Hepatocyte growth factor simulant prepared based on click chemistry method, preparation method and application

Abstract

The invention discloses a hepatocyte growth factor simulant prepared based on a click chemistry method, a preparation method and application, and belongs to the technical field of biological medicine. According to the invention, Ap-Met-N of which the tail end is marked with an azide group and Ap-Met-D of which the tail end is marked with a dibenzocyclooctylene alkyne group are subjected to strain-promoted alkyne-azide cycloaddition reaction to form a cyclic divalent nucleic acid aptamer for simulating the function of a hepatocyte growth factor, namely the ND-HCB; and carrying out annealing reaction on the ND-HCB and the short sequence of the modified N-acetylgalactosamine, which is complementarily paired with the ND-HCB, so as to prepare the ND-TCB. Experiments prove that ND-HCB has the capacity of activating a Met signal channel and promoting cell proliferation and migration, and ND-TCB further has the liver tissue targeting capacity and the liver targeting Met channel activating capacity on the basis, and can be used for treating acute liver injury. The invention provides a new growth factor medicine for treating acute liver injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a hepatocyte growth factor mimetic prepared based on click chemistry, a preparation method and an application thereof. Background Art

[0002] Liver injury is a common clinical condition, caused by various physical and chemical factors, both acutely and chronically. Acute liver injury (ALI) is a rapid hepatocyte necrosis and inflammatory response triggered by factors such as drug-induced injury, viral infection, excessive alcohol intake, or ischemia-reperfusion. It is characterized by marked elevations in serum transaminases and a dramatic deterioration in liver function. Severe or persistent liver injury can ultimately lead to acute liver failure, which carries a high mortality rate. Therefore, liver injury and its repair remain at the forefront of liver disease research.

[0003] The biological effects of growth factors on liver repair have been widely demonstrated, but their clinical translation still faces significant challenges. Currently, most growth factor-mediated liver injury repair strategies remain confined to the experimental research stage. For example, Fatima Rizvi's team used a lipid nanoparticle (LNP) delivery system to deliver nucleoside-modified mRNA for transient expression of HGF and EGF in hepatocytes. This strategy significantly improved liver function parameters and accelerated hepatocyte proliferation in a mouse model of non-alcoholic fatty liver disease (NAFLD). It also demonstrated the potential to promote tissue regeneration in an acetaminophen (APAP)-induced acute liver injury model. However, in clinical development, only recombinant hepatocyte growth factor (rh-HGF) has advanced to Phase II clinical trials for fulminant hepatitis. Phase I / II clinical trial data published by Ido et al. in 2011 demonstrated that rh-HGF did not cause serious adverse reactions under repeated intravenous administration and its pharmacokinetic profile met therapeutic needs. Despite this, rh-HGF has not yet been approved for marketing, highlighting the translational bottlenecks faced by growth factor drugs in terms of stability, targeting, and long-term safety.

[0004] As researchers continue to innovate and improve growth factors, aptamers as growth factor mimics have gained increasing attention. The programmability of aptamers allows the construction of multivalent complexes (such as dimers and nanorings), significantly improving receptor activation efficiency. However, unmodified linear aptamers face significant challenges in serum environments: on the one hand, the gap in the aptamer structure is susceptible to degradation by endogenous nucleases; on the other hand, due to insufficient molecular conformational stability, they are prone to target loss in complex biological environments. These inherent limitations not only limit the effective application of nucleic acid-based growth factors in vivo but also hinder their further clinical translation. Circularized aptamers, however, can eliminate the gap in the aptamer, resulting in higher serum stability and conformational stability. In 2023, Liang H et al. proposed using a T4 ligase cyclization strategy to develop a bivalent aptamer (CBA) with HGF-mimicking function. Compared to uncircularized bivalent aptamers, CBA exhibits superior stability and affinity, effectively activating the Met receptor-mediated regenerative signaling pathway. Furthermore, in vivo applications, CBA has demonstrated enhanced liver damage repair capabilities. However, T4 ligase constructs cyclic bivalent aptamers by catalyzing the formation of a phosphodiester bond between adjacent 5' phosphates and 3' hydroxyls in double-stranded DNA, thereby connecting two aptamers. This method is not suitable for aptamers with blunt ends. In addition, T4 ligase is sensitive to storage conditions, requiring low-temperature storage and reaction, and its catalytic activity is also dependent on specific buffer environments. These properties limit its development and application. Therefore, the exploration of new cyclic bivalent aptamers is of great significance for the use of growth factor drugs in the treatment of liver injury. Summary of the Invention

[0005] The present invention aims to provide a hepatocyte growth factor mimetic prepared by click chemistry, a preparation method and an application thereof, in order to solve the problems existing in the above-mentioned prior art. The strain-promoted alkyne-azide cycloaddition (SPAAC) reaction in the click chemistry method is adopted to prepare a hepatocyte growth factor mimetic based on a cyclic bivalent nucleic acid aptamer. The reaction process is mild, simple to operate, widely applicable and does not require the participation of enzymes. The developed hepatocyte growth factor mimetic ND-HCB has excellent stability and high affinity, enabling it to specifically bind to the Met receptor on the membrane surface and activate the Met signaling pathway. Furthermore, the hepatocyte growth factor mimetic ND-TCB developed in combination with GalNAc can play a role in specifically targeting the hepatocyte Met pathway, reducing off-target effects and alleviating nonspecific activation of adjacent tissues.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a hepatocyte growth factor mimetic ND-HCB prepared based on a click chemistry method, wherein a mesenchymal-epithelial transition factor nucleic acid aptamer Ap-Met-N terminally labeled with an azide group and a mesenchymal-epithelial transition factor nucleic acid aptamer Ap-Met-D terminally labeled with a dibenzocyclooctyne group are subjected to a strain-promoted alkyne-azide cycloaddition reaction to form a cyclic bivalent nucleic acid aptamer that simulates the function of hepatocyte growth factor, namely the ND-HCB;

[0008] The nucleotide sequence of Ap-Met-N is shown in any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, and SEQ ID NO. 8, and the nucleotide sequence of Ap-Met-D is shown in any one of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, and SEQ ID NO. 9. However, the nucleotide sequences of Ap-Met-N and Ap-Met-D of the present invention are not limited to the above sequences and may also be sequences with greater than 50% homology.

[0009] More preferably, the nucleotide sequence of the Ap-Met-N is shown as SEQ ID NO.6, and the nucleotide sequence of the Ap-Met-D is shown as SEQ ID NO.7.

