Inflammation targeting nano system based on genetic engineering modification as well as preparation method and application of inflammation targeting nano system

Abstract

The invention provides an inflammation targeting nano system based on genetic engineering modification as well as a preparation method and application thereof, and belongs to the field of medicines. Based on a transgenic cell membrane coating technology and a delivery system of tetrahedral framework nucleic acid, atorvastatin is efficiently delivered to the atherosclerotic plaque position, secretion of inflammatory factors and generation of active oxygen are effectively inhibited, cell uptake and the treatment effect of atorvastatin in atherosclerosis treatment are remarkably enhanced, and the treatment effect of atorvastatin in atherosclerosis treatment is improved. And the method has a good industrial application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to an inflammation-targeting nanosystem based on genetic engineering modification, and a preparation method and use thereof. Background Art

[0002] Cardiovascular disease is one of the most common fatal diseases in the world today. Atherosclerosis (AS) is the pathological basis of most serious cardiovascular diseases. In early atherosclerotic lesions, reactive oxygen species (ROS) can induce endothelial cell dysfunction and promote inflammation by activating nuclear factor-κB (NF-κB) and other signaling pathways, ultimately generating foam cells that accumulate in the vascular wall to form atherosclerotic plaques. Statins, as one of the main drugs currently used in the clinical treatment of AS, have the effects of lowering blood lipids and improving endothelial function. However, statins themselves have low bioavailability and can cause serious adverse reactions to other tissues. There is an urgent need to develop a new generation of nanomedicines that can control the location of plaques, release payloads, and enhance targeting effects. Summary of the invention

[0003] The purpose of the present invention is to provide an inflammation-targeting nanosystem based on genetic engineering modification, and a preparation method and use thereof.

[0004] The present invention provides a nanoparticle, which is composed of a mixture of a DNA tetrahedron complex and a fused cell membrane, wherein the mass ratio of the DNA tetrahedron complex to the fused cell membrane is 1:4-20;

[0005] The DNA tetrahedral complex is a product prepared using atorvastatin and tetrahedral framework nucleic acid as raw materials;

[0006] The fusion cell membrane is a product prepared by using active oxygen-responsive liposomes and cell membranes as raw materials, and the cell membranes are from cells that highly express VLA4.

[0007] Furthermore, the mass ratio of the DNA tetrahedral complex and the fused cell membrane was 1:10;

[0008] In the DNA tetrahedral complex, the molar ratio of atorvastatin to tetrahedral framework nucleic acid is 0.01-0.1 mmol:150-200 nmol, preferably 0.09 mmol:160 nmol;

[0009] In the fusion cell membrane, the mass ratio of the active oxygen responsive liposome to the cell membrane is 1:5 to 15, preferably 1:10.

[0010] Furthermore, the DNA tetrahedral complex is self-assembled by four DNA single strands, the four DNA single strands are S1, atorvastatin-S2, atorvastatin-S3 and atorvastatin-S4, and the sequences of S1, S2, S3 and S4 are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0011] Furthermore, the preparation method of the DNA tetrahedron complex comprises the following steps:

[0012] (1) reacting atorvastatin, a silanization agent and a base to obtain a solution 1;

[0013] (2) reacting solution 1, a carboxyl activator and a catalyst to obtain solution 2;

[0014] (3) reacting the single-stranded DNA, solution 2 and a base to obtain atorvastatin-single-stranded DNA; wherein the single-stranded DNA is S2, S3 or S4;

[0015] (4) S1, atorvastatin-S2, atorvastatin-S3 and atorvastatin-S4 are self-assembled to obtain a DNA tetrahedral complex.

[0016] Further, in step (1), the silanization agent is 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane; the base is an organic base, preferably imidazole; the molar ratio of atorvastatin, silanization agent and base is 1:1-1.5:2-4, preferably 1:1.08:2.97; the solvent of the reaction is an organic solvent, preferably N,N-dimethylformamide; the reaction conditions are: reacting at 20-40°C for 5-20 hours, preferably reacting at 30°C for 7-17 hours;

[0017] In step (2), the carboxyl activator is N,N'-disuccinimidyl carbonate; the catalyst is 4-dimethylaminopyridine; the molar ratio of the solution 1, the carboxyl activator and the catalyst is 1:7-9:14-16, preferably 1:8.3:15; the solvent of the reaction is an organic solvent, preferably N,N-dimethylformamide; the reaction conditions are: reacting at 10-40°C for 0.1-1 hour, preferably reacting at 15-35°C for 0.5 hour;

[0018] In step (3), the base is an organic base, preferably N,N-diisopropylethylamine; the molar ratio of the single-stranded DNA, solution 2 and the base is 1:1:1:10-30:70-80, preferably 1:20:75; the solvent of the reaction is an inorganic solvent, preferably a borate buffer; the reaction conditions are: reacting at 40-60°C for 0.1-1 hour, preferably reacting at 50°C for 0.5 hour.

[0019] Furthermore, the active oxygen responsive liposome is a thioketal-containing liposome; preferably, the thioketal-containing liposome is a product obtained by mixing DSPE-TK-PEG2000, lecithin and cholesterol in a solvent to form a transparent film, and then reacting with water; wherein the molar ratio of DSPE-TK-PEG2000, lecithin and cholesterol is 0.2-0.6:1-3:0.5-1.5, preferably 0.4:2:1;

[0020] The solvent is chloroform; the reaction temperature is 10-40° C., preferably 15-35° C.; the reaction time is 20-40 minutes, preferably 30 minutes.

