A nucleic acid tetrahedral complex co-loading nisin and quercetin, and a preparation method and anti-mrsa application thereof

Abstract

This invention discloses a nucleic acid tetrahedral complex co-loaded with nisin and quercetin, its preparation method, and its application in anti-MRSA. The complex provided by this invention is obtained by loading nisin and quercetin onto nucleic acid tetrahedra; the four single-stranded DNA sequences of the nucleic acid tetrahedron are shown in SQE ID NO. 1–4; the molar ratio of the nucleic acid tetrahedron to nisin and quercetin is (1–2):(400–600):(6500–7500). This complex can synergistically deliver nisin and quercetin, significantly improving the antibacterial activity of the active substances against MRSA at sub-inhibitory concentrations, overcoming the shortcomings of low antibacterial efficiency and poor stability when natural active substances are used alone; furthermore, this complex has good biocompatibility and can be efficiently taken up by MRSA, providing more efficient co-delivery systems based on nucleic acid tetrahedra, which has important scientific significance and application value for overcoming the application limitations of natural active substances and improving the control effect of MRSA infection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a nucleic acid tetrahedral complex co-loaded with nitroglycerin and quercetin, its preparation method, and its application against MRSA. Background Technology

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important drug-resistant pathogens in clinical settings and the food environment. Due to the long-term and widespread use of antibiotics, MRSA is increasingly evolving into multidrug-resistant and even pan-drug-resistant strains, posing a serious threat to public safety. Therefore, there is an urgent need to develop novel antimicrobial drugs and methods that do not rely on the mechanisms of action of traditional antibiotics.

[0003] Among currently available antibacterial drugs, quercetin, a natural flavonoid compound, possesses broad-spectrum antibacterial activity. Its mechanisms of action include disrupting bacterial cell membrane integrity, inhibiting biofilm formation, and interfering with the expression of virulence factors. Nisin, a natural antibacterial peptide, targets the bacterial cell wall precursor Lipid II, forming pores in the bacterial membrane, leading to intracellular ion leakage and osmotic imbalance, ultimately causing bacterial death. However, quercetin's poor water solubility and susceptibility to oxidative inactivation, coupled with nisin's susceptibility to protease degradation and insufficient stability, severely limit their application in antibacterial therapy. Therefore, overcoming the inherent limitations of natural active substances and synergistically leveraging their antibacterial potential has become an important research direction.

[0004] In recent years, nanodelivery systems (such as liposomes and polymer nanoparticles) have been explored to improve the stability of the aforementioned active substances. However, existing delivery systems still suffer from problems such as low drug loading efficiency, difficulty in achieving multi-component co-delivery, and potential biocompatibility risks. Especially for various antibacterial agents with different physicochemical properties, existing carriers struggle to achieve efficient loading and synergistic release. Tetrahedral framework nucleic acids (tFNAs) are three-dimensional nanostructures formed by the self-assembly of four specific DNA single strands through complementary base pairing. These structures possess advantages such as uniform size, controllable geometry, good biocompatibility, easy cellular uptake, and programmable modification, exhibiting unique advantages in drug delivery. Currently, there are studies using nucleic acid tetrahedra to load quercetin for the treatment of sepsis and the preparation of drugs for the treatment of retinal neovascularization diseases, but there are no reports of simultaneously loading nisin and quercetin onto nucleic acid tetrahedra for MRSA inhibition.

[0005] Therefore, developing a highly efficient co-delivery system based on nucleic acid tetrahedra to achieve the synergistic antibacterial effect of nisin and quercetin is of significant scientific importance and practical value for overcoming the limitations of natural active substances and improving the control of MRSA infection. This invention application is thus proposed. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of existing drugs that load nisin and quercetin with quercetin, which have poor water solubility and are easily oxidized and inactivated, and that nisin is easily degraded by proteases and has insufficient stability. The present invention provides a nucleic acid tetrahedral complex co-loaded with nisin and quercetin, its preparation method and its anti-MRSA application.

[0007] The first objective of this invention is to provide a nucleic acid tetrahedral complex co-loaded with nitroglycerin and quercetin.

[0008] A second objective of this invention is to provide a method for preparing a nucleic acid tetrahedral complex co-loaded with nisin and quercetin.

[0009] A third objective of this invention is to provide the application of a nucleic acid tetrahedral complex co-loaded with nisin and quercetin.

