DNA hydrogel with kiss hairpin cross-linked structure as well as preparation method and application of DNA hydrogel

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to DNA hydrogel with a kiss hairpin cross-linked structure as well as a preparation method and application thereof. The first tail end of the DNA single strand is connected with a first sticky tail end carrying a specific recognition TNF-alpha nucleic acid aptamer, and the second tail end of the DNA single strand is connected with a second sticky tail end; the two hairpin structures form an integrated hairpin structure through complementary pairing of bases at the adhesive tail ends, and the annular areas of the multiple integrated hairpin structures form the DNA hydrogel with the three-dimensional net-shaped framework through complementary pairing and crosslinking of the bases. According to the application, the anti-TNF-alpha nucleic acid aptamer is self-assembled in a DNA kiss hairpin cross-linking manner to form the hydrogel, so that controllable drug sustained release is realized. Compared with a plurality of TNF-alpha monoclonal antibodies and fusion protein biological agents, the anti-TNF-alpha nucleic acid aptamer has the advantages of good targeting property and low cost; the hydrogel is formed through self-assembly in a DNA kiss hairpin cross-linking mode, the slow release adjustable range is larger, and the safety is higher.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a DNA hydrogel with a kissing hairpin cross-linking structure, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease, and TNF-α plays a crucial role in its progression. Traditional anti-inflammatory drugs for arthritis treatment have significant side effects, increasing the risk of heart disease and other cardiovascular diseases. Novel monoclonal antibodies and fusion protein biologics targeting TNF-α are widely used clinically, but they also present challenges such as increased infection risk and high cost. Therefore, the development of new TNF-α inhibitors is of great clinical significance.

[0003] Nucleic acid aptamers are nucleic acid sequences of approximately 20-100 base pairs in length that can bind to targets with high affinity and specificity. The anti-TNF-α aptamer VR11 is a 25-base-long single-stranded DNA that can specifically bind to TNF-α and has shown promising therapeutic effects as a TNF-α antagonist in studies.

[0004] In recent years, DNA hydrogels have been extensively studied as novel materials for biomedical applications. Hydrogels are hydrophilic polymers with a three-dimensional network structure, formed by physical or chemical cross-linking. Their three-dimensional network structure is similar to biological tissues, and due to their high water content and large drug-carrying space, they are widely used in biomedicine and drug delivery. DNA hydrogels possess the dual properties of DNA molecules and hydrogels, achieving a fusion of hydrogel scaffold function and DNA biological function. Since nucleic acid aptamers are chemically based on DNA or RNA, they can be used as constituent materials of DNA hydrogels, making DNA hydrogels suitable for delivering nucleic acid aptamers.

[0005] However, existing DNA hydrogels for delivering nucleic acid aptamers have problems such as excessive cross-linking strength and difficulty in rapidly decomposing the aptamers into monomers or oligomers in vivo to exert their effects.

[0006] Therefore, providing an improved DNA hydrogel carrying anti-TNF-α nucleic acid aptamers that can rapidly respond to environmental changes after entering the human body and release free anti-TNF-α aptamer monomers or oligomers is a technical problem that needs to be solved. Summary of the Invention

[0007] To address the aforementioned technical problems, one objective of this invention is to provide a DNA hydrogel with a kissing hairpin cross-linking structure.

[0008] The present invention adopts the following technical solution: A DNA hydrogel with a kissing hairpin crosslinking structure includes a hairpin structure comprising two stem-loop units formed by the folding of a DNA single strand. The first end of the DNA single strand is connected to a first sticky end carrying a nucleic acid aptamer that specifically recognizes TNF-α, and the second end is connected to a second sticky end. Two hairpin structures with the same structure but opposite sticky end sequences form a structural unit through complementary base pairing at the sticky ends. The loop intervals of adjacent structural units are connected through complementary base pairing to form a kissing hairpin connection. Multiple structural units are connected through kissing hairpin connections to form a DNA hydrogel with a three-dimensional network backbone.