[0010] The present invention also provides a method for preparing the ND-HCB, comprising the following steps:

[0011] Ap-Met-N and Ap-Met-D are dissolved in water respectively, then mixed evenly in an equimolar ratio, and subjected to a strain-promoted alkyne-azide cycloaddition reaction under constant temperature oscillation to prepare the ND-HCB.

[0012] Preferably, the concentrations of Ap-Met-N and Ap-Met-D are both 10-15 μM;

[0013] And / or the constant temperature oscillation condition is: oscillation at 25° C. for 10-15 hours.

[0014] The present invention also provides a hepatocyte growth factor mimetic ND-TCB prepared based on the click chemistry method, which includes the ND-HCB and a short sequence complementary to the ND-HCB, the nucleotide sequence of the short sequence is CCCCGACCG, and the terminal of the short sequence is modified with N-acetylgalactosamine (GalNAc).

[0015] The present invention also provides a method for preparing the ND-TCB, comprising the following steps:

[0016] The ND-HCB and the short sequence were mixed at a molar ratio of 1:2, and then a DNA annealing buffer solution was added to perform an annealing reaction to prepare the ND-TCB.

[0017] Preferably, the annealing reaction is: heating at 95° C. for 5 minutes and then slowly cooling down.

[0018] The present invention also provides the use of the ND-HCB or the ND-TCB in any of the following:

[0019] (1) Application in the preparation of drugs for promoting cell proliferation and / or migration;

[0020] (2) Application in the preparation of drugs for repairing cell damage caused by APAP.

[0021] The present invention also provides use of the ND-HCB or the ND-TCB in preparing a medicine for treating acute liver injury.

[0022] The present invention also provides use of the ND-HCB or the ND-TCB in preparing HGF mimetics.

[0023] The present invention also provides a medicine for treating acute liver injury, comprising the ND-HCB or the ND-TCB.

[0024] The present invention discloses the following technical effects:

[0025] The present invention uses click chemistry to prepare two Met aptamers (Ap-Met-N and Ap-Met-D) with N3 groups or DBCO groups at the end through SPAAC into cyclic bivalent nucleic acid aptamers (ND-HCB) that can simulate the function of HGF. The sequence of ND-HCB is optimized by polyacrylamide gel electrophoresis and protein immunoblotting experiments, and ND-HCB-15 with the best effect of activating the Met pathway is screened out. Through serum and exonuclease stability experiments, it is confirmed that the ND-HCB synthesized by the SPAAC method has higher serum stability than the cyclic bivalent nucleic acid aptamer (T4-HCB) synthesized by the traditional T4 enzyme. Then, through protein immunoblotting experiments, it is proved that ND-HCB-15 can efficiently and long-term activate the Met downstream pathway; through CCK-8, RTCA, Transwell, scratch, and scattering experiments, the proliferation and migration promoting ability of ND-HCB-15 on cells are verified, and it is proved that ND-HCB-15 can reproduce the cell behavior induced by HGF. Through liver damage cell modeling experiments, it was preliminarily verified at the cellular level that ND-HCB-15 can effectively repair cell damage caused by APAP.

[0026] Furthermore, in order to achieve selective activation of the hepatic Met signaling pathway, avoid toxic side effects on other tissues, and better utilize ND-HCB for the treatment of liver damage in vivo, the present invention designed a short sequence modified with GalNAc so that it can bind to ND-HCB through complementary pairing to construct a liver-targeted cyclic bivalent functional nucleic acid aptamer. Flow cytometry experiments and mouse in vivo distribution imaging experiments confirmed that compared with ND-HCB, ND-TCB has a better ability to target hepatocytes both in vitro and in vivo. Finally, a mouse acute liver injury model was used to further confirm that the constructed click-chemistry-based cyclic bivalent nucleic acid aptamer has a good ability to repair damaged livers in vivo. The present invention provides new ideas for the application of functional nucleic acids in the field of regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 To test the construction effect of ND-HCB of different lengths by polyacrylamide;

[0029] Figure 2 To detect the Met activation effect of ND-HCB of different lengths by protein immunoassay;

[0030] Figure 3 To detect the stability of cyclic bivalent aptamers in exonucleases by polyacrylamide gel electrophoresis;

[0031] Figure 4 To analyze the stability of T4-HCB-15 and ND-HCB-15 in 10% serum by polyacrylamide gel electrophoresis;

[0032] Figure 5 To analyze the stability of monovalent nucleic acid aptamers of different lengths and corresponding cyclic bivalent nucleic acid aptamers in serum by polyacrylamide gel electrophoresis;

[0033] Figure 6 Western blotting was used to detect the activation of Met by HGF, Ap-Met-N-15, ND-HCB-15 and T4-HCB-15;

[0034] Figure 7 Western blotting was used to detect the activation ability of different concentrations of ND-HCB-15 on the Met pathway;

[0035] Figure 8Western blotting was used to test the ability of ND-HCB-15 to activate the Met downstream signaling pathway;

[0036] Figure 9 Western blotting was used to compare the activation ability of ND-HCB-15 and HGF at different reaction times on the Met signaling pathway;

[0037] Figure 10 To detect the cell proliferation promoting ability of different concentrations in different groups; A is the CCK-8 assay to detect the cell proliferation promoting effect of different concentrations of HGF, ND-HCB-15, and T4-HCB-15; B is the RTCA assay to detect the long-term cell proliferation promoting ability of HGF, ND-HCB-15, and T4-HCB-15; mean ± SD (n = 3), significant (****P < 0.0001, *P < 0.05);

[0038] Figure 11 The migration-promoting ability of HGF, Ap-Met-N-15, T4-HCB-15, and ND-HCB-15 was detected by wound healing assay; mean ± SD (n = 3), significance (****P < 0.0001, *P < 0.05), scale bar, 100 μm;

[0039] Figure 12 Transwell assay to investigate the migration-promoting abilities of HGF, Ap-Met-N-15, T4-HCB-15, and ND-HCB-15; mean ± SD (n = 3), significance (****P < 0.0001, ***P < 0.01), scale bar: 100 μm;

[0040] Figure 13 This is a cell scattering experiment to investigate the ability of HGF, Ap-Met-N-15, T4-HCB-15, and ND-HCB-15 to promote cell migration; scale bar: 100 μm;

[0041] Figure 14 CCK-8 assay to detect the repair effect of ND-HCB-15 on cell damage; mean ± SD (n = 3), significance (***P < 0.001, **P < 0.01, *P < 0.05);

[0042] Figure 15 To investigate the liver targeting performance of ND-TCB by flow cytometry;

[0043] Figure 16 To investigate the distribution of Cy5-labeled ND-HCB-15 and ND-TCB in the main organs of mice; in the figure, He represents the heart; Lu represents the lung; Lv represents the liver; Ki represents the kidney; Sp represents the spleen;

[0044] Figure 17 Western blotting was used to detect the effects of different treatment groups on the specific activation of Met in the liver;

[0045] Figure 18 The results of serum AST and ALT detection in mice in different treatment groups are mean ± standard deviation (n = 3), significant (****P < 0.0001, **P < 0.01, *P < 0.05);

[0046] Figure 19 The results of H&E staining of mouse liver tissue. DETAILED DESCRIPTION

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0049] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0050] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0051] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0052] The gene sequences involved in the present invention are shown in Table 1.

