DNA nano compound LGHL-101 as well as preparation method and application thereof

Abstract

The invention discloses a DNA nanocomposite LGHL-101 as well as a preparation method and application thereof, and belongs to the technical field of biology. The DNA nano compound LGHL-101 prepared by the invention can realize synergistic integration of triple treatment functions of active oxygen scavenging, gene silencing and gene substitution treatment, and effectively alleviates myocardial cell damage under an anoxic condition by realizing synergistic integration of triple functions of scavenging ROS in cells, regulating PTEN expression by miRNA-21 and regulating TGF-beta expression by miRNA-101, so that the myocardial cell injury is reduced, and the myocardial cell injury is reduced. Meanwhile, the DNA nano-composite LGHL-101 has good biocompatibility and biological stability, can stably exist in cells and play a role, and a new strategy for breaking vicious circle between miRNA expression abnormity and inflammatory microenvironment deterioration is provided for treatment of heart failure.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a DNA nanocomplex LGHL-101 and a preparation method and application thereof. Background Art

[0002] The development and progression of heart failure is often associated with pathological cardiac remodeling and cardiac dysfunction. Improving the prognosis of heart failure and reducing its mortality have become urgent public health issues. Current clinical treatments, including drug intervention, interventional therapy, and cardiac surgery, have improved the prognosis of heart failure, but residual risks remain. No treatments are currently available that can completely cure cardiac dysfunction and effectively reduce cardiac fibrosis. Therefore, the development of new approaches and strategies to target the treatment of heart failure, reverse cardiac fibrosis, and improve cardiac function is urgently needed.

[0003] Treatment strategies for heart failure based on miRNAs have attracted considerable attention, primarily focusing on innovative treatment approaches that regulate miRNA expression and activity. These include miRNA replacement therapy using miRNA mimics and gene silencing therapy that inhibits miRNAs through "antagomiRs" or "antimiRs." Both approaches aim to restore abnormally expressed miRNAs to normal levels. Although miRNA therapy can benefit failing hearts, it remains at the theoretical and laboratory stage, and clinical translation has been stagnant. Currently, only a few miRNA drugs have entered the clinical stage worldwide, and even fewer are for the treatment of heart failure. Some studies have been halted due to severe side effects. Furthermore, current research is limited to a single target, making it difficult to synergistically integrate multiple functions.

[0004] Reactive oxygen species (ROS) are a key factor influencing the inflammatory microenvironment and a hallmark of both acute and chronic inflammation. Ischemia / reperfusion injury also leads to ROS production. ROS deplete the endogenous antioxidant barrier, disrupting cellular signaling pathways in the heart, leading to cell death and replacement by fibrotic tissue, resulting in irreversible fibrosis. Furthermore, excessive ROS production and aberrant miRNA expression exacerbate this vicious cycle. Therefore, developing a drug that can simultaneously address both excessive ROS accumulation and aberrant miRNA expression is crucial for improving the prognosis of patients with heart failure. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a DNA nanocomplex LGHL-101 and its preparation method and application, so as to solve the technical problem that existing drugs have poor improvement effects on heart failure.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows: providing a preparation method of DNA nanocomplex LGHL-101, using Primer chain and Template chain to form a circular template CT under the action of T4 DNA ligase, and then amplifying CT into RCA long chain under the action of dNTP and Phi 29 enzyme, and finally the RCA long chain is amplified under the action of K + Under the action of , a G-quadruplex is formed in which Hemin is embedded to form DGAH, and the Linker chain and DNA-101 mimic chain are loaded successively to obtain the DNA nanocomplex LGHL-101.

[0007] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, the nucleotide sequence of the Primer chain is SEQ ID NO. 1, and the nucleotide sequence of the Template chain is SEQ ID NO. 2.

[0008] Furthermore, the nucleotide sequence of the linker chain is SEQ ID NO. 3, and the nucleotide sequence of the DNA-101 mimic chain is SEQ ID NO. 4.

[0009] The invention also discloses a DNA nanocomplex LGHL-101 prepared by the preparation method.

[0010] The present invention also discloses the use of the DNA nanocomplex LGHL-101 in preparing medicine for treating heart failure.

