A medicine for treating scald infection and its preparation method and use

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a drug for treating scald infection and a preparation method and application thereof. The application prepares a drug for treating scald infection, which is self-assembled from a tetrahedral framework nucleic acid and an antibiotic. The application preferably selects a tFNAs-NeoS drug, the drug has a simple preparation method, a high loading efficiency of neomycin sulfate, good stability in a complex biological medium, reduced cytotoxicity and effective dose of the antibiotic NeoS, better biological safety than tFNAs, and slowed generation of bacterial drug resistance. In terms of treating scald wound infection, the tFNAs-NeoS has better effects of promoting wound healing and biological safety than NeoS. Therefore, the prepared tFNAs-NeoS overcomes the problem of overuse of antibiotics, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a drug for treating burn infections, its preparation method, and its uses. Background Technology

[0002] Burn infection is the most common complication of burns, which can lead to sepsis, multiple organ failure, permanent scarring, and disfigurement. Globally, over ten million people seek treatment for burns each year, incurring hundreds of millions of dollars in costs, placing a huge burden on patients and society. Under the influence of high-temperature gases or liquids, localized overheating of the skin leads to irreversible damage characterized by changes in molecular conformation. As the temperature continues to rise, proteins denature, oxygen free radicals are released, and ultimately, cells die, forming an eschar. Furthermore, the body immediately initiates a series of physiological mechanisms to maintain homeostasis, including vasoconstriction, vasodilation, and coagulation; these responses can exacerbate the risk of systemic infection.

[0003] Topical antibiotics are crucial for burn treatment. Commonly used topical antibiotics include bacitracin, polymyxin B, and neomycin sulfate (NeoS), which are typically applied as ointments to the wound surface. However, they exhibit cytotoxicity to keratinocytes and fibroblasts and have difficulty penetrating eschar. Furthermore, the overuse of antibiotics accelerates infection by drug-resistant bacteria, exacerbating the challenges of infection control and leading to broader and more serious public health problems. Therefore, it is imperative to develop a strategy that minimizes antibiotic toxicity and effective dosage while maintaining its antibacterial efficacy.

[0004] Nanodelivery systems offer a promising solution to these problems. DNA polyhedra, first proposed by Turberfield and colleagues, include tetrahedral framework nucleic acids (tFNAs), which are self-assembled from four single-stranded DNA strands and are considered the most stable polyhedra. Unlike traditional nanomaterials, which suffer from cytotoxicity and low clearance defects, natural nucleic acid nanomaterials (tFNAs) have attracted significant attention due to their biocompatibility and biodegradability. Compared to simple double-stranded DNA, tFNAs possess ideal cell penetration capabilities, a more stable structure, and a richer array of drug loading sites. They can also modulate cellular biological behavior and can be arbitrarily changed in size and dimensions to meet the needs of various carriers. In this context, tFNAs have met the requirements for drug carriers. To date, numerous studies have demonstrated that tFNAs, as drug carriers, can carry oligonucleotides, small-molecule antibiotics, and peptides, and promote cell migration and proliferation. Nucleic acid nanocarrier technology offers a glimmer of hope for effectively improving NeoS sensitivity and reducing toxic side effects.

[0005] However, whether chemical drugs can self-assemble with nucleic acids depends not only on the configuration (e.g., A-type double-stranded, B-type double-stranded, triple-stranded, quadruple-stranded) and properties of the nucleic acids, but also on the structure and properties of the drug itself. Different drugs exhibit significant differences in their interactions with nucleic acids. Furthermore, biological media contain complex components that may compete with chemical drugs or nucleic acids for binding sites, disrupting the drug-tetrahedral framework nucleic acid complex structure and thus greatly reducing delivery efficiency. Therefore, whether tFNAs can effectively load and deliver burn medications remains a challenge.

[0006] Against this backdrop, exploring a composite material based on a tetrahedral framework for nucleic acid delivery of burn-related drugs, thereby reducing the toxic side effects and effective dosage of antibiotics and effectively treating burn wounds, is a problem that urgently needs to be solved in this field. Summary of the Invention

[0007] To address the problems of existing technologies, this invention provides a drug for treating burn infections and its preparation method, with the aim of safely and effectively treating burn wounds.

[0008] This invention provides a tetrahedral framework nucleic acid material for treating burn infections, the tetrahedral framework nucleic acid material being composed of tetrahedral framework nucleic acid and antibiotics, the antibiotics including neomycin and pharmaceutically acceptable salts thereof;

[0009] The ratio of the tetrahedral framework nucleic acid to the antibiotic is 9.5 μM: 30-420 μg.