[0053] Table 1

[0054]

[0055]

[0056]

[0057] Note: Ap-Met-N-0 and Ap-Met-D-0 are two nucleic acid aptamers obtained by modifying the terminal azide group (N3) and dibenzocyclooctyne group (DBCO) of the same nucleotide sequence, respectively.

[0058] Example 1 Preparation of HGF mimetics based on cyclic bivalent nucleic acid aptamers by click chemistry

[0059] 1. Test materials

[0060] 1.1 Cell culture

[0061] Human hepatoma cell line Hep-G2 and human prostate cancer cell line Du145 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Normal human hepatocytes L02 were purchased from the Wuhan University Cell Bank. Hep-G2 cells were cultured in MEM (with NEAA) supplemented with 10% FBS and a 1% penicillin-streptomycin-amphotericin B mixture (Vivacell: C3423-0100). L02 and Du145 cells were cultured in RPMI 1640 supplemented with 10% FBS, 1% triple antibody, and 1% sodium pyruvate. The cells were incubated at 37°C in 5% CO2 and washed with PBS.

[0062] 1.2 Preparation of experimental solutions and buffers

[0063] (1) RPMI 1640 complete culture medium: Add 50 mL of FBS, 5 mL of a mixture of penicillin-streptomycin-amphotericin B, and 5 mL of sodium pyruvate to 500 mL of RPMI 1640 culture medium to prepare a complete culture medium containing 10% serum.

[0064] (2) MEM complete culture medium: Add 50 mL of FBS, 5 mL of penicillin-streptomycin-amphotericin B mixture, and 5 mL of sodium pyruvate to 500 mL of MEM (NEAA) culture medium to prepare a complete culture medium containing 10% serum.

[0065] (3) RPMI 1640 starvation solution: 500 μL of BSA was added to 100 mL of RPMI 1640 culture medium to prepare a culture medium containing 0.5% BSA.

[0066] (4) MEM starvation solution: 500 μL of BSA was added to 100 mL of MEM culture medium to prepare a culture solution containing 0.5% BSA.

[0067] (5) DPBS buffer: DEPC solution was added to PBS solution at a volume ratio of 1:1000, mixed by inversion, and placed in the dark overnight before high-temperature sterilization to prepare DPBS (pH = 7.4) buffer.

[0068] (6) 5M NaCl buffer: Weigh 5.85 g of sodium chloride powder and add it to 20 mL of ddH2O and mix well.

[0069] (7) GelRed staining solution: Take 1 μL of 10000× GelRed staining solution and 1 mL of 5 M NaCl buffer, add them to 49 mL of ddH2O, mix well, and store at 4°C.

[0070] (8) 1×TBE buffer: Add 100 mL of 5×TBE buffer to 400 mL of ddH2O, mix well, and store at room temperature for later use.

[0071] 2. Test methods

[0072] 2.1 Preparation of ND-HCB

[0073] Preparation of monovalent nucleic acid aptamers: The DNA sequences used in this experiment are shown in Table 1. Dry powders of Met nucleic acid aptamers terminally labeled with an azide group (Ap-Met-N) and dibenzocyclooctyne group (Ap-Met-D) were dissolved in commercial DEPC water to prepare a 100 μM DNA stock solution, which was then stored at -20°C until use.

[0074] Preparation of HGF mimetic (ND-HCB): Ap-Met-N and Ap-Met-D were diluted to 10 μM in DPBS buffer. The two sequences were then mixed at a 1:1 molar ratio and placed in a constant temperature shaker at 25°C for 12 hours to allow the two single strands to fully bind through the SPAAC reaction, forming a cyclic bivalent functional nucleic acid aptamer structure. The sample was stored at 4°C.

[0075] 2.2 Preparation of T4-HCB

[0076] Preparation of T4-HCB using the T4 DNA ligase method: Ap-Met1-t4-15 and Ap-Met2-t4-15 were dissolved in commercial DEPC-treated water to prepare a 100 μM stock solution. DNA was diluted to 10 μM with DEPC-treated water, annealed by heating at 95°C for 5 minutes, and then rapidly cooled on ice. The annealed strands were then mixed at a 1:1 molar ratio and incubated in a 16°C PCR instrument for 6 hours. 0.5 U / μL Exonuclease I and 2.5 U / μL Exonuclease III were then added, and the mixture was incubated in a PCR instrument at 37°C for 1 hour to remove the uncircularized single strand. The enzymes were then inactivated by heating at 85°C for 10 minutes to obtain the cyclic bivalent nucleic acid aptamer T4-HCB.

[0077] Purification of T4-HCB: The synthesized T4-HCB was purified using a DNA rapid purification kit. The DNA product was separated from the reaction mixture through selective adsorption and elution, effectively removing impurities such as organic compounds, salt residues, and protein components in the reaction system.

[0078] 2.3 Preparation of liver-targeted HGF mimetic (ND-TCB)

[0079] The ND-HCB synthesized by the above method was mixed with the liver-targeting short sequence Ap-GN3 in a molar ratio of 1:2, and then 1× DNA annealing buffer solution was added to mix evenly. The mixture was placed in a metal analyzer and heated at 95°C for 5 minutes, and then slowly cooled to allow Ap-GN3 to complementarily pair with ND-HCB to form a liver-targeting cyclic bivalent nucleic acid aptamer (ND-TCB). The sample was stored at 4°C.

