Nucleic acid nanogel as well as preparation method and application thereof

Abstract

The invention provides a diagnosis and treatment integrated nucleic acid nanogel as well as a preparation method and application thereof, specifically, functional modification and programmable characteristics of nucleic acid are utilized to construct a multifunctional self-assembled nucleic acid nanogel, and the multifunctional self-assembled nucleic acid nanogel comprises an inflammation targeting ligand and NF-kB siRNA (siNF-kB), and optionally further comprises a contrast agent for protection and targeted delivery of siRNA to realize targeted therapy of inflammatory diseases, and can realize visualization and integration of diagnosis and treatment of inflammatory diseases.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedicine and relates to a multifunctional self-assembled integrated diagnosis and treatment nucleic acid nanogel and its preparation method and application. The multifunctional self-assembled nucleic acid nanohydrogel includes an inflammation targeting ligand and NF-κB siRNA (siNF-κB), and optionally further includes a contrast agent for protecting and targeted delivery of siRNA to achieve targeted treatment of inflammatory diseases, and can achieve visualization and integration of diagnosis and treatment of inflammatory diseases such as atherosclerosis. Background Art

[0002] Atherosclerosis (AS) is the main pathological mechanism of obstructive cardiovascular disease, which causes serious medical threats worldwide. Although a variety of highly effective anti-atherosclerotic treatments, such as cholesterol-lowering drugs and antiplatelet drugs, have been developed and optimized in the clinic, fatal cardiovascular events still exist. The limited efficacy of drugs may be due to insufficient target specificity, low bioavailability and high toxicity of drugs. In addition, some patients show characteristics of insensitivity to drug intervention, and there are still challenges in timely predicting acute events, resulting in unpredictable high-risk attacks. In addition, there are still unresolved issues in the accuracy of predicting vulnerable plaques with different morphologies. It is recognized that acute coronary syndrome (ACS) may be caused by superficial endothelial erosion, which has significant differences in anatomical characteristics compared with thin-shell and lipid-rich vulnerable plaques. Therefore, it is urgent to use substantial targets to effectively diagnose and treat plaques, and to monitor the medication process in real time, so as to timely and accurately predict the treatment effect and reduce the occurrence of acute events.

[0003] To date, the inflammatory environment inside the plaque has been considered an ideal target for the treatment and imaging of inflammatory diseases such as atherosclerosis. A variety of anti-inflammatory therapies have been developed, including traditional anti-inflammatory drugs (such as methotrexate and colchicine), cytokine-targeted biological therapies (such as the anti-IL-1β inhibitor Canakinumab and the anti-IL-6 inhibitor Tocilizumab), and phospholipase A2 inhibitors to reduce the occurrence of acute cardiovascular events. However, clinical trial results have shown that these drugs have limited efficiency or increased fatal infections due to their inability to be effectively delivered to the lesion area. In order to image inflammation in plaques, radionuclide-based positron emission tomography (PET) methods have been developed to image immune cells within plaques, providing an important supplementary description for high-risk plaques. However, radionuclides may have safety issues, and imaging sensitivity may be affected by highly metabolically active tissues. In addition, imaging of immune cells in plaques cannot provide spatial information on epidermal erosion associated with the endothelium. As a chronic inflammatory disease, ankylosing spondylitis occurs on the dysfunctional aortic endothelium and is accompanied by inflammatory disorders, which subsequently promotes the accumulation of immune cells in plaques. Studies have shown that inhibition of individual monocytes does not provide a significant benefit in advanced atherosclerosis, which may be attributed to the continued infiltration of monocytes.

[0004] However, there are limited therapeutic approaches targeting the endothelium, and researchers have mainly used nucleic acid therapy as a therapeutic approach to restore diseased endothelial function. For example, it has been reported that the use of nucleic acid therapeutics to inhibit endothelial transforming growth factor β (TGF-β) signaling or delete the transcription factor BACH1 can alleviate atherosclerosis by reducing endothelial inflammation, providing solid evidence for the potential role of the endothelium in the effective diagnosis and treatment of plaques. However, currently developed strategies cannot achieve visualized treatment of inflammatory diseases such as atherosclerosis, and the development of endothelial targeted therapeutic nanoplatforms remains stagnant, which may be attributed to the complex manufacturing of therapeutic materials and the technical difficulty of plaque imaging. Although nucleic acid therapy has been approved for clinical treatment of various diseases and has shown promise in the treatment of atherosclerosis, toxicity issues, tissue targeting disabilities, manufacturing complexity, limited thermal stability, and reproducibility difficulties due to the use of cationic materials as carriers remain concerns.

[0005] Therefore, the development of therapeutically safe nucleic acid therapies for endothelial-targeted diagnosis and treatment of plaques is a promising strategy to effectively combat inflammatory diseases such as atherosclerosis. Summary of the invention

[0006] In view of the various problems existing in the anti-inflammatory treatment of inflammatory diseases such as atherosclerosis, such as lack of lesion targeting, low bioavailability, high toxicity, and limited clinical treatment effect; lack of effective drugs targeting inflammatory endothelium; inability to achieve visualized treatment; toxicity of nucleic acid drug carriers, lack of tissue targeting disability, complex preparation, poor thermal stability and reproducibility, etc., the purpose of the present disclosure is to provide a multifunctional self-assembled nucleic acid nanogel, which is preferably a three-in-one nucleic acid nanogel, which specifically co-delivers an inflammatory targeting ligand, an anti-inflammatory drug NF-κB siRNA, and a contrast agent, and uses imaging equipment to achieve plaque imaging, thereby realizing integrated diagnosis and treatment and visualized treatment.

[0007] Specifically, the purpose of the present disclosure is to provide a multifunctional nucleic acid nanogel, which includes side chain modified DNA, inflammation targeting ligand, and NF-κB targeting siRNA, wherein the side chain modified DNA is used as a carrier and is grafted with the inflammation targeting ligand to form a Y2 structural unit.

[0008] In some embodiments of the present disclosure, the multifunctional self-assembled nucleic acid nanogel further comprises one or more contrast agents, and the contrast agent and the side-chain modified DNA are grafted to form a Y1 structural unit.

[0009] In some embodiments of the present disclosure, the inflammation targeting ligand is selected from proinflammatory cell adhesion molecules and chemokines, preferably selected from vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, monocyte chemoattractant protein-1 (MCP-1), scavenger receptor (SR), chemokine (CCL), and more preferably vascular cell adhesion molecule-1 (VCAM-1, hereinafter referred to as VP).