[0010] Preferably, the antibiotic includes neomycin, neomycin sulfate, or neomycin B sulfate.

[0011] Preferably, the antibiotic is neomycin sulfate.

[0012] Preferably, the ratio of the tetrahedral framework nucleic acid to the antibiotic is 9.5 μM: 277-278 μg.

[0013] Preferably, the tetrahedral framework nucleic acid is composed of four single-stranded nucleic acids S1, S2, S3 and S4 through base complementary pairing;

[0014] The molar ratio of S1 to S2, S3, and S4 is 1:1:1:1.

[0015] Preferably, the nucleotide sequence of S1 includes SEQ ID NO.1; and / or, the nucleotide sequence of S2 includes SEQ ID NO.2; and / or, the nucleotide sequence of S3 includes SEQ ID NO.3; and / or, the nucleotide sequence of S4 includes SEQ ID NO.4.

[0016] This invention provides a method for preparing the tetrahedral framework nucleic acid material according to any one of the above claims, comprising the following steps:

[0017] The tetrahedral framework nucleic acid and antibiotics are mixed to obtain the product.

[0018] Preferably, the mixing is performed by shaking at 0-4°C for 4-8 hours.

[0019] The present invention provides the use of the tetrahedral framework nucleic acid material described in any of the above claims in the preparation of drugs for treating burns and / or bacterial infections.

[0020] This invention provides a drug for treating burn infections, which is made by using the tetrahedral framework nucleic acid material described in any one of the above-mentioned claims as the active ingredient and adding pharmaceutically acceptable excipients.

[0021] The term "pharmaceutically acceptable salt" refers to acidic and / or basic salts formed by the aforementioned antibiotics with inorganic and / or organic acids and bases, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkyl ammonium salts. The salts described in this invention can be hydrochlorides, sulfates, citrates, benzenesulfonates, hydrobromides, hydrofluoric acids, phosphates, acetates, propionates, succinates, oxalates, malates, succinates, fumarates, maleates, tartrates, or trifluoroacetates of the compounds.

[0022] This invention prepares a drug for treating burn infections, which is self-assembled from tetrahedral framework nucleic acids and antibiotics. This invention screened methods for preparing antibiotics and nanoscale nucleic acid antibiotics, and preferred the tFNAs-NeoS drug. This drug preparation method is simple; it has high loading efficiency for neomycin sulfate; it exhibits good stability in complex biological media; tFNAs-NeoS reduces the cytotoxicity and effective dose of the antibiotic NeoS, and has better biocompatibility than tFNAs; it also slows down the development of bacterial resistance. In treating burn wound infections, tFNAs-NeoS has better wound healing promotion and biocompatibility than NeoS. Therefore, the tFNAs-NeoS prepared in this invention overcomes the problem of antibiotic overuse and has promising clinical application prospects.

[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0025] Figure 1 Figure A shows the experimental results of agarose gel electrophoresis to verify the synthesis of tetrahedral framework nucleic acids (tFNAs). Figure 1 Figure B shows the experimental results of polyacrylamide gel electrophoresis (PAGE) to verify the synthesis of tFNAs-NeoS.

[0026] Figure 2 The results show the particle size distribution and zeta potential of tFNAs and tFNAs-NeoS under dynamic light scattering detection.

[0027] Figure 3 Atomic force microscopy (AFM) images of tFNAs and tFNAs-NeoS.

[0028] Figure 4 Transmission electron microscopy (TEM) images of tFNAs and tFNAs-NeoS.

[0029] Figure 5 PAGE electrophoresis results and quantitative results of the stability of tFNAs and tFNAs-NeoS in biological media;

[0030] Figure 6 The graph shows the effects of tFNAs, NeoS, and tFNAs-NeoS on cell proliferation as determined by the CCK8 assay; the left graph shows the results after 24 hours; and the right graph shows the results after 48 hours.

[0031] Figure 7 Figure showing the results of Calcein / PI staining to verify the biosafety of tFNAs-NeoS.

[0032] Figure 8 The image shows the results of fluorescence staining to detect the cell entry effect of tFNAs-NeoS.

[0033] Figure 9 Figures showing the scratch test results for tFNAs, NeoS, and tFNAs-NeoS.

[0034] Figure 10 The graph shows the results of the minimum inhibitory concentration (MIC) determination.

[0035] Figure 11 The graph shows the antibacterial results of tFNAs, NeoS, and tFNAs-NeoS determined by the plating method.