[0021] Furthermore, the cells highly expressing VLA4 are C1498-VLA cells, and the preparation of C1498-VLA cells comprises the following steps: incubating plasmid DNA and transfection reagent to form a complex, transfecting retroviral cells, digesting, centrifuging, and taking the supernatant to suspend C1498 cells to obtain C1498-VLA cells.

[0022] Furthermore, the sequence of the plasmid DNA is shown in SEQ ID No. 5; the transfection reagent is Lipofectamine 3000, and the incubation time is 10 to 30 minutes, preferably 20 minutes;

[0023] The retrovirus cells are AmphoPhoenix cells, and the transfection time is 3 to 5 hours, preferably 4 hours.

[0024] The present invention also provides a method for preparing the nanoparticles, which comprises the following steps: mixing the DNA tetrahedron complex and the fused cell membrane, and ultrasonicating to obtain the nanoparticles.

[0025] Furthermore, the ultrasonic conditions are: ultrasonication at a frequency of 30 to 50 kHz and a power of 50 to 150 w for 2 to 4 minutes;

[0026] Preferably, the ultrasonic conditions are: ultrasound at a frequency of 42 kHz and a power of 100 W for 3 minutes.

[0027] The present invention also provides the use of the nanoparticles in preparing medicines for preventing and / or treating cardiovascular diseases.

[0028] Furthermore, the cardiovascular disease is atherosclerosis.

[0029] Furthermore, the drug is a drug for reducing atherosclerotic plaques.

[0030] The present invention also provides a pharmaceutical composition, which is a pharmaceutical preparation prepared by using the above-mentioned nanoparticles as active ingredients and adding pharmaceutically acceptable excipients.

[0031] The present invention has achieved the following beneficial effects:

[0032] The present invention is based on transgenic cell membrane coating technology and tetrahedral framework nucleic acid (tFNA) delivery system. By efficiently delivering atorvastatin to the location of atherosclerotic plaques, the secretion of inflammatory factors and the generation of reactive oxygen species (ROS) are effectively inhibited, and the cellular uptake and therapeutic effect of atorvastatin (AT) in the treatment of atherosclerosis are significantly enhanced, which has good prospects for industrial application.

[0033] The present invention further optimizes the reaction raw material ratio and screens out the preferred raw material ratio shown in Example 1.

[0034] (1) A novel nanodrug system TK-VLA@TAT was constructed for the first time and demonstrated excellent targeting ability to the affected area and rapid foam cell internalization.

[0035] (2) In in vitro experiments, TK-VLA@TAT nanoparticles inhibited the formation of foam cells, the expression of a series of proinflammatory factors (IL-1β, IL-18, IL-6, TNF-α), the apoptosis of macrophages, and enhanced the clearance of ROS, thereby reducing the inflammatory response and lipid production in atherosclerosis.

[0036] (3) In vivo experiments, TK-VLA@TAT treatment demonstrated protective effects on aortic vessels by tail vein injection in atherosclerotic mouse models, such as reducing inflammatory cell infiltration, collagen degradation, and lipid formation, and alleviating the formation of atherosclerotic plaques.

[0037] (4) TK-VLA@TAT showed no obvious toxic effects in both in vitro and in vivo experiments.

[0038] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0039] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1This is the plasmid map of pQCXIP-VLA4.

[0041] Figure 2 Characterization and in vivo targeting of TK@VLA@TAT: (A) Schematic diagram of the synthesis of TAT; (B) TEM image of TAT synthesis; (C) AFM image of TAT; (D) PAGE gel electrophoresis of TAT; (E) HPCE spectrum of TAT; (F) WT-C1498 flow cytometry verification; (G) VLA-1498 flow cytometry verification; (H) WB detection of integrins α4 and β1; (I) potential of TK-VLA@TAT; (J) particle size of TK-VLA@TAT; (K) TEM image of TK-VLA@TAT; (L) TEM image of TK-VLA@TAT in 100uM H2O2; (M) TEM image of TK-VLA@TAT in 0uM H2O2; (N) release rate of TK-VLA@TAT in different concentrations of H2O2; (O) targeting of nanomaterials in mice; (P) statistical graph of in vivo targeting fluorescence in mice.

[0042] Figure 3 Figure 3 shows the inhibitory effect of TK-VLA@TAT on apoptosis induced by oxidized low-density lipoprotein (Ox-LDL), antioxidant activity, and attenuation of foam cell formation: (A) Schematic diagram showing the targeted drug delivery of TK-VLA@TAT to RAW 264.7 and the cellular uptake of TK-VLA@TAT under Ox-LDL treatment; (B) Cellular uptake of TAT and TK-VLA@TAT was detected by confocal fluorescence microscopy. Scale bar = 30 μm; (C) Cellular uptake of TAT and TK-VLA@TAT was detected by flow cytometry. Scale bar = 30 μm; (D) Oil red O (ORO) staining microscopy images and their quantitative analysis showed the effects of various nanoparticles on foam cell development. Scale bar = 80 μm; (E) β-galactosidase (Sa-β-Gal) staining microscopy images and their quantitative analysis showed the effects of various nanoparticles on foam cell development. Scale bar = 80 μm; (F) Flow cytometry was used to assess apoptosis of RAW 264.7 cells under different treatment settings.

