A targeted ros nanodelivery system

Abstract

The application discloses a ROS-targeted nano-drug delivery system, which uses a biomimetic hybrid nanovesicle and montmorillonite as a composite carrier and loads ROS-responsive micelles prepared by mixing a PD-L1 antagonistic peptide complex and an RGD complex. The biomimetic hybrid nanovesicle is prepared by modifying a cell-derived nanovesicle with a liposome composed of lecithin, cholesterol and rhamnolipid. The system realizes the synergy of four functions through the EPR effect of the biomimetic hybrid nanovesicle, the active targeting of RGD, the immunomodulation of the PD-L1 antagonistic peptide and the ROS-responsive rupture of the micelles. Experiments show that the system has a significant structural response under a high ROS environment, can quickly disintegrate to release drugs, has high selectivity and high uptake rate for high-ROS tumor cells, has low toxicity to normal cells and good blood compatibility. The system provides an efficient and safe nano-drug delivery platform for the targeted treatment of ROS-related diseases such as tumors.

Description

Technical Field

[0001] This application belongs to the field of drug carrier and formulation technology, specifically relating to a targeted ROS nanodelivery system. Background Technology

[0002] Reactive oxygen species (ROS) are a class of oxygen-containing molecules with high reactivity. Under normal physiological conditions, ROS in the body are in dynamic equilibrium and participate in a variety of physiological processes such as cell signal transduction and immune regulation.

[0003] However, in pathological states such as tumors and inflammation, cellular metabolism is disordered and mitochondrial respiratory chain function is abnormal within diseased tissues. This is accompanied by overactivation of the oxidase system, leading to a significant increase in ROS levels and the formation of a high-ROS microenvironment. ROS has become a highly selective biomarker for the disease microenvironment. This specific microenvironment is not only an important characteristic of diseased tissues but also provides a key target for targeted therapy.

[0004] Currently, treatment strategies targeting the high ROS microenvironment of diseased tissues mainly fall into two categories: one is to directly kill diseased cells using the cytotoxicity of ROS; the other is to construct ROS-responsive drug delivery systems to achieve precise drug release at the lesion site. Among these, ROS-responsive drug delivery systems have become a research hotspot in the biomedical field because they can effectively reduce the toxic side effects of drugs on normal tissues and increase local drug concentrations. However, single liposome-modified nanovesicles have weak targeting properties, making it difficult to simultaneously achieve passive and active targeting of diseased tissues.

[0005] Therefore, developing a composite nano-drug delivery system that combines high stability, precise targeting, and ROS responsiveness to achieve efficient accumulation and controllable release of drugs at the lesion site is of great significance for improving the treatment effect of ROS-related diseases. Currently, there is no publicly available ternary composite drug delivery system of "bionic hybrid nanovesicles + montmorillonite + ROS-responsive micelles". Summary of the Invention

[0006] To provide a drug delivery system that combines high stability, precise targeting, and ROS responsiveness, this application aims to provide a targeted ROS nano-drug delivery system that integrates biomimetic hybrid nanovesicles, montmorillonite, and ROS-responsive micelles (a mixture of PD-L1 antagonistic peptide complex and RGD complex) into one system, thereby achieving four functions: passive targeting (EPR), active targeting (RGD), immune regulation (PD-L1), and ROS response.

[0007] To achieve the above objectives, this application provides a targeted ROS nanodelivery system, which employs the following technical solution: This application provides a targeted ROS nanodelivery system, wherein the drug delivery system uses biomimetic hybrid nanovesicles and montmorillonite as carriers to load ROS-responsive micelles.

[0008] Preferably, the biomimetic hybrid nanovesicles are liposome-modified nanovesicles.

[0009] Preferably, the ROS-responsive micelles are prepared by mixing a PD-L1 antagonistic peptide complex and an RGD complex.

[0010] Preferably, the mass ratio of the biomimetic hybrid nanovesicles to montmorillonite is 1:1-2.

[0011] Preferably, the liposomes are lecithin, cholesterol, and rhamnolipin.

[0012] Preferably, the mass ratio of liposomes to nanovesicles is 5:1.

[0013] Preferably, the mass ratio of the PD-L1 antagonistic peptide complex to the RGD complex is 1:1.

[0014] Preferably, the PD-L1 antagonist peptide complex is a PD-L1 antagonist peptide and PCL. 3k The molar ratio of -TK-NHS is 1-1.5:1.

[0015] Preferably, the RGD complex is PCL. 3k -NH2 and PEG 5k The molar ratio of -RGD is 2:1.

[0016] Preferably, the delivery system is used in a ROS microenvironment.

