Tetrahedral framework multifunctional nano-composite as well as preparation method and application thereof

Abstract

The invention provides a tetrahedral framework multifunctional nano-composite as well as a preparation method and application thereof, and belongs to the field of nucleic acid molecule medicines. According to the invention, micromolecule drug loading is realized on a miRNA delivery system based on tetrahedral framework DNA for the first time, the miRNA and antibiotic co-delivery nano-composite is prepared, and the nano-composite has high stability, good cell and tissue permeability and remarkable biocompatibility, can be combined with anti-inflammatory action and antibacterial activity at the same time, and can be used for preparing the nano-composite for co-delivery of the miRNA and the antibiotic. The compound is applied to combined treatment of infected wound healing, and a new treatment strategy is provided for treating complex and refractory diseases.

Description

Technical Field

[0001] The invention belongs to the field of nucleic acid molecule drugs, and in particular relates to a tetrahedral framework multifunctional nanocomposite and a preparation method and application thereof. Background Art

[0002] The skin is an important organ of the body that is in direct contact with the external environment. It senses external stimuli, regulates body temperature, and protects the body from external damage. Impaired skin wound healing can significantly reduce the quality of life and is becoming a growing concern worldwide. Infections caused by bacterial contamination remain one of the most important obstacles to wound healing; they can also lead to cellulitis, organ dysfunction, and in severe cases, death. Infected wounds are a typical complex disease involving a series of pathological processes, including bacterial infection and dysregulated inflammation. Bacteria produce endotoxins and promote the expression of inflammatory factors, thereby exacerbating the inflammatory storm. Therefore, clinical strategies for infected wounds should simultaneously inhibit bacterial growth and severe inflammation.

[0003] Currently, antibiotics are the main strategy for treating infections, among which levofloxacin (LEV) has been widely used as a broad-spectrum chemotherapeutic antibiotic. However, frequent and high-dose systemic antibiotics are the leading cause of drug resistance and can lead to serious side effects. In the face of these challenges, it is necessary to develop a new antimicrobial strategy that reduces antibiotic-resistant bacteria, reduces the dose and frequency of antibiotics, and can allow antibiotics to be applied locally at the wound site rather than systemically, thereby preventing the formation of drug-resistant bacteria.

[0004] MicroRNAs (miRNAs, miR for short) regulate almost all biological processes in the human body and have attracted considerable attention in the field of nucleic acid therapy. A large number of studies have shown that the expression of miRNA is dynamically regulated at multiple stages of wound healing. Therefore, miRNA-based treatment methods have attracted increasing interest in the field of wound healing. 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 clinical application of RNA therapy has been hindered by problems such as easy degradation, difficulty in cellular entry, and poor targeting. Therefore, the development of miR delivery vectors is the main challenge for miR therapy.

[0005] Current antibiotic and 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. Summary of the invention

[0006] In order to solve the defects of the prior art, the object of the present invention is to provide a tetrahedral framework multifunctional nanocomposite and a preparation method and use thereof.

[0007] The present invention provides a nanocomplex, which is prepared from siRNA-modified tetrahedral framework nucleic acid and antibiotics as raw materials, wherein the molar ratio of siRNA-modified tetrahedral framework nucleic acid to antibiotics is 1:50-70, and the siRNA is miRNA with antibacterial and / or anti-inflammatory effects.

[0008] Furthermore, the molar ratio of the siRNA-modified tetrahedral framework nucleic acid to the antibiotic is 1:60.

[0009] Furthermore, the siRNA-modified tetrahedral framework nucleic acid is a product prepared using siRNA and tetrahedral framework nucleic acid as raw materials, wherein the molar ratio of siRNA to tetrahedral framework nucleic acid is 1-5:1, preferably 3:1.

[0010] Furthermore, the siRNA-modified tetrahedral framework nucleic acid is self-assembled from four single strands, and the sequences of the four single strands are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0011] Furthermore, the method for preparing the siRNA-modified tetrahedral framework nucleic acid comprises the following steps: mixing the siRNA and the tetrahedral framework nucleic acid and reacting them to obtain the siRNA.

[0012] Furthermore, the reaction conditions are to first heat the reaction and then cool the reaction;

[0013] The heating reaction temperature is 80-100°C and the time is 5-15min;

[0014] The temperature of the cooling reaction is 0-10°C and the time is 10-30 minutes.

[0015] Furthermore, the heating reaction temperature is 95°C and the time is 10 minutes;

[0016] The temperature of the cooling reaction is 4°C and the time is 20 minutes.

[0017] Furthermore, the heating reaction temperature is 95°C and the time is 10 minutes;

[0018] The temperature of the cooling reaction is 4°C and the time is 20 minutes.

[0019] Furthermore, the siRNA is miRNA-146; and the antibiotic is levofloxacin.

[0020] The present invention also provides a method for preparing the nanocomplex, which comprises the following steps: mixing the siRNA-modified tetrahedral framework nucleic acid and the antibiotic and reacting them to obtain the nanocomplex.

[0021] Furthermore, the reaction temperature is 0 to 10°C and the reaction time is 1 to 5 hours;

[0022] Preferably, the reaction temperature is 4° C. and the reaction time is 3 hours.

[0023] The present invention also provides the use of the nanocomposite in preparing a drug with antibacterial and / or anti-inflammatory effects.

[0024] The present invention also provides use of the nanocomplex in preparing a medicine for promoting healing of infected wounds.