[0009] Preferably, each stem-loop unit consists of an unpaired single-stranded loop region and a complementary double-stranded stem region. The DNA sequence of the single-stranded loop region is shown in SEQ ID No:1. The DNA sequences of the double-stranded stem regions of the first stem-loop unit are shown in SEQ ID No:2 and SEQ ID No:3, respectively. The DNA sequences of the double-stranded stem regions of the second stem-loop unit are shown in SEQ ID No:4 and SEQ ID No:5, respectively. The sequences of the first sticky end and the second sticky end are shown in SEQ ID No:6 and SEQ ID No:7, respectively, or in SEQ ID No:8 and SEQ ID No:9, respectively. The sequence of the TNF-α aptamer is shown in SEQ ID No:10.

[0010] The second objective of this invention is to provide a method for preparing a DNA hydrogel with a kissing hairpin cross-linking structure as described above, comprising the following steps: S1. Based on the hairpin structure, design a single-stranded DNA sequence and integrate the selected anti-TNF-α nucleic acid aptamer into the stem region of the hairpin structure to form a single-stranded DNA sequence containing the hairpin cross-linking structure to be cross-linked and the anti-TNF-α nucleic acid aptamer. S2. Preparation of hydrogel solution containing kissing hairpin cross-linking structure and anti-TNF-α nucleic acid aptamer: Take 0.4 mM of the DNA single strand solution and add enzyme-free water to obtain a DNA single strand solution with a final concentration of 0.375 mM; S3. Assembly: The obtained DNA single-stranded solution was annealed in a PCR instrument. After annealing, it was removed from the PCR instrument and placed in an ice bath for 5 min. Then, 10 mM MgCl2 was added to complete the cross-linking reaction, resulting in the DNA hydrogel with the desired kissing hairpin cross-linking structure.

[0011] Preferably, the annealing conditions are 95°C for 5 minutes.

[0012] Preferably, the ratio of MgCl2 to DNA single-strand solution is 2.78 μL: 25 μL.

[0013] A third objective of this invention is to provide an application of a DNA hydrogel with a kissing hairpin cross-linking structure as described above in the preparation of products for treating rheumatoid arthritis.

[0014] The fourth objective of this invention is to provide a product for treating rheumatoid arthritis, the product comprising a DNA hydrogel having a kissing hairpin cross-linking structure as described above.

[0015] The beneficial effects of this invention are as follows: 1) This invention provides a DNA hydrogel with a kissing hairpin cross-linking structure and carrying an anti-TNF-α nucleic acid aptamer. The hydrogel is formed by self-assembly of the anti-TNF-α nucleic acid aptamer via DNA kissing hairpin cross-linking, achieving controlled drug release. Compared to various TNF-α monoclonal antibodies and fusion protein biopharmaceuticals, the anti-TNF-α nucleic acid aptamer has the advantages of both good targeting and low cost; the self-assembly of the hydrogel via DNA kissing hairpin cross-linking allows for a wider range of adjustable release and higher safety.

[0016] 2) Existing DNA hydrogels for delivering nucleic acid aptamers have excessively strong cross-linking properties, making it difficult for them to rapidly decompose into monomers or oligomers in vivo to exert their effects. This application creatively employs a kissing hairpin cross-linked DNA hydrogel to carry nucleic acid aptamers and provides a feasible preparation method: cross-linking the loop region of a hairpin structure carrying a single-stranded DNA sequence that specifically recognizes TNF-α, combined with complementary pairing of sticky ends, to form an injectable hydrogel that can rapidly decompose and exfoliate drugs.

[0017] 3) The hydrogel material provided in this application can rapidly respond to environmental changes after entering the body. The principle of rapid response is that after the hydrogel enters the body, due to changes in the concentration of ions in the physiological environment (such as Mg), 2+ (Due to decreased concentration or the presence of competing ions), the cross-linking between the kiss-shaped hairpins weakens, leading to rapid dispersion of the hairpin structure.