[0036] Figure 12 The graph shows the effects of tFNAs, NeoS, and tFNAs-NeoS on bacterial growth curves.

[0037] Figure 13 This is a diagram showing the morphological changes of bacteria as detected by scanning electron microscopy.

[0038] Figure 14 The figure shows the effects of tFNAs, NeoS, and tFNAs-NeoS on bacterial metabolic activities.

[0039] Figure 15 Photographs of burn wounds on rats over time, records of wound extent, and results of wound area.

[0040] Figure 16 Images of hematoxylin and eosin (HE) staining of wound tissues from rats in each group.

[0041] Figure 17 The graph shows the wound scoring results for each group of rats.

[0042] Figure 18 The images show the blood routine results of rats in each group.

[0043] Figure 19 The image shows the immunohistochemical staining results at the wound sites of rats in each group.

[0044] Figure 20 The results show the expression of CD86 and CD206 at the wound site of rats in each group. Detailed Implementation

[0045] In the following examples and experimental cases, reagents and materials not specifically described are all commercially available products.

[0046] All oligonucleotide sequences were synthesized by Sangon Biotech Co., Ltd. (Shanghai).

[0047] Example 1: A drug tFNAs-NeoS for treating burn infections and its preparation method

[0048] This embodiment provides a drug for treating burn infections, which is prepared according to the following method:

[0049] (1) Synthesis method of tFNAs

[0050] Four single-stranded DNA strands (S1, S2, S3, and S4) were dissolved at a concentration of 1 μM per strand in TM buffer (10 nM Tris-HCl and 50 nM MgCl2·6H2O, pH = 8.0). The sequences of each strand are shown in Table 1. The mixture was then heated at 95 °C for 10 min, rapidly cooled to 4 °C, and held for 20 min.

[0051] (2) Synthesis method of tFNAs-NeoS

[0052] tFNAs (500 nM) and NeoS (400 μg / ml) were mixed at a volume ratio of 19:1 and shaken at 4 °C for 6 h.

[0053] Table 1 Single-stranded DNA sequences

[0054]

[0055]

[0056] The technical solution of the present invention will be further explained through experiments below.

[0057] To facilitate experimental observation, the 5′ of S1 was modified with Cy5.

[0058] Example 1: Characterization of tFNAs-NeoS

[0059] This experimental example characterizes the tFNAs and tFNAs-NeoS prepared in Example 1.

[0060] I. Experimental Methods

[0061] 1. Synthesis of tFNAs-NeoS and characterization of its structure, size, and other properties.

[0062] The successful synthesis and structure of tFNAs-NeoS were verified and observed using agarose gel electrophoresis, 8% polyacrylamide gel electrophoresis (PAGE), a Zetasizer Nano ZS90 system (Malvern Panalytical, UK), atomic force microscopy (AFM, Shimadzu, Kyoto, Japan), and transmission electron microscopy (TEM, Hitachi Ltd., Tokyo, Japan).

[0063] When determining tFNAs-NeoS using 8% polyacrylamide gel electrophoresis (PAGE), NeoS at concentrations of 0, 50, 100, 200, 400, and 800 μg / mL were mixed at a volume ratio of 19:1 and shaken at 4°C for 6 h. Then, the PAGE experiment was performed.

[0064] 2. Determination of drug loading efficiency of tFNAs-NeoS

[0065] The drug loading efficiency of tFNAs-NeoS was determined using the standard curve method.

[0066] 500 nM tFNAs were mixed with NeoS at volume ratios of 50, 100, 200, 400, 600, and 800 μg / mL at a ratio of 19:1, and the mixtures were shaken at 4 °C for 6 h. Then, the concentration of free NeoS was measured, and the drug loading efficiency was calculated using the following formula:

[0067] 3. Stability testing of tFNAs-NeoS in biological environments

[0068] tFNAs and tFNAs-NeoS were stored in Eagle's Minimum Essential Medium (eMEM) containing 0-10% FBS for 16 hours; or incubated in eMEM containing 10% FBS for 0-24 hours; or incubated in Luria-Bertani (LB) for 0-24 hours. The degradation of tFNAs and tFNAs-NeoS was detected by 6% PAGE, and the fluorescence intensity was detected by a gel imaging system.

[0069] II. Experimental Results

[0070] 1. Synthesis of tFNAs-NeoS and its structure, size, and other properties.

