A miR delivery complex based on DNA tetrahedral framework and its preparation method and application in promoting wound healing

Abstract

The present invention provides a miR delivery complex based on a DNA tetrahedral framework, a preparation method thereof, and an application thereof in promoting wound healing, which belongs to the field of nucleic acid molecular medicine. The miR delivery complex of the present invention is a tetrahedral framework nucleic acid obtained by base complementary pairing of three oligonucleotide chains and three micro RNAs with restriction sites connected at the ends; the sequences of the three oligonucleotide chains are shown in SEQ ID NO.1 to 3, and the sequences of the three micro RNAs with restriction sites connected at the ends are shown in SEQ ID NO.4. The miR delivery complex of the present invention has an excellent effect of promoting wound healing.

Description

Technical Field

[0001] The present invention belongs to the field of nucleic acid molecule drugs, and in particular relates to a miR delivery complex based on a DNA tetrahedral framework, a preparation method thereof, and an application thereof in promoting wound healing. Background Art

[0002] MicroRNA (miR) plays a pivotal role in the field of nucleic acid drugs because it regulates almost all life processes of the human body. In recent years, miR therapy has become the focus of research in the field of gene therapy. Simply put, miR therapy is to use miR mimics to perform miR replacement therapy or to inhibit the function of miR through miR inhibitors. Due to its long-term effect and low toxicity, miR therapy has shown its advantages over small molecules and antibodies in the fields of tumors, infectious diseases, genetic diseases, rare diseases, etc., and the speed of nucleic acid drugs to market has also accelerated in recent years. However, due to some difficulties in the delivery of nucleic acid drugs (such as biosafety issues and drug degradation during delivery), the final effect of nucleic acid drugs is uncertain. In addition, the full development of clinical application of RNA therapy is hindered by problems such as easy degradation, difficulty in cell entry and poor targeting. Therefore, the development of miR delivery vectors is the main challenge of miR therapy. At present, a variety of delivery systems such as viral vectors, liposomes, and dendrimers have been developed. However, the above delivery systems have problems such as low biosafety, high toxicity and low transfection rate, which makes most miR therapies remain in the preclinical stage.

[0003] Current RNA delivery systems are limited in their large-scale production and clinical application due to problems such as biological toxicity, uneven encapsulation, poor targeting, and poor degradability. Finding a highly controllable and repeatable way to achieve efficient delivery of RNA drugs is a bottleneck for RNA therapy.

[0004] Based on the Watson-Crick hybridization principle, DNA has good editability, which has enabled DNA nanotechnology to develop rapidly and be widely used in various fields since it was first proposed in 1982. Due to the good biological stability and biocompatibility, editability and excellent ability to penetrate cell membranes, DNA nanostructures have great application potential in the field of drug delivery. It has been confirmed that the DNA tetrahedral framework structure (tFNA) can transport both positively charged polypeptides and negatively charged DNA and RNA. As the simplest DNA nanostructure that has been successfully synthesized, tFNA can be internalized by cells in a caveolin-dependent manner, and ultimately transport the loaded "cargo" into the cell.

[0005] Although miRNA delivery and miR therapy are currently achieved by modifying nucleic acid drugs at the vertices of tFNA or hanging them on the side arms, these methods not only destroy the structural characteristics of the tetrahedron, but also increase the size of the nanostructure, which will make tFNA unable to achieve the best cell entry efficiency and tissue penetration ability. This delivery mode exposes miR outside the carrier, bringing more instability to the delivery of miR. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a miR delivery complex based on a DNA tetrahedral framework, a preparation method thereof, and an application thereof in promoting wound healing.

[0007] The present invention provides a miR delivery complex based on a DNA tetrahedral framework, which is a tetrahedral framework nucleic acid obtained by base complementary pairing of three oligonucleotide chains and three micro RNAs with restriction sites connected to their ends; the sequences of the three oligonucleotide chains are shown in SEQ ID NO.1 to 3, and the sequences of the three micro RNAs with restriction sites connected to their ends are shown in SEQ ID NO.4.

[0008] Furthermore, the miR delivery complex is prepared by the following method: the oligonucleotide chains shown in SEQ ID NO.1 to 3 and the miRNA shown in SEQ ID NO.4 are dissolved in a solvent, then placed at 90 to 95°C for 10 to 15 minutes, and then the temperature is lowered to 2 to 8°C for more than 20 minutes.

[0009] Furthermore, the molar ratio of the oligonucleotide chains shown in SEQ ID NOs. 1 to 3 and the miRNA shown in SEQ ID NO. 4 is 1:1:1:3.

[0010] Furthermore, the solvent is TM buffer, wherein the concentration of Tris-HCl is 10 mM, the concentration of MgCl2 is 50 mM, and the pH is 8.0.

[0011] The present invention also provides a method for preparing the aforementioned miR delivery complex, which comprises the following steps:

[0012] The oligonucleotide chains shown in SEQ ID NO.1 to 3 and the miRNA shown in SEQ ID NO.4 are dissolved in a solvent, and then placed at 90 to 95°C for 10 to 15 minutes, and then the temperature is lowered to 2 to 8°C and maintained for more than 20 minutes to obtain the product.

[0013] Furthermore, the molar ratio of the oligonucleotide chains shown in SEQ ID NOs. 1 to 3 and the miRNA shown in SEQ ID NO. 4 is 1:1:1:3.

[0014] Furthermore, the solvent is TM buffer, wherein the concentration of Tris-HCl is 10 mM, the concentration of MgCl2 is 50 mM, and the pH is 8.0.

[0015] The present invention also provides use of the aforementioned miR delivery complex in preparing a drug for promoting wound healing.

