Nano-carrier system for targeting aging alveolar epithelial cells as well as preparation method and application of nano-carrier system

Abstract

The invention relates to the field of biological medicine, in particular to a nano-carrier system targeting aging alveolar epithelial cells as well as preparation and application of the nano-carrier system. The preparation method comprises the following steps: carrying out self-assembly on chemically synthesized PEI-Se-Se-BAI1 and si-cGAS to form BAI1-cGAS particles; the alveolar epithelial cells with high expression of Anti-DPP4-Scfv are obtained through gene editing, and the extracellular vesicles alpha-DPP4-MNs are obtained through centrifugation; finally, the BAI1-cGAS and the alpha-DPP4-MNs are subjected to membrane extrusion, and the nano carrier system BAI1-cGAS (at) DNs is prepared. The nano-carrier system can specifically target aged alveolar epithelial cells in pulmonary fibrosis through Anti-DPP4-Scfv, BAI1 is released, mitochondrial outer membrane permeability is inhibited, and mtDNA is prevented from being released to cytoplasm; meanwhile, si-cGAS is released, and activation of a cGAS / STING pathway and a downstream inflammation pathway is inhibited, so that inflammatory aging of alveolar epithelial cells is resisted, and pulmonary fibrosis is treated. The nano-carrier system integrates the advantages of aging cell membrane positioning, responsive drug release, dual inflammation pathway inhibition and the like, specifically delays the aging of alveolar epithelial cells, and provides an efficient, safe and accessible method for treating pulmonary fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a nano-carrier system targeting senescent alveolar epithelial cells, a preparation method thereof and an application thereof. Background Art

[0002] Cell senescence is a response to sub-lethal damage caused by a series of stimuli, which is characterized by indefinite growth arrest and the production of a large number of senescence-associated secretory phenotypes (SASP), thereby inducing inflammation in the body through autocrine and paracrine pathways, and further causing tissue and organ dysfunction and inflammatory aging. More and more evidence shows that the abnormally accumulated senescent cells are an important cause of senescence-related diseases and pathological changes.

[0003] Pulmonary fibrosis is a chronic, progressive and invasive interstitial lung disease, which is characterized by irreversible destruction of alveolar structure and interstitial scar formation, can damage lung function and lead to death, and its pathogenesis has not been fully understood, and there is currently no effective treatment method. Research shows that the senescence of alveolar type II epithelial cells and fibroblasts is a key factor in the occurrence and development of pulmonary fibrosis. The senescence of alveolar type II epithelial cells can promote its differentiation disorder and reduced stemness, and promote inflammatory cell infiltration, collagen deposition and fibrosis through the production of a large amount of SASP. Therefore, targeting the clearance of senescent cells or blocking the secretion of SASP has become a new strategy for the treatment of pulmonary fibrosis, and seeking relevant intervention targets and specific therapeutic drugs is the key among them.

[0004] Mitochondria are important organelles that control cell energy metabolism, proliferation, apoptosis and senescence, and the stability of their structure and function is of great significance for maintaining the normal physiological functions of cells. Research shows that the mitochondria of senescent alveolar epithelial cells or fibroblasts are damaged by many factors such as inflammation and injury, and their integrity is destroyed, especially the increase in the outer membrane permeability (MOMP), resulting in the release of mitochondrial DNA (mtDNA) into the cytoplasm. The mtDNA in the cytoplasm further activates the cGAS-STING pathway, thereby triggering the release of SASP and senescence-related factors. Therefore, blocking the cGAS-mediated inflammatory cascade reaction is considered to be an important path to inhibit inflammaging; on the other hand, the level of MOMP mainly depends on the formation of membrane pores mediated by BAX and BAK. As a small molecule inhibitor of BAX, BAI1 can block the activity of BAX, inhibit the increase in senescence-related MOMP, reduce the leakage of mtDNA into the cytoplasm, and thus effectively inhibit the activation of the cGAS-STING pathway. The combined application of the above two inhibition techniques can effectively ensure the abnormal and continuous activation of the inflammatory signaling pathway in senescent cells, reduce the release of SASP, and improve senescence phenotypes such as pulmonary fibrosis.

[0005] How to specifically block the pathological activation of the cGAS-STING pathway related to senescent cells without affecting the physiological functions of normal cells is a key problem to be solved in the research and development of anti-aging drugs. Research has confirmed that the high expression of dipeptidyl peptidase 4 (DPP4) is closely related to the process of pulmonary fibrosis. In the ACE2 cells of patients with pulmonary fibrosis, the expression and activity of DPP4 increase with the progression of the disease. DPP4 is a transmembrane serine polypeptide enzyme that is selectively expressed in senescent cells, including senescent lung epithelial cells and fibroblasts. Therefore, DPP4, as a surface marker of senescent cells, has important potential in the sorting and targeted therapy of senescent cells. Currently, most anti-pulmonary fibrosis drugs used clinically inhibit collagen synthesis, but they cannot reverse existing fibrosis, promote lung repair, or reduce mortality. Targeting senescent cells with DPP4 and specifically blocking the release of inflammatory pathways and SASP is expected to become a new and promising strategy for the research and development of drugs for the prevention and treatment of pulmonary fibrosis. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a preparation method of a nano-carrier system targeting senescent alveolar epithelial cells in view of the unclear anti-pulmonary fibrosis target and the lack of specific therapeutic drugs in the prior art.