[0071] The results of the agarose gel electrophoresis experiment are as follows: Figure 1 As shown in Figure A: tFNAs were synthesized stepwise from four single-stranded DNA strands (S1-S3). The PAGE results are as follows... Figure 1 As shown in B: When NeoS at different concentrations was assembled with tFNAs, the structure of the tetrahedral framework nucleic acid was not destroyed. Dynamic light scattering (DLS) results are shown below. Figure 2 As shown, the particle size of tFNAs-NeoS is significantly larger than that of tFNAs, and the zeta potential also changes from negative to positive. This indicates the successful synthesis of tFNAs-NeoS. The AFM plot (...) Figure 3 ) and TEM image ( Figure 4 It was observed that tFNAs exhibited a triangular nanostructure, and tFNAs-NeoS possessed a similar nanostructure.

[0072] 2. Drug loading efficiency of tFNAs-NeoS

[0073] The drug loading efficiency results are shown in Table 2: a high loading efficiency can be obtained when the initial concentration of NeoS is 50-400 μg / mL; the loading efficiency decreases significantly when the initial concentration of NeoS continues to increase. Therefore, this invention preferably uses a NeoS concentration of 400 μg / mL for material preparation.

[0074] Table 2 Drug loading efficiency

[0075]

[0076] 3. Stability of tFNAs-NeoS

[0077] Stability test results are as follows Figure 5As shown, the fluorescence signal of tetrahedral framework nucleic acids decreased when FBS was added to the cell culture medium, but after incubation for 24 hours in cell culture medium containing 10% FBS, they still showed fluorescence intensity comparable to tFNAs. When tFNAs-NeoS were incubated in Luria-Bertani medium for 24 hours, they also showed fluorescence intensity comparable to tFNAs. This indicates that tFNAs-NeoS can maintain a certain degree of stability in complex biological media, providing a basis for potential antibacterial and in vivo applications.

[0078] The above results indicate that tFNAs-NeoS, exhibiting a nanocomposite structure, was successfully prepared using the method described in Example 1. It possesses properties such as small size, ease of synthesis, high drug loading capacity, and good stability, and has the potential for clinical application.

[0079] Experimental Example 2: Effects of tFNAs-NeoS on Cells

[0080] The tFNAs and tFNAs-NeoS samples used in this experimental example were prepared according to the method in Example 1.

[0081] I. Experimental Methods

[0082] 1. Cell Culture

[0083] L929 (mouse fibroblast cell line) CRL1730 TM L929 cells were cultured in high-glucose DMEM (0.1 mM non-essential amino acids, 4 mM L-glutamine, 10% FBS, and 1% penicillin-streptomycin antibiotics). 5000 L929 cells were seeded into each well of a 96-well plate and treated for 24 and 48 hours with NeoS (100 μg / mL), tFNA (50, 100, 200, 300, 400, 500 nM), and tFNAs-NeoS (100 nM).

[0084] 2. Experimental Grouping

[0085] The experiment was divided into four groups: control group, tFNAs group, NeoS group, and tFNAs-NeoS group. The control group received PBS, the tFNAs group received 100 nM tFNAs, the NeoS group received 100 μg / mL NeoS, and the tFNAs-NeoS group received 100 nM tFNAs-NeoS.

[0086] 3. Measurement of cell proliferation

[0087] In the experimental groups, the drugs in the tFNAs groups were 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, and 500 nM, respectively. After treatment with the drugs for 24 h or 48 h, cell viability was measured using the CCK8 reagent.

[0088] 4. Live and dead staining experiment

[0089] To further determine the biosafety of the drug, cells were treated with the drug for 24 hours according to their groupings, and then stained with Calcein / PI and observed under a microscope. Green indicates live cells, and red indicates dead cells.

[0090] 5. Measurement of cellular uptake

[0091] In the experimental groups, the control group received single-stranded DNA S1. To facilitate experimental observation, the 5′ of S1 was modified with Cy5.

[0092] Cells were seeded in confocal culture dishes (Corning Inc., NY, USA) and cultured for 24 h. Cells were treated with different drugs according to their groupings, and after 24 h of culture in the dark, they were fixed with 4% paraformaldehyde (Servicebio, China) for 60 min, stained with phalloidin (Servicebio, China) for 30 min, and stained with 4',6-diamidinyl-2-phenylindole (DAPI) (Servicebio, China) for 10 min. Cells were washed three times with PBS between each step, and finally blocked with 10% glycerol (Servicebio, China). The intracellular nanomaterial infiltration was observed using a laser confocal microscope (FV3000, Olympus, Japan).

[0093] 6. Cell migration experiment

[0094] The effects of different drugs on cell migration were investigated using a scratch assay.