[0016] The present invention also provides a medicine for promoting wound healing, which is a preparation prepared by taking the aforementioned miR delivery complex as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a miR delivery complex (BiRDs) based on a DNA tetrahedral framework, which uses a DNA tetrahedral framework as a nucleic acid skeleton and surrounds miR mimics on the outside. This structure integrates miR into the interior of the DNA tetrahedral framework, maintaining the small size of the DNA tetrahedral framework while maintaining the structural characteristics of the DNA tetrahedral framework. Moreover, compared with existing miR delivery methods, BiRDs have tissue permeability and show extremely outstanding stability in complex serum environments. The present invention uses BiRDs to carry miR-132 and explores its application in promoting wound healing. Studies have shown that it can exert its effect through skin penetration and exert an excellent, synergistic effect in promoting wound healing.

[0019] The present invention constructs a vector for carrying miR, which has excellent stability, biocompatibility, cell-entry ability and tissue permeability. The construction of BiRDs makes the vector no longer a major shortcoming of miR therapy, and has a good application prospect in miR therapy, and also provides a new idea for the delivery of other nucleic acid analogs (LNA, PNA, etc.).

[0020] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0021] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the synthesis of BiRDS.

[0023] Figure 2 The agarose gel electrophoresis results of BiRDS: the left picture shows BiRDS with only the 5' end of miR-132mimic modified with Cy5; the right picture shows BiRDS with the 5' end and 3' end of miR-132mimic modified with Cy5 and BHQ-2 respectively; GelRed represents the nucleic acid backbone.

[0024] Figure 3 The fluorescence intensity results during the preparation of BiRDS; data are expressed as mean ± SD (n = 3); ****P < 0.0001 compared with +R (t test).

[0025] Figure 4 Fluorescence intensity results of BiRDS and BiRDS-BHQ; ****P<0.0001 compared with BiRDS (t test).

[0026] Figure 5 Zeta potential distribution and particle size distribution of BiRDS.

[0027] Figure 6 Atomic force microscopy (AFM) results of BiRDS.

[0028] Figure 7 Transmission electron microscopy (TEM) results of BiRDS.

[0029] Figure 8 The fluorescence images are of AGE detection after BiRDS and tFNAs were incubated with different concentrations of FBS at 37°C.

[0030] Fig. 9 Fluorescence images of AGE detection after BiRDS and tFNAs were exposed to DNase I.

[0031] Fig.10 The fluorescence statistical results of AGE detection after BiRDS and tFNAs were incubated with different concentrations of FBS at 37°C.

[0032] Fig.11 Fluorescence statistical results of AGE detection after BiRDS and tFNAs were exposed to DNase I.

[0033] Fig.12Figure 2 is the result of cellular uptake and transdermal ability test of BiRDS: A is the result of cellular uptake of miRNA, BiRDS and tFNAs during 12 hours of culture; B is the result of flow cytometry test of cellular uptake of miRNA, BiRDS and tFNAs; C is the result of quantitative evaluation of fluorescence intensity detected by flow cytometry, and the data are shown as mean ± standard deviation (n = 3), **** is p < 0.0001 compared with the miRNA group, ns indicates no significance (t test); D is a schematic diagram of BiRDS transdermal administration; E is the fluorescence signal after topical application of tFNAs, BiRDS and miRNA for 24 hours; F is the result of quantitative analysis of fluorescence signal after topical application of tFNAs, BiRDS and miRNA for 24 hours, and the data are shown as mean ± standard deviation (n = 3), **** is p < 0.0001 compared with the miRNA group, # P < 0.05 compared with the tFNAs group (t test); G is the confocal fluorescence image of miRNA, tFNAs and BiRDS transdermal delivery for 24 hours, the scale bar is 100 μm; H is the fluorescence intensity of miRNA, tFNAs and BiRDS transdermal delivery for 24 hours, the data are expressed as mean ± standard deviation (n = 3), **** is P < 0.001 compared with the miRNA group, ns indicates no significance (t test); I is the fluorescence depth of miRNA, tFNAs and BiRDS transdermal delivery for 24 hours, the data are expressed as mean ± standard deviation (n = 3), **** is P < 0.001 compared with the miRNA group, ns indicates no significance (t test).

[0034] Fig.13 The results of the effect of BiRDS on cell migration are shown in the upper figure. The scratch images of HaCaT cells at 0, 4 and 8 hours, the scale bar is 200 μm; the lower figure is the quantitative analysis result; the data are expressed as mean ± standard deviation (n = 3), **** compared with the control group P < 0.0001, ** compared with the control group P < 0.01, * compared with the control group P < 0.05, #### Compared with the miRNA group, P < 0.0001, ### Compared with the miRNA group, P < 0.001, # Compared with the miRNA group, P<0.05, &&& Compared with the tFNAs group, P < 0.001, && Compared with the tFNAs group, P < 0.01 (t test).

[0035] Fig.14 The results of CCK8 cell viability test; data are expressed as mean ± standard deviation (n = 3), **** compared with the control group P < 0.0001, *** compared with the control group P < 0.001, * compared with the control group P < 0.05, ####Compared with the miRNA group, P < 0.0001, ## Compared with the miRNA group, P < 0.01, # Compared with the miRNA group, P<0.05, & Compared with the tFNAs group, P<0.05 (t test).

[0036] Fig.15 Figure 2 Relative gene expression levels of HB-EGF, NF-κB, PTEN and AKT in HaCaT cells after exposure to miRNA (250 nM), tFNAs (250 nM) and BiRDS (250 nM) for 48 h: A is HB-EGF; B is NF-κb; C is PTEN; D is AKT; Data are expressed as mean ± SD (n = 3), ****P < 0.0001 compared with the control group, ***P < 0.001 compared with the control group, **P < 0.01 compared with the control group, *P < 0.05 compared with the control group, #### Compared with the miRNA group, P < 0.0001, ### Compared with the miRNA group, P < 0.001, ## Compared with the miRNA group, P < 0.01, # Compared with the miRNA group, P<0.05, && Compared with the tFNAs group, P < 0.01, & Compared with the tFNAs group, P < 0.05; ns: not significant (t test).