[0043] Figure 4TK-VLA@TAT inhibits the expression of inflammatory cytokines in RAW264.7 cells stimulated by oxidized low-density lipoprotein (Ox-LDL): (A) Western blotting was used to detect the expression of inflammatory cytokines in RAW264.7 cells under different treatment conditions. Expression of inflammation-related proteins in RAW264.7 cells; (B) Western blot results of IL-6; (C) Western blot results of IL-1β; (D) Western blot results of TNF-α; (E) Western blot results of MCP-1; Concentrations of inflammatory factors (F) TNF-α, (G) IL-6, (H) IL-1β and (I) MCP-1 in the culture supernatant of RAW264.7 cells under different treatment conditions; (JM) Immunofluorescence results of (J) TNF-α, (K) IL-1β, (L) IL-6 and (M) MCP-1 expression in RAW264.7 cells under different treatment conditions; (N) CCK-8 detection of RAW264.7 cell viability after treatment with different nanoformulations.

[0044] Figure 5 Figure 3 TK-VLA@TAT inhibits the generation of reactive oxygen species (ROS) in RAW264.7 cells stimulated by oxidized low-density lipoprotein (Ox-LDL): (A) Schematic diagram of TK-VLA@TAT regulating ROS generation in RAW 264.7 cells through the INOS / NF-kb / IKB-α / NRF2 and HO-1 pathways; (B) Protein expression (iNOS, NF-kB, IKB-α, NRF2 and HO-1) in RAW 264.7 cells under different treatment conditions was detected by Western blotting; (C) Quantitative analysis of iNOS measured by Western blotting, n=3; (D) Quantitative analysis of IKB-α measured by Western blotting, n=3; (E) Quantitative analysis of NF-kB measured by Western blotting, n=3; (F) Quantitative analysis of NRF2 measured by Western blotting, n=3; RAW 264.7 cells under different treatment conditions Immunofluorescence data of (H) iNOS, (I) NF-kB, (J) NRF2, (K) HO-1, and (K) HO-1 expression in 264.7 cells. Scale bar = 20 μm. (L) Confocal laser scanning microscopy (CLSM) was used to visualize ROS levels in RAW264.7 cells under different treatment conditions.

[0045] Figure 6The therapeutic effect of TK-VLA@TAT in atherosclerotic mice: (A) Schematic diagram depicting the development of the atherosclerotic mouse model and TK-VLA@TAT treatment; (B) DHE staining and quantitative analysis of sections of the aortic root, arch, and brachiocephalic artery after atherosclerotic mice were treated with different formulations (saline, AT, TAT, TK-WT@TAT, and TK-VLA@TAT). Scale bar = 200 μm; Quantification of the expression levels of (C) TNF-α, (D) IL-1β, and (E) IL-6 obtained from the serum of atherosclerotic mice after different treatments; Quantification of the expression levels of (F) IL-6, (G) IL-1β, and (H) TNF-α obtained from the aorta of atherosclerotic mice after different treatments; (I) total cholesterol (TC), (J) high-density lipoprotein cholesterol (HDL-C), and (K) low-density lipoprotein cholesterol (LDL-C) levels in blood serum; (L) results and (M) images of Oil Red O (ORO) staining sections of aorta of atherosclerotic mice after different treatments.

[0046] Figure 7 TK-VLA@TAT alleviated plaque formation and inflammation in atherosclerotic mice: (A) Representative images of aortic root sections stained with hematoxylin-eosin (H&E), (B) Masson's trichrome, and (C) Oil Red O (ORO) and their corresponding quantitative analysis. Scale bars = 500 μm and 50 μm; Representative microscopic images of aortic root sections stained with (D) CD-14, (E) MMP-9, and (F) α-SMA antibodies and their corresponding quantitative analysis. DETAILED DESCRIPTION

[0047] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0048] In the present invention, "room temperature" refers to 25±10°C, and "overnight" refers to 12±5 hours.

[0049] Atorvastatin, Atorvastatin, referred to as AT.

[0050] The full name of VLA4 in Chinese is late-appearing antigen-4.

[0051] Example 1. Preparation of Inflammation Targeting Nanosystem Based on Genetic Engineering

[0052] 1. Synthesis of tetrahedral framework nucleic acid-atorvastatin complex (tFNA-AT)

[0053] 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSICl2) (124 mg, 0.39 mmol) and imidazole (73 mg, 1.07 mmol) were added to a DMF solution (2.0 mL) containing (200 mg, 0.36 mmol). The mixed solution was stirred at 30 ° C overnight. The mixed solution was diluted with water, extracted with DCM (10 mL), dried with Na2SO4 after extraction, and concentrated under reduced pressure to obtain 170 mg of solution 1. Solution 1 (170 mg, 0.21 mmol), N,N'-disuccinimidyl carbonate (DSC) (65.3 mg, 0.175 mmol) and 4-dimethylaminopyridine (DMAP) (38.8 mg, 0.315 mmol) were stirred in DMF (1.7 mL) at room temperature for 0.5 hours. The mixture was then diluted with water, the organic phase was extracted with ethyl acetate, the extracted organic phase was washed three times with water (3 mL), and then dried over Na2SO4. The solution was then concentrated under reduced pressure to obtain Solution 2.

