A nucleic acid aptamer targeting M1 macrophages, its screening method and application

Abstract

This invention discloses a nucleic acid aptamer targeting M1 macrophages, its screening method, and its applications. Using Cell-SELEX technology, through multiple rounds of negative and positive screening, the aptamer APT-Che, exhibiting high affinity and specificity for macrophages, was successfully obtained. The spatial conformation of APT-Che was simulated and analyzed using Discovery Studio software, and its specificity was experimentally verified using inverted fluorescence microscopy and flow cytometry. The results confirmed that APT-Che specifically binds to macrophages, exhibits weak binding affinity to other types of normal cells, and possesses drug-carrying capacity while still targeting macrophages. The nucleic acid aptamer screened in this invention not only provides a novel cell-targeted delivery tool for research related to atherosclerosis and other inflammatory diseases but will also effectively accelerate the development of targeted drugs, improving their therapeutic efficacy and safety.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nucleic acid aptamer targeting M1 macrophages, its screening method, and its application. Background Technology

[0002] Aptamers, or single-stranded oligonucleotide sequences, are obtained through multiple rounds of in vitro screening and enrichment using the systematic evolution of chegands by exponential enrichment (SELEX) system. They are capable of specifically and with high affinity binding to targets. Compared to antibodies, aptamers offer advantages such as smaller molecular weight, shorter preparation time, lower cost, greater stability, easier modification, and lower immunogenicity. Therefore, they show broad application potential in cell imaging, disease detection and diagnosis, targeted drug delivery, and disease treatment. Approximately 30 years after their discovery, thousands of aptamers have been discovered and applied in various fields. In medicine, aptamers are primarily used for disease diagnosis, treatment, and drug delivery. However, currently, only Pegaptanib (trade name Macugen), approved by the US FDA for the treatment of age-related macular degeneration, is used for clinical treatment. In addition, several other aptamers have entered clinical trials. The DNA aptamer AGRO100, targeting prostate-specific membrane antigen (PSMA) and conjugated with doxorubicin, is used for prostate cancer treatment and has entered Phase I clinical trials. NOX-A12, targeting CXCL12 (CXC Chemokine Ligand 12), is adapted to myelofibrosis and malignant tumors, blocking the CXCL12 / CXCR4 signaling pathway and inhibiting tumor cell migration and angiogenesis; it has entered Phase II / III clinical trials. Furthermore, aptamers, as molecular probes, are used for highly sensitive detection of disease biomarkers, such as the detection of tumor biomarkers in blood (e.g., PSA, HER2), and some technologies have entered the clinical validation stage. All of these indicate that nucleic acid aptamers have excellent clinical application prospects.

[0003] Macrophages are a type of immune cell with multiple functions, capable of exhibiting various functional states depending on the environment. Macrophage polarization can influence the course of many human diseases, including but not limited to atherosclerosis, cystic fibrosis, asthma, allergic inflammation, chronic inflammation, infection, tissue repair, and cancer. The development of atherosclerosis is closely related to the polarization, infiltration, and functional abnormalities of M1 macrophages. From the perspective of atherosclerosis pathogenesis, M1 macrophages are involved in the entire process of plaque formation, progression, and rupture, serving as a key target driving the disease. Therefore, directly targeting M1 macrophages with drugs can block the pathological process of atherosclerosis at its source. While interventions such as nanomedicines and surgery exist, there is a lack of universally recognized and specifically effective chemical drugs and biological agents. Furthermore, existing drug therapies have large molecular weights and poor targeting, failing to reach the lesion site effectively. Therefore, screening for drugs that can precisely target macrophages or effective drug delivery tools is crucial for the prevention and treatment of atherosclerosis and inflammatory diseases.

[0004] Therefore, screening a nucleic acid aptamer that can target M1 macrophages to carry drugs specifically into the body is of profound significance for regulating the polarization of M1 macrophages and for preparing therapeutic drugs for treating atherosclerosis and related inflammation, as well as for targeted therapy of inflammatory diseases. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a nucleic acid aptamer targeting M1 macrophages, its screening method, and its application. The nucleic acid aptamer APT-Che, which can target M1 macrophages, was screened and obtained. This not only provides a novel cell-targeted delivery tool for research on atherosclerosis and inflammation-related diseases, but also effectively accelerates the development of targeted drugs and improves their therapeutic efficacy and safety.