[0010] The fourth objective of this invention is to provide a product.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a nucleic acid tetrahedral complex co-loaded with nisin and quercetin, obtained by loading nisin and quercetin onto a nucleic acid tetrahedron; the four single-stranded DNA sequences of the nucleic acid tetrahedron are shown in SQE ID NO. 1-4; the molar ratio of the nucleic acid tetrahedron to nisin and quercetin is (1-2):(400-600):(6500-7500).

[0012] This invention utilizes nucleic acid tetrahedra to load nisin and quercetin, with both substances non-intercalated into the double-stranded grooves of the nucleic acid tetrahedra. The resulting nucleic acid tetrahedron-nisin-quercetin complex (tFNQ) exhibits significant antibacterial effects. tFNQ significantly reduces the minimum inhibitory concentration (MIC) of both nisin and quercetin, demonstrating a synergistically enhanced antibacterial effect against methicillin-resistant Staphylococcus aureus (MRSA) at sub-inhibitory concentrations. Simultaneously, the co-loading of the two active substances in the nucleic acid tetrahedra results in a high loading rate, enhancing the stability of the active substances. It also possesses nuclease-responsive release capability, triggering drug release in the presence or activity of specific nucleases, enabling precise treatment and targeted delivery. Furthermore, it can be efficiently taken up by MRSA, resulting in better antibacterial effects. This overcomes the shortcomings of low antibacterial efficiency and poor stability when natural active substances are used alone, demonstrating promising application prospects.

[0013] Preferably, the molar ratio of the nucleic acid tetrahedron to nifedipine and quercetin is 1:(400-600):(6500-7500).

[0014] More preferably, the molar ratio of the nucleic acid tetrahedron to nifedipine and quercetin is 1:(400-500):(6500-7000).

[0015] More preferably, the molar ratio of the nucleic acid tetrahedron to nifedipine and quercetin is 1:500:7000.

[0016] Preferably, the four single-stranded DNA sequences are: S1 (SQE ID NO.1): 5'-ACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTA-3'; S2 (SQE ID NO.2): 5'-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCCAATAC-3'; S3 (SQE ID NO.3): 5'-TCAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTC-3'; S4 (SQE ID NO. 4): 5'-TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCTCGCAT-3'.

[0017] Preferably, the molar ratio of the four single-stranded DNA strands is (1-3):(1-3):(1-3):(1-3).

[0018] More preferably, the molar ratio of the four single-stranded DNA strands is 1:1:1:1.

[0019] Furthermore, the potency of the lactobacillus peptide is ≥ 900,000 IU g. -1 .

[0020] This invention provides a method for preparing a nucleic acid tetrahedral complex co-loaded with nisin and quercetin. Four single-stranded DNA strands are mixed and incubated to self-assemble into a nucleic acid tetrahedron. Subsequently, the nucleic acid tetrahedron, nisin, and quercetin are mixed in proportion and incubated with shaking at 25–37°C for 6–24 h. After the reaction is completed, the complex is purified by ultrafiltration.

[0021] Preferably, the self-assembly conditions of the four single-stranded DNA strands are: maintaining a temperature of 90–98°C for 5–15 minutes, followed by rapid cooling to 0–8°C and maintaining the temperature for 10–30 minutes.

[0022] Furthermore, the four DNA single strands were maintained at 94°C for 10 min, and then at 4°C for 20 min.

[0023] Preferably, ultrafiltration purification is performed using a 10–50 kDa ultrafiltration membrane.

[0024] More preferably, the reaction conditions for mixing nucleic acid tetrahedrons, nisin, and quercetin are 25°C and incubated with shaking for 12 hours, followed by purification using a 30 kDa ultrafiltration membrane to obtain the complex.

[0025] This invention provides the application of the above-mentioned complex in the preparation of antibacterial drugs.

[0026] Preferably, the drug can inhibit methicillin-resistant Staphylococcus aureus.

[0027] This invention provides the use of the above-mentioned complex in the preparation of products that inhibit methicillin-resistant Staphylococcus aureus.

[0028] The present invention also provides a product comprising the above-described complex.

[0029] Preferably, the product further contains pharmaceutically acceptable excipients or formulations.