[0054] Three ssDNA (S2, S3, S4, 160nmol) solutions (30uL) were added to a borate (BBS) buffer, and then solution 2 (DMSO, concentration (c) = 20mM, 160uL, 3.2umol) and N,N-diisopropylethylamine (DIPEA) (c = 400mM, 30uL, 12umol) were added respectively. The reaction mixture was stirred at 50°C for 0.5 hours. Then 5M NaCl aqueous solution (1v / v) and absolute ethanol (3v / v) were added, shaken, placed at -20°C for 20 minutes, centrifuged at 12000rpm for 15 minutes, ethanol was decanted and evaporated. Eluted with DMSO (160uL), and then tetrabutylammonium fluoride (TBAF) (100uL) was added. The reaction mixture was stirred at 50°C for 2 hours. Then, 5 M NaCl aqueous solution (1 v / v) and absolute ethanol (3 v / v) were added, shaken and stored at -20°C for 20 minutes, centrifuged at 12000 rpm for 15 minutes, and the ethanol was decanted and evaporated to obtain the final ssDNA@AT: S2@AT. The final ssDNA@AT was purified by RP-HPLC.

[0055] Referring to the preparation method of S2@AT, the only difference is that S2 is replaced by S3, and ssDNA@AT:S3@AT is prepared; referring to the preparation method of S2@AT, the only difference is that S2 is replaced by S4, and ssDNA@AT:S4@AT is prepared.

[0056] Four custom-designed single-stranded oligonucleotides S1, S2@AT, S3@AT and S4@AT (as shown in Table S1) were added in equimolar amounts (1uM) to a 200uL microtube containing 96uL TM buffer (composed of 10mM Tris, 50mM MgCl2, pH 8.0), followed by vortexing and centrifugation. Next, the mixture was subjected to one annealing cycle (95°C for 10 minutes, then 4°C for 20 minutes) to assemble the tetrahedral framework nucleic acid-atorvastatin complex (tFNA-AT, abbreviated as TAT) ( Figure 2 A).

[0057] Table 1. The ssDNA sequences are as follows:

[0058]

[0059]

[0060] 2. Preparation of C1498-VLA and C1498-VLA Cell Membrane

[0061] The specific operations are as follows:

[0062] 1) Cell membrane genetic engineering and detection

[0063] The plasmid DNA pQCXIP-VLA4 ( Figure 1 ), pQCXIP-VLA4 contains the gene encoding integrin α4 (MiaoLing Biology, China). Lipofectamine 3000 (a transfection reagent) and plasmid DNA were diluted in Opti-MEM medium, then mixed and incubated for 20 minutes to form DNA-Lipo complex. The complex was added to AmphoPhoenix cells (a retroviral cell line) and incubated for 4 hours to achieve transfection. The transfected cells were digested and centrifuged, and the wild-type C1498 (C1498-WT) cells were resuspended with the supernatant to obtain C1498-VLA cells with high VLA4 expression.

[0064] The cells were labeled with FITC-conjugated anti-mouse CD49d antibody (BioLegend, USA) and Alexa Fluor 647-conjugated anti-mouse / rat CD29 antibody (BioLegend, USA) and verified using Attune NxT flow cytometer (Thermo Scientific, USA) that the obtained cells were indeed C1498-VLA cells with high VLA4 expression.

[0065] Among them, the pQCXIP-VLA4 sequence (SEQ ID No.5) is as follows:

[0066]

[0067] 2) Preparation of C1498-VLA cell membrane

[0068] The present invention uses cell lysis and differential centrifugation to separate the cell membrane (VLA) of C1498-VLA cells.

[0069] The specific operations are as follows:

[0070] The selected C1498-VLA cells were washed with PBS buffer and resuspended in separation buffer. Subsequently, the cell suspension was homogenized 15 times using a Kinematica Polytron PT 10 / 35 probe homogenizer at 70% power. The homogenized cells were centrifuged at 10000g for 25 minutes, and the supernatant was ultracentrifuged at 150000g for 35 minutes to obtain C1498-WT cell membranes. The membranes were collected and stored in DEPC water containing 0.2mM EDTA.

[0071] 3. Preparation of thioketal liposomes (TK-liposomes)

[0072] The present invention mixes DSPE-TK-PEG2000, lecithin and cholesterol in a round-bottom flask in a molar ratio of 0.4:2:1 in chloroform. After evaporating chloroform under low pressure, a transparent film is formed at the bottom of the flask. The film is hydrated with 1 mL of ddH2O solution and incubated at room temperature for 30 minutes. The mixture is then ultrasonically treated to form a clear suspension, and extruded using a liposome extruder to obtain TK liposomes (TK-liposomes).

[0073] 4. Synthetic fusion cell membrane TK-VLA

[0074] The VLA cell membrane from step (2) was mixed with TK-liposome at a ratio of 1:10 (w / w) and co-extruded to form a fusion membrane TK-VLA.

[0075] 5. Preparation of composite nanoparticles TK-VLA@TAT

[0076] The TAT obtained in step 1 and the TK-VLA obtained in step 4 were mixed at a mass ratio of 1:10, and ultrasonicated at a frequency of 42 kHz and a power of 100 W for 3 minutes to obtain TK-VLA@TAT.

[0077] The following is a control sample preparation.

[0078] Comparative Example 1: Preparation of Nanostructured Sample TK-WT@TAT

[0079] 1. Synthesis of tetrahedral framework nucleic acid-atorvastatin complex (tFNA-AT): as described in Example 1.

[0080] 2. Preparation of C1498-WT and C1498-WT cell membranes

[0081] Referring to step 2) of Example 1, wild-type C1498 cell membranes were directly prepared using wild-type C1498 cells.