[0018] 4) Theoretically, the DNA hydrogel with a kissing hairpin cross-linking structure carrying an anti-TNF-α nucleic acid aptamer provided in this application has a good effect on diseases affected by TNF-α levels. Mouse experiments have confirmed that the DNA hydrogel with a kissing hairpin cross-linking structure and carrying an anti-TNF-α nucleic acid aptamer provided in this application can achieve the effect of treating rheumatoid arthritis. Attached Figure Description

[0019] Figure 1This is a schematic diagram of the DNA chain design in the kissing hairpin crosslinking hydrogel of the present invention. In the figure, A is a schematic diagram of the principle of crosslinking at the kissing hairpin site; B is a single-stranded DNA sequence diagram of the hydrogel containing kissing hairpin crosslinking and anti-TNF-α aptamer.

[0020] Figure 2 This is a schematic diagram of the cross-linking of the kiss-shaped hair clip cross-linking hydrogel of the present invention. In the figure, A is the cross-linking design diagram of the kiss-shaped hair clip cross-linking hydrogel, and B and C are schematic diagrams of the cross-linking of the kiss-shaped hair clip cross-linking hydrogel.

[0021] Figure 3 This is a scanning electron microscope image of the cross-linked hydrogel of the kiss-shaped hair clip of the present invention. In the figure, A and B are different magnifications.

[0022] Figure 4 Electrophoretic image of the kiss-shaped hairpin crosslinked hydrogel prepared in Example 1.

[0023] Figure 5 The results are from the in vitro cytotoxicity experiment of Example 2.

[0024] Figures 6-8 The results of the hydrogel decomposition experiment in Example 3 are as follows. Figure 6 The results are from decomposition at 4 ℃. Figure 7 The results are from the decomposition at 22 °C. Figure 8 The results are from decomposition at 37 °C. The arrows in the figure represent the undecomposed hydrogel portions.

[0025] Figure 9 This is a flowchart of the CIA's animal experimentation process.

[0026] Figure 10 Photographs of the joints of mice in different drug administration groups.

[0027] Figure 11 Figure 1 shows the changes in body weight of mice in different drug administration groups. P <0.0001 compared with the first group.

[0028] Figure 12 The results of arthritis index in mice in different drug administration groups, ##P <0.01 compared with the second group.

[0029] Figure 13 Results of joint swelling in mice in different drug administration groups. ##P <0.01 compared with the second group.

[0030] Figure 14 * TNF-α concentration in the serum of mice in different drug administration groups P <0.05, **** P <0.0001.

[0031] Figure 15The results of H&E staining of liver, spleen and kidney tissues of mice in different drug administration groups. Detailed Implementation

[0032] The technical solution of the present invention will be described in more detail below with reference to experiments.

[0033] Experimental cells: Human rheumatoid arthritis fibroblast synovial cells (MH7A), purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0034] Experimental animals: SPF-grade DBA / 1 male mice, 6–8 weeks old, weighing 20±1 g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The animal housing was kept at an ambient temperature of 25±1℃, with free access to food. Experiments began after 7 days of acclimatization.

[0035] Main drug: DNA strand, purchased from Genewiz (Jiangsu) Co., Ltd.

[0036] Example 1 Design and preparation of DNA hydrogels See Figures 1-2 A DNA hydrogel containing a kissing hairpin cross-linking structure and an anti-TNF-α nucleic acid aptamer includes a hairpin structure comprising two stem-loop units formed by the folding of a single-stranded DNA. Each stem-loop unit consists of an unpaired single-stranded loop and a complementary double-stranded stem. The single-stranded DNA has two ends: a first end connects to a nucleic acid aptamer carrying specific recognition of TNF-α and a first sticky end, and a second end connects to a second sticky end. Two hairpin structures with identical structures but opposite sticky end sequences form a structural unit through complementary base pairing at their sticky ends. The loop intervals of adjacent structural units are connected by complementary base pairing to form a kissing hairpin connection. Multiple structural units are connected by kissing hairpins and expand in three-dimensional space to form a DNA hydrogel with a three-dimensional network backbone.

[0037] The single-stranded circular DNA sequence of the hairpin structure is: 5'-TGTACATTTTTACATGT-3' (SEQ ID No:1); the sequences of the double-stranded stem region of the first stem-loop unit are 5'-CTTCGGTGACTGGAGG-3' (SEQ ID No:2) and 5'-CCTCCAGTCACCGAAG-3' (SEQ ID No:3); the sequences of the double-stranded stem region of the second stem-loop unit are 5'-CTCGTCCAAGCACGAC-3' (SEQ ID No:4) and 5'-GTCGTGCTTGGACGAG-3' (SEQ ID No:5).