[0030] The present invention has the following beneficial effects: This invention provides a nucleic acid tetrahedral complex co-loaded with nisin and quercetin, exhibiting significant antibacterial effects. It significantly reduces the minimum inhibitory concentration (MIC) of both nisin and quercetin, demonstrating a synergistically enhanced antibacterial effect against MRSA at sub-inhibitory concentrations. Simultaneously, the co-loading of the two active substances in the nucleic acid tetrahedron results in a high loading rate, enhancing the stability of the active substances. Furthermore, it possesses nuclease-responsive release characteristics, enabling drug release to be triggered in the presence or under conditions of specific nuclease activity changes, achieving precise treatment and targeted delivery. The nucleic acid tetrahedral co-loaded complex of nisin and quercetin provided by this invention solves the problems of insufficient stability of nisin, poor water solubility and easy oxidation and inactivation of quercetin. Furthermore, it fully utilizes the loading function of nucleic acid tetrahedra, enabling efficient loading of nisin and quercetin through binding to the double-stranded grooves of the nucleic acid tetrahedron. This improves the stability and solubility of the active substances while increasing their local concentration, thus enhancing the antibacterial effect. It also possesses advantages such as good biocompatibility and easy cellular uptake, allowing for efficient uptake by MRSA and achieving highly effective antibacterial action. This invention provides more efficient anti-MRSA co-delivery drug systems based on nucleic acid tetrahedra, which has significant scientific and application value in overcoming the application limitations of natural active substances and improving the control of MRSA infection. Attached Figure Description

[0031] Figure 1 Synthesis and characterization of the nucleic acid tetrahedral-lactam peptide-quercetin complex tFNQ (Figure a shows the synthesis schematic of tFNAs and tFNQ; b shows the PAGE analysis of tFNAs; c shows the morphological structure of tFNAs observed by TEM, scale bar 200 nm; d shows the UV-Vis absorption spectra of tFNAs and tFNQ; e shows the particle size of tFNAs and tFNQ; f shows the zeta potential of tFNAs and tFNQ; g shows the docking results of tFNAs with lactam peptide (left) and tFNAs with quercetin (right); h shows the competitive binding of Hoechst33342 to tFNAs / tFNQ).

[0032] Figure 2 The loading rates of tFNAs and Nisin / Quercetin under different conditions are shown in the figure (a is the concentration-absorbance standard curve of Nisin; b is the concentration-absorbance standard curve of Quercetin; c is the loading rate of tFNAs and Nisin at different molar concentration ratios; d is the loading rate of tFNAs and Quercetin at different molar concentration ratios; e is the loading rate of tFNAs and Nisin / Quercetin at different temperatures; f is the loading rate of tFNAs and Nisin / Quercetin at different incubation times).

[0033] Figure 3The stability of tFNQ is shown in the figure (a is the PAGE electrophoresis diagram of tFNQ structure stability; b is the change in loading rate of tFNQ within 48h).

[0034] Figure 4 The release characteristics and release rate of tFNQ are shown in the figures (a) melt curves of tFNQ treated with different concentrations of DNase I; b) melt temperature changes of tFNQ treated with different concentrations of DNase I; c) release rate of lactobacillus peptides after treatment with tFNQ using DNase I (10U); d) release rate of quercetin after treatment with tFNQ using DNase I 1 (10U). Data are expressed as mean ± standard deviation (Mean ± SD).

[0035] Figure 5 The uptake of tFNQ by MRSA was shown in the figure (a) as the mean fluorescence intensity of the blank control group; b) as the mean fluorescence intensity of the sNQ-treated group; c) as the mean fluorescence intensity of the tFNQ-treated group; d) as the significance analysis of the difference in mean fluorescence intensity among different treatment groups; e) as CLSM characterization of tFNQ uptake by MRSA. Data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using two-way ANOVA, and inter-group differences were analyzed using the Bonferroni multiple comparison test. p <0.05,** p <0.01, *** p <0.001).

[0036] Figure 6 The antibacterial effect of tFNQ on MRSA is shown in the figure (a) is the antibacterial effect of tFNAs loaded with Nisin / Quercetin (tFN / tFQ); b is the growth curve of each treatment group after 24 h of co-culture with MRSA; c is the significance analysis of the difference in the average inhibition rate among the treatment groups; d is the plate colony count; e is the SEM morphology observation results of MRSA after different treatments, scale bar: 2 μm; f is the staining results of live and dead bacteria after different treatments, scale bar: 0 μm; control represents the control group (untreated bacteria), blank group represents blank without light and no bacteria, tFN represents nucleic acid tetrahedron loaded with nisin, tFQ represents nucleic acid tetrahedron loaded with quercetin, tFNQ represents nucleic acid tetrahedron co-loaded with nisin and quercetin, NQ represents a mixture of nisin and quercetin, sNQ represents single-chain loaded with nisin and quercetin; data are expressed as mean ± standard deviation (Mean ± standard deviation). SD indicates that statistical analysis was performed using one-way ANOVA, and differences between groups were analyzed using the Bonferroni multiple comparison test.* p <0.05,** p <0.01, *** p<0.001). Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0038] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0039] The lactobacillus peptide Nisin used in this example was obtained by purchasing a commercially available product. It was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. (potency ≥ 900,000 IU g). - ¹), Quercetin was purchased from Beijing Solarbio Science & Technology Co., Ltd. (HPLC ≥ 98%).

