Nucleic acid aptamer-loaded ruthenium (II) complex nano net as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nucleic acid aptamers, and particularly relates to a nucleic acid aptamer-loaded ruthenium (II) complex nano net as well as a preparation method and application thereof. The aptamer-loaded ruthenium (II) complex nano net provided by the invention can specifically recognize cancer cells in a targeting manner, and can reduce cytotoxicity. The ruthenium (II) complex is combined with the nucleic acid aptamer, so that the defects that the nucleic acid aptamer is unstable in metabolism and easy to remove by nuclease are overcome, the structural stability and the biological activity of the nucleic acid aptamer loaded ruthenium (II) complex nano net are improved, the compound can specifically recognize nucleolin in tumor cells, G4DNA conformation is formed, and the tumor cells can be specifically recognized. And targeted recognition of tumor cells is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleic acid aptamers, and particularly relates to a nucleic acid aptamer-loaded ruthenium (II) complex nanonet and a preparation method and application thereof. Background Art

[0002] Early diagnosis of cancer can promote timely medical intervention and effective treatment, thereby inhibiting tumor growth and development. Accurate diagnosis and treatment have become effective means to alleviate tumor development and improve patient survival. Molecular imaging can non-invasively display key molecular targets and host responses associated with different stages of cancer, thereby promoting accurate diagnosis and treatment of cancer, such as fluorescent bioimaging, which is a promising non-invasive, real-time and high-resolution imaging method for early diagnosis of tumors.

[0003] Fluorescence bioimaging mainly relies on small molecule probes, which are mainly composed of three parts: a reactive group that specifically interacts with proteins, a linker that regulates the activity and specificity of the reactive group, and a reporter group that is used to enrich, purify and detect target molecules, usually also called a tag. The role of the reporter group is to facilitate people to simply and quickly identify or purify the labeled protein. Introducing the reporter group into active small molecules is a key step in the discovery of drug targets using small molecule probes in chemical proteomics. The most commonly used reporter groups are fluorescent groups and biotin.

[0004] In practical applications, the morphology of fluorescent molecular probes is usually granular or fibrous, which is related to the molecular structure, synthesis method and application requirements. According to the expected application and the characteristics of the target molecule, the present invention adjusts the ligand structure to design a fluorescent probe with a reticular structure, achieves targeted recognition of tumor cells, and overcomes the problem that the ligand is difficult to be absorbed by tumor cells and has high toxicity to tumor cells. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a nucleic acid aptamer loaded ruthenium (II) complex nanonet and its preparation method and application. The nucleic acid aptamer loaded ruthenium (II) complex nanonet provided by the present invention can achieve targeted recognition of tumor cells.

[0006] The present invention provides a nucleic acid aptamer-loaded ruthenium (II) complex nanonet, comprising a nucleic acid aptamer and a ruthenium (II) complex chemically combined with the nucleic acid aptamer, wherein the nucleic acid aptamer and the ruthenium (II) complex are assembled to form a nano-net structure;

[0007] The ruthenium (II) complex comprises a central metal ion ruthenium (II), a primary ligand and an auxiliary ligand;

[0008] The main ligand includes 1-4'-bromobutyl-2-phenylvinyl imidazole [4,5f] [1,10] phenanthroline, and the auxiliary ligand includes 2,2-bipyridine or 1,10-phenanthroline.

[0009] Preferably, the nucleic acid aptamer is AS1411.

[0010] Preferably, the chemical bonding is coordination bonding and / or covalent bonding.

[0011] Preferably, when the auxiliary ligand is 2,2-bipyridine, the 1-4'-bromobutyl-2-phenylvinylimidazole [4,5f][1,10]phenanthroline is replaced by 1-4'-butyl-2-phenylvinylimidazole [4,5f][1,10]phenanthroline coupled with Erianin, or a mixture of 1-4'-bromobutyl-2-phenylvinylimidazole [4,5f][1,10]phenanthroline and 1-4'-butyl-2-phenylvinylimidazole [4,5f][1,10]phenanthroline coupled with Erianin.

[0012] Preferably, the molar ratio of the ruthenium (II) complex to the nucleic acid aptamer is 0.5 to 1:1.

[0013] Preferably, the diameter of the fibers forming the network in the nano-network structure is 0.10 to 35.4 nm;

[0014] The size of the mesh in the nano-net structure is 0.5-30 nm.

[0015] Preferably, the height of the nano-network structure is preferably 0.5-30 nm.

[0016] The present invention also provides a method for preparing the nucleic acid aptamer-loaded ruthenium (II) complex nanonet described in the above technical solution, comprising the following preparation steps:

[0017] The ruthenium (II) complex and the nucleic acid aptamer are mixed and incubated to obtain the nucleic acid aptamer-loaded ruthenium (II) complex nanonet.

[0018] Preferably, the incubation temperature is 30-50° C. and the incubation time is 12-48 hours.

[0019] The present invention also provides the use of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet described in the above technical solution or the nucleic acid aptamer-loaded ruthenium (II) complex nanonet prepared by the preparation method in the preparation of tumor-targeted fluorescent probes.

[0020] The nucleic acid aptamer-loaded ruthenium (II) complex nanonet provided by the present invention comprises a nucleic acid aptamer and a ruthenium (II) complex chemically combined with the nucleic acid aptamer, wherein the nucleic acid aptamer and the ruthenium (II) complex are assembled to form a nano-net structure; the ruthenium (II) complex comprises a central metal ion ruthenium (II), a main ligand and an auxiliary ligand; the main ligand comprises 1-4'-bromobutyl-2-phenylvinylimidazole [4,5f] [1,10] phenanthroline, and the auxiliary ligand comprises 2,2-bipyridine or 1,10-phenanthroline.

[0021] The nucleic acid aptamer-loaded ruthenium (II) complex nanonet provided by the present invention has a nano-net structure, and the net structure has a very high specific surface area, so that the nucleic acid aptamer-loaded ruthenium (II) complex nanonet has a stronger ability to bind to the tumor cell membrane and the cell nucleus; in addition, the net structure has good stability, can resist the erosion of cell fluid and biological enzymes, and after being prepared into a fluorescent probe, the biocompatibility and sensitivity of the fluorescent probe are improved, the targeted recognition of tumor cells is achieved, and the cytotoxicity can be reduced. The central metal ion and ligand in the ruthenium (II) complex can interact with the base on the nucleic acid aptamer to form a variety of covalent bonds or coordination bonds, forming a variety of G-quadruplex conformations, which can be spontaneously folded to form a nano-net structure. The present invention uses a ruthenium (II) complex as a chromophore of the fluorescent probe, which has a characteristic fluorescence emission peak. The present invention introduces an alkane chain and a styrene group into the ruthenium (II) complex, which can improve the transmembrane absorption ability of the ruthenium (II) complex into cancer cells. The present invention adopts nucleic acid aptamers as the targeting recognition group of tumor cells, which can form a G4 DNA conformation, and then combine with a ruthenium (II) complex to achieve targeted recognition of tumor cells. In the present invention, the nano-network structure formed after the nucleic acid aptamer combines with the ruthenium (II) complex is more stable, which not only overcomes the defects of the nucleic acid aptamer being metabolically unstable and easily removed by nucleases, but also improves the biological activity of the nucleic acid aptamer loaded with the ruthenium (II) complex, thereby achieving targeted recognition of tumor cells.

