Inflammation-responsive DNAzyme delivery system, preparation method and application thereof

Abstract

This invention discloses an inflammation-responsive DNAzyme delivery system, comprising a DNA tetrahedron and an inflammation-responsive DNAzyme. At least one vertex of the DNA tetrahedron carries an extended strand with a nucleotide sequence as shown in SEQ ID NO:2. The inflammation-responsive DNAzyme is loaded onto the DNA tetrahedron through partial or complete complementation to the extended strand, and an apurinyl / pyrimidine (AP) site is designed on the inflammation-responsive DNAzyme. The delivery system provided by this invention can effectively improve the uptake efficiency of DNAzyme by cells and achieve targeted delivery of DNAzyme to inflammatory cells and controlled release within inflammatory cells. The hgd40-tFNA provided by this invention can effectively improve colitis by reducing the inflammatory response of inflammatory cells and restoring intestinal barrier function, and can be used to prepare drugs for treating inflammatory bowel diseases, especially ulcerative colitis.

Description

Inflammation-responsive DNA zyme delivery system, its preparation method and application Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an inflammation-responsive DNAzyme delivery system, its preparation method, and its application. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease that is currently incurable, placing a heavy burden on patients' quality of life. Immune dysregulation and the dysregulation of pro-inflammatory cytokine expression play a crucial role in the pathogenesis of UC. Dysfunctional helper T cells 2 (Th2) in UC can exacerbate the disease by secreting cytokines such as IL-5 and IL-13. GATA3 is a major transcriptional regulator in the Th2-driven immune response in UC, controlling Th2 cell differentiation, activation, and cytokine expression. Its expression level is significantly increased in the mucosa of UC patients. In mouse models, T cell-specific overexpression of GATA3 enhances IL-13 production and significantly aggravates DSS-induced colitis. Conversely, gene knockdown of GATA3 in T cells or targeted degradation of GATA3 using DNase technology can effectively reduce mucosal inflammation and alleviate UC symptoms. Therefore, GATA3 has become a promising therapeutic target for UC.

[0003] Deoxyribozymes (DNAzymes) are catalytic oligonucleotides with highly efficient catalytic activity and structure recognition capabilities. DNAzymes with RNA cleavage activity catalyze the cleavage of specific RNA sites, mediating mRNA cleavage and inhibiting protein expression at the mRNA level. hgd40 is a GATA3-specific DNAzyme, a 10-23 type DNAzyme with a 15-nucleotide catalytic domain flanked by two GATA3 mRNA substrate-binding domains. The nucleotide sequence of hgd40 is shown in SEQ ID NO:1, and its functional schematic diagram is shown in Figure 1. In an oxazolone (CAS:15646-46-5) and trinitrobenzenesulfonic acid (TNBS)-induced colitis model, hgd40 has been shown to inhibit GATA3 expression and significantly improve colitis through a TNF-independent mechanism after rectal administration.

[0004] Although DNAzymes have shown great therapeutic potential in the treatment of ulcerative colitis (UC), their clinical efficacy remains unsatisfactory due to their short half-life, non-specific tissue distribution, and low cellular uptake. Further research and development are needed to create a DNAzyme delivery system capable of intestinal-targeted delivery and controlled release to improve the clinical efficacy of DNAzymes in UC treatment. Summary of the Invention

[0005] The main objective of this invention is to provide a DNAzyme delivery system to solve at least one of the above-mentioned technical problems.

[0006] According to one aspect of the present invention, an inflammation-responsive DNAzyme delivery system is provided, comprising a DNA tetrahedron and an inflammation-responsive DNAzyme, wherein at least one vertex of the DNA tetrahedron has an extended strand with a nucleotide sequence as shown in SEQ ID NO:2; the inflammation-responsive DNAzyme is loaded on the DNA tetrahedron by being partially or completely complementary to the extended strand, and the inflammation-responsive DNAzyme is designed with a depurinyl / depyrimidine (AP) site.

[0007] In the inflammatory-responsive DNAzyme delivery system provided by this invention, DNA tetrahedra can effectively penetrate biological barriers through a "corner attack" mechanism, thereby significantly improving the cellular uptake efficiency of DNAzymes. The apurinyl / depyrimidine endonuclease 1 (APE1) can recognize the AP site and cleave the DNA sugar-phosphate backbone at the 5' end of the AP site to release the DNAzyme. Furthermore, while APE1 is located only in the nucleus in normal cells, it translocates to the cytoplasm during inflammation. Therefore, by utilizing APE1's recognition and cleavage of the AP site, and its different distribution in normal and inflammatory cells, targeted delivery of DNAzymes to inflammatory cells and controlled release within inflammatory cells can be achieved.

[0008] In some embodiments, the DNA tetrahedron is synthesized by hybridization of eight DNA single strands; wherein at least one DNA single strand contains a nucleotide sequence as shown in SEQ ID NO:2, such that at least one vertex of the hybridized DNA tetrahedron has an extended strand with a nucleotide sequence as shown in SEQ ID NO:2.