[0006] To achieve the above objectives, the present invention provides a nucleic acid aptamer targeting M1 macrophages, wherein the sequence of the nucleic acid aptamer is any one of SEQ ID NO:1, SEQ ID NO:16 and SEQ ID NO:26.

[0007] Preferably, the sequence of the nucleic acid aptamer is SEQ ID NO:26.

[0008] This invention also provides a method for screening nucleic acid aptamers targeting M1 macrophages, comprising the following steps:

[0009] (1) Construct a random oligonucleotide library, upstream primers, and downstream primers;

[0010] (2) After denaturation, the nucleotide library was subjected to negative screening, positive screening, PCR amplification and recovery, alkaline denaturation and verification in sequence to obtain the first round of screening products;

[0011] (3) Repeat step (2) multiple times with the first round of screening products to obtain nucleic acid aptamers;

[0012] (4) Sequencing and cytological verification screened nucleic acid aptamers targeting M1 macrophages.

[0013] Preferably, the sequence of the random oligonucleotide library in step (1) is SEQ ID NO:27, the sequence of the upstream primer is SEQ ID NO:28, and the sequence of the downstream primer is SEQ ID NO:29.

[0014] Preferably, the negative screening cells in step (2) are human primary multinucleated cells; the positive screening cells are human primary macrophages.

[0015] More preferably, the human primary macrophages are of type M0.

[0016] Preferably, the cells used in the cytological verification screening in step (3) are one or more of M1 macrophages, hepatocytes, and kidney cells.

[0017] More preferably, the hepatocytes are human hepatic stellate cells (LX-2) and / or C57BL / 6J mouse primary hepatocytes.

[0018] More preferably, the kidney cells are human renal epithelial cells (293T).

[0019] The present invention also provides an application of a nucleic acid aptamer targeting M1 macrophages, wherein the application is in the preparation of a drug for biomolecular transport carriers.

[0020] Preferably, the biomolecule is one or more of nucleic acids, oligopeptides, polypeptides, carbohydrates, lipids, nanoparticles, and nanoblocks.

[0021] Further preferably, the nucleic acid is selected from microRNA or siRNA that are effective against atherosclerosis or other inflammatory diseases.

[0022] Preferably, the drug is a drug for intervening in atherosclerosis and other inflammatory diseases.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention successfully screened and obtained the aptamer APT-Che, which exhibits high affinity and specificity for M-type macrophages, using Cell-SELEX technology. It specifically binds to M1-type macrophages and has weak binding affinity to other types of normal cells. The discovery of this aptamer not only provides a novel cell-targeted delivery tool for research on atherosclerosis and inflammation-related diseases, but will also effectively accelerate the development of targeted drugs, improving their therapeutic efficacy and safety. The development of APT-Che will provide a highly efficient and specific targeted delivery tool for research fields (such as targeted drug delivery in in vitro cell culture) and clinical diagnosis and treatment (such as delivery of bioactive molecules or drug carriers like microRNA and siRNA), possessing significant scientific research value and industrial application potential. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the process of the present invention.

[0026] Figure 2 The image shows the morphology of macrophages in Example 1. The left image shows M0 type macrophages, and the right image shows M1 type macrophages.

[0027] Figure 3 This is a screenshot of the screening results from Example 1.

[0028] Figure 4 This is a screening fluorescence image of LX2 cells from Example 2.

[0029] Figure 5 This is a screening fluorescence image of primary hepatocytes from C57BL / 6J mice in Example 2.

[0030] Figure 6 This is a screening fluorescence image of 293T cells from Example 2.

[0031] Figure 7 This is a screening fluorescence image of M1 macrophages in Example 2.

[0032] Figure 8 The images show fluorescence images of the effects of APT-Che on different cells in Example 2.