[0017] It needs to be explained that: the biomimetic hybrid nanovesicles are composed of liposomes made of lecithin, cholesterol, and rhamnolipin in a specific ratio, which are then modified into nanovesicles. The liposome membrane structure is similar to that of cell membranes, exhibiting good biocompatibility. This reduces the recognition and clearance by the reticuloendothelial system, prolonging the circulation time of the drug delivery system in vivo. Through high permeability and retention effects, it passively accumulates in lesion sites such as tumors. Montmorillonite, as a layered silicate nanomaterial, when combined with the biomimetic hybrid nanovesicles at a mass ratio of 1:1-2, enhances the mechanical stability and drug loading capacity of the carrier. Simultaneously, its layered structure physically encapsulates the ROS-responsive micelles, preventing premature leakage in the bloodstream and ensuring the structural integrity of the drug delivery system. The RGD complex in the ROS-responsive micelles is composed of PCL-NH2 and NHS-PEG-RGD coupled in a 2:1 molar ratio, guiding the drug delivery system to precisely anchor to diseased cells, further improving the drug accumulation efficiency at the target site. The beneficial effects of the embodiments in this application are as follows: First, a ternary composite drug delivery system consisting of "biomimetic hybrid nanovesicles + montmorillonite + ROS-responsive micelles" was constructed, demonstrating significant structural responsiveness in a high-ROS microenvironment. Experiments showed that under simulated high-ROS conditions, the particle size of Examples 1-3 of this application significantly increased, the absolute value of the Zeta potential decreased (10-15 mV), and TEM observation revealed micelle disintegration and a loose carrier structure, confirming its excellent ROS-triggered release capability, superior to single-carrier systems.

[0018] Secondly, the system integrates the EPR effect (passive targeting), RGD-mediated active targeting, and the immunomodulatory function of PD-L1 antagonistic peptides, achieving highly efficient recognition and endocytosis of high ROS diseased cells (such as HepG2). Flow cytometry and fluorescence microscopy results showed that the fluorescence intensity of Example 2 in HepG2 cells was 2.3 times that of Comparative Example 3, with a cell uptake rate of 85.6%, significantly higher than all comparative systems. At the same time, the uptake rate in normal hepatocytes (LO2) was low (10-13%), demonstrating good targeting specificity.

[0019] Finally, the samples maintained low cytotoxicity (cell viability > 85%) in the cytotoxicity test. The hemolysis test results showed that the hemolysis rate of each sample was ≤ 5% at a concentration ≤ 400 μg / mL, which met the safety standards for biomaterials. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of cell viability in Example 2 of the experimental cases of this application.

[0021] Figure 2 This is a schematic diagram of the cell viability of Comparative Example 3 in the experimental examples of this application. Detailed Implementation

[0022] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0023] In the following examples, CVRARTR was purchased from Anhui Guotai Biotechnology Co., Ltd.; PCL 3k -TK-NHS was purchased from Shanghai Jinpan Biotechnology Co., Ltd.; PCL 3K -NH2 and PEG 5K-RGD was purchased from Guangzhou Weihua Biotechnology Co., Ltd.; TrypLE™ Express Enzyme was purchased from Gibco; DMEM high glucose medium was purchased from Gibco; fetal bovine serum (FBS) was purchased from Procell; human hepatocellular carcinoma cells (HepG2, high ROS expression cells) and human normal hepatocytes (LO2, low ROS expression cells) were purchased from China Center for Type Culture Collection; all other reagents not specifically noted are commonly available commercial products.

[0024] Preparation Example 1 ROS-responsive micelle preparation (1) Preparation of PD-L1 antagonistic peptide complex The stimuli-responsive polymeric PD-L1 antagonistic peptide complex, denoted as PCL, was synthesized by amidation reaction. 3k -TK-CVRARTR, accurately weigh 17.2 mg (0.02 mM) of CVRARTR and PCL. 3k -TK-NHS 49.96 mg (0.015 mM) was placed in a dry beaker, and 5 mL of DMF was added at room temperature. The mixture was stirred at room temperature for 4 h. After stirring, the reaction solution was transferred to a dialysis bag (MWCO: 1000 Da). The solution was dialyzed first in PBS (pH=7.4) for 12 h, and then dialyzed in pure water for 12 h. The dialyzed product was then lyophilized to obtain PCL. 3k -TK-CVRARTR white solid powder, store for future use.