[0038] In the same hairpin structure, the adhesive ends are partially complementary. The first adhesive end sequence is: 5'-GCTAAGAATCTA-3' (SEQ ID No:6), and the second adhesive end sequence is: 3'-CGATTCTTAGATTGAAGTCCGGACTTCA-5' (SEQ ID No:7, in the sequence list according to the direction from the 5' end to the 3' end). The first 12-nt are complementary to the first adhesive end. The first adhesive end sequence of another hairpin structure that is complementary to it to form a structural unit is: 5'-ACTTCAGGCCTGAAGTTAGATTCTTAGC-3' (SEQ ID No:8), and the second adhesive end sequence is: 3'-ATCTAAGAATCG-5' (SEQ ID No:9, in the sequence list according to the direction from the 5' end to the 3' end). Thus, the adhesive ends of the same hairpin structure are partially complementary, and the adhesive ends of the two hairpin structures that make up the same structural unit are complementary.

[0039] In the specific design, two hairpin structures are interconnected to form a structural unit. The connecting region sequence is composed of a palindromic sequence (5'-ACTTCAGGCCTGAAGT-3'), which can self-pair complementaryly. To ensure the correct folding of the DNA single strand, two additional regions are designed in the hairpin structure to pair and form a double strand. The sequences a and c are 5'-GCTAAG-3', 5'-AATCTA-3', 5'-CTTAGC-3', and 5'-TAGATT-3', respectively. Among them, a and c pair complementaryly to form a double-stranded region, and b and d pair complementaryly to form a double-stranded region. Thus, the DNA single strand folds into a secondary structure with hairpin structures and sticky ends.

[0040] For ease of description, sequences a and b are defined as the first sticky end 5'-GCTAAGAATCTA-3' (SEQ ID No:6), and sequences c, d, and the connecting region sequence are defined as the second sticky end 3'-CGATTCTTAGATTGAAGTCCGGACTTCA-5' (SEQ ID No:7). The first sticky end sequence of another hairpin structure that complements and forms a structural unit is 5'-ACTTCAGGCCTGAAGTTAGATTCTTAGC-3' (SEQ ID No:8), and the second sticky end sequence is 3'-ATCTAAGAATCG-5' (SEQ ID No:9, in the sequence list according to the direction from the 5' end to the 3' end).

[0041] The anti-TNF-α aptamer is a single-stranded DNA that can specifically bind to tumor necrosis factor α (TNF-α), with the sequence 5'-TGGTGGATGGCGCAGTCGGCGACAA-3' (SEQ ID No:10).

[0042] The method for preparing DNA hydrogels includes the following steps: First, a basic stem-loop unit is designed. Each stem-loop unit consists of an unpaired single-stranded loop region and a complementary double-stranded stem region. The single-stranded loop region is a 17-nt long loop with a 5-base-pair (T5) region located in the center of the loop structure, flanked by two 6-base-pair (bp) interaction regions. The 6 bp regions are composed of palindromic sequences and can self-pair. The T5 regions remain unpaired. See [link to documentation]. Figure 1 In the hairpin structure, single-stranded loop regions in different hairpin structures can pair up to form connections. The 3' end of the DNA single strand connects to the first sticky end, and the 5' end connects to the second sticky end. Then, the DNA single strand of the anti-TNF-α aptamer is integrated into the second sticky end of the hairpin structure, endowing it with structural targeting function.

[0043] DNA oligonucleotides containing kissing hairpin cross-links and anti-TNF-α aptamers are typically prepared using a solid-phase phosphoramide method. The process involves sequential deprotection, coupling, oxidation, and blocking cycles on a universal vector, with nucleotides added one at a time. After each nucleotide addition, the 5' DMT protecting group is removed with acid to expose the hydroxyl group for the next nucleotide to link. After synthesis, the oligonucleotides are cleaved from the vector via ammonolysis, and all protecting groups are removed. Finally, high-purity DNA oligonucleotides are obtained using Genewiz's proprietary DHPLC purification method.