[0033] Figure 9 This is a flow cytometry result of nucleic acid aptamers in Example 3.

[0034] Figure 10 This is a bar chart of flow cytometry analysis of nucleic acid aptamers in Example 3.

[0035] Figure 11 The image shows the prediction diagrams for APT-Che in Example 3. The left image is the prediction diagram for the secondary structure, and the right image is the prediction diagram for the tertiary structure.

[0036] Figure 12 This is a predicted secondary structure diagram of APT-Che-NF-κB decoy ODN in Example 4.

[0037] Figure 13 The images show fluorescence images of the effects of APT-Che-NF-κB decoy ODN on different cells in Example 4.

[0038] Figure 14 The figure shows the flow cytometry results of APT-Che-NF-κB decoy ODN on peripheral blood macrophages of C57BL / 6J mice in Example 5. Figure A is the flow cytometry results and B is the flow cytometry bar chart.

[0039] Figure 15 The figure shows the flow cytometry results of APT-Che-NF-κB decoy ODN on macrophages in the spleen of C57BL / 6J mice in Example 5. Figure A is the flow cytometry results and B is the flow cytometry bar chart. Detailed Implementation

[0040] The technical solution of the present invention will be further explained and described below with reference to the accompanying drawings and specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present invention and should not be construed as limiting the present invention. The scope of protection of the present invention should be determined by the contents of the claims. Modifications and substitutions made by those skilled in the art to the technical solution of the present invention without creative effort all fall within the scope of protection of the present invention.

[0041] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative and qualitative experiments in the following examples were performed in triplicate or at least, and the results were averaged.

[0042] Main reagents:

[0043] DMEM medium and 1640(+) complete medium: Gibco, USA;

[0044] DNA purification and recovery kit: Tiangen Biotech (Beijing) Co., Ltd.;

[0045] Nucleic acid dye: Shanghai Yuanye Biotechnology Co., Ltd.;

[0046] UNIQ-10 oligonucleotide purification kit, affinity chromatography column: Shanghai Sangon Biotech Co., Ltd.;

[0047] AGAROSE: BioFroxx (Germany);

[0048] DNA Ladder: Wuhan Saiweier Biotechnology Co., Ltd.

[0049] Taq PCR Master Mix: Wuhan Saiwei Biotechnology Co., Ltd.;

[0050] Macrophage colony-stimulating factor (M-CSF), interferon-γ (IFN-γ): PEPROTECH, USA;

[0051] Lipopolysaccharide (LPS): MCE Corporation, USA;

[0052] Trypsin: Wuhan Saiweier Biotechnology Co., Ltd.

[0053] Library and primer synthesis:

[0054] aptamer library and primers: Shanghai Sangon Biotech Co., Ltd.

[0055] Cell lines:

[0056] M0 and M1 macrophages: isolated, extracted, and induced from discarded blood samples after routine peripheral blood tests of normal individuals;

[0057] Primary multinucleated cells from normal individuals: extracted from discarded blood samples after routine peripheral blood tests in normal individuals;

[0058] Hepatocytes: isolated and extracted from the livers of six-week-old male C57BL / 6J mice;

[0059] Human kidney epithelial cells (293T): provided by Professor Wu Jiangfeng's laboratory at Three Gorges University;

[0060] Human hepatic stellate cells (LX-2): provided by Professor Wu Jiangfeng's laboratory at Three Gorges University.

[0061] Core formula:

[0062] Wash buffer (WB): 4.948 g glucose, 1.016 g MgCl.6H2O, dissolved in 1000 mL sterile PBS (pH=7.4), cooled to room temperature, dialyzed through a 0.22 μm filter membrane, and stored at 4℃ for later use.

[0063] Binding buffer (BB): 0.1 g tRNA, 1 g BSA, dissolved in 1000 mL WB solution, dialyzed through a 0.22 μm filter membrane, and stored at 4 °C for later use.