[0025] The present invention has achieved the following beneficial effects:

[0026] The present invention realizes small molecule drug loading on the tetrahedral framework DNA-based miRNA delivery system for the first time, and prepares a nanocomplex (B / L) for co-delivery of miRNA and antibiotics. B / L has good serum stability, excellent cell and bacterial transport capacity, and significant transdermal ability. B / L downregulates the factor kappa-B (NF-κB) pathway by binding to TNF receptor-associated factor 6 (TRAF6) and IL1 receptor-associated kinase 1 (IRAK1), thereby inhibiting the expression of downstream inflammatory factors such as tumor necrosis factor α (TNF-α), interleukin 1β (IL1β) and interleukin-6 (IL-6). In addition, B / L can also scavenge reactive oxygen species (ROS), thereby reducing the level of inflammation in human keratinocytes (HaCaTs).

[0027] The present invention further discovered that after B / L treatment, the anti-inflammatory effect exerted a synergistic effect, achieving unexpected technical effects.

[0028] The present invention utilizes an infectious wound model infected with Staphylococcus aureus and Escherichia coli to demonstrate that the nanocomplex has dual antibacterial and anti-inflammatory effects, effectively promotes the healing of infected wounds, and provides a new therapeutic strategy for the treatment of complex and refractory diseases.

[0029] 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.

[0030] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. 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

[0031] Figure 1 (A) Schematic diagram of the synthesis of B / L and (B) the agarose gel electrophoresis results of B / L and the fluorescence intensity results during the preparation of B / L: the data are expressed as mean ± SD (n = 3). **** Compared with the B / L group, P<0.0001; *** P < 0.001 compared with the B / L group; ** Compared with the B / L group, P<0.01; * Compared with the B / L group, P<0.05; #### Compared with the BiRDS group, P<0.0001; ### Compared with the BiRDS group, P<0.001; ## Compared with the BiRDS group, P<0.01; # Compared with the BiRDS group, P<0.05 (t test) (GelRed represents the nucleic acid backbone).

[0032] Figure 2 (A) Atomic force microscopy (AFM) results of B / L and (B) Transmission electron microscopy (TEM) results of B / L.

[0033] Figure 3 These are the fluorescence images and fluorescence statistical results of AGE detection after BiRDS and B / L were incubated with different concentrations of FBS at 37°C.

[0034] Figure 4 The results of cell uptake detection of B / L: (A) The results of cell uptake flow cytometry detection of LEV, miR-146 mimic (miRNA), tFNA, BiRDS and B / L; (B) The results of cell uptake of LEV, miR-146 mimic (miRNA), tFNA, BiRDS and B / L during 12 hours of culture; (C) The results of quantitative evaluation of fluorescence intensity, data are shown as mean ± standard deviation (n = 3), **** Compared with the B / L group, P<0.0001; *** P < 0.001 compared with the B / L group; ** Compared with the B / L group, P<0.01; * Compared with the B / L group, P<0.05; #### Compared with the BiRDS group, P<0.0001; ### Compared with the BiRDS group, P<0.001;## Compared with the BiRDS group, P<0.01; # Compared with the BiRDS group, P<0.05 (t test).

[0035] Figure 5 The results of the B / L uptake detection of Staphylococcus aureus and Escherichia coli and the quantitative evaluation of fluorescence intensity are shown as mean ± standard deviation (n = 3). **** Compared with the B / L group, P<0.0001; *** P < 0.001 compared with the B / L group; ** Compared with the B / L group, P<0.01; * Compared with the B / L group, P<0.05; #### Compared with the BiRDS group, P<0.0001; ### Compared with the BiRDS group, P<0.001; ## Compared with the BiRDS group, P<0.01; # Compared with the BiRDS group, P<0.05 (t test).

[0036] Figure 6 Schematic diagram of transdermal administration of B / L; (A) is the fluorescence signal and quantitative analysis results of the skin 24 hours after topical application of LEV, miR-146 mimic (miRNA), tFNA, BiRDS and B / L; (B) is the fluorescence signal and quantitative analysis results of frozen sections 24 hours after topical application of LEV, miR-146 mimic (miRNA), tFNA, BiRDS and B / L.

[0037] Figure 7 24-hour growth curves of Staphylococcus aureus and Escherichia coli.

[0038] Figure 8 Colonies and quantitative analysis of Staphylococcus aureus and Escherichia coli (data shown as mean ± standard deviation (n = 3), **** Compared with the B / L group, P<0.0001; *** P < 0.001 compared with the B / L group; ** Compared with the B / L group, P<0.01; * Compared with the B / L group, P<0.05; #### Compared with the BiRDS group, P<0.0001; ### Compared with the BiRDS group, P<0.001; ## Compared with the BiRDS group, P<0.01; # Compared with the BiRDS group, P<0.05 (t test).

[0039] Fig. 9Scanning electron microscope images of Staphylococcus aureus and Escherichia coli.

[0040] Fig.10 B / L inhibits the inflammatory response of HaCaTs by directly targeting TRAF6 and IRAK1: (A) Schematic diagram showing the effects of B / L on different molecules in HaCaT cells; (B) Relative gene expression levels of TRAF6 and IRAK1 in HaCaT cells; (C) Western blot analysis of the expression levels of GADPH, TRAF6, and IRAK1 proteins; (D) Immunofluorescence images of TRAF6 and IRAK1 contents in HaCaT cells and three-dimensional reconstruction of fluorescence microscopy images based on fluorescence intensity. Data are shown as mean ± standard deviation (n = 3), **** Compared with the B / L group, P<0.0001; *** P < 0.001 compared with the B / L group; ** Compared with the B / L group, P<0.01; * Compared with the B / L group, P<0.05; #### Compared with the BiRDS group, P<0.0001; ### Compared with the BiRDS group, P<0.001; ## Compared with the BiRDS group, P<0.01; # Compared with the BiRDS group, P<0.05 (t test).