[0025] (2) Preparation of RGD composite The same amidation reaction was used to synthesize the targeted polymer RGD complex, denoted as PCL. 3K -PEG 5K -RGD, accurately weigh PCL 3K -NH2 30mg (0.01 mM) and PEG 5K -RGD 25 mg (0.005 mM) was placed in a dry beaker and dissolved in 5 mL LMF at room temperature. The mixture was stirred for 4 h, and then the reaction solution was transferred to a dialysis bag (MWCO: 1000 Da). The solution was dialyzed first in PBS (pH=7.4) for 12 h, and then dialyzed in pure water for 12 h. The dialyzed product was then lyophilized to obtain PCL. 3K -PEG 5K -RGD is a white solid powder; store for future use.

[0026] (3) Preparation of ROS-responsive micelles Accurately weigh 5.0 mg PCL 3K -TK-CVRARTR and 5.0mg PCL 3K -PEG 5K-RGD was placed in a dry beaker, and 3 mL of DMF was added. The mixture was stirred until the solid was completely dissolved. The dissolved liquid was then slowly added dropwise to 7 mL of PBS (pH=7.4) while stirring to form a micelle solution. After the addition was complete, the solution was filtered through a 0.45 μm disposable filter. The filtrate was transferred to a dialysis bag (MWCO: 10000 Da) and dialyzed in PBS (pH=7.4) for 8 h to remove the organic solvent DMF, followed by dialyzing in purified water for 12 h. After dialysis, ROS-responsive micelles were obtained. The prepared ROS-responsive micelles were stored at 4 °C for future use.

[0027] (4) Characterization of ROS-responsive micelles: Characterization was performed using transmission electron microscopy. 10 μL of ROS-responsive micelle solution was dropped into a 200-mesh copper grid. After standing for 3 min, the liquid on the surface was absorbed with disposable filter paper. Then, 10 μL of phosphotungstic acid solution was added for negative staining. After standing for 3 min, the liquid was absorbed with filter paper and placed under a baking lamp for 3 min to dry the liquid. After the sample was dry, the micromorphology and size of the nano micelles were observed using transmission electron microscopy. The nano micelle particle size was about 120 nm.

[0028] Preparation Example 2 Biomimetic hybrid nanovesicle preparation (1) Cell culture and passage 1) Mouse embryonic fibroblasts (NIH / 3T3) were seeded in DMEM high glucose + 10% FBS medium and passaged when the cell density reached 80%. 2) Discard the old culture medium and gently rinse the cells twice with warmed sterile PBS; 3) Add warmed pancreatic enzyme digestion solution, 25cm 2 Add 1 mL to the culture flask, and so on, and digest at 37°C; 4) When the cells are observed to shrink and become round, tap the bottle horizontally, add an equal amount of complete culture medium containing trypsin to stop digestion, and gently pipette to dissociate and detach the cells. 5) Centrifuge at 1000 rpm for 4-5 minutes at 4℃; 6) Discard the supernatant, add fresh complete culture medium (DMEM high glucose + 10% FBS), mix well, and passage according to cell density.

[0029] (2) Cell resuscitation 1) Preheat the hot water bath to a constant temperature of 37℃, and ensure the clean bench is ready for use; 2) Quickly remove the frozen cells stored in the liquid nitrogen tank and rapidly warm them by shaking them in a water bath; 3) After complete melting, add 4 times the volume of complete culture medium to neutralize, gently pipette, centrifuge and seed cells as in step (1).

[0030] (3) Cell cryopreservation 1) Observe the cell growth status and density, and prepare the cell gradient cryopreservation box and other equipment to be ready for use; 2) Cell digestion and centrifugation are performed in the same manner as step (1); 3) Prepare cell cryopreservation solution: 90% FBS + 10% DMSO, mix the cells by pipetting, transfer them into cell cryopreservation tubes labeled with cell information, and place them in a cell cryopreservation box at -80℃; 4) After 24 hours, the cell cryopreservation tubes were transferred to liquid nitrogen tanks.

[0031] (4) Vesicle preparation 1) Cells are digested using commercially available TrypLE™ Express Enzyme, supplemented by pipetting with a pipette tip; 2) Centrifuge the cells and dilute them with sterile PBS to approximately 5 × 10⁻⁶. 6 mL -1 Place in an ice bath; 3) Prepare, inspect and assemble a sterile Mini-extruder, adapt it to the track etching polycarbonate membrane with corresponding aperture, and check for leaks; 4) Mix the cells by pipetting and then squeeze them through a syringe to each pore size (10, 5, 1, 0.4 μm). Each was incubated 10 times; stabilized at 37℃ for 1 hour. 5) Centrifuge at low temperature and high speed using a differential centrifuge, discard the precipitate, and remove dead cells and cell debris in sequence (3000g, 4℃, 30min) and microvesicles (10000g, 4℃, 30min). 6) The obtained supernatant was centrifuged using an ultra-high speed centrifuge (140000g, 4℃, 70min) to obtain the precipitate. The operation was repeated twice for washing. The precipitate was the target vesicle structure. 7) The protein concentration of the vesicles resuspended in PBS was measured using a BCA kit, and the vesicles were aliquoted and stored at -80°C.

