FACOD (at) DSPE nano-particle, preparation method and application of FACOD (at) DSPE nano-particle in antibiosis / sterilization

Abstract

The invention relates to the technical field of biological preparation, in particular to FACOD (at) DSPE nano-particles, a preparation method and application of the FACOD (at) DSPE nano-particles in animal antibiosis / sterilization, the preparation method comprises the steps that 1, a compound IV and a compound COD are used, then N, N-diisopropylethylamine is added, the mixture is mixed and stirred overnight at the room temperature, extraction, drying, filtering, separation and purification are conducted through a separating funnel, and FACOD is obtained; and (2) wrapping the FACOD by using distearoyl phosphatidyl ethanolamine-methoxyl polyethylene glycol, so as to construct the FACOD (at) DSPE (Distearoyl Phosphatidyl Ethanolamine) nano particles. Compared with the prior art, the prepared FACOD (at) DSPE nano-particles with the light response characteristic can synergistically release FA with the yield of about 85.02% and CO with the yield of 50.3% under the illumination condition, the sterilization rate on escherichia coli is 91.36-99.80%, the sterilization rate on staphylococcus aureus is 94.11-99.85%, and the sterilization rate on drug-resistant staphylococcus aureus is 86.83-98.92%; and the FACOD (at) DSPE nanoparticles have the characteristics of low toxicity to cells and mice and good biological safety.

Description

Technical Field

[0001] This invention relates to the field of biopreparation technology, specifically to a FACOD@DSPE nanoparticle, its preparation method, and its application in antibacterial applications. Background Technology

[0002] Gas therapy is an emerging disease intervention strategy. It primarily utilizes gas molecules, such as nitric oxide (NO), CO, and hydrogen sulfide (H2S), to regulate key signaling pathways in the pathological microenvironment. These gases have been shown to inhibit neutrophil infiltration, alleviate ischemia-reperfusion injury, and antagonize oxidative stress by regulating pathways such as sGC / cGMP, HO-1, and Nrf2 / KEAP1. Gas therapy has made significant progress in the treatment of inflammatory diseases and has shown great potential in the treatment of major diseases in recent years. Typical examples include nitric oxide (NO), hydrogen sulfide (H2S), oxygen (O2), carbon monoxide (CO), and formaldehyde (FA). Compared with traditional chemotherapy, gas therapy has significant advantages. First, gas molecules do not induce drug resistance during treatment and can even enhance the sensitivity of drug-resistant cells to chemotherapeutic drugs.3 Second, most of the gases involved in this method are important endogenous signaling molecules in the body, playing crucial roles in various biological processes; therefore, gas therapy is considered a highly biocompatible treatment strategy.4 Regardless of the type of gas, its reactivity and diffusivity make it difficult to use directly for treatment. Therefore, gas therapy requires a donor that can release the gas in situ to achieve the therapeutic effect.

[0003] In recent years, studies have discovered a class of flavonol compounds that release CO2 and produce a gradually weakening fluorescence signal under light irradiation. The hydroxyl groups on flavonols are modifiable and can serve as linking sites for gas release groups. Therefore, these flavonol compounds represent a highly promising platform for developing dual-gas donors. Thus, developing dual-gas molecular donors based on flavonol compounds, enabling controlled gas release and in-situ fluorescence signal generation, and making the gas release process visible, will provide a new technical approach for dual-gas combined therapy and offer important molecular tools for studying the combined action mechanisms of dual gases. Summary of the Invention

[0004] The purpose of this invention is to provide FACOD@DSPE nanoparticles, their preparation method, and their application in antibacterial / bactericidal processes. The aim is to prepare novel photoresponsive antibacterial and / or bactericidal compounds that can synergistically release formaldehyde and carbon monoxide under light irradiation using a new preparation method.

[0005] To achieve the above objectives, the present invention provides a method for preparing FACOD@DSPE nanoparticles, the method comprising: (1) Dissolve 314.68 mg of compound IV and 300 mg of compound COD in dichloromethane as solvent, then add 268.98 mg of N,N-diisopropylethylamine and mix well. Mix and stir overnight at room temperature, extract using a separatory funnel, dry, filter, separate and purify to obtain FACOD; (2) Weigh 3.07 mg of the FACOD and dissolve it in CHCl3 to prepare FACOD(CHCl3) solution. Add the FACOD(CHCl3) solution dropwise to a 1 mg / mL distearylphosphatidylethanolamine-methoxy polyethylene glycol solution to fully encapsulate FACOD with DSPE-PEG2000, remove chloroform, and construct FACOD@DSPE nanoparticles.

[0006] Preferably, in the above technical solution, the preparation method of the compound COD is as follows: 3.90 g of compound III was weighed and dissolved in 60 mL of ethanol solution. 21 mL of sodium hydroxide aqueous solution was added to the compound III solution, and the mixture was stirred at room temperature for 30 minutes to obtain the first reaction mixture. 2.14 mL of benzaldehyde was then added to the first reaction mixture, and the mixture was stirred at room temperature for 5 hours to obtain the second reaction mixture. The second reaction mixture was cooled to 0 °C in an ice bath, and 4.6 g of K2O2 was added. The mixture was then heated to room temperature and stirred overnight. The solution was acidified to pH 6.5 with hydrochloric acid, and the precipitate was collected by filtration. The precipitate was washed with 3 x 10 mL of ethanol to obtain compound COD.