[0080] 3. Polyacrylamide gel electrophoresis

[0081] The DNA samples to be tested were diluted to a working concentration of 1 μM using DPBS buffer. The diluted samples were mixed with 5× Loading Buffer containing bromophenol blue indicator at a volume ratio of 5:1 and thoroughly mixed by vortexing. Each group of DNA samples was added to a pre-cast 16% polyacrylamide gel, and the electrophoresis parameters were set to a constant voltage of 110 V. Separation was continued for 100 minutes in 1× TBE electrophoresis buffer. After the electrophoresis was completed, the gel was transferred to GelRed staining solution and stained in the dark for 30 minutes. After staining, it was placed on a chemiluminescence imaging system (ChemiDocT XRS + ) for imaging.

[0082] 4. Stability test

[0083] 4.1 Exonuclease stability test:

[0084] 1 μM ND-HCB and T4-HCB were added to 0.5 U / μL ExoI buffer solution and incubated in a 37°C incubator for different time periods (0 h, 6 h, and 12 h). DNA samples from each group were loaded onto a prepared 16% polyacrylamide gel and electrophoresed in 1× TBE buffer at 110 V for 100 min. The gel was stained in GelRed staining solution in the dark for 30 min and then developed and analyzed using an imager.

[0085] 4.210% serum stability test:

[0086] T4-HCB-15 and ND-HCB-15 were incubated at 1 μM in RPMI 1640 medium containing 10% FBS in a 37°C incubator for various times (0, 1, 6, 12, 24, 32, 48, and 72 h) or (0, 0.5, 1, 2, 4, and 6 days). The samples were loaded onto a 16% polyacrylamide gel and electrophoresed in 1× TBE buffer at 110 V for 100 min. The gels were then stained with GelRed stain for 30 min in the dark and analyzed using an imaging device.

[0087] 5. Western Blotting

[0088] For experiments examining protein changes at the cellular level, 1.5×10 6 L02 cells were plated at a concentration of 100 cells / well in a 60 mm dish and cultured in a 37°C, CO2 incubator until the cell density reached 80%. The cells were then starved for 6 hours using RPMI 1640 starvation medium. 5 nM HGF, Ap-Met-N-15, and ND-HCB were then added to the dish and allowed to react for 30 minutes or for varying durations (0 min, 30 min, 1 h, 6 h, 12 h, and 24 h). The cells were then harvested using a scraper.

[0089] To investigate changes in mouse tissue protein levels, BALB / C mice were injected via the tail vein with PBS and 0.05 nmol / L of HGF, T4-HCB-15, ND-HCB-15, and ND-TCB. Thirty minutes after injection, the mice were dissected, and the liver and lungs were removed. Ten milligrams of tissue were sheared on ice and added to RIPA lysis buffer containing 1× protease inhibitor and 1× phosphatase inhibitor. The tissue was then sheared into the smallest possible volume using scissors and then disrupted on ice using a handheld tissue disruptor, each time for 5 seconds, until no tissue particles were visible. The tissue homogenate was then centrifuged at 13,000 rpm for 15 minutes at 4°C, and the supernatant was collected for BCA protein quantification. Subsequently, SDS loading buffer was added to the protein, mixed thoroughly, and boiled at 100°C for 10 minutes to denature the protein.

[0090] Cells were lysed using Western / IP lysis buffer (containing 1× PMSF and 1× phosphatase inhibitors), sonicated on ice, and centrifuged at 12,000 rpm for 15 minutes in a pre-cooled centrifuge at 4°C. The protein supernatant was collected, mixed with 5× SDS buffer, and denatured in 100°C water for 10 minutes. The samples were then stored at -20°C. Before electrophoresis, protein samples were removed from -20°C and thawed on ice. Vortexed to mix thoroughly, the samples were then added to a pre-prepared 8% SDS-PAGE. A voltage of 90 V was initially applied, and after the marker was removed, the voltage was switched to 120 V to separate the proteins. For transfer, electrophoresis was performed at 100 V for 80 minutes to transfer the proteins to a PVDF membrane soaked in anhydrous methanol. Blocking was performed with 5% skim milk for 90 minutes. The membrane was then incubated with the primary antibody corresponding to the target for 60 minutes on a shaker at room temperature, then refrigerated at 4°C overnight. The primary antibody was then rewarmed for 1 hour the following day. The membrane was washed with 1×TBST buffer for 3 times, 5 min each time, and then incubated with the corresponding secondary antibody for 1 h. Finally, the chemiluminescence imaging system (ChemiDocT XRS + ) for imaging.

[0091] 6. Cell Proliferation Analysis

[0092] CCK-8 assay: L02 cells were plated at 4000 cells / well in a 96-well plate. After overnight attachment, the complete medium was replaced with RPMI 1640 starvation medium. Different concentrations (0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.2 nM) of HGF or ND-HCB-15 or different concentrations (0, 0.2, 0.4, 8, 1, 1.5, and 2 nM) of T4-HCB-15 were added to the cells for 48 h. CCK-8 reagent was then added and incubated for 1 h. The absorbance at 450 nm was measured using a microplate reader.

[0093] RTCA experiment: First, L02 cells were plated at a density of 4000 cells / well in a test plate and cultured in a 37°C, 5% CO2 incubator for 12 hours. After the cells adhered, the complete culture medium was replaced with RPMI 1640 starvation solution, and 0.5nM HGF, Ap-Met-N-15, ND-HCB-15, and T4-HCB-15 were added to the cells, respectively. Real-time cell analysis (RTCA) was used to monitor the growth of L02 cells in each group within 100 hours, and growth curves were drawn.

[0094] Hep-G2 cells were plated at 8,000 cells / well in a 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator. 4 mM acetaminophen (APAP) was added for 24 hours, then the medium was replaced with MEM starvation solution. 0.5 nM HGF, T4-HCB-15, ND-HCB-15, and ND-TCB were added, respectively, and the cells were incubated for 36 hours. Finally, cell viability was measured using a CCK-8 assay.

[0095] 7. Cell migration assay

[0096] Scratch test: L02 cells were cultured at 6×10 5 Cells were plated at a concentration of 100 cells / well in a 6-well plate and cultured in a 37°C, 5% CO2 incubator for 36 h. When the cell density reached about 90%, the cells were scratched with a 10 μL pipette tip and washed three times with PBS to remove non-adherent cells. RPMI 1640 starvation solution containing 2 nM HGF, T4-HCB-15, Ap-Met-N-15, and ND-HCB-15 were added for culture. The cell growth status was observed at different time periods (0 h, 24 h, 48 h) and photographed using a 20× objective lens.