[0044] The above design involves a single-stranded DNA cell containing a kissing hairpin cross-linking structure and an anti-TNF-α aptamer. This single strand interacts with the DNA through the kissing hairpin structure to form a DNA hydrogel containing the kissing hairpin cross-linking structure and the anti-TNF-α aptamer. The preparation method is as follows: S1. Preparation of hydrogel solution containing kissing hairpin cross-linking structure and anti-TNF-α nucleic acid aptamer: Take 23.44 μL of 0.4 mM DNA solution, add 1.56 μL of enzyme-free water, and finally obtain 25 μL of DNA solution with a concentration of 0.375 mM.

[0045] S2. Assembly: A 25 μL solution of 0.375 mM DNA was annealed in a PCR instrument at 95°C for 5 min. Immediately after annealing, the solution was removed from the PCR instrument and incubated on ice for 5 min. Then, 2.78 μL of 10 mM MgCl2 was added to the 25 μL DNA solution to support the kissing hairpin cross-linking process. The final product was a 27.78 μL, 0.34 mM self-assembled hydrogel of anti-TNF-α aptamers based on a kissing hairpin cross-linking structure.

[0046] verify A non-kissing hairpin-crosslinked anti-TNF-α aptamer DNA hydrogel was prepared and designated as the non-kissing hairpin-crosslinked hydrogel, serving as the control group. The preparation method was as follows: S1. Design a non-kissing hairpin cross-link structure and a DNA sequence for the anti-TNF-α aptamer. Each hairpin cross-link structure contains a 17-nt loop, with two 6-base-pair (bp) regions on each side of the T5 region. The 6-bp regions are designed to be unpairable sequences, and the two T5 regions also remain unpaired. Therefore, the two hairpin structures cannot cross-link.

[0047] Non-kissing hairpin cross-linked DNA sequence (loop region); 5'-CACACATTTTTACACAC-3'; DNA sequence of anti-TNF-α aptamer: 5'-TGGTGGATGGCGCAGTCGGCGACAA-3'; S2: Preparation of hydrogel solution containing non-kissing hairpin cross-linked structure and anti-TNF-α nucleic acid aptamer: Take 23.44 μL of 0.4 mM non-kissing hairpin cross-linked DNA solution, add 1.56 μL of enzyme-free water, and finally obtain a DNA solution with a volume of 25 μL and a concentration of 0.375 mM.

[0048] S3. Assembly: A 25 μL solution of 0.375 mM DNA was annealed at 95°C for 5 min in a PCR instrument. Immediately after annealing, the solution was removed from the PCR instrument and incubated on ice for 5 min. Then, 2.78 μL of 10 mM MgCl2 was added to the 25 μL DNA solution to support the kissing hairpin cross-linking. The final product was a 27.78 μL, 0.34 mM non-kissing hairpin cross-linked structure and a self-assembled hydrogel of anti-TNF-α aptamers prepared based on the kissing hairpin cross-linking structure.

[0049] 1. Appearance Comparison The prepared hairpin crosslinked hydrogels (experimental group) and non-hairpin crosslinked products (control group) were transferred to 200 μL EP tubes and pre-frozen at -80℃ for 24 h. The next day, they were removed and placed into the chamber of a freeze dryer, the chamber door was closed, the freeze-drying program was started, the vacuum degree was set to 0.1 mber, and freeze-drying was carried out for at least 24 h. After freeze-drying was completed, the products of the experimental group and the control group were removed and observed.

[0050] The prepared kiss-shaped hairpin crosslinked hydrogel was white in color and had a loose structure; the control group's non-kiss-shaped hairpin crosslinked product was white in color and had a sheet-like structure. This is because the kiss-shaped hairpin crosslinked hydrogel, due to its special base pairing crosslinking, has a large network gap and a loose structure, resulting in a larger specific surface area and porosity, and thus a stronger swelling capacity. In contrast, the control group's non-kiss-shaped hairpin product was densely packed and had a low swelling rate.