[0007] Another technical problem to be solved by the present invention is to provide the nano-carrier system prepared by the above preparation method.

[0008] Another technical problem to be solved by the present invention is to provide the application of the above nano-carrier system.

[0009] The last technical problem to be solved by the present invention is to provide a pharmaceutical composition containing the nano-carrier system.

[0010] To solve the above technical problems, the present invention discloses the following technical solutions:

[0011] In the first aspect, the present invention discloses a preparation method of a nano-carrier system targeting senescent alveolar epithelial cells, comprising the following steps:

[0012] (1) Polyethyleneimine-biselenide-carboxyl (PEI-Se-Se-COOH) obtained by esterification reaction of carboxyl-biselenide-carboxyl (COOH-Se-Se-COOH) and polyethyleneimine (PEI) is dialyzed and freeze-dried, and then reacted with inhibitor BAI1 to obtain a high molecular polymer PEI-Se-Se-BAI1;

[0013] (2) Self-assemble the polymer PEI-Se-Se-BAI1 obtained in step (1) with si-cGAS to obtain polymer nanoparticles BAI1-cGAS loaded with si-cGAS;

[0014] (3) Add the Anti-DPP4 plasmid cloned with the coding sequence of the anti-DPP4 chimeric antigen receptor Anti-DPP4 Scfv to the MLE-12 alveolar epithelial cell culture medium and culture to obtain the MLE-12 alveolar epithelial cell line specifically expressing Anti-DPP4-Scfv. After digestion, resuspension, and extrusion, alveolar epithelial cell-derived extracellular vesicles α-DPP4-MNs are obtained;

[0015] (4) Mix the polymer nanoparticles BAI1-cGAS obtained in step (2) with the alveolar epithelial cell-derived extracellular vesicles α-DPP4-MNs obtained in step (3) and extrude to obtain the nano-drug delivery system BAI1-cGAS@DNs.

[0016] Among them, in step (1), the molar ratio of the carboxyl-bis-selenium bond-carboxyl to polyethyleneimine is 1:1 to 3:1; the preferred molar ratio is 1:1.

[0017] Among them, in step (1), the carboxyl-bis-selenium bond-carboxyl and polyethyleneimine are dissolved in a methanol aqueous solution, and the concentration of the methanol aqueous solution is 200 to 300 mL / L (i.e., 20 to 30 vt%), and the preferred concentration is 200 mL / L (i.e., 20 vt%).

[0018] Among them, in step (1), for the esterification reaction, the conditions are: under nitrogen protection, stirring for 12 to 36 h in the dark at 40 to 60 °C, and the preferred conditions are: under nitrogen protection, stirring for 24 h in the dark at 60 °C.

[0019] Among them, in step (1), the polyethyleneimine-bis-selenium bond-carboxyl contains a cationic compound with a bis-selenium bond.

[0020] Among them, in step (1), for dialysis, dialysis is carried out in ultrapure water using a 1000 Da dialysis bag.

[0021] Among them, in step (1), the molar ratio of the polyethyleneimine-bis-selenium bond-carboxyl to the inhibitor BAI1 is 2:1 to 2:3, and the preferred molar ratio is 1:1.

[0022] Among them, in step (1), for the reaction, the reaction system includes an anhydrous dimethyl sulfoxide solution (anhydrous DMSO solution) containing dicyclohexylcarbodiimide (DCC) and dimethylacetamide (DMAC).

[0023] Specifically, in the anhydrous DMSO solution, the concentrations of DCC and DMAC are 5-10 mg / mL, and the preferred concentration is 10 mg / mL.

[0024] Among them, in step (1), the reaction conditions are as follows: under nitrogen protection, stirring for 12-36 h under dark conditions at 40-60 °C, and the preferred conditions are: under nitrogen protection, stirring for 24 h under dark conditions at 60 °C.

[0025] Among them, in step (1), after the reaction is completed, the solution of the polymer PEI-Se-Se-BAI1 is diluted with water, acidified with hydrochloric acid to pH 3.5-4.5, dialyzed in ultrapure water using a 1000 Da dialysis bag to remove unreacted impurities, and then the obtained solution is frozen and dried to obtain the polymer PEI-Se-Se-BAI1.

[0026] Among them, in step (1), the polymer PEI-Se-Se-BAI1 is a ROS-sensitive cationic lipid PEI-Se-Se-BAI1 containing a diselenide bond.

[0027] Among them, in step (2), the mass ratio of the polymer PEI-Se-Se-BAI1 to si-cGAS is 2625:60-78, and the preferred mass ratio is 2625:69.

[0028] Among them, in step (2), in the self-assembly, si-cGAS is encapsulated by an electrostatic adsorption drug-loading method to form polymer nanoparticles BAI1-cGAS.

[0029] Among them, in step (2), the si-cGAS includes a sense strand and an antisense strand.

[0030] Specifically, the base sequence of the sense strand is as follows: CAAAGAAGGACUACCUAUU(dT)(dT); the base sequence of the antisense strand is as follows: AAUAGGUAGUCCUUCUUUG(dT)(dT).