[0022] When the main ligand of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet provided by the present invention is 1-4'-bromobutyl-2-phenylvinyl imidazole [4,5f][1,10] phenanthroline, the ruthenium (II) complex with a bromobutyl group is both hydrophilic and lipophilic, and can combine with the base pairs in the nucleic acid aptamer, which is conducive to the formation of a network structure. The present invention also found that when the auxiliary ligand is 2,2-bipyridine, it is easier to form a network structure, and the network structure formed by the combination of the bipyridine-modified ruthenium (II) complex and the nucleic acid aptamer is clearer and more complete, because bipyridine has good planarity, and it is easy to have an effective π-π stacking effect with the base pairs in the nucleic acid aptamer, which is more conducive to promoting the formation of a network structure (such as Example 2 Figure 5). In the present invention, when the ruthenium (II) complex is grafted with the Erianin molecule, the fluorescence intensity of the ruthenium (II) complex can be enhanced, and the cytotoxicity of the ruthenium (II) complex can be reduced, which is more conducive to the targeted recognition of tumor cells; due to the increase of volume steric hindrance and spatial steric hindrance of Erianin, the formation of the network structure will be hindered to a certain extent, but it can still be seen from the atomic force microscopy (AFM) image that the Erianin-modified bipyridine ruthenium (II) complex and the nucleic acid aptamer are connected (such as Example 3 Fig. 9 ), which can be used to prepare fluorescent probes for targeted recognition and detection of tumor cells. In the present invention, when the auxiliary ligand is 1,10-phenanthroline with high stereoactivity, the ruthenium (II) complex formed after combining with the main ligand 1-4'-bromobutyl-2-phenylvinyl imidazole [4,5f] [1,10] phenanthroline also binds to the nucleic acid aptamer (as shown in Example 5 Fig.17 ), and can also be used to prepare fluorescent probes. However, when the main ligand is replaced with 1-4'-butyl-2-phenylvinyl imidazole [4,5f][1,10] phenanthroline to couple Erianin, due to the large steric hindrance of phenanthroline, the steric hindrance and volume hindrance effects are accumulated after the grafting of Erianin molecules, resulting in a decrease in the binding affinity and selectivity of the ruthenium (II) complex with the nucleic acid aptamer, and it is unable to insert between the base pairs of the nucleic acid aptamer, and basically cannot form a network structure (such as Comparative Example 1 Fig.21 ) and cannot be used to prepare fluorescent probes.

[0023] The present invention proves that the structure of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet provided by the present invention is stable through fluorescence resonance energy transfer experiments (FRET), melting point experiments and fluorescence titration experiments, and proves that the fluorescence intensity of the ruthenium (II) complex combined with the nucleic acid aptamer increases by nearly 3 times through CD spectrum and atomic force microscopy tests, which greatly improves the targeted recognition of tumor cells. The nucleic acid aptamer-loaded ruthenium (II) complex nanonet provided by the present invention has great potential for application in early anti-tumor diagnosis and treatment, and has important clinical application value.

[0024] The present invention also provides a method for preparing the nucleic acid aptamer-loaded ruthenium (II) complex nanonet, comprising the following preparation steps: mixing the ruthenium (II) complex and the nucleic acid aptamer, and incubating to obtain the nucleic acid aptamer-loaded ruthenium (II) complex nanonet. The preparation method combines the incubation of the nucleic acid aptamer and the ruthenium (II) complex, and the obtained nucleic acid aptamer-loaded ruthenium (II) complex nanonet can effectively reduce the cytotoxicity of the ruthenium (II) complex and improve the targeted recognition ability of tumor cells. Moreover, the preparation method provided by the present invention is simple to operate, has a high yield, and has potential market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1This is an AFM image of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 1E (RBLR31-AS1411) prepared in Example 1;

[0026] Figure 2 This is a fluorescence titration spectrum of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 1E (RBLR31-AS1411) prepared in Example 1;

[0027] Figure 3 This is a graph showing the fluorescence resonance energy transfer (FRET) melting point results of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 1E (RBLR31-AS1411) prepared in Example 1;

[0028] Figure 4 This is the CD spectrum of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 1E (RBLR31-AS1411) prepared in Example 1;

[0029] Figure 5 This is an AFM image of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 2E (RBLR31-BBr-AS1411) prepared in Example 2;

[0030] Figure 6 This is a fluorescence titration spectrum of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 2E (RBLR31-BBr-AS1411) prepared in Example 2;

[0031] Figure 7 This is a graph showing the fluorescence resonance energy transfer (FRET) melting point results of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 2E (RBLR31-BBr-AS1411) prepared in Example 2;

[0032] Figure 8 The CD spectrum of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 2E (RBLR31-BBr-AS1411) prepared in Example 2;

[0033] Fig. 9 This is an AFM image of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 3E (RBLR31-MLS-AS1411) prepared in Example 3;

[0034] Fig.10 This is a fluorescence titration spectrum of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 3E (RBLR31-MLS-AS1411) prepared in Example 3;

[0035] Fig.11This is a graph showing the fluorescence resonance energy transfer (FRET) melting point results of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 3E (RBLR31-MLS-AS1411) prepared in Example 3;

[0036] Fig.12 This is the CD spectrum of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 3E (RBLR31-MLS-AS1411) prepared in Example 3;

[0037] Fig.13 This is an AFM image of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 4E (RPLR31-AS1411) prepared in Example 4;

[0038] Fig.14 This is a fluorescence titration spectrum of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 4E (RPLR31-AS1411) prepared in Example 4;

[0039] Fig.15 This is a graph showing the fluorescence resonance energy transfer (FRET) melting point results of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 4E (RPLR31-AS1411) prepared in Example 4;

[0040] Fig.16 This is the CD spectrum of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 4E (RPLR31-AS1411) prepared in Example 4;

[0041] Fig.17 This is an AFM image of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 5E (RPLR31-BBr-AS1411) prepared in Example 5;

[0042] Fig.18 This is a fluorescence titration spectrum of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 5E (RPLR31-BBr-AS1411) prepared in Example 5;

[0043] Fig.19 This is a graph showing the fluorescence resonance energy transfer (FRET) melting point results of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 5E (RPLR31-BBr-AS1411) prepared in Example 5;

[0044] Fig. 20 This is the CD spectrum of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 5E (RPLR31-BBr-AS1411) prepared in Example 5;

[0045] Fig.21This is an AFM image of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 6E (RPLR31-MLS-AS1411) prepared in Comparative Example 1;

[0046] Fig. 22 This is a fluorescence titration spectrum of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 6E (RPLR31-MLS-AS1411) prepared in Comparative Example 1;

[0047] Fig.23 This is a graph showing the fluorescence resonance energy transfer (FRET) melting point results of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 6E (RPLR31-MLS-AS1411) prepared in Comparative Example 1;

[0048] Fig.24 This is the CD spectrum of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 6E (RPLR31-MLS-AS1411) prepared in Comparative Example 1. DETAILED DESCRIPTION

[0049] The present invention provides a nucleic acid aptamer-loaded ruthenium (II) complex nanonet, comprising a nucleic acid aptamer and a ruthenium (II) complex chemically combined with the nucleic acid aptamer, wherein the nucleic acid aptamer and the ruthenium (II) complex are assembled to form a nano-net structure;

[0050] The ruthenium (II) complex comprises a central metal ion ruthenium (II), a primary ligand and an auxiliary ligand;

[0051] The main ligand includes 1-4'-bromobutyl-2-phenylvinyl imidazole [4,5f] [1,10] phenanthroline, and the auxiliary ligand includes 2,2-bipyridine or 1,10-phenanthroline.

[0052] In the present invention, unless otherwise specified, the raw materials used in the present invention are preferably commercially available products.