[0032] (5) Liposome preparation 1) Accurately weigh lecithin, cholesterol, and rhamnolipin, and dissolve them fully in anhydrous ethanol at a ratio of 2:1:1.156 (mol%). Heat in a sterile, light-protected water bath and vortex to mix. 2) Prepare a thermostatic magnetic stirrer in the clean bench and stir an appropriate amount of sterile PBS at a constant temperature and speed until it is stable (300~500rpm, 50~65℃). 3) The above mixture was rapidly injected into the constant-temperature PBS using a sterile insulin needle at a ratio of 2 mg / mL relative to the PBS solution, and stirred for 20-30 minutes. 4) Use a rotary evaporator for reduced pressure evaporation to remove residual ethanol and stabilize the structure; be careful to avoid drying out. 5) The product was filtered through a 0.22 μm filter to remove impurities and sediment, yielding liposomes.

[0033] (6) Preparation of biomimetic hybrid nanovesicles 1) Thoroughly mix the obtained vesicles and liposomes at a ratio of 1:5 (wt%, protein / total lipid); 2) Vortex, ice water bath ultrasound (30%, 30son, 2minoff, 4~6 cycles); 3) Stabilize in a 37℃ water bath for 1 hour, then extrude through a 0.4μm pore size polycarbonate membrane once; 4) Centrifuge at 140,000g, 4℃, for 70 min to obtain the precipitate. Wash twice with PBS to obtain biomimetic hybrid nanovesicles.

[0034] (7) Transmission electron microscopy morphological detection 1) Gently pick up the copper mesh with sterile forceps, rinse the copper mesh with double-distilled water, and keep the carbon support membrane side facing up; 2) Use a pipette to take 10 μL of freshly separated vesicle solution (dissolved in PBS) and drop it onto the copper mesh carbon support membrane. Wait for the vesicles to adsorb for about 90 seconds, then use filter paper to absorb the residual liquid and air dry in the dark and wind-proof environment. 3) Add 10 μL of 3% uranium acetate solution for negative staining for 30 seconds, blot dry with filter paper and air dry in the dark and wind-proof environment; 4) Imaging by transmission electron microscopy, the vesicles, liposomes and biomimetic hybrid nanovesicles prepared in this example all have typical roundish tea saucer-like morphology and exhibit obvious bilayer membrane structure. This verifies the nanoscale vesicle structure of the biomimetic hybrid nanovesicles. In addition, transmission electron microscopy can be used to observe that the liposomes have a diameter of about 80 nm, the vesicles have a diameter of about 150~200 nm, and the fused hybrid biomimetic hybrid nanovesicles have a diameter slightly larger than that of vesicles, about 200 nm.

[0035] Preparation Example 3 Preparation of biomimetic hybrid nanovesicles and montmorillonite hybrid carriers (1) Material pretreatment 1) Montmorillonite activation: Weighing and dissolving: Weigh 1 mg of montmorillonite and prepare a suspension: Add sterile ultrapure water to prepare a suspension of 1 mg / mL; 2) Magnetic stirring: Stir magnetically at room temperature for 2 hours to ensure that the montmorillonite fully swells; 3) Filtration and impurity removal: Use a 0.22μm sterile filter membrane to filter and remove large particles that are not fully dispersed, to obtain activated montmorillonite. Store at 4℃ for later use to avoid the particles from re-aggregating at low temperatures.

[0036] (2) Biomimetic hybrid nanovesicle resolution 1) Slow thawing: Take out the frozen vesicle sample from Preparation Example 2 and slowly place it in an ice bath to thaw, avoiding rupture caused by drastic temperature differences; 2) Dilution and homogenization: Dilute with sterile PBS to a protein concentration of 1 mg / mL, gently pipette or swirl to mix, and keep on ice. (3) Complex reaction 1) Take 1 mL of biomimetic hybrid nanovesicle suspension with a concentration of 1 mg / mL (containing 1 mg of biomimetic hybrid nanovesicles), and add 1 mg of activated montmorillonite suspension. The montmorillonite suspension should be slowly added to the vesicle suspension while maintaining magnetic stirring in an ice bath (100-200 rpm) to avoid local high concentrations that could lead to agglomeration and result in a mixed solution.