[0082] 3. Preparation of thioketal liposomes (TK-liposomes): as described in Example 1.

[0083] 4. Synthetic fusion cell membrane TK-WT

[0084] The wild-type C1498 cell membrane was fused with TK-liposome at a ratio of 1:10 (w / w) and co-extruded to form the fusion membrane TK-WT.

[0085] 5. Preparation of composite nanoparticles TK-WT@TAT

[0086] The TAT obtained in step 1 and the TK-WT obtained in step 4 were mixed at a mass ratio of 1:10, and ultrasonicated at a frequency of 42 kHz and a power of 100 W for 3 min to obtain TK-WT@TAT.

[0087] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0088] Experimental Example 1: Screening of the ratio of fusion membrane TK-VLA and TAT complex

[0089] In order to verify the optimal ratio of the fusion membrane TK-VLA and TAT complex, the present invention detected the encapsulation rate and drug loading efficiency of the fusion membrane TK-VLA and TAT complex at different ratios. The specific results are shown in Table 2.

[0090] The encapsulation efficiency calculation formula is:

[0091] Encapsulation rate (%) = (mass of TAT encapsulated / total mass of TAT input) × 100%

[0092] The drug loading efficiency calculation formula is:

[0093]

[0094] Table 2. Effects of different ratios of fusion membrane TK-VLA and TAT complex on encapsulation efficiency and drug loading efficiency

[0095]

[0096] As can be seen from Table 2, when the mass ratio of the fusion membrane TK-VLA to the TAT complex is 10:1, the encapsulation rate is the highest, and compared with other ratios, an unexpected technical effect is achieved. Therefore, the fusion membrane TK-VLA and TAT complex are selected to prepare the composite nanoparticles TK-WT@TAT at a mass ratio of 10:1.

[0097] Experimental Example 2: Characterization of inflammation-targeted nanosystem and in vitro and in vivo effect testing

[0098] 1. Experimental methods

[0099] To verify the successful synthesis of TAT and TK-VLA@TAT, the study used 8% PAGE gel electrophoresis, HPCE, DLS, AFM and TEM for characterization. PAGE gel electrophoresis (8%) and HPCE were used to determine the approximate relative molecular weight of TAT in these structures. The surface structure and properties of TAT TK-VLA@TAT were determined using TEM (HT770, Hitachi, Japan) and AFM (Shimadzu, Kyoto, Japan). The hydrodynamic diameter and zeta potential of the particles were measured using DLS on a Zetasizer Nano (ZS90, Malvern, UK).

[0100] 2. Experimental results

[0101] (1) Characterization of TAT

[0102] Figure 2 BE are the synthetic TEM image, AFM image, PAGE gel electrophoresis image and HPCE spectrum of TAT, respectively. These results show that the present invention successfully synthesized TAT.

[0103] (2) Characterization and in vivo targeting effect of nanoparticles TK-VLA@TAT and TK-WT@TAT

[0104] VLA4 is a heterodimer composed of integrin α4 and integrin β1. As a ligand, it has a satisfactory targeting ability for the inflammatory factor VCAM-1 on the surface of endothelial cells. C1498 cells express high levels of integrin β1 but do not express integrin α4. The present invention engineered C1498 cells through viral transfection and obtained a genetically modified cell line (C1498-VLA) that can stably express α4 and β1 ( Figure 2 F, 2G).

[0105] The present invention evaluated the two integrins of VLA4 on TK-VLA and TK-WT by Western blotting. α4 and β1 were observed on TK-VLA, while only β1 was found on TK-WT ( Figure 2H). Subsequently, the present invention coated the nanoparticle core TAT with TK-VLA and TK-WT to obtain membrane-coated nanoparticles TK-VLA@TAT and TK-WT@TAT. Then the encapsulation efficiency (EE%) and drug loading efficiency (LE%) of TK-VLA on TAT were investigated, and the EE% and LE% reached 58.21% and 5.3%, respectively. Particle size analysis showed that the average hydrodynamic diameter of TK-VLA@TAT was about 200 nanometers, and the Zeta potential was about -27mV ( Figure 2 I, 2J). TEM results showed that TK-VLA@TAT exhibited a typical phospholipid bilayer structure ( Figure 2 K).

[0106] During the progression of atherosclerosis (AS), atherosclerotic plaques secrete inflammatory cytokines and chemokines, including a significantly upregulated specific surface marker VCAM-1. VLA-4 expressed by TK-VLA can enable it to actively migrate to the site of inflammation, and TK-VLA may serve as an ideal drug targeting system. The present invention uses Cy5-labeled TK-VLA@TAT to study the in vivo targeting ability of nanomedicines. 6 hours after intravenous injection in mice, fluorescence can be detected in both the aortic arch and the abdominal aorta. The fluorescence intensity increased significantly and reached a peak at 12 hours. Compared with TK-WT@TAT, the accumulation of TK-VLA@TAT in the aorta was more obvious at the 6h and 12h time points ( Figure 2 O, 2P). This indicates that the effective targeting ability of the TK-VLA membrane enables the intravenously injected TK-VLA@TAT to accumulate more effectively in atherosclerotic lesions.