[0040] Example 1: Preparation of nucleic acid tetrahedron-lactam peptide-quercetin complex (tFNQ) 1. Preparation of nucleic acid tetrahedra (tFNAs) The four DNA single strands used in this embodiment are described in the prior art (Zhang Q, Lin S, Wang L, et al. Tetrahedral framework nucleic acids act as antioxidants inacute kidney injury treatment[J]. Chemical Engineering Journal, 2020:127426.). Equimolar concentrations of DNA single strands S1-S4, whose sequences are shown in Table 1, were dissolved in 1×TM buffer, and then heated at 94°C for 10 minutes and kept at 4°C for 20 minutes to obtain nucleic acid tetrahedra (tFNAs).

[0041] Table 1 Information on the four single-stranded sequences

[0042] 2. Preparation of a complex of nucleic acid tetrahedral co-loaded with nitroglycerin and quercetin (tFNQ) Nisin and quercetin were dissolved separately in sterile 2% DMSO to prepare 10 mM stock solutions. At a molar ratio of tFNAs, nisin, and quercetin of 1:500:7000, 10 μL of nisin solution (10 mM) and 14 μL of quercetin solution (10 mM) were added to 200 μL of tFNAs solution (100 nM), and the mixture was incubated at 25 °C with shaking for 12 h. The resulting solution was then purified by 30 kDa ultrafiltration centrifugation to obtain a complex (tFNQ) of nisin and quercetin co-loaded with nucleic acid tetrahedra, as illustrated in the diagram below. Figure 1 As shown in a.

[0043] Under the same molar ratio and conditions, nisin and quercetin were incubated with four unassembled single chains to prepare single-chain loaded complexes (sNQ); at the same time, nisin and quercetin were incubated to obtain control complexes (NQ) for later use.

[0044] Example 2 Characterization of the nucleic acid tetrahedron-lactam peptide-quercetin complex The tFNAs and tFNQ prepared in Example 1 were measured according to the following methods: (1) The molecular weight of tFNAs was characterized using 8% PAGE; the voltage was set to 110 V and the electrophoresis time was set to 50 min. After electrophoresis, the gel was placed in 1000 times diluted TS-GelRed nucleic acid staining solution and stained by shaking for 15 min. Then it was observed in the gel imaging system.

[0045] The measurement results are as follows Figure 1 As shown in b, the synthesized tFNAs molecules are approximately 500 bp in size, and their migration rate in an 8% PAGE gel is much lower than that of the other bands.

[0046] (2) Observation of nanomorphology using transmission electron microscopy (TEM): The concentration of tFNAs was diluted to 100 nM. 10 μL of tFNAs was dropped onto a copper mesh substrate and negatively stained with 100 μL of 2% phosphotungstic acid solution. After drying, it was observed under TEM.

[0047] The measurement results are as follows Figure 1 As shown in Figure c, the tFNAs exhibit a triangular outline as observed by transmission electron microscopy, indicating that the tFNAs self-assembled successfully.

[0048] (3) The changes in the characteristic absorption peak of tFNQ were measured using a UV-Vis spectrophotometer; after shaking and incubating the tFNQ solution for 12 h, it was ultrafiltered for 10 min at 10000 rpm using a 30 Kda ultrafiltration tube. 1 mL of the solution from the inner tube was then measured at 190 nm-400 nm using a UV-Vis spectrophotometer. 1 mL of the solution from the outer tube was also measured at 194 nm and 370 nm.

[0049] The measurement results are as follows Figure 1 As shown in d, the UV-Vis absorption spectrum shows that the characteristic absorption peaks of tFNQ overlap at 194 nm and 370 nm, indicating that nisin and quercetin were successfully loaded onto tFNAs.