[0064] Example 1: Screening of nucleic acid aptamers

[0065] (1) Construction of random oligonucleotide library: Design and synthesize single-stranded DNA library (ssDNA library, 60 bases in total) and upstream and downstream primers (Table 1); the single-stranded DNA in the single-stranded DNA library consists of a random sequence in the middle and constant sequences at both ends, and is chemically synthesized at the laboratory level;

[0066] Table 1 Library and primer sequences

[0067]

[0068] (2) The synthesized library and primers were centrifuged at 4℃, 5000×g for 1 min, and then ddH2O was added to obtain a storage solution with a concentration of 100μM. The solution was then frozen at -20℃ for later use.

[0069] (3) Peripheral blood mononuclear cells were obtained from normal human peripheral blood using density gradient centrifugation, and the mononuclear cell concentration was adjusted to 2×10⁻⁶. 6 / mL was injected into 6-well plates, and macrophage colony-stimulating factor M-CSF was administered. After 7 days, primary human M0 macrophages were generated by polarization and used as positive selection cells. Figure 2 Then, normal human primary multinucleated cells were used as negative selection cells and cultured in a 37°C constant temperature incubator.

[0070] (4) Pre-denaturation: Take 30 μL of library stock solution, dilute it 40 times with sterile enzyme-free water to obtain 2500 nM stock solution, take half of the volume to resuspend it and place it in 100℃, slowly cool it overnight to room temperature to obtain denatured library;

[0071] Rapid annealing: Take half the volume of the library stock solution, resuspend it, place it in a 95℃ metal bath for 5 minutes, and then immediately remove it and place it on ice for 10 minutes to obtain the annealed library.

[0072] The denatured library and the annealed library were mixed at a volume ratio of 1:1 to obtain the library to be screened.

[0073] (5) Positive screening: M0 macrophages cultured to good polarization and density of about 85% were taken, the culture medium was discarded, and the cells were washed twice with washing buffer. 1 mL of the library to be screened was added, and the cells were incubated at 37℃ in a 5% CO2 cell culture incubator for 60 min. The supernatant was discarded, 200 μL of trypsin was added to the bottom of the dish for 10 min of digestion, 1640(+) complete culture medium was added to stop the digestion, and the cell suspension was collected in a 1.5 mL EP tube. The cells were washed twice with washing buffer, and the cell suspension was collected. The cells were centrifuged at 800 rpm for 3 min, the supernatant was discarded, and 1 mL of washing buffer was added to resuspend and wash. The cells were centrifuged at 800 rpm for 3 min. The process was repeated twice to obtain positive screening cells.

[0074] (6) Add 100 μL of SDS solution to the positive selection cells, heat in a metal bath at 95°C for 10 min, then centrifuge at 12000 rpm and 22°C for 20 min, and retain the supernatant as a PCR template.

[0075] (7) PCR amplification: Using the supernatant obtained in step (6) as a PCR template, and combining it with the primers synthesized in step (1), PCR amplification was performed. The PCR amplification system is shown in Table 2, and the PCR program is shown in Table 3.

[0076] Table 2 PCR amplification system

[0077]

[0078] Table 3 PCR amplification program

[0079]

[0080] In the first round of screening, the annealing temperature of PCR was 59.5℃, and the number of cycles was 20; in the second round of screening, the annealing temperature of PCR was 57.9℃, and the number of cycles was 18; in the third round of screening, the annealing temperature of PCR was 57.9℃, and the number of cycles was 22.

[0081] (8) Take the PCR product obtained in step (7) for electrophoresis, remove the gel and purify it to obtain a DNA solution; then use alkaline denaturing affinity column chromatography to separate the DNA solution to obtain a positive strand ssDNA solution, and then purify it with a UNIQ-10 column to obtain a secondary library (FAM-SSDNA).