[0095] In the scratch assay, cells were first evenly seeded into 6-well plates. After the cells had fully grown to confluence, a vertical line was drawn in the center of each well to represent the scratch. Different drugs were added according to the group assignments, and cell migration was observed and recorded under an inverted optical microscope at 0h, 24h, 48h, and 72h to determine the number of cells at the scratch site.

[0096] II. Experimental Results

[0097] 1. Biosafety of tFNAs and tFNAs-NeoS

[0098] The results of the viability test are as follows Figure 6As shown, cell viability decreased with increasing tFNA concentration. Cell viability significantly decreased after treatment with NeoS or 500 nM tFNAs for 48 h; however, cell viability was not significantly affected after treatment with tFNAs-NeoS for 24 h or 48 h. This indicates that tFNAs-NeoS reduced the cytotoxicity of NeoS and its biocompatibility was superior to that of tFNAs.

[0099] Calcein / PI staining results are as follows: Figure 7 As shown, a certain number of dead cells were observed in cells treated with NeoS, while almost no dead cells were observed in the tFNAs and tFNAs-NeoS groups, further demonstrating that tFNAs-NeoS has excellent biocompatibility.

[0100] 2. Cellular uptake of tFNAs-NeoS

[0101] Confocal microscopy experimental results are as follows Figure 8 As shown, compared with the control group, the ability of tFNAs and tFNAs-NeoS to enter cells was significantly enhanced. This indicates that tFNAs can enter cells efficiently, outperforming single-stranded DNA entry.

[0102] 3. Effects of tFNAs-NeoS on cell migration

[0103] The results of the scratch test are as follows Figure 9 As shown, compared with the control group, the number of cells at the wound site in the tFNAs and tFNAs-NeoS groups increased significantly over time, demonstrating excellent cell migration and wound healing capabilities.

[0104] These results indicate that tFNAs-NeoS reduces the cytotoxicity of NeoS and has better biocompatibility than tFNAs. tFNAs-NeoS can efficiently enter cells and effectively promote cell migration.

[0105] Experimental Example 3: In vitro antibacterial properties of tFNAs-NeoS

[0106] The tFNAs and tFNAs-NeoS samples used in this experiment were prepared according to the method in Example 1. The bacteria used in this experiment were Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC25923.

[0107] I. Experimental Methods

[0108] 1. Experimental Grouping

[0109] The experiment was divided into four groups: control group, tFNAs group, NeoS group, and tFNAs-NeoS group. The control group received PBS, the tFNAs group received 200 nM tFNAs, the NeoS group received 1.25 μg / mL NeoS, and the tFNAs-NeoS group received 200 nM tFNAs-NeoS.

[0110] 2. Determination of minimum inhibitory concentration

[0111] Different concentrations of neomycin sulfate and tFNAs-NeoS drug solutions were prepared using LB medium, with NeoS concentrations of 0.2 μg / mL, 0.4 μg / mL, 0.8 μg / mL, 1.2 μg / mL, 1.6 μg / mL, 2.0 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL. 10 μL of the diluted bacterial suspension was added to each well for each drug concentration. The 96-well plate was incubated at 37°C for 24 hours. The lowest drug concentration at which no bacterial growth was observed was recorded as the minimum inhibitory concentration (MIC) of the drug for that bacterium.

[0112] 3. Bacterial colony count

[0113] The corresponding concentrations of drugs for each group were prepared using LB culture medium. The bacterial suspensions were mixed with the drugs for each group for 24 hours, and the bacterial colonies were counted using the plating method.

[0114] 4. Determination of bacterial growth curve

[0115] The corresponding concentrations of drugs for each group were prepared using LB culture medium. The bacterial suspensions were mixed with the drugs for each group and cultured in a bacterial incubator at 37°C for 0, 2, 4, 6, 8, 16 and 24 hours, respectively, and the OD value at 600 nm was measured.

[0116] 5. Determination of the influence of bacterial morphology

[0117] According to the grouping, each group of drugs and bacteria were cultured for 24 hours, then the cells were fixed with 4% formaldehyde, dehydrated, and observed under a scanning electron microscope.

[0118] 6. Determination of the effect on bacterial metabolic activity

[0119] tFNAs, NoeS, and tFNAs-NeoS were respectively compared with 5×10 5CFU / mL Escherichia coli and Staphylococcus aureus were mixed and incubated at 37°C for 3 hours, followed by centrifugation to collect the supernatant. The supernatant was then subjected to an o-nitrophenyl-β-D-glucopyranoside (ONPG) test: the supernatant was incubated with 4 mM ONPG reagent at 37°C, and the OD value at 420 nm was measured after 20 hours.