[0107] (3) ROS-responsive release, cellular uptake and in vitro therapeutic effects of nanoparticles TK-VLA@TAT

[0108] During the progression of atherosclerosis (AS), oxidized low-density lipoprotein (Ox-LDL) accumulates in the intimal region, causing local inflammation and generating excessive reactive oxygen species (ROS). Drug release analysis showed that high concentrations of ROS stimulated the decomposition of TK-VLA@TAT. Transmission electron microscopy (TEM) images showed the collapse of the original TK-VLA structure after incubation in 100 μM / L H2O2 for 10 h ( Figure 2 L). In contrast, vesicles insensitive to ROS (VLA) maintained their vesicle structure ( Figure 2M). In addition, the present invention verifies the sensitivity of TK-VLA@TAT to ROS. TK-VLA@TAT was incubated with PBS with different concentrations of hydrogen peroxide (H2O2) (0, 50, 100 and 200 μM / L) for 20 hours, and the drug release rate was measured at different time points. In the absence of H2O2, the release rate of TK-VLA@TAT was approximately 15%. As the concentration of H2O2 increased, the drug release rate of TK-VLA@TAT gradually increased, and when the concentration of H2O2 was 50, 100 and 200 μM / L, the release rate reached approximately 84%, 92% and 97%, respectively ( Figure 2 N). These results suggest that the fusion of TK-liposomes with VLA enables targeted release in environments with high ROS levels, thereby accelerating the release of TAT in the inflammatory microenvironment.

[0109] The present invention speculates that TK-VLA@TAT can target the inflammatory microenvironment through its VLA-4 and release TAT under high ROS conditions, and then TAT is internalized by cells to exert its biological effects ( Figure 3 A). The present invention used confocal microscopy and flow cytometry to observe the cellular uptake efficiency of Cy5-labeled TAT and TK-VLA@TAT into foam cells. The results showed that both TAT and TK-VLA@TAT could enter macrophages in large quantities without the use of any transfection agent. It is worth noting that the significant internalization of TK-VLA@TAT was earlier than that of TAT, starting from the sixth hour ( Figure 3 B). Flow cytometry analysis further confirmed the massive internalization of TK-VLA@TAT by foam cells without the need for any adjuvant ( Figure 3 C), The results are consistent with those of immunofluorescence studies.

[0110] In the early stages of atherosclerosis, fatty streaks are rich in senescent foam cells, which drive the degradation of elastic fibers and the thinning of the fibrous cap by secreting inflammatory cytokines, chemokines and matrix remodeling enzymes, thereby increasing the risk of plaque rupture. The experimental results of the present invention show that TK-VLA@TAT has excellent anti-apoptosis and ROS scavenging effects. The present invention further explores the effect of TK-VLA@TAT on the generation of macrophage foam cells. Oil red O (ORO) staining results showed that compared with the cells in the oxidized low-density lipoprotein (Ox-LDL) treated model group, the cells in the TK-VLA@TAT group significantly reduced red lipid droplets in the cytoplasm, indicating that the composite nanoparticle drug effectively inhibited the development of macrophage foam cells. Semi-quantitative analysis of the ORO staining positive area showed that TK-VLA@TAT significantly inhibited the formation of macrophage foam cells ( Figure 3 D).

[0111] Inflammatory responses exacerbate the progression of atherosclerosis, and senescent cells, as a key source of inflammatory cytokines, play a key role in exacerbating the instability of atherosclerotic plaques. The present invention created a senescent foam cell model using Ox-LDL and evaluated the inhibitory effect of TK-VLA@TAT on their formation using SA-β-galactosidase (SA-β-gal) activity staining ( Figure 3 E). The staining results showed that cells in the TK-VLA@TAT group displayed the lowest β-galactosidase activity, indicating that TK-VLA@TAT can effectively inhibit the formation of senescent cells.

[0112] Foam cells store a large amount of oxidized cholesterol, and excessive accumulation of oxidized cholesterol can induce apoptosis. Therefore, inhibiting apoptosis is a potential therapeutic strategy for treating atherosclerosis. The present invention uses flow cytometry to evaluate the therapeutic response of TK-VLA@TAT, TAT or TK-WT@TAT to ox-LDL-induced macrophage apoptosis ( Figure 3 F). The study found that compared with the control group, Ox-LDL enhanced the apoptosis of macrophages, while TK-VLA@TAT, TAT and TK-WT@TAT all inhibited apoptosis, among which TK-VLA@TAT showed the strongest inhibitory effect.

[0113] (4) Inhibitory effect of nanoparticles TK-VLA@TAT on inflammatory factors

[0114] Inflammation is also a powerful driving force in the progression of atherosclerosis. Excessive ROS production and persistent oxidative stress not only mediate tissue and cell damage, but also further trigger inflammatory cascades. Foam cells in plaques can secrete a large number of proinflammatory factors, such as IL-1β, IL-6, MCP-1, TNF-α, and TNF-α. In order to detect the expression of these four typical proinflammatory factors, the present invention uses Western blotting and immunofluorescence to determine the inflammatory response of foam cells ( Figure 4 AE and Figure 4 JM). In the Ox-LDL-treated group, the levels of these four pro-inflammatory factors were significantly higher than those in the control group. An inhibitory effect on inflammatory response was observed in foam cells treated with TK-VLA@TAT, TAT or TK-WT@TAT, among which TK-VLA@TAT showed the strongest effect compared with other nanomaterials. In addition, the present invention also examined the effect of TK-VLA@TAT on cytokine secretion by macrophages. An ELISA kit was used to quantify the levels of inflammatory factors in the cell culture supernatant. The results showed that in the Ox-LDL-treated model group, the levels of TNF-α, IL-1β and IL-6 were significantly increased ( Figure 4JL). In contrast, the TK-VLA@TAT group showed significantly lower concentrations of 186.2, 96.3, 61.5, and 319.4 pg / mL, respectively. CCK-8 results showed that TK-VLA@TAT, TK-WT@TAT, and TAT had higher cell viability compared with the Ox-LDL group ( Figure 4 N). In conclusion, TK-VLA@TAT can alleviate inflammation in the atherosclerotic microenvironment by effectively reducing the secretion of proinflammatory factors by foam cells.

