Exosome-coated response type drug-loaded dendrimer nanogel system as well as preparation method and application of exosome-coated response type drug-loaded dendrimer nanogel system

Abstract

The invention relates to an exosome-coated responsive drug-loaded dendrimer nanogel system as well as a preparation method and application thereof. The preparation method comprises the following steps: grafting a drug on the surface of a phenylboronic acid modified third-generation PAMAM (Polyamidoamine) dendrimer through a boric acid ester bond; then, synthesizing active oxygen sensitive drug-loaded nanogel by using a cross-linking agent containing a double-selenium bond through a reversed-phase microemulsion method, and wrapping the surface of the drug-loaded nanogel with exosome through a physical effect. The drug-loaded nanogel system prepared by the invention can effectively improve the bioavailability of small-molecule drugs, realizes targeted delivery and responsive release of the drugs at focus parts, shows an excellent synergistic treatment effect in an acute lung injury model, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of preparation and application of responsive nanogel platforms, and particularly relates to an exosome-coated responsive drug-loaded dendrimer nanogel system and its preparation method and application. Background Art

[0002] Acute lung injury (ALI) refers to a disease characterized by extensive inflammatory responses in the lungs caused by various direct or indirect factors such as trauma, infection, and inhalation of harmful gases, which may lead to respiratory failure and threaten life. Currently, one of the main clinical means for treating acute lung injury is chemotherapy. Chemotherapy drugs can reduce lung tissue damage by regulating the production of various inflammatory mediators and cytokines in the pathological process of acute lung injury. However, traditional chemotherapy drugs have low bioavailability, large toxic and side effects, and due to high drug doses, healthy organs and tissues are also easily damaged, so it is difficult to achieve ideal therapeutic effects. Drug carrier technology provides a new approach for the treatment of acute lung injury. Through specific targeting mechanisms, drug carriers can directly deliver chemotherapy drugs to the damaged areas of the lungs, thereby increasing the drug concentration in the lesion area, reducing the toxic and side effects of drugs on normal tissues, and at the same time controlling the drug release rate, thereby extending the drug's effective period and achieving better therapeutic effects.

[0003] Taxifolin (Tax) is a natural product extracted from the roots of larch in alpine regions, with strong anti-inflammatory and antioxidant activities. Studies have shown that Tax can inhibit inflammatory responses by regulating the protein kinase B / IκB kinase / nuclear factor κB (Akt / IKK / NF-κB) and mitogen-activated protein kinase / cytosolic phospholipase A2 (MAPKs / cPLA2) signaling pathways (Mediators Inflammation 2019, 2019, 3740867). Tax can effectively scavenge free radicals, and its numerous polyphenol groups on the surface can reduce oxidation reactions by chelating with metals. Tax itself can also regulate the expression of related antioxidant proteins in the antioxidant system and play an antioxidant role by reducing the activity of xanthine oxidase, thereby synergistically scavenging the excessive accumulation of reactive oxygen species (ROS) levels in activated macrophages. However, Tax has disadvantages such as poor water solubility, short half-life, difficulty in being phagocytosed by target cells, and low bioavailability, which limit the widespread application of Tax in the treatment of ALI. Therefore, developing a nanodelivery system for targeted delivery and controlled release of Tax is crucial for improving the bioavailability of Tax to enhance the therapeutic effect of ALI. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an exosome-coated responsive drug-loaded dendrimer nanogel system, its preparation method and application, so as to provide new ideas for constructing a safe, intelligent and efficient drug carrier.

[0005] The present invention provides an exosome-coated responsive drug-loaded dendrimer nanogel system. The drug is grafted onto the surface of a phenylboronic acid-modified third-generation PAMAM (polyamide-amine) dendrimer through a borate ester bond. Subsequently, an active oxygen-sensitive drug-loaded nanogel is synthesized by a reverse microemulsion method using a cross-linker containing a diselenide bond, and exosomes are physically wrapped on the surface of the drug-loaded nanogel.

[0006] Preferably, the drug includes but is not limited to taxifolin Tax; the exosomes include but are not limited to mesenchymal stem cell-derived exosomes MSC-Exo.

[0007] The present invention provides a preparation method for an exosome-coated responsive drug-loaded dendrimer nanogel system, comprising the following steps:

[0008] (1) A 4-bromomethylphenylboronic acid BPBA solution is dropped into a third-generation PAMAM dendrimer G3.NH 2 solution, and after stirring reaction, purification and freeze-drying, a third-generation PAMAM dendrimer G3-PBA modified with phenylboronic acid PBA is obtained;

[0009] (2) The G3-PBA prepared in step (1) is formulated into a solution and mixed with a drug solution, and after stirring reaction, purification and freeze-drying, a third-generation PAMAM dendrimer G3-drug grafted with the drug is obtained;

[0010] (3) The G3-drug prepared in step (2) and the cross-linker containing a diselenide bond are respectively dissolved in water, mixed and stirred to form an aqueous phase; a surfactant and an emulsifier are respectively added to an organic solvent, mixed and stirred to form an oil phase; the aqueous phase is added dropwise to the oil phase to obtain a W / O polymer emulsion. After ultrasonic fragmentation, a triethylamine solution is added dropwise, stirred, centrifuged, and the lower-layer gel is collected, dispersed with a solvent and purified by dialysis to obtain a responsive drug-loaded dendrimer nanogel DT-NGs;

[0011] (4) Cells are cultured in a medium, and then replaced with a cell culture medium dedicated to exosomes and cultured continuously; the medium rich in exosomes is collected and cell-derived exosomes are obtained after centrifugation and purification;

[0012] (5) The solution of the responsive dendrimer nanogel DT-NGs prepared in step (3) is ultrasonically mixed with the exosomes prepared in step (4), and after extrusion and centrifugation, an exosome-coated responsive drug-loaded dendrimer nanogel system is obtained.