[0120] II. Experimental Results

[0121] 1. Minimum inhibitory concentration

[0122] MIC experimental results are as follows Figure 10 As shown: for *Escherichia coli*, the MIC of NeoS was 5 μg / mL, and the MIC of tFNAs-NeoS was 1.2 μg / mL; for *Staphylococcus aureus*, the MIC of NeoS was 2.5 μg / mL, and the MIC of tFNAs-NeoS was 1.2 μg / mL. The results indicate that tFNAs-NeoS reduces the effective dose of antibiotics and increases bacterial sensitivity to antibiotics.

[0123] 2. The antibacterial effect of the drug

[0124] The experimental results of the plate counting method are as follows: Figure 11 As shown, for *Escherichia coli* and *Staphylococcus aureus*, the number of bacteria in the NeoS group and the tFNAs-NeoS group was significantly reduced, especially in the tFNAs-NeoS group. This indicates that tFNAs-NeoS has superior antibacterial properties compared to NeoS.

[0125] Growth curves as follows Figure 12 As shown, compared with the control group and the tFNAs group, the growth rate of bacteria treated with NeoS slowed down with prolonged culture time; while the growth of the tFNAs-NeoS group was significantly inhibited, showing a low OD value even after 24 hours of culture. These results further indicate that tFNAs-NeoS has superior antibacterial activity compared to NeoS.

[0126] 3. Effects of drugs on bacterial morphology

[0127] Scanning electron microscopy results as follows Figure 13 As shown, compared with the control group, the bacteria in the tFNAs group still maintained an intact bacterial morphology, some bacteria in the NeoS group shrank and ruptured, while the bacteria in the tFNAs-NeoS group showed extensive and obvious damage and shrinkage, resulting in incomplete bacterial structure. The results indicate that tFNAs-NeoS leads to the rupture of bacterial structure.

[0128] 4. Effects of drugs on bacterial metabolic activity

[0129] The results are as follows Figure 14 As shown, the solutions of the tFNAs-NeoS group are darker in color and have higher OD values, especially in E. coli, indicating that the galactosidase reaction of the tFNAs-NeoS group is stronger, and more galactosidase permeates out of the bacteria, further proving that tFNAs-NeoS makes the bacteria have higher membrane permeability.

[0130] The above results indicate that the MIC of tFNAs-NeoS against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC25923 is 1.2 μg / mL, reducing the effective dose of antibiotics and increasing bacterial sensitivity to antibiotics. tFNAs-NeoS exhibits superior antibacterial activity compared to NeoS.

[0131] Experimental Example 4: tFNAs-NeoS applied to scalded wounds in rats

[0132] The tFNAs and tFNAs-NeoS samples used in this experimental example were prepared according to the method in Example 1.

[0133] I. Experimental Methods

[0134] 1. Establishment of a rat scald wound model

[0135] The animal preparation protocol was approved by the Ethics Committee of the Department of Experimental Animal Science, Sichuan University. Male Sprague Dawley rats (220-300g) aged 7-9 weeks were used for adaptive culture, with unrestricted access to water and food in a light-dark environment for 12 hours. The rats were euthanized after the experiment. Forty rats were randomly divided into four groups. After anesthesia, all rats had their backs shaved and were injected with tenoxacin (15mg / kg body weight) for pain management. A metal block was placed in a 98℃ water bath for 0.5h, and then applied to the shaved skin on the back for 9s. Ten minutes after the burn, 50μl of 2×10⁻⁶ sodium hydroxide solution was applied. 7 The solution of Escherichia coli and Staphylococcus aureus at CFU / mL was administered subcutaneously, as close as possible to the burn area.

[0136] 2. Experimental Grouping

[0137] The experiment was divided into four groups: a control group, a tFNAs group, a NeoS group, and a tFNAs-NeoS group. The control group received physiological saline; the tFNAs group received 200 nM tFNAs; the NeoS group received 100 μg / ml NeoS; and the tFNAs-NeoS group received 200 nM tFNAs-NeoS. The dosage was 500 μl per animal per administration.

[0138] On day 0, each group of wounds was treated with medication, and then medication was administered every other day, with photos taken of the wounds during this period. Medication was discontinued after day 14.

[0139] 3. Tissue staining

[0140] After treatment, the wound tissues of rats in each group were dissected, fixed with formaldehyde, and then stained with hematoxylin and eosin (HE).