[0031] Specifically, in the self-assembly process, the polymer PEI-Se-Se-BAI1 is dissolved in an aqueous NaCl solution, si-cGAS is added in proportion, and incubated at 32-42 °C for 20-40 min.

[0032] Among them, in step (3), the Anti-DPP4 plasmid cloned with the coding sequence of the anti-DPP4 chimeric antigen receptor Anti-DPP4 Scfv is prepared by GenePharma.

[0033] Among them, in step (3), the coding sequence of the anti-DPP4 chimeric antigen receptor Anti-DPP4 Scfv is as shown in SEQ ID NO: 1, including an extracellular region and a transmembrane region. Specifically, the extracellular region includes a single-chain antibody and a hinge region. The C-terminus of the single-chain antibody is connected to the N-terminus of the hinge region, and the single-chain antibody specifically recognizes murine DPP4 antigen; the N-terminus of the transmembrane region is connected to the C-terminus of the hinge region of the extracellular region and is subsequently embedded in the cell membrane of MLE-12 cells.

[0034] Among them, in step (3), the MLE-12 alveolar epithelial cell medium has the following formulation: 10% v / v fetal bovine serum + 1% v / v penicillin / streptomycin + 89% v / v high-glucose DMEM medium.

[0035] Among them, in step (4), the mass ratio of the polymer nanoparticles BAI1-cGAS to the alveolar epithelial cell extracellular vesicles α-DPP4-MNs is 50-100:1, and the preferred mass ratio is 100:1.

[0036] Among them, in steps (3) and (4), the extrusion is carried out by reciprocating extrusion using an extruder with a 200 nm filter membrane, and the number of extrusion times is 5-20 times back and forth, preferably 20 times.

[0037] In a second aspect, the present invention provides a nanocarrier system BAI1-cGAS@DNs targeting senescent alveolar epithelial cells, and the nanocarrier system BAI1-cGAS@DNs is prepared by the preparation method of the nanocarrier system targeting senescent alveolar epithelial cells.

[0038] Specifically, the nanocarrier system BAI1-cGAS@DNs includes polymer nanoparticles BAI1-cGAS and alveolar epithelial cell extracellular vesicles α-DPP4-MNs;

[0039] Furthermore, the polymer nanoparticles BAI1-cGAS are polymer nanoparticles containing diselenide bonds, including inhibitor BAI1 and short interfering RNA of cyclic GMP-AMP synthase (si-cGAS, that is, cGAS-specific small interfering RNA); the alveolar epithelial cell extracellular vesicles α-DPP4-MNs include a chimeric antigen receptor Anti-DPP4 Scfv that specifically binds to DPP4.

[0040] Specifically, the nano-vector system BAI1-cGAS@DNs specifically targets senescent alveolar epithelial cells during pulmonary fibrosis through Anti-DPP4-Scfv, and then releases PEI-Se-Se-BAI1 and si-cGAS. The former accumulates in mitochondria, and the high ROS environment in senescent cells causes the cleavage of the diselenide bond (-Se-Se-), releasing BAI1, an inhibitor of BAX, thereby inhibiting the permeability of the mitochondrial outer membrane (MOMP), preventing the release of mtDNA into the cytoplasm, and inhibiting the activation of the cGAS / STING pathway and downstream inflammatory pathways caused by it; at the same time, si-cGAS specifically degrades the abnormally activated cGAS-STING pathway in the cytoplasm, further inhibits the inflammatory response in cells and tissues, thereby counteracting the inflammatory aging of alveolar epithelial cells and treating the occurrence of pulmonary fibrosis.

[0041] In a third aspect, the application of the nano-vector system prepared by the preparation method of the present invention and the application of the nano-vector system of the present invention in the preparation of drugs for preventing and / or treating senile pulmonary fibrosis diseases are also within the scope protected by the present invention.

[0042] Specifically, the nano-vector system BAI1-cGAS@DNs has targeting performance. It specifically targets senescent alveolar epithelial cells with high expression of DPP4 through a multifunctional nano-material system, releases BAI1 and si-cGAS, regulates BAX in the mitochondria of alveolar epithelial cells, improves the leakage of mtDNA, down-regulates the cGAS-STING pathway, regulates its own chronic inflammation, counteracts inflammatory aging, restores the normal function of alveolar epithelial cells, reduces the stress burden of cells, and alleviates senescent phenotypes.

[0043] In a fourth aspect, the present invention provides a pharmaceutical composition, which contains the nano-vector system prepared by the preparation method of the present invention or contains the nano-vector system of the present invention.

[0044] Among them, the dosage form of the pharmaceutical composition can exist in any form of pharmaceutical preparation, such as an injection, such as a freeze-dried injection.