[0050] (4) The particle size and zeta potential of tFNAs and tFNQ were measured using a laser particle size analyzer based on dynamic light scattering and electrophoretic light scattering, respectively. The concentrations of tFNAs and tFNQ were set to 100 nM, and a buffer solution (TE buffer containing 10 mM MgCl2) consistent with the assembly conditions was used. Before measurement, air bubbles were removed by sonication, and 1 mL of tFNAs / tFNQ was measured at a constant temperature of 25℃.

[0051] The measurement results are as follows Figure 1 As shown in e and f, the particle size of tFNAs increased from 5 nm to 22 nm after drug loading; the potential increased from -9.8 mV to -2.7 mV; these results indicate that tFNQ was successfully prepared.

[0052] (5) Molecular docking was used to simulate the binding site of Nisin and Quercetin; Discovery Studio 2019 software was used to select one side of the tetrahedron for de novo modeling; AutoDock 4.2 software was used for semi-flexible docking, with the DNA double strand as the rigid acceptor and the active small molecule as the flexible ligand; Pymol 2.1 software was used for result analysis and visualization, and residue sites within 4 Å of the ligand were selected for interaction analysis.

[0053] The measurement results are as follows Figure 1 As shown in g, molecular docking simulations show Nisin binding to the double-stranded large groove of tFNAs and Quercetin binding to the double-stranded small groove.

[0054] (6) Verify the binding site using a fluorescence competitive binding experiment; 100 μL of Hoechst 33342 dye was incubated with 100 μL of tFNAs and tFNQ in a black microplate at room temperature for 15 min, and the absorption peaks of the continuous wavelength were measured using a multi-functional continuous wavelength microplate reader.

[0055] The measurement results are as follows Figure 1As shown in h, the fluorescence competitive binding experiment indicates that both substances bind to the double-stranded groove in a non-intercalated manner.

[0056] Example 3: tFNQ Load Rate Measurement and Optimization 1. Load rate measurement The loading efficiency of Nisin / Quercetin was analyzed using a microplate reader. First, the absorbance of nisin solution (gradient concentration: 10–100 μM) at 194 nm and the absorbance of quercetin solution (gradient concentration: 10–50 μM) at 370 nm were recorded to obtain a concentration-absorbance standard curve.

[0057] Following the method in Example 1, the molar ratios of nucleic acid tetrahedrons and nisin were set to 1:200, 1:300, 1:400, 1:500, and 1:600, respectively, and the molar ratios of nucleic acid tetrahedrons and quercetin were set to 1:6500, 1:7000, 1:7500, 1:8000, and 1:8500, respectively (tFNAs concentration was 100 nM, 200 μL; nisin concentration was 10 mM, 0.4 μL, 0.6 μL, 0.8 μL, 1 μL, and 1.2 μL were added, respectively; quercetin concentration was 10 mM, 13 μL, 14 μL, 15 μL, 16 μL, and 17 μL were added, respectively). The mixture was incubated at room temperature with shaking for 12 h. After incubation, free nisin and quercetin were removed using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The absorbance of free nisin and quercetin was then measured, and the loading rate of tFNAs on nisin and quercetin at different concentration ratios was calculated.

[0058] Nisin load factor formula

[0059] In the formula: y1 represents the absorbance of Nisin at 194nm before loading, and y2 represents the absorbance of free Nisin at 194nm after loading; Quercetin's load factor formula is:

[0060] In the formula: y1 represents the absorbance of Quercetin at 370nm before loading, and y2 represents the absorbance of free Quercetin at 370nm after loading.

[0061] The concentration-absorbance standard curves of Nisin and Quercetin are as follows: Figure 2 As shown in a and b, the standard curve formula for Nisin at 194 nm is Y = -0.11254 + 0.00215X, and the standard curve formula for Quercetin at 370 nm is Y = 0.03919 + 0.00272X.

[0062] The calculated load rate results are as follows Figure 2 As shown in c and d, the optimal incubation concentrations of nucleic acid tetrahedron with nisin and quercetin are 1:(400-500):(6500-7000), with a loading rate of 69.17% for nisin and 93.98% for quercetin.

[0063] 2. Incubation temperature optimization The optimal incubation concentration ratio was selected, and tFNAs were incubated with nisin and quercetin at 4℃, 25℃ and 37℃ for 12 h with shaking. After incubation, free nisin and quercetin were removed using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The concentrations of free nisin and quercetin were then measured, and the loading rates of tFNAs on nisin and quercetin at different temperatures were calculated.