[0010] In some embodiments of the present disclosure, the NF-κB is selected from NF-κB p65, NF-κB p50, NF-κB IKK and NF-κB NEMO, preferably NF-κB p65.

[0011] In some embodiments of the present disclosure, the side chain modified DNA is dibenzocyclooctyne (DBCO) modified DNA.

[0012] In some embodiments of the present disclosure, the contrast agent is selected from MRI contrast agents, DSA contrast agents, PET contrast agents, CT contrast agents, PET-CT contrast agents, preferably MRI contrast agents, and more preferably Gd-DOTA (gadoteric acid).

[0013] In some embodiments of the present disclosure, the MRI contrast agent is selected from gadolinium contrast agents, preferably selected from but not limited to Gd-DOTA (gadolinic acid) and Gd-DTPA (magenvestat; magnetically displayed dimeglumine).

[0014] In some embodiments of the present disclosure, the DSA contrast agent, PET contrast agent, CT contrast agent, PET-CT contrast agent is selected from but not limited to iodine preparations (iohexol, iodixanol) and barium sulfate.

[0015] In some embodiments of the present disclosure, the Y2 structural unit is formed by grafting an inflammation targeting ligand having an azide group on the side chain with dibenzocyclooctyne (DBCO)-modified DNA.

[0016] In some embodiments of the present disclosure, the Y2 structural unit is formed by grafting an inflammatory targeting ligand with an azide group on the side chain and three DBCO-modified DNA chains; the three DNA chains satisfy pairing complementarity in pairs, and preferably, the grafting includes covalent coupling reaction between two DNA chains without sticky ends and the inflammatory targeting ligand, and then self-assembly with a DNA chain with a sticky end.

[0017] In some embodiments of the present disclosure, the Y1 structural unit is formed by grafting a contrast agent having an azide group on the side chain with dibenzocyclooctyne-modified DNA.

[0018] In some embodiments of the present disclosure, the Y1 structural unit is formed by grafting a contrast agent having an azide group on the side chain with three DBCO-modified DNA chains; preferably, the grafting comprises three DBCO-modified DNA chains chemically reacting with the contrast agent and then self-assembling, wherein each DNA chain has a single-stranded overhang as a sticky end.

[0019] In some embodiments of the present disclosure, the Y2 building block contains a sticky end.

[0020] In some embodiments of the present disclosure, the Y1 structural unit contains three sticky ends.

[0021] In some embodiments of the present disclosure, the siRNA is both a functional nucleic acid and a cross-linking agent.

[0022] In some embodiments of the present disclosure, the end of the siRNA has an additional gene sequence that can pair with DNA bases.

[0023] On the other hand, the purpose of the present disclosure is to provide a method for preparing the multifunctional self-assembled nucleic acid nanogel described in any of the above schemes, which comprises the following steps:

[0024] Step 1. Using the principle of complementary base pairing, a contrast agent with a first group modified on the side chain is chemically reacted with DNA modified with a second group, and then a Y1 structural unit is obtained through self-assembly of the DNA chain;

[0025] Step 2. Using the complementary base pairing principle, the inflammation targeting ligand with the first group modified on the side chain is covalently coupled with the DNA modified with the second group, and then the Y2 structural unit is obtained through the self-assembly of the DNA chain;

[0026] Step 3. NF-κB targeting siRNA is used as a cross-linking agent and is uniformly mixed with the Y1 structural unit and the Y2 structural unit respectively, and then allowed to stand to obtain the multifunctional self-assembled nucleic acid nanogel;

[0027] Alternatively, NF-κB targeting siRNA is used as a cross-linking agent and is uniformly mixed with the Y1 structural unit and the Y2 structural unit and then allowed to stand to obtain the multifunctional self-assembled nucleic acid nanogel;

[0028] Wherein, preferably, the first group is selected from azide, NHS, amino, maleimide, thiol, more preferably azide;

[0029] Preferably, the second group is selected from dibenzocyclooctyne (DBCO), amino, carboxyl, and thiol, more preferably dibenzocyclooctyne (DBCO).

[0030] In some embodiments of the present disclosure, the Y1 structural unit comprises three DBCO-modified DNA chains chemically reacting with the contrast agent to obtain three DNA conjugates, and then the three DNA conjugates are self-assembled through base complementary pairing, and the Y1 structural unit contains three sticky ends.

[0031] In some embodiments of the present disclosure, wherein obtaining the Y2 structural unit comprises coupling the targeted inflammatory ligand and two DNA chains without sticky ends, and then self-assembling with a DNA chain containing a sticky end, the Y2 structural unit contains a sticky end.

[0032] In some embodiments of the present disclosure, the contrast agent is selected from MRI contrast agents, DSA contrast agents, PET contrast agents, CT contrast agents, PET-CT contrast agents, preferably MRI contrast agents, and more preferably Gd-DOTA (gadolinic acid).

[0033] In some embodiments of the present disclosure, the molar ratio of the Y1 structural unit and the Y2 structural unit to the NF-κB siRNA cross-linking agent is (3Y1+Y2):NF-κB siRNA cross-linking agent=2:1

[0034] In some embodiments of the present disclosure, the molar ratio of the Y1 structural unit, the Y2 structural unit and the NF-κB targeting siRNA cross-linking agent is preferably 4:1:6.5.

[0035] In some embodiments of the present disclosure, the molar ratio of (Y1 structural unit and Y2 structural unit): NF-κB siRNA cross-linking agent is preferably 1:1.5.

[0036] In some embodiments of the present disclosure, the molar ratio of the Y1 structural unit to the Y2 structural unit is selected from 1:1 to 10:1.

[0037] In some embodiments of the present disclosure, the size of the multifunctional self-assembled nucleic acid nanogel is between 50 nm and 200 nm.

[0038] In some embodiments of the present disclosure, the method for preparing the multifunctional self-assembled nucleic acid nanogel, wherein the chemical reaction or covalent coupling reaction includes all achievable technical means well known to those skilled in the art, such as click chemistry reaction, esterification reaction, amidation reaction, preferably click chemistry reaction.

[0039] On the other hand, the purpose of the present disclosure is to provide a use of the multifunctional self-assembled nucleic acid nanogel described in any of the above schemes or the multifunctional self-assembled nucleic acid nanogel prepared by the preparation method described in any of the above schemes in the preparation of drugs for treating and / or preventing inflammatory diseases, preferably in the preparation of drugs for treating and / or preventing atherosclerosis.