[0013] Preferably, the solvent of the solution in step (1) is dimethyl sulfoxide (DMSO); the molar ratio of BPBA to G3.NH 2 is 10-15:1; the stirring reaction temperature is 70-80 °C and the time is 24-36 h; the purification process parameters are: dialysis against water for 3 days using a dialysis bag with a molecular weight cut-off of 3500 Da.

[0014] Preferably, the solvent of the G3-PBA solution in step (2) is ultrapure water, and the solvent of the drug solution is methanol and ultrapure water; the molar ratio of G3-PBA to the drug is 1:5-10; the stirring reaction temperature is 20-25 °C and the time is 36-72 h; the purification process parameters are: dialysis against water for 3 days using a dialysis bag with a molecular weight cut-off of 3500 Da.

[0015] Preferably, the molar ratio of G3-drug to the cross-linking agent containing a diselenide bond in step (3) is 1:3-7; the cross-linking agent containing a diselenide bond includes but is not limited to 2-bromoethyl acrylate diselenide; the mass ratio of the surfactant to the emulsifier is 3-8:1, the surfactant includes but is not limited to Span 80, and the emulsifier includes but is not limited to Tween 80; the volume ratio of the organic solvent in the oil phase, water in the water phase to triethylamine is 10-14:1:0.5-1, and the organic solvent includes but is not limited to cyclohexane.

[0016] Preferably, the ultrasonic fragmentation time in step (3) is 5-10 min and the stirring time is 10-15 h; the centrifugation process parameters are: 10000-12000 rpm, 10-15 min; the dispersion solvent includes but is not limited to acetone; the dialysis process parameters are: dialysis against water for 3 days using a dialysis bag with a molecular weight cut-off of 8000-14000 Da.

[0017] Preferably, the specific process of step (4) includes: extracting mesenchymal stem cells MSCs from the femurs and tibias of 2-week-old Sprague-Dawley (SD) rats and culturing them in DMEM / F12 medium containing 10% serum; when the MSCs grow to passages 3-6 and the confluence rate reaches 75%, changing to an exosome-specific mesenchymal stem cell medium and continuing to culture for 48 h; collecting the medium rich in exosomes, first centrifuging at 200-300 g for 10-15 min, collecting the supernatant and centrifuging at 2000-3000 g for 15-20 min, then centrifuging the supernatant at 9000-10000 g for 1-1.5 h, and finally centrifuging the supernatant at 90000-100000 g for 1-1.5 h, collecting the precipitate and resuspending it with phosphate buffer (PBS) to obtain mesenchymal stem cell-derived exosomes MSC-Exo.

[0018] Preferably, in the step (5), the solvents of DT-NGs and exosomes are both phosphate buffer solution; the mass ratio of DT-NGs to exosomes is 5:1 to 2; the ultrasonic mixing time is 10 to 20 min; the concentration of the exosomes is 0.3 to 0.5 mg / mL; the extrusion process parameters are to repeatedly extrude 10 to 15 times using an Avanti mini-extruder with a filter membrane pore size of 400 nm; the centrifugation process parameters are 10000 to 12000 rpm and 5 to 10 min.

[0019] The present invention also provides an application of an exosome-coated responsive drug-loaded dendrimer nanogel system in the anti-inflammatory / antioxidant synergistic treatment of acute lung injury.

[0020] Polyamidoamine dendrimer (PAMAM) has abundant amino groups on its surface, and has a highly branched three-dimensional structure and excellent monodispersity. Nanogels (NGs) are nanoscale hydrogel particles with a three-dimensional network structure composed of hydrophilic or amphiphilic polymers through physical or chemical cross-linking methods, and have advantages such as good colloidal stability, biocompatibility, high loading capacity, and easy phagocytosis by cells. In addition, NGs can improve the drug delivery efficiency, endow the material with environmental responsiveness and better anti-tumor effects by modifying metal ions on the monomer surface, loading drugs internally or designing cross-linking agents. Therefore, using PAMAM dendrimer as a raw material to prepare nanogels is expected to combine the dual advantages of dendrimers and nanogels, providing new ideas for constructing responsive drug delivery carriers.

[0021] Exosomes are cell-derived nanovesicles, and the proteins, lipids and nucleic acids carried by them can be used as transmission carriers of important signals for cell immune regulation. Among them, mesenchymal stem cell-derived exosomes (MSC-Exo) can promote the polarization of macrophages into the M2 anti-inflammatory type by targeting the nuclear factor κB (NF-κB) signaling pathway or cell metabolism pathway in cells, thereby weakening the over-activated inflammatory response. In addition, MSC-Exo plays an important role in restoring damaged alveolar epithelial cells and is expected to be used to repair lung tissue damage mediated by inflammatory mediators.

[0022] The exosome-coated responsive drug-loaded dendrimer nanogel system provided by the present invention, its preparation method and application have good biosafety, can effectively improve the bioavailability of small molecule drugs, and at the same time target and deliver drugs to the lesion site and release drugs responsively, showing excellent synergistic treatment effects in the ALI mouse model, providing new ideas for constructing safe, intelligent and efficient drug carriers.

[0023] Beneficial effects

[0024] (1) The reaction conditions of the present invention are simple, easy to operate and separate, and have good development prospects.

[0025] (2) The drug-loaded nanogel system prepared by the present invention can effectively improve the bioavailability of Tax, and at the same time can respondently release Tax in the inflammatory microenvironment of the lesion site, providing a new idea for constructing a safe, efficient and intelligent drug carrier.