[0011] The present invention utilizes DNA nanotechnology to construct a synthetically simple nanocomplex using long RCA chains as carriers. The resulting DNA nanocomplex, LGHL-101, achieves the synergistic integration of the triple therapeutic functions of reactive oxygen species scavenging, gene silencing, and gene replacement therapy. This DNA nanocomplex facilitates the entry of free linker chains, DNA-101 mimic chains, and hemin into cells, enabling the controlled release of the DNA-101 mimic under intracellular miRNA-21 stimulation. By synergistically integrating the triple functions of intracellular ROS scavenging, miRNA-21 regulation of PTEN expression, and miRNA-101 regulation of TGF-β expression, it effectively mitigates myocardial cell damage under hypoxic conditions and improves the prognosis of heart failure. Furthermore, the DNA nanocomplex, LGHL-101, exhibits excellent biocompatibility and biostability, allowing it to stably exist and function within cells. This provides a novel strategy for the treatment of heart failure, breaking the vicious cycle between abnormal miRNA expression and a worsening inflammatory microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1Schematic diagram of the synthesis of DNA nanocomplex LGHL-101; Figure 2 This is the application principle diagram of DNA nanocomplex LGHL-101; Figure 3 This is the gel electrophoresis image of DNA nanocomplex LGHL-101; Figure 4 PAGE characterization of DNA nanocomplex LGHL-101; Figure 5 This is the fluorescence result of DNA nanocomplex LGHL-101; Figure 6 For particle size characterization of LGHL-101; Figure 7 AFM morphology and structural characterization of RCA long chains and LGHL-101; Figure 8 For the corresponding height analysis of RCA long chain and LGHL-101; Figure 9 Circular dichroism characterization of LGL-101 and LGHL-101; Figure 10 The color change of ABTS oxidative discoloration catalyzed by LGL-101 and LGHL-101; Figure 11 The absorbance changes of ABTS oxidation catalyzed by LGL-101 and LGHL-101; Figure 12 The effect of different Hemin concentrations on the absorbance of ABTS after oxidation; Figure 13 The standard curve corresponding to the absorbance change after ABTS oxidation catalyzed by different concentrations of Hemin; Figure 14 The GPX activity results of LGHL-101 are shown; Figure 15 is the effect of incubation time on LGHL-101; Figure 16 This is the PAGE verification result of DNA nanocomplex LGHL-101; Figure 17 is the fluorescence intensity result of DNA nanocomplex LGHL-101; Figure 18 The biocompatibility of DNA nanocomplex LGHL-101; Figure 19 Confocal microscopy imaging of the internalization of the DNA nanocomplex LGHL-101 into cells; Figure 20To characterize the efficiency of DNA nanocomplex LGHL-101 internalization into cells; Figure 21 Confocal microscopy imaging of the strand displacement reaction of DNA nanocomplex LGHL-101 in cells; Figure 22 To characterize the efficiency of strand displacement reaction of DNA nanocomplex LGHL-101 in cells; Figure 23 Confocal microscopy imaging of the DNA nanocomplex LGHL-101 clearing ROS after entering cells; Figure 24 Comparison of the ability of DNA nanocomplex LGHL-101 to scavenge ROS after entering cells; Figure 25 PTEN mRNA expression in H9C2 cells after treatment with DNA nanocomplex LGHL-101; Figure 26 The expression of TGF-β mRNA in H9C2 cells after the action of DNA nanocomplex LGHL-101; Figure 27 is the protein expression level of PTEN and TGF-β in H9C2 cells after the action of DNA nanocomplex LGHL-101; Figure 28 is the survival rate of cardiomyocytes after treatment with LGHL-101 under hypoxia; Figure 29 This is the state of myocardial cells after treatment with LGHL-101 under hypoxic environment. DETAILED DESCRIPTION

[0013] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. In the examples, where specific conditions are not specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. It is obvious to those skilled in the art that various modifications are within the spirit and scope of the present invention as defined and determined by the appended claims, and all inventions and creations utilizing the present invention are protected.

[0014] Example A method for preparing a DNA nanocomplex LGHL-101, such as Figure 1 As shown, the following steps are included: S1. Synthesis of circular template CT: CT is formed by using Primer chain and Template chain under the action of T4 DNA ligase, specifically: 20 μL of Primer (SEQ ID NO. 1) and Template (SEQ ID NO. 2) and 20 μL of 10×T4 Buffer are mixed, and the volume is supplemented to 197 μL with ultrapure water. After mixing, the final concentration of Primer and Template in the mixture is 2 μM, and then heated at 95°C for 10 minutes, and then gradually cooled and annealed to 25°C for 2 hours; after the reaction is completed, 3 μL of T4 DNA ligase is added to the reaction system, mixed, and circularized at 16°C for 16 hours, and finally heated at 65°C for 10 minutes to inactivate the enzyme to obtain CT.