[0040] In some embodiments of the present disclosure, the use of the multifunctional self-assembled nucleic acid nanogel in the preparation of drugs for treating and / or preventing inflammatory diseases also includes one or more imaging devices to achieve integrated diagnosis and treatment and visualization; preferably, the imaging devices include MRI, DSA, PET, CT, and PET-CT.

[0041] In some embodiments of the present disclosure, the sequences of the three DBCO-modified DNA chains are selected from Ya, Yb and Yc.

[0042] In some embodiments of the present disclosure, the sequences of the two DNA chains without sticky ends are selected from Y-a' and Y-b'.

[0043] In some embodiments of the present disclosure, the sequence of the one DNA strand containing a sticky end is selected from Ya, Yb and Yc, preferably Yc.

[0044] In some embodiments of the present disclosure, the Ya, Yb, Yc, Y-a' and Y-b' sequences are all known sequences (ACS Appl. Mater. Interfaces, 2020, 12, 21441-21449), purchased from Shanghai Sangon Biotechnology Co., Ltd. (Order No. 112697264). The sequences are listed as follows:

[0045] Table 1

[0046]

[0047] In some embodiments of the present disclosure, the sequences of the NF-κB targeting siRNA and the sequences mentioned in the examples are known sequences (Biomaterials, 2017Apr:122:10-22), purchased from Shanghai Sangon Biotechnology Co., Ltd. (Order No. 112694742), and the sequences are listed as follows:

[0048] Table 2

[0049]

[0050]

[0051] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0052] By utilizing the programmed self-assembly ability of functional nucleic acids and the functional modification of nucleic acids, a multifunctional self-assembled nucleic acid nanogel integrating targeting peptides, MRI contrast agents, and siRNA is constructed to achieve the embedding and protection of siRNA. At the same time, it can effectively deliver siRNA to the plaque lesion site and combine with MRI to achieve the integrated diagnosis and treatment of atherosclerosis. The multifunctional self-assembled nucleic acid nanogel has targeting, magnetic resonance imaging, and gene silencing effects. The multifunctional self-assembled nucleic acid nanogel provides a promising strategy for constructing a new functional nucleic acid assembly structure to achieve the integrated diagnosis and treatment of atherosclerosis, and lays a good foundation for potential clinical application transformation.

[0053] The multifunctional self-assembled nucleic acid nanogels disclosed in the present disclosure performed well in both in vitro and in vivo experiments:

[0054] 1. In vitro experiments can effectively target inflammatory endothelial cells and be taken up, and then significantly downregulate the mRNA and protein expression levels of NF-κB, thereby effectively reducing the content of subsequent inflammation-related indicators;

[0055] 2. In in vivo experiments, it can effectively target atherosclerotic plaque areas, produce lasting retention and enrichment effects, and thus significantly alleviate the inflammatory state within the plaque, improve the composition of the plaque, and cause the plaque to regress significantly;

[0056] 3. Imaging equipment such as MRI can be used to monitor the effect of plaque treatment in real time, realizing integrated diagnosis and treatment and visualization, providing timely treatment prognosis information, and improving the accuracy of drug treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 It is a 20% denaturing PAGE gel image of DNA chain and conjugate;

[0058] Figure 2 middle, Figure 2 A shows the 10% non-denaturing PAGE gel electrophoresis of DNA single strand and Y-shaped structural unit. Figure 2 B is the 3% agarose gel electrophoresis of NF-κB siRNA nanohydrogel;

[0059] Figure 3 DLS characterization of NF-κB siRNA nanohydrogel;

[0060] Figure 4 middle, Figure 4 A to Figure 4 C is the targeting performance of NF-κB siRNA nanohydrogel in inflammatory endothelial cells, Figure 4 A is the CLSM image of VCAM-1 expression in MAECs after treatment with TNF-α (10 ng / mL, 6 h), scale bar: 25 μm; Figure 4 B. Figure 4 C is flow cytometry ( Figure 4 B) and CLSM( Figure 4 C) Fluorescence intensity of cells used to characterize non-targeted Cy5.5 / NF-NG and targeted Cy5.5 / VP-NF-NG treatment of iMAECs, scale bar: 25 μm. Statistical analysis: **p<0.01, ***p<0.001;

[0061] Figure 5 middle, Figure 5 B to Figure 5 G is the evaluation of mRNA expression levels in iMAECs after NF-κB siRNA nanohydrogel treatment: NF-κB (p65) ( Figure 5 B) IL-8 ( Figure 5 C) IL-6 ( Figure 5 D) MCP-1( Figure 5 E), ICAM-1 ( Figure 5 F) and E-selectin ( Figure 5 G) Relative mRNA expression levels. Statistical analysis: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001;

[0062] Figure 6 middle, Figure 6 A. Figure 6 B. Figure 6 C respectively shows the expression of NF-κB (p65) in MAEC and iMAEC after treatment with different drugs ( Figure 6 A), ICAM-1 ( Figure 6 B) and E-selectin ( Figure 6 C) Western blot image; Figure 6 D. Figure 6 E. Figure 6 F are the IL-6 secreted by MAEC and iMAEC after treatment with different drugs ( Figure 6 D) IL-8 ( Figure 6 E) and MCP-1( Figure 6 F) ELISA test results. Statistical analysis: *p<0.05, **p<0.01, ***p<0.001;

[0063] Figure 7 middle, Figure 7 A is the in vitro fluorescence image of the aorta after injection of Linker-Cy5.5, Cy5.5 / NF-NG and Cy5.5 / VP-NF-NG at different time intervals (1, 4, 8 and 24 h); Figure 7 B is the mean fluorescence intensity of the imaged aorta; Figure 7 C is a CLSM image of the cross section of the aortic root after tail vein injection of Linker-Cy5.5, Cy5.5 / NF-NG and Cy5.5 / VP-NF-NG at different times. Scale bar: 100 μm. The white arrow indicates the cavity area of ​​the aortic valve. Statistical analysis: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001;

[0064] Figure 8 middle, Figure 8 A is the T1 mapping image of VP-Gd-NF-NG at different Gd concentrations and the regression curve between 1 / T1 and Gd concentration; Figure 8 B is a T1-weighted MRI image of atherosclerotic mouse plaques before, 1, 4, 8, and 24 h after injection of VP-Gd-NF-NG, 1: brachiocephalic trunk, 2: left common carotid artery, 3: left subclavian artery, 4: trachea;

[0065] Fig. 9 middle, Fig. 9 A is the magnetic resonance imaging of the anti-atherosclerotic effect of VP-Gd-NF-NG; Fig. 9B is the Oil Red O staining image of aorta after treatment with PBS, siNF-κB, SCR-NG, NF-NG, and VP-NF-NG; Fig. 9 C is the statistics of the plaque lesion area in the Oil Red O staining image using Image Pro Plus software. Statistical analysis: **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0066] The present disclosure is further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present disclosure and are not used to limit the scope of protection of the present disclosure. In practical applications, adjustments and improvements made by those skilled in the art based on the present disclosure still fall within the scope of protection of the present disclosure.