[0026] (3) For the drug-loaded nanogel platform coated with exosomes prepared by the present invention, on the one hand, the MSC-Exo wrapped on its surface can target and deliver the nanogel to the lesion site, and enhance the phagocytosis effect of alveolar macrophages on the nanogel; on the other hand, the ROS responsiveness of the nanogel system enables it to scavenge the accumulated ROS at the lesion site and respondently release Tax to exert antioxidant activity, thereby synergistically restoring the oxidative stress homeostasis; at the same time, the drug-loaded nanogel system can also synergistically exert the anti-inflammatory activities of MSC-Exo and Tax, induce the polarization of alveolar macrophages from the M1 type to the M2 type, inhibit the excessive immune activation at the lesion site, and down-regulate the expression of pro-inflammatory cytokines, achieving an enhanced anti-inflammatory treatment effect, and having potential clinical application value. Description of the Drawings

[0027] Figure 1 It is a schematic diagram of the synthesis and therapeutic application of MSC@DT-NGs in Example 1 of the present invention.

[0028] Figure 2 It is the 1 1H NMR spectra of G3-PBA (A) and G3-Tax (B) prepared in Example 2 of the present invention.

[0029] Figure 3 It is the hydrated kinetic size (A) and Zeta potential (B) diagrams of G3-Tax, DT-NGs, MSC-Exo, and MSC@DT-NGs in Example 3 of the present invention.

[0030] Figure 4 It is the TEM images of DT-NGs (A) and MSC@DT-NGs (B) in Example 4 of the present invention.

[0031] Figure 5 It is the curve diagram of the change of the hydrated kinetic size of MSC@DT-NGs dispersed in water, PBS, and DMEM medium over time in Example 5 of the present invention.

[0032] Figure 6 It is the WB results of MSCs, MSC-Exo, and MSC@DT-NGs in Example 6 of the present invention.

[0033] Figure 7This is the in vitro drug-responsive release curve of MSC@DT-NGs under different conditions in Example 7 of the present invention.

[0034] Figure 8 This is the cell viability graph after Tax, DT-NGs, and MSC@DT-NGs were co-incubated with MH-S cells for 24 h respectively in Example 8 of the present invention.

[0035] Figure 9 This is the flow cytometry graph (A) and the average fluorescence intensity quantification graph (B) after DT-NGs and MSC@DT-NGs were co-incubated with MH-S cells for 4 hours respectively in Example 9 of the present invention.

[0036] Figure 10 This is the flow cytometry analysis graph (A) and the relative fluorescence intensity quantification graph (B) of the reactive oxygen species expression level in cells after Tax, DT-NGs, and MSC@DT-NGs were co-incubated with LPS-activated MH-S cells for 24 h respectively in Example 10 of the present invention.

[0037] Figure 11 This is the flow cytometry analysis graph (A) of the expression levels of CD86 and CD206 in cells, the percentage of M1 macrophages (B), the percentage of M2 macrophages (C), and the relative M2 / M1 macrophage ratio (D) after Tax, DT-NGs, and MSC@DT-NGs were co-incubated with LPS-activated MH-S cells for 24 h respectively in Example 11 of the present invention.

[0038] Figure 12 This is the WB test result graph (A) of the intracellular NF-κB protein expression after Tax, DT-NGs, and MSC@DT-NGs treated LPS-activated MH-S cells, and the relative quantification result graphs of NF-κB expression in the nucleus (B) and cytoplasm (C) in Example 12 of the present invention.

[0039] Figure 13 This is the ELISA test result graph of pro-inflammatory cytokines interleukin (IL)-1β (A), interleukin-6 (B), interleukin-10 (C), and tumor necrosis factor (TNF)-α (D) in the bronchoalveolar lavage fluid of mice in each experimental group in Example 13 of the present invention.

[0040] Figure 14 This is the histological section analysis of the lungs of mice in each experimental group in Example 13 of the present invention. Detailed implementation mode

[0041] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0042] Unless otherwise specified, all chemical reagents in the present invention can be directly used without further purification. G3.NH 2 was purchased from Dendritech, USA. 2-Bromoethyl acrylate diselenide (a crosslinker containing a diselenide bond (SeSeCrosslinker)) was purchased from Shanghai Mujin Biotechnology Co., Ltd. Tax was purchased from Shanghai Macklin Biochemical Co., Ltd. MH-S cells (mouse alveolar macrophage cell line) were from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. RPMI-1640 complete medium and DMEM / F12 medium were purchased from Shanghai Siaolesen Biomedical Co., Ltd. Cell Counting Kit-8 (CCK-8) and ROS detection kit were purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). Monoclonal antibodies such as Anti-CD86-PE and Anti-CD206-FITC were purchased from Thermo Fisher Scientific (Waltham, MA). BALB / c mice were purchased from Shanghai SLAC Laboratory Animal Center. Water with a resistivity higher than 18.2 MΩ·cm used in all experiments was purified through a laboratory water purification system (PURIST UV Ultrapure, Shanghai Leagene Biotechnology Co., Ltd.).

[0043] The present invention uses nuclear magnetic resonance hydrogen spectroscopy ( 1 1H NMR), Zeta potential and dynamic light scattering analysis (DLS), transmission electron microscopy (TEM), Western blot (WB) and other means to characterize the physical and chemical properties of the exosome-coated responsive drug-loaded dendrimer nanogel system. Then, the CCK-8 method was used to evaluate the cytotoxicity of the exosome-coated responsive drug-loaded dendrimer nanogel system and related control materials; flow cytometry was used to evaluate the phagocytosis effect of cells on the materials; flow cytometry was used to evaluate the effect of the materials on the intracellular ROS level; flow cytometry was used to evaluate the effect of the materials on macrophage transformation; WB was used to evaluate the effect of the materials on intracellular related inflammatory signaling pathways; finally, an ALI mouse model was established to verify the in vivo anti-inflammatory and antioxidant therapeutic effect of the nanogel system.