[0082] (9) According to the system in Table 2 and the procedure in Table 3, amplify the secondary library obtained in step (8), verify it by electrophoresis, and complete the first round of screening;

[0083] (10) Repeat steps (4)-(9) to perform 3 rounds of screening. Figure 3 Finally, the selected libraries were sent for high-throughput sequencing, and 26 library sequences with high enrichment content and many motif sequence repetitions were selected as the nucleic acid aptamer screening results (Table 4). Before each round of screening, the number of PCR cycles and temperature needed to be optimized. Negative screening was required during the second and third rounds of screening. The method was to take half a volume of the secondary library, prepare the library to be screened according to step (4), and then place it in a 1×10 7 / mL of normal human primary multinucleated cells were mixed and incubated in a 37℃, 5% CO2 cell culture incubator. The culture dish was shaken every 20 min. After incubation for 60 min, the culture dish was centrifuged at 800 rpm, 22℃, for 4 min. The precipitate was discarded and the supernatant was retained to complete the negative screening. In the subsequent step (5), the supernatant obtained from the negative screening was used to replace the library to be screened in step (5) to complete the positive screening. Finally, nucleic acid aptamers were obtained. Figure 3 ).

[0084] Table 4. High-throughput sequencing results of 26 sequences

[0085]

[0086] Example 2

[0087] (1) Peripheral blood was collected from normal individuals, and peripheral blood mononuclear cells were obtained by density gradient centrifugation. The cell concentration in each well was adjusted to 5 × 10⁻⁶. 5 The cells were placed in 24-well plates with 50 ng / mL M-CSF and induced for 7 days to obtain M0 macrophages. Then, they were induced for 1 day with 100 ng / mL LPS and 20 ng / mL IFN-γ to obtain M1 macrophages.

[0088] (2) Take the 26 nucleic acid aptamers obtained in Example 1, prepare them into a solution with a concentration of 250 nM, and then co-incubate them with the M1 macrophages obtained in step (1). Use the M1 macrophages without added nucleic acid aptamers as the control group. After co-incubation for 1 h, observe the cell fluorescence under an inverted fluorescence microscope.

[0089] (3) Logarithmic growth phase LX-2 and 293T cells were seeded in 24-well plates. When the cell density was about 80%, 26 nucleic acid aptamers obtained in Example 1 were taken and prepared into a solution with a concentration of 250 nM. Then, they were added to 24-well plates containing LX-2 and 293T cells respectively. LX-2 and 293T cells without nucleic acid aptamers were used as control groups. After incubation for 1 h, cell fluorescence was observed under an inverted fluorescence microscope.

[0090] (4) The extracted C57BL / 6J mouse primary hepatocytes were seeded in 24-well plates. When the cell density was about 90%, 26 nucleic acid aptamers obtained in Example 1 were taken and prepared into a solution with a concentration of 250 nM. Then, C57BL / 6J mouse primary hepatocytes containing nucleic acid aptamers were added. C57BL / 6J mouse primary hepatocytes without nucleic acid aptamers were used as the control group. After incubation for 1 h, cell fluorescence was observed under an inverted fluorescence microscope.

[0091] The results showed that aptamers 1, 16, 24, and 26 entered M1 macrophages in greater numbers. Figure 7 ); Article 24: A relatively large number of nucleic acid aptamers entered primary hepatocytes of C57BL / 6J mice ( Figure 6 Almost none of the 26 nucleic acid aptamers entered LX-2 cells. Figure 4 ) and 293T cells ( Figure 6 Finally, nucleic acid aptamers 1, 16, and 26 were selected as candidate aptamers and named APT-1, APT-16, and APT-26, respectively. Figure 8 ).

[0092] Example 3

[0093] (1) M1 macrophages were induced according to the method described in step (1) of Example 2, and then APT-1, APT-16 and APT-26 (250 nM) were added and co-incubated with them for 1 h. The cells were then observed under an inverted fluorescence microscope. Cells without nucleic acid aptamers were incubated for 1 h as a control group.

[0094] (2) Flow cytometry detection: M1 macrophages were induced according to the method described in step (1) of Example 2, and then APT-1, APT-16 and APT-26 (250 nM) were added and co-incubated with them for 1 h, and then flow cytometry detection was performed; cells without nucleic acid aptamers were incubated for 1 h as the control group;

[0095] (3) Take the best nucleic acid aptamer and add it to a 24-well plate containing M1 macrophages, C57BL / 6J mouse primary hepatocytes LX-2 and 293T cells respectively, so that the final concentration is 200 nM. After incubation for 1 h, observe the cell fluorescence under an inverted fluorescence microscope.