[0067] Example 1: Synthesis of Y1 building block (Gd-DOTA-DNA conjugate)

[0068] Step 1: Synthesis reaction routes of three DNA conjugates: Gd-DOTA-Ya, Gd-DOTA-Yb and Gd-DOTA-Yc:

[0069]

[0070] The DNA chains used in this experiment are Ya, Yb and Yc, and their specific sequences are shown in Table 1. The specific experimental process is as follows: 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-(2-thioethyl)acetamide (DOTA-N 3 , 2 equivalents) and DNA-DBCO (1 equivalent), the DNA concentration was controlled to 200 μM, and the reaction was shaken at 50°C for 24 hours. After the reaction, the unreacted DOTA-N was removed by ultrafiltration. 3 The water was then removed by vacuum concentration to obtain the purified DOTA-DNA conjugate, which was characterized by 20% denaturing PAGE gel electrophoresis. Then, the obtained DOTA-DNA conjugate (1 equivalent, 110 μM) and gadolinium chloride (GdCl) were reacted in N'-α-hydroxyethylpiperazine-N'-ethanesulfonic acid buffer (HEPES, pH = 5.5). 3 , 200 equivalents) and shaken at 37°C overnight. After completion, excess GdCl was removed by ultrafiltration. 3 Then, the three purified Gd-DOTA-DNA conjugates, namely Gd-DOTA-Ya, Gd-DOTA-Yb and Gd-DOTA-Yc, were obtained by vacuum concentration and then characterized and analyzed by 20% denaturing PAGE gel electrophoresis and mass spectrometry.

[0071] Step 2: Synthesis of Y1 structural unit (i.e. Gd-Y1)

[0072]

[0073] In this experiment, the three DNA chains obtained in step 1 were self-assembled into a Y1 structural unit with three sticky ends by using complementary base pairing, which was used for the subsequent assembly of nucleic acid nanohydrogels. Specifically, a nucleic acid sequence as a sticky end was introduced at the 3' end of the DNA chain, and Gd-DOTA was grafted and modified at the 5' end, and then the Gd-Y1 structural unit was obtained through nucleic acid self-assembly. It contains three sticky ends and is a key component in the construction of nanohydrogels. The introduction of Gd can provide MRI capabilities for nucleic acid nanohydrogels.

[0074] Experimental steps: Gd-DOTA-Ya, Gd-DOTA-Yb and Gd-DOTA-Yc DNA conjugates were mixed in TAE / Mg 2+ Buffer solution was added and heated to 90°C for 5 minutes, and then cooled to room temperature to obtain the Y1 structural unit containing Gd-DOTA (ie, Gd-Y1). The assembly was characterized and analyzed by 10% non-denaturing PAGE gel electrophoresis.

[0075] If the three DNA chains (Ya, Yb and Yc) mentioned in step 1 do not need to be modified, step 1 can be omitted and the three DNA chains can be directly self-assembled according to the method of step 2 to obtain the unmodified Y1 structural unit, which is referred to as the Y1a structural unit in the present disclosure.

[0076] Example 2: Synthesis of Y2 structural unit (i.e. VP-Y2)

[0077] Step 1: Synthesis of VP-DNA conjugate

[0078] Reaction route:

[0079]

[0080] Vascular cell adhesion molecule-1-N 3 (VP-N 3 ) is covalently bound to DNA-DBCO to prepare VP-DNA conjugate. The DNA chains used in this experiment are Y-a' and Y-b', and the specific sequences are shown in Table 1. The specific experimental steps are as follows: VP-N 3 (2 equivalents) and DNA-DBCO (1 equivalent, 200 μM) were mixed in DMSO and shaken at 50°C for 24 h. The excess VP-N was removed by ultrafiltration. 3Then, the water was removed by vacuum concentration to obtain VP-DNA conjugates, namely VP-Y-a' and VP-Y-b' conjugates. The conjugates were characterized and analyzed by 20% denaturing PAGE gel electrophoresis and mass spectrometry.

[0081] Step 2: Synthesis of Y2 building block

[0082]

[0083] In this experiment, the two conjugates obtained in step 1 and Yc were self-assembled into a Y2 structural unit with only one sticky end by using complementary base pairing, which was used for the subsequent assembly of nucleic acid nanohydrogels. The Y2 structural unit has two targeting peptide ligands and only one sticky end, which can be used as an end-capping structural unit when assembling nucleic acid nanohydrogels and endow nucleic acid nanohydrogels with endothelial cell targeting ability.

[0084] Experimental steps: Mix the three DNA chains VP-Y-a', VP-Y-b' and Yc in equal volumes and at equal molar concentrations in TAE / Mg 2+ Buffer solution was added and heated to 90°C for 5 minutes, and then cooled to room temperature to obtain the VP-modified Y2 structural unit (VP-Y2). The assembly was characterized and analyzed by 10% non-denaturing PAGE gel electrophoresis.

[0085] If the three DNA chains (Y-a', Y-b' and Yc) mentioned in step 1 and step 2 do not need to be modified, step 1 can be omitted and the three DNA chains can be directly self-assembled according to the method of step 2 to obtain the unmodified Y2 structural unit, which is referred to as the Y2a structural unit in the present disclosure.

[0086] Example 3: Preparation of NF-κB siRNA Nanohydrogel (VP-Gd-NF-NG)

[0087] The two structural units of DOTA-Gd-modified Y1 and VP-modified Y2 and the siNF-κB crosslinker (sequence see Table 2 siNF-κB linker-sense / anti-sense strand) were uniformly mixed at a molar ratio of 4:1:6.5, and allowed to stand at 4°C for a period of time to obtain a well-assembled nucleic acid nanohydrogel, and the assembled nucleic acid nanohydrogel was characterized and evaluated by agarose gel electrophoresis.