[0015] S2. Synthesis of RCA long chains: CT was amplified into RCA long chains under the action of dNTP and Phi 29 enzyme. Specifically, 60 μL CT, 20 μL dNTP (10 mM), 10 μL 10× phi 29 DNA polymerase buffer and 4 μL phi 29 DNA polymerase were added to an EP tube, and the volume was made up to 200 μL with ultrapure water. After mixing, the tube was incubated at 30°C for 30 min, and then heated at 65°C for 10 min to inactivate the RCA long chain.

[0016] S3. Synthesis of DNA nanocomplex LGHL-101: The RCA long chain was placed in K + Under the action of , a G-quadruplex is formed and Hemin is embedded to form DGAH, and the Linker chain and DNA-101 mimic chain are loaded successively. Specifically, 100 μL RCA long chain, 15 μL 20×PBS, 18 μL KCl (1M), 90 μL Hemin (20 μM) and a Linker chain (SEQ ID NO. 3) with a final concentration of 1 μM are mixed, and the mixture is supplemented to 300 μL with ultrapure water. The mixture is incubated at 37°C for 1 hour, and then a DNA-101 mimic chain (SEQ ID NO. 4) with a final concentration of 1 μM is added. The mixture is incubated at 37°C for another 1 hour to finally obtain the DNA nanocomplex LGHL-101. Figure 1 The letter “n” in represents a large number of repeating units, specifically the loading ratio of Hemin to LGL-101, which is a positive integer.

[0017] Comparative Example The difference between this comparative example and the example is that Hemin in S3 is omitted, and the other implementation conditions are the same as those of the example to prepare the compound LGL-101.

[0018] The sequence information of the primers used in the present invention is shown in Table 1.

[0019] Table 1 Primer sequence list

[0020] MicroRNAs (miRNAs) are a class of highly conserved endogenous noncoding RNAs (21–25 nucleotides in length) that exert pathological and physiological functions by regulating the expression of target genes. miRNA-21 is a profibrotic factor overexpressed in cardiac fibrotic tissue and activated fibroblasts. It downregulates phosphatase and tensin homolog deleted on chromosome 10 (PTEN), activates protein kinase B, and inhibits the nuclear translocation of transcription factor EB, thereby inhibiting autophagy and promoting myocardial fibrosis. Furthermore, miRNA-21 plays an important role in regulating inflammatory gene expression signatures; reduced miRNA levels can reverse cardiac fibrosis. Furthermore, miRNA-21 is a promoter of transforming growth factor-β (TGF-β)-mediated endothelial-mesenchymal transition, which can exacerbate cardiac fibrosis.

[0021] The present invention mainly designs the DNA sequence of the Template chain and uses the RCA reaction to amplify the long DNA chain rich in G-quadruplex sequence. + Under the action of , G-quadruplex is formed and embedded in Hemin to form DGAH, which enhances the peroxidase activity of Hemin. After entering the cell, it eliminates excessive ROS and alleviates the deterioration of the inflammatory microenvironment. At the same time, multiple linker chains and DNA-101 mimic chains are loaded on the RCA product. Figure 2 As shown, the linker strand in LGHL-101 is fully complementary to the miRNA-21 sequence. Upon entry into cells, it hybridizes with miRNA-21, hindering its regulation of downstream signaling pathways and achieving gene silencing. The DNA-101 mimic strand released by the strand displacement reaction has the same sequence as miRNA-101, potentially enabling gene replacement therapy. The linker strand and DNA-101 mimic strand are used to regulate the expression of fibrosis-related factors PTEN and TGF-β, thereby ameliorating metabolic disorders caused by abnormal miRNA expression. This DNA nanocomplex effectively combines the functions of DGAH to scavenge intracellular ROS, miRNA-21 to regulate PTEN expression, and miRNA-101 to regulate TGF-β expression, thereby breaking the vicious cycle between abnormal miRNA expression and aggravated inflammatory microenvironment.