[0096] The results showed that fluorescence and cell loss detection indicated that APT-26 entered the largest number of M1 macrophages, and the APT-26 group had the strongest average fluorescence intensity. Figure 9-10 Therefore, APT-26 was selected as the final nucleic acid aptamer and named APT-Che.

[0097] The nucleic acid aptamer APT-Che was used, and its secondary and tertiary structures were predicted using computer-aided methods. The results are as follows: Figure 11 As shown, the upper part represents the secondary structure, and the lower part represents the tertiary structure, indicating that the APT-Che structure is stable and will not degrade easily.

[0098] Example 4

[0099] (1) Peripheral blood was collected from normal individuals, and peripheral blood mononuclear cells were obtained by density gradient centrifugation. The concentration of mononuclear cells was adjusted to 2 × 10⁻⁶.6 M1 macrophages were polarized after 7 days by administering 50 ng / mL M-CSF to 6-well plates and administering 100 ng / mL LPS and 20 ng / mL IFN-γ for 6-8 weeks. M1 macrophages were obtained 24 hours later.

[0100] (2) The nucleic acid aptamer APT-Che was linked to the small nucleic acid NF-κB decoy ODN to form APT-Che-NF-κBdecoy ODN, the sequence of which is: SEQ ID NO:30 ( Figure 12 );

[0101] (3) APT-Che-NF-κB decoy ODN was added to primary hepatocytes, 293T, LX2 and M1 macrophages of C57BL / 6J mice to a final concentration of 200 nM. After incubation for 1 day, cell fluorescence was observed under an inverted fluorescence microscope. M1 macrophages without any APT-Che-NF-κB decoy ODN were used as blank control group, and M1 macrophages with NF-κB decoy ODN were used as control group.

[0102] The results showed that APT-Che-NF-κB decoy ODN could enter M1 macrophages, but could not enter C57BL / 6J mouse primary hepatocytes, 293T cells, or LX2 cells. Figure 12 This indicates that APT-Che can carry drugs that specifically target M1 macrophages.

[0103] Example 5

[0104] (1) Take normal 6-8 week old male C57BL / 6J mice weighing 20-24g, inject them intraperitoneally with 5mg / kg LPS, and feed them normally for 24h to obtain an inflammatory mouse model;

[0105] (2) Drug injection:

[0106] Experimental group: 150 µL (200 µM) Cy5-labeled APT-Che-NF-κB decoy ODN was injected via the tail vein;

[0107] Control group 1: 150 µL (200 µM) Cy5-labeled NF-κB decoy ODN was injected via the tail vein;

[0108] Control group 2: 150 µL of normal saline was injected via the tail vein;

[0109] (3) One hour after injection, the mice were sacrificed and peripheral blood and spleen were collected. Macrophages were extracted from them and detected by flow cytometry.

[0110] The results showed that APT-Che-NF-κB decoy ODN in the experimental group could enter M1 macrophages. Figure 14-15 ).

Claims

1. A nucleic acid aptamer targeting M1 macrophages, characterized in that: The sequence of the nucleic acid aptamer is any one of SEQ ID NO:1, SEQ ID NO:16 and SEQ ID NO:

26.

2. The nucleic acid aptamer targeting M1 macrophages according to claim 1, characterized in that: The sequence of the nucleic acid aptamer is SEQ ID NO:

26.

3. The application of a nucleic acid aptamer targeting M1 macrophages as described in claim 1 or 2, characterized in that: The application is in the preparation of drugs using biomolecular transport carriers.

4. The application according to claim 3, characterized in that: The biomolecules are one or more of the following: nucleic acids, oligopeptides, polypeptides, carbohydrates, lipids, nanoparticles, and nanoblocks.

5. The application according to claim 4, characterized in that: The nucleic acids are selected from microRNAs or siRNAs that are effective against atherosclerosis or other inflammatory diseases.

6. The application according to claim 3, characterized in that: The drug is used to treat atherosclerosis and other inflammatory diseases.

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