[0045] Beneficial effects:

[0046] The present invention designs the following anti-aging nano-drugs for senescent alveolar epithelial cells in pulmonary fibrosis: First, a ROS-sensitive cationic lipid polymer compound containing a diselenide bond (PEI-Se-Se-BAI1) is synthesized, and then si-cGAS is electrostatically adsorbed and self-assembled to form core polymer nanoparticles (BAI1-cGAS). Then, engineered Escherichia coli is specifically prepared using molecular cloning technology to obtain an Anti-DPP4 plasmid, which is added to the mouse alveolar epithelial cell line MLE-12 and electrotransformed to produce MLE-12 cells that specifically express anti-DPP4. Engineered extracellular vesicles α-DPP4-MNs are produced by membrane extrusion and fused with BAI1-cGAS to finally form a nano-prodrug BAI1-cGAS@DNs that targets senescent cells. This drug can effectively regulate mitochondrial BAX in alveolar epithelial cells, improve the leakage of mtDNA, and thus down-regulate the activity of the cGAS-STING pathway by specifically targeting and recognizing senescent alveolar epithelial cells in pulmonary fibrosis and releasing BAI1 and si-cGAS. This process helps to regulate its own chronic inflammation and thus counteract inflammation-related aging. In addition, this intervention can restore the normal function of alveolar epithelial cells, reduce the stress burden of cells, protect cells from overactivation, and thus maintain their function and finally alleviate the senescent phenotype. The present invention helps to analyze and explore new targets and new mechanisms for anti-aging treatment and provides new methods and new technologies for the development of biological agents for various aging-related diseases. Brief Description of the Drawings

[0047] The following further specifically describes the present invention in conjunction with the drawings, and the above and / or other advantages of the present invention will become clearer.

[0048] Figure 1 It is a chemical synthesis process diagram of PEI-Se-Se-BAI1.

[0049] Figure 2 It is a transmission electron microscope image of the BAI1-cGAS@DNs nano-drug delivery system complex.

[0050] Figure 3 It is a dynamic light scattering particle size distribution diagram of the BAI1-cGAS@DNs nano-drug delivery system.

[0051] Figure 4 It is the detection of the drug loading and encapsulation efficiency of si-cGAS in the BAI1-cGAS@DNs nano-drug delivery system.

[0052] Figure 5 It is the responsive in vitro drug release of si-cGAS in the BAI1-cGAS@DNs nano-drug delivery system.

[0053] Figure 6 To study the stability of BAI1-cGAS@DNs nanodrug delivery system towards RNase.

[0054] Figure 7 Immunofluorescence staining of p21 in senescent MLE-12 cells after treatment with the BAI1-cGAS@DNs nanodrug delivery system.

[0055] Figure 8 H&E, Masson, α-SMA and β-Gal staining of lung tissue sections of aged mice after treatment with the BAI1-cGAS@DNs nanodrug delivery system.

[0056] Figure 9 Expression of proteins related to lung inflammation, aging, and pulmonary fibrosis in mice with pulmonary fibrosis after treatment with the BAI1-cGAS@DNs nanodrug delivery system.

[0057] Figure 10 Schematic diagram of the nanocarrier system BAI1-cGAS@DNs targeting senescent alveolar epithelial cells for the treatment of pulmonary fibrosis. DETAILED DESCRIPTION

[0058] The present invention will be further described in detail below in conjunction with specific implementations, and the above and / or other advantages of the present invention will become more clear.

[0059] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0060] In the following embodiments, the polyethyleneimine (PEI), the carboxyl-diselenide bond-carboxyl (COOH-Se-Se-COOH), the dicyclohexylcarbodiimide (DCC), and the dimethylacetamide (DMAC) were purchased from Xi'an Qiyue Biotechnology Co., Ltd.; the MLE-12 cells were purchased from Wuhan Pronosai Life Science Technology Co., Ltd.; the si-cGAS and the RNase were purchased from Beijing Qingke Biotechnology Co., Ltd.; and the C57BL / 6J mice were purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd.

[0061] Example 1: Preparation of BAI1-cGAS@DNs

[0062] 1. Preparation of polymer nanoparticles BAI1-cGAS

[0063] (1) Preparation of polyethyleneimine-diselenide bond-carboxyl (PEI-Se-Se-COOH)

[0064] 20 mg of polyethyleneimine (PEI, molecular weight 2000) and 2.48 mg of carboxyl-diselenide-carboxyl (COOH-Se-Se-COOH) were placed in a round-bottom flask, and 5.0 mL of 25 vt% methanol aqueous solution (the molar ratio of PEI to COOH-Se-Se-COOH was 1:1) was added. The air in the reaction system was removed using a three-way valve, and the mixture was stirred under nitrogen protection in the dark at 60 °C for 24 h to prepare a cationic compound solution of PEI-Se-Se-COOH containing diselenide bonds. The solution was placed in a dialysis bag with a molecular weight cut-off of 1000 Da, dialyzed in ultrapure water for 24 h, and then freeze-dried to obtain a solid powder of polyethyleneimine-diselenide-carboxyl (PEI-Se-Se-COOH).