[0037] 2) Ultrasonic Assistance: Using micro-shock waves to enhance interaction and promote the embedding of montmorillonite into the vesicle membrane, the mixture is transferred to a sterile centrifuge tube and placed in an ice-water bath sonicator to prevent the temperature from rising and damaging the vesicles. Parameter settings: power 30%, working time 30s, interval 2min, cycle 4-6 times; standing: after sonication, stand at room temperature for 30min to promote electrostatic adsorption and hydrophobic interaction.

[0038] 3) Purification and characterization pretreatment: Differential centrifugation was used to purify and remove unbound free montmorillonite to obtain a high-purity composite system; Centrifugation: Centrifuge at 140,000g and 4℃ for 70 min and collect the precipitate; Washing: The precipitate was resuspended with sterile PBS and centrifuged and washed once to ensure the removal of free particles; Resuspension: The final precipitate was resuspended with an appropriate amount of PBS to obtain a biomimetic hybrid nanovesicle and montmorillonite mixed carrier.

[0039] (4) Result characterization Transmission electron microscopy revealed that montmorillonite was successfully attached to the surface of vesicles, and particle size analysis showed that the composite system had a particle size of approximately 210 nm.

[0040] Preparation Example 4 Preparation of biomimetic hybrid nanovesicles and montmorillonite hybrid carriers (1) Material pretreatment 1) Montmorillonite activation: Weighing and dissolving: Weigh 2mg of montmorillonite and prepare a suspension: Add sterile ultrapure water to prepare a suspension of 1mg / mL; 2) Magnetic stirring: Stir magnetically at room temperature for 2 hours to ensure that the montmorillonite fully swells; 3) Filtration and impurity removal: Use a 0.22μm sterile filter membrane to filter and remove large particles that are not fully dispersed, to obtain activated montmorillonite. Store at 4℃ for later use to avoid the particles from re-aggregating at low temperatures.

[0041] (2) Biomimetic hybrid nanovesicle resolution 1) Slow thawing: Take out the frozen vesicle sample from Preparation Example 2 and slowly place it in an ice bath to thaw, avoiding rupture caused by drastic temperature differences; 2) Dilution and homogenization: Dilute with sterile PBS to a protein concentration of 1 mg / mL, gently pipette or swirl to mix, and keep on ice. (3) Complex reaction 1) Take 1 mL of biomimetic hybrid nanovesicle suspension with a concentration of 1 mg / mL (containing 1 mg of biomimetic hybrid nanovesicles), and add 2 mg of activated montmorillonite suspension. The montmorillonite suspension should be slowly added to the vesicle suspension while maintaining magnetic stirring in an ice bath (100-200 rpm) to avoid local high concentrations that could lead to agglomeration and result in a mixed solution.

[0042] 2) Ultrasonic Assistance: Using micro-shock waves to enhance interaction and promote the embedding of montmorillonite into the vesicle membrane, the mixture is transferred to a sterile centrifuge tube and placed in an ice-water bath sonicator to prevent the temperature from rising and damaging the vesicles. Parameter settings: power 30%, working time 30s, interval 2min, cycle 4-6 times; standing: after sonication, stand at room temperature for 30min to promote electrostatic adsorption and hydrophobic interaction.

[0043] 3) Purification and characterization pretreatment: Centrifuge at 140,000g, 4℃ for 70min and collect the precipitate; Wash: Resuspend the precipitate with sterile PBS and repeat centrifugation and washing once to ensure removal of free particles; Resuspend: The final precipitate is resuspended with an appropriate amount of PBS to obtain a biomimetic hybrid nanovesicle and montmorillonite mixed carrier.

[0044] (4) Biomimetic hybrid nanovesicles have components similar to those of vesicles and liposomes. Fourier transform infrared spectroscopy can detect characteristic functional groups contained in substances. Data shows that at a range close to 3440 cm⁻¹... -1 and 1643cm -1 Near the wavenumber, all three vesicle structures exhibit strong infrared absorption peaks, which are the absorption peaks of the OH and C=O bonds, while the peak at 1124 cm⁻¹... -1 Up to 879cm -1 The infrared absorption range roughly reflects the absorption peaks of some single bonds (CO, CC, CH, etc.) within the glycosyl group. Because cell-derived vesicles contain a large number of membrane proteins and content proteins, their protein structures contain abundant OH and C=O bonds; rhamnolipid (RHL) liposomes contain glycolipid components, and each RHL molecule contains one or two glycosyl groups, thus exhibiting a distinct infrared absorption range for glycosyl groups (CO, CC, CH). Infrared spectroscopy results show that biomimetic hybrid nanovesicles also exhibit characteristic structural groups similar to those of vesicles and liposomes, fully demonstrating that biomimetic hybrid nanovesicles do indeed possess structural components inherited from vesicles and liposomes.