[0037] Fig.16 Western blot analysis results of HB-EGF, NF-κB, PTEN and AKT in HaCaT cells after treatment with miRNA (250 nM), tFNAs (250 nM) and BiRDS (250 nM) for 48 hours: A is the result of western blot analysis; B is the relative protein expression intensity of HB-EGF; C is the relative protein expression intensity of NF-κB; D is the relative protein expression intensity of PTEN; E is the relative protein expression intensity of AKT; Data are expressed as mean ± SD (n = 3), ****P < 0.0001 compared with the control group, ***P < 0.001 compared with the control group, #### Compared with the miRNA group, P < 0.0001, ### Compared with the miRNA group, P < 0.001, &&&& Compared with the tFNAs group, P < 0.0001, &&& Compared with the tFNAs group, P < 0.001, && Compared with the tFNAs group, P < 0.01; ns: not significant (t test).

[0038] Fig.17The results of protein immunofluorescence analysis of HB-EGF, NF-κB, PTEN and AKT in HaCaT cells after treatment with miRNA (250nM), tFNAs (250nM) and BiRDS (250nM) for 48 hours: A is the protein immunofluorescence image of HB-EGF; B is the protein immunofluorescence image of NF-κB; C is the protein immunofluorescence image of PTEN; D is the protein immunofluorescence image of AKT; E is the relative protein expression intensity of HB-EGF; F is the relative protein expression intensity of NF-κB; G is the relative protein expression intensity of PTEN; H is the relative protein expression intensity of AKT; Data are expressed as mean ± standard deviation (n=3), ****P<0.0001 compared with the control group, ***P<0.001 compared with the control group, **P<0.01 compared with the control group, *P<0.05 compared with the control group, #### Compared with the miRNA group, P < 0.0001, ### Compared with the miRNA group, P < 0.001, &&&& Compared with the tFNAs group, P < 0.0001, &&& Compared with the tFNAs group, P < 0.001, & Compared with the tFNAs group, P < 0.05; ns: not significant (t test).

[0039] Fig.18 The effect of BiRDS on wound healing: A is a schematic diagram of skin wounds treated with BiRDS in vivo; B is a typical photo of wounds in each group treated at different times; C is the recovery status of wounds in each group.

[0040] Fig.19 Figure 2 shows the histological effect of BiRDS on wound healing: A is the HE staining and results of the injured tissues of each group on the 7th and 14th days, red arrows: blood vessels, blue arrows: fibroblasts, green arrows: inflammatory cells, black arrows: hair follicles; B is the Masson trichrome staining results of the injured tissues of each group on the 7th and 14th days; C is the wound healing rate of each group; D is the quantitative result of the percentage of collagen fibers of each group on the 7th day of treatment; E is the quantitative result of the percentage of collagen fibers of each group on the 14th day of treatment; Data are expressed as mean ± standard deviation (n = 3); ****P < 0.0001 compared with the control group, ***P < 0.001 compared with the control group, #### Compared with the miRNA group, P < 0.0001, ### Compared with the miRNA group, P < 0.001, ## Compared with the miRNA group, P < 0.01, # Compared with the miRNA group, P<0.05, &&&& Compared with the tFNAs group, P < 0.0001, &&&Compared with the tFNAs group, P < 0.001, && Compared with the tFNAs group, P < 0.01, & Compared with the tFNAs group, P < 0.05; ns: not significant (t test).

[0041] Fig. 20 The results of the in vivo anti-inflammatory and proliferative properties of BiRDS: A is the expression results of HB-EGF, NF-κB, PTEN and p-AKT in the regenerated skin of different treatment categories by WB examination on day 14; B is the relative protein expression level of HB-EGF; C is the relative protein expression level of NF-κB; D is the relative protein expression level of PTEN; E is the relative protein expression level of AKT; Data are expressed as mean ± SD (n = 3); ****P < 0.0001 compared with the control group, ***P < 0.001 compared with the control group, **P < 0.01 compared with the control group, #### Compared with the miRNA group, P < 0.0001, ## Compared with the miRNA group, P < 0.01, # Compared with the miRNA group, P<0.05, &&&& Compared with the tFNAs group, P < 0.0001, &&& Compared with the tFNAs group, P < 0.001, & Compared with the tFNAs group, P < 0.05; ns: not significant (t test).

[0042] Fig.21 The results of in vivo anti-inflammatory and proliferative properties of BiRDS: A is the image obtained in the regenerated skin samples of each treatment group using immunofluorescence labeling of Ki-67 on the 7th and 14th days, scale bar: 100 μm; B is the image obtained in the regenerated skin samples of each treatment group using immunofluorescence labeling of HB-EGF on the 7th and 14th days, scale bar: 100 μm; C is the relative fluorescence intensity of HB-EGF; D is the relative fluorescence intensity of NF-κB; E is the relative fluorescence intensity of PTEN; F is the relative fluorescence intensity of AKT; data are expressed as mean ± standard deviation (n = 3); ****P < 0.0001 compared with the control group, ***P < 0.001 compared with the control group, **P < 0.01 compared with the control group, #### Compared with the miRNA group, P < 0.0001, ## Compared with the miRNA group, P < 0.01, # Compared with the miRNA group, P<0.05, &&&& Compared with the tFNAs group, P < 0.0001, &&& Compared with the tFNAs group, P < 0.001, &Compared with the tFNAs group, P < 0.05; ns: not significant (t test). DETAILED DESCRIPTION

[0043] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0044] Example 1. Synthesis of BiRDS

[0045] All oligonucleotide chain sequences (S1, S2 and S3 single chains, and miR-132 mimic) were manufactured by Sangon (Sangon Biotechnology, Shanghai). All oligonucleotide chains (specific sequences are shown in Table 1) were dissolved in TM buffer (10mM Tris-HCl, 50mM MgCl2, pH = 8.0), the final concentrations of S1, S2 and S3 single-stranded oligonucleotide chains were 1000nM, and the final concentration of miR-132 mimic was 3000nM. After thorough mixing, they were quickly heated to 95°C for 10 minutes, then quickly cooled to 4°C and maintained for more than 20 minutes, that is, the self-assembly synthesis of the tetrahedral framework DNA-based miRNA delivery system (BiRDS) was obtained.