[0041] Fig.11 B / L inhibits the inflammatory response of HaCaTs by downregulating the NF-κB pathway and clearing the excess ROS accumulated in HaCaT cells: (A) Relative gene expression levels of NF-κB p65, NF-κB p-p65, TNF-α, IL-1β, and IL-6 in HaCaT cells; (B) Western blot analysis of the protein expression levels of GADPH, NF-κB p65, NF-κB p-p65, TNF-α, IL-1β, and IL-6; (C) Immunofluorescence images of NF-κB p65, NF-κB p-p65, TNF-α, IL-1β, and IL-6 in HaCaT cells and three-dimensional reconstruction of fluorescence microscopy images based on fluorescence intensity. Data are shown as mean ± standard deviation (n = 3). **** Compared with the B / L group, P<0.0001; *** P < 0.001 compared with the B / L group; ** Compared with the B / L group, P<0.01; * Compared with the B / L group, P<0.05; #### Compared with the BiRDS group, P<0.0001; ### Compared with the BiRDS group, P<0.001; ##Compared with the BiRDS group, P<0.01; # Compared with the BiRDS group, P<0.05 (t test).

[0042] Fig.12 B / L clears excess ROS accumulated in HaCaT cells to inhibit the inflammatory response of HaCaTs: (A) Flow cytometry detection results of ROS of LEV, miR-146 mimic (miRNA), tFNA, BiRDS and B / L; (B) Immunofluorescence images of ROS in HaCaT cells and three-dimensional reconstruction of fluorescence microscopy images based on fluorescence intensity (data are shown as mean ± SD (n = 3), **** Compared with the B / L group, P<0.0001; *** P < 0.001 compared with the B / L group; ** Compared with the B / L group, P<0.01; * Compared with the B / L group, P<0.05; #### Compared with the BiRDS group, P<0.0001; ### Compared with the BiRDS group, P<0.001; ## Compared with the BiRDS group, P<0.01; # Compared with the BiRDS group, P<0.05 (t test).

[0043] Fig.13 To promote the healing of infected wounds in vivo by B / L, photos of infected wounds infected with Staphylococcus aureus or Escherichia coli after 0, 3, 7, and 14 days and the relative percentage of wound area after treatment.

[0044] Fig.14 H&E staining and Masson staining of the wound surface 7 days (A, B) and 14 days (C, D) after treatment of infected wounds infected with Staphylococcus aureus or Escherichia coli with LEV, miR-146 mimic (miRNA), tFNA, BiRDS and B / L (scale bars are 2 mm and 0.2 mm (red arrows: inflammatory cells; blue arrows: fibroblasts; black arrows: hair follicles)).

[0045] Fig.15 Photographs and analysis of surviving bacterial colonies after treating infected wounds infected with Staphylococcus aureus or Escherichia coli with LEV, miR-146 mimic (miRNA), tFNA, BiRDS and B / L (data are shown as mean ± SD (n = 3), **** Compared with the B / L group, P<0.0001; *** P < 0.001 compared with the B / L group; ** Compared with the B / L group, P<0.01; *Compared with the B / L group, P<0.05; #### Compared with the BiRDS group, P<0.0001; ### Compared with the BiRDS group, P<0.001; ## Compared with the BiRDS group, P<0.01; # Compared with the BiRDS group, P<0.05 (t test).

[0046] Fig.16 B / L inhibits skin inflammatory response in vivo: (A) and (B) are immunoblot analysis of the expression levels of GADPH, TRAF6, IRAK1 and NF-xB p65 proteins in infected wounds infected with Staphylococcus aureus or Escherichia coli (data are shown as mean ± SD (n = 3), **** Compared with the B / L group, P<0.0001; *** P < 0.001 compared with the B / L group; ** Compared with the B / L group, P<0.01; * Compared with the B / L group, P<0.05; #### Compared with the BiRDS group, P<0.0001; ### Compared with the BiRDS group, P<0.001; ## Compared with the BiRDS group, P<0.01; # Compared with the BiRDS group, P<0.05 (t test).

[0047] Fig.17 B / L is B / L inhibiting skin inflammatory response in vivo: (A) and (B) are immunofluorescence images of TNF-α, IL-1β, and IL-6 in infected wounds infected with Staphylococcus aureus or Escherichia coli, respectively (data are shown as mean ± SD (n = 3), **** Compared with the B / L group, P<0.0001; *** P < 0.001 compared with the B / L group; ** Compared with the B / L group, P<0.01; * Compared with the B / L group, P<0.05; #### Compared with the BiRDS group, P<0.0001; ### Compared with the BiRDS group, P<0.001; ## Compared with the BiRDS group, P<0.01; # Compared with the BiRDS group, P<0.05 (t test). DETAILED DESCRIPTION

[0048] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0049] All BiRDS oligonucleotide chain sequences (S1, S2 and S3 single chains, and miR-146 mimic) used in the present invention are manufactured by Sangon (Sangon Biotechnology, Shanghai); all tFNA oligonucleotide chain sequences (S1, S2, S3 and S4 single chains) used in the present invention are manufactured by Sangon (Sangon Biotechnology, Shanghai).

[0050] Levofloxacin (LEV) was purchased from Sigma-Aldrich.