[0051] 2. Scanning electron microscopy characterization Take 10 μL of the prepared kiss-shaped hair clip crosslinked hydrogel, freeze-dry it, cut it into appropriately sized pieces, spread it evenly on an 8 mm × 8 mm square silicon wafer, spray the silicon wafer with platinum, fix it on the sample stage, and measure it on the instrument.

[0052] like Figure 3 As shown in the figure, A and B are scanning electron microscope images of the kiss-shaped hair clip crosslinked hydrogel under different field of view sizes. It can be observed that after freeze-drying, the kiss-shaped hair clip crosslinked hydrogel exhibits a dense and porous network structure under the electron microscope, indicating that due to the special crosslinking method of the kiss-shaped hair clip, a stable network with a large number of pores is constructed, which verifies the formation of the kiss-shaped hair clip crosslinked hydrogel.

[0053] 3. Non-denaturing polyacrylamide gel electrophoresis characterization The cross-linked structure of the kissing hair clip was characterized by 8% non-denaturing polyacrylamide gel electrophoresis. Figure 4 As shown, the DNA single strands form a kissing hairpin structure through complementary base pairing. The bands in the figure indicate that kissing hairpin crosslinking is involved in the formation of larger structures.

[0054] Example 2 In vitro cytotoxicity assay The cell viability of MH7A cells in hydrogel culture medium containing gradient concentrations of kiss-hairpin cross-linked DNA was detected using the CCK-8 assay.

[0055] The cultivation method is as follows: 1) MH7A cells were digested with trypsin to prepare a cell suspension, and seeded into 96-well culture plates at a density of approximately 5000 cells per well, with 3 replicates for each concentration condition. The plates were then incubated overnight at 37°C in a 5% CO2 incubator. 2) After the cells have adhered (12 hours), aspirate the culture medium and wash thoroughly with PBS. Discard the PBS, then add 100 μL of culture medium to each well. Dilute the 0.34 mM hydrogel to 1×, 2×, 4×, and 8×, respectively. 3) Incubate the 96-well plate in an incubator for 24 hours, then wash MH7A three times with PBS. Add 10 μL of CCK-8 solution and 100 μL of DMEM to each well and co-incubate with the cells for 1 to 2 hours. Detect the cells at 450 nm using a microplate reader.

[0056] See Figure 5 The results showed that there were no significant differences between the experimental groups and the control group, and the cell viability did not change, confirming that the DNA hydrogel with the kiss-shaped hairpin cross-linking structure prepared in this application has excellent biocompatibility.

[0057] Example 3 Decomposition Experiment The decomposition rate of the kissing hairpin cross-linked structure prepared in this application was characterized by 8% non-denaturing polyacrylamide gel electrophoresis. The DNA hydrogel cross-linked structure for traditional delivery of nucleic acid aptamers is a G quadruple cross-linked structure, so the G quadruple cross-linked structure was selected as the control group.

[0058] Following the method described in Example 1, a 0.34 mM hydrogel with a hairpin crosslinking structure and a volume of 10 μL were prepared, as well as a 0.34 mM hydrogel with a G-quadruplex crosslinking structure (purchased from Suzhou Genewiz Biotechnology Co., Ltd.). Both hydrogel samples were subjected to polyacrylamide gel electrophoresis at three different temperatures: 4°C, 22°C, and 37°C.

[0059] See Figure 6 At 4℃, both the kiss-shaped hairpin crosslinked structure and the G-quadruplex crosslinked structure hydrogels were stable, and the gel images showed that the hydrogels were predominantly undissociated. Figure 7 As shown, at 22°C, the kissing hairpin cross-linked hydrogel decomposes, generating single-stranded DNA or oligomers. These smaller fragments migrate more rapidly in polyacrylamide gel electrophoresis, appearing as new bands located at lower positions; the G-quadruplex cross-linked hydrogel remains stable, with no change in band position, and does not enter the polyacrylamide gel. Figure 8 As shown, at 37℃, most of the kissing hairpin cross-linked structure decomposed, and the hydrogel dissociated into anti-TNF-α aptamer dimers located at the 150 bp position; the G quadruple cross-linked structure hydrogel remained stable and showed no decomposition.