[0053] In the present invention, the nucleic acid aptamer-loaded ruthenium (II) complex nanonet includes a nucleic acid aptamer and a ruthenium (II) complex chemically bonded to the nucleic acid aptamer, and the nucleic acid aptamer and the ruthenium (II) complex are assembled to form a nano-mesh structure. In the present invention, the chemical bonding is preferably a coordination bond and / or a covalent bond. In the present invention, the molar ratio of the ruthenium (II) complex to the nucleic acid aptamer is preferably 0.5 to 1:1, more preferably 0.6 to 0.8:1, and further preferably 0.72:1. In the present invention, the fibrous diameter of the net formed in the nano-mesh structure is preferably 0.10 to 35.4 nm. In the present invention, the size of the mesh in the nano-mesh structure is preferably 0.5 to 30 nm. In the present invention, the height of the nano-network structure induced by the nucleic acid aptamer-loaded ruthenium (II) complex nano-network is preferably 0.5 to 30 nm, specifically 0.5 nm, 0.9 nm, 2 nm, 2.8 nm or 20 nm.

[0054] The mesh structure formed by the nucleic acid aptamer-loaded ruthenium (II) complex nanonet provided by the present invention has a very high specific surface area and a stronger ability to bind to tumor cell membranes and cell nuclei; in addition, the mesh structure has good stability and can resist the erosion of cell fluid and biological enzymes. After being prepared into a fluorescent probe, the biocompatibility and sensitivity of the fluorescent probe are improved, and tumor cells can be better targeted and identified.

[0055] The nucleic acid aptamer-loaded ruthenium (II) complex nanonet provided by the present invention includes nucleic acid aptamers. In the present invention, the nucleic acid aptamer is preferably AS1411. The AS1411 used in the present invention is a nucleic acid aptamer targeting nucleolin, which can specifically bind to nucleolin (NCL) on the surface of cancer cells. Nucleolin is highly expressed on the surface of various cancer cell membranes and is also present in the nucleoplasm, cytoplasm and cell membrane. During tumor growth, NCL shuttles from the nucleus to the cell surface and is overexpressed on the surface of cancer cells. However, nucleic acid aptamers are metabolically unstable and easily removed by nucleases. The nano-mesh structure formed by combining AS1411 nucleic acid aptamers with ruthenium (II) complexes in the present invention is more stable, which not only overcomes the defects of nucleic acid aptamers being metabolically unstable and easily removed by nucleases, but also improves the biological activity of nucleic acid aptamer-loaded ruthenium (II) complexes, thereby achieving targeted recognition of tumor cells.

[0056] The nucleic acid aptamer-loaded ruthenium (II) complex nanonet provided by the present invention includes a ruthenium (II) complex. In the present invention, the ruthenium (II) complex includes a central metal ion ruthenium (II), a primary ligand and an auxiliary ligand. In the present invention, the primary ligand preferably includes 1-4'-bromobutyl-2-phenylvinyl imidazole [4,5f][1,10] phenanthroline. In the present invention, the auxiliary ligand preferably includes 2,2-bipyridine or 1,10-phenanthroline, more preferably 2,2-bipyridine. In the present invention, the primary ligand and the auxiliary ligand are bonded to the central metal ion, and the bonding mode is preferably a coordination bond and / or a covalent bond.

[0057] In the present invention, when the auxiliary ligand is 2,2-bipyridine, the 1-4'-bromobutyl-2-phenylvinylimidazole [4,5f][1,10]phenanthroline can be replaced by 1-4'-butyl-2-phenylvinylimidazole [4,5f][1,10]phenanthroline coupled with Erianin, or a mixture of 1-4'-bromobutyl-2-phenylvinylimidazole [4,5f][1,10]phenanthroline and 1-4'-butyl-2-phenylvinylimidazole [4,5f][1,10]phenanthroline coupled with Erianin. When the nucleic acid aptamer-loaded ruthenium (II) complex nanonet provided by the present invention uses 1-4'-bromobutyl-2-phenylvinyl imidazole [4,5f] [1,10] phenanthroline as the main ligand, the ruthenium (II) complex with a bromobutyl group has both hydrophilicity and lipophilicity, can be combined with the base pairs in the nucleic acid aptamer, and is conducive to the formation of a network structure. When 1-4'-butyl-2-phenylvinyl imidazole [4,5f] [1,10] phenanthroline is coupled with Erianin as the main ligand, an Erianin molecule is coupled to the ruthenium (II) complex molecule, the fluorescence intensity of the ruthenium (II) complex can be enhanced, and the cytotoxicity of the ruthenium (II) complex can be reduced. However, after the Erianin molecule is grafted, the steric hindrance and volume steric hindrance effects will be increased, which is not conducive to the insertion of the ruthenium (II) complex between the base pairs of the nucleic acid aptamer, and to a certain extent, the formation of the network structure will be hindered. When 2,2-bipyridine is used as an auxiliary ligand in the present invention, 2,2-bipyridine has good planarity and is easy to effectively generate π-π stacking effect with base pairs in nucleic acid aptamers, which is more conducive to promoting the formation of a network structure; while the auxiliary ligand 1,10-phenanthroline has high stereochemistry and will weaken the affinity and binding strength of the ruthenium (II) complex with the nucleic acid aptamer, but the ruthenium (II) complex (RPLR31-BBr) formed by the combination of the auxiliary ligand 1,10-phenanthroline and the main ligand 1-4'-bromobutyl-2-phenylvinylimidazole [4,5f] [1,10] phenanthroline can still combine with the nucleic acid aptamer and can be used to prepare a fluorescent probe.

[0058] The present invention also provides a method for preparing the nucleic acid aptamer-loaded ruthenium (II) complex nanonet described in the above technical solution, comprising the following preparation steps:

[0059] The ruthenium (II) complex and the nucleic acid aptamer are mixed and incubated to obtain the nucleic acid aptamer-loaded ruthenium (II) complex nanonet.

[0060] In the present invention, the incubation temperature is preferably 30-50° C., more preferably 37° C., and the incubation time is preferably 12-48 h, more preferably 24 h.

[0061] The present invention also provides a method for preparing the ruthenium (II) complex described in the above technical solution, preferably comprising the following preparation steps:

[0062] Firstly, 1,10-phenanthroline-5,6-dione, 3-phenylacrolein, ammonium acetate and glacial acetic acid are mixed and subjected to a condensation reaction to obtain ligand A;

[0063] The ligand A, the ruthenium (II) compound and the first solvent are mixed to perform a coordination reaction to obtain a ligand B;

[0064] The ligand B, 1,4-dibromobutane, potassium carbonate and the second solvent are thirdly mixed and subjected to a first substitution reaction to obtain a ligand C.

[0065] In the present invention, 1,10-phenanthroline-5,6-dione, 3-phenylacrolein, ammonium acetate and glacial acetic acid are first mixed and subjected to a condensation reaction to obtain ligand A. The reaction formula of the condensation reaction is shown in Formula 1:

[0066]

[0067] In the present invention, the molar ratio of 1,10-phenanthroline-5,6-dione, 3-phenylacrolein and ammonium acetate is preferably 1:1.5:43-44. In the present invention, the temperature of the condensation reaction is preferably 100-120°C, and the time is preferably 4-6h. In the present invention, the condensation reaction is preferably carried out under heating reflux.

[0068] After obtaining ligand A, the present invention mixes the ligand A, the ruthenium (II) compound and the first solvent for a second coordination reaction to obtain ligand B.

[0069] In the present invention, the first solvent is preferably a mixed solvent of ethylene glycol and water, and the volume ratio of ethylene glycol to water is preferably 9:1. In the present invention, the molar ratio of the ligand A to the ruthenium (II) compound is preferably 1:1.4-1.6. In the present invention, the temperature of the coordination reaction is preferably 110-120°C, more preferably 120°C, and the time is preferably 6-24h, more preferably 6h. In the present invention, the coordination reaction is preferably carried out under reflux conditions. In the present invention, the ruthenium (II) compound is preferably a dichlorobipyridylruthenium (II) compound or a dichlorobis(1,10-phenanthroline)ruthenium (II) compound. In the present invention, the ruthenium (II) compound is preferably a ruthenium (II) compound with crystal water, and the present invention does not limit the amount of crystal water in the ruthenium (II) compound. In a specific embodiment of the present invention, the ruthenium (II) compound may be a dihydrated dichlorobipyridylruthenium (II) compound or a dihydrated dichlorobis(1,10-phenanthroline)ruthenium (II) compound.