[0044] Example 1

[0045] This example provides a reactive drug-loaded dendrimer nanogel system encapsulated with exosomes (MSC@DT-NGs). Taxifolin Tax is grafted onto the surface of the third-generation PAMAM dendrimer modified with phenylboronic acid PBA through a borate ester bond. Subsequently, a reactive oxygen species (ROS)-sensitive drug-loaded nanogel is synthesized using a crosslinker containing a diselenide bond through an inverse microemulsion method. Furthermore, mesenchymal stem cell-derived exosomes MSC-Exo are physically encapsulated on the surface of the drug-loaded nanogel. The synthesis schematic diagram and its therapeutic application are as Figure 1 shown.

[0046] The preparation method of the reactive drug-loaded dendrimer nanogel system encapsulated with exosomes (MSC@DT-NGs) in this example includes the following steps:

[0047] (1) Weigh 30 mg of G3.NH 2 and 7.46 mg of BPBA, and dissolve them separately in 2 mL of DMSO. Subsequently, add the BPBA solution to the G3.NH 2 solution, place it in a water bath at 70 °C, and stir for 24 h to obtain the G3-PBA crude product; transfer the crude product to a dialysis bag with a molecular weight cut-off of 3500 Da, dialyze it in ultrapure water for three days, and freeze-dry to obtain the G3-PBA powder.

[0048] (2) Weigh 20 mg of G3-PBA and dissolve it in 5 mL of ultrapure water; weigh 4 mg of Tax and dissolve it in 100 μL of methanol, then add 5 mL of ultrapure water to fully dissolve Tax; add the G3-PBA solution to the Tax solution and stir for 36 h to obtain the G3-Tax crude product; finally, transfer the crude product to a dialysis bag with a molecular weight cut-off of 3500 Da, dialyze it in ultrapure water for 3 days, and obtain the product G3-Tax after freeze-drying.

[0049] (3) Dissolve 20 mg of G3-Tax and 3 mg of 2-bromoethyl acrylate diselenide (a cross-linking agent containing diselenide bonds) in 1 mL of deionized water and mix and stir for 15 min; then, dropwise add the mixed solution into 12 mL of a cyclohexane solution containing Span 80 and Tween 80 (the mass ratio of Span 80 to Tween 80 is 5:1, and the total mass of Span 80 and Tween 80 is 280 mg), stir for 5 min to initially form a W / O emulsion. Subsequently, place the W / O emulsion in an ultrasonic crusher and ultrasonicate for 5 min, and add 1 mL of triethylamine dropwise, and continue to stir overnight. Centrifuge the reacted solution at 12,000 rpm for 15 min to collect the precipitate. Redisperse the precipitate with 10 mL of acetone and then place it in a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da and dialyze in ultrapure water for three days, and obtain responsive drug-loaded dendritic macromolecule nanogels (DT-NGs) after freeze-drying.

[0050] (4) Extract MSCs from the femurs and tibias of 2-week-old Sprague-Dawley (SD) rats, and then culture them in DMEM / F12 medium containing 10% serum; when the MSCs are at passages 3 - 6 and the confluence rate reaches 75%, change to a mesenchymal stem cell medium dedicated to exosomes and continue to culture for 48 h; then, collect the medium rich in exosomes and centrifuge at 300 g for 10 min to remove dead cells. Then, centrifuge the supernatant at 3000 g for 15 min to remove cell debris; subsequently, centrifuge the supernatant at 10,000 g for 1 h to remove microvesicles; finally, centrifuge the supernatant at 100,000 g for 1 h, collect the precipitate and resuspend it with PBS to obtain mesenchymal stem cell-derived exosomes MSC-Exo.

[0051] (5) Ultrasonicate 2 mg of DT-NGs and 1 mL of MSC-Exo (0.4 mg / mL) in an ice bath at 4 °C for 10 min to fully mix, and then, use an Avanti mini-extruder with a filter membrane pore size of 400 nm to extrude the mixed solution back and forth 11 times, centrifuge at 10,000 g for 5 min to remove free MSC-Exo, and then collect the precipitate to obtain an exosome-coated responsive drug-loaded dendritic macromolecule nanogel system (MSC@DT-NGs).

[0052] Example 2

[0053] Weigh 5 mg each of G3-PBA and G3-Tax in Example 1, and dissolve them separately in 500 μL of D 2 O for nuclear magnetic resonance hydrogen spectrum analysis ( Figure 2 ). As Figure 2 shown in A, the benzene ring proton peak of the nuclear magnetic resonance hydrogen spectrum of G3-PBA is at 7.0 - 8.0 ppm, indicating that PBA has been successfully grafted onto G3.NH2 Integral calculation shows that each G3.NH 2 On it, 5 PBAs are grafted. Compared with G3-PBA, the proton nuclear magnetic resonance spectrum of G3-Tax shows resonance peaks at 6.4 - 6.9 ppm, indicating that taxifolin is successfully modified onto G3-PBA. Integral calculation shows that 2.8 Tax are grafted onto each G3-PBA.