[0046] Table 1. Oligonucleotide chain sequences of the present invention

[0047]

[0048] Note: In Table 1, capital letters represent DNA and lowercase letters represent RNA.

[0049] In order to facilitate the use of fluorescence to observe the status of miR-132mimic, the 5' end of miR-132mimic can be modified with Cy5 (Cy5-uaacagucuacagccauggucgcagg), and then BiRDS can be prepared according to the above method.

[0050] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0051] Test Example 1: Physical and Chemical Characterization of BiRDS of the Present Invention

[0052] 1. Experimental methods

[0053] The product prepared in Example 1 (BiRDS, modified with Cy5) was analyzed by agarose gel electrophoresis. The morphology of BiRDS prepared in Example 1 (without Cy5 modification) was observed by atomic force microscopy and transmission electron microscopy (Cypher VRS, Oxford Instruments, United Kingdom; Libra200, Zeiss, Oberkochen, Germany). The size and potential of BiRDS prepared in Example 1 (without Cy5 modification) were measured by Zetasizer Nano ZS90.

[0054] Functional verification of BiRDS biological switch: In order to determine that the biological switch is in a closed state after BiRDS is produced, the present invention modified the 5' and 3' ends of miR-132mimic with Cy5 and BHQ-2 groups, respectively, and then prepared BiRDS (BiRDS-BHQ) according to the method described in Example 1. According to the design principle of BiRDS, if the miR is fused inside the tetrahedral framework, the fluorescence will be quenched because the 5' end of each miR is close to the 3' end of the adjacent miR. Finally, the quenching results were analyzed using Varioska LUX (Thermo Scientific, VLOLOTD0).

[0055] 2. Experimental results

[0056] The present invention designs and synthesizes a miRNA vector (BiRDS), which can carry miRNA mimics into cells and tissues. The vector is composed of an external miRNA mimic and an internal nucleic acid core. The synthesis diagram is shown in FIG. Figure 1 As shown. The fragment sequences of S1, S2 and S3 are all 5'-RNA1-DNA1-RNA2-DNA2-3', S1, S2 and S3 form the internal nucleic acid core, RNA1, RNA2 and DNA2 in S1, S2 and S3 bind to miRNA, where the 3' end of miRNA binds to DNA2 of one S chain, and the 5' end binds to RNA2 of another S chain, ultimately generating a DNA-RNA hybrid domain to form a miRNA carrier (BiRDS).

[0057] To demonstrate the efficient synthesis of BiRDS, the 5' end of the miRNA was modified with a sulfo-cyano 5 (Cy5) residue, which exhibits fluorescence. As the miRNA binds to the backbone, the fluorescence intensity gradually increases ( Figure 2 and Figure 3); Subsequently, in order to confirm that the prepared BiRDS has the assumed structure, the 3' end of the miRNA was modified with quencher-2 (BHQ-2) to obtain BIRDS-BHQ. When the framework was successfully closed, the BiRDS-BHQ fluorescence decreased from 100% to 9.57%, indicating the presence of the expected structure ( Figure 2 and Figure 4 ), indicating that the BiRDS structure was successfully synthesized.

[0058] The zeta potential and size distribution of the produced BiRDS were evaluated using dynamic light scattering (DLS), which yielded a zeta of -3.61 ± 4.61 mV and a size of 6.536 ± 1.745 nm ( Figure 5 ). The results showed that the size of BiRDS was similar to that of a 20-bp tetrahedral framework and was significantly smaller than the nucleic acid drug suspended within the vertices of the tFNA.

[0059] The morphology of BiRDS was evaluated using transmission electron microscopy (TEM) and atomic force microscopy (AFM). It can be seen that in addition to its small size and uniform distribution, BiRDS also has a triangular structure ( Figure 6 and Figure 7 ).

[0060] Test Example 2: Stability of BiRDS of the present invention

[0061] 1. Experimental methods

[0062] The BiRDS (modified with Cy5) prepared in Example 1 was mixed with different concentrations (0-10%) of FBS (corning, New York, USA) (BiRDS to FBS volume ratio of 1:1), incubated at 37°C for 1 hour, and the product was analyzed by AGE.

[0063] The BiRDS prepared in Example 1 (modified with Cy5) was mixed with different concentrations (0-16 U / mL) of DNase I (Sangon Biotechnology, China) and MgCl2 reaction buffer (Sangon Biotechnology, China) at 37°C for 1 hour to observe the stability of BiRDS in DNase I (BiRDS to MgCl2 reaction buffer volume ratio 1:1). After that, ethylenediaminetetraacetic acid (EDTA) (50×10 -3 M) to terminate the reaction (Sangon Biotechnology, China). The finished product was studied by AGE (120 V, 20 min).