[0070] In a specific embodiment of the present invention, when a dihydrated dichlorobipyridylruthenium (II) compound is used, ligand 1B (RBLR31) is obtained; when a dihydrated dichlorobis(1,10-phenanthroline)ruthenium (II) compound is used, ligand 2B (RPLR31) is obtained.

[0071] After obtaining ligand B, the present invention thirdly mixes the ligand B, 1,4-dibromobutane, potassium carbonate and the second solvent to perform a first substitution reaction to obtain ligand C.

[0072] In a specific embodiment of the present invention, when ligand 1B (RBLR31) is used, ligand 1C (RBLR31-BBr) is obtained, that is, a ruthenium (II) complex when the main ligand is 1-4'-bromobutyl-2-phenylvinylimidazole [4,5f][1,10]phenanthroline and the auxiliary ligand is 2,2-bipyridine; when ligand 2B (RPLR31) is used, ligand 2C (RPLR31-BBr) is obtained, that is, a ruthenium (II) complex when the main ligand is 1-4'-bromobutyl-2-phenylvinylimidazole [4,5f][1,10]phenanthroline and the auxiliary ligand is 1,10-phenanthroline.

[0073] In the present invention, the second solvent is preferably N,N-dimethylformamide. In the present invention, the amount ratio of the ligand B, 1,4-dibromobutane and potassium carbonate is preferably 190mg:1mL:2g. In the present invention, the temperature of the third mixing is preferably 50-80°C, more preferably 60°C, and the time is preferably 20-60min, more preferably 30min. In the present invention, the temperature of the first substitution reaction is preferably 40-80°C, more preferably 60°C, and the time is preferably 12-40h, more preferably 24h.

[0074] In the present invention, the method for preparing the Erianin-modified ruthenium (II) complex D preferably further comprises: fourthly mixing the ligand C prepared by the above technical solution, Erianin, sodium methoxide and a third solvent, and performing a second substitution reaction to obtain the ligand D.

[0075] In a specific embodiment of the present invention, when ligand 1C (RBLR31-BBr) is used, ligand 1D (RBLR31-MLS) is obtained, which is a ruthenium (II) complex 1D modified with bipyridine by Erianin, that is, a ruthenium (II) complex when the main ligand is 1-4'-butyl-2-phenylvinyl imidazole [4,5f][1,10]phenanthroline coupled with Erianin and the auxiliary ligand is 2,2-bipyridine.

[0076] In the present invention, the third solvent is preferably N,N-dimethylformamide. In the present invention, preferably, the molar ratio of the ligand C, Erianin and sodium methoxide is preferably 0.1:0.1:1. In the present invention, the temperature of the second substitution reaction is preferably 90°C, and the time is preferably 30 minutes.

[0077] In a specific embodiment of the present invention, the ruthenium (II) complex can be ligand 1B (RBLR31), ligand 2B (RPLR31), ligand 1C (RBLR31-BBr), ligand 2C (RPLR31-BBr) or ligand 1D (RBLR31-MLS), and can further be ligand 1B (RBLR31) or ligand 1C (RBLR31-BBr).

[0078] The present invention also provides the use of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet described in the above technical solution or the nucleic acid aptamer-loaded ruthenium (II) complex nanonet prepared by the preparation method in the preparation of tumor-targeted fluorescent probes.

[0079] The present invention uses the aptamer-loaded ruthenium (II) complex nanonet directly as a fluorescent probe, and utilizes the property of the aptamer that can target and bind to nucleolin overexpressed on the surface of tumor cell membrane to achieve tumor targeted fluorescent diagnosis. The aptamer-loaded ruthenium (II) complex nanonet provided by the present invention can also be used to load drugs, thereby achieving targeted treatment of tumor cells.

[0080] The technical solution of the present invention is further described below through specific implementation methods. Those skilled in the art should understand that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0081] Example 1

[0082] Preparation of dichlorobipyridylruthenium (II) compound: 2mmol 2,2-bipyridine, 9.6mmol lithium chloride, 1mmol ruthenium trichloride, 10mL water and 150mL N,N'-dimethylformamide were mixed, and reacted in an argon atmosphere at 140°C for 8h to obtain dichlorobipyridylruthenium (II) compound.

[0083] Preparation of ligand A: Weigh 1 mmol of 1,10-phenanthroline-5,6-dione, 1.5 mmol of 3-phenylpropenal, and 44 mmol of ammonium acetate, add them into glacial acetic acid and mix them. Carry out condensation reaction at 120° C. for 6 h to obtain ligand A.

[0084] Preparation of ligand 1B: Weigh ligand A (0.3 mmol), dihydrate dichlorobipyridylruthenium (II) compound (0.2 mmol), and a mixed solvent of ethylene glycol and water (ethylene glycol and water volume ratio is 9:1), add them into a three-necked flask, and heat and reflux at 120°C for 6 hours to obtain ligand 1B, referred to as RBLR31, with a yield of 89.5%. The reaction equation is:

[0085]

[0086] The structural formula of ligand 1B (RBLR31) is

[0087] The characterization data of ligand 1B (RBLR31) are as follows:

[0088] The analysis results of the prepared ligand 1B (RBLR31) by electrospray mass spectrometry were: In CH3CN, m / z: 735.16 ([M-2ClO4 - -H] + ,cal:735.16);368.08([M-2ClO4] 2+ / 2,cal:368.08).

[0089] The analysis results of the hydrogen nuclear magnetic resonance spectrum of the prepared ligand 1B (RBLR31) are: 500 MHz, DMSO: δ8.98 (dd, J=8.3, 1.3 Hz, 2H), 8.86 (ddt, J=19.3, 8.3, 1.0 Hz, 4H), 8.22 (td, J=7.9, 1.5 Hz, 2H), 8.11 (td, J=7.9, 1.5 Hz, 2H), 7.98 (dd, J=5.3, 1.3 Hz, 2H), 7.89-7.71 (m, 8H), 7.61-7.58 (m, 3H), 7.46--7.34 (m, 4H), 7.13--7.05 (m, 2H).

[0090] The analysis results of the carbon NMR spectrum of the prepared ligand 1B (RBLR31) are: 126 MHz, DMSO: δ156.87, 156.65, 151.45, 148.96, 144.63, 137.95, 137.79, 137.25, 136.09, 130.26, 129.45, 129.08, 128.85, 127.94, 127.82, 127.05, 126.02, 125.78, 124.50, 124.43, 19.43.

[0091] The ligand 1B (RBLR31) prepared in Example 1 was used to prepare the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 1E: 72 μM of the complex 1B (RBLR31) was mixed with 100 μM of the nucleic acid aptamer AS1411, and incubated at 37°C for 24 h to obtain the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 1E, referred to as RBLR31-AS1411.

[0092] Figure 1 This is an AFM image of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 1E (RBLR31-AS1411) prepared in Example 1. Figure 1 It can be seen that the aptamer-loaded ruthenium (II) complex nanonet 1E (RBLR31-AS1411) presents a complete mesh structure, and the thickness of the formed nanostructure is about 0.9nm, indicating that the ligand 1B (RBLR31) and AS1411 have a good binding effect. Bipyridine has good planarity and is easy to bind to the base pairs in the aptamer, which is conducive to the formation of the mesh structure. The meshes in the mesh structure present a variety of irregular shapes, the size of the meshes ranges from about 0.5 to 30nm, and the diameter of the fibers forming the mesh structure is about 0.10 to 35.4nm.