[0009] In some embodiments, the nucleotide sequences of the eight DNA single strands used for hybridization to synthesize the DNA tetrahedron are shown in SEQ ID NO:8~15, respectively.

[0010] In some embodiments, the nucleotide sequence of the inflammatory response DNAzyme consists of a Linker chain, a (polyT)n sequence composed of n T-base nucleotides, and a DNAzyme chain from the 5' end to the 3' end; the Linker chain is partially or completely complementary to the extended chain, and an AP site is provided on the Linker chain at a position 1 to 4 bp away from the (polyT)n sequence.

[0011] In some implementations, n is selected from natural numbers between 2 and 5. This increases the degrees of freedom of the DNAzyme strand.

[0012] In some embodiments, the DNAzyme is hgd40, and the nucleotide sequence of the DNAzyme chain is shown in SEQ ID NO:1.

[0013] In some implementations, the nucleotide sequence of the inflammatory-responsive DNAzyme is shown in SEQ ID NO:5.

[0014] The inflammatory-responsive hgd40 delivery system (hgd40-tFNA) provided by this invention exhibits effective cellular uptake and intestinal accumulation after intravenous injection. Furthermore, it selectively releases hgd40 into inflammatory cells under APE1-mediated cytoplasmic regulation, effectively knocking down GATA3 in inflammatory cells while maintaining low activity in normal cells. The hgd40-tFNA provided by this invention can effectively improve colitis by reducing the inflammatory response of inflammatory cells and restoring intestinal epithelial homeostasis. It can be used in the preparation of drugs for treating inflammatory bowel diseases, especially ulcerative colitis.

[0015] According to another aspect of the present invention, a method for preparing an inflammation-responsive DNA zyme delivery system is provided, comprising the following steps:

[0016] The DNA single strands used for hybridization to synthesize DNA tetrahedra and the inflammatory-responsive DNAzyme are added to TM buffer, mixed, heated at 95°C for 10-20 min, and then cooled to 4°C to obtain the final product.

[0017] In some implementations, the TM buffer has a pH of 8.0 and comprises 2 mM Tris and 5 mM MgCl2.

[0018] In some embodiments, the molar ratio of any two DNA single strands used for hybridization to synthesize DNA tetrahedra is 1:1.

[0019] In some implementations, the molar ratio of the inflammatory-responsive DNAzyme to any single strand of DNA used for hybridization to synthesize the DNA tetrahedron is 1:1. Attached Figure Description

[0020] Figure 1 is a schematic diagram of the sequence and function of GATA3-specific DNA zyme hgd40;

[0021] Figure 2 shows the characterization and release results of hgd40-tFNA, where a is a schematic diagram of the structure of hgd40-tFNA; b is a schematic diagram of the cell-selective release of hgd40 from hgd40-tFNA mediated by APE1 and the process of achieving GATA3 mRNA silencing; c is the PAGE analysis results of tFNA, tFNA-linker, and hgd40-tFNA; d is a representative AFM image of hgd40-tFNA, scale bar = 50 nm; e is a schematic diagram of the release process of hgd40 from hgd40-tFNA mediated by APE1 and the fluorescence kinetic curve; f is a schematic diagram of the cellular release of hgd40 from hgd40-tFNA mediated by APE1; g is a representative immunofluorescence staining image of APE1 (red) and the cell nucleus (Hoechst, blue) in Jurkat cells, scale bar = 10 nm. μm; h represents the fluorescence image of hgd40-tFNA released in Jurkat cells pretreated with or without LPS;

[0022] Figure 3 shows the dynamic light scattering (DLS) analysis results of tFNA and hgd40-tFNA, where: a is the size plot; b is the zeta potential plot;

[0023] Figure 4 shows the results of the gene silencing effect analysis of hgd40-tFNA; where a is a representative fluorescence image of free hgd40 and hgd40-tFNA after co-incubation with Caco-2 cells for 6 hours, Hoechst labeled the cell nucleus, Cellmaskgreen labeled the cell membrane, scale bar = 10 μm; b is a schematic diagram of the inhibition of GATA3 expression by hgd40-tFNA with or without AP lock in LPS pretreated Jurkat cells and GATA3. Figure c shows the RT-qPCR analysis results of mRNA; c is a schematic diagram of hgd40-tFNA inhibiting GATA3 expression in Jurkat cells pretreated with or without LPS pretreatment, along with the RT-qPCR analysis results of GATA3 mRNA; d is the WB analysis results of GATA3 protein in Jurkat cells pretreated with LPS after treatment with free hgd40, tFNA, and hgd40-tFNA; ef and ef are the RT-qPCR analysis results of IL-5 and IL-13 mRNA in Jurkat cells pretreated with LPS after treatment with hgd40-tFNA; compared with the PBS group, * P<0.05, ** P<0.01, ***P<0.001; compared with the hgd40-tFNA group without AP lock, ## P<0.01;