[0065] (2) Preparation of ROS-sensitive cationic lipid PEI-Se-Se-BAI1 containing diselenide bonds (polymer PEI-Se-Se-BAI1)

[0066] 22 mg of the polyethyleneimine-diselenide-carboxyl (PEI-Se-Se-COOH) obtained in step (1) was dissolved in 5.0 mL of anhydrous dimethyl sulfoxide (DMSO) containing 50 mg of dicyclohexylcarbodiimide (DCC) and 50 mg of dimethylacetamide (DMAC), and 4.67 mg of inhibitor BAI1 was added for reaction (the molar ratio of PEI-Se-Se-COOH to BAI1 was 1:1). The air in the reaction system was removed using a three-way valve, and the mixture was stirred under nitrogen protection in the dark at 60 °C for 24 h to obtain a PEI-Se-Se-BAI1 solution. The PEI-Se-Se-BAI1 solution was diluted with water, acidified with hydrochloric acid to pH 3.5 - 4.5, dialyzed in ultrapure water using a 1000 Da dialysis bag to remove unreacted impurities, and then the resulting solution was frozen and dried to obtain a solid powder of the polymer PEI-Se-Se-BAI1. Figure 1 Figure for the chemical synthesis process of PEI-Se-Se-BAI1.

[0067] (3) Preparation of polymer nanoparticles BAI1-cGAS

[0068] The solid powder of PEI-Se-Se-BAI1 obtained in step (2) was dissolved in 2 mL of 150 mM NaCl, and short interfering RNA of cyclic GMP-AMP synthase (si-cGAS) was mixed with it in a ratio (the mass ratio of PEI-Se-Se-BAI1 to si-cGAS was 2625:69), and vortexed for 5 min to ensure uniform contact between the two. Then, it was incubated at 37 °C for 30 min, and si-cGAS was encapsulated by electrostatic adsorption drug-loading method to self-assemble into polymer nanoparticles BAI1-cGAS.

[0069] 2. Preparation of α-DPP4-MNs

[0070] (1) Preparation and amplification of Anti-DPP4 plasmid

[0071] The Anti-DPP4 plasmid cloned with the coding sequence of the anti-DPP4 chimeric antigen receptor Anti-DPP4 Scfv was prepared by GenePharma. The anti-DPP4 chimeric antigen receptor Anti-DPP4 Scfv, whose coding sequence is shown in SEQ ID NO: 1, includes an extracellular region and a transmembrane region. Among them, the extracellular region includes a single-chain antibody and a hinge region. The C-terminus of the single-chain antibody is connected to the N-terminus of the hinge region, and the single-chain antibody specifically recognizes murine DPP4 antigen; the N-terminus of the transmembrane region is connected to the C-terminus of the hinge region of the extracellular region and is subsequently embedded in the cell membrane of MLE-12 cells.

[0072] The Anti-DPP4 plasmid was added to the competent cells of Escherichia coli. After mixing, it was placed on ice for 5 min, then reacted in a 42 °C water bath for 90 s, and quickly inserted on ice and placed for 5 min. Then 800 μL of LB liquid medium without antibiotics was added, and it was shaken and cultured at 37 °C and 180 rpm for 45 - 60 min. An appropriate amount of the bacterial solution was spread on an LB plate containing 100 μg / mL ampicillin and cultured overnight in an inverted manner in a 37 °C incubator. One single colony was selected, placed in an LB liquid medium, and shaken and cultured overnight at 37 °C and 180 rpm. The Escherichia coli suspension obtained by overnight culture was centrifuged, and the Anti-DPP4 plasmid DNA was extracted using an endotoxin-free plasmid extraction kit.

[0073] (2) Preparation of MLE-12 alveolar epithelial cells specifically expressing Anti-DPP4 Scfv and α-DPP4-MNs of MLE-12 alveolar epithelial cell extracellular vesicles

[0074] The Anti-DPP4 plasmid extracted in step (1) was added to the MLE-12 alveolar epithelial cell medium (medium formula: 10% v / v fetal bovine serum + 1% v / v penicillin / streptomycin + 89% v / v high-glucose DMEM medium). The MLE-12 alveolar epithelial cells were electrotransfected using an electroporator, and then cultured for another 24 h to obtain an MLE-12 alveolar epithelial cell line specifically expressing Anti-DPP4 Scfv. The cells were digested with trypsin, resuspended in PBS with pH = 7.4, the cell suspension was collected, and the MLE-12 alveolar epithelial cell extracellular vesicles α-DPP4-MNs were obtained by repeatedly extruding 20 times using an extruder with a 200 nm filter membrane.

[0075] 3. Preparation of the Nano-Drug Delivery System BAI1-cGAS@DNs Targeting Senescent Alveolar Epithelial Cells with High Expression of DPP4

[0076] Mix the polymer nanoparticles BAI1-cGAS prepared in step 1 with the MLE-12 alveolar epithelial cell extracellular vesicles α-DPP4-MNs prepared in step 2 at a mass ratio of 100:1, and repeatedly extrude 20 times using an extruder with a 200 nm filter membrane to obtain the BAI1-cGAS@DNs nano-drug delivery system.

[0077] Example 2: Characterization of the BAI1-cGAS@DNs Nano-Drug Delivery System

[0078] 1. Morphological Characteristics of the BAI1-cGAS@DNs Nano-Drug Delivery System

[0079] Drop the α-DPP4-MNs, BAI1-cGAS, and BAI1-cGAS@DNs sample solutions obtained in Example 1 onto the copper grid of the electron microscope. Among them, the α-DPP4-MNs sample is negatively stained with uranyl acetate, and its morphology and particle size are observed using a transmission electron microscope (TEM).