[0097] Transwell assay: L02 cells with a density of about 80% were starved with RPMI 1640 solution for 6 h, then trypsinized and plated at 4 × 10 4 Plate cells per well in a Transwell chamber and add 1 nM HGF, Ap-Met-N-15, T4-HCB-15, or ND-HCB-15, respectively. Add RMPI 1640 complete medium to the bottom of the chamber and incubate in a 37°C, 5% CO2 incubator for 24 hours. Wash three times with PBS, fix the cells with pre-chilled paraformaldehyde at 4°C for 15 minutes, wash three times with PBS, stain with 1% crystal violet solution for 15 minutes, and wash three times with PBS. Gently wipe the cells in the upper chamber with a cotton swab. Allow the cells to dry and then photograph them under a microscope (10x objective).

[0098] Cell scattering assay: L02 cells were plated at 1000 cells / well in a 6-well plate and cultured in a 37°C, 5% CO2 incubator for 2 days. When single cells were observed to grow into cell clusters, the complete medium was replaced with RPMI 1640 starvation solution, and 5 nM HGF, Ap-Met-N-15, T4-HCB-15, and ND-HCB-15 were added, respectively, to incubate the cells. Microscope images were taken at 0 h and again 24 h after drug exposure (20× objective lens).

[0099] 8. Flow cytometry

[0100] Hep-G2 and DU145 were cultured at 6×10 5 Cells were plated at a concentration of 100 cells / well in a 6-well plate and cultured overnight in a 37°C, 5% CO2 incubator. When the cell density reached approximately 80%, 50 nM Cy5-labeled ND-HCB-15 and ND-TCB were added to the cells for 1 hour, respectively. The cells were trypsinized and centrifuged at 1500 rpm for 5 minutes at 4°C. The cells were then resuspended in PBS and analyzed using flow cytometry.

[0101] 9. In vitro injury modeling experiment

[0102] Hep-G2 cells were plated at 8,000 cells / well in a 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator. 4 mM acetaminophen (APAP) was added for 24 hours, followed by MEM starvation solution containing 0.5 nM HGF, T4-HCB-15, or ND-HCB-15 for 36 hours. Cell viability was assessed using a CCK-8 assay.

[0103] 10. Biodistribution experiment

[0104] BALB / C female nude mice were purchased from Wu's Laboratory Animal Co., Ltd. Six-week-old BALB / C mice weighing approximately 20 g were injected via the tail vein with 0.2 nmol Cy5-labeled ND-HCB-15 and ND-TCB. The mice were dissected at different time points (0.5 h, 2 h, 4 h, 8 h, 24 h, 48 h, and 72 h) to obtain major organs (heart, liver, spleen, lung, and kidney), and fluorescence imaging was performed using a small animal fluorescence imager.

[0105] 11. Construction and treatment of acute liver injury model in mice

[0106] Six-week-old BALB / C mice weighing approximately 20g were selected. First, the mice were fasted for 15 hours, but not water. After weighing, they were intraperitoneally injected with 390mg / kg of acetaminophen (APAP) to establish an acute liver injury model. Twelve hours later, PBS and 0.6nmoL of Ap1-Ap2, ND-HCB-15, and ND-TCB were administered via the tail vein, with injections occurring 12 hours apart for a total of two doses. Twelve hours after the second dose, blood was collected from the orbital vein in a 1.5mL EP tube, allowed to stand at room temperature for 2 hours, and then centrifuged at 2000rpm in a pre-cooled centrifuge for 10 minutes. Serum was collected and assayed using alanine aminotransferase / glutamic oxaloacetic transaminase (ALT / GPT) and aspartate aminotransferase (AST / GOT) assays. Mice were dissected, and liver tissue was collected and fixed in 4% paraformaldehyde for 48 hours before being embedded in paraffin.

[0107] 12. H&E staining

[0108] Mice were euthanized, and liver and lung tissues were obtained by dissection and fixed in 4% paraformaldehyde. The tissues were then sent to KexueDog Biotechnology Co., Ltd. for paraffin embedding, sectioning, and H&E staining. The sections were photographed using an inverted fluorescence microscope.

[0109] 13. Statistical analysis

[0110] All experiments were performed with 3-6 replicates per group, and the data are presented as mean ± standard deviation (SD). Differences between groups were assessed using one-way analysis of variance (ANOVA) and student's t-test. Results are expressed as ns, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. P < 0.05 was considered statistically significant.

[0111] 14. Results and Analysis

[0112] 14.1 Design and Sequence Optimization of ND-HCB

[0113] Studies have shown that cyclic bivalent aptamers, formed by cyclizing aptamers targeting the Met receptor through T4 ligase, can induce dimerization of two Met proteins, triggering receptor autophosphorylation and activating downstream regenerative signaling pathways, thereby driving the repair of damaged liver tissue. Compared with uncyclized single-stranded or bivalent aptamers, cyclic bivalent aptamers have better stability and higher Met receptor binding affinity in biological media and have demonstrated therapeutic potential in a mouse model of acute liver injury. However, T4 ligase constructs cyclic bivalent aptamers by catalyzing the formation of a phosphodiester bond between adjacent 5' phosphate and 3' hydroxyl groups in double-stranded DNA, thereby connecting two aptamers. This method is not suitable for aptamers with blunt ends. In addition, T4 ligase is sensitive to storage conditions and requires low temperature for storage and reaction. Its catalytic activity also depends on specific buffer environments, which limits its development and application. Click chemistry is a simple, efficient, and highly selective chemical reaction used to construct complex molecular structures. One of the most classic reactions is SPAAC. Compared to enzyme-catalyzed cyclization, SPAAC has broad applicability and can be applied to a variety of nucleic acid aptamers. Its mild reaction conditions, the absence of a catalyst, and high yields produce products with excellent stability and biocompatibility. Based on this, the present invention utilizes SPAAC to construct ND-HCB that mimics the function of HGF.

[0114] The present invention designs cyclic bivalent nucleic acid aptamers with different stem lengths (ND-HCB-0, 5, 10, 15, 20). Figure 1 Polyacrylamide gel electrophoresis results showed that cyclic bivalent nucleic acid aptamers with different stem lengths (ND-HCB-0, 5, 10, 15, 20) all exhibited a higher migration hysteresis than the linear single-stranded nucleic acid aptamer at a molecular weight of around 100 kD. The difference in molecular weight confirmed the successful construction of the cyclic bivalent nucleic acid aptamer.