[0115] (5) Scavenging effect of nanoparticles TK-VLA@TAT on reactive oxygen species

[0116] A key mechanism by which mitochondrial dysfunction leads to cellular aging is the excessive production of ROS during mitochondrial respiration. When the level of ROS production exceeds the natural antioxidant capacity of the organism, it can induce oxidative stress and cause substantial damage to tissues and cells, ultimately leading to endothelial dysfunction. In the vulnerable plaque microenvironment, ROS can promote the formation of Ox-LDL. After being phagocytosed by macrophages, excess Ox-LDL can activate NOX and produce a large amount of intracellular ROS, thereby promoting the formation of senescent foam cells. Therefore, the present invention studies the role of TK-VLA@TAT in scavenging ROS ( Figure 5 A). First, the protein expressions of INOS, NF-κB, HO-1, IκB-α, and NRF2 were investigated using Western blotting and immunofluorescence ( Figure 5 BK). The study found that compared with the Ox-LDL-treated group, the foam cells treated with TK-VLA@TAT showed the most significant decrease in INOS and NF-κB expression, while in the TK-VLA@TAT group, the expression of HO-1, IκB-α and NRF2 was most significantly upregulated. In addition, the present invention studied the ROS scavenging effect of TK-VLA@TAT in foam cells by flow cytometry. Cells treated with PBS and Ox-LDL were used as the normal control group and the model group with excessive ROS production, respectively. The cells in the model group showed strong green fluorescence, indicating that ROS production was high. In the TK-VLA@TAT, TAT and TK-WT@TAT groups, the green fluorescence intensity was low, indicating the significant antioxidant ability of TAT nanomedicine ( Figure 5 L). In particular, cells in the TK-VLA@TAT group showed the weakest green fluorescence signal. These experimental results indicate that TK-VLA@TAT can effectively inhibit the production of intracellular ROS and help achieve better therapeutic effects in the treatment of atherosclerosis.

[0117] (6) In vivo therapeutic effect of nanoparticles TK-VLA@TAT

[0118] The effects of different drugs (saline, AT, TAT, TK-WT@TAT, and TK-VLA@TAT) on the progression of atherosclerosis were evaluated. Six-week-old female APOE- / - mice were fed a high-fat diet for one month and then randomly divided into five groups by a blinded investigator. The mice received weekly intravenous infusions of different formulations ( Figure 6 A). The biosafety of TK-VLA@TAT was evaluated by H&E staining. After intravenous injection of TK-VLA@TAT into atherosclerotic mice, five organs (heart, liver, spleen, lung, and kidney) were collected and subjected to H&E staining. The results of H&E staining showed that TK-VLA@TAT did not cause significant damage in atherosclerotic mice, thus demonstrating the in vivo safety of the nanoparticles.

[0119] In addition, the present invention also studies the effect of these formulations on clearing ROS in the aorta of atherosclerotic mice. ROS levels were quantified by staining the aortic root, aortic arch, and brachiocephalic artery sections with dihydroethidium (DHE). Figure 6 B). The saline-treated group (control group) showed significant red fluorescence, reflecting the increased ROS production in these aortic tissues. The red fluorescence intensities of the other four groups were lower, with the lowest fluorescence intensity detected in the TK-VLA@TAT group, indicating that TK-VLA@TAT had the best ROS scavenging effect.

[0120] ELISA assay results showed that the expression levels of major pro-inflammatory cytokines in aortic tissue and serum of APOE- / - atherosclerotic mice in the TK-VLA@TAT treatment group were the lowest compared with all other groups ( Figure 6 CH). These data support that TK-VLA@TAT effectively alleviates systemic inflammation. Meanwhile, compared with the saline group, the serum total cholesterol (TC) level of mice treated with TK-VLA@TAT was not significantly reduced, while the high-density lipoprotein cholesterol (HDL-C) content was moderately enhanced in all treatment groups, with the most significant increase observed in the TK-VLA@TAT group ( Figure 6 IK). In addition, the levels in the TK-VLA@TAT group were significantly reduced compared to non-HDL cholesterol levels. In summary, these findings suggest that TK-VLA@TAT has a satisfactory therapeutic effect on atherosclerosis in mice. As shown in the ORO staining, aortic intimal lesions showed that the saline group had the largest plaque area, accounting for 15% of the aortic tissue. The free drug AT partially reduced the plaque area to 8% ( Figure 6 L, 6M, Figure 7C). Compared with free AT, TAT and TK-WT@TAT showed slightly better therapeutic effects due to enhanced cellular uptake. TK-VLA@TAT had the best therapeutic effect on inflammatory plaques by targeting its delivery and release to atherosclerotic plaques, reducing the plaque area to 3%.