[0080] The results are as Figure 2 shown, among which, Figure 2 The left is the image of α-DPP4-MNs, Figure 2 The middle is the image of BAI1-cGAS, Figure 2 The right is the image of BAI1-cGAS@DNs. It can be seen that the extracellular vesicles α-DPP4-MNs synthesized based on the method of Example 1 have a bilayer membrane structure and uniform particle size; compared with BAI1-cGAS and BAI1-cGAS@DNs, it can be clearly observed that the membrane structure coated on the surface of the nanoparticles after extrusion, and the nanoparticles are spherical with uniform particle size.

[0081] 2. Determination of Particle Size Distribution

[0082] Take 1 mL of the α-DPP4-MNs, BAI1-cGAS, and BAI1-cGAS@DNs sample solutions prepared in Example 1 and add them to the sample cell, and measure the particle size distribution of the three using a Malvern nanoparticle size analyzer.

[0083] The dynamic light scattering particle size distribution diagram is as Figure 3As shown, the particle sizes of α-DPP4-MNs, BAI1-cGAS, and BAI1-cGAS@DNs are 82.83±16.37 nm, 75.26±21.07 nm, and 88.74±13.69 nm, respectively. It can be seen from the figure that the particle size distributions of α-DPP4-MNs, BAI1-cGAS, and BAI1-cGAS@DNs prepared by the method of Example 1 are relatively narrow, indicating that the particle sizes are relatively uniform. BAI1-cGAS is successfully encapsulated by α-DPP4-MNs, and the particle size slightly increases after fusion.

[0084] 3. Determination of drug loading and encapsulation efficiency

[0085] Using the FITC labeling kit, the green fluorescent dye FITC was used to label si-cGAS to obtain FITC-si-cGAS. The si-cGAS in Example 1 was replaced with FITC-si-cGAS, and then the BAI1-FITC-cGAS@DNs nano-drug delivery system was synthesized according to the preparation method of Example 1. It was centrifuged at 6000 rpm for 5 min, and the supernatant was taken to detect the drug loading and encapsulation efficiency of FITC-si-cGAS in the nano-drug delivery system by a fluorescence microplate reader.

[0086] The results are as Figure 4 shown. Only 5.89% of the total amount of FITC-si-cGAS added was detected in the supernatant, and the remaining 90.36% was successfully encapsulated. On average, each 1 g of BAI1-FITC-cGAS@DNs contained 21.85 μg of FITC-si-cGAS, indicating that the nano-drug delivery system prepared by the preparation method of Example 1 has a high encapsulation efficiency.

[0087] Example 3: Responsive in vitro drug release of BAI1-cGAS@DNs

[0088] Take 100 μL of BAI1-cGAS@DNs solution and dissolve it in aqueous solutions with pH = 7.4, pH = 5.4, and pH = 5.4 + 10 nM H2O2 (hydrogen peroxide) respectively. At 3, 6, 12, 24, 48, and 72 h, take the supernatant and detect its si-cGAS concentration with an ultra-micro nucleic acid detector, and calculate the release efficiency of each group.

[0089] Figure 5As a result, it can be seen that under neutral conditions (pH = 7.4), si-cGAS can be slowly released from BAI1-cGAS@DNs, and the total release amount only reaches about 17.8% within 72 h, indicating that the release of si-cGAS is inhibited in a neutral environment; under acidic conditions (pH = 5.4), rapid release behavior of si-cGAS can be observed, and the drug release reaches 53.04% at 24 h and 67.22% at 72 h, indicating that the release efficiency of si-cGAS is significantly improved in an acidic environment; under acidic conditions in the presence of H2O2, BAI1-cGAS@DNs have the fastest drug release behavior, and the final release amount at 72 h is about 78.93%, indicating that the presence of H2O2 further promotes the release of si-cGAS, showing the responsiveness of the nanodrug delivery system to reactive oxygen species (ROS). Thus, it can be seen that the nanocarrier system has pH responsiveness and ROS responsiveness and can rapidly release si-cGAS under acidic and oxidative stress conditions.

[0090] Example 4: Study on the stability of BAI1-cGAS@DNs against RNase

[0091] Free si-cGAS (Naked si-cGAS) and the nanodrug delivery system containing an equal amount of si-cGAS (BAI1-cGAS@DNs) were incubated with 10 μg / mL Rnase at 37 °C for 0, 15, and 30 min, respectively, and then the nanoparticles were collected by centrifugation at 12,000 rpm for 10 min. The nanoparticles were dissolved in 0.5 M NaCl containing H2O2 and under acidic (pH = 5.5) conditions to extract si-cGAS. Subsequently, 1% agarose gel electrophoresis containing the nucleic acid dye GelRed was used for detection, and the results were imaged with ultraviolet light (UV).

[0092] Figure 6 The results showed that the concentration of free si-cGAS decreased significantly after 30 min. Correspondingly, after the nanoparticles encapsulated si-cGAS, they could effectively resist the degradation by RNase within 30 min.