[0054] Example 3

[0055] 1 mL of G3-Tax, DT-NGs, MSC-Exo, and MSC@DT-NGs with a concentration of 1 mg / mL are respectively taken for hydrodynamic size and Zeta potential tests. The results are as Figure 3 shown in Figures 3A and 3B. The hydrodynamic diameters of G3-Tax, DT-NGs, MSC-Exo, and MSC@DT-NGs are 128.4 nm, 100.7 nm, 226.3 nm, and 78.3 nm respectively, and the surface potentials are 22.7 mV, 11.1 mV, -6.8 mV, and -5.2 mV respectively. After being encapsulated by exosomes, the surface potential of the nanogel decreases from 11.1 mV to -5.2 mV, approaching the potential of the individual exosomes. In addition, after being encapsulated by exosomes, the hydrodynamic diameter of the nanogel decreases from 100.7 nm to 78.3 nm, and the aggregation state of its aqueous solution changes. The above results indicate that exosomes are successfully encapsulated on the surface of the nanogel.

[0056] Example 4

[0057] Weigh the DT-NGs and MSC@DT-NGs drug-loaded nanogel systems prepared in Example 1 and configure them into a 1 mg / mL solution. Drop the solution onto the surface of a copper mesh with a carbon film and let it stand at room temperature for drying to obtain the sample. For the copper mesh with the carbon film dropped with MSC@DT-NGs drug-loaded nanogel, after dropping, let it stand for 2 min, then drop phosphotungstic acid for negative staining, and let it stand at room temperature for drying to obtain the negatively stained MSC@DT-NGs sample. Use a JEM-2010F transmission electron microscope to detect the sample. The TEM image of DT-NGs is as Figure 4 shown in Figure 4A, and its morphology presents as uniform spheres with a size of about 87.2 nm. The particle size of MSC@DT-NGs is slightly smaller than that of DT-NGs, with a size of about 60.1 nm ( Figure 4 Figure 4B). In addition, the negatively stained TEM image of MSC@DT-NGs ( Figure 4 the upper left corner of Figure 4B) shows that a clearly defined membrane structure covers the surface of MSC@DT-NGs, further indicating that MSC-Exo is successfully encapsulated on the surface of DT-NGs, proving the successful preparation of MSC@DT-NGs.

[0058] Example 5

[0059] Disperse 0.1 mg of the MSC@DT-NGs prepared in Example 1 in 1 mL of H 2 O, PBS, and DMEM medium respectively. The hydrodynamic size of MSC@DT-NGs in various solutions did not change significantly within one week (as Figure 5 shown), indicating that the prepared MSC@DT-NGs have good colloidal stability.

[0060] Example 6

[0061] To verify the successful extraction of exosomes and the retention of exosome activity in the nanogel, Western blotting (WB) was used to detect the expression of exosome positive markers CD63 and TSG101, as well as the negative marker Calnexin. Mesenchymal stem cells (MSCs) and MSC-Exo were used as controls. The results are as Figure 6 shown. MSC@DT-NGs have the same high expression of CD63 and TSG101 proteins as MSC-Exo, and neither expresses the intracellular marker protein Calnexin, demonstrating the successful encapsulation of exosomes on the surface of the nanogel system without affecting the bioactive components of exosomes.

[0062] Example 7

[0063] To investigate the in vitro drug-responsive release performance of the material, take the MSC@DT-NGs (1 mg) prepared in Example 1 and dissolve it in 5 mL of phosphate buffer in different systems and transfer it to a dialysis bag. Subsequently, immerse the dialysis bag in 45 mL of phosphate buffer in different systems and incubate it in a shaker at 37 °C and 90 rpm. The phosphate buffers in different systems are: pH = 7.4, pH = 6.5, pH 7.4 buffer containing 0.1 mM H 2 O 2 (pH = 7.4 + H 2 O 2 ), pH 6.5 buffer containing 0.1 mM H 2 O 2 (pH = 6.5 + H 2 O 2 ). At different time points, take 5.0 mL from the external solution of the dialysis bag and supplement it with the same volume of fresh buffer. The cumulative release amount of Tax was determined by measuring the absorbance of the solution at 320 nm by UV-vis spectroscopy. The results are as Figure 7 shown. MSC@DT-NGs only release 38.7% of Tax within 72 h under the condition of pH = 7.4, proving that the prepared material can maintain good stability in the normal physiological environment and avoid excessive drug leakage. And the nanogel releases more Tax under the conditions of pH = 6.5 and pH = 7.4 + H 2 O 2In the buffer systems, the drug release rates within 72 h were 50.1% and 61.3% respectively. The increased drug release amounts were due to the acid responsiveness of the borate ester bond and the excellent reactive oxygen sensitivity of the nanogel itself. It should be noted that the nanogel has a drug release rate of Tax reaching 64.3% at 24 h in the buffer system of pH = 6.5 + H 2 O 2 . When the incubation time was extended to 72 h, the drug release rate of Tax was as high as 74.0%. This is because in the presence of both an acidic environment and hydrogen peroxide, the simultaneous cleavage of the phenylborate ester bond and the diselenide bond in MSC@DT-NGs was triggered, increasing the drug release of Tax, which provides an important guarantee for the responsive drug release of the prepared nanogel at the ALI lesion site.

[0064] Example 8

[0065] The effects of Tax, DT-NGs, and MSC@DT-NGs on the viability of MH-S cells were evaluated using CCK-8. MH-S cells in the logarithmic growth phase were collected and seeded in a 96-well cell culture plate at a density of 1×10 4 cells per well and cultured in an environment of 37 °C and 5% CO 2 for 24 h. Then, the old medium was removed, and fresh medium containing Tax, DT-NGs, MSC@DT-NGs (Tax concentrations were 0, 10, 25, 50, 100, 200, 400, 600, 800 μM respectively) was added, and the cells were co-incubated for 24 h. The original medium was discarded, and the cells were washed three times with PBS. Then, fresh medium containing 10% (v / v) CCK-8 was added, and the cells were further incubated in the incubator for 3 h. The absorbance values of each well were measured at a wavelength of 450 nm using a multifunctional microplate reader. The cells treated with PBS were used as the blank control, and the cell viability was recorded as 100%. The results are as Figure 8 shown. When the Tax concentration was 100 μM, the cell activities of the Tax, DT-NGs, and MSC@DT-NGs treatment groups were all greater than 90%. When the Tax concentration was 200 μM, the cell activities of the Tax, DT-NGs, and MSC@DT-NGs treatment groups were 98.8%, 69.0%, and 59.2% respectively. This is because the surface amino groups of G3.NH 2 resulted in a relatively high surface potential of the nanogel, so it had certain cytotoxicity. In addition, after being encapsulated by mesenchymal stem cell-derived exosomes, the intracellular delivery efficiency of the nanogel was enhanced, thus showing more obvious cytotoxicity. At the same time, as the Tax concentration increased, the toxicity of the drug to the cells became more significant. To avoid the toxic effects of the drug and the gel on the cells, a Tax concentration of 100 μM was selected for subsequent experiments.