[0007] Preferably, in the above technical solution, the chemical formula of compound III is as follows: The chemical formula of the compound COD is as follows: The chemical name of the compound COD is 7-hydroxy-4-phenyl-3H-naphtho[2,3-c]furan-1-one.

[0008] Preferably, in the above technical solution, the preparation method of compound IV is as follows: (1) Add 1.5g of compound I to 50mL of anhydrous acetonitrile, mix and cool to 0℃, add 4.8g of dimethyl sulfide, purge with nitrogen for protection, add 9.2g of benzoyl peroxide and react at 0℃ for 6 hours, adjust pH to 9, react overnight, extract, dry, filter and take the filtrate, evaporate to dryness, purify to obtain compound II; (2) Add 500 mg of the compound II to 10 mL of anhydrous dichloromethane, cool to 0 °C, slowly add 1.58 g of sulfonyl chloride, and continue the reaction at room temperature for 8 hours under nitrogen protection. Extract the organic phase, dry it, filter it, evaporate the filtrate to dryness, separate and purify it to obtain compound IV.

[0009] Preferably, in the above technical solution, the chemical formula of compound I is as follows: The name of compound I is 2-nitrobenzyl alcohol; The chemical formula of compound II is as follows: ; The chemical formula of compound IV is as follows: The chemical name of compound IV is 2-nitrobenzylchloromethyl.

[0010] This invention also provides a method for preparing FACOD@DSPE nanoparticles, wherein the FACOD structure in the FACOD@DSPE nanoparticles is as follows: .

[0011] The present invention also provides the application of the FACOD@DSPE nanoparticles as described above in the preparation of photoresponsive antibacterial and / or bactericidal formulations.

[0012] Preferably, in the above technical solution, the antibacterial or bactericidal strain is drug-resistant Staphylococcus aureus, and when the concentration of FACOD@DSPE nanoparticles is 30-50 μM, the bactericidal rate against drug-resistant Staphylococcus aureus is 86.83-98.92%.

[0013] Preferably, in the above technical solution, the antibacterial or bactericidal strain is Staphylococcus aureus, and when the concentration of FACOD@DSPE nanoparticles is 10-50 μM, the bactericidal rate against Staphylococcus aureus is 94.11-99.85%.

[0014] Preferably, in the above technical solution, the antibacterial or bactericidal bacteria is Escherichia coli, and when the concentration of FACOD nanoparticles is 10-50 μM, the bactericidal rate against Escherichia coli is 91.36-99.80%.

[0015] A formulation containing FACOD@DSPE nanoparticles as described above at a concentration of 30-50 μM, characterized in that the formulation is an animal photosensitizing antibacterial and / or bactericidal agent, wherein the bacteria killed or / and inhibited by the formulation are Escherichia coli and / or drug-resistant Staphylococcus aureus and / or Staphylococcus aureus.

[0016] Compared with the prior art, the beneficial effects of this application are as follows: (1) For the first time, FACOD with photoresponsive properties was synthesized using 7-hydroxy-4-phenyl-3H-naphtho[2,3-c]furan-1-one and 2-nitrobenzylchloromethyl. FACOD@DSPE nanoparticles were constructed using FACOD and could synergistically release carbon monoxide (CO) and formaldehyde (FA) under light irradiation. When the concentration of FACOD@DSPE nanoparticles was 50 μM, the FA release yield was about 85.02% and the CO release yield was 50.3%.

[0017] (2) FACOD@DSPE nanoparticles can be used to prepare photoresponsive bactericides or antibacterial agents. When the concentration of FACOD nanoparticles is 10-50 μM, the bactericidal rate against Escherichia coli is 91.36-99.80%, and the bactericidal rate against Staphylococcus aureus is 94.11-99.85%. When the concentration of FACOD nanoparticles is 30-50 μM, the bactericidal rate against Escherichia coli is 91.36-99.80%, and the bactericidal rate against Staphylococcus aureus is 94.11-99.85%. At μM, the bactericidal rate against drug-resistant Staphylococcus aureus (MRSA) was 86.83-98.92%. The principle behind the good bactericidal rate of FACOD@DSPE nanoparticles against MRSA is as follows: When FACOD@DSPE nanoparticles are dissolved in water and applied to the skin, they can synergistically release 50.3% carbon monoxide and 85.02% formaldehyde under light conditions. Formaldehyde and carbon monoxide destroy the structural integrity of MRSA, causing it to die. Formaldehyde gas has strong penetrating power, and the groups on the PBP2a protein on which MRSA depends for survival react with the carbonyl group of formaldehyde to form a covalent bond, causing the three-dimensional structure of the PBP2a protein to denature and become inactive. Carbon monoxide interferes with the core energy metabolism of bacteria and induces oxidative stress. The two work synergistically to kill MRSA. Attached Figure Description

[0018] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings.