[0141] 4. Wound scoring

[0142] The wound healing status was scored based on four aspects: black eschar, wound depth, edema, and resurfacing epithelium.

[0143] The scoring criteria are shown in the table below:

[0144]

[0145] 5. Determination of the effect of drugs on rat blood routine tests

[0146] Fourteen days after treatment, blood was collected from each group of rats for routine blood tests, including white blood cell count (WBC), lymphocyte count (Lymph), granulocyte percentage (Gran%), and monocyte percentage (Mon%).

[0147] 6. Immunohistochemical staining experiment

[0148] (1) Take paraffin sections and place them in xylene I for 15 min, xylene II for 15 min, xylene III for 15 min, anhydrous ethanol I for 5 min; anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and wash with distilled water. (2) Antigen retrieval: Immerse the sections in citrate buffer (pH 6.0), retrieval instrument at 80℃ for 20 min; after cooling, wash with PBS 3 times, 5 min each time. (3) Block endogenous peroxidase: Place the sections in 3% hydrogen peroxide, room temperature for 10 min; wash with PBS 3 times, 5 min each time. (4) Serum blocking: Add goat serum blocking solution, room temperature for 20 min; add primary antibody, incubate at 4℃ overnight; wash with PBS 3 times, 5 min each time; add secondary antibody, incubate at 37℃ for 30 min; wash with PBS 3 times, 5 min each time. (5) DAB staining: Prepare fresh DAB staining solution, add it to the tissue, and stain at room temperature. Control the staining time under a microscope. Positive results are brownish-yellow. Wash the sections with distilled water to stop the staining. (6) Counterstaining cell nuclei: Counterstain with hematoxylin for 3 minutes, wash with tap water, rinse with running water after the stain has turned blue again. (7) Dehydration and mounting: Soak the sections in 75%, 85%, 95%, anhydrous ethanol, and xylene for 10 minutes each, and mount with neutral resin. Observe the content of TNFα, IL1β, VEGF, and αSMA in the immunohistochemical sections under an optical microscope.

[0149] 7. Experiments to explore the anti-inflammatory mechanism

[0150] After dewaxing, the sections were immersed in 0.01M citrate buffer (pH 6.0), heated to boiling in a microwave oven on high, then the power was turned off. This process was repeated once after 5 minutes, followed by cooling. The sections were washed three times with PBS for 5 minutes each time. 10% serum blocking solution was added and the sections were incubated at room temperature for 30 minutes. Primary antibody was added and the sections were incubated overnight at 4°C. The sections were washed three times with PBS. Secondary antibody was added and the sections were incubated at 37°C for 30 minutes. DAPI was added and the sections were incubated at room temperature for 10 minutes. The sections were washed three times with PBS. The sections were then mounted with anti-fluorescence attenuation mounting medium.

[0151] All specimens were examined according to the standard operating procedure (SOP) for pathological examination. This included dehydration, trimming, embedding, sectioning, staining, and mounting. Finally, microscopic examination was performed to detect the expression of CD86 and CD206.

[0152] II. Experimental Results

[0153] 1. Healing status of burn wounds

[0154] The wound healing status of scalded rats in each group is as follows: Figure 15As shown, with the extension of time, compared with the control group, the wound size of each experimental group decreased significantly, and the eschar was reduced. In particular, after 14 days of treatment, the wounds in the tFNAs-NeoS group were almost completely healed without eschar. The results indicate that tFNAs-NeoS has a very good therapeutic effect on burn wounds, and its therapeutic effect is superior to that of tFNAs and NeoS.

[0155] 2. HE staining results

[0156] HE staining results are as follows Figure 16 As shown: the stained tissue sections of the control group contained a large amount of cell debris, while the wounds of the tFNAs-NeoS group had intact newly formed epithelial tissue and no cell debris. This indicates that tFNAs-NeoS effectively promotes wound healing.

[0157] 3. Scoring Results

[0158] The scoring results are as follows Figure 17 As shown, tFNAs-NeoS avoids the formation of black eschar, reduces wound depth, alleviates edema at the wound site, and promotes the regeneration of epithelial tissue at the wound site, thereby effectively promoting the healing of burn wounds.

[0159] 4. Effects on blood routine tests

[0160] The effects on blood routine test results are as follows: Figure 18 As shown, compared with the control group, the number of white blood cells, lymphocytes, granulocytes, and monocytes decreased, especially in the tFNAs-NeoS group, where the levels of these immune cells in the blood decreased the most. This indicates that burns in rats trigger a series of immune responses, leading to an increase in immune cell levels. As the wound heals, the levels of immune cells return to normal, further confirming that tFNAs-NeoS can promote wound healing and reduce the level of immune cells in living organisms.