[0066] Example 9

[0067] The MH-S cells were used as a cell model to verify the phagocytosis effect of cells on DT-NGs and MSC@DT-NGs. Cells in the logarithmic growth phase were collected and seeded in a 12-well cell culture plate at a density of 1×10 5 cells per well, and cultured at 5% CO 2 2, 37 °C for 24 h. Subsequently, the medium was replaced with fresh serum-free RPMI-1640 medium, and DT-NGs and MSC@DT-NGs were added respectively, and co-incubated with the cells for 4 h (DT-NGs and MSC@DT-NGs were labeled with Cy5.5, and the Tax concentration was 100 μM). The normal group was treated with PBS. After discarding the medium, the cells were digested with trypsin, centrifuged, and collected. After washing three times with PBS, the intracellular fluorescence intensity was detected by flow cytometry. As Figure 9 shown, the fluorescence intensity of MSC@DT-NGs was significantly higher than that of DT-NGs and the PBS group, indicating that the encapsulation of exosomes endows the nanogel system with excellent cell targeting.

[0068] Example 10

[0069] To verify the effect of MSC@DT-NGs on the scavenging of reactive oxygen species (ROS) in macrophages, mouse alveolar macrophages (MH-S cells) in the logarithmic growth phase were collected and seeded in a 12-well plate at a density of 1×10 5 cells per well, and cultured at 5% CO 2 2, 37 °C for 24 h. Subsequently, the medium was replaced with RPMI-1640 medium containing lipopolysaccharide (LPS) (concentration 2 μg / mL) and incubated for 24 h. PBS was added to the control group. Subsequently, the medium was replaced with fresh serum-free RPMI-1640 medium containing Tax, DT-NGs or MSC@DT-NGs (Tax concentration 100 μM) and incubated for 4 h. After discarding the old medium, RPMI-1640 medium containing serum was added and co-incubated with the cells for 20 h. After collecting the cells by trypsin digestion, the cell pellet was resuspended with 1 mL PBS, and the ROS fluorescent probe DCFH-DA was added and co-incubated with the cells at 37 °C for 20 min. After washing three times with PBS to remove the probe not bound to the cells, the cell pellet was resuspended with 300 μL PBS and transferred to a flow tube, and the effect of the nanomaterials on the scavenging of intracellular ROS in macrophages was evaluated by flow cytometry. The results are as Figure 10As shown, Tax can reduce the level of a large amount of intracellular ROS caused by LPS to a certain extent. Importantly, since DT-NGs are ROS-responsive and can improve the bioavailability of Tax, they exhibit antioxidant properties superior to free Tax. Compared with the DT-NGs group, MSC@DT-NGs showed a more significant ROS scavenging effect, indicating that the exosome coating can further amplify the antioxidant activity of Tax by enhancing the cell uptake performance of the material, showing excellent ability to alleviate macrophage over-oxidative stress.

[0070] Example 11

[0071] To verify the effect of the MSC@DT-NGs drug-loaded nanogel system on macrophage polarization, MH-S cells in the logarithmic growth phase were collected and seeded in 12-well plates at a density of 1×10 5 cells per well, and cultured at 5% CO 2 ₂, 37 °C for 24 h. The medium was discarded and replaced with RPMI-1640 medium containing LPS (concentration 2 μg / mL), and incubated for 24 h. PBS was added to the control group. Subsequently, it was replaced with fresh serum-free RPMI-1640 medium containing Tax, DT-NGs, and MSC@DT-NGs (Tax concentration 100 μM) and incubated for 4 h. The old medium was discarded, and RPMI-1640 medium containing serum was added and co-incubated with the cells for 20 h. Subsequently, the medium was discarded, the cells were washed twice with PBS, digested with trypsin and collected, and the cell pellet was resuspended with 200 μL PBS. Anti-CD206-FITC and Anti-CD86-PE antibodies were added and co-incubated with the cells in the dark at 4 °C for 30 min. After washing three times with PBS to remove the antibodies not bound to the cells, the cell pellet was resuspended with 300 μL PBS and transferred to a flow tube. The effect of the nanogel on macrophage polarization was evaluated by detecting the changes in the expression levels of CD86 and CD206 in the cells. As Figure 11As shown in A-D, in the comparative analysis with the LPS group, a decrease in the CD86 expression level was observed in the Tax group, while the CD206 expression level did not show a significant difference from that of the LPS group. At the same time, the number of M1 macrophages in the Tax group was reduced compared to the LPS group. This result indicates that Tax can effectively inhibit the polarization process of LPS-induced macrophages into the M1 type. In the group with Tax loaded on nanogels (DT-NGs), the expression level of CD86 decreased significantly, while the expression level of CD206 increased slightly, confirming that the delivery efficiency of Tax through nanogels was improved, thereby more effectively regulating the phenotypic transformation of MH-S cells. Further, compared with the DT-NGs group, the MSC@DT-NGs group showed the highest CD206 expression level and the lowest CD86 expression level, and its M2 / M1 ratio was 1.3 times that of the DT-NGs group. This phenomenon is attributed to the strong anti-inflammatory activity of mesenchymal stem cell exosomes (MSC-Exo) itself and its ability to enhance the uptake of nanogels by cells, thus effectively promoting the polarization of MH-S cells into the M2 type and demonstrating a synergistic enhanced immunomodulatory effect.