[0019] Figure 1 This is a graph showing the FA and CO release amounts of FACOD@DSPE nanoparticles at different concentrations; Figure 2 This is a particle size distribution of FACOD@DSPE obtained by the present invention; Figure 3 This is a dynamic light scattering (DLS) particle size distribution diagram of FACOD@DSPE obtained by the present invention; Figure 4 These are the UV and fluorescence spectra of FACOD in DMF before and after light treatment; Figure 5 This is the ultraviolet fluorescence change spectrum of the DSPE-PEG2000 solution; Figure 6 These are the FA standard curve and the curve showing the relationship between FACOD concentration and FA concentration; Figure 7 These are the CO standard curve and the curve showing the relationship between FACOD concentration and CO concentration; Figure 8 This is a graph showing the colony growth of Staphylococcus aureus in different groups; Figure 9 This is a graph showing the colony growth of Escherichia coli in different groups; Figure 10 This is a graph showing the colony growth of drug-resistant Staphylococcus aureus in different groups; Figure 11 The graph shows the biofilm formation after treatment with FACOD@DSPE on E. coli, S. aureus, and MRSA, and the bar chart shows the inhibition of the three bacterial species. Figure 12 Figure showing cell hemolysis at different concentrations of FACOD@DSPE particles; Figure 13 A bar chart showing cell viability in FACOD@DSPE particles at different concentrations; Figure 14 Images showing the wound conditions in the control group and the FACOD@DSPE treatment group in mouse experiments; Figure 15 Image of HE section of skin from untreated mice; Figure 16 Image of HE section of skin from mice in the FACOD@DSPE formulation group. Detailed Implementation

[0020] The technical solutions in the embodiments of this invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0021] I. Preparation of 2-nitrobenzylchloromethyl (1) In a round-bottom flask containing 50 mL of anhydrous acetonitrile, 1.5 g of compound I (2-nitrobenzyl alcohol) was added. After cooling to 0 °C, 4.8 g of dimethyl sulfide was added, and under nitrogen protection, 9.2 g of benzoyl peroxide (BPO) was added in four portions, each 30 minutes apart. The reaction was continued at 0 °C for 6 hours. 1 M sodium hydroxide was added to adjust the pH to 9. The mixed solution was reacted overnight. After the reaction was completed, the mixture was extracted using a separatory funnel to obtain the organic phase. The organic phase was dried using anhydrous sodium sulfate, filtered through a funnel, and then evaporated to dryness to obtain the crude product. The crude product was purified by column chromatography to obtain 1.15 g of compound II, with a yield of 55.1%.

[0022] (2) In a round-bottom flask containing 10 mL of anhydrous dichloromethane, 500 mg of compound II was added. After cooling to 0 °C, an excess of 1.58 g of sulfonyl chloride was slowly added under nitrogen protection. The reaction was continued at room temperature for 8 hours. After the reaction was completed, the mixture was extracted using a separatory funnel to obtain the organic phase. The organic phase was dried using anhydrous sodium sulfate, filtered through a funnel, and then evaporated to dryness to obtain the crude product. The crude product was purified by column chromatography to obtain 391 mg of compound IV, with a yield of 82.51%. Compound IV was named 2-nitrobenzylchloromethyl ether (also known as "o-nitrobenzylchloromethyl ether").

[0023] The chemical synthesis route of 2-nitrobenzylchloromethyl is as follows:

[0024] II. Preparation of compound COD 21 mL of sodium hydroxide aqueous solution (5 M, 104 mmol) was added to 3.90 g of compound III (21 mmol) in 60 mL of ethanol solution, and the mixture was stirred at room temperature for 30 minutes to obtain the first reaction mixture. 2.14 mL of benzaldehyde (21 mmol) was added to the first reaction mixture, and the mixture was stirred at room temperature for 5 hours to form a deep red solution, which was the second reaction mixture. The second reaction mixture was then cooled to 0 °C in an ice bath, 4.6 g of K₂O₂ was added, and the resulting mixture was stirred overnight while heated to room temperature. The solution was acidified to pH 6.5 with 1 M hydrochloric acid, resulting in a bright yellow precipitate. The precipitate was filtered and washed with 3 x 10 mL of ethanol to give 4.1 g of compound COD, with a yield of 67.9%. The chemical name of compound COD is 7-hydroxy-4-phenyl-3H-naphtho[2,3-c]furan-1-one (also known as "hydroxyphenylnaphthofuranone").

[0025] The chemical synthesis route for 7-hydroxy-4-phenyl-3H-naphtho[2,3-c]furan-1-one is as follows:

[0026] III. Preparation of FACOD 300 mg of compound COD (1.04 mmol) and 314.68 mg of compound IV (1.56 mmol) were mixed in 20 mL of dichloromethane (DCM). 268.98 mg of N,N-diisopropylethylamine (DIEPA, 2.08 mmol) was added to the reaction mixture as a deacidification base to promote the reaction. The reactants were stirred overnight at room temperature to allow sufficient reaction. The reaction progress was monitored by thin-layer chromatography (TLC) until completion. After the reaction was complete, the mixture was extracted using a separatory funnel to obtain the organic phase. The organic phase was dried over anhydrous sodium sulfate, filtered through a funnel, and the filtrate was evaporated to dryness to obtain the crude product. The crude product was purified by column chromatography to give 271.00 mg of FACOD, with a yield of 57.43%.