[0051] Example 1. Synthesis of BiRDS without Cy5 modification

[0052] Four oligonucleotide chains (S1-S4, the specific sequences are shown in Table 1) were dissolved in TM Buffer (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0), the final concentrations of S1, S2 and S3 oligonucleotide chains were all 1000 nM, and the final concentration of miR-146 mimic was 3000 nM. 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, thereby self-assembly synthesizing a tetrahedral framework DNA-based miRNA delivery system that was not modified with Cy5 (BiRDS that was not modified with Cy5).

[0053] Table 1. Oligonucleotide chain sequences of the present invention

[0054]

[0055]

[0056] Note: Capital letters in the table represent DNA monomers, and lowercase letters represent RNA monomers.

[0057] Example 2: Synthesis of B / L without Cy5 modification

[0058] Levofloxacin (60 μM) was mixed with BiRDS (1 μM) not modified with Cy5 prepared in Example 1 and shaken at 4° C. for 3 hours to obtain B / L not modified with Cy5.

[0059] In order to facilitate the experiment, the present invention also used the following method to perform Cy5 modification.

[0060] Example 3. Synthesis of BiRDS

[0061] Refer to the method of Example 1, the only difference is that S4 is replaced by Cy5-modified miR-146 to obtain BiRDS.

[0062] Example 4, Synthesis of B / L

[0063] Levofloxacin (60 μM) was mixed with BiRDS (1 μM) prepared in Example 3 and shaken at 4° C. for 3 hours to obtain B / L.

[0064] The following is a control sample preparation.

[0065] Comparative Example 1: Preparation of Tetrahedral Framework Nucleic Acids

[0066] All oligonucleotide chains (specific sequences of S5-S8 are shown in Table 1) were dissolved in TM Buffer (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0), and the final concentrations of S5, S6, S7 and S8 single-stranded oligonucleotide chains were all 1000 nM. After thorough mixing, they were rapidly heated to 95°C and maintained for 10 minutes, and then rapidly cooled to 4°C and maintained for more than 20 minutes, thereby self-assembling and synthesizing tetrahedral framework nucleic acids (tFNAs).

[0067] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0068] Experimental Example 1: Physical and Chemical Characterization of B / L

[0069] 1. Experimental methods

[0070] The product prepared in Example 3 (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.

[0071] 2. Experimental results

[0072] In the present invention, BiRDS consists of three external miRNAs and an internal nucleic acid core. The internal nucleic acid core consists of three single DNA / RNA strands (S1-S3) and nucleic acid sequences according to Watson-Crick base pairing. The three external miRNAs bind to the core. Then, LEV is loaded into BiRDS by vibration to form B / L( Figure 1A). The successful synthesis of BiRDS and B / L was verified by 2% agarose gel electrophoresis (AGE). In order to better verify the successful preparation of BiRDS and B / L, the present invention modified the 5' end of miRNA with fluorescent sulfo-cyano 5 (Cy5) residue. Figure 1 As shown in Figure B, with the gradual loading of miRNA, the relative fluorescence intensity gradually increased. The molecular weight of the material after LEV loading was slightly higher, indicating that the synthesis of BiRDS and B / L was successful. The morphology, size and potential of B / L prepared in Example 2 without Cy5 modification are shown in Figure Figure 2 As shown in A and 2B.

[0073] The above experimental results show that the present invention successfully synthesized BiRDS and B / L.

[0074] Experimental Example 2: Stability Test of B / L

[0075] 1. Experimental methods

[0076] The nanomaterial BiRDS prepared in Example 3 and B / L prepared in Example 4 were mixed with different concentrations (0-10%) of FBS (corning, New York, USA), incubated at 37°C for 1 hour, and then analyzed by AGE. The finished product was studied by AGE (120V, 20 minutes).

[0077] 2. Experimental results

[0078] The structural stability of nanomaterials is the key to their effectiveness. A qualified drug carrier must maintain the stability of its overall structure while protecting the drug from serum degradation. BiRDS maintains sufficient stability due to its structural similarity, similar to tFNA. However, tFNA cannot provide protection against apical RNA binding. The stability results of B / L and BiRDS are shown in Figure 2. Figure 3 As shown, with the increase of serum concentration, B / L and BiRDS showed almost no degradation and no secondary structure formation, proving that both substances exhibited excellent stability.

[0079] The above experimental results show that the B / L and BiRDS prepared by the present invention have excellent stability.

[0080] Experimental Example 3: Test of co-delivery ability of B / L

[0081] 1. Experimental methods

[0082] 1.1 Experimental Grouping

[0083] To test the co-delivery ability of B / L, the treatments of each experimental group were as follows:

[0084] Blank group: treated with PBS phosphate buffer (0.1 mol / L);

[0085] LEV group: treated with 15000nM LEV;

[0086] miRNA group: treated with 750 nM Cy5-labeled miR-146 mimic;

[0087] tFNAs group: treated with 250 nM tFNAs prepared in Control Example 1;

[0088] BiRDS group: treated with 250 nM BiRDS prepared in Example 3;

[0089] B / L group: treated with 250 nM B / L prepared in Example 4.

[0090] 1.2 Cellular uptake capacity

[0091] In order to verify the cellular uptake ability of B / L prepared in Example 4, HaCaT cells (50,000 cells / mL) were incubated with blank group, LEV group, miRNA group, tFNAs group, BiRDS group and B / L group at 37°C for 12 hours, and analyzed by immunofluorescence and flow cytometry.

[0092] 1.3 Bacterial uptake capacity

[0093] In order to verify the bacterial uptake ability of the B / L prepared in Example 4, Staphylococcus aureus or Escherichia coli (1×10 6 CFU / mL) were incubated with the blank group, LEV group, miRNA group, tFNAs group, BiRDS group and B / L group at 37°C for 12 h and analyzed by immunofluorescence.

