture of AQ4N and HCPT, AH-Exos nanoparticles, or AH-Exos@CPM nanoparticles (HCPT 20 μM) at pH 7.4 or pH 6.5 was added. The plates were then incubated in a 37°C CO2 cell culture incubator for 12 h to obtain cells (iI). After incubation, the drug-containing medium was discarded, and the plates were incubated again with blank medium for 12 h to allow for complete apoptosis, yielding cells (iA). After incubation, apoptotic bodies were extracted from the cell supernatant and co-cultured with blank cells from 6-well plates (ii). This process was repeated to obtain cells on plate (iii). Cells (iI, ii-I, iii-I) in 6-well plates were fixed with 4% paraformaldehyde for 10 min, and the nuclei were stained with Hoechst 33342. Intracellular drug fluorescence was observed under a confocal microscope. In the AH-Exos and AH-Exos@CPM groups, significant fluorescence was observed in cells on plates (ii) and (iii). The red fluorescence intensity representing AQ4N increased relative to the green fluorescence representing HCPT, exhibiting a programmed delivery phenomenon. However, almost no fluorescence was observed in plates (ii) and (iii) of the AQ4N solution group, HCPT solution group, and the AQ4N and HCPT mixed solution group. Apoptotic bodies induced by AH-Exos@CPM nanoparticles were collected and uniformly dispersed in PBS (pH 7.4). Cells were disrupted by five freeze-thaw cycles. Free drugs were separated using ultrafiltration centrifuge tubes with a pore size of 3 kDa. The proportions of free AQ4N and HCPT in the total drug were determined by high performance liquid chromatography and a multi-mode microplate reader. We found that the proportion of free HCPT decreased, while the proportion of free AQ4N remained basically unchanged. The amount of AQ4N relative to HCPT increased continuously during the delivery process. In addition, cells in the iI and iA stages of the AH-Exos@CPM group were collected. After cell disruption, the intracellular content of AQ4N and the cytotoxic drug AQ4N was detected by HPLC. The content of two intracellular apoptosis marker proteins, γH2AX and cleaved caspase-3, was detected by Western blot. It was observed that AQ4N in apoptotic cells maintained its own state and was not converted into the toxic drug AQ4. The continuous consumption of HCPT caused apoptosis, produced apoptotic bodies, and promoted drug penetration.

[0093] Example 9: Investigation on the ability of inhibiting cell burial to enhance apoptotic body permeability

[0094] Mature bone marrow-derived macrophages (BMDMs) were obtained by extracting bone marrow-derived macrophages from mice and culturing them in 20 ng / ml mouse M-CSF at 37°C for 7 days. M2-type macrophages (M2Φ) were obtained by polarizing the cells with interleukin-4, and the distribution of the cell membrane protein aTIM-4 was detected by Western blot. AH-Exos-induced apoptotic bodies and a mixed solution of AH-Exos-induced apoptotic bodies and aTIM-4 were added and co-cultured for 4 h. After cells were aspirated with PBS, they were collected and analyzed by quantitative flow cytometry. Figure 9 As shown, macrophages have a strong ability to take up apoptotic bodies, but this uptake can be blocked by aTIM-4. Using 1×10 4 4T1 cells and 5×10 3 BMDM cells were dispersed in 15 μl of cell culture medium and seeded in 96-well plates containing agarose. The culture medium was changed every 2 days. On day 6, culture media containing AQ4N solution, HCPT solution, a mixture of AQ4N and HCPT, AH-Exos nanoparticles, and AH-Exos@CPM nanoparticles at pH 7.4 and pH 6.5 were added, and the plates were incubated at 37°C in a CO2 cell culture incubator for 24 h. The old culture medium was then discarded, the cell spheroids were washed, and drug penetration was observed under a confocal microscope using Z-axis scanning at 40 μm scan intervals. Due to the inhibitory effect of aTIM-4 on macrophage cytotoxicity, AH-Exos@CPM showed the longest penetration distance and exhibited significant programmed penetration. For the tumor spheroid growth inhibition experiment, the culture medium containing the above drugs was changed every 2 days. On day 6, images were taken using an optical microscope, and the tumor spheroid volume was calculated. Similarly, the biomimetic mineralized exosomes with the strongest permeability showed the strongest tumor inhibitory effect.