[0027] The chemical synthesis route of FACOD is as follows:

[0028] FACOD is a newly synthesized compound, and its proton, carbon, and ultra-high resolution mass spectra were measured.

[0029] 1HNMR(500MHz, CDCl3)δ8.82(s,1H),8.01(d,J=8.5Hz,1H),7.93(dd,J1=9.0Hz,J2=1Hz,1H),7.92(s,1H),7.87(m,3H),7.56(td,1H, J=7Hz),7.47(td,J=7Hz,1H),7.41(m,2H),7.34-7.26(m,4H),5.37(s,2H),4.61(s,2H).13CNMR(126MHz,CDCl3)δ174.47,157.23,15 0.71,145.49,135.12,134.86,133.46,132.54,129.84,129.78,129.09,128.56,128.00,127.82,127.14,127.07,126.63,126.19,1 26.00,125.01,123.52,122.04,113.20,94.68,66.94.HRMS(ESI):m / z[M+H+]calcd.for:C27H19NO6K+:492.0849;found:492.0860.

[0030] The release pathways of formaldehyde and carbon monoxide from FACOD are as follows:

[0031] As can be seen from the above release pathway, FACOD decomposes into 7-carboxy-4-phenyl-3H-naphtho[2,3-c]furan-1-one and 2-nitrophenol under light (hv) conditions. The 7-carboxy-4-phenyl-3H-naphtho[2,3-c]furan-1-one further decomposes into formaldehyde (FA) and 7-hydroxy-4-phenyl-3H-naphtho[2,3-c]furan-1-one (COD). The 7-hydroxy-4-phenyl-3H-naphtho[2,3-c]furan-1-one decomposes into carbon monoxide and phenyl 1-hydroxy-2-naphthocarboxylate under light (hv) conditions, thereby releasing formaldehyde and carbon monoxide. IV. Preparation of FACOD@DSPE Nanoparticles FACOD was encapsulated with the amphiphilic phospholipid molecule DSPE-PEG2000 (distearate phosphatidylethanolamine-methoxy polyethylene glycol) to construct FACOD@DSPE nanoparticles. DSPE molecules have a hydrophilic head and a hydrophobic tail, and can self-assemble into micelles or vesicles under high-speed rotation, encapsulating the hydrophobic FACOD within its hydrophobic core, thereby improving the water solubility and stability of FACOD. The specific preparation method of the FACOD nanoparticles is as follows: (1) Preparation of solution Accurately weigh 3.07 mg of FACOD solid into a centrifuge tube and dissolve it in N,N-dimethylformamide (DMF) to a concentration of 10 mM, obtaining a stock solution of FACOD dissolved in DMF. Aliquot the solution into small centrifuge tubes, 50 μM per tube, and store in the dark at -20°C.

[0032] Accurately weigh 3.07 mg of solid FACOD into a centrifuge tube and dissolve it in chloroform (CHCl3) to a concentration of 10 mM, obtaining a mother liquor of FACOD dissolved in CHCl3. Then, dilute the mother liquor to different concentrations using CHCl3 and store it in 4 mL centrifuge tubes at -20°C, protected from light.

[0033] Weigh 10 mg of DSPE-PEG2000 and dissolve it in pure water to obtain a 10 mg / mL DSPE-PEG2000 stock solution, which is then stored at -4°C.

[0034] (2) Preparation of FACOD@DSPE nanoparticles A 10 mg / mL DSPE-PEG2000 stock solution was diluted to 1 mg / mL with pure water. Under vigorous shaking, FACOD(CHCl3) solutions of different concentrations were added dropwise to the 1 mg / mL DSPE-PEG2000 solution to ensure thorough contact and encapsulation. A thermoelectric blower was used to remove the dissolved chloroform until the solution became clear, thus producing FACOD@DSPE nanoparticles of different concentrations. The specific addition of FACOD(CHCl3) solution to the different concentrations of FACOD@DSPE nanoparticles is shown in Table 1 below.

[0035] Table 1. Raw material information for the preparation of FACOD@DSPE nanoparticles at different concentrations. <![CDATA[Concentration of FACOD (CHCl3) solution (μM)]]> 50 100 150 250 375 500 <![CDATA[Volume of FACOD (CHCl3) solution added (μL)]]> 200 200 200 200 200 200 FACOD@DSPE nanoparticle concentration (μM) 10 20 30 50 75 100 FACOD at different concentrations (50-500 μM) was encapsulated into DSPE-PEG2000 to form nanoparticles. The yields of carbon monoxide and formaldehyde in the FACOD@DSPE nanoparticles at different concentrations were then measured and calculated. The results are as follows: Figure 1 As shown.

[0036] Figure 1 This is a graph showing the carbon monoxide and formaldehyde yields of 10-100 μM FACOD@DSPE nanoparticles. From... Figure 1 It can be seen that the yields of carbon monoxide and formaldehyde both show a trend of first increasing and then decreasing with the concentration of FACOD@DSPE nanoparticles. When the concentration of FACOD@DSPE nanoparticles is 50 μM, the yields of the two gases are the highest, with FA at 85.02% and CO at 50.3%.