[0045] Example 1 A targeted ROS nanodelivery system (1) The ROS-responsive micelles of Preparation Example 1 and the biomimetic hybrid nanovesicles and montmorillonite mixed carrier of Preparation Example 3 were mixed at a mass ratio of 1:2. The mixture was magnetically stirred (150 rpm) for 2 h under ice bath conditions, with ultrasonic assistance every 30 min at 20% power for 10 s to promote the physical encapsulation and electrostatic adsorption of micelles and carrier. (2) Purification: Ultra-high speed centrifugation (140000g, 4℃, 70min), discard the supernatant (to remove unloaded free micelles), resuspend the precipitate in sterile PBS, repeat centrifugation and washing once, and finally resuspend in 2mL PBS to obtain the targeted ROS nano-delivery system.

[0046] (3) Characterization: The obtained nano-drug delivery system was subjected to dynamic light scattering (DLS) analysis using a Malvern Zetasizer Nano ZS. The results showed that its hydrated particle size was about 230 nm.

[0047] Example 2 A targeted ROS nanodelivery system (1) The ROS-responsive micelles of Preparation Example 1 and the biomimetic hybrid nanovesicles and montmorillonite mixed carrier of Preparation Example 4 were mixed at a mass ratio of 1:3. The mixture was magnetically stirred (150 rpm) for 2 h under ice bath conditions, with ultrasonic assistance every 30 min at 20% power for 10 s to promote the physical encapsulation and electrostatic adsorption of micelles and carrier. (2) Purification: Ultra-high speed centrifugation (140000g, 4℃, 70min), discard the supernatant (to remove unloaded free micelles), resuspend the precipitate in sterile PBS, repeat centrifugation and washing once, and finally resuspend in 2mL PBS to obtain the targeted ROS nano-delivery system.

[0048] (3) Characterization: The obtained nano-drug delivery system was subjected to dynamic light scattering (DLS) analysis using a Malvern Zetasizer Nano ZS. The results showed that its hydrated particle size was about 230 nm.

[0049] Example 3 A targeted ROS nanodelivery system (1) The ROS-responsive micelles of Preparation Example 1 and the biomimetic hybrid nanovesicles and montmorillonite mixed carrier of Preparation Example 3 were mixed at a mass ratio of 1:4. The mixture was magnetically stirred (150 rpm) for 2 h under ice bath conditions, with ultrasonic assistance every 30 min at 20% power for 10 s to promote the physical encapsulation and electrostatic adsorption of micelles and carrier. (2) Purification: Ultra-high speed centrifugation (140000g, 4℃, 70min), discard the supernatant (to remove unloaded free micelles), resuspend the precipitate in sterile PBS, repeat centrifugation and washing once, and finally resuspend in 2mL PBS to obtain the targeted ROS nano-delivery system.

[0050] (3) Characterization: The obtained nano-drug delivery system was subjected to dynamic light scattering (DLS) analysis using a Malvern Zetasizer Nano ZS. The results showed that its hydrated particle size was about 240 nm.

[0051] Comparative Example 1 Only biomimetic hybrid nanovesicles loaded with ROS-responsive micelles (1) The ROS-responsive micelles of Preparation Example 1 and the biomimetic hybrid nanovesicles of Preparation Example 2 were mixed at a mass ratio of 1:2 and magnetically stirred (150 rpm) for 2 h under ice bath conditions. (2) Purification: Centrifuge at ultra-high speed (140000g, 4℃, 70min), discard the supernatant, resuspend the precipitate in sterile PBS, wash once, and finally resuspend in 2mL PBS to obtain the nano-drug delivery system.

[0052] Comparative Example 2 Only montmorillonite-loaded ROS-responsive micelles (1) The ROS-responsive micelles of Preparation Example 1 and the activated montmorillonite suspension (1 mg / mL) of Preparation Example 3 were mixed at a mass ratio of 1:2 and magnetically stirred in an ice bath for 2 h. (2) Purification: Centrifuge (140000g, 4℃, 70min), wash and resuspend to obtain nano-drug delivery system.

[0053] Comparative Example 3 Nano micelles Same as in Example 4, but without a carrier.