[0064] During the study, tFNAs were used as controls. The preparation method of tFNAs included the following steps:

[0065] All oligonucleotide chain sequences (S1', S2', S3' and S4' single chains) were manufactured by Sangon (Sangon Biotechnology, Shanghai). All oligonucleotide chains (specific sequences are shown in Table 2) were dissolved in TM buffer (10mM Tris-HCl, 50mM MgCl2, pH = 8.0), and the final concentration of S1', S2', S3' and S4' single-stranded oligonucleotide chains was 1000nM. After being fully mixed, they were quickly heated to 95°C and maintained for 10 minutes, and then quickly cooled to 4°C and maintained for more than 20 minutes, that is, tFNAs were synthesized by self-assembly.

[0066] Table 2. tFNAs oligonucleotide chain sequences

[0067]

[0068] 2. Experimental results

[0069] Good biological stability is essential for effective drug delivery. Studies have shown (such as J. He, W. Chen, X. Chen, Y. Xie, Y. Zhao, T. Tian, ​​B. Guo, X. Cai. Tetrahedral framework nucleic acid loaded with glabridin: A transdermal delivery system applicated to anti-hyperpigmentation. Cell Prolif. 56 (12), e13495 (2023).) that tFNAs maintain sufficient stability in the serum environment and protect the loaded RNA from enzymatic degradation before releasing the RNA. However, tFNAs cannot provide sufficient protection for RNA extending beyond the top. In order to verify the ability of BiRDS to maintain appropriate stability in a serum environment, agarose gel electrophoresis (AGE) was performed for drug-serum stability experiments. In order to replicate the conditions of cell culture or human body, different concentrations of fetal bovine serum (FBS) were added to BiRDS and tFNAs, and then incubated at 37°C. Figure 8 and Fig.10As shown: In the range of 0-10% FBS concentration, RNA maintained excellent stability in FBS, while the BiRDS structure was not destroyed. In addition, the position of the system did not change during AGE, indicating that no new secondary structure was developed. Subsequently, the stability of BiRDS and tFNAs in DNase I was evaluated. DNase I (DNase I) is an endonuclease abundant in plasma that can randomly hydrolyze double-stranded DNA. After incubating BiRDS and tFNAs with different concentrations of DNase I, it was found that both substances showed excellent stability ( Fig. 9 and Fig.11 ).

[0070] The above results indicate that the BiRDS prepared by the present invention has excellent stability in vivo.

[0071] Experimental Example 3: Cellular Uptake and Transdermal Ability of BiRDS of the Present Invention

[0072] 1. Experimental methods

[0073] 1.1 Cellular uptake capacity

[0074] In order to verify the cellular uptake ability of BiRDS prepared in Example 1, HaCaT cells (5×10 5 After culturing for 24 hours, the cells were incubated with Cy5-labeled tFNAs, miRNA or BiRDS (the concentrations of tFNAs, miRNA and BiRDS were all 250 nM) at 37°C for 12 hours and analyzed by immunofluorescence and flow cytometry. The preparation method of tFNAs was the same as that of Experimental Example 2; the miRNA was miR-132 mimic. A blank control group (Control) was set up in the experiment.

[0075] 1.2 Transdermal ability

[0076] After thoroughly mixing BiRDS, tFNAs, and miRNA (BiRDS, tFNAs, and miRNA were all linked to Cy5) with Aquaphor cream (Eucerin, Germany), Balb / c mice (6-8 weeks old; 18-22 g) were treated. BiRDS was the nanomaterial BiRDS prepared in Example 1; the preparation method of tFNAs was the same as that of Experimental Example 2; and miRNA was miR-132 mimic. The normal saline treatment was used as the control group (Control).

[0077] The back of Balb / c mice was shaved with an electric razor and depilatory cream was used. Drugs (BiRDS, tFNAs, miRNA) or saline were thoroughly mixed with Aquaphor cream (100 μl of drug or saline was mixed with 1 g of Aquaphor, 100 μl of drug contained 1000 nM BiRDS, tFNAs or miRNA, and the solvent was saline) and evenly applied to the back of mice (1 g / mouse). Tegaderm (3M, USA) was applied to the area where the drug was applied. The mice were kept in a dark environment, and after 24 hours, the back of the mice was cleaned with baby wipes to prevent false positive results. The fluorescence of the back of each mouse was evaluated using an in vivo imaging system (PerkinElmer, USA).

[0078] 2. Experimental results

[0079] According to the confocal imaging results ( Fig.12 A) After 12 hours, a large number of BiRDS and tFNAs particles penetrated the cell membrane, while HaCaT cells only absorbed a small amount of miR-132 mimic. Flow cytometry results further demonstrated that BiRDS particles can be successfully taken up by cells, thus confirming the immunofluorescence results ( Fig.12 B and 12C).

[0080] Transdermal administration is a promising drug delivery method because of its non- / minimally invasive nature and high level of patient compliance. The ability of BiRDS to cross the epidermal barrier and deeply penetrate the dermis was evaluated based on in vivo fluorescence imaging (Lumina III IVIS; PerkinElmer). After 24 hours of drug penetration, miRNA was not detected, while BiRDS showed a strong fluorescence signal similar to tFNAs ( Fig.12 E and 12F). The successful transdermal ability of BiRDS was then verified histologically using frozen sections of skin tissue. Cy5-labeled BiRDS and tFNAs (red) significantly penetrated the epidermis to reach the dermis, while miRNA did not accumulate in the cortex ( Fig.12 G, 12H and 12I).

[0081] These results indicate that the BiRDS prepared in the present invention can be regarded as a stable, efficient and intelligent miRNA transdermal delivery system that can quickly penetrate HaCaT cells and skin, while also avoiding the shortcomings of traditional miRNA delivery systems such as low delivery efficiency, poor tissue permeability, low biosafety and high cost.