[0094] 1.4 Transdermal ability

[0095] The Blank, LEV, miRNA, tFNAs, BiRDS, and B / L groups were mixed thoroughly with Aquaphor cream (Eucerin, Germany), and then treated with albino laboratory (Balb / c) mice (6-8 weeks old; 18-22 g).

[0096] Balb / c mice were shaved on the back with an electric razor and depilatory cream was used to remove hair. The above-prepared drug was evenly applied to the back of the mouse (1 g / mouse), and 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).

[0097] 2. Experimental results

[0098] The drug delivery ability is crucial for evaluating the efficacy of drug delivery systems. The present invention determines the delivery effect of B / L on cells, bacteria and skin. First, HaCaT cells were used as a model to determine the transport effect of B / L in cells. After 12 hours of treatment with LEV, miRNA, tFNA, BiRDS and B / L, flow cytometry results showed that more than 90% of the substances in the B / L, BiRDS and tFNA groups entered the cells, but only 16. The LEV group was 16% ( Figure 4 A). Confocal fluorescence imaging confirmed this observation. The fluorescence signals of the B / L group, BiRDS group, and tFNA group were similar. However, only a weak fluorescence signal was observed in the miRNA group, while no fluorescence signal was detected in the LEV group ( Figure 4 B and 4C).

[0099] Next, the present invention selected Staphylococcus aureus and Escherichia coli to represent Gram-positive bacteria and Gram-negative bacteria, respectively. Confocal laser microscopy was used to measure the absorption of B / L by Staphylococcus aureus and Escherichia coli. The results showed that B / L, BiRDS and tFNA could enter Staphylococcus aureus and Escherichia coli ( Figure 5 ).

[0100] Transdermal delivery (TDD) is an excellent drug delivery method and is considered a good drug delivery system for gene therapy due to its non-invasiveness and high patient compliance. However, the structure and barrier function of the skin greatly hinder transdermal delivery. Therefore, the present invention evaluated whether B / L has skin penetration ability. According to the 24h fluorescence signal intensity based on in vivo fluorescence imaging, the B / L group, BiRDS group and tFNA group showed similar strong fluorescence signals, while no miRNA group was detected ( Figure 6 A). These results were confirmed histologically using frozen sections. B / L, BiRDS, and tFNA formulations significantly penetrated the epidermis to reach the dermis, while miRNA did not accumulate in the cortex. These results suggest that B / L has good skin penetration, allowing the drug to be applied topically to the wound surface rather than systemically, thus preventing side effects.

[0101] ( Figure 6 B).

[0102] The above experimental results show that B / L has good permeability in cells, bacteria and skin, can deliver drugs to cells, bacteria and skin, and has good drug delivery ability.

[0103] Experimental Example 4: Antibacterial ability test of B / L

[0104] 1. Experimental methods

[0105] 1.1 Experimental Grouping

[0106] In order to test the antibacterial ability of B / L, the treatment of each experimental group is as follows:

[0107] Blank group: treated with PBS phosphate buffer (0.1 mol / L);

[0108] LEV group: treated with 200 nM LEV;

[0109] miRNA group: treated with 3.75 nM Cy5-labeled miR-146 mimic;

[0110] tFNAs group: treated with 1.25 nM tFNAs prepared in Control Example 1;

[0111] BiRDS group: treated with 1.25 nM BiRDS prepared in Example 3;

[0112] B / L group: treated with 1.25 nM B / L prepared in Example 4.

[0113] Staphylococcus aureus or Escherichia coli bacterial suspension (1×10 6 CFU / ml) were added into the blank group, LEV group, miRNA group, tFNAs group, BiRDS group and B / L group, and cultured at 37°C for 24 hours.

[0114] 1.2 Growth curve

[0115] Each mixture was transferred to a 96-well plate and the OD value at 600 nm was read for each group between 0 and 24 hours using an automated spectrophotometer (BioTek Instruments, VT). A growth curve was constructed by plotting the data.

[0116] 1.3 Colony count

[0117] Each group of suspensions was placed on a standard LB agar plate and cultured for 24 h. Finally, the number of colony forming units was counted and images of the agar plates were taken.

[0118] 1.4 Scanning electron microscopy

[0119] Each group of mixtures was placed in a 12-well plate and a glass cover was placed in each well to prevent bacterial adhesion. After incubation at 37°C for 24 hours, the supernatant was carefully removed and washed with sterile PBS three times. After adding 2.5% glutaraldehyde (v / v), the biofilm was fixed at 4°C for 12 hours. After dehydrating the biofilm using repeated ethanol concentrations (30-100%, v / v), it was sputter-coated with gold and examined with a scanning electron microscope (InspectF, FEI, Holanad).

[0120] 2. Experimental results

[0121] Currently, antibiotics are the main strategy for treating infections. However, frequent and high-dose systemic antibiotics may lead to drug resistance and severe side effects. Another strategy is to use tFNAs to improve antibiotic delivery through endocytosis and reduce antibiotic efflux in bacteria. Growth curve results such as Figure 7 As shown, B / L inhibited the growth of Staphylococcus aureus and Escherichia coli. From the 10th hour, the bacteria in the LEV group began to grow actively, while there was no bacterial growth in the B / L group throughout the 24 hours. Figure 8 As shown, no colonies of Staphylococcus aureus and Escherichia coli were found in the B / L group, while obvious colonies were found in the LEV group on the plate. In addition, scanning electron microscopy (TEM) was used to observe Escherichia coli and Staphylococcus aureus after treatment with different materials. Normal Staphylococcus aureus has a spherical morphology, while Escherichia coli usually has a rod-shaped morphology. The Escherichia coli in the blank group has a complete and smooth cell wall, while the cell wall of the B / L treatment group is wrinkled and broken, indicating morphological damage ( Fig. 9 ). Overall, B / L significantly enhanced bacterial entry and disrupted the cell membranes of both Gram-positive and Gram-negative bacteria, thereby inhibiting bacterial proliferation and killing them. Notably, the antibacterial effect of B / L was superior to that of antibiotics alone, thereby reducing the dosage and frequency of antibiotic use and avoiding the serious side effects of antibiotics.