[0064] The results are as follows Figure 2 As shown in e, the optimal incubation temperature is 25℃, at which the loading rate of nisin is 68.34% and the loading rate of quercetin is 91.73%.

[0065] 3. Optimization of incubation time At the optimal incubation concentration and temperature, tFNAs were incubated with nisin and quercetin by shaking for 6 h, 12 h, 24 h, and 48 h, respectively. After incubation, free nisin and quercetin were removed using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The concentrations of free nisin and quercetin were then measured, and the loading rates of tFNAs on nisin and quercetin at different incubation times were calculated.

[0066] The results are as follows Figure 2 As shown in f, the optimal incubation time is 12-24 h, at which time the loading rate of nisin is 67.95% and the loading rate of quercetin is 94.19%.

[0067] In summary, the optimal loading efficiency was achieved when the molar ratio of tFNAs, nisin, and quercetin was 1:500:7000, with incubation at 25°C for 12 h under shaking conditions. The average loading rate of quercetin was 95.7% ± 1.3%, and the average loading rate of nisin was 69.7% ± 3.3%, indicating that tFNAs can co-load two active substances with a high loading rate.

[0068] Example 4: Determination of the stability and release characteristics of tFNQ 1. Stability tFNQ was incubated with LB liquid medium at 37°C for different times (6 h, 12 h, 24 h, 48 h). The structural stability was analyzed by PAGE gel electrophoresis, and the tFNQ bands were observed using a gel imaging system. tFNQ was stored at 4°C for 48 h, and the absorbance at 194 nm and 370 nm was recorded every 6 h. The loading rate change at different times was calculated based on the concentration-absorbance standard curve.

[0069] The results are as follows Figure 3 As shown in figure a, it indicates that tFNQ did not show significant degradation within 48 hours and its structure remained stable; the loading rate results are statistically shown in figure a. Figure 3 As shown in b, the loading rate of the active substance did not decrease significantly within 48 h, indicating that the loading of the active substance was stable.

[0070] 2. Release characteristics tFNAs were incubated with different concentrations of DNase I (2, 4, and 10 U) at 37°C for 30 min, and the reaction was terminated by adding 5 mM EDTA. Subsequently, 2×SBYR Green solution (10 mM Tris, 1 mM EDTA, pH 8.0) was added to TE buffer at a ratio of 1:1. Melting curves and melting point peaks were measured using a CFX96 real-time system. Simultaneously, after terminating the reaction, the reaction mixture was added to a spin column (MWCO 3000) and the supernatant was collected by centrifugation (15000×g, 15 min). Abs at 194 nm and 370 nm were measured, and the release amounts of nisin and quercetin were calculated using absorbance-concentration standard curves.

[0071] The measurement results are as follows Figure 4 As shown in a and b, tFNAs undergo unwinding and degradation under the action of nucleases, resulting in a decrease in overall thermal stability. In the presence of DNase I (10 U), the release rates of both active substances significantly increased with prolonged action time, ultimately reaching over 90%. Figure 4 As shown in c and d, tFNQ possesses nuclease-responsive release characteristics, enabling it to trigger drug release in the presence or activity of specific nucleases, thus achieving precision treatment and targeted delivery.

[0072] Example 5: MRSA's uptake of tFNQ (1) Flow cytometry detection: MRSA suspension was inoculated into 96-well plates and incubated at 37°C for 8 h. The OD value was measured at 600 nm. After dilution by 100 times, the suspension was inoculated into 24-well plates. The same concentrations of sNQ and tFNQ containing Cy5 fluorescent single strands were added, and the plates were incubated at 37°C in the dark on a shaker for 3 h. After centrifugation at 4000 rpm for 5 min, the bacteria were collected into 2 mL EP tubes. The supernatant was discarded, and the tubes were washed with PBS. After centrifugation, the tubes were washed with PBS again and transferred to flow cytometry tubes. The final bacterial concentration was 1×10⁻⁶. 6 The concentration of CFU / mL was measured using flow cytometry to detect the proportion of bacteria containing Cy5 and their intracellular fluorescence levels, followed by data analysis. Each sample contained 10,000 bacteria, while the control group consisted of simple bacteria.

[0073] The results are as follows Figure 5 As shown in the figure, the fluorescence intensity of sNQ in MRSA is only 0.84%, while tFNQ shows a significantly enhanced fluorescence signal in MRSA, with an average fluorescence intensity as high as 72.1%, indicating that MRSA efficiently takes up tFNQ.