[0093] Figure 2 This is the fluorescence titration spectrum of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 1E (RBLR31-AS1411) prepared in Example 1. Figure 2 It can be seen that the fluorescence intensity of ligand 1B (RBLR31) increased after binding to AS1411, indicating that ligand 1B (RBLR31) can bind to AS1411 and function as a fluorescent probe.

[0094] Figure 3 This is the fluorescence resonance energy transfer (FRET) melting point result of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 1E (RBLR31-AS1411) prepared in Example 1. Figure 3It can be seen that the combination of ligand 1B (RBLR31) and AS1411 can significantly increase the melting point of AS1411, indicating that the combination of ligand 1B (RBLR31) and AS1411 can significantly improve its stability.

[0095] Figure 4 CD spectrum of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 1E (RBLR31-AS1411) prepared in Example 1. Figure 4 It can be seen that ligand 1B (RBLR31) and AS1411 can bind and self-assemble.

[0096] Example 2

[0097] Preparation of ligand 1C: 190 mg of ligand 1B prepared in Example 1, 15 mL of N,N-dimethylformamide (DMF) and 2 g of K2CO3 were mixed, heated at 60°C for 30 min, 1 mL of 1,4-dibromobutane was added and the reaction continued for 24 h to obtain ligand 1C, referred to as RBLR31-BBr, which is a ruthenium (II) complex with a main ligand of 1-4'-bromobutyl-2-phenylvinyl imidazole [4,5f] [1,10] phenanthroline and an auxiliary ligand of 2,2-bipyridine, with a yield of 69%. The reaction equation is:

[0098]

[0099] The structural formula of ligand 1C (RBLR31-BBr) is

[0100] The characterization data of ligand 1C (RBLR31-BBr) are as follows:

[0101] The analysis results of the prepared ligand 1C (RBLR31-BBr) by electrospray mass spectrometry were: In CH3CN, m / z: 436.06 ([M-ClO4 - ] 2+ ,cal:435.40);494.09([M-ClO4 - +NH4 + ] 2+ ,cal:494.10);552.11([M+2NH4 + ] 2+ ,cal:552.80).

[0102] The analysis results of the H NMR spectrum of the prepared ligand 1C (RBLR31-BBr) are as follows: 500 MHz, DMSO: δ9.09 (ddd, J = 22.7, 11.2, 5.2 Hz, 2H), 8.93-8.82 (m, 4H), 8.26--8.18 (m, 2H), 8.16--8.01 (m, 5H), 7.96-7.89 (m, 4H), 7.85 (t, J = 5.0 Hz, 2H), 7.80-7.71 (m, 1H), 7.61(dd,J=11.6,5.4Hz,4H),7.50(t,J=7.5Hz,2H),7.43(t,J=7.3Hz,1H),7.39-7.31(m,2H),4.10(dt,J=6 1.2, 6.3Hz, 2H), 3.62 (d, J = 6.2Hz, 1H), 3.59-3.44 (m, 1H), 1.92 (dd, J = 28.3, 22.6Hz, 2H), 1.83-1.54 (m, 2H).

[0103] The analysis results of the carbon NMR spectrum of the prepared ligand 1C (RBLR31-BBr) are: 126MHz, DMSO: δ157.12 (s), 155.01 (s), 153.16 (s), 151.89 (s), 150.89 (s), 149.74 (s), 145.78 (s), 145.51 (s), 138.40 (s), 137.70 (s), 136.16 (s), 130.95 (s) ,130.34(s),129.96(s),129.85(s),129.38(s),128.68(s),128.37(s),127.10(s),126.66(s),12 6.23(s),125.78(s),124.96(s),121.92(s),113.87(s),67.26(s),35.02(s),29.42(s),29.09(s).

[0104] The prepared ligand 1C (RBLR31-BBr) was used to prepare the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 2E, referred to as RBLR31-BBr-AS1411.

[0105] Figure 5 AFM image of the aptamer-loaded ruthenium (II) complex nanonet 2E (RBLR31-BBr-AS1411) prepared in Example 2. Figure 5It can be seen that the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 2E (RBLR31-BBr-AS1411) presents a mesh structure, the thickness of the formed nanostructure is about 2nm, the meshes in the mesh structure present a variety of irregular shapes, the size range of the meshes is about 0.5-30nm, and the fiber diameter of the mesh structure is about 0.10-35.4nm. In addition, the molecular structure of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 2E (RBLR31-BBr-AS1411) has both hydrophilicity and lipophilicity due to the bromobutyl group, which is more conducive to inserting into the base pair of the nucleic acid aptamer and more conducive to the formation of the mesh structure. Therefore, the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 2E (RBLR31-BBr-AS1411) of Example 2 has a clearer and more complete mesh structure than the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 1E (RBLR31-AS1411) of Example 1.

[0106] Figure 6 This is the fluorescence titration spectrum of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 2E (RBLR31-BBr-AS1411) prepared in Example 2. Figure 6 It can be seen that the fluorescence intensity of ligand 1C (RBLR31-BBr) increased after binding to AS1411, indicating that ligand 1C (RBLR31-BBr) can bind to AS1411 and function as a fluorescent probe.

[0107] Figure 7 This is the fluorescence resonance energy transfer (FRET) melting point result of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 2E (RBLR31-BBr-AS1411) prepared in Example 2. Figure 7 It can be seen that the melting point of AS1411 can be increased after the ligand 1C (RBLR31-BBr) combines with AS1411, indicating that the combination of ligand 1C (RBLR31-BBr) with AS1411 can improve its stability.

[0108] Figure 8 CD spectrum of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 2E (RBLR31-BBr-AS1411) prepared in Example 2. Figure 8 It can be seen that: it indicates that ligand 1C (RBLR31-BBr) can bind to AS1411 and that self-assembly may occur.

[0109] Example 3

[0110] Preparation of ligand 1D: Add 31.8 mg (0.1 mmol) of Erianin into a microwave tube, add ligand 1C (RBLR31-BBr) (0.1 mmol) prepared in Example 2, 54 mg (1 mmol) of sodium methoxide, add 15 mL of DMF, heat at 90°C for 30 min, and obtain ligand 1D, i.e., Erianin-modified bipyridine ruthenium (II) complex 1D, referred to as RBLR31-MLS, i.e., the main ligand is 1-4'-butyl-2-phenylvinyl imidazole [4,5f] [1,10] phenanthroline coupled Erianin, and the auxiliary ligand is 2,2-bipyridine, the yield is 40%. Its chemical reaction formula is:

[0111]

[0112] The structural formula of ligand 1D (RBLR31-MLS) is

[0113] The characterization data of Erianin modified ruthenium (II) bipyridine complex 1D (RBLR31-MLS) are as follows:

[0114] The analysis results of the prepared Erianin modified bipyridine ruthenium (II) complex 1D (RBLR31-MLS) by electrospray mass spectrometry were: In CH3CN, m / z: 554.17 ([M-ClO4 - ] 2+ ,cal:554.13);612.20([M-ClO4 - +NH4 + ] 2+ ,cal:612.83);369.78([M-2ClO4 - +H + ] 3+ ,cal:369.78).