[0082] Experimental Example 4: BiRDS of the present invention promotes the transition of HaCaT cells from inflammation to proliferation

[0083] 1. Experimental methods

[0084] (1) Effects of BiRDS on cell migration and cell activity

[0085] In order to evaluate the migration ability of HaCaT cells in various environments, a scratch test was performed. HaCaT cells (5×10 5 After 24 hours of culture, a scratch was made on the bottom of each plate using a sterile pipette tip. Afterwards, the cells were treated with 50 μL TM buffer or tFNAs, miRNA or BiRDS dissolved in TM buffer (all at 250 nM, without Cy5 modification) in serum-free RPMI-1640 medium for 12 hours. The extent of cell migration was measured using an Olympus microscope after 0, 4 and 8 hours.

[0086] In addition, the effect of BiRDS on cell viability was evaluated: suspended HaCaT cells (5000 cells / well) were seeded into 96-well plates. After treating the cells with 50 μL TM buffer or tFNAs, miRNA or BiRDS dissolved in TM buffer (all at 250 nM, without Cy5 modification) for 24 hours, CCK-8 (10 μL / well) was added and incubated at 37 ° C for 4 hours. After incubation, the absorbance at 450 nm of each cell was obtained using a Tecan microplate reader.

[0087] BiRDS is the nanomaterial BiRDS prepared in Example 1; the preparation method of tFNAs is the same as that in Experimental Example 2; miRNA is miR-132mimic; and the TM buffer group is Control.

[0088] (2) RNA extraction and real-time PCR

[0089] The suspended HaCaT cells (5×10 5 / well) were planted in 6-well plates. After treating HaCaT cells with BiRDS, tFNAs, and miRNA (all at a concentration of 250nM) for 48 hours, HaCaT cells were collected and total RNA was extracted using an RNA extraction kit (TAKARA, Japan) according to the manufacturer's instructions, and the RNA quality was verified using a NanoDrop instrument (Thermo Scientific). Complementary DNA (cDNA) was generated from RNA using the PrimeScript RT kit (TAKARA). Quantitative real-time (qRT)-PCR was performed using the ABIQuantStudio 3PCR system (Applied Biosystems, USA), which used SYBR Premix ExTaq II (TAKARA). Using 2 -ΔΔCT Method to evaluate relative gene expression levels.

[0090] BiRDS is the nanomaterial BiRDS prepared in Example 1; the preparation method of tFNAs is the same as that in Experimental Example 2; miRNA is miR-132 mimic. Cells not treated with drugs are Control.

[0091] (3) Western blot analysis

[0092] The suspended HaCaT cells (5×10 5 100 μg / well) were seeded into 6-well plates. After treating HaCaT cells with BiRDS, tFNAs, and miRNA (all at a concentration of 250 nM) for 48 h, HaCaT cells were collected and all proteins were extracted using a whole protein extraction kit (KGB5303-100, Keygen Biotechnology). After adding a quarter volume of 5× sodium dodecyl sulfate (SDS) loading buffer, the mixture was heated to boiling to denature the proteins. 8-12% SDS-PAGE was used to separate the target proteins. After transfer to a PVDF membrane, the samples were blocked with 5% bovine serum albumin for one hour at 4°C, and the primary antibodies were allowed to penetrate the membrane. These antibodies are as follows: HB-EGF antibody (Catalog AF8239, R&D Systems), NF-κB antibody (8242S, CST, USA), PTEN antibody (ab267787, Abcam, UK), p-AKT antibody (Ser473, CST (USA)), and GAPDH antibody (5174S, CST). The next day, the membrane was incubated with appropriate secondary antibodies (ZSGB-BIO, China) for 1 hour and then rinsed with TBST (TBS with Tween-20). Next, the membrane was exposed using a gel and blot imaging device (Syngene, Bangalore, India). The results were semi-quantitatively evaluated using ImageJ software.

[0093] BiRDS is the nanomaterial BiRDS prepared in Example 1; the preparation method of tFNAs is the same as that in Experimental Example 2; miRNA is miR-132mimic.

[0094] (4) Immunofluorescence staining

[0095] To further observe protein expression, immunofluorescence staining was performed. Suspended HaCaT cells (5×10 5 / well) were seeded into 6-well plates. After HaCaT cells were treated with BiRDS, tFNAs, and miRNA (all at a concentration of 250nM) for 48 hours, the target protein expression was visualized and evaluated using immunofluorescence staining. After fixation for 30 minutes, the samples were treated with 0.5% Triton X-100 for 10 minutes and 5% sheep serum for 1 hour. After incubation with diluted primary antibodies (as described above) at 4°C for another night, the samples were stained with appropriate fluorescent secondary antibodies (ZSGB-BIO, China). After DAPI staining for 10 minutes and fluorescein isothiocyanate staining for 20 minutes, the cell nucleus and cytoskeleton were examined. 5 minutes × 3 PBS rinses were performed between each stage. Images were captured using a laser scanning confocal microscope (Olympus FV3000).

[0096] BiRDS is the nanomaterial BiRDS prepared in Example 1; the preparation method of tFNAs is the same as that in Experimental Example 2; miRNA is miR-132 mimic. Cells not treated with drugs are Control.

[0097] 2. Experimental results

[0098] The scratch assay and CCK-8 assay were used to evaluate the effect of BiRDS on cell migration and proliferation. Fig.13 ), in the presence of BiRDS, the migration of HaCaT cells increased after 8 h, whereas this increase was not observed in the other groups. The results of the CCK-8 assay showed that ( Fig.14 ), BiRDS stimulated the growth of HaCaT cells and was beneficial to the growth and proliferation of HaCaT cells, which was consistent with the scratch experiment.