[0037] Based on the above experiments, the yields of the two gases were highest when the concentration of FACOD@DSPE nanoparticles was 50 μM. Therefore, FACOD@DSPE nanoparticles with a concentration of 50 μM were used as the experimental object for particle size characterization. Figure 2 This is a particle size image of FACOD@DSPE nanoparticles, from... Figure 2 Several nanoparticles were observed to have a relatively rounded shape.

[0038] The particle size characterization of the FACOD@DSPE nanoparticles is shown in Table 2 below. Figure 3 As shown.

[0039] Table 2. Particle size characterization of FACOD@DSPE nanoparticles Z-Average (d.nm) Size (d.nm) % intensity St Dev(d.nm) 240.9 202.3 99.3 78.48 Table 2 combined with Figure 3It can be seen that the average particle size of FACOD@DSPE nanoparticles is 240.9±78.48 nm, and 99.3% of the particle size is within this range, indicating that the prepared nanoparticles are relatively stable.

[0040] The 50 μM FACOD was treated with 350-450 nm light, and the UV and fluorescence spectra of FACOD in DMF before and after light treatment were detected to detect the changes in UV fluorescence of FACOD. The detection results are as follows: Figure 4 As shown.

[0041] Figure 4 The right-hand figure shows the absorbance of FACOD under different wavelengths of light. As can be seen from the figure, under light treatment at a wavelength of 350 nm, the absorbance of 50 μM FACOD at 0 min is 3.0 × 10⁻⁶. -1 The absorbance was around 2.0 × 10⁻⁶ at 55 min. -1 Around 350nm light, the absorbance of FACOD showed a significant decrease, indicating that FACOD was significantly depleted after processing with 350nm light.

[0042] Figure 4 The top left of the right-hand graph shows the change in absorbance of 50 μM FACOD under 410 nm light over time. The graph shows that as the illumination time increases, the absorbance of FACOD first decreases and then reaches 1.5 × 10⁻⁶. -1 The study showed that FACOD decomposes rapidly under 410nm illumination and then tends to reach equilibrium in about 20 minutes.

[0043] Figure 4 The left figure shows the fluorescence intensity of FACOD under different wavelengths of light. As can be seen from the figure, FACOD fluctuates at 0 after 55 minutes of light exposure, indicating that FACOD is almost completely consumed after 55 minutes of light exposure. The fluctuation at 0 minutes may be due to fluctuations caused by the instrument, because both FACOD@DSPE and FACOD show similar fluctuation lines at 0 minutes.

[0044] like Figure 5 The image shows the UV fluorescence change spectrum of 50 μM FACOD encapsulated in a 0.5 mg / mL DSPE-PEG2000 solution under 410 nm light. The UV spectrum illustrates the UV absorption of the FACOD@DSPE solution at different time points. V. Detecting FA release status A standard curve was plotted with the standard concentration of the FA standard solution on the x-axis and absorbance on the y-axis, and the following was obtained: Figure 6The FA standard curve on the left has the equation y = 0.0032x + 0.009 and R² = 0.9903. The formaldehyde concentration can be calculated based on the absorbance from this curve.

[0045] according to Figure 6 The standard curve on the right shows the relationship between the concentration of FACOD@DSPE nanoparticles and the concentration of FA, as shown in the figure. Figure 6 As shown in Figure F, the regression equation for the relationship between the concentration of FACOD@DSPE solution and the concentration of FA is y = 0.2973x - 1.7565, with R² = 0.9988. After mixing 50 μM FACOD@NP solution with the FA kit and irradiating it with light, the absorbance was measured to be 0.145. Substituting this into the regression equation, the calculated FA production amount was 42.51 μM, representing a yield of approximately 85.02%.

[0046] Based on y = 0.2973x - 1.7565, the FA release rate using 30 μL of 50 μM FACOD@DSPE nanoparticles is calculated as: (0.2973 × 50 - 1.7565) × 3 × 10⁻¹⁰ -5 L = 13.1085 μmol / L × 3 × 10 -5 L = 3.93 × 10 -10 Since the molar mass of FA is 30.03 g / mol, the amount of FA released is 1.180 × 10⁻⁶ mol. −8 g. VI. Monitoring CO Release A standard curve was plotted with the standard concentration of CO standard solution on the x-axis and fluorescence intensity on the y-axis, and the following was obtained: Figure 7 The left side shows the CO standard curve, and the equation for the relationship between CO concentration and fluorescence is y = 167.96x + 1268.8, R0. 2 =0.9832, and the CO concentration can be calculated based on the absorbance from this curve. The concentration of FACOD@DSPE nanoparticles tested was 50 μM, obtained by... Figure 7 The right-hand graph shows a CO fluorescence intensity value of 5.7*10. 3 Substituting the values ​​into the CO standard curve, we obtain the CO emission amount, with a CO yield of 50.3%. VII. Application Research of FACOD Nanoparticles Since the donor releases gases FA and CO, both of which are highly effective antibacterial molecules, we will explore the antibacterial effects of FACOD. We will select Gram-positive, Gram-negative, and drug-resistant bacteria for experiments to explore the effects of FACOD on these three types of bacteria.