[0024] Figure 5 shows the biodistribution analysis results of hgd40-tFNA in mice, where a is a schematic diagram of the experimental procedure; bc are representative fluorescence images and quantitative fluorescence intensity analysis results of normal mice and DSS-induced colitis mice at different time points after tail vein injection of ssDNA and hgd40-tFNA; de are representative in vitro fluorescence images and quantitative fluorescence intensity analysis results of major organs (heart, liver, spleen, lung, kidney, pancreas, and intestine) of normal mice and DSS-induced colitis mice 24 hours after tail vein injection of ssDNA and hgd40-tFNA; fg is representative fluorescence images and quantitative fluorescence intensity analysis results of colon tissue sections of normal mice and DSS-induced colitis mice 24 hours after tail vein injection of ssDNA and hgd40-tFNA, scale bar = 50 μm; compared with normal mice injected with tail vein ssDNA, *** P<0.001, ### P<0.001;

[0025] Figure 6 shows the in vivo biosafety evaluation results of hgd40-tFNA injected into mice via the tail vein. In Figure 6, a is a representative H&E staining image of tissue sections from the heart, liver, spleen, lung, and kidney, with a scale bar of 100 μm; b and c are the detection results of key liver and kidney function serum biochemical indicators: alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and urea (UREA); compared with the PBS group, ns indicates no significant difference.

[0026] Figure 7 shows the analysis results of hgd40-tFNA improving DSS-induced colitis in mice; where a is a schematic diagram of the experimental procedure; b is a graph of the weight change of mice in each group; c is a representative image of the colonic tissue of mice in each group; d is the colonic length of mice in each group; e is the disease activity index of mice in each group; f is a representative H&E staining image of colonic tissue sections of mice in each group, scale bar = 100 μm; gh is the CD3 concentration in colonic tissue sections of mice in each group. + T cells and F4 / 80 + Representative immunohistochemical staining images and quantitative cell counting results of macrophages, scale bar = 50 μm; ik represents the RT-qPCR analysis results of IL-1β, IL-6, and TNF-α mRNA expression levels in colon tissue of mice in each group; l represents the histopathological score of colon tissue of mice in each group; ns indicates no significant difference compared with the PBS group. *** P<0.001; compared with the free hgd40 group, ###P<0.001;

[0027] Figure 8 shows the analysis results of hgd40-tFNA in restoring intestinal barrier function. Figure a shows the RT-qPCR analysis results of GATA3 mRNA expression levels in colon tissues of mice in each group; Figure b shows representative immunohistochemical staining of GATA3 in colon tissue sections of mice in each group (scale bar = 25 μm); Figure c shows the RT-qPCR analysis results of IL-13 mRNA expression levels in colon tissues of mice in each group; Figure d shows the quantitative analysis results of FITC-glucan concentration in serum of mice in each group; Figure e shows the WB analysis results of ZO-1, Occludin, Cleaved-caspase 3 (CC 3), and Cleaved PARP (C-PARP) in colon tissues of mice in each group; Figure f shows representative immunohistochemical staining of Cleaved-caspase 3 (CC 3) and ZO-1 in colon tissue sections of mice in each group (scale bar = 50 μm); Figure g shows the quantitative analysis results of Cleaved-caspase 3 (CC 3) positive cells in colon tissues of mice in each group; compared with the PBS group, * P<0.05, ** P<0.01, *** P<0.001; compared with the free hgd40 group, # P<0.05, ## P<0.01, ### P<0.001. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the embodiments. The embodiments are for illustrative purposes only and do not limit the invention in any way. Unless otherwise specified, the raw materials and reagents used in the embodiments are conventional products that can be obtained commercially; experimental methods that do not specify specific conditions in the embodiments are generally performed under conventional conditions in the art or according to the conditions recommended by the manufacturer.

[0029] The nucleotide sequences used in this invention and associated with the synthesis of hgd40-tFNA are shown in Table 1. All sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0030] Table 1. Nucleotide sequences associated with the synthesis of hgd40-tFNA

[0031]

[0032] In this invention, Jurkat cells were cultured in RPMI 1640 medium, and Caco-2 cells (human colorectal adenocarcinoma cells) were cultured in DMEM medium. All media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.

[0033] Male C57BL / 6J mice (23–25 g) were purchased from Spifort (Beijing) Biotechnology Co., Ltd. The mice were housed in a 12 / 12-hour light / dark cycle and a specific pathogen-free environment with free access to food and water.

[0034] Relevant experimental data are expressed as mean ± standard deviation (n≥3). Statistical significance was determined using GraphPad Prism 8.0. One-way ANOVA was used for analyses among multiple groups, and two-tailed Student's t-test was used for paired comparisons. Significance level: "ns" indicates no significant difference. * This indicates that P < 0.05. ** This indicates that P < 0.01. *** This indicates that P < 0.001.

[0035] Example 1: Preparation of hgd40-tFNA

[0036] Includes the following steps:

[0037] Take oligonucleotide chains S1, A1, A2, B1, B2, C1, C2, D1, and D2 as shown in Table 1, mix them in equimolar amounts in TM buffer (2 mM Tris, 5 mM MgCl2, pH=8.0), then heat the mixture to 95°C, maintain for 15 min, and then cool to 4°C to obtain hgd40-tFNA.