[0115] Using c-Met phosphorylation (p-Met) as an indicator of Met pathway activation, the activation performance of ND-HCB with different stem lengths on the Met pathway was investigated by Western blotting. The present invention selected L02 cells that highly express c-Met receptors as a cell model, and added ND-HCB with different stem lengths to co-incubate the cells for 30 minutes. Figure 2 As shown, ND-HCB activated Met phosphorylation to varying degrees, with the degree of phosphorylation increasing with increasing stem length. When the stem length was 15 base pairs, the phosphorylation level was optimal, similar to that of HGF. Further increasing the stem length to 20 base pairs decreased the p-Met level. Therefore, the present invention selected ND-HCB-15 with a stem length of 15 base pairs for subsequent experiments.

[0116] 14.2 Stability Analysis of ND-HCB

[0117] Aptamers are easily degraded by exonucleases in physiological environments, thus affecting their application in physiological environments. Compared with single-stranded aptamers, cyclic bivalent aptamers lack free ends, which can avoid degradation by exonucleases, thereby improving their stability in physiological environments. Therefore, the present invention then investigates the stability of ND-HCB-15 in exonucleases by polyacrylamide gel electrophoresis and compares it with the cyclic bivalent aptamer (T4-HCB-15) synthesized by the T4 ligase method. Figure 3 As shown in the figure, ND-HCB-15 synthesized by click chemistry method still maintained structural stability in the presence of exonuclease for 12 hours, which was similar to T4-HCB-15, indicating that ND-HCB-15 synthesized by click chemistry method had good anti-exonuclease activity.

[0118] The ability of nucleic acid aptamers to maintain structural integrity in serum is one of the important factors in evaluating their potential for in vivo application. Therefore, the present invention investigated the stability of DN-HCB15 in serum and compared it with T4-HCB-15. Figure 4 As shown, the stability of T4-HCB-15 in serum decreased over time, being almost completely degraded by 48 hours. However, ND-HCB-15 remained structurally stable after 72 hours in serum. These experimental data demonstrate that the stability of cyclic bivalent aptamers synthesized via SPAAC is significantly superior to that achieved via T4 enzyme ligation in serum.

[0119] The present invention investigates the effect of different stem lengths on serum stability. Figure 5 As shown, the structure of single-chain Ap-Met-N-0 changed at day 0.5, Ap-Met-N-5 began to degrade significantly at day 1, and both Ap-Met-N-10 and Ap-Met-N-15 were significantly degraded at day 4, while 50% of Ap-Met-N-20 remained structurally stable at day 6. At the same time, it can be seen that the stability of the corresponding ND-HCB in serum also increased with the extension of the stem length, and even maintained good stability at day 6. In summary, the inventors can reasonably speculate that with this stem-loop structure-based nucleic acid aptamer design method, the longer the stem length, the greater the stability in serum.

[0120] 14.3ND-HCB efficiently and long-lastingly activates the Met signaling pathway

[0121] After confirming that ND-HCB-15 has better stability, we then investigated its effect on Met pathway activation and compared it with HGF, Ap-Met-N-15, and T4-HCB-15. Figure 6 As can be seen, the single-chain Ap-Met-N-15 structure is comparable to the blank control, indicating that the single-chain structure cannot effectively activate the Met pathway. Compared with T4-HCB-15, the ND-HCB-15 group has significantly increased p-Met levels, indicating that it has stronger activation of the Met pathway. This may be due to the fact that ND-HCB-15 is more stable than T4-HCB-15 under physiological conditions, resulting in stronger pathway activation.

[0122] Western blotting was used to investigate the activation of Met pathway by different concentrations of ND-HCB-15 (0nM, 1nM, 2.5nM, 5nM, 10nM, 20nM). Figure 7 It can be seen that with the increase of ND-HCB-15 concentration, the expression of p-Met protein showed an arc-shaped trend of first increasing and then decreasing, and reached the maximum Met activation level when the ND-HCB-15 concentration was 2.5 nM.

[0123] After HGF binds to the Met receptor, it drives receptor dimerization and activates autophosphorylation of the intracellular tyrosine kinase domain, forming binding sites that can be recognized by downstream effector molecules. This process leads to dynamic changes in downstream signaling, including cascade reactions in the PI3K / AKT / mTOR pathway and the ERK / MAKP pathway. Therefore, the present invention next examined the activation performance of ND-HCB-15 on the Met downstream signaling pathway using p-Met, p-Akt, and p-Erk1 / 2 as indicators.

[0124] from Figure 8As can be seen, compared with the blank control group, the expression of p-Akt and p-Erk1 / 2 in both the HGF- and ND-HCB-15-treated groups increased significantly. This result suggests that ND-HCB-15 can activate Met downstream signaling pathways (PI3K / AKT / mTOR pathway and ERK / MAKP pathway) by inducing Met dimerization. Notably, the activation effect of ND-HCB-15 on Met downstream pathways is similar to that of HGF, indicating that ND-HCB-15 can effectively mimic the function of HGF.

[0125] One of the main limitations of recombinant growth factors is their poor stability and rapid degradation in physiological environments, resulting in loss of pathway activation ability. The above study confirmed that ND-HCB-15 has excellent stability in physiological environments. Furthermore, the ability of ND-HCB-15 to continuously activate the Met pathway was evaluated by Western blotting and compared with HGF. The results are as follows: Figure 9 As shown, HGF induced high Met phosphorylation levels at 10 minutes of stimulation. With prolonged incubation, p-Met levels decreased, and by 12 hours, p-Met was almost undetectable, likely due to HGF's gradual degradation and inactivation in the serum environment. In contrast, ND-HCB-15 exhibited the highest Met phosphorylation activation level at 30 minutes. Over time, p-Met levels decreased more slowly than HGF, and p-Met was still detectable even after 12 hours of incubation.

[0126] The above experimental results show that compared with HGF, ND-HCB-15 has a more sustained pathway activation performance.