[0047] (1) Effects of different concentrations of FACOD@DSPE solution on Staphylococcus aureus Take several sterile culture dishes and label them with the corresponding groups: normal control group, methicillin group, 50 μM FACODNP, 0 μM FACODNP+hv, 10 μM FACODNP+hv, 30 μM FACOD+hv, and 50 μM FACODNP+hv, where hv represents the irradiation intensity of 100 mW / cm². 2 Irradiation was performed using a 100W high-pressure mercury lamp. 0.1 mL of different concentrations of FACOD@DSPE solution was added to each petri dish, followed by 0.1 mL of the corresponding Staphylococcus aureus bacterial suspension. The FACOD@DSPE bacterial suspension was then evenly spread onto the surface of LB solid medium using a sterile spreader. Three replicates were set for each concentration gradient. Colony counts and sterilization rates are shown in Table 3, where sterilization rate (%) = (blank control - experimental group) / blank control × 100%.

[0048] Table 3. Colony composition and bactericidal rate of Staphylococcus aureus in different groups Group Colony count Sterilization rate Blank control 1937 —— Methicillin 271 86.01% 50 μM FACOD NP 1992 -2.84% 0 μM FACOD NP+hv 1065 45.02% 10 μM FACOD NP+hv 114 94.11% 30 μM FACOD NP+hv 12 99.38% 50 μM FACOD NP+hv 3 99.85% Figure 8 This is a graph showing the colony growth of Staphylococcus aureus in different groups. Figure 8 As shown in Table 3, the number of colonies in the culture dishes containing 10 μM FACOD NP+hv was significantly lower than that in the blank control and the culture dishes containing 0 μM FACOD NP+hv, indicating that 10 μM FACOD NP can significantly kill Staphylococcus aureus under light conditions. Furthermore, the bactericidal rate of 10 μM FACOD NP+hv (94.11%) and that of methicillin (86.01%) show that 10 μM FACOD NP is slightly more effective than methicillin in killing Staphylococcus aureus under light conditions. Figure 7 It can be seen that there are almost no colonies in the culture dishes of 30 μM FACOD NP+hv and 50 μM FACOD NP+hv. As shown in Table 2, the bactericidal rates of 30 μM FACOD NP and 50 μM FACOD NP against Staphylococcus aureus under light conditions reached 99.38% and 99.85%, respectively.

[0049] (2) Effects of different concentrations of FACOD@DSPE solution on Escherichia coli Take several sterile culture dishes and label them with the corresponding groups: normal control group, methicillin group, 50 μM FACODNP, 0 μM FACODNP+hv, 10 μM FACODNP+hv, 30 μM FACOD+hv, and 50 μM FACODNP+hv, where hv represents the irradiation intensity of 100 mW / cm². 2Irradiation was performed using a 100W high-pressure mercury lamp. 0.1 mL of different concentrations of FACOD@DSPE solution was added to each petri dish, followed by 0.1 mL of the corresponding E. coli suspension. The FACOD@DSPE solution was then evenly spread onto the surface of LB solid medium using a sterile spreader. Three replicates were set up for each concentration gradient. Colony counts and sterilization rates are shown in Table 4, where sterilization rate (%) = (blank control - experimental group) / blank control × 100%.

[0050] Table 4. Colony composition and sterilization rate of Escherichia coli in different groups Group Colony count Sterilization rate Blank control 1980 —— Methicillin 96 95.15% 50 μM FACOD NP 1527 36.11% 0 μM FACOD NP+hv 1265 22.88% 10 μM FACOD NP+hv 171 91.36% 30 μM FACOD NP+hv 18 99.09% 50 μM FACOD NP+hv 4 99.80% Figure 9 The graph shows the colony growth of *E. coli* in different groups. It can be seen that the colony counts in the 30 μM FACOD NP+hv and 50 μM FACOD NP+hv culture dishes were extremely low, significantly fewer than those in the methicillin group. Table 4 shows that the lower colony count in the 50 μM FACOD NP group compared to the blank control group may be due to operational errors or low activity of the added *E. coli*, possibly indicating insufficient *E. coli* count or low activity. The lower colony count in the 0 μM FACOD NP+hv group compared to the blank control group may be due to the white light used in this experiment containing ultraviolet light, which could have a certain killing effect on *E. coli*. The bactericidal rate of 10 μM FACOD NP+hv was 91.36%, slightly lower than that of methicillin (95.15%). However, the bactericidal rates of 30 μM FACOD NP and 50 μM FACOD NP under light conditions were 99.09% and 99.80%, respectively, significantly higher than those of the methicillin group. This indicates that FACOD NP+hv at 30 μM and 50 μM had a significantly better bactericidal effect on Escherichia coli than methicillin.

[0051] (3) Effects of different concentrations of FACOD@DSPE solution on drug-resistant Staphylococcus aureus Take several sterile culture dishes and label them with the corresponding groups: normal control group, methicillin group, 50 μM FACODNP, 0 μM FACODNP+hv, 10 μM FACODNP+hv, 30 μM FACOD+hv, and 50 μM FACODNP+hv, where hv represents the irradiation intensity of 100 mW / cm². 2Irradiation was performed using a 100W high-pressure mercury lamp. 0.1 mL of different concentrations of FACOD@DSPE solution was added to each petri dish, followed by 0.1 mL of the corresponding drug-resistant Staphylococcus aureus suspension. The FACOD@DSPE solution was then evenly spread onto the surface of LB solid medium using a sterile spreader. Three replicates were set up for each concentration gradient. Colony counts and sterilization rates are shown in Table 5, where sterilization rate (%) = (blank control - experimental group) / blank control × 100%.