[0161] 5. Immunohistochemical results

[0162] Immunohistochemical results as follows Figure 19 As shown, compared with the control group, the levels of TNFα and IL1β in the tFNAs-NeoS group were significantly reduced, indicating that the inflammatory response in rats was weakened and the immune system's defense against pathogens was reduced after treatment with tFNAs-NeoS, demonstrating that the drug effectively prevented bacterial infection. Compared with the control group, the levels of VEGF and αSMA in the tFNAs-NeoS group were increased, indicating enhanced angiogenesis, cell contraction, and tissue repair capabilities in rats. Therefore, tFNAs-NeoS greatly promoted wound healing.

[0163] 6. Anti-inflammatory mechanism

[0164] The expression of CD86 and CD206 is as follows: Figure 20 As shown: Compared with the control group, the NeoS group showed increased CD86 expression, indicating that NeoS led to an enhanced inflammatory response, but there may be overactivation of the immune system, resulting in inflammation and tissue damage; while the tFNAs-NeoS group showed increased CD206 expression, indicating that tFNAs-NeoS led to the activation of M2 macrophages, which helps to reduce inflammation and promote tissue repair, indicating that the body is repairing damaged tissue through an anti-inflammatory response.

[0165] These results indicate that tFNAs-NeoS promotes the healing of burn wounds in rats, with significantly better effects than tFNAs and NeoS alone. Furthermore, tFNAs-NeoS does not induce an excessive immune response during wound healing, demonstrating better biocompatibility.

[0166] As demonstrated by the above experimental examples, this invention successfully prepared tFNAs-NeoS, which possesses properties such as small size, ease of synthesis, high drug loading capacity, and good stability in biological media. tFNAs-NeoS reduces the cytotoxicity of the antibiotic NeoS, exhibiting superior biocompatibility compared to tFNAs. tFNAs-NeoS can efficiently enter cells and effectively promote cell migration. For *Escherichia coli* and *Staphylococcus aureus*, the minimum inhibitory concentration (MIC) of tFNAs-NeoS is 1.2 μg / mL, reducing the effective dose of the antibiotic and increasing bacterial sensitivity. tFNAs-NeoS exhibits superior antibacterial activity compared to NeoS. In treating burn wounds in rats, tFNAs-NeoS showed significantly better therapeutic effects than both tFNAs and NeoS. Furthermore, during wound healing, tFNAs-NeoS does not induce an excessive immune response, demonstrating better biocompatibility.

[0167] As can be seen from the above embodiments and experimental examples, this invention prepares a drug for treating burn infections. This drug is self-assembled from tetrahedral framework nucleic acids and the antibiotic neomycin sulfate. Neomycin sulfate exhibits high loading efficiency and good stability in complex biological media. tFNAs-NeoS reduces the cytotoxicity and effective dose of the antibiotic NeoS, demonstrating better biocompatibility than tFNAs and slowing the development of bacterial resistance. In treating burn wound infections, tFNAs-NeoS shows better wound healing promotion and biocompatibility than NeoS. Therefore, the tFNAs-NeoS prepared in this invention overcomes the problem of antibiotic overuse and has promising clinical application prospects.

Claims

1. The use of tetrahedral framework nucleic acid materials in the preparation of drugs for treating Staphylococcus aureus and Escherichia coli, characterized in that: The tetrahedral framework nucleic acid material is composed of tetrahedral framework nucleic acid and antibiotics; The ratio of the tetrahedral framework nucleic acid to the antibiotic was 9.5 μM: 277-278 μg; The antibiotic is neomycin sulfate; The tetrahedral framework nucleic acid is composed of four single-stranded nucleic acids S1, S2, S3 and S4 through complementary base pairing; the molar ratio of S1 to S2, S3 and S4 is 1:1:1:1, the nucleotide sequence of S1 is shown in SEQ ID NO. 1, the nucleotide sequence of S2 is shown in SEQ ID NO. 2, the nucleotide sequence of S3 is shown in SEQ ID NO. 3 and the nucleotide sequence of S4 is shown in SEQ ID NO.

4.

2. The use according to claim 1, characterized in that, The preparation method of the tetrahedral framework nucleic acid material includes the following steps: The tetrahedral framework nucleic acid and antibiotics are mixed to obtain the product.

3. The use according to claim 2, characterized in that: The mixing process involves shaking at 0-4°C for 4-8 hours.

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