[0074] (2) Characterization by confocal laser scanning microscopy (CLSM): MRSA suspension was inoculated into 96-well plates and incubated at 37℃ for 8 h. The OD value was measured at 600 nm. After dilution by 100 times, the suspension was inoculated into 24-well plates. A final concentration of 1 μM Cy5-ssDNA, Cy5-tFNAs, and Cy5-tFNQ was added, and the plates were incubated at 37℃ in the dark for 40 min. 10 μL of SYTO 9 dye was added to each well, and the plates were stained in the dark for 10 min. After centrifugation at 4000 rpm for 5 min, the samples were collected in 1 mL EP tubes. After washing with PBS, the plates were centrifuged again, and the supernatant was discarded. The suspension was resuspended in PBS to a concentration of 1×10⁶ CFU / mL. 2-5 µL of the suspension was dropped onto a glass slide, covered with a coverslip, and allowed to dry before being examined by 100x oil immersion using a confocal laser scanning microscope. The control group consisted of simple bacteria. SYTO 9 has an excitation wavelength of 488 nm and an emission wavelength of 503 nm, while Cy5 has an excitation wavelength of 650 nm and an emission wavelength of 670 nm.

[0075] CLSM image results as follows Figure 5 As shown in f, the proportion of red fluorescence signal in the sNQ treatment group is extremely low, while obvious red fluorescence signal can be observed in the tFNQ treatment group. Furthermore, the red signal highly overlaps with the outline of green bacteria, and significant yellow colocalization signal is present in the merged image; indicating that MRSA can efficiently take up tFNQ.

[0076] Experimental Example 6: In vitro antibacterial experiment of tFNQ against MRSA Activated methicillin-resistant Staphylococcus aureus (MRSA, ST239, isolated from commercially available pork, with a drug tolerance spectrum including β-lactam penicillin, aminoglycoside gentamicin, chloramphenicol and macrolide clarithromycin) was inoculated into 96-well plates.

[0077] 1. Antibacterial experiments in different drug delivery systems At sub-inhibitory concentrations of nisin and quercetin (Nisin MIC =100 μM, Quercetin MIC Under conditions of 1400 μM, nisin (tFN) and quercetin (tFQ) were monoloaded using nucleic acid tetrahedra, prepared using the same method as in Example 1. Simultaneously, nisin and quercetin (tFNQ) were co-loaded using nucleic acid tetrahedra. The three different delivery systems were co-cultured with MRSA for 24 h to determine the optimal monoload / co-load method. The specific procedure was as follows: A 96-well plate was prepared. 100 μL of tFN was added to the first column of wells, 100 μL of tFQ to the second column, and 100 μL of tFNQ to the third column. These were mixed with an equal volume of bacterial culture in the logarithmic growth phase. The 96-well plate was placed in a constant temperature incubator and incubated at 37°C for 24 h. The absorbance at 600 nm was measured after 24 h. Three replicates were set up for each group.

[0078] Under sub-inhibitory concentration conditions, the antibacterial effects of three different delivery systems (tFN, tFQ, tFNQ) on MRSA differed as follows: Figure 6 a. Compared with the tFNQ treatment group, the bacterial growth OD values ​​of both the tFN and tFQ treatment groups were higher and the differences were statistically significant, indicating that the single-load system had a weaker inhibitory effect on MRSA than the co-load system.

[0079] 2. Growth curve measurement At sub-inhibitory concentrations of nisin and quercetin, three groups were set up: single-chain loaded complex (sNQ), control complex (NQ), and tFNQ. A 96-well plate was prepared. 100 μL of NQ was added to the first row of wells, sNQ to the second row, and tFNQ to the third row, mixed with an equal volume of bacterial culture in the logarithmic growth phase. The 96-well plate was placed in a growth curve analyzer and incubated at 37℃ for 24 h. Absorbance was measured at 600 nm every h to plot bacterial growth curves for each treatment group. The inhibition rate of each treatment group was calculated based on the absorbance values. Untreated bacteria growth observation served as the control group, and LB liquid medium alone served as the blank control. Each group was divided into three replicates. The inhibition rate was calculated using the following formula:

[0080] Where: OD controlThis represents the absorbance value of the control group at 600 nm, OD blank This represents the absorbance value at 600 nm for the blank control group, OD. treatment This indicates the absorbance value at 600 nm for the treatment group.