[0122] It can be seen from the above experimental results that B / L inhibits the growth of Staphylococcus aureus and Escherichia coli and has a good antibacterial effect.

[0123] Experimental Example 5: Anti-inflammatory ability test of B / L

[0124] 1. Experimental methods

[0125] 1.1 Experimental Grouping

[0126] To test the ability of B / L to target TRAF6 and IRAK1 to suppress inflammation in HaCaTs, the treatments of each experimental group were as follows:

[0127] Blank group: treated with PBS phosphate buffer (0.1 mol / L);

[0128] LPS group: treated with 10ug / ml LPS;

[0129] LEV group: treated with 200 nM LEV;

[0130] miRNA group: treated with 3.75 nM Cy5-labeled miR-146 mimic;

[0131] tFNAs group: treated with 1.25 nM tFNAs prepared in Control Example 1;

[0132] BiRDS group: treated with 1.25 nM BiRDS prepared in Example 3;

[0133] B / L group: treated with 1.25 nM B / L prepared in Example 4.

[0134] Suspended HaCaT cells (50,000 cells / mL) were seeded into 6-well plates. In the Blank group, LPS group, LEV group, miRNA group, tFNAs group, BiRDS group, and B / L group, HaCaT cells were treated at 37° C. for 48 hours and then collected.

[0135] 1.2 RNA extraction and real-time PCR

[0136] Total RNA from each group 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 a PrimeScript RT kit (TAKARA). Quantitative real-time (qRT)-PCR was performed using an ABIQuantStudio 3PCR system (AppliedBiosystems, USA) using SYBR Premix Ex Taq II (TAKARA). -ΔΔCT Method to evaluate relative gene expression levels.

[0137] 1.3 Western blot analysis

[0138] Each group of proteins was extracted using a whole protein extraction kit (KGB5303-100, Keygene 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 two different sets of primary antibodies were allowed to penetrate the membrane. The next day, the membrane was incubated with a suitable secondary antibody (ZSGB-BIO, China) for 1 hour and then rinsed with TBST (TBS with Tween-20). Next, a gel and blot imaging device (Syngene, Bangalore, India) was used to expose the membrane. The results were semi-quantitatively evaluated using ImageJ software.

[0139] Among them, the primary antibodies used in the first group were as follows: TRAF6 antibody (R1311, Shanghai, China), IRAK1 antibody (HA500377, Anhui), and GAPDH antibody (5174S, CST).

[0140] The primary antibodies used in the second group were as follows: NF-κB antibody (8242S, CST), IL-6 antibody (R1412, Anhui), IL1βantibody (HA601035, Anhui), TNF-α antibody (ab 183218, Abcam, Cambridge, UK) and GAPDH antibody (5174S, CST).

[0141] 1.4 Immunofluorescence staining

[0142] To further observe protein expression, immunofluorescence staining was performed. Immunofluorescence staining was used to visualize and evaluate protein expression in each group. 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 (the same primary antibodies were used before and after each group as mentioned 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).

[0143] 1.5 Flow cytometry detection of ROS expression in each group after treatment

[0144] To further investigate the expression of ROS after treatment in each group, flow cytometry was performed. The cells in the 12-well plate were digested with EDTA-free trypsin, washed with PBS phosphate buffer (0.1 mol / L), and finally incubated with the DCFH-DA probe at 37C for 20 minutes and then washed with serum-free cell culture medium. The collected cells were quantitatively analyzed using a flow cytometer (FC500, Beckman Coulter). The data were analyzed using FLOW JO X software.

[0145] 2. Experimental results

[0146] (1) B / L inhibits inflammation in HaCaTs by directly targeting TRAF6 and IRAK1

[0147] The healing of skin wounds involves many mechanisms, such as hemostasis, inflammation, proliferation, wound remodeling, and scar tissue formation. Among them, inflammation is considered to be the key to creating an environment conducive to wound regeneration and tissue repair. Bacterial infection often causes excessive inflammation in skin wounds. For example, when bacteria dissolve or die, lipopolysaccharide (LPS) is released from their surface. Endotoxin is a free lipopolysaccharide that can hinder wound healing and cause severe inflammation. It has been found that miR146 controls the expression of many genes and proteins involved in inflammatory responses, including IRAK1 and TRAF6, which are important adapter molecules targeting downstream inflammatory pathways. After the activation of cell surface receptors (such as TLR4), the molecular cascade of IRAK1 and TRAF6 induces the phosphorylation and degradation of i-κBα, thereby activating the NF-κB signaling pathway. This pathway plays an important role in many inflammatory processes in various disease phenotypes. Activation of NF-κB leads to increased transcription of genes related to inflammation and enhances the production of cytokines such as TNF-α, IL-1β, and IL-6. Although miR146 has been identified as a key component of HaCaTs skin wound healing due to the delivery limitations of miRNA, as mentioned above, it is still difficult to use this determinant to address skin wound healing. Therefore, considering the pathological characteristics of infected wounds, the present invention assembled three equivalent miR146 mimics into B / L.