[0060] It can be seen that the G quadruple crosslinked structure remains stable within the experimental temperature range due to its strong crosslinking effect, without significant decomposition. In contrast, the kissing hair clip crosslinked structure has a lower crosslinking strength, and its decomposition rate increases significantly with increasing temperature, enabling it to respond quickly to environmental changes after entering the human body.

[0061] Example 4 Treatment of experimental arthritis in mice 1. Prepare 800 μL of 8 μM DNA hydrogel containing a kissing hairpin cross-linked structure and an anti-TNF-α aptamer according to the method in Example 1. Simultaneously prepare 800 μL of 8 μM single-stranded anti-TNF-α aptamer solution.

[0062] 2. Establishment and administration of a mouse model of collagen-induced arthritis (CIA) The experimental animals were 32 male DBA / 1 mice aged 6 weeks. The 32 mice were randomly divided into four groups of 8 mice each: model group, normal group, anti-TNF-α aptamer single chain group, and hydrogel group containing kissing hairpin cross-linking structure and anti-TNF-α aptamer.

[0063] Modeling process: One day in advance, the fur on the backs of mice was shaved. An equal volume of 2 mg / mL chicken type II collagen and 2 mg / mL complete Freund's adjuvant were mixed and emulsified on ice to prepare a type II collagen emulsion. 0.1 mL of this emulsion was injected intradermally into the base of the tail of each mouse to induce inflammation; this was recorded as day 0. On day 21, another 0.1 mL of the emulsion was injected intradermally into the base of the tail of each mouse as a booster injection. Modeling was completed when the number of swollen joints and the arthritis inflammatory index in the mice reached their peak, and drug administration began.

[0064] Mice in the normal group were not subjected to modeling and were only fed according to standard procedures; this group was designated as Group 1. CIA mice were randomly divided into three groups: Group 2: Model group, injected with the same volume of physiological saline via tail vein; Group 3: Anti-TNF-α aptamer single-stranded group; Group 4: Hydrogels containing kissing hairpin cross-linking structures and anti-TNF-α nucleic acid aptamers; Experimental flowchart as follows Figure 9 As shown, mice in the anti-TNF-α aptamer single-chain group were administered 100 μL of 8 μM solution once every two days for a total of ten doses; mice in the hydrogel group containing the kissing hairpin cross-linking structure and the anti-TNF-α aptamer were administered 100 μL of 8 μM solution once every two days for a total of ten doses.

[0065] 3. Evaluation of therapeutic effect 1) Mouse body weight, arthritis index, and number of swollen joints. The paws of mice in each group were photographed and recorded at the peak of modeling and at the end of drug administration. Figure 10 From day 28 onwards, the mice's body weight, arthritis index, and number of swollen joints were recorded every three days. Figure 11 The first group of mice had a relatively stable body weight with a slight increase; the second group of mice initially showed a decreasing body weight; and the third and fourth groups began to gradually increase their body weight from day 37. This indicates that the development of arthritis may be accompanied by changes in basic vital signs such as weight loss in mice.

[0066] like Figure 12 and Figure 13As shown, compared with the second group, the third and fourth groups of mice showed a significant decrease in arthritis index and number of swollen joints after treatment; the fourth group also showed a faster decrease in scores compared with the third group. This demonstrates that the hydrogel containing the kissing hairpin cross-linking structure and the anti-TNF-α aptamer is more effective than the single-chain anti-TNF-α aptamer group.

[0067] 2) ELISA detection of TNF-α in serum Whole blood was collected from all mice via ocular sampling at the end of the experiment. After standing at room temperature, the blood samples were centrifuged to obtain the supernatant serum. The concentration of TNF-α in the serum was detected using a commercially available ELISA kit, following the manufacturer's instructions. After the colorimetric reaction was terminated, the absorbance of each well was measured using a microplate reader at a detection wavelength of 450 nm and a reference wavelength of 610 nm, and the concentration of TNF-α in the sample was calculated accordingly.

[0068] like Figure 14 As shown, the serum TNF-α concentration in mice in each treatment group was significantly lower than that in the model group (Group 2), with statistically significant differences. The decrease was more pronounced in Group 4, demonstrating that the hydrogel containing the kissing hairpin cross-linking structure and the anti-TNF-α nucleic acid aptamer had a better therapeutic effect than the single-chain group containing the anti-TNF-α nucleic acid aptamer.