[0099] Wound healing is a complex process that involves many interactions between cells and matrix and includes a series of overlapping events such as inflammation, proliferation, new tissue formation, and tissue remodeling. In addition, during the process of skin wound healing, it is necessary to minimize inflammation because severe and chronic inflammation prevents cells from entering the proliferation phase and ultimately hinders their recovery. Early studies have shown that overexpression of HB-EGF in keratinocytes can significantly reduce cell proliferation, which may be due to the degradation of internalized EGFR by HB-EGF. It has also been reported that HB-EGF is a key upstream gene of the NF-κB pathway, which is an important pathway of inflammatory response during wound healing. Therefore, after treating HaCaT with BiRDS, quantitative polymerase chain reaction (qPCR) was used to detect the expression levels of HB-EGF and NF-κB to evaluate the effect of BiRDS on wound healing. Fig.15As shown in A and 15B, BiRDS successfully reduced the expression of HB-EGF mRNA and further inhibited the expression of NF-κB. In addition, Western blot analysis ( Fig.16 ) and immunofluorescence staining ( Fig.17 ) The protein expression level of NF-κB was determined, and it was found that BiRDS significantly reduced the protein expression of HB-EGF and NF-κB compared with the levels in other groups.

[0100] The PTEN / AKT signaling pathway regulates many biological processes, including wound healing, migration, cell proliferation, and tissue homeostasis. Activating this pathway can promote faster wound recovery in the skin. The present invention found that treatment of HaCaT cells with BiRDS downregulated PTEN and upregulated p-AKT ( Fig.15 C and 15D). Western blot was also used to investigate ( Fig.16 A, 16D and 16E) and immunofluorescence staining ( Fig.17 C, 17D, 17G and 17H) detected the expression levels of PTEN and p-AKT proteins. Compared with the other groups, BiRDS significantly reduced the expression of PTEN and activated p-AKT.

[0101] Based on these results, it is clear that BiRDS can inhibit inflammation by targeting NF-κB signaling via HB-EGF, while upregulating the PTEN / AKT pathway. Overall, this promotes keratinocyte growth and proliferation, helping to promote the switch from inflammatory to proliferative HaCaT cells and promote wound healing.

[0102] Experimental Example 5: BiRDS of the present invention promotes wound healing

[0103] 1. Experimental methods

[0104] (1) Animal modeling and treatment

[0105] Four groups of male BALB / c mice (age 6-8 weeks, body weight 18-22 g) were randomly assigned. Full-thickness skin defects were created using a 5 mm diameter circular punch. The prepared drugs (BiRDS, tFNAs, miRNA not modified with Cy5) or saline were then thoroughly mixed with Aquaphor cream (100 μl of drug or saline was mixed with 1 g Aquaphor, 100 μl of drug contained 1000 nM BiRDS, tFNAs or miRNA, and the solvent was saline) and evenly applied around the wound (100 μl of drug / mouse). Tegaderm (3M, USA) was also covered in the area, and the dressing was changed every 24 hours. As described above. After treatment, the wound site was evaluated and recorded from day 0 to day 14. Skin tissue samples were collected on days 7 and 14 for further study. The following is the calculation method of wound closure rate (%): Wound closure rate (%) = (1-wound size on day X / original wound size on day 0) x 100%.

[0106] (2) Histological analysis

[0107] Skin tissue samples were collected from mouse specimens on days 7 and 14 after surgery. For comparison, samples of normal skin were collected. After fixation with 4% paraformaldehyde solution, the samples were dried and paraffin-embedded, and then cut into 4 μm sections. After H&E and Masson's trichrome staining, these sections were photographed on a camera using an Olympus FSX100 microscope (Japan). All samples were stained on the same day and then randomly analyzed. The fiber percentage of each sample was calculated by measuring and averaging three different fields of view per slide.

[0108] (3) RNA extraction and real-time PCR

[0109] Total RNA was extracted from mouse skin tissue samples using an RNA extraction kit (TAKARA, Japan) on days 7 and 14 following the manufacturer's instructions, and the RNA quality was verified using a NanoDrop instrument (Thermo Scientific). Complementary DNA (cDNA) was generated from mRNA using a PrimeScript RT reagent kit (TAKARA). Quantitative real-time (qRT)-PCR has been performed using an ABIQuantStudio3PCR system (Applied Biosystems, USA) using SYBR PremixEx Taq II (TAKARA). 2 -ΔΔCT Method to evaluate relative gene expression levels.

[0110] (4) Western blot analysis

[0111] Mouse skin tissue samples were collected on days 7 and 14, and all proteins were extracted using a whole protein extraction kit (KGB5303-100, Keygen Biotechnology). After adding a quarter volume of 5× sodium dodecyl sulfate (SDS) loading buffer, the mixture was heated to boiling to denature the proteins. 8-12% SDS-PAGE was used to separate the target proteins. After transfer to a PVDF membrane, the samples were blocked with 5% bovine serum albumin for one hour at 4°C, and the primary antibodies were allowed to penetrate the membrane. These antibodies are as follows: HB-EGF antibody (Catalog AF8239, R&D Systems), NF-κB antibody (8242S, CST, USA), PTEN antibody (ab267787, Abcam, UK), p-AKT antibody (Ser473, CST (USA)), and GAPDH antibody (5174S, CST). The next day, the membrane was incubated with appropriate secondary antibodies (ZSGB-BIO, China) for 1 hour and then rinsed with TBST (TBS with Tween-20). Next, the membrane was exposed using a gel and blot imaging device (Syngene, Bangalore, India). The results were semi-quantitatively evaluated using ImageJ software.