[0088] Example 3a

[0089] According to the method of Example 3, the molar ratio of the two structural units Y1 and Y2 and the siNF-κB cross-linker was changed, and Y1:Y2:siNF-κB cross-linker was uniformly mixed at a molar ratio of 1:1:2, 2:1:3.5, 3:1:5, 5:1:8 or 10:1:15.1, respectively, and well-assembled nucleic acid nanohydrogels could still be prepared.

[0090] Example 4 Preparation of NF-NG

[0091] The two structural units Y1a (unmodified) and Y2a (unmodified) and the siNF-κB crosslinker (sequence see Table 2 siNF-κB linker-sense / anti-sense strand) were uniformly mixed at a molar ratio of 4:1:6.5, and allowed to stand at 4°C for a period of time to obtain a well-assembled nucleic acid nanohydrogel, and the assembled nucleic acid nanohydrogel was characterized and evaluated by agarose gel electrophoresis.

[0092] Example 4a

[0093] According to the method of Example 4, the molar ratio of the two structural units Y1a and Y2a and the siNF-κB cross-linker was changed, and Y1a:Y2a:siNF-κB cross-linker was uniformly mixed at a molar ratio of 1:1:2, 2:1:3.5, 3:1:5, 5:1:8 or 10:1:15.1, respectively, and well-assembled nucleic acid nanohydrogels could still be prepared.

[0094] Example 5 Preparation of Gd-DOTA modified NF-NG (Gd-NF-NG)

[0095] The two structural units of Gd-DOTA-modified Y1 (Gd-Y1) and Y2a and the siRNA crosslinker (sequence see Table 2 siNF-κB linker-sense / anti-sense strand) were uniformly mixed in a molar ratio of 4:1:6.5, and allowed to stand at 4°C for a period of time to obtain a well-assembled nucleic acid nanohydrogel, and the assembled nucleic acid nanohydrogel was characterized and evaluated by agarose gel electrophoresis.

[0096] Example 5a

[0097] According to the method of Example 5, the molar ratio of the two structural units Y1 and Y2a and the siNF-κB crosslinker (sequence see Table 2 siNF-κB linker-sense / anti-sense strand) was changed, and Y1:Y2a:siNF-κB crosslinker was uniformly mixed at a molar ratio of 1:1:2, 2:1:3.5, 3:1:5, 5:1:8 or 10:1:15.1, respectively, and well-assembled nucleic acid nanohydrogels could still be prepared.

[0098] Example 6 Preparation of VP-modified NF-NG (VP-NF-NG)

[0099] The two structural units Y1a and VP-modified Y2 and the siRNA crosslinker (sequence see Table 2 siNF-κBlinker-sense / anti-sense strand) were uniformly mixed at a molar ratio of 4:1:6.5, and allowed to stand at 4°C for a period of time to obtain a well-assembled nucleic acid nanohydrogel, and the assembled nucleic acid nanohydrogel was characterized and evaluated by agarose gel electrophoresis.

[0100] Example 6a

[0101] According to the method of Example 6, the molar ratio of the two structural units Y1a and Y2 and the siNF-κB crosslinker (sequence see Table 2 siNF-κB linker-sense / anti-sense strand) was changed, and Y1a:Y2:siNF-κB crosslinker was uniformly mixed at a molar ratio of 1:1:2, 2:1:3.5, 3:1:5, 5:1:8 or 10:1:15.1, respectively, and well-assembled nucleic acid nanohydrogels could still be prepared.

[0102] Example 7 Preparation of SCR-NG Nanogel

[0103] The two structural units Y1 and Y2 and the SCR siRNA crosslinker (sequence see Table 2 siSCR linker-sense / anti-sense strand) were uniformly mixed at a molar ratio of 4:1:6.5, and allowed to stand at 4°C for a period of time to obtain a well-assembled nucleic acid nanohydrogel, and the assembled nucleic acid nanohydrogel was characterized and evaluated by agarose gel electrophoresis.

[0104] Example 7a

[0105] According to the method of Example 7, the molar ratio of the two structural units Y1 and Y2 and the SCR siRNA crosslinker (sequence see Table 2 siSCR linker-sense / anti-sense strand) was changed, and Y1:Y2:SCR siRNA crosslinker was uniformly mixed at a molar ratio of 1:1:2, 2:1:3.5, 3:1:5, 5:1:8 or 10:1:15.1, respectively, and a well-assembled nucleic acid nanohydrogel was still prepared.

[0106] Example 8 Characterization Analysis

[0107] 1. Characterization of three DNA conjugates Gd-DOTA-Ya, Gd-DOTA-Yb and Gd-DOTA-Yc: Figure 1As shown in Figure 2, the migration speed of the DOTA-DNA conjugate is slower than that of the unmodified DNA chain, and the band is single and sharp, indicating that DOTA is successfully grafted onto the DNA chain. 3+ After coordination with DOTA, a new bright and single band appeared above the band of DOTA-DNA conjugate, which can be attributed to Gd-DOTA-DNA conjugate. This shows that DOTA-DNA and Gd-DOTA-DNA conjugates have been successfully synthesized and have good hydrophilicity and purity.

[0108] 2. Characterization of VP-DNA conjugates: Figure 1 As shown, in the PAGE image, it can be seen that compared with the unmodified DNA chain, the migration rate of the VP-DNA conjugate slows down and appears as a sharp, single band, which shows that VP is successfully grafted onto the DNA chain.

[0109] 3. Characterization of Y1 and Y2 structural units

[0110] like Figure 2 As shown in A, both Y1 and Y2 structural units appear as single sharp bands, and their migration speeds are slower than those of the unmodified Y1 and Y2 structural units, proving that we have successfully prepared well-assembled Gd-Y1 and VP-Y2 structural units.

[0111] 4. Characterization of four nucleic acid nanohydrogels, including NF-NG prepared in Example 4, Gd-DOTA-modified NF-NG (Gd-NF-NG) prepared in Example 5, VP-modified NF-NG (VP-NF-NG) prepared in Example 6, and Gd-DOTA and VP-modified NF-NG (VP-Gd-NF-NG) prepared in Example 3

[0112] The results are as follows Figure 2 As shown in B, the four nucleic acid nanohydrogels showed similar migration rates in the 3% agarose gel electrophoresis, but slower than the migration rates of the Y-shaped structural unit and the siRNA crosslinker, indicating that the nucleic acid nanohydrogels were successfully prepared. In addition, dynamic light scattering (DLS) was used to detect and analyze the hydrated particle size of the four nucleic acid nanohydrogels. The results are shown in Figure 3 As shown, similar to the results of agarose gel electrophoresis, the four nanohydrogels showed relatively close sizes, and the hydrated particle sizes of NF-NG, Gd-NF-NG, VP-NF-NG and VP-Gd-NF-NG were 94.4nm, 105.2nm, 116.2nm and 126.7nm, respectively.