[0069] 3) Biosafety testing H&E staining analysis is a core safety assessment tool in animal experiments. By visually displaying changes in organ and tissue structure and cell morphology, it can comprehensively reveal the potential toxicity of experimental interventions, verify model stability, and provide morphological evidence for mechanism research.

[0070] At the end of the experiment, mice were anesthetized and euthanized. Liver, spleen, and kidneys were harvested, and each tissue was immediately placed in 4% paraformaldehyde for dehydration, paraffin embedding, sectioning, and H&E staining. Tissue sections were observed using a tissue slide scanner; see [link to documentation]. Figure 15 No organic changes were observed in the major organs of mice in each treatment group, demonstrating that the hydrogel containing a kissing hairpin cross-linking structure and an anti-TNF-α nucleic acid aptamer has good biocompatibility and can ensure a balance between efficacy and safety.

[0071] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A DNA hydrogel with a kissing hairpin cross-linking structure, characterized in that, The device includes hairpin structures comprising two stem-loop units formed by the folding of a single DNA strand. The first end of the single DNA strand is connected to a first sticky end carrying a nucleic acid aptamer that specifically recognizes TNF-α, and the second end is connected to a second sticky end. Two hairpin structures with identical structures but opposite sticky end sequences form a structural unit through complementary base pairing at their sticky ends. The loop regions of adjacent structural units form a kissing hairpin connection through complementary base pairing. Multiple structural units are connected through kissing hairpin connections to form a DNA hydrogel with a three-dimensional network backbone.

2. The DNA hydrogel with a kissing hairpin cross-linking structure as described in claim 1, characterized in that, Each stem-loop unit consists of an unpaired single-stranded loop region and a complementary double-stranded stem region. The DNA sequence of the single-stranded loop region is shown in SEQ ID No:

1. The DNA sequences of the double-stranded stem regions of the first stem-loop unit are shown in SEQ ID No:2 and SEQ ID No:3, respectively. The DNA sequences of the double-stranded stem regions of the second stem-loop unit are shown in SEQ ID No:4 and SEQ ID No:5, respectively. The sequences of the first sticky end and the second sticky end are shown in SEQ ID No:6 and SEQ ID No:7, respectively, or in SEQ ID No:8 and SEQ ID No:9, respectively. The sequence of the TNF-α aptamer is shown in SEQ ID No:

10.

3. A method for preparing a DNA hydrogel with a kissing hairpin cross-linking structure as described in claim 1, characterized in that, Includes the following steps: S1. Based on the hairpin structure, design a single-stranded DNA sequence and integrate the selected anti-TNF-α nucleic acid aptamer into the stem region of the hairpin structure to form a single-stranded DNA sequence containing the hairpin cross-linking structure to be cross-linked and the anti-TNF-α nucleic acid aptamer. S2. Preparation of hydrogel solution containing kissing hairpin cross-linking structure and anti-TNF-α nucleic acid aptamer: Take 0.4 mM of the DNA single strand solution and add enzyme-free water to obtain a DNA single strand solution with a final concentration of 0.375 mM; S3. Assembly: The obtained DNA single-stranded solution was annealed in a PCR instrument. After annealing, it was removed from the PCR instrument and placed in an ice bath for 5 minutes. Then, 10 mM MgCl2 was added to complete the cross-linking reaction, resulting in the DNA hydrogel with the desired kissing hairpin cross-linking structure.

4. The preparation method according to claim 3, characterized in that, The annealing conditions were 95℃ for 5 minutes.

5. The preparation method according to claim 3, characterized in that, The ratio of MgCl2 to DNA single-strand solution was 2.78 μL: 25 μL.

6. The use of a DNA hydrogel with a kissing hairpin cross-linking structure as described in claim 1 or 2 in the preparation of a product for treating rheumatoid arthritis.

7. A product for treating rheumatoid arthritis, characterized in that, A DNA hydrogel comprising a kissing hairpin crosslinking structure as described in claim 1 or 2.

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