[0022] Experimental Example 1 1. Polypropylene gel electrophoresis (PAGE) characterization Figure 3 In the PAGE gel electrophoresis image shown, the bands corresponding to the Primer and Template chains in lane 4 disappear, and new, higher-molecular-weight bands appear, indicating successful hybridization of the two chains to form CT. Lane 5 shows the product of CT after RCA amplification, which is retained in the sample loading chamber, indicating the generation of a higher-molecular-weight product, i.e., successful synthesis of the RCA long chain.

[0023] The product prepared by S3 was characterized by PAGE. Figure 4 As shown, the bands representing the Linker chain and DNA-101 mimic chain in lane 6 disappeared, indicating that the two were successfully loaded onto the RCA long chain to form LGHL-101. Figure 4 Lane 8 in the middle is the product of the reaction between LGHL-101 and miRNA-21. The band represented by miRNA-21 disappears, and the band represented by DNA-101 mimic appears, indicating that the strand displacement reaction has been successfully carried out.

[0024] To further verify whether the strand displacement reaction was successful, Cy3 (SEQ ID NO. 5) was modified on the linker chain and BHQ2 (SEQ ID NO. 6) was modified on the DNA-101 mimic chain to enable LGHL-101 to have the ability to respond to miRNA-21 fluorescence. Cy3 and BHQ2 represent the fluorescent group and quencher group modifications. The results are shown in Figure 2. Figure 5 As shown in the fluorescence results, the significant decrease in LGHL-101 fluorescence indicated the successful loading of the DNA-101 mimic chain, and the obvious recovery of fluorescence after reaction with miRNA-21 further proved its specific response to miRNA-21 and the release of DNA-101 mimic.

[0025] 2. Particle size characterization The hydrated particle size of the relevant products during the synthesis process was further measured, and the results were as follows: Figure 6 As shown, the particle size of the RCA product is 38.57 nm, the particle size of the linker-loaded product LGHL is 63.88 nm, and the particle size of the product LGHL-101, which is further loaded with the DNA-101 mimic chain, is 131.9 nm. The gradual increase in particle size during the synthesis process also confirms the successful completion of each reaction step, indicating the successful synthesis of LGHL-101.

[0026] 3. Comparison of atomic force microscopy (AFM) characterization of RCA long chain and LGHL-101 The structure and morphology of RCA long chain and LGHL-101 were characterized by AFM. Figure 7As shown in the figure, it can be seen that the RCA long chain is in a linear long chain shape, while LGHL-101 is loaded with Linker chain and DNA-101 mimic chain, and + After forming G-quadruplexes under the action of , they appear in clusters.

[0027] The line height analysis of the two is carried out, such as Figure 8 As shown in Figure 3, it can be found that the height of LGHL-101 is significantly higher than that of the RCA long chain. These results further prove the successful synthesis of LGHL-101.

[0028] 4. Hemin successfully embedded into DGAH The formation of G-quadruplex in the product LGHL-101 was verified by circular dichroism spectroscopy. Figure 9 As shown, LGL-101 (comparative example) and LGHL-101 (example) before and after loading Hemin have obvious negative peaks near 240nm and obvious positive peaks near 270nm, which are consistent with the characteristic peaks of typical G-quartet, indicating that G-quartet has a strong affinity to K-terminal. + It is successfully formed under the action of , and the loading of Hemin will not affect LGHL-101.

[0029] The ABTS method was used to verify whether Hemin was successfully embedded and formed DGAH. When the DNA nanocomplex exhibited peroxidase activity, H2O2 could convert ABTS into ABTS free radicals, causing the solution color to change from colorless to green. The stronger the peroxidase activity, the darker the color. Figure 10 and Figure 11 As shown, the sample used in number "1" is H2O, the sample used in number "2" is Hemin, the sample used in number "3" is LGL-101, and the sample used in number "4" is LGHL-101. The LGL-101 group does not contain Hemin and therefore has no peroxidase activity. Its physical and absorbance results are consistent with those of the blank group (H2O group). Compared with the LGL-101 group, the LGHL-101 group caused ABTS to appear dark green ( Figure 10 ), the absorbance increased significantly ( Figure 11 ), indicating that Hemin is embedded in the G-quadruplex and forms DGAH, which significantly increases its peroxidase activity, that is, LGHL-101 is successfully synthesized.