[0052] Table 5. Colony composition and bactericidal rate of drug-resistant Staphylococcus aureus in different groups Group Colony count Sterilization rate Blank control 835 —— Methicillin 989 -18.44% 50 μM FACOD NP 997 -17.37% 0 μM FACOD NP+hv 980 -19.40% 10 μM FACOD NP+hv 393 52.93% 30 μM FACOD NP+hv 110 86.83% 50 μM FACOD NP+hv 9 98.92% As is well known, the resistance of MRSA (methicillin-resistant Staphylococcus aureus) to β-lactam antibiotics (penicillins, cephalosporins, carbapenems, etc.) is the core reason for the increased difficulty in killing it. Essentially, it is due to the structural mutation of bacterial penicillin-binding protein (PBP) and the stable expression of resistance genes. Figure 10 This is a graph showing the colony growth of drug-resistant Staphylococcus aureus in different groups. Figure 10 As shown in Table 5, the number of colonies in the 30 μM FACOD NP+hv culture dish was significantly less than that in the blank control culture dish. The bactericidal rate of 30 μM FACOD NP against drug-resistant Staphylococcus aureus under light conditions was 86.83%, indicating that 30 μM FACOD NP has a good bactericidal effect against drug-resistant Staphylococcus aureus under light conditions. The number of colonies in the 50 μM FACOD NP+hv culture dish was extremely low, and the bactericidal rate against drug-resistant Staphylococcus aureus reached 98.92%, indicating an excellent bactericidal effect. VIII. Effects of FACOD NP on biofilm formation and growth of three bacteria Bacterial biofilms represent a highly organized, environmentally resistant social survival mode in which bacteria transform from individual organisms. Once a biofilm forms, treating related bacterial infections becomes extremely difficult. Therefore, obtaining antibacterial drugs with strong biofilm-disrupting capabilities is crucial for treating bacterial infections. This study investigated the effects of FACOD@DSPE on biofilm formation and growth in three types of bacteria. The specific experimental method is as follows: (1) Preparation of bacterial suspension: Single colonies of the three types of bacteria were picked and inoculated into test tubes containing 5 mL of sterile LB liquid medium. The test tubes were placed in a shaker at 37℃ and 180 r / min for 24 hours to allow the bacteria to enter the logarithmic growth phase. After the culture was completed, 1 mL of bacterial suspension was transferred to a test tube containing 9 mL of LB medium, and the suspension was vortexed to disperse the bacteria evenly. The suspension was then serially diluted 10-fold to adjust the bacterial concentration to 1×10⁻⁶. 6 -1×10 7CFU / mL.

[0053] (2) Biofilm formation: In a 96-well cell culture plate, 100 μL of FACOD@DSPE solution of different concentrations was added to each well, followed by 100 μL of bacterial suspension adjusted to the desired concentration. Each concentration gradient was set up in triplicate. In the control group, 100 μL of LB was added to each well instead of FACOD@NPs solution, followed by 100 μL of bacterial suspension. The 96-well plate was placed in a 37°C incubator and incubated statically for 24-48 hours to allow bacteria to form a biofilm.

[0054] (3) Biofilm treatment: Gently pour off the culture medium in the culture plate, slowly rinse 2-3 times with sterile PBS buffer, soaking for 3-5 minutes each time to remove unattached airborne bacteria, and air dry. Add 200 μL of anhydrous methanol to each well, fix for 15-20 minutes, then pour off the methanol and air dry naturally. Add 100 μL of 0.1% crystal violet staining solution to each well, and stain for 15-20 minutes. Slowly rinse 2-3 times with sterile PBS to remove excess staining solution, and air dry naturally. Add 200 μL of ethanol to each well, shake for 10-15 minutes to ensure complete elution of crystal violet.

[0055] The absorbance (OD) value of each well was measured at 570 nm using a microplate reader. The OD value is directly proportional to the amount of biofilm. Based on the OD values ​​of the experimental and control groups, the inhibition rate of different concentrations of FACOD@DSPE on the biofilm of the three bacteria was calculated as follows: Inhibition rate = (OD value of control group - OD value of experimental group) ÷ OD value of control group × 100%. Statistical software was used to analyze the experimental data, comparing the differences between the different concentrations of FACOD@DSPE treatment groups and the control group to determine whether the effect of FACOD@DSPE on the formation and growth of biofilm of the three bacteria was statistically significant.