[0093] Example 5: Evaluation of the anti-aging effect of BAI1-cGAS@DNs on alveolar epithelial cell line MLE-12 cells

[0094] MLE-12 cells were induced with DMEM medium containing 0.01 U / mL bleomycin for 2 days to construct senescent MLE-12 cells.

[0095] Normal and senescent MLE-12 cells were respectively seeded at 10 5The number of cells / holes was seeded in a 6-well cell culture plate. After the cells adhered, 2 mL of DMEM medium containing PBS, si-cGAS, BAI1, BAI1-cGAS, and BAI1-cGAS@DNs solution was added to the cells respectively and co-incubated with the cells for 24 h. After the incubation ended, 4% paraformaldehyde (Beyotime, P0099) was added for fixation, and then the expression of the senescence marker p21 in senescent cells in each group of solutions was detected by immunofluorescence experiment.

[0096] The experimental results are as Figure 7 shown. It can be seen that the expression level of p21 in the PBS group was relatively high, indicating that the cells were in a senescent state; the expression level of p21 in the si-cGAS group decreased, but was still relatively high; the expression level of p21 in the BAI1 group decreased further; the expression level of p21 in the BAI1-cGAS group decreased significantly; the expression level of p21 in the BAI1-cGAS@DNs group was the lowest, indicating that this group had the most significant inhibitory effect on the p21 expression of senescent cells. The above experimental results show that BAI1-cGAS@DNs significantly down-regulated the p21 expression of senescent MLE-12 cells and had a good anti-aging effect.

[0097] Example 6: Treatment of mice with pulmonary fibrosis using the BAI1-cGAS@DNsF nanodrug delivery system

[0098] 1. Experiment

[0099] C57BL / 6J mice at 6 - 8 weeks old were injected with bleomycin through tracheal intubation at a dose of 2 U / kg to exhibit pulmonary fibrosis-like diseases. On the 5th day, 11th day, and 17th day, PBS, si-cGAS, BAI1, BAI1-cGAS, and BAI1-cGAS@DNs were injected through the tail vein at a dose of 200 μL per mouse respectively to achieve the treatment of pulmonary fibrosis. Finally, the mice were sacrificed on the 21st day, and the lung tissues were collected.

[0100] 2. Detection

[0101] (1) H&E, Masson, α-SMA, and β-Gal staining of lung sections after treatment

[0102] The lung tissues of the mice with pulmonary fibrosis collected were fixed with 4% paraformaldehyde (Beyotime, P0099), embedded in paraffin after 24 h, sectioned, and stained with H&E, Masson, α-SMA, and β-Gal (SA-β-Gal).

[0103] Figure 8 The H&E staining results showed that the alveolar structure of the mice treated with BAI1-cGAS@DNs was intact. Compared with the PBS group and other treatment groups, the symptoms of alveolar wall thickening and inflammatory cell infiltration were alleviated.

[0104] Figure 8 The Masson staining results showed that the blue positive area in the lungs of mice after BAI1-cGAS@DNs treatment decreased, and the collagen content decreased compared with the PBS group and other treatment groups.

[0105] Figure 8 The α-SMA staining results showed that the immunohistochemical positive area of the lung fibrosis-related protein α-SMA in the lungs of mice after BAI1-cGAS@DNs treatment decreased, and the lung fibrosis condition was greatly improved.

[0106] Figure 8 The β-Gal staining results showed that the blue positive area in the mice after BAI1-cGAS@DNs treatment decreased compared with the PBS group and other treatment groups, and the senescent phenotype was alleviated.

[0107] (2) Expression of lung senescence-related proteins after treatment

[0108] After sacrificing the mice, fresh lung tissues were collected, and Western blot was used to detect the expression of senescence-related proteins DPP4, p16, p21, the expression of inflammation-related proteins cGAS, p65, BAX, and the expression of lung fibrosis-related protein α-SMA. GAPDH was used as an internal reference protein.

[0109] Figure 9 The results showed that the expressions of senescence-related proteins DPP4, p16, p21, inflammation-related proteins cGAS, p65, BAX, and lung fibrosis-related protein α-SMA in the mice after BAI1-cGAS@DNs treatment decreased significantly compared with the PBS group. This indicated that BAI1-cGAS@DNs improved the process of pulmonary fibrosis through the following mechanisms: 1) Targeting senescent cells: By downregulating the expression of senescence-related proteins, reducing the senescent phenotype of cells; 2) Downregulating the expression of BAX: Reducing cell apoptosis and protecting lung tissue; 3) Regulating the cGAS-STING pathway: Reducing chronic cell inflammation and inhibiting inflammatory responses; 4) Reducing chronic cell inflammation: By downregulating the expression of inflammation-related proteins, alleviating inflammatory damage; 5) Alleviating the senescent phenotype: By downregulating the expression of senescence-related proteins, delaying cell senescence. These results demonstrated that BAI1-cGAS@DNs had significant potential in the treatment of pulmonary fibrosis. Figure 10 It is a schematic diagram of the treatment of pulmonary fibrosis with the nanocarrier system BAI1-cGAS@DNs targeting senescent alveolar epithelial cells.