[0115] The analysis results of the H NMR spectrum of the prepared Erianin modified bipyridine ruthenium (II) complex 1D (RBLR31-MLS) are as follows: 500 MHz, DMSO: δ9.16-9.10 (m, 1H), 9.09-8.99 (m, 1H), 8.89 (d, J = 8.2 Hz, 2H), 8.86 (d, J = 6.6 Hz, 2H), 8.23 ​​(t, J = 7.9 Hz, 3H), 8.17-8.09 (m, 3H), 8.09-8.05 (m, 2H), 7.95-7.88 (m, 3H), 7.85 (t, J = 6.3 Hz, 4H ),7.61(dd,J=10.5,4.9Hz,6H),7.45-7.39(m,2H),6.84-6.71(m,3H),6.50(d,J=13.9Hz,2H),5.11(d,J=7.2Hz,2H),4.02(s,2H) ,3.76-3.66(m,7H),3.58(d,J=18.5Hz,3H),3.51(d,J=11.0Hz,2H),2.75(d,J=15.0Hz,4H),2.13-2.07(m,2H),2.00-1.93(m,2H).

[0116] The analysis results of the prepared Erianin modified bipyridine ruthenium (II) complex 1D (RBLR31-MLS) are as follows: 126MHz, DMSO: δ155.01(s), 151.01(s), 149.75(s), 148.76(s), 147.59(s), 146.03(s), 145.49(s), 143.64(s), 143.38(s), 136.27(s), 135.60(s), 133.95(s), 132.50(s), 132.16(s), 128.79(s), 128.09(s), 127.82(s), 127.66(s),127.23(s),126.52(s),126.16(s),124.88(s),124.42(s) ),124.09(s),123.67(s),122.83(s),119.80(s),118.88(s),116.80 (s),112.22(s),111.76(s),110.45(s),103.99(s),66.33(s),58.26(s),54.08(s),53.78(s),36.02(s),34.96(s),25.43(s),23.78(s).

[0117] Preparation of nucleic acid aptamer-loaded ruthenium (II) complex nanonet 3E: The Erianin-modified bipyridine ruthenium (II) complex 1D (RBLR31-MLS) prepared in Example 3 was used to prepare nucleic acid aptamer-loaded ruthenium (II) complex nanonet 3E, referred to as RBLR31-MLS-AS1411.

[0118] Fig. 9 This is an AFM image of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 3E (RBLR31-MLS-AS1411) prepared in Example 3. Fig. 9 It can be seen that the network structure presented by the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 3E (RBLR31-MLS-AS1411) has some breakpoints. This is because the Erianin molecules are grafted into the ruthenium (II) complex structure. Due to the increase in volume steric hindrance and spatial steric hindrance, the formation of the network structure is hindered. However, it can still be seen from the AFM image that the Erianin-modified bipyridine ruthenium (II) complex is connected to the nucleic acid aptamer and can be used to prepare fluorescent probes; however, the overall molecular structure of the formed nucleic acid aptamer-loaded ruthenium (II) complex nanonet 3E (RBLR31-MLS-AS1411) is larger than the nucleic acid aptamer-loaded ruthenium (II) complex nanonet obtained in Example 1 and Example 2, so the thickness of the nanostructure is about 20nm.

[0119] Fig.10 This is the fluorescence titration spectrum of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 3E (RBLR31-MLS-AS1411) prepared in Example 3. Fig.10 It can be seen that the fluorescence intensity of Erianin-modified ruthenium (II) bipyridine complex 1D (RBLR31-MLS) is significantly improved after binding with AS1411, indicating that Erianin-modified ruthenium (II) bipyridine complex 1D can bind to AS1411 with high binding strength and can function as a fluorescent probe.

[0120] Fig.11 This is the fluorescence resonance energy transfer (FRET) melting point result of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 3E (RBLR31-MLS-AS1411) prepared in Example 3. Fig.11 It can be seen that the Erianin-modified ruthenium (II) bipyridine complex 1D (RBLR31-MLS) can increase the melting point of AS1411 after binding with the nucleic acid aptamer AS1411, indicating that the Erianin-modified ruthenium (II) bipyridine complex 1D can improve its stability after binding with AS1411.

[0121] Fig.12CD spectrum of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 3E (RBLR31-MLS-AS1411) prepared in Example 3. Fig.12 It can be seen that the Erianin-modified bipyridine ruthenium (II) complex 1D can bind to AS1411 and that self-assembly may occur.

[0122] Example 4

[0123] The difference from Example 1 is that 1,10-phenanthroline is used instead of 2,2-bipyridine to prepare dihydrate dichlorobis(1,10-phenanthroline)ruthenium(II) compound.

[0124] Preparation of ligand 2B: Use dihydrated dichlorobis(1,10-phenanthroline)ruthenium(II) compound instead of dichlorobipyridylruthenium(II) compound. Obtain ligand 2B, referred to as RPLR31, with a yield of 86.4%. The reaction equation is:

[0125]

[0126] The structural formula of ligand 2B (RPLR31) is

[0127] The characterization data of ligand 2B (RPLR31) are as follows:

[0128] The detection result of the prepared ligand 2B (RPLR31) by electrospray mass spectrometry was: In CH3CN, m / z: 783.16 ([M-2ClO4 - -H] + ,cal:783.16);392.08([M-2ClO4] 2+ / 2,cal:392.08).

[0129] The analysis results of the hydrogen nuclear magnetic resonance spectrum of the prepared ligand 2B (RPLR31) are: 500 MHz, DMSO: δ8.91-8.88 (m, 3H), 8.77 (ddd, J = 8.3, 2.8, 1.2 Hz, 6H), 8.39 (s, 6H), 8.12-8.11 (m, 4H), 7.80-7.76 (m, 8H), 7.42 (d, J = 7.9 Hz, 3H), 7.13-7.04 (m, 2H).

[0130] The analysis results of the carbon NMR spectrum of the prepared ligand 2B (RPLR31) are: 126MHz, DMSO: δ152.68, 152.56, 147.77, 147.42, 147.32, 144.22, 137.25, 137.11, 136.68, 136.64, 136.09, 130.50, 130.46, 129.9 3,129.45,128.91,128.84,128.17,128.13,128.08,127.74,126.55,126.37,126.02,125.78,125.14,40.10,39.93,39.77,39.60,39.43,39.27,39.10,21.02,19.43.

[0131] The ligand 2B (RPLR31) prepared in Example 4 was used to prepare the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 4E, referred to as RPLR31-AS1411.

[0132] Fig.13 This is an AFM image of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 4E (RPLR31-AS1411) prepared in Example 4. Fig.13 It can be seen that the network structure formed by the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 4E (RPLR31-AS1411) is relatively complete, indicating that the binding ability of ligand 2B (RPLR31) and AS1411 is strong, but its auxiliary ligand is 1,10-phenanthroline with stronger stereoactivity, so the surface of the formed network structure is closer to the atomic force microscope probe, and the color of the picture is darker than that of the network structure formed by the auxiliary ligand 2,2-bipyridine in Example 1, and the thickness of the formed nanostructure is about 0.5 nm.

[0133] Fig.14 This is the fluorescence titration spectrum of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 4E (RPLR31-AS1411) prepared in Example 4. Fig.14 It can be seen that the fluorescence intensity of ligand 2B (RPLR31) increased after binding to AS1411, indicating that ligand 2B (RPLR31) can bind to AS1411 and function as a fluorescent probe.

[0134] Fig.15 This is the fluorescence resonance energy transfer (FRET) melting point result of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 4E (RPLR31-AS1411) prepared in Example 4. Fig.15It can be seen that the combination of ligand 2B (RPLR31) and AS1411 can significantly increase the melting point of AS1411, indicating that the combination of ligand 2B (RPLR31) and AS1411 can significantly improve its stability.

[0135] Fig.16 CD spectrum of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 4E (RPLR31-AS1411) prepared in Example 4. Fig.16 It can be seen that the conformation of AS1411 can be changed after the ligand 2B (RPLR31) binds to AS1411, which once again shows that the ligand 2B (RPLR31) can bind to AS1411 and that self-assembly may occur.