[0054] Test case Performance evaluation of targeted ROS nanodelivery systems (1) Samples: Targeted ROS nanodelivery systems prepared in Examples 1-3 and nanodelivery systems / nanomicelles prepared in Comparative Examples 1-3; (2) ROS responsiveness evaluation 1) Sample preparation: Dilute the samples of each example and comparative example to a concentration of 100 μg / mL with serum-free DMEM medium; 2) Divided into two groups: control group (conventional culture conditions) and ROS-induced group (H2O2 was added to make the final concentration 100μM to simulate a high ROS microenvironment), and incubated in a 37℃, 5%CO2 incubator for 4h; 3) Detection of particle size and potential changes: The hydrated particle size and zeta potential of the samples before and after incubation were detected using a Malvern laser particle size analyzer; Observation of morphological changes: The microstructure of the samples after incubation was observed by TEM. 4) Experimental results: As shown in Table 1; Control group: The particle size of each sample was stable (particle size fluctuation of Examples 1-3 ≤ 10%, fluctuation of Comparative Examples 1-3 ≤ 15%), the potential did not change significantly, and the samples showed regular spherical or near-spherical structures under TEM; ROS-induced group: The particle size of Examples 1-3 increased significantly (35%-50%), the absolute value of the potential decreased (10-15mV), and micelle disintegration and loose carrier structure were visible under TEM; Comparative Example 1 (bionic hybrid nanovesicles only) increased particle size by 20%-25%, Comparative Example 2 (montmorillonite only) increased by 15%-20%, and Comparative Example 3 (pure micelles) increased by 25%-30%, and the morphological integrity was better than that of the Examples. The ternary composite drug delivery system of the present application has more significant ROS responsiveness and can rapidly disintegrate its structure under high ROS conditions, providing conditions for drug release.

[0055] Table 1. Results of ROS response tests (n=10)

[0056] (3) Targeting evaluation (cell uptake experiment) 1) Sample fluorescent labeling: Each sample was fluorescently labeled using FITC, with a labeling efficiency ≥90%; 2) Cell seeding: HepG2 cells and LO2 cells were seeded into 24-well plates, 1×10⁶ cells per well. 5 10 cells, cultured for 24 hours until adherent; 3) Uptake experiment: Add fluorescently labeled sample (final concentration 100 μg / mL), incubate at 37℃ for 2 h, and wash 3 times with PBS to remove free sample; 4) Detection: The intracellular fluorescence intensity was detected by flow cytometry as shown in Table 2, and the cell uptake was observed by fluorescence microscopy as shown in Table 3. 5) Results: Table 2 shows that in HepG2 cells, the intracellular fluorescence intensity of Examples 1-3 was significantly higher than that of Comparative Examples 1-3, with Example 2 showing the highest fluorescence intensity (2.3 times that of Comparative Example 3). Fluorescence microscopy revealed significant intracellular fluorescence aggregation in the Example groups, while the fluorescence distribution in the Comparative Examples was dispersed. This indicates that the carrier (especially montmorillonite / vesicles) provided additional adsorption sites or improved the cell adhesion of the nanoparticles. Specificity maintenance: In normal LO2 cells, the FI values ​​of each group did not differ significantly, indicating that the addition of the carrier did not significantly increase non-specific uptake of non-target cells. The intracellular fluorescence intensity of all samples was low, with no significant difference between the Examples and Comparative Examples. Table 3 shows that the carrier-assisted uptake rate (78-86%) of Examples 1-3 was significantly higher than that of Comparative Example 1 (57%) and Comparative Example 2 (51%), indicating that the carrier combination (especially the system containing montmorillonite) significantly improved the uptake efficiency of the nano-drug delivery system in tumor cells. Targeting maintenance: The uptake rate in LO2 cells remained at a low level (10-13%), verifying the good targeting specificity of the system.

[0057] Table 2. Flow cytometry detection of intracellular fluorescence intensity (FI value) (n=10)

[0058] Table 3. Statistics on cell uptake rate (n=10)

[0059] (4) Cytotoxicity evaluation (CCK-8 assay) 1) This experiment used the CCK-8 assay to investigate the in vitro toxicity of the drug delivery systems of Example 2 and Comparative Example 3 to HepG2 cells. HepG2 cells with a cell density of approximately 80% were digested with trypsin and counted. The cells were then seeded evenly into 96 wells at a density of 5000 cells / well, and culture medium was added. The cells were then cultured in the medium for 24 hours. The old culture medium was then discarded, and RMPI-1640 medium with and without nanomicelles of different drug concentrations was added to each well. Medium without fetal bovine serum was added to bring the final volume of each well to 100 μL. After 24 hours, the old culture medium was discarded, and 90 μL of medium and 10 μL of CCK-8 reagent were added. The plates were then incubated for 2 hours until the 96-well plate turned orange-red. The plates to be tested were removed, shaken on a microplate reader for 30 seconds, and the OD value of each well was measured at 450 nm. The following formula was used to calculate various cell viability rates. The negative control group was represented by the OD value of the wells in the culture plate without cell inoculation, and the control group was represented by the OD value of the wells in the culture plate with added blank culture medium. Each concentration was measured repeatedly, and the calculated viability rates for each group were compared with those for the control group to calculate relative cell viability. Cell viability (%) ×100%, where A is the absorbance value of samples with different concentrations, A1 is the absorbance value of the negative control group, and A0 is the absorbance value of the blank control group.