[0110] In summary, after intravenous injection, the BAI1-cGAS@DNsF nanomedicine delivery system of the present invention actively targets DPP4 overexpressed on the surface of senescent alveolar epithelial cells through α-DPP4 Scfv in the BAI1-cGAS@DNsF nanomedicine delivery system, thereby achieving active enrichment of the nanomedicine in senescent cells. Under the action of the intracellular mitochondrial high-ROS microenvironment, the diselenide bond connection in the core of the nanomedicine delivery system is broken, exposing the small molecule inhibitor BAI1 of BAX, which can effectively regulate mitochondrial BAX in alveolar epithelial cells, improve the leakage of mtDNA, and thus synergistically downregulate the activity of the cGAS-STING pathway with si-cGAS. This process helps to regulate its own chronic inflammation and then combat inflammation-related aging. In addition, this intervention can restore the normal function of alveolar epithelial cells, reduce the stress burden of cells, protect cells from overactivation, thereby maintaining their function and ultimately alleviating the senescent phenotype. The present invention helps to analyze and explore new targets and new mechanisms for anti-aging treatment, and provides new methods and new technologies for the development of biological agents for various aging-related diseases.

[0111] The present invention provides a nanocarrier system targeting senescent alveolar epithelial cells, as well as ideas and methods for its preparation and application. There are many specific methods and ways to implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. A preparation method of a nano-carrier system targeting senescent alveolar epithelial cells, characterized in that, It includes the following steps: (1) Polyethyleneimine-diselenide-carboxyl obtained by the esterification reaction of carboxyl-diselenide-carboxyl and polyethyleneimine is dialyzed and freeze-dried, and then reacted with inhibitor BAI1 to obtain the polymer PEI-Se-Se-BAI1; (2) The polymer PEI-Se-Se-BAI1 obtained in step (1) is self-assembled with si-cGAS to obtain polymer nanoparticles BAI1-cGAS loaded with si-cGAS; (3) Anti-DPP4 plasmid cloned with the encoding sequence of anti-DPP4 chimeric antigen receptor Anti-DPP4 Scfv is added to the MLE-12 alveolar epithelial cell culture medium to culture the MLE-12 alveolar epithelial cell line specifically expressing Anti-DPP4-Scfv. After digestion and resuspension, it is extruded to obtain alveolar epithelial cell extracellular vesicles α-DPP4-MNs; (4) The polymer nanoparticles BAI1-cGAS obtained in step (2) are mixed with the alveolar epithelial cell extracellular vesicles α-DPP4-MNs obtained in step (3) and then extruded to obtain the nano-drug delivery system.

2. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of carboxyl-diselenide-carboxyl to polyethyleneimine is 1:1 to 3:1; carboxyl-diselenide-carboxyl and polyethyleneimine are dissolved in a methanol aqueous solution, and the concentration of the methanol aqueous solution is 200 to 30 mL / L; for the esterification reaction, the conditions are: under nitrogen protection, stirring at 40 to 60 °C in the dark for 12 to 36 h.

3. The preparation method according to claim 1, wherein In step (1), the molar ratio of polyethyleneimine-diselenide-carboxyl to inhibitor BAI1 is 2:1 to 2:3; for the reaction, the reaction system includes an anhydrous dimethyl sulfoxide solution containing dicyclohexylcarbodiimide and dimethylacetamide; for the reaction, the conditions are: under nitrogen protection, stirring at 40 to 60 °C in the dark for 12 to 36 h.

4. The preparation method according to claim 1, wherein In step (2), the mass ratio of the polymer PEI-Se-Se-BAI1 to si-cGAS is 2625:60 to 78.

5. The preparation method according to claim 1, characterized in that, In step (3), the encoding sequence of the anti-DPP4 chimeric antigen receptor Anti-DPP4 Scfv is as shown in SEQ ID NO: 1; the formula of the MLE-12 alveolar epithelial cell culture medium is: 10% v / v fetal bovine serum + 1% v / v penicillin / streptomycin + 89% v / v high-glucose DMEM medium.

6. The preparation method according to claim 1, wherein In step (4), the mass ratio of the polymer nanoparticles BAI1-cGAS to the alveolar epithelial cell extracellular vesicles α-DPP4-MNs is 50 to 100:

1.

7. A nanocarrier system targeting senescent alveolar epithelial cells, characterized in that, The nano-carrier system is prepared by the preparation method described in any one of claims 1 to 6.

8. The nanocarrier system according to claim 7, characterized in that, The nano-carrier system includes polymer nanoparticles BAI1-cGAS and alveolar epithelial cell extracellular vesicles α-DPP4-MNs; Among them, the polymer nanoparticle BAI1-cGAS is a polymer nanoparticle containing diselenide bonds, including the inhibitor BAI1 and short interfering RNA of cyclic GMP-AMP synthase; the alveolar epithelial cell extracellular vesicle α-DPP4-MNs includes a chimeric antigen receptor Anti-DPP4 Scfv that specifically binds to DPP4.

9. Use of the nanocarrier system prepared by the preparation method according to any one of claims 1 to 6, or the nanocarrier system according to any one of claims 7 to 8, in the preparation of a drug for preventing and / or treating pulmonary fibrosis senescence diseases.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the nanocarrier system prepared by the preparation method according to any one of claims 1 to 6, or contains the nanocarrier system according to any one of claims 7 to 8.

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