[0038] A schematic diagram of the hgd40-tFNA structure is shown in Figure 2a. The tetrahedral shape and nanoscale size of hgd40-tFNA enable it to effectively penetrate biological barriers via a "corner attack" mechanism. hgd40 is secured to the vertices of the tFNA via an "AP lock" using AP as the lock and APE1 as the key, allowing for cell-selective release of hgd40 by utilizing the different distributions of APE1 in normal and inflammatory cells (Figure 2b).

[0039] in addition:

[0040] Cy3-labeled hgd40-tFNA was prepared by replacing S1 with Cy3-S1, and is denoted as Cy3-hgd40-tFNA.

[0041] The AP-locking fluorescent quencher (Cy3-BHQ2) labeled hgd40-tFNA was prepared by replacing S1 with Cy3-S1 and A1 with BHQ2-A1, and is denoted as hgd40-tFNA (Cy3-BHQ2).

[0042] hgd40-tFNA, labeled with Cy3 and Cy5, was prepared by replacing S1 with Cy3-S1 and C1 with Cy5-C1. This was denoted as hgd40-tFNA (Cy3-Cy5).

[0043] hgd40-tFNA (Cy3-BHQ2-Cy5) was prepared by replacing S1 with Cy3-S1, A1 with BHQ2-A1, and C1 with Cy5-C1.

[0044] AP-free hgd40-tFNA was prepared by replacing S1 with S2;

[0045] hgd40-tFNA labeled with Cy5 was prepared by replacing C1 with Cy5-C1, and denoted as hgd40-tFNA-Cy5; similarly, hgd40-tFNA-Cy5.5 was prepared by replacing C1 with Cy5.5-C1.

[0046] Cy5-labeled tFNA was prepared using A1-0, A2, B1, B2, Cy5-C1, C2, D1, and D2, and denoted as Cy5-tFNA. Similarly, Cy5.5-tFNA was prepared using Cy5.5-C1 instead of Cy5-C1.

[0047] tFNA-linkers were prepared using Linker chains A1, A2, B1, B2, C1, C2, D1, and D2 for the following experimental studies.

[0048] Example 1: Characterization of tFNA and hgd40-tFNA

[0049] The zeta potential, particle size, and polydispersity index of tFNA and hgd40-tFNA were measured by dynamic light scattering (DLS) using a Malvern Panaco nanoparticle size potentiometer, Zetasizer Pro (Malvern).

[0050] The morphological features of hgd40-tFNA were characterized using atomic force microscopy (AFM).

[0051] The synthesis process of tFNA and hgd40-tFNA was evaluated by polyacrylamide gel electrophoresis (PAGE). Samples were electrophoresed on 6% polyacrylamide gels at 100V for 60 min with 1×TAE buffer, followed by gel red staining for 10 min, and finally imaged using a gel imaging system (ChemiDocMP, Bio-rad).

[0052] The results are shown in Figures 2 and 3.

[0053] PAGE analysis showed that the migration rates of hgd40-tFNA, tFNA-linker, and tFNA decreased sequentially with increasing molecular weight. Furthermore, the Cy3-labeled hgd40 chain (red) and the GelRed-stained tFNA (green) showed good overlap, confirming that hgd40 was successfully loaded onto tFNA and that hgd40-tFNA successfully self-assembled (c in Figure 2).

[0054] AFM imaging results also show that hgd40-tFNA exhibits a typical uniform-sized three-dimensional tetrahedral structure (d in Figure 2).

[0055] Dynamic light scattering (DLS) results showed that hgd40-tFNA had a larger hydrodynamic size (22.75 ± 0.08 nm) and a lower zeta potential (-19.83 ± 1.16 mV) compared to tFNA (Figure 3).

[0056] Experimental Example 2: Verification of APE1-mediated controlled release of hgd40

[0057] The hgd40-tFNA labeled with Cy3-BHQ2 (Cy3-BHQ2) (200 nM) was mixed with different concentrations of APE1 (0, 0.5, 1, 2.5 U / mL) and incubated at 37°C. The fluorescence intensity of Cy3 was measured using a microplate reader (BioTek, SYNERGY H1).

[0058] The result is shown in Figure 2e.

[0059] In hgd40-tFNA, due to the short distance (<10 nm) between Cy3 and BHQ2 molecules, the Cy3 fluorescence in the AP lock is initially quenched by BHQ2. After incubation with APE1, the Cy3 fluorescence gradually increases over time, and the intensity is positively correlated with the APE1 concentration (e in Figure 2), proving that APE1 can cleave the AP site, leading to the release of hgd40.

[0060] Experiment 3: Verification of Selective Cellular Release of HGD40

[0061] A schematic diagram of APE1-mediated hgd40 release at the cellular level is shown in Figure 2f.

[0062] 50,000 Jurkat cells were pretreated with 500 ng / mL LPS for 6 hours to induce an inflammatory state, resulting in inflammatory cells with high GATA3 expression.