[0030] Experimental Example 2 Hemin concentration optimization In order to make full use of the G-quadruplex to improve the ROS scavenging efficiency, the Hemin concentration in the synthesis process was further optimized using the ABTS method, and a standard curve equation between the free Hemin concentration and the ABTS absorbance was constructed. The results are shown in Figure 2. Figure 12 and Figure 13 When the concentration of Hemin is in the range of 1.3-9.3 μM, the absorbance and Hemin concentration satisfy the linear relationship, and the standard curve equation is Abs=0.087 c Hemin +0.049 (R 2 =0.994).

[0031] An excess of hemin was subsequently added during the synthesis of LGHL-101. After completion, the mixture was ultrafiltrated using an ultrafiltration tube (8000 rpm, 10 minutes). The hemin that was not incorporated into the G-quadruplex due to the excess was recovered from the ultrafiltrate. The ultrafiltrate was then subjected to an ABTS colorimetric reaction, and its absorbance was measured. Using the standard curve equation, the excess hemin concentration was calculated to be 2 μM, resulting in a hemin-to-LGL-101 loading ratio of 30:1. The amount of loaded hemin (loaded hemin = added amount minus residual amount) was determined to be 6 μM. Subsequent LGHL-101 synthesis was also performed using 6 μM hemin to maximize the utilization of the G-quadruplexes in the long RCA chain.

[0032] Experimental Example 3 Peroxidase Activity and H2O2 Scavenging Ability of DNA Nanocomplex LGHL-101 The following experiment was conducted using Hemin, LGL-101 (Comparative Example), and LGHL-101 (Example) at a concentration of 200 nM. The glutathione peroxidase (GPX) activity of LGHL-101 was measured using a glutathione peroxidase (GPX) activity assay kit. Unreacted GSH in the kit reacts with the chromogenic substrate DTNB to form a yellow compound, TNB, which exhibits a characteristic absorption peak at 412 nm. Therefore, GPX activity can be determined by measuring the absorbance of TNB at 412 nm, which is negatively correlated with GPX activity. Figure 14 The results showed that the GPX activity of LGHL-101 was significantly higher than that of free Hemin, reaching 141U / mL, indicating that the DNA nanocomplex LGHL-101 has excellent GPX activity.

[0033] The LGL-101 group was used as a comparison to further explore the ability of LGHL-101 to remove H2O2. LGL-101 / LGHL-101 at a concentration of 200nM was incubated with 1000μM H2O2 for 2h, and then the remaining H2O2 concentration was detected using a H2O2 detection kit. The results are shown in Figure 2. Figure 15 As shown in the figure, it can be found that with the increase of incubation time, the H2O2 concentration gradually decreased, and dropped to about 40% after 2 hours, while the H2O2 concentration in the LGL-101 group decreased very little and remained basically stable, which indicates that LGHL-101 completes the removal of ROS by exerting GPX activity.

[0034] Experimental Example 4 Biological Stability of DNA Nanocomplex LGHL-101 The cellular environment is more complex than the solution environment and may affect the structure and function of LGHL-101. For example, intracellular nucleases may degrade DNA. Therefore, it is necessary to investigate the serum stability and nuclease stability of LGHL-101 before conducting cell experiments.

[0035] This experiment used 2% FBS and 0.5 U / mL DNase I enzyme to simulate the cell culture environment and intracellular environment, respectively, to preliminarily investigate the biological stability of LGHL-101. LGHL-101 was incubated with 2% FBS and 0.5 U / mL DNase I enzyme at 37°C for 6 hours, and then the structural stability of the treated samples was verified by PAGE. The results are shown in Figure 2. Figure 16 As shown in the figure, it can be seen that after 6 hours of treatment with 2% FBS and 0.5U / mL DNase I, the sample still remained in the sample tank and no new bands appeared, indicating that LGHL-101 can exist stably under the above conditions.

[0036] LGHL-101 was further modified with the fluorescent group Cy3 and the quenching group BHQ2 to verify its stability by the change of fluorescence intensity. Figure 17 As shown, the fluorescence intensity of the sample remained almost unchanged over 6 hours, indicating that the DNA nanocomplex was stable and not degraded. These results demonstrate that LGHL-101 possesses good biostability and resistance to enzymatic degradation, likely due to the protective effect of the steric hindrance and rigid structure of the long RCA chain, which underlies its stable existence and function within cells.