[0113] Biological evaluation

[0114] The present disclosure is further described and explained below in conjunction with test examples, but these embodiments are not intended to limit the scope of the present disclosure.

[0115] Test Example 1: In vitro endothelial cell targeting ability study

[0116] This experiment was used to verify that VP-modified nucleic acid nanohydrogels can be endowed with the ability to target inflammatory aortic endothelial cells.

[0117] First, TNF-α inflammatory cytokine at a concentration of 10 ng / mL was co-incubated with normal mouse aortic endothelial cells (MAEC) for 6 hours to construct an inflammatory MAEC (iMAEC) pathological model. Subsequently, immunofluorescence staining was used to characterize and evaluate the expression level of VCAM-1 on MAEC cells after TNF-α stimulation, using normal MAEC as a control. The results are shown in Figure 4 As shown in A, the expression level of VCAM-1 in MAEC is very low under normal conditions. On the contrary, after 6 h of TNF-α stimulation, the expression level of VCAM-1 on MAEC was significantly upregulated according to CLSM images, confirming that the iMAEC pathological model was successfully constructed by TNF-α stimulation and can be used for subsequent experiments.

[0118] In order to verify the ability of VP-NF-NG to target inflammatory aortic endothelial cells, the nucleic acid nanohydrogel (NF-NG) with unmodified VP was used as a control, and the in vitro targeting effects of the two were evaluated by flow cytometry and CLSM. By using a linker labeled with Cy5.5 fluorescence, two nucleic acid nanohydrogels, Cy5.5 / NF-NG and Cy5.5 / VP-NF-NG, were prepared, in which the Cy5.5 concentration was 500nM. Subsequently, the two nucleic acid nanohydrogels were incubated with iMAEC for 1 hour, and after the incubation, they were detected and analyzed by flow cytometry and CLSM imaging. The test results of flow cytometry and CLSM are shown in Figure 2. Figure 4 As shown in B and C, iMAECs treated with targeted Cy5.5 / VP-NF-NG exhibited higher average fluorescence intensity and stronger red fluorescence signal than iMAECs treated with non-targeted Cy5.5 / NF-NG, indicating that VP-modified nucleic acid nanohydrogels can indeed effectively target iMAECs and be taken up, indicating that they have a strong ability to target inflammatory aortic endothelial cells.

[0119] Test Example 2: In vitro anti-inflammatory study

[0120] This experiment was used to study the anti-inflammatory effect of nucleic acid nanohydrogel in vitro.

[0121] a. Analysis of mRNA knockdown in iMAECs after nucleic acid nanohydrogel treatment

[0122] MAECs were stimulated by TNF-α to induce them to transform into an inflammatory state, and then free siNF-κB (sequence see Table 2 siNF-κB sense / anti-sense strand), SCR-NG, NF-NG and VP-NF-NG prepared in the examples were used for anti-inflammatory treatment, and the siRNA concentration was uniformly 150nM. After the treatment, normal MAECs were used as controls and iMAECs were used as pathological models. qRT-PCR was used to measure the mRNA expression levels of six indicators, NF-κB (p65), IL-6, IL-8, MCP-1, ICAM-1 and E-selectin, in iMAECs after treatment with different nucleic acid nanohydrogels. The results are shown in Figure 5 As shown in BG, after incubation with TNF-α, the mRNA expression levels of six inflammation-related indicators, NF-κB (p65), IL-6, IL-8, MCP-1, ICAM-1 and E-selectin, were significantly upregulated, among which the mRNA expression of IL-6, IL-8 and MCP-1 reached twice or more than that of normal MAEC, indicating that the inflammatory state of iMAEC was significantly activated. At the same time, after incubation with iMAEC for 48 hours using different drug preparations, its inflammatory state was alleviated, especially in the VP-NF-NG treatment group.

[0123] like Figure 5 As shown in Figure B, the mRNA expression level of NF-κB (p65) in iMAECs treated with NF-NG and VP-NF-NG was significantly downregulated, among which VP-NF-NG was able to reduce its expression level from about 140% in iMAECs to about 80%, revealing the efficient degradation of target genes by VP-NF-NG, indicating that it may have powerful anti-inflammatory therapeutic potential.

[0124] like Figure 5 As shown in CG, the mRNA expression levels of inflammation-related markers (IL-6, IL-8, MCP-1, E-selectin, ICAM-1) in iMAEC treated with nucleic acid nanohydrogels were also significantly downregulated, indicating that after the mRNA expression level of NF-κB (p65) was downregulated, the expression of its downstream inflammation-related indicators would also be significantly inhibited. In addition, since VP-NF-NG has the ability to target VCAM-1, it can better target iMAEC and be taken up, so it shows the most significant mRNA expression level downregulation effect in all six analysis indicators, indicating that it has excellent anti-inflammatory ability and also lays the foundation for the subsequent anti-inflammatory effect in vivo.

[0125] b. Analysis of protein downregulation in iMAECs after NF-κB siRNA nanohydrogel treatment

[0126] In this experiment, protein expression levels of relevant indicators were determined and analyzed using Western blot test and enzyme-linked immunosorbent assay (ELISA). Figure 6 As shown in A, the results of Western blot test showed that the expression level of NF-κB (p65) in iMAEC was 5.36 times that in MAEC. Figure 6 BF showed that the protein expression levels of ICAM-1, E-selectin, IL-8, IL-6 and MCP-1 in iMAEC were significantly increased. These results further proved that TNF-α stimulation of MAEC can activate its inflammatory state. However, after iMAEC was treated with different drug preparations, its inflammatory state was alleviated, and the expression levels of related proteins were inhibited and downregulated. Figure 6 As shown in AC, the results of Western blotting test showed that NF-NG and VP-NF-NG can effectively downregulate the expression levels of NF-κB (p65), ICAM-1 and E-selectin. Among them, VP-NF-NG achieved the most excellent protein downregulation effect due to its targeting ability, and the content of NF-κB (p65), ICAM-1 and E-selectin was downregulated from 5.36 times, 1.63 times and 1.88 times in the pathological state of iMAEC to 1.28 times, 0.16 times and 0.60 times respectively. At the same time, the ELISA test results showed a trend similar to the Western blotting test results. Compared with other groups, VP-NF-NG showed the most significant ability to downregulate protein expression. The content of three secreted proteins, IL-6, IL-8 and MCP-1, secreted into the cell culture medium by iMAEC treated with it were significantly downregulated ( Figure 6 DF). The above results indicate that VP-NF-NG can effectively inhibit the inflammatory response in iMAECs, effectively relieve them from the pathological state, and then gradually restore them to the steady-state balance.