[0063] (1) Immunofluorescence staining of APE1

[0064] Inflammatory cells and untreated Jurkat cells were fixed in 4% paraformaldehyde (15 min), permeabilized with 0.1% Triton X-100 (10 min), and blocked with 5% BSA (1 h). Incubation with primary antibodies (APE1, Abcam, ab189474) was performed overnight at 4°C, followed by incubation with FITC-conjugated secondary antibodies at room temperature for 2 h. Cell nuclei were counterstained with Hoechst. Images were captured using a fluorescence microscope (Leica Thunder Imager DMI8, Germany).

[0065] The result is shown as g in Figure 2.

[0066] Fluorescence imaging results showed that in normal cells, APE1 is located only in the nucleus; however, when cells transition to an inflammatory state, some of APE1 translocates into the cytoplasm.

[0067] (2) Cellular selective release of HGD40

[0068] hgd40-tFNA (Cy3-BHQ2-Cy5) (200 nM) was co-incubated with normal or LPS-pretreated Jurkat cells for 4 hours. Cells were collected, stained with Hoechst, and then images were captured using a fluorescence microscope (Leica Thunder Imager DMI8, Germany) to observe the distribution of Cy3 and Cy5 fluorescence signals in the cells.

[0069] The result is shown in h in Figure 2.

[0070] Fluorescence imaging results showed that Cy5 fluorescence signals were observed in both normal Jurkat cells and LPS-pretreated Jurkat cells, with comparable signal intensities, indicating similar uptake levels of tFNA in both cell types. However, the Cy3 fluorescence signal in inflammatory cells was significantly stronger than that in normal cells, suggesting that the hgd40-tFNA provided by this invention can achieve selective release of hgd40 from inflammatory cells.

[0071] Experiment 4: Verification of effective GATA3 gene silencing effect of hgd40-tFNA in inflammatory cells

[0072] (1) Cellular uptake of hgd40-tFNA

[0073] Caco-2 cells were grown at 3 × 10⁻⁶ per dish. 4 Cells were seeded at a density of [number] cells per 35 mm confocal dish. Cells were incubated for 6 hours with either Cy3-labeled free hgd40 (Cy3-hgd40, 200 nM) or Cy3-hgd40-tFNA (200 nM). Subsequently, they were treated with CellMask. TM Cell membranes were stained with Green (2 µg / mL) for 5 minutes, followed by counterstaining of the nuclei with Hoechst. Fluorescence images were acquired using a fluorescence microscope (Leica Thunder Imager DMI8).

[0074] The result is shown in Figure 4a.

[0075] Therapeutic oligonucleotides typically struggle to cross the cell membrane barrier and enter the cytoplasm to exert their gene-silencing function during delivery due to strong electrostatic repulsion. However, tFNA can rapidly enter cells via a "corner attack" mechanism. Fluorescence imaging results showed negligible Cy3 fluorescence in Caco-2 cells with free hgd40, while Cy3 exhibited a strong signal in Caco-2 cells with hgd40-tFNA, indicating minimal uptake of free hgd40 by cells, and efficient internalization of hgd40-tFNA.

[0076] (2) Gene silencing efficiency of hgd40-tFNA

[0077] The tetrahedral structure of tFNA may create steric hindrance, preventing hgd40 from binding to GATA3 mRNA with high affinity. To investigate whether gene silencing efficiency is affected by steric hindrance, LPS-pretreated Jurkat cells were seeded in 24-well plates at 200,000 cells per well. PBS, AP-free hgd40-tFNA (400 nM), and hgd40-tFNA (400 nM) were then added to the LPS-pretreated Jurkat cells, respectively, and incubated for 12 h. Cells were then collected and GATA3 mRNA knockdown efficiency was analyzed by RT-qPCR.

[0078] Total RNA was extracted from cells using Trizol reagents according to the manufacturer's protocol. cDNA was synthesized using cDNA SynthnesisSuperMix. Quantitative real-time PCR (RT-qPCR) was performed using the SsoAdvanced Universal SYBR Green-based detection system. Primer sequences are listed in Table 2 below.

[0079] Table 2 RT-qPCR primers

[0080]

[0081] The result is shown in Figure 4b.

[0082] RT-qPCR analysis showed that hgd40-tFNA containing the AP lock (APE1-mediated release) achieved approximately 69% knockdown efficiency of GATA3 mRNA in LPS-pretreated Jurkat cells. In contrast, hgd40-tFNA without the AP lock exhibited a lower knockdown efficiency of only about 28% for GATA3 mRNA. These results indicate that APE1-mediated hgd40 release can enhance the gene silencing efficiency of hgd40-tFNA for GATA3.

[0083] (3) Gene silencing efficiency of hgd40-tFNA in normal and inflammatory cells

[0084] Normal Jurkat cells and LPS-pretreated Jurkat cells were seeded into 24-well plates, 200,000 cells per well. PBS and hgd40-tFNA (400 nM) were added to the cells, and the cells were incubated for 12 h. The knockdown efficiency of GATA3 mRNA was analyzed by RT-qPCR.