[0095] Example 10: Pharmacokinetic Study of AH-Exos@CPM

[0096] Male SD rats (220-250g) were randomly divided into groups and administered HCPT solution, AH-Exos nanoparticles, or AH-Exos@CPM nanoparticles via tail vein injection. The equivalent dose of HCPT was 2 mg / kg. Blood samples were collected from the fundus venous plexus of the rats at predetermined time points (0.03, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24 h) and placed in heparinized EP tubes. The blood samples were centrifuged to obtain plasma. After centrifugation and protein precipitation, the supernatant was added to a 96-well black plate, and the plasma concentration of HCPT was measured using a multi-functional microplate reader. Figure 10As shown, HCPT solution is rapidly cleared from the blood, while exosome-loaded or mineralized biomimetic exosomes can significantly prolong the half-life of HCPT in the blood. This is because the mineralized, hard calcium phosphate shell can effectively block the shear force of the blood and maintain the stability of the nanoparticles.

[0097] Example 11: Investigation of the tissue distribution of AH-Exos@CPM

[0098] Healthy 4T1 cells were digested with 0.05% trypsin and then redispersed uniformly in PBS (pH 7.4) at a concentration of 5 × 10⁻⁶. 7 Cells / ml. 100 μl of the above cell suspension was injected subcutaneously into the right posterior back of female BALB / c mice. The tumor volume of the mouse model of 4T1 mammary cancer ectopic tumors reached approximately 300 mm². 3 At that time, AQ4N solution, AH-Exos nanoparticles, and AH-Exos@CPM nanoparticles were administered via tail vein injection. The equivalent dose of AQ4N was 8 mg / kg. Mice were anesthetized and in vivo imaging was performed using a small animal imaging system 2, 4, 8, 12, and 24 hours after intravenous injection. Mice were sacrificed 24 hours after injection, and organs such as the heart, liver, spleen, lung, kidney, and tumor were collected for in vitro tissue fluorescence imaging, such as... Figure 11 As shown, the solution AQ4N was rapidly eliminated from the body and did not accumulate at the tumor site, while exosome-encapsulated or mineralized biomimetic exosome preparations could specifically accumulate at the tumor site for a long time, which is consistent with the pharmacokinetic results of nano-formulations.

[0099] Example 12: In vivo permeability detection of AH-Exos@CPM

[0100] 4T1 tumor cells in good growth condition and in the logarithmic growth phase were digested with 0.05% trypsin, and the digestion was terminated with fresh RPMI 1640 cell culture medium. The cells were then centrifuged and collected, and uniformly dispersed in PBS (pH 7.4) to a concentration of 2 × 10⁻⁶ cells / mL. 7 Cells / mL, stored in an ice box. 100 μl of well-suspended 4T1 cells were subcutaneously seeded into the right posterior back of mice. When the tumor volume reached 500 mm², [the cells were incubated]. 3 At approximately 48 hours post-mortem injection, mice were administered AQ4N solution, HCPT solution, a mixture of AQ4N and HCPT, AH-Exos nanoparticles, or AH-Exos@CPM nanoparticles via tail vein injection. The equivalent dose of HCPT was 2.8 mg / kg. Mice were then sacrificed, tumors were collected, and frozen sections were prepared. Cell nuclei were stained with DAPI, and drug fluorescence was observed using a confocal microscope. Figure 12As shown, AH-Exos@CPM exhibits the strongest tumor penetration ability, which is due to the strong tumor enrichment ability of the nanoparticles and the fact that the inhibition of cell burial by aTIM-4 further amplifies the penetration efficiency of apoptotic bodies.

[0101] Example 13: Pharmacodynamic assay of AH-Exos@CPM

[0102] Establish a subcutaneous ectopic breast cancer tumor-bearing model in Balb / c mice (4-5 weeks old, average weight 18-22g), until the tumor volume in the tumor-bearing mice reaches 100mm. 3 Day 0 was designated as the approximate time of birth, and mice were randomly divided into 6 groups of 5 mice each. On days 0, 2, 4, 6, and 8, mice were injected via the tail vein with saline, AQ4N solution, HCPT solution, a mixture of AQ4N and HCPT, AH-Exos nanoparticles, or AH-Exos@CPM nanoparticles. The equivalent dose of HCPT was 2.8 mg / kg. After administration began, tumor size and body weight were measured daily, and a weight change curve over time was plotted to evaluate weight changes after administration. Mice were sacrificed on day 12, and blood was collected by removing the eyes. Serum was obtained by centrifugation and separation. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CREA) were measured as indicators of liver and kidney function. After all mice were sacrificed, the heart, liver, spleen, lungs, and kidneys were fixed in 4% paraformaldehyde and stained with Hematoxylin and eosin (H&E) for histological evaluation. Simultaneously, the tumor tissue was dissected, photographed, and weighed. The weighed tumor tissue was fixed with 4% paraformaldehyde and sectioned. Following the section staining guidelines provided by the reagent kit company, the paraffin sections were dewaxed with xylene and ethanol solutions of varying concentrations. The tumor tissue sections were stained using the TUNEL apoptosis detection kit and the Ki67 cell proliferation detection kit, and photographed using a confocal microscope. Results are as follows: Figure 13 As shown, the tumors in mice injected with the solution grew rapidly, second only to those injected with saline, which was due to the rapid clearance of the solution in vivo. Because of the rapid accumulation at the tumor site and the strongest tumor penetration capacity, the biomimetic mineralized exosomes produced the best tumor treatment effect, almost completely inhibiting tumor growth. Furthermore, H&E staining, Ki 67 staining, and TUNEL staining results indicated that AH-Exos@CPM could induce widespread apoptosis and inhibit cell proliferation, but with good safety. Except for mice treated with HCPT solution and the AQ4N and HCPT mixed solution, which showed some fluctuation in body weight, the body weight of mice in other groups did not decrease significantly. No significant abnormalities were observed in liver and kidney function or H&E staining of major organs in all groups of mice.