[0127] Test Example 3: In vivo plaque targeting analysis

[0128] This experiment was mainly used to evaluate the in vivo plaque targeting ability of VP-NF-NG.

[0129] Apolipoprotein E gene-deficient (ApoE- / -) mice were selected as an atherosclerosis model to evaluate the targeting ability of VP-modified nucleic acid nanohydrogels to plaques in vivo. Cy5.5-modified linker (Linker-Cy5.5), Cy5.5 / NF-NG and Cy5.5 / VP-NF-NG were injected into atherosclerotic mice through the tail vein, respectively, where the concentration of Cy5.5 was uniformly 100μg Cy5.5 / kg mouse. At predetermined time points (1h, 4h, 8h and 24h) after injection of the preparation, the mice were anesthetized and cardiac perfused, and then the aorta was dissected and isolated, and in vitro fluorescence imaging was performed on it using the IVIS fluorescence imaging system (PerkinElmer). Figure 7 As shown in A and B, compared with Linker-Cy5.5, mice treated with Cy5.5 / NF-NG and Cy5.5 / VP-NF-NG showed stronger fluorescence signals at each time point, which may be due to the passive targeting properties of the nanoparticles, thereby increasing the accumulation of nucleic acid nanohydrogels in the plaque lesion area. Among them, the aorta of mice in the Cy5.5 / VP-NF-NG treatment group showed the strongest fluorescence signal, indicating that the modification of the targeting peptide VP can give the nucleic acid nanohydrogel excellent in vivo plaque targeting ability. In addition, the aorta of mice injected with Cy5.5 / VP-NF-NG still showed extremely strong fluorescence signals and average fluorescence intensity 24 hours later, indicating that Cy5.5 / VP-NF-NG has excellent retention and enrichment capabilities in the plaque area. In order to gain a deeper understanding of the distribution of nanohydrogels in the plaque, the dissected aortic root was prepared into slices and stained with DAPI and anti-CD31 fluorescent antibodies for CLSM microscopy imaging. The results are shown in Figure 7 As shown in Figure C, consistent with the results of in vitro aortic fluorescence imaging, Cy5.5 / VP-NF-NG showed a stronger Cy5.5 signal than mice treated with Linker-Cy5.5 and Cy5.5 / NF-NG; and the aortic root sections of mice in the Cy5.5 / VP-NF-NG treatment group still showed significant Cy5.5 signals after 24 hours, indicating that VP-modified nucleic acid nanohydrogels have significant endothelial cell targeting and retention capabilities.

[0130] Test Example 4: Magnetic Resonance Imaging

[0131] This experiment is mainly used to evaluate the MRI properties of the nucleic acid nanogel described in the present disclosure.

[0132] The in vitro relaxation properties of Gd-DOTA and VP-modified nucleic acid nanohydrogels (VP-Gd-NF-NG) were characterized and analyzed by T1mapping on a 3T MAGNETOM Prisma MRI scanner (Siemens Healthineers). Figure 8 As shown in A, when the Gd concentration in VP-Gd-NF-NG gradually increases, its relaxation time will gradually decrease, and by making a linear regression curve of 1 / T1 and Gd concentration, it is found that the relaxation rate of VP-Gd-NF-NG is 5.7614mM-1s-1, indicating that the Gd-modified nucleic acid nanohydrogel does have excellent MRI capabilities. In addition, to further explore the in vivo MRI capabilities of VP-Gd-NF-NG, VP-Gd-NF-NG was injected into atherosclerotic mice through the tail vein, and after different time intervals (1h, 4h, 8h and 24h), the mice were scanned using an 11.7T BioSpec MRI system to observe the in vivo MRI effect of VP-Gd-NF-NG. Figure 8 As shown in B, before the injection of VP-Gd-NF-NG into the carotid artery, there was no obvious enhanced signal on the carotid artery wall. However, after the injection of VP-Gd-NF-NG, uneven enhanced highlight signals appeared on the carotid artery wall, which can be attributed to the atherosclerotic plaques that have formed on the carotid artery wall. In addition, even 24 hours after injection, significantly enhanced plaque signals can still be observed, indicating that VP-Gd-NF-NG has a persistent drug retention and accumulation ability in the plaque area, which is consistent with the in vitro fluorescence imaging results. The above results show that VP-Gd-NF-NG has significant plaque targeting, imaging and retention capabilities, indicating that it may have potential diagnostic and therapeutic effects on atherosclerosis.

[0133] Test Example 5: In vivo visualization of atherosclerosis treatment capabilities and evaluation of anti-therapeutic efficacy

[0134] ApoE- / - mice aged 6-8 weeks were used to establish an atherosclerosis mouse model through a 4-week high-cholesterol diet. Subsequently, five drug preparations, PBS, siNF-κB, SCR-NG, NF-NG and VP-Gd-NF-NG, were injected into the mice through the tail vein for anti-atherosclerosis treatment. The drugs were administered twice a week for a total of five times, and the siRNA dosage was 1 mg / kg mouse. On the first day (d1), the eighth day (d8) and the fifteenth day (d15), the mouse aorta was scanned and imaged using the uMR 9.4T MRI system to visualize the anti-atherosclerosis therapeutic effect of VP-Gd-NF-NG. Fig. 9As shown in A, at d1 after the injection of VP-Gd-NF-NG, the right common carotid artery showed a significantly enhanced signal, revealing the presence of plaques in the mouse artery. At d8 and d15, no obvious enhanced signal was observed in the right common carotid artery area, indicating that the plaques regressed and stabilized after treatment with VP-NF-NG. After the treatment, the mouse carotid arteries were collected and stained with Oil Red O to observe the plaque lesion area after treatment. Fig. 9 As shown in A (right), compared with the control group mice, the plaque area in the carotid artery of the VP-Gd-NF-NG treated mice was significantly reduced, which was consistent with the MRI results, confirming the feasibility of using multifunctional nucleic acid nanohydrogels for the integrated diagnosis and treatment of atherosclerosis.