[0121] The present invention also performed histological and immunohistochemical analysis of atherosclerotic plaques in the aortic root. Hematoxylin and eosin (H&E) staining of the aortic root showed that plaques in the saline-treated group and the AT group were mainly composed of necrotic cores. In contrast, significantly reduced plaque area and necrotic core size ( Figure 7 A). From another perspective, the collagen level around the plaques in the TK-VLA@TAT treatment group was evaluated using Masson's trichrome staining, and the results showed a higher collagen concentration and thicker fibrous cap ( Figure 7 B). Anti-CD14 and anti-matrix metalloproteinase-9 (MMP-9) antibody staining showed that TK-VLA@TAT significantly reduced the number of monocytes and MMP-9 levels in aortic arch plaques ( Figure 7 D, 7E). As total necrotic core area and mononuclear cell infiltration were positively correlated with plaque development and disease severity, TK-VLA@TAT was shown to effectively inhibit the atherosclerotic process, with a stronger effect than TAT and TK-WT@TAT. As the expansion of vascular smooth muscle cells (VSMCs) is known to affect AS progression, the present invention also used α-smooth muscle actin (α-SMA) to stain plaques to measure VSMC aggregation ( Figure 7 F). The highest VSMC aggregation rate was seen in the TK-VLA@TAT treatment group. Therefore, the higher collagen concentration and increased VSMC aggregation around the plaques suggest that TK-VLA@TAT can prevent AS development by stabilizing the plaques, thereby preventing further progression of the plaques.

[0122] The above results show that the nanoparticles TK-VLA@TAT of the present invention have excellent ability to target the affected area, can be quickly taken up by cells in a short time, can significantly inhibit the expression of pro-inflammatory factors (IL-1β, IL-18, IL-6, TNF-α), enhance the scavenging effect of reactive oxygen species, and achieve a good effect in treating atherosclerosis.

[0123] In summary, the present invention provides an inflammation-targeted nanosystem based on genetic engineering modification, and its preparation method and use. The present invention is based on the transgenic cell membrane coating technology and the tetrahedral framework nucleic acid delivery system, and by efficiently delivering atorvastatin to the atherosclerotic plaque, the secretion of inflammatory factors and the generation of reactive oxygen species are effectively inhibited, and the cell uptake and therapeutic effect of atorvastatin in the treatment of atherosclerosis are significantly enhanced, and it has a good prospect for industrial application.

Claims

1. A nanoparticle, characterized in that: It is composed of a mixture of a DNA tetrahedron complex and a fused cell membrane, wherein the mass ratio of the DNA tetrahedron complex to the fused cell membrane is 1:4-20; The DNA tetrahedral complex is a product prepared using atorvastatin and tetrahedral framework nucleic acid as raw materials; The fusion cell membrane is a product prepared by using active oxygen-responsive liposomes and cell membranes as raw materials, and the cell membranes are from cells that highly express VLA4.

2. The nanoparticle according to claim 1, characterized in that The mass ratio of DNA tetrahedral complex and fused cell membrane was 1:10; In the DNA tetrahedral complex, the molar ratio of atorvastatin to tetrahedral framework nucleic acid is 0.01-0.1 mmol:150-200 nmol, preferably 0.09 mmol:160 nmol; In the fusion cell membrane, the mass ratio of the active oxygen responsive liposome to the cell membrane is 1:5 to 15, preferably 1:

10.

3. The nanoparticle according to claim 1, characterized in that The DNA tetrahedron complex is self-assembled by four DNA single strands, the four DNA single strands are S1, atorvastatin-S2, atorvastatin-S3 and atorvastatin-S4, and the sequences of S1, S2, S3 and S4 are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively.

4. The nanoparticle according to claim 1, characterized in that The active oxygen responsive liposome is a thioketal-containing liposome; preferably, the thioketal-containing liposome is a product obtained by mixing DSPE-TK-PEG2000, lecithin and cholesterol in a solvent to form a transparent film, and then reacting with water; wherein the molar ratio of DSPE-TK-PEG2000, lecithin and cholesterol is 0.2-0.6:1-3:0.5-1.5, preferably 0.4:2:1; The solvent is chloroform; the reaction temperature is 10-40° C., preferably 15-35° C.; the reaction time is 20-40 minutes, preferably 30 minutes.

5. The nanoparticle according to claim 1, characterized in that The cells that highly express VLA4 are C1498-VLA cells, and the preparation of C1498-VLA cells comprises the following steps: The plasmid DNA and the transfection reagent are incubated to form a complex, which is then transfected into retroviral cells, digested, centrifuged, and the supernatant is taken to suspend C1498 cells to obtain C1498-VLA cells.

6. The nanoparticle according to claim 5, characterized in that The sequence of the plasmid DNA is shown in SEQ ID No. 5; the transfection reagent is Lipofectamine 3000, and the incubation time is 10 to 30 minutes, preferably 20 minutes; The retrovirus cells are AmphoPhoenix cells, and the transfection time is 3 to 5 hours, preferably 4 hours.

7. A method for preparing the nanoparticles according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: mixing the DNA tetrahedron complex and the fused cell membrane, and ultrasonicating to obtain nanoparticles.

8. Use of the nanoparticles according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or treating cardiovascular diseases.

9. The use according to claim 8, characterized in that The cardiovascular disease is atherosclerosis.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition is a pharmaceutical preparation prepared by using the nanoparticles according to any one of claims 1 to 6 as active ingredients and adding pharmaceutically acceptable excipients.

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