[0037] Experimental Example 5 Biocompatibility of DNA Nanocomplex LGHL-101 H9C2 cells under normal oxygen conditions were incubated with different concentrations (0, 10, 50, 100, 150 and 200 nM) of LGHL-101 for 24 h, and then the cell viability was detected using the CCK-8 kit. Figure 18 As shown, when the concentration of LGHL-101 was in the range of 10-200nM, the cell survival rate was above 90%, indicating that LGHL-101 had good biocompatibility and had no effect on normal H9C2 cells. It can be used to treat heart failure, providing feasibility for its clinical transformation.

[0038] Experimental Example 6 Internalization of DNA Nanocomplex LGHL-101 into Cells and Efficiency of Strand Displacement Reaction Since the DNA nanocomplex LGHL-101 ultimately needs to enter cells to function, this experimental example investigated the efficiency of LGHL-101 internalization into cells. Specifically, LGHL-101 was labeled with Cy3 to make it have red fluorescence. Then, LGHL-101 was incubated with H9C2 cells pretreated with hypoxia for different time periods (0, 1, 2, 4, 6, and 8 hours). The changes in red fluorescence in the cells were observed by confocal microscopy. Figure 19 and Figure 20 As shown in the figure, as the action time prolonged, the red fluorescence in the cells gradually increased, indicating that the internalization of LGHL-101 into the cells was time-dependent.

[0039] The efficiency of the strand displacement reaction of LGHL-101 after hybridization with miRNA-21 after entering the cells was further investigated. The linker chain was labeled with Cy3 and the DNA-101 mimic chain was labeled with BHQ2 to enable fluorescence recovery that specifically responded to miRNA-21. LGHL-101 was then incubated with H9C2 cells pretreated with hypoxia for different times (0, 2, 4, 6, 8, and 10 hours), and the intracellular Cy3 fluorescence intensity was observed using confocal microscopy. The results are shown in Figure 2. Figure 21 and Figure 22 As shown, with the increase of incubation time, the red fluorescence intensity in the cells gradually increased, indicating that LGHL-101 gradually entered the cells and reacted with miRNA-21 to release DNA-101 mimic.

[0040] Experimental Example 7 Ability of DNA Nanocomplex LGHL-101 to Scaveng ROS DCFH-DA is a commonly used fluorescent probe for ROS. DCFH itself is nonfluorescent. After penetrating the cell membrane, it is hydrolyzed by esterases within the cell to form DCF, which is then oxidized by ROS to produce green-fluorescent DCF. Therefore, the fluorescence intensity of DCF reflects the level of intracellular ROS. Cells cultured under normoxia have low ROS levels, generally not exceeding 20 nM. Cells cultured under hypoxia experience oxidative stress, leading to a significant increase in intracellular ROS levels.

[0041] To verify whether the DNA nanocomplex LGHL-101 can eliminate excessive ROS in cells, H9C2 cells cultured under hypoxia were treated with PBS, Hemin, LGL-101 and LGHL-101 at a concentration of 200 nM for 24 h, and then the treated cells were stained with DCFH-DA. Figure 23 and Figure 24As shown, under normoxic conditions, green fluorescence was weak and ROS levels were low. However, hypoxic treatment significantly increased ROS levels, with deeper green fluorescence (PBS group). Cells treated with LGHL-101 showed a significant decrease in green fluorescence, indicating a significant reduction in ROS levels, suggesting that LGHL-101 has a peroxidase-like ROS scavenging function. This also demonstrates that the tetramer in LGHL-101G is crucial for the internalization of free hemin into cells, increasing its peroxidase activity and resistance to enzymatic degradation.

[0042] Experimental Example 8: Evaluation of the Ability of DNA Nanocomplex LGHL-101 to Silence miRNA-21 and Gene Replacement Therapy for miRNA-101 in Cells miRNA-21 mainly inhibits the expression of PTEN and related pathways, and miRNA-101 mainly inhibits the expression of TGF-β and related signaling pathways. Therefore, the effect of LGHL-101 on PTEN and TGF-β expression was explored at the transcriptional and translational levels.

[0043] H9C2 cells were pretreated in hypoxia for 24 hours to simulate myocardial cells in heart failure, and then treated with PBS, LGH (without Linker and DNA-101 mimic), LGHL (without DNA-101 mimic), and LGHL-101 at a concentration of 200 nM for 24 hours. RT-qPCR results showed that for PTEN mRNA ( Figure 25 ). The LGH group, which only had ROS scavenging function, showed no significant change compared to the control group (PBS group). However, the LGHL group, which had ROS scavenging and miRNA-21 gene silencing functions, showed a significant increase in PTEN mRNA expression. The LGHL-101 group, which had a triple effect, saw further increases in PTEN mRNA expression. This suggests that the added linker chain successfully hybridized with miRNA-21, blocking its inhibitory effects on downstream signaling and silencing miRNA-21.