[0127] 14.4ND-HCB promotes cell proliferation and migration

[0128] HGF induces Met receptor dimerization and autophosphorylation, activating downstream pathways, among which the PI3K / AKT / mTOR pathway can promote cell survival and enhance cell migration. Triggering the ERK / MAKP pathway can drive the cell cycle process and promote cell proliferation and differentiation. It has been verified that ND-HCB-15 can activate Met and its downstream PI3K / AKT / mTOR pathway and ERK / MAKP pathway. Next, we examined whether ND-HCB-15 can reproduce HGF-induced cell behavior. Cell proliferation is one of the typical biological phenotypes activated by the HGF / Met signaling pathway. First, the CCK-8 method was used to detect the proliferation activity of L02 cells after 36 hours of treatment with different concentrations of HGF, ND-HCB-15 and T4-HCB-15. Figure 10As shown in A, HGF and ND-HCB-15 both exhibited maximum proliferative activity at a concentration of 0.4 nM, while T4-HCB-15 needed to increase its concentration to 1 nM to achieve the same effect level. The above results indicate that ND-HCB-15 can achieve efficient Met signal activation at low concentrations, and its proliferative effect is significantly better than that of T4-HCB-15 prepared by the traditional T4 DNA ligation method. Furthermore, the effects of different treatment groups (blank control group, HGF, T4-HCB-15 and ND-HCB-15) on cell proliferation were monitored in real time by real-time label-free dynamic cell analysis technology (Real Time Cell Analysis, RTCA), and growth curves were drawn. Figure 10 As can be seen in Figure B, during the 100-hour real-time monitoring process, the cell growth trends of the HGF, T4-HCB-15, and ND-HCB-15 treatment groups were significantly higher than those of the blank control group 24 hours after drug addition. Among them, the cell growth curve of ND-HCB-15 had the largest proliferation slope, and it maintained its proliferation-promoting ability as the observation time prolonged. The growth curve proliferation slopes of the HGF-treated and T4-HCB-15-treated groups began to slow down in the 60-80 hour period. This may be because the stability of HGF and T4-HCB-15 in serum is lower than that of ND-HCB-15, and they are gradually degraded and become ineffective.

[0129] Cell migration is one of the typical biological phenotypes of HGF / Met signaling pathway activation. In order to verify the migration-promoting ability of ND-HCB-15, the wound healing rate of cells treated with HGF, T4-HCB-15 and ND-HCB-15 was investigated by a scratch test. Figure 11 As can be seen in the results, at the 0-hour starting point, the scratch area of ​​each cell group was essentially the same. At the 48-hour measurement endpoint, the healing rate reached 48% for T4-HCB-15 and 58% for ND-HCB-15, respectively, approaching the healing rate of HGF (61%). The blank control group and the single-chain Ap-Met-15-treated group had the smallest healing areas. This suggests that ND-HCB-15 exerts a similar effect to HGF by binding to the Met receptor, causing dimerization and activating the Met signaling pathway, promoting cell migration.

[0130] Furthermore, the cell migration promoting ability of ND-HCB-15 was investigated by Transwell assay and compared with HGF, Ap-Met-N-15, and T4-HCB-15. Figure 12As shown, in the control group (Ctrl), cells seeded in the upper chamber gradually migrated to the filter membrane after 24 hours of culture, stimulated by the serum in the lower chamber. These migrated cells were then stained with crystal violet and imaged and counted. Compared to the control group, the number of cells migrating to the filter membrane in the ND-HCB-15-treated group was 2.6 times higher, similar to that in the HGF group and greater than that in the T4-HCB-15 group. These experimental results demonstrate that ND-HCB-15 significantly promotes cell migration. As expected, no significant pro-migratory effect was observed in the single-chain Ap-Met-N-15 group.

[0131] Finally, the cell scattering experiment was used to examine the ability of ND-HCB-15 to promote cell migration. Figure 13 As shown, at the initial observation time of 0 hours, cells in each group were tightly clustered together. After 24 hours of treatment, the cells in the blank control group still grew in tight colonies. However, after 24 hours of treatment with HGF, T4-HCB-15 or ND-HCB-15 (5nM), the cells were significantly discretized, the colony area increased, and the proportion of single cells increased. This experiment once again proves that ND-HCB-15 can exert HGF-like migration-promoting properties. In summary, the above series of phenotypic results can be concluded that the efficacy of ND-HCB-15 as an HGF mimetic has been verified, and it can reproduce HGF-induced cell behavior.

[0132] Acetaminophen is a widely used antipyretic and analgesic. Although considered safe at therapeutic doses, at higher doses, acetaminophen can lead to reduced glutathione (GSH) depletion, causing mitochondrial oxidative stress and ultimately cell necrosis. Activation of the Met signaling pathway can drive cell proliferation and promote the regeneration and repair of damaged hepatocytes. Therefore, we next conducted a preliminary cellular investigation of the regenerative and repair effects of ND-HCB-15 on APAP-induced hepatocyte injury.

[0133] The present invention selected Hep-G2 as a cell model. After APAP-induced cell damage, different treatment groups (PBS, HGF, T4-HCB-15, ND-HCB-15) were added and treated for 24 hours. The cell activity was detected by CCK-8 method. Figure 14 As shown in the figure, the cell viability of the APAP-treated group was significantly reduced compared to the blank control group, indicating that APAP caused cell death. However, after treatment with HGF, T4-HCB-15, and ND-HCB-15, cell viability recovered to varying degrees, with the ND-HCB-15-treated group showing significantly stronger activity than the T4-HCB-15-treated group (P<0.05), achieving a therapeutic effect similar to that of HGF.

[0134] 14.5 Construction of HGF Mimetics with Liver Targeting Properties and Analysis of Targeting Performance

[0135] The present invention designs a short sequence (Ap-GN3) modified with N-acetylgalactosamine (GalNAc), which enables it to bind to the ring of ND-HCB-15 through complementary pairing, thereby constructing a liver-targeted cyclic bivalent functional nucleic acid aptamer - ND-TCB, which specifically targets hepatocytes, activates the liver Met signaling pathway, and promotes the regeneration and repair of damaged livers.

[0136] First, the present invention selected Hep-G2 cells that express Met and highly express ASGPR (asialoglycoprotein receptor) and DU145 cells that express Met but do not express ASGPR as cell models, and added Cy5 fluorescently labeled ND-HCB-15 and ND-TCB respectively and incubated for 1 hour, and the targeting of ND-TCB was investigated by flow cytometry. Figure 15 As shown, in Hep-G2 cells, the fluorescence peak position of ND-TCB is significantly shifted to the right compared to ND-HCB, indicating that more ND-TCB binds to Hep-G2 cells. In DU145 cells, the fluorescence peak positions of ND-TCB and ND-HCB-15 almost overlap. These experimental results show that the ability of Ap-GN3 to specifically target the ASGPR receptor enables ND-TCB to bind to HepG2 cells with high ASGPR expression, while it has no targeting ability for DU145 cells with low ASGPR expression.