[0060] 2) Results: The cell viability in Example 2 was as follows Figure 1 As shown, the cell viability of Comparative Example 3 is as follows: Figure 2 As shown; pass Figure 1 and Figure 2 It was found that neither Example 2 nor Comparative Example 3 showed significant killing effect on HepG2 cells at a drug concentration of 100 μg / mL, and the relative cell viability was above 85%.

[0061] (5) Biocompatibility evaluation (hemolysis test) 1) Red blood cell preparation: Take whole blood from healthy mice, add anticoagulant, centrifuge at 1000 rpm for 10 min, discard the supernatant, wash 3 times with PBS, and prepare a 2% (v / v) red blood cell suspension; 2) Sample preparation: Dilute each sample with PBS to different concentrations (50, 100, 200, 400 μg / mL), mix 0.5 mL of sample with 0.5 mL of red blood cell suspension, and set up a positive control group (0.1% Triton X-100) and a negative control group (PBS). 3) Incubation and detection: Incubate at 37℃ for 1 hour, centrifuge at 3000 rpm for 10 minutes, collect the supernatant, and detect the OD value at 540 nm using an ELISA reader. Calculate the hemolysis rate (Hemolysis rate = (OD value of experimental group - OD value of negative control group) / (OD value of positive control group - OD value of negative control group) × 100%). 4) Results: The hemolysis rate of each example and comparative sample was ≤5% when the concentration was ≤400μg / mL, which met the standard for hemolysis rate of biological materials (≤5%); the hemolysis rate of Examples 1-3 was not significantly different from that of the comparative example; the targeted ROS nano-delivery system of this application has good blood compatibility and no obvious risk of hemolysis.

[0062] In summary, the targeted ROS nanodelivery system prepared in this application achieves synergistic optimization of ROS responsiveness, precise targeting, and biocompatibility through a ternary composite structure of "biomimetic hybrid nanovesicles + montmorillonite + ROS-responsive micelles": compared with single carriers or pure micelle systems (comparative examples 1-3), its ROS responsiveness is more significant, and it can rapidly disintegrate and release drugs at the lesion site; with the dual effects of RGD active targeting and EPR passive targeting, it has stronger targeting recognition ability for high ROS diseased cells and higher cellular uptake efficiency; while effectively killing diseased cells, it has low toxicity to normal cells and good blood compatibility, providing an efficient and safe drug delivery platform for targeted therapy of ROS-related diseases (such as tumors and inflammation).

[0063] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. All equivalent changes and improvements made within the scope of this application shall still fall within the patent coverage of this application.

Claims

1. A targeted ROS nanodelivery system, characterized in that, The drug delivery system uses biomimetic hybrid nanovesicles and montmorillonite as carriers to load ROS-responsive micelles.

2. The targeted ROS nanodelivery system as described in claim 1, characterized in that, The biomimetic hybrid nanovesicles are liposome-modified nanovesicles.

3. The targeted ROS nanodelivery system as described in claim 1, characterized in that, The ROS-responsive micelles were prepared by mixing a PD-L1 antagonistic peptide complex and an RGD complex.

4. The targeted ROS nanodelivery system as described in claim 1, characterized in that, The mass ratio of the biomimetic hybrid nanovesicles to montmorillonite is 1:1-2.

5. The targeted ROS nanodelivery system as described in claim 2, characterized in that, The liposomes are lecithin, cholesterol, and rhamnolipin.

6. The targeted ROS nanodelivery system as described in claim 2, characterized in that, The mass ratio of liposomes to nanovesicles is 5:

1.

7. The targeted ROS nanodelivery system as described in claim 3, characterized in that, The mass ratio of the PD-L1 antagonistic peptide complex to the RGD complex is 1:

1.

8. The targeted ROS nanodelivery system as described in claim 3, characterized in that, The PD-L1 antagonist peptide complex is a PD-L1 antagonist peptide and PCL. 3k The molar ratio of -TK-NHS is 1-1.5:

1.

9. The targeted ROS nanodelivery system as described in claim 3, characterized in that, The RGD complex is PCL. 3k -NH2 and PEG 5k The molar ratio of -RGD is 2:

1.

10. A targeted ROS nanodelivery system according to any one of claims 1-9, characterized in that, Application of the delivery system in the ROS microenvironment.

Images