[0112] (5) Immunofluorescence staining

[0113] To further observe protein expression, immunofluorescence staining was performed. Immunofluorescence staining was used to visualize and evaluate the expression of target proteins in sections of mouse skin tissue samples. After fixation for 30 minutes, the samples were treated with 0.5% Triton X-100 for 10 minutes and 5% sheep serum for 1 hour. After incubation with diluted primary antibodies (as described above) at 4°C for another night, the samples were stained with appropriate fluorescent secondary antibodies (ZSGB-BIO, China). After DAPI staining for 10 minutes and fluorescein isothiocyanate staining for 20 minutes, the cell nucleus and cytoskeleton were examined. 5 minutes × 3 PBS rinses were performed between each stage. Images were captured using a laser scanning confocal microscope (Olympus FV3000).

[0114] 2. Experimental results

[0115] Digital photographs were taken at 0, 3, 7, and 14 days after surgery ( Fig.18 B and 18C), and according to Fig.19C Calculation of wound closure rate (%). Wound closure rate (%) = (1-wound size on day X / original wound size on day 0) × 100%. On days 3, 7, and 14, the wound closure rate (%) of BiRDS was found to be significantly higher than that of the other groups (p<0.05). The wounds of BiRDS seemed to be almost completely healed after 14 days of medication, but the wounds of other categories still had obvious scars, indicating delayed wound recovery. Between days 7 and 14, histological studies were performed to further evaluate the quality of the newly regenerated skin. More precisely, hematoxylin and eosin (H&E) staining of epithelial cells showed that the BiRDS group had less inflammatory cell infiltration and a thicker epidermis compared with the other groups. In addition, in the BiRDS group, granular tissue containing collagen fibers was found to fill or almost fill the entire wound bed on day 7. BiRDS-type lesions were completely re-epithelialized after 14 days of medication, showing corresponding morphological characteristics and typical skin layer structure ( Fig.19 A). In addition, Masson's trichrome staining showed that the epidermis after BiRDS treatment had more dense and organized collagen deposition than the other categories. This suggests that BiRDS accelerates skin regeneration by promoting wound contraction, collagen deposition, and re-epithelialization—all of which are critical for wound recovery ( Fig.19 B, 19D and 19E).

[0116] To further verify the effective regulation of target genes by BiRDS in vivo, the expression levels of related proteins have been detected. During the 14-day observation period, it was found that the expression of HB-EGF, NF-κB and PTEN proteins in the BiRDS group was significantly downregulated, and AKT phosphorylation was promoted; these factors contribute to the transition from inflammation to proliferation during wound healing ( Fig. 20 A-20E).

[0117] Ki-67 is one of the most popular cell proliferation markers used by histologists because it is specific for proliferating cells. With this in mind, the immunofluorescence intensity of key target proteins of HB-EGF and Ki-67 was evaluated in regenerated skin treated with BiRDS. Fig.21 As shown in A-21F, immunofluorescence images confirmed that the key target proteins of HB-EGF were blocked. In contrast, the relative immunofluorescence intensity of Ki-67 was significantly enhanced. Therefore, these results demonstrate the significant potential of BiRDS to promote wound healing by simultaneously inhibiting inflammation and promoting proliferation.

[0118] In summary, the present invention provides a miR delivery complex (BiRDs) based on a DNA tetrahedral framework, which uses a DNA tetrahedral framework as a nucleic acid skeleton and surrounds miR mimics on the outside. This structure integrates miR into the interior of the DNA tetrahedral framework, maintaining the small size of the DNA tetrahedral framework while maintaining the structural characteristics of the DNA tetrahedral framework. Compared with existing miR delivery methods, BiRDs have tissue permeability and show extremely outstanding stability in complex serum environments. The present invention uses BiRDs to carry miR-132 and explores its application in promoting wound healing. Studies have shown that it can play an excellent, synergistic role in promoting wound healing through skin penetration.

Claims

1. A miR delivery complex based on a DNA tetrahedral framework, characterized in that: It is a tetrahedral framework nucleic acid obtained by base complementary pairing of three oligonucleotide chains and three micro RNAs with restriction sites connected to the ends; the sequences of the three oligonucleotide chains are shown in SEQ ID NO.1-3, and the sequences of the three micro RNAs with restriction sites connected to the ends are shown in SEQ ID NO.4; the molar ratio of the oligonucleotide chains shown in SEQ ID NO.1-3 and the miRNA shown in SEQ ID NO.4 is 1:1:1:

3.

2. The miR delivery complex according to claim 1, characterized in that: The miR delivery complex is prepared by the following method: the oligonucleotide chains shown in SEQ ID NO.1-3 and the miRNA shown in SEQ ID NO.4 are dissolved in a solvent, then placed at 90-95°C for 10-15 minutes, and then the temperature is lowered to 2-8°C for more than 20 minutes.

3. The miR delivery complex according to claim 2, characterized in that: The solvent is TM buffer, wherein the concentration of Tris-HCl is 10 mM, the concentration of MgCl2 is 50 mM, and the pH is 8.

0.

4. A method for preparing the miR delivery complex according to any one of claims 1 to 3, characterized in that: It includes the following steps: The oligonucleotide chains shown in SEQ ID NO.1-3 and the miRNA shown in SEQ ID NO.4 are dissolved in a solvent, and then placed at 90-95°C for 10-15 minutes, and then the temperature is lowered to 2-8°C for more than 20 minutes to obtain; The molar ratio of the oligonucleotide chains shown in SEQ ID NO.1 to 3 and the miRNA shown in SEQ ID NO.4 is 1:1:1:

3.

5. The method according to claim 4, characterized in that: The solvent is TM buffer, wherein the concentration of Tris-HCl is 10 mM, the concentration of MgCl2 is 50 mM, and the pH is 8.

0.

6. Use of the miR delivery complex according to any one of claims 1 to 3 in the preparation of a medicament for promoting wound healing.

7. A drug for promoting wound healing, characterized in that: It is a preparation prepared by taking the miR delivery complex described in any one of claims 1 to 3 as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

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