[0072] Example 12

[0073] To verify the effect of the MSC@DT-NGs nanogel system on the expression of NF-κB protein in the cytoplasm and nucleus, MH-S cells in the logarithmic growth phase were collected and seeded in a 6-well plate at a density of 2×10 5 cells per well, and cultured at 5% CO 2 , 37 °C for 24 h. The culture medium was discarded, and replaced with RPMI-1640 medium containing LPS (concentration 2 μg / mL), and incubated for 24 h. PBS was added to the control group. Subsequently, it was replaced with fresh serum-free RPMI-1640 medium containing Tax, DT-NGs, MSC@DT-NGs (Tax concentration 100 μM) and incubated for 6 h. The old culture medium was discarded, and RPMI-1640 medium containing serum was added and co-incubated with the cells for 20 h. Subsequently, the culture medium was discarded, the cells were washed twice with PBS, and after trypsin digestion and cell collection, nuclear protein extraction reagent was used to separate nuclear proteins and cytoplasmic proteins. The protein concentration was measured, and then SDS-PAGE electrophoresis, membrane transfer, immunoreaction, and ECL chemical developer fixing experiments were carried out in sequence, and the gel images were analyzed. As Figure 12As shown, the LPS group served as the negative control group, with the highest expression level of NF-κB in the nucleus and the lowest in the cytoplasm, which is consistent with the process that NF-κB translocates to the nucleus after activation, thereby triggering relevant inflammatory responses. NF-κB showed a decreasing trend in nuclear expression and an increasing trend in cytoplasmic expression in the Tax group, DT-NGs group, and MSC@DT-NGs group, which is consistent with the aforementioned experimental results, that is, Tax, DT-NGs, and MSC@DT-NGs showed gradually enhanced anti-inflammatory effects. This is because the nuclear translocation of NF-κB is inhibited, thereby reducing the transcription and expression of inflammation-related genes. Especially in the MSC@DT-NGs group, the expression of NF-κB in the nucleus was the lowest, while the expression in the cytoplasm was the highest. This phenomenon may be related to the anti-inflammatory activity of MSC-Exo. MSC-Exo can enhance the uptake of nanogels by cells, thereby effectively inhibiting the nuclear translocation of NF-κB and reducing the release of inflammatory mediators, showing a synergistically enhanced immune regulation ability.

[0074] Example 13

[0075] All animal experiments were strictly conducted in accordance with the standards of the Experimental Animal Ethics Committee of Donghua University. Six-week-old male BALB / c mice used in the experiments were purchased from Shanghai SLAC Laboratory Animal Center. An acute lung injury animal model was established 24 hours after intratracheal atomization of LPS (5 mg / kg), and an equal volume of PBS was atomized intratracheally in the control group. The white mice were randomly divided into 5 groups (PBS control group, LPS treatment group, Tax treatment group, DT-NGs treatment group, and MSC@DT-NGs treatment group), with 7 mice in each group. 100 μL of PBS, Tax, DT-NGs, or MSC@DT-NGs solution (Tax concentration was 1 mM / kg) was administered to the lungs of white mice in each group through an atomizing dispenser. At 24 hours after treatment, the lungs were perfused with PBS, and bronchoalveolar lavage fluid was extracted. After centrifugation at 4°C (4000 rpm, 5 min), the supernatant was transferred to a new centrifuge tube to obtain bronchoalveolar lavage fluid, which was stored at -80°C. Subsequently, ELISA was used to detect the expression levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and anti-inflammatory cytokine (IL-10) in the bronchoalveolar lavage fluid. As Figure 13 shown, compared with the LPS group, the expression levels of pro-inflammatory cytokines in the bronchoalveolar lavage fluid of mice in the Tax and DT-NGs treatment groups were significantly downregulated. Since MSC@DT-NGs improved the biological activity of Tax and the immune regulation performance of coexistent exosomes, the bronchoalveolar lavage fluid of mice treated with MSC@DT-NGs had the lowest level of pro-inflammatory cytokines and the highest level of anti-inflammatory cytokine expression, effectively achieving an enhanced anti-inflammatory treatment effect on acute lung injury mice.

[0076] At 24 h after treatment, one mouse was selected from each of the 5 experimental groups, and its lung tissue was immersed in tissue fixative for 24 h, and H&E staining was performed to analyze the degree of lung tissue damage. The experimental results are as Figure 14 shown (black arrows indicate alveolar wall congestion, green arrows indicate inflammatory cells, and the scale bar in the figure is 50 μm). Compared with the model group, in the mice treated with Tax and DT-NGs, the degree of alveolar wall congestion and the degree of inflammatory cell infiltration were both reduced, but there was still a small amount of inflammatory cell infiltration and alveolar wall congestion. In the mice treated with MSC@DT-NGs, the alveolar wall congestion, the degree of pulmonary inflammatory infiltration, and the alveolar structure almost returned to the level of normal mice. This may be mainly attributed to the fact that MSC@DT-NGs can improve the bioavailability of Tax while targeting and delivering it to alveolar macrophages and achieving the responsive release of Tax, and finally combining with the immune regulation and tissue repair characteristics of MSC-Exo itself to achieve the anti-inflammatory and antioxidant synergistic treatment of ALI.