Claims

1. A tumor-inhibiting programmed drug permeation biomimetic mineralized exosome, characterized in that, The drug was prepared by co-loading two small molecule chemotherapy drugs that act at different spatial locations of the tumor into a biomimetic exosome, and then coating the surface of the biomimetic exosome with a mineralized shell containing a monoclonal antibody that inhibits cell burial through biomineralization. The monoclonal antibody that inhibits cell burial is aTIM-4, a monoclonal antibody that binds to the TIM-4 receptor on the surface of macrophages; the two small molecule chemotherapeutic drugs that act at different spatial locations of the tumor refer to a hydrophobic small molecule drug that is cytotoxic to all tumor regions, and a hydrophilic hypoxia-activating prodrug that is cytotoxic only to hypoxic sites within the tumor. The hydrophobic small molecule drug that is cytotoxic to all tumor regions is 10-hydroxycamptothecin, and the hydrophilic hypoxia-activating prodrug that is cytotoxic only to hypoxic sites within the tumor is banoxantrone; the biomimetic exosomes refer to exosomes homologous to tumor cells; and the mineralized shell refers to a calcium phosphate shell. The calcium phosphate is prepared by calcium chloride and disodium hydrogen phosphate in a weight ratio of (1-4):(2-6).

2. The tumor-inhibiting programmed drug permeation biomimetic mineralized exosomes according to claim 1, characterized in that, The biomimetic exosomes mentioned refer to exosomes secreted by 4T1 mouse breast cancer cells.

3. The tumor-inhibiting programmed drug permeation biomimetic mineralized exosomes according to claim 1, characterized in that, The components of the tumor-inhibiting programmed drug permeating the biomimetic mineralized exosomes are in the following weight ratio: mineralized shell: biomimetic exosome: hydrophilic, hypoxia-activating prodrug that is cytotoxic only to hypoxic sites inside the tumor: hydrophobic, small molecule drug that is cytotoxic to all tumor regions: aTIM-4 = 2.28 mg: 1 mg: 0.4 mg: 0.14 mg: 0.2 mg.

4. A method for preparing tumor-infiltrating biomimetic mineralized exosomes that inhibit cell burial, as described in claim 1, characterized in that, Includes the following steps: Serum-free cell culture medium from mouse cancer cells was collected. Tumor cells, dead cells, and cell debris were removed by repeated centrifugation. The cells were resuspended in PBS containing protease inhibitors, and the supernatant was removed by ultracentrifugation to obtain blank exosomes. Hydrophobic small-molecule drugs cytotoxic to all tumor regions and hydrophilic hypoxia-activating prodrugs cytotoxic only to hypoxic sites within the tumor were dissolved in organic solvents and deionized water, respectively, and mixed with the prepared blank exosomes. The mixture was sonicated in an ice bath. After sonication, the exosomes co-loaded with the hydrophobic small-molecule drugs cytotoxic to all tumor regions and the hydrophilic hypoxia-activating prodrugs cytotoxic only to hypoxic sites within the tumor were incubated in a constant temperature incubator to restore the integrity of the exosome membrane. Then, the secretions were dispersed by stirring, and an aqueous solution containing the monoclonal antibody aTIM-4 (which inhibits cell death) and CaCl2 was added dropwise. After thorough stirring, an aqueous solution of Na2HPO4 was added, and stirring continued to obtain biomimetic mineralized exosomes. The organic solvent is one of methanol, anhydrous ethanol, and dimethyl sulfoxide; the ultrasonication is performed in an ice bath with an ultrasonic power of 100 W-200 W.

5. The method for preparing tumor-infiltrating biomimetic mineralized exosomes that inhibit cell burial according to claim 4, characterized in that, The organic solvent is dimethyl sulfoxide; the ultrasonication is performed in an ice bath with an ultrasonic power of 180W.

6. The use of the tumor-inhibiting programmed drug permeation biomimetic mineralized exosomes as described in claim 1 in the preparation of tumor therapeutic agents.

Images