[0085] The result is shown in Figure 4c.

[0086] RT-qPCR analysis showed that GATA3 mRNA in LPS-pretreated cells was significantly silenced by hgd40-tFNA (approximately 65% ​​knockdown efficiency) compared to normal cells (approximately 24% knockdown efficiency), indicating that hgd40-tFNA can function efficiently in inflammatory cells while maintaining low activity in normal cells.

[0087] (4) Western blot (WB) analysis of GATA3 protein expression level in inflammatory cells

[0088] Jurkat cells pretreated with LPS were seeded into 6-well plates with 800,000 cells per well. Then, PBS, free hgd40 (400 nM), tFNA (400 nM), and hgd40-tFNA (400 nM) were added to the LPS-pretreated Jurkat cells and incubated for 36 h. The cells were then collected for Western blot analysis.

[0089] Cells were lysed in RIPA buffer, and tissue proteins were extracted by homogenization with RIPA buffer. Protein concentration was determined using the BCA method. Proteins were separated by 10% SDS-PAGE and then transferred to a PVDF membrane. After blocking with 5% skim milk powder, the membrane was incubated with primary antibody overnight at 4°C, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature (RT) for 1 hour. Finally, protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit.

[0090] The result is shown in d of Figure 4.

[0091] Western blot analysis showed that, compared with the free hgd40 group, the expression level of GATA3 protein in inflammatory cells of the hgd40-tFNA group was effectively reduced.

[0092] (5) Effects on the expression levels of inflammatory cytokine mRNA

[0093] Normal Jurkat cells and LPS-pretreated Jurkat cells were seeded into 24-well plates, with 200,000 cells per well. PBS was added to the normal Jurkat cells, while PBS and hgd40-tFNA (400 nM) were added to the LPS-pretreated Jurkat cells. The cells were incubated for 12 h. Cells were then collected and the expression levels of IL-5 and IL-13 mRNA were analyzed by RT-qPCR.

[0094] The results are shown in e and f in Figure 4.

[0095] Inhibition of the GATA3 factor can alleviate intracellular inflammatory responses by downregulating the expression of a range of genes, including IL-5 and IL-13, through binding to a conserved promoter sequence.

[0096] RT-qPCR analysis showed that hgd40-tFNA treatment significantly reduced the expression levels of IL-5 and IL-13 mRNA in inflammatory cells.

[0097] The above results indicate that the hgd40-tFNA provided by this invention can effectively silence the GATA3 gene and inhibit GATA3 expression in inflammatory cells through APE1-mediated hgd40 release, while maintaining low activity in normal cells.

[0098] Experimental Example 5: Biodistribution and Biosafety Evaluation of hgd40-tFNA

[0099] (1) Tracking the distribution of organisms in the body

[0100] To induce ulcerative colitis (UC), 3% sodium dextran sulfate (DSS) was added to the drinking water of mice for 7 days to construct DSS-induced colitis mice.

[0101] Normal male C57BL / 6J mice were randomly divided into two groups of three. Each group received either 200 μL of Cy5.5-labeled ssDNA (Cy5.5-C1, 1 μM) or Cy5.5-labeled hgd40-tFNA (Cy5.5-hgd40-tFNA, 1 μM) via tail vein injection. Three DSS-induced colitis mice were randomly selected and injected with 200 μL of Cy5.5-hgd40-tFNA (1 μM) via tail vein injection. At 0.5, 1, 8, and 24 hours post-injection, abdominal signals were captured using an in vivo fluorescence imaging system (IVIS) to assess the biodistribution of ssDNA and hgd40-tFNA. A schematic diagram of the experimental procedure is shown in Figure 5a. Twenty-four hours post-injection, the mice were sacrificed, and major organs (heart, liver, spleen, lung, kidney, pancreas, and intestine) were removed for in vitro fluorescence imaging.

[0102] A 0.5 cm section of distal rectal tissue was taken, fixed in 4% paraformaldehyde at 4°C for 48 hours, dehydrated with a series of ethanol solutions, cleared with xylene, and finally embedded in paraffin. Sections were then prepared to obtain colon tissue sections. Fluorescence imaging was performed on the colon tissue sections.

[0103] The results are shown in Figure 5.

[0104] In vivo fluorescence imaging in mice showed that both ssDNA and hgd40-tFNA exhibited strong Cy5.5 fluorescence signals in the mouse abdomen (where the intestines are located). Specifically, hgd40-tFNA showed prolonged retention in the abdomens of both healthy and colitis-affected mice, maintaining a strong fluorescence signal for 24 hours. In contrast, ssDNA underwent rapid systemic clearance, with its fluorescence signal decreasing to almost undetectable levels at 24 hours (Figure 5, bc). Ex vivo fluorescence imaging of organs further revealed that, 24 hours post-injection, the fluorescence signals of both ssDNA and hgd40-tFNA were primarily distributed in the intestines, with hgd40-tFNA showing the strongest fluorescence intensity in colitis-affected mice (Figure 5, de). Fluorescence imaging of colon tissue sections showed that hgd40-tFNA fluorescence was located within intestinal cells, and the signal intensity in colitis-affected mice was stronger than in normal mice (Figure 5, fg).