[0137] After confirming that ND-TCB has the ability to target and bind to liver cells with high expression of ASGPR, the performance of ND-TCB in targeting liver tissue in vivo was further investigated. First, the present invention investigated the biodistribution of ND-HCB-15 and ND-TCB in the main organs of mice. Fluorescently labeled ND-HCB-15 and ND-TCB were injected through the tail vein, and the main organs (heart, liver, spleen, lung, kidney) were dissected after different time periods and imaged using a small animal imaging device. Figure 16 As shown, ND-HCB-15 and ND-TCB rapidly accumulated in the liver 30 minutes after tail vein injection. Over time, fluorescence gradually decreased, reaching near-undetectable levels 72 hours after injection, indicating that ND-HCB-15 and ND-TCB were completely metabolized and excreted. However, it is noteworthy that some ND-HCB-15 fluorescence was also distributed in the lungs at 30 minutes and 2 hours, while ND-TCB fluorescence was virtually undetectable in the lungs under the same conditions, demonstrating ND-TCB's ability to be targeted and enriched in the liver. Furthermore, compared to ND-HCB-15, ND-TCB exhibited stronger liver retention.

[0138] Subsequently, we further investigated whether ND-TCB has the ability to specifically activate the Met signaling pathway in the liver. The present invention injected PBS, HGF, T4-HCB-15, ND-HCB-15 and ND-TCB into the tail vein of mice, and 30 minutes later, the liver and lung tissues were collected for protein immunoblotting. The results are as follows. Figure 17 As shown, in the liver, p-Met protein expression was significantly increased in the HGF, ND-HCB-15, and ND-TCB treatment groups compared to the blank control group. In the lung, only the HGF, T4-HCB-15, and ND-HCB-15 treatment groups showed significant increases in p-Met expression compared to the blank control group, while ND-TCB treatment did not significantly increase p-Met protein expression. This experiment demonstrates that ND-TCB has liver-targeting properties, capable of specifically activating the Met signaling pathway in liver tissue, improving the safety of its in vivo application.

[0139] 14.6 Application of Mouse Model of Acute Liver Injury

[0140] Studies have confirmed that after liver damage occurs, the concentration of HGF in plasma increases significantly. It activates downstream signaling pathways by specifically binding to the c-Met receptor on the surface of liver cells, driving the liver regeneration process. Under normal circumstances, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum are very low. When liver cells are damaged, the permeability of the liver cell membrane increases, and ALT and AST in the cytoplasm are released into the blood, causing an increase in ALT and AST in the blood. After confirming that ND-TCB can effectively target and activate the liver Met signaling pathway, the present invention constructed an APAP-induced acute liver injury model in mice, using AST and ALT as indicators to evaluate the therapeutic effect of ND-TCB on acute liver injury in mice, and compared it with the single-chain simple mixed non-cyclized group (Ap1-Ap2) and ND-HCB-15. The results are as follows. Figure 18 As shown in the results, the APAP modeling group detected significantly higher AST and ALT levels in the mouse serum compared with the blank control group, and there was a significant difference (P<0.001), indicating that the acute liver injury model was successfully established. The single-chain simple mixed group (Ap1-Ap2) had no significant reduction effect compared with the APAP modeling group after treatment (P>0.05). However, after treatment with ND-HCB-15 and ND-TCB, ALS and AST levels were significantly reduced (P<0.001), and the reduction effect of ND-TCB was better than that of ND-HCB-15. The above results show that ND-TCB can better target and activate the liver Met signaling pathway in mice and play a role in liver damage repair.

[0141] The liver tissue sections of each group of mice were stained with H&E. Figure 19As shown in the staining results, the nuclei of the liver tissue cells in the blank control group were centrally located and clearly visible, the cytoplasm was abundant, and a small number of blood cells were visible in the sinusoids. The APAP-treated group showed focal necrosis, showing inflammatory cell infiltration and dilated and congested liver sinusoids. The Ap1-Ap2 group also showed large areas of cell necrosis, indicating that simple mixing of the double chains cannot activate the Met pathway to promote tissue repair. In both the ND-HCB-15 and ND-TCB-treated groups, liver structures similar to those of normal mice were observed, liver damage was significantly weakened, and fibrosis was reduced or disappeared. These results indicate that the designed HGF mimetic has a strong therapeutic potential for repairing liver damage in vivo.

[0142] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A hepatocyte growth factor mimetic ND-HCB prepared based on click chemistry, characterized in that: The method comprises the following steps: a strain-promoted alkyne-azide cycloaddition reaction of a mesenchymal-epithelial transition factor nucleic acid aptamer Ap-Met-N terminally labeled with an azide group and a mesenchymal-epithelial transition factor nucleic acid aptamer Ap-Met-D terminally labeled with a dibenzocyclooctyne group to form a cyclic bivalent nucleic acid aptamer that simulates the function of hepatocyte growth factor, namely the ND-HCB; Wherein, the nucleotide sequence of the Ap-Met-N is shown as any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8, and the nucleotide sequence of the Ap-Met-D is shown as any one of SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.

9.

2. A method for preparing ND-HCB according to claim 1, characterized in that: The following steps are involved: Ap-Met-N and Ap-Met-D are dissolved in water respectively, then mixed evenly in an equimolar ratio, and subjected to a strain-promoted alkyne-azide cycloaddition reaction under constant temperature oscillation to prepare the ND-HCB.

3. The preparation method according to claim 2, wherein The concentrations of Ap-Met-N and Ap-Met-D are both 10-15 μM; And / or the constant temperature oscillation condition is: oscillation at 25° C. for 10-15 hours.

4. A hepatocyte growth factor mimetic ND-TCB prepared based on click chemistry, characterized in that: The method comprises the ND-HCB according to claim 1 and a short sequence complementary to the ND-HCB, wherein the nucleotide sequence of the short sequence is CCCCGACCG, and the terminal of the short sequence is modified with N-acetylgalactosamine.

5. A method for preparing ND-TCB according to claim 4, characterized in that: The following steps are involved: The ND-HCB and the short sequence were mixed at a molar ratio of 1:2, and then a DNA annealing buffer solution was added to perform an annealing reaction to prepare the ND-TCB.

6. The preparation method according to claim 5, wherein The annealing reaction is as follows: heating at 95° C. for 5 minutes and then slowly cooling down.

7. Use of the ND-HCB according to claim 1 or the ND-TCB according to claim 4 in any of the following: (1) Application in the preparation of drugs for promoting cell proliferation and / or migration; (2) Application in the preparation of drugs for repairing cell damage caused by APAP.

8. Use of the ND-HCB according to claim 1 or the ND-TCB according to claim 4 in the preparation of a medicament for treating acute liver injury.

9. Use of the ND-HCB according to claim 1 or the ND-TCB according to claim 4 in preparing an HGF mimetic.

10. A drug for treating acute liver injury, characterized in that: The method comprises the ND-HCB according to claim 1 or the ND-TCB according to claim 4.

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