[0081] Bacterial growth curve as shown Figure 6 As shown in b, under 1 / 2 MIC conditions, the logarithmic growth phase of bacteria in the tFNQ treatment group was delayed from 6 h to 12 h, with an average inhibition rate of 68.16% ± 3.32%, significantly higher than that of the NQ treatment group (5.24% ± 2.28%) and the sNQ treatment group (8.26% ± 2.79%), indicating that tFNQ has significantly enhanced antibacterial activity (e.g., Figure 6 c).

[0082] 3. Plate colony count Take 20 μL of bacterial culture from each well in the above growth curve assay and drop it onto LB agar medium. Spread it evenly using a disposable spreader and incubate at 37°C for 24 h. Then, count the colonies on the plate. The untreated simple bacterial growth observation serves as the control group, and the sterile plate serves as the blank control.

[0083] The results are as follows Figure 6 As shown in d, the number of colonies in the tFNQ treatment group was reduced by 10 times compared to the sNQ and NQ treatment groups.

[0084] 4. Characterization by scanning electron microscopy (SEM) Bacterial samples were collected after treatment, and 2.5% glutaraldehyde fixative was added until the bacteria were submerged. The samples were fixed at 4°C for 12 h, dehydrated using an alcohol gradient, and dried. Samples were prepared for electron microscopy observation, and then analyzed to observe the morphological changes in the spatial structure of bacteria after treatment with NQ, sNQ, and tFNQ. Untreated bacteria were used as a positive control group.

[0085] The results are as follows Figure 6 As shown in Figure e, the MRSA cells in the tFNQ-treated group showed obvious shrinkage on their surface, and some cells were observed to rupture and leak their contents, indicating that tFNQ has a significant antibacterial effect.

[0086] 5. Characterization by laser confocal scanning microscopy (CLSM) MRSA bacterial suspensions from different treatment groups were collected, washed three times with PBS, and used as bacterial suspensions with 0.85% NaCl solution. SYTO 9 and PI dyes were mixed at a 1:1 volume ratio, and then 3 μL of the mixture was added to 1 mL of PBS. Samples from different treatment groups were incubated in the dark for 15 min with the staining solution. Stained samples were observed using a CLSM. SYTO 9 was excited at 488 nm and emitted at 503 nm, while PI was excited at 490 nm and emitted at 635 nm.

[0087] The results are as follows Figure 6 As shown in f, red fluorescence signal dominates in the tFNQ treatment group, while green fluorescence is significantly reduced, indicating that the integrity of a large number of cell membranes is disrupted. This suggests that tFNQ can significantly enhance the antibacterial activity of the active substance against MRSA at sub-inhibitory concentrations.

[0088] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A nucleic acid tetrahedral complex co-loaded with nisin and quercetin, characterized in that, It is obtained by loading nisin and quercetin onto a nucleic acid tetrahedron; the four single-stranded DNA sequences of the nucleic acid tetrahedron are shown in SQE ID NO.1-4; the molar ratio of the nucleic acid tetrahedron to nisin and quercetin is (1-2):(400-600):(6500-7500).

2. The complex according to claim 1, characterized in that, The molar ratio of the nucleic acid tetrahedron to nifedipine and quercetin is 1:(400-600):(6500-7500).

3. The complex according to claim 1, characterized in that, The molar ratio of the four single-stranded DNA strands is (1-3):(1-3):(1-3):(1-3).

4. A method for preparing the complex according to any one of claims 1 to 3, characterized in that, Four single-stranded DNA molecules were mixed and incubated to self-assemble into a nucleic acid tetrahedron. The nucleic acid tetrahedron, nisin, and quercetin were then mixed in a specific ratio and incubated at 25–37°C with shaking for 6–24 h. After the reaction was completed, the complex was purified by ultrafiltration.

5. The preparation method according to claim 4, characterized in that, The self-assembly conditions for the four single-stranded DNA strands are: maintaining a temperature of 90–98°C for 5–15 minutes, followed by rapid cooling to 0–8°C and maintaining the temperature for 10–30 minutes.

6. The preparation method according to claim 4, characterized in that, Purification was performed using a 10–50 kDa ultrafiltration membrane.

7. The use of the complex according to any one of claims 1 to 3 in the preparation of antibacterial drugs.

8. The use of the complex according to any one of claims 1 to 3 in the preparation of a product that inhibits methicillin-resistant Staphylococcus aureus.

9. A product characterized in that, Contains the complex according to any one of claims 1 to 3.

10. The product according to claim 9, characterized in that, The product also contains pharmaceutically acceptable excipients or preparations.

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