[0105] (2) In vivo biosafety

[0106] Normal male C57BL / 6J mice were randomly divided into two groups of four each. Each group received a tail vein injection of either 200 μL PBS or hgd40-tFNA (2 μM), three times a week. On day 8, major tissues (heart, liver, spleen, lung, and kidney) and serum were collected for analysis. Tissue samples were prepared into sections and stained with H&E according to standard procedures for histopathological evaluation. Key blood biochemical parameters—alanine transaminase (ALT), aspartate transaminase (AST), creatinine (CREA), and urea (UREA)—were measured in serum using an automated biochemical analyzer according to the manufacturer's protocol.

[0107] The results are shown in Figure 6. H&E staining results showed no abnormalities in the major organs of the mice, while serum biochemistry confirmed that the liver / kidney function of the mice was normal.

[0108] The above results demonstrate that the hgd40-tFNA provided by this invention exhibits high intestinal accumulation and biocompatibility.

[0109] Experiment 6: Evaluation of the in vivo therapeutic effect of hgd40-tFNA

[0110] Ulcerative colitis (UC) was induced in mice by adding 3% sodium dextran sulfate (DSS) to their drinking water for 7 consecutive days.

[0111] Normal male C57BL / 6J mice were randomly divided into 5 groups: a normal control group of 5 mice and each of the other groups of 6 mice.

[0112] Normal control group (Control): Normal feeding, drinking sterile water;

[0113] PBS group: Drink sterile water containing 3% DSS and receive 200 μL PBS via tail vein injection on days 1 and 4;

[0114] hgd40 group: Drink sterile water containing 3% DSS and receive 200 μL of free hgd40 (2 μM) via tail vein injection on days 1 and 4.

[0115] tFNA group: Drink sterile water containing 3% DSS and receive 200 μL tFNA (2 μM) via tail vein injection on days 1 and 4.

[0116] hgd40-tFNA group: Drink sterile water containing 3% DSS and receive 200 μL hgd40-tFNA (2 μM) via tail vein injection on days 1 and 4.

[0117] The experimental procedure diagram is shown in Figure 7a.

[0118] Mouse weight and fecal characteristics were recorded daily. On day 8 of the experiment, mice were sacrificed, and their colons were collected for subsequent analysis. Relevant analyses included:

[0119] (1) The Disease Activity Index (DAI) score was calculated to quantify the severity of intestinal inflammation, following the method described in “Kihara, N. et al. Vanilloid receptor-1 containing primary sensory neurones mediate dextran sulphate sodium induced colitis in rats. Gut52, 713–719 (2003)”. The DAI value represents the average of three parameters: percentage weight loss, stool consistency score, and rectal bleeding score.

[0120] (2) Measure the length of the colon.

[0121] (3) The colon tissue was prepared into paraffin sections, and then subjected to H&E staining and immunohistochemical staining, respectively. Immunohistochemical staining included the following steps:

[0122] Colon tissue sections were dewaxed and rehydrated using a gradient of ethanol. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide (15 min). Antigen retrieval was performed using a microwave method in citrate buffer (pH 6.0). Sections were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h, then incubated overnight at 4°C with the following primary antibodies: GATA3 (Cell Signaling Technology, 5852), CD3 (Abcam, ab16669), F4 / 80 (Abcam, ab300421), APE1 (Abcam, ab189474), Cleaved-caspase 3 (Cell Signaling Technology, 9664), and ZO-1 (Proteintech, 21773-1-AP). After washing, sections were processed using the Zen BioScience IHC Detection Kit (#18002) according to the manufacturer's instructions.

[0123] (4) RT-qPCR analysis of the mRNA expression levels of IL-1β, IL-6, TNF-α, GATA3 and IL-13 in colon tissue was performed using the same method as before, and the primer sequences are shown in Table 2.

[0124] (5) Score the colonic tissue pathology based on intestinal injury and inflammatory infiltration.

[0125] (6) Western blot (WB) analysis of the expression levels of tight junction proteins and apoptosis-related markers in colon tissue.

[0126] In addition, the method described in the reference "Plant green pigment of chlorophyllin attenuates inflammatory bowel diseases by suppressing autophagy activation in mice (DOI:10.1152 / ajpgi.00291.2021)" was used to evaluate the effect of hgd40-tFNA on the intestinal barrier using a FITC-glucan permeability assay.

[0127] The results are shown in Figures 7 and 8. From the results in Figures 7 and 8, we can see that:

[0128] 1) As shown in Figure 7bd, compared with the PBS, tFNA, and hgd40 treatment groups, the hgd40-tFNA treatment group showed significant recovery in body weight and colon length. Consistently, at the end of the experiment, hgd40-tFNA treatment significantly reduced the Disease Activity Index (DAI) score of mice compared with the PBS and hgd40 treatment groups (Figure 7e).