[0148] In the present invention, HaCaTs were stimulated with LPS to simulate an inflammatory environment. After B / L treatment, the gene expression levels of miR146 target proteins TRAF6 and IRAK1 were detected by quantitative polymerase chain reaction (qPCR). Fig.10As shown in B, B / L treatment successfully reduced the expression of TRAF6 and IRAK1 mRNA. In addition, the inhibitory effect of B / L on TRAF6 and IRAK1 was determined at the protein level by Western blot (WB) quantitative analysis. After LPS treatment, the activation of these proteins could be clearly observed compared with the blank group. In the B / L treatment group, the target proteins TRAF6 and IRAK1 were significantly downregulated in HaCaTs, which was basically consistent with the blank group ( Fig.10 C and 10D). The present invention confirmed these results by immunofluorescence staining. Immunofluorescence imaging and quantitative analysis showed that LPS stimulation caused strong red fluorescence in protein expression, while the red fluorescence in the BiRDS and B / L groups was significantly downregulated, indicating that the expression of target proteins TRAF6 and IRAK1 was downregulated.

[0149] (2) B / L downregulates the NF-κB pathway and reduces the expression of inflammatory factors

[0150] NF-κB is a key nuclear transcription factor that is involved in regulating gene expression and mediating inflammation and stress responses in the body. The activity of the NF-κB pathway further enhances the expression of cytokines such as TNF-α, IL-1β, and IL-6. Therefore, the present invention further uses qPCR to detect the expression levels of NF-κB p65, NF-κB p-p65, TNF-α, IL-1β, and IL-6. Fig.11 As shown in A, B / L successfully reduced the expression of NF-κBp65, NF-κB p-p65, TNF-α, IL-1β, and IL-6 mRNA. In addition, the inhibitory effect of B / L on NF-κB p65, NF-κB p-p65, TNF-α, IL-1β, and IL-6 was determined by WB protein quantitative analysis. The results showed that in the B / L-treated group, the expression of NF-κB in HaCaTs was significantly downregulated, and the levels of downstream inflammatory factors were also significantly reduced. ( Fig.11 B). Then, the present invention confirmed the expression of NF-κB p65, NF-κB p-p65, TNF-α, IL-1β and IL-6 again by immunofluorescence staining. Immunofluorescence imaging showed that the red fluorescence of BiRDS group and B / L group was significantly downregulated, indicating that the expression of inflammation-related proteins was significantly downregulated ( Fig.11 C).

[0151] (3) B / L removes excess ROS accumulated in HaCaT cells to inhibit the inflammatory response of HaCaTs

[0152] Fig.12 The results of B / L clearing excess ROS accumulated in HaCaT cells to inhibit the inflammatory response of HaCaTs are shown. Fig.12A), LEV, miRNA, tFNA, BiRDS and B / L treatment reduced the ROS content in HaCaT cells treated with LPS. The ROS reduction effect after B / L treatment was higher than the sum of the ROS reduction effects after LEV and BiRDS treatments, indicating that B / L played a synergistic effect in reducing the expression of ROS. Fig.12 B results showed that the green fluorescence of the BiRDS group and the B / L group was significantly downregulated, indicating that the ROS level was significantly reduced. From the above experimental results, it can be seen that B / L significantly downregulated the expression of target proteins TRAF6 and IRAK1 in HaCaTs by directly targeting TRAF6 and IRAK1, and B / L had a significant inhibitory effect on TRAF6, IRAK1, NF-κBp65 and NF-κB p-p65, inhibited the expression of TNFα, IL-1β and IL-6, significantly downregulated the NF-κB pathway, and reduced the expression of inflammatory factors, thereby reducing the in vitro inflammatory response. Moreover, the anti-inflammatory effect after B / L treatment was higher than the sum of the anti-inflammatory effects after LEV and BiRDS treatment, and played a synergistic effect.

[0153] Experimental Example 6: Animal experiment on the effect of B / L on promoting healing of infected wounds

[0154] 1. Experimental methods

[0155] 1.1 Animal modeling and treatment

[0156] Six groups of female BALB / c mice (age 6-8 weeks, weight 18-22 g) were randomly assigned. Full-thickness skin defects were created using a 5 mm diameter circular punch. Then, 50 μl of Escherichia coli or Staphylococcus aureus suspension (1×10 6 CFU / mL) was evenly applied around the wound to establish an infected wound. The treatment of each experimental group is as follows:

[0157] Blank group: treated with PBS phosphate buffer (0.1 mol / L);

[0158] LEV group: treated with 15000nM LEV;

[0159] miRNA group: treated with 750 nM Cy5-labeled miR-146 mimic;

[0160] tFNAs group: treated with 250 nM tFNAs prepared in Control Example 1;

[0161] BiRDS group: treated with 250 nM BiRDS prepared in Example 3;

[0162] B / L group: treated with 250 nM B / L prepared in Example 4.

[0163] The above groups were then thoroughly mixed with Aquaphor cream (Eucerin, Germany) (1 g Aquaphor was dissolved in every 100 ul of drug or saline) and evenly applied around the wound (100 ul drug / mouse). Tegaderm (3M, USA) was also covered in the area, and the dressing was changed every 24 hours. 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) × 100%. On day 7, the antibacterial activity was evaluated in vivo. The bacteria were dispersed on LB agar plates and cultured in a static environment at 37°C for 12 hours.