Claims

1. An exosome-coated responsive drug-loaded dendrimer nanogel system, characterized in that: The drug was grafted onto the surface of the third-generation PAMAM dendrimer modified with phenylboronic acid through a boronate bond, and then a cross-linker containing a diselenide bond was used to synthesize active oxygen-sensitive drug-loaded nanogels through a reverse microemulsion method, and exosomes were encapsulated on the surface of the drug-loaded nanogels through physical action.

2. The responsive drug-loaded dendrimer nanogel system according to claim 1, characterized in that: The drug includes Taxol; and the exosomes include exosomes MSC-Exo derived from mesenchymal stem cells.

3. A method for preparing an exosome-coated responsive drug-loaded dendrimer nanogel system, comprising the following steps: (1) adding 4-bromomethylphenylboronic acid BPBA solution dropwise to the third-generation PAMAM dendrimer G3.NH2 solution, stirring for reaction, purifying, and freeze-drying to obtain the third-generation PAMAM dendrimer G3-PBA modified with phenylboronic acid PBA; (2) preparing the G3-PBA prepared in step (1) into a solution and mixing it with the drug solution, stirring the reaction, purifying, and freeze-drying to obtain the drug-grafted third-generation PAMAM dendrimer G3-drug; (3) dissolving the G3-drug and the cross-linking agent containing a diselenide bond prepared in step (2) in water, respectively, mixing and stirring to form an aqueous phase; adding a surfactant and an emulsifier to an organic solvent, respectively, mixing and stirring to form an oil phase; adding the aqueous phase dropwise to the oil phase to obtain a W / O polymer emulsion, and after ultrasonic crushing, adding a triethylamine solution dropwise, stirring, centrifuging, collecting the lower layer of gel, dispersing with a solvent, and dialysis purification to obtain a responsive drug-loaded dendrimer nanogel DT-NGs; (4) culturing cells in a culture medium, then replacing it with an exosome-specific cell culture medium and continuing the culture; collecting the exosome-rich culture medium and purifying it by centrifugation to obtain cell-derived exosomes; (5) The responsive dendrimer nanogel DT-NGs solution prepared in step (3) is ultrasonically mixed with the exosomes prepared in step (4), and after extrusion and centrifugation, an exosome-coated responsive drug-loaded dendrimer nanogel system is obtained.

4. The preparation method according to claim 3, characterized in that: The solvent of the solution in step (1) is dimethyl sulfoxide; the molar ratio of BPBA to G3.NH2 is 10-15:1; the stirring reaction temperature is 70-80°C and the time is 24-36h; the purification process parameters are: dialyzing against water for 3 days using a dialysis bag with a molecular weight cutoff of 3500Da.

5. The preparation method according to claim 3, characterized in that: In the step (2), the solvent of the G3-PBA solution is ultrapure water, and the solvent of the drug solution is methanol and ultrapure water; the molar ratio of G3-PBA to the drug is 1:5-10; the stirring reaction temperature is 20-25°C, and the time is 36-72h; the purification process parameters are: dialyzing water for 3 days using a dialysis bag with a molecular weight cutoff of 3500Da.

6. The preparation method according to claim 3, characterized in that: In the step (3), the molar ratio of the G3-drug to the cross-linking agent containing a diselenide bond is 1:3-7; the cross-linking agent containing a diselenide bond includes 2-bromoethyl acrylate diselenide; the mass ratio of the surfactant to the emulsifier is 3-8:1, the surfactant includes Span 80, and the emulsifier includes Tween 80; the volume ratio of the organic solvent in the oil phase, the water in the aqueous phase and triethylamine is 10-14:1:0.5-1, and the organic solvent includes cyclohexane.

7. The preparation method according to claim 3, characterized in that: In the step (3), the ultrasonic crushing time is 5 to 10 minutes, and the stirring time is 10 to 15 hours; the centrifugation process parameters are: 10000 to 12000 rpm, 10 to 15 minutes; the dispersion solvent includes acetone; the dialysis process parameters are: dialyzing water for 3 days using a dialysis bag with a molecular weight cutoff of 8000 to 14000 Da.

8. The preparation method according to claim 3, characterized in that: The specific process of step (4) includes: extracting mesenchymal stem cells from the femur and tibia of 2-week-old Sprague-Dawley rats, and culturing them in DMEM / F12 medium containing 10% serum; when the mesenchymal stem cells grow to the 3rd to 6th generation and the fusion rate reaches 75%, replacing it with an exosome-specific mesenchymal stem cell medium, and continuing to culture for 48 hours; collecting the exosome-rich medium, first centrifuging it at 200-300g for 10-15 minutes, collecting the supernatant and centrifuging it at 2000-3000g for 15-20 minutes, then centrifuging the supernatant at 9000-10000g for 1-1.5 hours, and finally centrifuging the supernatant at 90000-100000g for 1-1.5 hours, collecting the precipitate and resuspending it with phosphate buffer to obtain mesenchymal stem cell-derived exosomes MSC-Exo.

9. The preparation method according to claim 3, characterized in that: In the step (5), the solvents for DT-NGs and exosomes are both phosphate buffer; the mass ratio of DT-NGs to exosomes is 5:1-2; the ultrasonic mixing time is 10-20 min; the concentration of the exosomes is 0.3-0.5 mg / mL; the extrusion process parameters are repeated extrusion 10-15 times using an Avanti micro-extruder with a filter membrane pore size of 400 nm; and the centrifugation process parameters are 10000-12000 rpm, 5-10 min.

10. Use of the exosome-coated responsive drug-loaded dendrimer nanogel system as claimed in claim 1 in the synergistic anti-inflammatory / antioxidant treatment of acute lung injury.

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