[0129] 2) Key histopathological features of ulcerative colitis include intestinal epithelial cell damage, mucosal ulceration, inflammatory cell infiltration and crypt structure destruction. H&E staining analysis showed that ulcerative colitis in mice was significantly improved after hgd40-tFNA treatment (f in Figure 7).

[0130] 3) Immunohistochemical staining results of CD3 (T cell marker) and F4 / 80 (macrophage marker) further verified the improvement in the degree of inflammatory cell infiltration. Quantitative analysis showed that after hgd40-tFNA treatment, the infiltration of T lymphocytes and macrophages was significantly reduced (gh in Figure 7).

[0131] 4) RT-qPCR analysis showed that after hgd40-tFNA treatment, the mRNA expression levels of IL-1β, IL-6 and TNF-α in colon tissue were significantly downregulated (ik in Figure 7).

[0132] 5) Colon histopathological scores showed that after hgd40-tFNA treatment, the colon histopathological scores of mice were significantly reduced (l in Figure 7).

[0133] 6) RT-qPCR analysis showed that, compared with the control group, the expression level of GATA3 mRNA in the PBS and hgd40 treatment groups was significantly upregulated. However, after treatment with hgd40-tFNA, the upregulation of GATA3 mRNA expression level was effectively inhibited (Figure 8a). At the same time, immunohistochemical staining results also showed that the expression level of GATA3 protein in the hgd40-tFNA treatment group was significantly reduced (Figure 8b).

[0134] 7) GATA3 is a major transcription factor regulating Th2 cell differentiation and drives the production of IL-5 and IL-13 by binding to its promoter. IL-13 has been identified as a key effector cytokine in UC, primarily exacerbating disease progression by impairing epithelial barrier function. RT-qPCR analysis showed that IL-13 mRNA expression was significantly upregulated in colon tissue of the UC model, and this upregulation was effectively inhibited after treatment with hgd40-tFNA (Figure 8c).

[0135] 8) Compared with the PBS group, the serum FITC-glucan concentration of mice in the hgd40-tFNA treatment group was significantly reduced, indicating that hgd40-tFNA treatment can significantly improve intestinal permeability in UC mice (d in Figure 8).

[0136] 9) Western blot analysis and immunohistochemical staining results showed that hgd40-tFNA treatment significantly reduced the expression of the apoptosis marker Cleaved-caspase 3 (CC 3) and its downstream substrate Cleaved PARP (C-PARP), while restoring the expression of intestinal tight junction proteins ZO-1 and Occludin (eg in Figure 8), indicating that hgd40-tFNA treatment can improve intestinal barrier function.

[0137] The above results demonstrate that the hgd40-tFNA provided by this invention exhibits significant therapeutic effects in a mouse UC model. Specifically, hgd40-tFNA can alleviate UC and restore intestinal barrier homeostasis by downregulating the GATA3 / IL-13 axis.

[0138] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. An inflammation-responsive DNA zyme delivery system, characterized in that, The invention comprises a DNA tetrahedron and an inflammatory-responsive DNAzyme, wherein: the DNA tetrahedron is synthesized by hybridization of eight single-stranded DNA strands; wherein at least one single-stranded DNA strand contains a nucleotide sequence as shown in SEQ ID NO:2 such that at least one vertex of the DNA tetrahedron has an extended strand with a nucleotide sequence as shown in SEQ ID NO:2; the nucleotide sequence of the inflammatory-responsive DNAzyme consists of a Linker strand, a (polyT)n sequence, and a DNAzyme strand sequentially from the 5' end to the 3' end; wherein n is selected from natural numbers from 2 to 5, the Linker strand is partially or completely complementary to the extended strand, and an AP site is provided at a position 1 to 4 bp away from the (polyT)n sequence; the nucleotide sequences of the eight single-stranded DNA strands are shown in SEQ ID NO:8 to 15 respectively; the nucleotide sequence of the inflammatory-responsive DNAzyme is shown in SEQ ID NO:

5.

2. The method for preparing the inflammation-responsive DNA zyme delivery system according to claim 1, characterized in that, The process includes the following steps: adding single-stranded DNA used for hybridization to synthesize DNA tetrahedrons and inflammatory-responsive DNAzyme to TM buffer, mixing, heating at 95°C for 10-20 min, and then cooling to 4°C to obtain the final product.

3. The preparation method according to claim 2, characterized in that, The TM buffer has a pH of 8.0 and consists of 2 mM Tris and 5 mM MgCl2.

4. The preparation method according to claim 2 or 3, characterized in that, In the DNA single strands used for hybridization to synthesize DNA tetrahedra, the molar ratio of any two DNA single strands is 1:

1.

5. The preparation method according to claim 4, characterized in that, The molar ratio of the inflammatory-responsive DNAzyme to any single strand of DNA used for hybridization to synthesize a DNA tetrahedron is 1:

1.

6. The use of the inflammatory-responsive DNAzyme delivery system according to claim 1 in the preparation of a medicament for treating inflammatory bowel disease.

7. The application according to claim 6, characterized in that, The inflammatory bowel disease mentioned is ulcerative colitis.

Images