[0136] Example 5

[0137] Preparation of ligand 2C: The difference from Example 2 is that ligand 2B (RPLR31) prepared in Example 4 is used to replace ligand 1B (RBLR31), and ligand 2C is obtained, referred to as RPLR31-BBr, that is, a ruthenium (II) complex when the main ligand is 1-4'-bromobutyl-2-phenylvinyl imidazole [4,5f] [1,10] phenanthroline and the auxiliary ligand is 1,10-phenanthroline, and the yield is 55%. The reaction equation is:

[0138]

[0139] The structural formula of ligand 2C (RPLR31-BBr) is

[0140] The characterization data of ligand 2C (RPLR31-BBr) are as follows:

[0141] The analysis results of the prepared ligand 2C (RPLR31-BBr) by electrospray mass spectrometry were: In CH3CN, m / z: 460.06 ([M-2ClO4 - ] 2+ ,cal:459.42);518.09([M-ClO4 - +NH4 + ] 2+ ,cal:518.12).

[0142] The analysis results of the H NMR spectrum of the prepared ligand 2C (RPLR31-BBr) are as follows: 500 MHz, DMSO: δ9.06 (ddd, J = 21.6, 10.6, 5.0 Hz, 2H), 8.79 (ddd, J = 6.8, 5.2, 1.1 Hz, 4H), 8.41 (s, 4H), 8.17-8.01 (m, 8H), 7.92 (d, J = 7 .4Hz,2H),7.84-7.72(m,7H),7.50(t,J=7.5Hz,2H),4.08(ddd,J=12.7,11.1,6.3Hz,2 H), 3.62 (d, J = 6.1Hz, 1H), 3.45 (t, J = 6.6Hz, 1H), 2.02-1.85 (m, 2H), 1.79-1.58 (m, 2H).

[0143] The analysis results of the prepared ligand 2C (RPLR31-BBr) carbon nuclear magnetic resonance spectrum are: 126MHz, DMSO: δ154.95 (s), 153.20 (s), 151.39 (s), 150.22 (s), 147.62 (s), 146.20 (s), 145.93 (s), 137.58 (s), 137.33 (s), 136.16 (s), 130.94 (s) ,130.22(s),130.05(s),129.84(s),129.39(s),128.63(s),128.28(s),126.80(s),126.52( s),126.21(s),125.72(s),121.86(s),113.89(s),67.07(s),35.00(s),29.40(s),29.09(s).

[0144] The ligand 2C (RPLR31-BBr) prepared in Example 5 was used to prepare the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 5E, referred to as RPLR31-BBr-AS1411.

[0145] Fig.17 This is an AFM image of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 5E (RPLR31-BBr-AS1411) prepared in Example 5. Fig.17 It can be seen that the nucleic acid aptamer-loaded ruthenium (II) complex nanonet 5E (RPLR31-BBr-AS1411) presents a network structure, but there are some broken fragments in the network structure, indicating that the binding strength of the ruthenium (II) complex with the auxiliary ligand 1,10-phenanthroline to the nucleic acid aptamer is weaker than that in Example 2, but it can still bind and can be used to prepare fluorescent probes; the thickness of the formed nanostructure is about 2.8nm.

[0146] Fig.18 This is the fluorescence titration spectrum of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 5E (RPLR31-BBr-AS1411) prepared in Example 5. Fig.18 It can be seen that the fluorescence intensity of ligand 2C (RPLR31-BBr) increased after binding to AS1411, indicating that ligand 2C (RPLR31-BBr) can bind to AS1411 and function as a fluorescent probe.

[0147] Fig.19 This is the fluorescence resonance energy transfer (FRET) melting point result of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 5E (RPLR31-BBr-AS1411) prepared in Example 5. Fig.19 It can be seen that the melting point of AS1411 can be increased after the ligand 2C (RPLR31-BBr) combines with AS1411, indicating that the combination of ligand 2C (RPLR31-BBr) with AS1411 can improve its stability.

[0148] Fig. 20 CD spectrum of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 5E (RPLR31-BBr-AS1411) prepared in Example 5. Fig. 20 It can be seen that the ligand 2C (RPLR31-BBr) can change the conformation of AS1411 after binding with AS1411, which once again shows that the ligand 2C (RPLR31-BBr) can bind with AS1411 and that self-assembly may occur.

[0149] Comparative Example 1

[0150] Preparation of ligand 2D: The difference from Example 3 is that ligand 2C (RPLR31-BBr) prepared in Example 5 is used to obtain ligand 2D, i.e., Erianin-modified phenanthroline ruthenium (II) complex 2D, referred to as RPLR31-MLS, i.e., the main ligand is 1-4'-butyl-2-phenylvinyl imidazole [4,5f] [1,10] phenanthroline coupled Erianin, and the auxiliary ligand is 1,10-phenanthroline when the ruthenium (II) complex has a yield of 55%. Its chemical reaction formula is:

[0151]

[0152] The structural formula of ligand 2D (RPLR31-MLS) is

[0153] The characterization data of Erianin modified phenanthroline ruthenium (II) complex 2D (RPLR31-MLS) are as follows:

[0154] The electrospray mass spectrometry analysis results of the prepared Erianin modified phenanthroline ruthenium (II) complex 2D (RPLR31-MLS) were: In CH3CN, m / z: 578.17 ([M-2ClO4 - ] 2+ ,cal:578.18);636.20([M-ClO4 - +NH4 + ] 2+ ,cal:636.88).

[0155] The analysis results of the H NMR spectrum of the prepared Erianin modified phenanthroline ruthenium (II) complex 2D (RPLR31-MLS) are as follows: 500 MHz, DMSO: δ9.15-8.96 (m, 2H), 8.83-8.75 (m, 4H), 8.40 (d, J = 3.1 Hz, 4H), 8.08 (dddd, J = 20.4, 19.1, 8.7, 5.8 Hz, 8H), 7.89-7.69 (m, 9H), 7.46-7.3 6(m,2H),6.85-6.69(m,3H),6.50(d,J=17.1Hz,2H),5.18-4.98(m,2H),4.10-3.88(m,2H),3.76-3.64( m,7H),3.58(dd,J=19.6,2.5Hz,3H),3.49(d,J=10.0Hz,2H),2.75(t,J=11.3Hz,4H),2.16-1.85(m,4H).

[0156] The analysis results of the prepared Erianin modified phenanthroline ruthenium (II) complex 2D (RPLR31-MLS) are as follows: 126MHz, DMSO: δ153.17(s), 151.31(s), 150.09(s), 148.15(s), 147.60(s), 146.19(s), 145.94(s), 137.61(s), 137.33(s), 136.08(s), 135.74(s), 134.62(s), 130.94(s), 130.04(s), 129.7 5(s),129.35(s),128.61(s),128.19(s),126.84(s),126.20(s),125.75(s),121.88(s),120.91(s),118.90(s),114.33(s) ),113.93(s),112.55(s),106.11(s),68.48(s),60.39(s),56.20(s),55.88(s),38.14(s),37.09(s),27.60(s),25.88(s).

[0157] Preparation of nucleic acid aptamer-loaded ruthenium (II) complex nanonet 6E: Nucleic acid aptamer-loaded ruthenium (II) complex nanonet 6E was prepared using Erianin-modified phenanthroline ruthenium (II) complex 2D (RPLR31-MLS) and nucleic acid aptamer AS1411, abbreviated as RPLR31-MLS-AS1411.