[0135] In addition, to evaluate the anti-atherosclerotic therapeutic effects of all preparations in vivo, all mice were killed after the treatment process, and the whole aorta was dissected and collected for Oil Red O staining to observe the lesion area. Fig. 9 B and Fig. 9 As shown in C, the aorta of mice in the PBS group had the most obvious Oil Red O staining, showing the largest plaque lesion area, accounting for about 30.2% of the total aorta area. siNF-κB and SCR-NG had a weaker inhibitory effect on plaque development, with plaque proportions of 27.7% and 22.9%, respectively. However, after NF-NG and VP-NF-NG treatment of atherosclerotic mice, the plaque lesion area in the aorta that could be stained with Oil Red O was reduced to 17.8% and 5.5%, respectively, indicating that the nucleic acid nanohydrogel loaded with siNF-κB has excellent therapeutic effects. It is worth noting that VP-NF-NG with endothelial cell targeting ability showed the most significant anti-atherosclerotic therapeutic effect, with the plaque lesion area reduced to 5.5%, which was 5.5 times, 5.1 times, 4.2 times and 3.3 times lower than that of PBS, siNF-κB, SCR-NG and NF-NG groups, respectively. This excellent therapeutic effect may be because VP-NF-NG can significantly target the plaque area and produce a lasting retention and enrichment effect.

Claims

1. A multifunctional self-assembled nucleic acid nanogel, characterized in that: It comprises side chain modified DNA, inflammation targeting ligand and NF-κB targeting siRNA, wherein the side chain modified DNA is used as a carrier and is grafted with the inflammation targeting ligand to form a Y2 structural unit.

2. The multifunctional self-assembled nucleic acid nanogel according to claim 1, characterized in that: The multifunctional self-assembling nucleic acid nanogel further comprises one or more contrast agents, and the contrast agent and the side chain-modified DNA are grafted to form a Y1 structural unit.

3. The multifunctional self-assembled nucleic acid nanogel according to claim 1 or 2, characterized in that: The inflammation targeting ligand is selected from pro-inflammatory cell adhesion molecules and chemokines, preferably selected from vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E-selectin, monocyte chemoattractant protein-1, scavenger receptor, chemokine, and more preferably vascular cell adhesion molecule-1.

4. The multifunctional self-assembled nucleic acid nanogel according to any one of claims 1 to 3, characterized in that: The NF-κB is selected from NF-κB p65, NF-κB p50, NF-κB IKK and NF-κB NEMO, preferably NF-κB p65.

5. The multifunctional self-assembled nucleic acid nanogel according to any one of claims 1 to 4, characterized in that: The side chain modified DNA is dibenzocyclooctyne modified DNA.

6. The multifunctional self-assembling nucleic acid nanogel according to any one of claims 1 to 5, characterized in that: The contrast agent is selected from MRI contrast agents, DSA contrast agents, PET contrast agents, CT contrast agents, and PET-CT contrast agents, preferably MRI contrast agents, and more preferably Gd-DOTA.

7. The multifunctional self-assembling nucleic acid nanogel according to any one of claims 1 to 6, characterized in that: The Y2 structural unit is formed by grafting an inflammation targeting ligand with an azide group on the side chain and DNA modified with dibenzocyclooctyne; and / or the Y1 structural unit is formed by grafting a contrast agent with an azide group on the side chain and DNA modified with dibenzocyclooctyne.

8. The multifunctional self-assembling nucleic acid nanogel according to any one of claims 1 to 7, characterized in that: The Y2 structural unit contains a sticky end.

9. The multifunctional self-assembling nucleic acid nanogel according to any one of claims 1 to 8, characterized in that: The Y1 structural unit contains three sticky ends.

10. The multifunctional self-assembling nucleic acid nanogel according to any one of claims 1 to 9, characterized in that: The siRNA is both a functional nucleic acid and a cross-linking agent.

11. A method for preparing a multifunctional self-assembled nucleic acid nanogel according to any one of claims 1 to 10, characterized in that: The following steps are involved: Step 1. Using the principle of complementary base pairing, a contrast agent with a first group modified on the side chain is chemically reacted with DNA modified with a second group, and then a Y1 structural unit is obtained through self-assembly of the DNA chain; Step 2. Using the complementary base pairing principle, the inflammation targeting ligand with the first group modified on the side chain is covalently coupled with the DNA modified with the second group, and then the Y2 structural unit is obtained through the self-assembly of the DNA chain; Step 3. NF-κB targeting siRNA is used as a cross-linking agent and is uniformly mixed with the Y1 structural unit and the Y2 structural unit respectively, and then allowed to stand to obtain the multifunctional self-assembled nucleic acid nanogel; Alternatively, NF-κB targeting siRNA is used as a cross-linking agent and is uniformly mixed with the Y1 structural unit and the Y2 structural unit and then allowed to stand to obtain the multifunctional self-assembled nucleic acid nanogel; Wherein, preferably, the first group is selected from azide, NHS, amino, maleimide, thiol, more preferably azide; Preferably, the second group is selected from dibenzocyclooctyne, amino, carboxyl, and thiol, more preferably dibenzocyclooctyne.

12. The method for preparing the multifunctional self-assembled nucleic acid nanogel according to claim 11, wherein: The Y1 structural unit comprises three DBCO-modified DNA chains reacting chemically with the contrast agent to obtain three DNA conjugates, and then the three DNA conjugates are self-assembled through base complementary pairing. The Y1 structural unit contains three sticky ends.

13. The method for preparing the multifunctional self-assembled nucleic acid nanogel according to claim 11, wherein: The method of obtaining the Y2 structural unit includes covalently coupling the targeted inflammatory ligand and two DNA chains without sticky ends, and then self-assembling with a DNA chain containing a sticky end to form a Y2 structural unit, wherein the Y2 structural unit contains a sticky end.

14. The multifunctional self-assembling nucleic acid nanogel according to any one of claims 1 to 13, wherein The molar ratio of Y1 structural unit and Y2 structural unit to NF-κB siRNA crosslinker is (3Y1+Y2):NF-κB siRNA crosslinker=2:1; preferably, the molar ratio of Y1 structural unit and Y2 structural unit to NF-κB siRNA crosslinker is 4:1:6.

5.

15. Use of the multifunctional self-assembled nucleic acid nanogel according to any one of claims 1 to 10 or the multifunctional self-assembled nucleic acid nanogel prepared by the preparation method according to any one of claims 11 to 14 in preparing a drug for treating and / or preventing inflammatory diseases, preferably in preparing a drug for treating and / or preventing atherosclerosis.

16. The use according to claim 15, characterized in that It also includes one or more imaging devices to achieve integrated diagnosis and treatment and visualization.

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