[0164] 1.2 Histological analysis

[0165] 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 embedded in paraffin, 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. Immunohistochemistry was used to detect the relative expression levels of TNFα, IL-1β, and IL-6, and the degree of inflammation and osteogenesis in different groups were analyzed. All samples were observed under an Olympus FSX100 microscope.

[0166] 2. Experimental results

[0167] like Fig.13 As shown, B / L significantly accelerated the healing rate of wounds infected with Escherichia coli or Staphylococcus aureus. The B / L group showed significant wound healing ability from day 3 to day 7 compared with the LEV, miRNA, tFNA, and BiRDS groups. After 14 days, the wounds treated with B / l showed obvious healing, while the wounds of the other categories still showed unhealed wound areas and visible scars, indicating a slower recovery rate. B / L showed the highest healing rate of infected wounds throughout the wound healing stage. Hematoxylin and eosin (H&E) staining of each group can further study the histological changes within the wounds on days 7 and 14. As Fig.14As shown, the epidermal layer of the LEV, miRNA, and tFNA groups was still incomplete, while epithelialization in the B / L-treated group was complete and the wound was completely covered by a full layer of epidermis. In addition, a small number of new hair follicles appeared in the B / L group on the 14th day. Masson trichrome staining showed that the collagen repair rate of the wound tissue in the B / L group was greater than that in the other groups on the 7th and 14th days. In general, the B / L ratio improves wound healing by promoting wound contraction, collagen repair, and re-epithelialization. In addition, after B / L treatment, the number of Escherichia coli or Staphylococcus aureus around the wound was significantly reduced, while the LEV group had almost no antibacterial effect ( Fig.15 A significant antimicrobial effect was also observed in the BiRDS group, which may be due to BiRDS downregulating inflammation levels in the body, making it more difficult for pathogens to invade and multiply.

[0168] In addition to bacterial infection, dead bacteria promote extreme skin inflammation by releasing toxins upon death / dissolution, inducing inflammation and toxicity to host tissues. The expression of key related proteins was analyzed by WB and immunofluorescence staining to detect the inflammatory status of the wounds. After a 14-day observation period, in the B / L treatment group, the target proteins TRAF6 and IRAK1 of Escherichia coli or Staphylococcus aureus infected wounds and the key inflammatory-related factor NF-KB p65 were significantly downregulated, and inflammation in the injured area was reduced ( Fig.16 ). In addition, in immunofluorescence staining, red fluorescence was rarely observed in the B / L group, indicating that inflammatory factors such as TNF-α, IL-1β, and IL-6 were significantly inhibited ( Fig.17 ). These results indicate that bacteria can cause severe inflammatory responses, and B / L can effectively inhibit bacterial growth, relieve inflammatory responses, and promote skin wound healing in vivo.

[0169] From the above experimental results, it can be seen that B / L significantly improved the healing rate of wounds infected with Escherichia coli or Staphylococcus aureus during the entire wound healing stage. B / L can effectively inhibit bacterial growth, relieve inflammatory response and promote skin wound healing in the body.

[0170] In summary, the present invention provides a tetrahedral framework multifunctional nanocomposite and its preparation method and use. The present invention realizes small molecule drug delivery on the miRNA delivery system based on tetrahedral framework DNA for the first time, and prepares a nanocomposite for co-delivery of miRNA and antibiotics. The nanocomposite has high stability, good cell and tissue permeability and significant biocompatibility, and can combine anti-inflammatory and antibacterial activity at the same time, and is applied to the combined treatment of infected wound healing, providing a new treatment strategy for the treatment of complex and refractory diseases.

Claims

1. A nanocomposite, characterized in that: The nanocomplex is prepared using siRNA-modified tetrahedral framework nucleic acid and antibiotics as raw materials, wherein the molar ratio of the siRNA-modified tetrahedral framework nucleic acid to the antibiotic is 1:50-70, and the siRNA is miRNA with antibacterial and / or anti-inflammatory effects.

2. The nanocomposite according to claim 1, characterized in that The molar ratio of the siRNA-modified tetrahedral framework nucleic acid to the antibiotic is 1:

60.

3. The nanocomposite according to claim 1, characterized in that The siRNA modified tetrahedral framework nucleic acid is a product prepared using siRNA and tetrahedral framework nucleic acid as raw materials, wherein the molar ratio of siRNA to tetrahedral framework nucleic acid is 1-5:1, preferably 3:

1.

4. The nanocomposite according to claim 3, characterized in that The siRNA modified tetrahedral framework nucleic acid is self-assembled from four single strands, and the sequences of the four single strands are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively.

5. The nanocomposite according to claim 3, characterized in that: The method for preparing the siRNA-modified tetrahedral framework nucleic acid comprises the following steps: mixing the siRNA and the tetrahedral framework nucleic acid and reacting them to obtain the siRNA.

6. The nanocomposite according to any one of claims 1 to 5, characterized in that: The siRNA is miRNA-146; the antibiotic is levofloxacin.

7. A method for preparing the nanocomposite according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: mixing the siRNA-modified tetrahedral framework nucleic acid and the antibiotic and reacting them to obtain a nanocomplex.

8. The method according to claim 7, characterized in that The reaction temperature is 0 to 10°C and the reaction time is 1 to 5 hours; Preferably, the reaction temperature is 4° C. and the reaction time is 3 hours.

9. Use of the nanocomposite according to any one of claims 1 to 6 in the preparation of a drug having antibacterial and / or anti-inflammatory effects.

10. Use of the nanocomplex according to any one of claims 1 to 6 in the preparation of a medicament for promoting healing of infected wounds.

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