[0158] Fig.21 This is an AFM image of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 6E (RPLR31-MLS-AS1411) prepared in Comparative Example 1. Fig.21 It can be seen that the obtained nucleic acid aptamer and ruthenium (II) complex cannot form a network structure. There are many molecular fragments in the figure, and the thickness of the nanostructure is about 10 to 30 nm. Obviously, the ruthenium (II) complex after the auxiliary ligand 1,10-phenanthroline and the main ligand 1-4'-butyl-2-phenylvinyl imidazole [4,5f][1,10]phenanthroline are coupled with Erianin cannot be assembled with the nucleic acid aptamer. The reason is that 1,10-phenanthroline has a large stereochemistry. After the Erianin molecule is grafted, the steric hindrance and volume steric hindrance effects are accumulated, resulting in a decrease in the binding affinity and selectivity of the ruthenium (II) complex with the nucleic acid aptamer, and it cannot be inserted between the base pairs, and basically cannot form an effective connection structure, so it cannot be used to prepare fluorescent probes.

[0159] Fig. 22 This is the fluorescence titration spectrum of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 6E (RPLR31-MLS-AS1411) prepared in Comparative Example 1. Fig. 22 It can be seen that the fluorescence intensity of Erianin-modified phenanthroline ruthenium (II) complex 2D is high, but it is basically unable to bind to the nucleic acid aptamer AS1411, indicating that Erianin-modified phenanthroline ruthenium (II) complex 2D cannot bind to AS1411, so it cannot be used to prepare a fluorescent probe.

[0160] Fig.23 This is the fluorescence resonance energy transfer (FRET) melting point result of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 6E (RPLR31-MLS-AS1411) prepared in Comparative Example 1. Fig.23 It can be seen that the melting point of Erianin-modified phenanthroline ruthenium (II) complex 2D and the nucleic acid aptamer AS1411 is basically unchanged after being mixed, indicating that Erianin-modified phenanthroline ruthenium (II) complex 2D and AS1411 cannot effectively bind.

[0161] Fig.24 CD spectrum of the nucleic acid aptamer loaded ruthenium (II) complex nanonet 6E (RPLR31-MLS-AS1411) prepared in Comparative Example 1. Fig.24 It can be seen that the Erianin-modified phenanthroline ruthenium (II) complex 2D cannot change the conformation of the nucleic acid aptamer AS1411, which once again shows that the Erianin-modified phenanthroline ruthenium (II) complex 2D cannot bind to AS1411.

[0162] Description of the detection and analysis method:

[0163] The fluorescence titration results of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet obtained in the embodiment and the comparative example were detected by a fluorescence spectrophotometer. The specific operation included: in the fluorescence spectrophotometer, 3 mL of 10 μM ruthenium (II) complex prepared in Example 1 was added to the sample pool, which had a strong fluorescence emission peak at about 595 nm under 420 nm excitation. Subsequently, 2 μL of AS1411 was added every 2 min for scanning, and the peak height was positively correlated with the binding concentration of the ruthenium (II) complex and AS1411. The change of the fluorescence emission peak of the ruthenium (II) complex with the addition of AS1411 was monitored, and the titration was stopped when saturation occurred.

[0164] The fluorescence resonance energy transfer (FRET) melting point experiment of the nucleic acid aptamer-loaded ruthenium (II) complex nanonet prepared in the embodiment and the comparative example was detected by using a Bio-Rad real-time PCR detection system. 2 μL of AS1411 (5′-GGTGGTGGTGGTTGTGGTGGTGGTGG-3′, SEQ ID NO.1, wherein FAM is added to the 5′ end of the sequence shown in SEQ ID NO.1, and TAMRA-3′ is added to the 3′ end, so that the melting point change of AS1411 can be seen on a fluorescence quantitative instrument) with a fluorescent label was placed in a buffer solution (100 mM KCl, 10 mM Tris-HCl, 10 mM Na3AsO4 (pH 7.2)), heated at 90°C for 5 min, cooled and stored at 4°C overnight for use. Under the condition of a total reaction system of 25 μL, AS1411 (0.20 μM) and different concentrations of ruthenium (II) complexes (specific concentrations are 1, 2, 3, and 6 μM) were added to a buffer solution, and the fluorescence change curve of FAM at 30-100°C was detected.

[0165] The CD spectra of the nucleic acid aptamer-loaded ruthenium (II) complex nanonets prepared in the examples and comparative examples were detected by a Jasco I810 circular dichroism spectrometer. A quartz cuvette with a volume of 3 mL was used. At room temperature, the prepared ruthenium (II) complex (0-72 μM) was gradually added to a Tris-HCl buffer solution containing 2 μM AS1411 G4 DNA. After mixing, the mixture was allowed to stand for 2 min. The CD spectra in the wavelength range of 200-600 nm were scanned and collected, and the buffer solution baseline was deducted from the sample spectrum.

[0166] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A nucleic acid aptamer-loaded ruthenium (II) complex nanonet, comprising a nucleic acid aptamer and a ruthenium (II) complex chemically bonded to the nucleic acid aptamer, wherein the nucleic acid aptamer and the ruthenium (II) complex are assembled to form a nano-net structure; The ruthenium (II) complex comprises a central metal ion ruthenium (II), a primary ligand and an auxiliary ligand; The main ligand includes 1-4'-bromobutyl-2-phenylvinyl imidazole [4,5f] [1,10] phenanthroline, and the auxiliary ligand includes 2,2-bipyridine or 1,10-phenanthroline.

2. The nucleic acid aptamer-loaded ruthenium (II) complex nanonet according to claim 1, characterized in that: The nucleic acid aptamer is AS1411.

3. The nucleic acid aptamer-loaded ruthenium (II) complex nanonet according to claim 1, characterized in that: The chemical bonding is coordination bonding and / or covalent bonding.

4. The nucleic acid aptamer-loaded ruthenium (II) complex nanonet according to claim 1, characterized in that: When the auxiliary ligand is 2,2-bipyridine, the 1-4'-bromobutyl-2-phenylvinyl imidazole [4,5f] [1,10] phenanthroline is replaced by 1-4'-butyl-2-phenylvinyl imidazole [4,5f] [1,10] phenanthroline coupled with Erianin, or a mixture of 1-4'-bromobutyl-2-phenylvinyl imidazole [4,5f] [1,10] phenanthroline and 1-4'-butyl-2-phenylvinyl imidazole [4,5f] [1,10] phenanthroline coupled with Erianin.

5. The nucleic acid aptamer-loaded ruthenium (II) complex nanonet according to claim 1, characterized in that: The molar ratio of the ruthenium (II) complex to the nucleic acid aptamer is 0.5 to 1:

1.

6. The nucleic acid aptamer-loaded ruthenium (II) complex nanonet according to claim 1, characterized in that: The diameter of the fibers forming the network in the nano-network structure is 0.10 to 35.4 nm; The size of the mesh in the nano-net structure is 0.5-30 nm.

7. The nucleic acid aptamer-loaded ruthenium (II) complex nanonet according to claim 1, characterized in that: The height of the nano-network structure is preferably 0.5 to 30 nm.

8. The method for preparing the nucleic acid aptamer-loaded ruthenium (II) complex nanonet according to any one of claims 1 to 7, characterized in that: The method comprises the following preparation steps: The ruthenium (II) complex and the nucleic acid aptamer are mixed and incubated to obtain the nucleic acid aptamer-loaded ruthenium (II) complex nanonet.

9. The preparation method according to claim 8, characterized in that: The incubation temperature is 30 to 50° C. and the incubation time is 12 to 48 hours.

10. Use of the aptamer-loaded ruthenium (II) complex nanonet according to any one of claims 1 to 7 or the aptamer-loaded ruthenium (II) complex nanonet prepared by the preparation method according to any one of claims 8 to 9 in the preparation of tumor-targeted fluorescent probes.

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