[0044] In the changes of TGF-β mRNA expression level ( Figure 26 ), with little change in the LGH group. TGF-β mRNA expression levels were significantly decreased in the LGHL group, suggesting that blocking miRNA-21 has a certain inhibitory effect on TGF-β mRNA. TGF-β mRNA expression levels further decreased in the LGHL-101 group, indicating that the released DNA-101 mimic chain successfully affected the cfos / TGF-β axis, acting as a gene replacement therapy for miRNA-101.

[0045] The expression of the two mRNA downstream target proteins was further verified by WB. Figure 27 As shown, compared with the PBS group, PTEN protein expression in the LGH group remained almost unchanged. However, PTEN protein expression was significantly upregulated in the LGHL and LGHL-101 groups to 157.6% and 216.2%, respectively. Regarding TGF-β, TGF-β protein expression was significantly downregulated in the LGHL and LGHL-101 groups to 41.2% and 15.6%, respectively, compared with the PBS and LGH groups. This result mirrored the RT-qPCR results, further demonstrating that LGHL-101 successfully silenced miRNA-21 and replaced it with miRNA-101. Furthermore, these results suggest that miRNA-21 silencing and miRNA-101 gene replacement therapy mutually promote and synergize, further modulating metabolic disorders caused by cellular miRNA abnormalities.

[0046] Experimental Example 9: Investigation of the ability of the triple-functional DNA nanocomplex LGHL-101 to alleviate hypoxia-induced cardiomyocyte apoptosis With normal oxygen as the control group, H9C2 cells treated with PBS, LGH, LGHL and LGHL-101 at a concentration of 200 nM were treated, and the cell survival rate of each group was detected by CCK-8. Figure 28 As shown, the cell survival rate decreased by nearly 50% after hypoxia treatment, and increased after treatment with DNA complexes. Among them, the LGHL-101 group with triple therapeutic effects had the highest cell survival rate, reaching 77.1%, which was significantly higher than the LGH group with only ROS scavenging ability (58.7%) and the LGHL group with ROS scavenging and gene silencing functions (65.5%). This was due to the synergistic effect of LGHL-101's triple functions of ROS scavenging ability, gene silencing and gene replacement therapy.

[0047] Annexin V-FITC / PI apoptosis kit was further used to verify the apoptosis of H9C2 cells under the above treatment to verify the triple therapeutic effect of DNA nanocomplex. Figure 29 As shown, the survival rates of H9C2 cells treated with PBS, LGH, LGHL, and LGHL-101 after hypoxic culture were 58.1%, 62.2%, 63.2%, and 70.3%, respectively, which are generally consistent with the results of CCK-8 cell survival measurements. LGHL-101 significantly reduced apoptosis in cells exposed to oxidative stress, demonstrating the best therapeutic effect. These results also demonstrate that the triple treatment modality exerts a synergistic effect, further reducing cardiomyocyte apoptosis and enhancing therapeutic efficacy.

Claims

1. A method for preparing a DNA nanocomplex LGHL-101, characterized in that: Primer chain and Template chain are used to form circular template CT under the action of T4 DNA ligase, and then CT is amplified into RCA long chain under the action of dNTP and Phi 29 enzyme. + Under the action of , a G-quadruplex is formed in which Hemin is embedded to form DGAH, and the Linker chain and DNA-101 mimic chain are loaded successively to obtain the DNA nanocomplex LGHL-101.

2. The method for preparing the DNA nanocomplex LGHL-101 according to claim 1, characterized in that: The nucleotide sequence of the Primer chain is SEQ ID NO. 1, and the nucleotide sequence of the Template chain is SEQ ID NO.

2.

3. The method for preparing the DNA nanocomplex LGHL-101 according to claim 1, characterized in that: The nucleotide sequence of the linker chain is SEQ ID NO. 3, and the nucleotide sequence of the DNA-101 mimic chain is SEQ ID NO.

4.

4. A DNA nanocomplex LGHL-101, characterized in that The method is prepared according to any one of claims 1 to 3.

5. Use of the DNA nanocomplex LGHL-101 according to claim 4 in the preparation of a drug for treating heart failure.

Images