[0056] Figure 11 These are graphs showing biofilm formation after treatment with FACOD@DSPE on E. coli, S. aureus, and MRSA, and bar charts showing the inhibition of the three bacterial species. From... Figure 11As can be seen, the blank control group of the three bacteria was dark purple, indicating that all three bacteria formed dense and thick biofilms in the absence of treatment. Compared with the blank control group, the purple color of 50 µM FACOD NP remained very deep with little change, indicating that the destructive effect of 50 µM FACOD alone on the biofilm formation of the three bacteria was very limited in the absence of light. Compared with the blank control group and 50 µM FACOD, the purple colors of 20 µM FACOD+hv and 30 µM FACOD+hv were significantly lighter, indicating that 20 µM FACOD and 30 µM FACOD could effectively destroy the biofilm formation of the three bacteria under light conditions. Compared with the blank control group and 50 µM FACOD, the purple color of 50 µM FACOD+hv almost disappeared, indicating that the biofilm was almost completely removed / inhibited. In conclusion, FACOD@DSPE has a certain antibacterial effect under light treatment, and the antibacterial effect increases with increasing concentration. Figure 11 The bar chart on the right shows that at 10 µM FACOD+hv, the OD values ​​of the three bacterial strains decreased by 25-46%, while at 20 µM FACOD+hv and 30 µM FACOD+hv, the decrease was approximately 45-63%. When the concentration reached 50 µM, the OD decreased by more than 80%, with MRSA decreasing by as much as 95%. IX. Safety Assessment of FACOD@DSPE Nanoparticles Cell hemolysis assay: Hemolytic activity was detected using 4% porcine erythrocytes. 100 μL of PBS was added to centrifuge tubes as a negative control; 1.0% Triton X-100 was added to centrifuge tubes as a positive control; FACOD@DSPE nanoparticles at concentrations of 0 μM, 10 μM, 20 μM, 30 μM, and 50 μM were added to centrifuge tubes respectively, and 100 μL of 4% erythrocyte suspension was added to each tube. The tubes were incubated at 37℃ for 3 h, centrifuged at 1500 rpm for 10 min, and the results are as follows. Figure 11 As shown; 100 μL of PBS was added to the centrifuge tube as a negative control; 1.0% Triton X-100 was added to the centrifuge tube as a positive control; FACOD@DSPE nanoparticles at concentrations of 0 μM, 10 μM, 20 μM, 30 μM, and 50 μM were added to 10 centrifuge tubes respectively; 100 μL of 4% red blood cell suspension was added to each of the above centrifuge tubes; and the mixture was irradiated with an intensity of 100 mW / cm². 2 After irradiation with a 100W high-pressure mercury lamp for 10 minutes, the mixture was incubated in a 37℃ incubator for 3 hours, followed by centrifugation at 1500 rpm for 10 minutes. The results are as follows: Figure 12 As shown.

[0057] Figure 12 This image shows the hemolysis of cells at different concentrations of FACOD@DSPE particles. From... Figure 12 It can be seen that no hemolysis was observed in the erythrocytes of the blank control group and the 0 μM FACOD NP group. Very minor hemolysis was observed in the erythrocytes of the 10 μM FACOD NP group and the 20 μM FACO group. A small amount of hemolysis was observed in the erythrocytes of the 30 μM FACOD group and the 50 μM FACOD NP group. No hemolysis was observed in the erythrocytes of the blank control group, the 0 μM FACOD NP+hv group, the 10 μM FACOD NP+hv group, and the 20 μM FACO+hv group. Very minor hemolysis was observed in the erythrocytes of the 30 μM FACOD NP+hv group and the 50 μM FACO+hv group. The small amount of hemolysis observed in the above cell hemolysis experiments is mild and within the expected range, insufficient to cause large-scale hemolysis, and falls within the scope of biosafety.

[0058] Cytotoxicity assay: Cells were introduced at a rate of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well into 96-well plates and incubated in a standard CO2 incubator for 24 hours. After incubation, 0 μM FACOD NP, 10 μM FACOD NP, 20 μM FACOD NP, 50 μM FACOD NP, 80 μM FACOD NP, and 100 μM FACOD NP were added to the 96-well plates, and incubation continued for another 24 hours. The viability of each group was then measured using a CCK-8 cell viability assay kit.

[0059] Figure 13 Bar chart showing cell viability at different concentrations of FACOD@DSPE nanoparticles. Figure 13 It can be seen that the cell viability of 0 μM FACOD NP, 10 μM FACOD NP, 20 μM FACOD NP and 50 μM FACOD NP is 100%, and the cell viability of 80 μM FACOD NP and 100 μM FACOD NP is higher than 90%.

[0060] The hemolysis and cytotoxicity experiments demonstrate that FACOD@DSPE nanoparticles exhibit low toxicity and high biocompatibility at concentrations below 100 μM, indicating that 10-50 μM FACOD@DSPE nanoparticles can be used to treat drug-resistant Staphylococcus aureus, Staphylococcus aureus, and Escherichia coli. In the application process, FACOD@DSPE nanoparticles are dissolved in water and then applied to the skin. Under light exposure, the applied FACOD@DSPE nanoparticles release carbon monoxide and formaldehyde to kill surrounding drug-resistant Staphylococcus aureus, Staphylococcus aureus, and Escherichia coli. Based on the hemolysis and cytotoxicity experiments, this method of application of FACOD@DSPE nanoparticles is safe and acceptable. 10. Mouse animal experiments Experimental Methods: Mice were anesthetized with a SurgiVetCOS9000 air anesthesia system using a face mask with 2% isoflurane (oxygen flow rate 1.0 L / min). After the corneal reflex disappeared and the muscles relaxed, the hair on the back of the mice was initially removed with electric clippers. Then, a hair removal cream was evenly applied and left to stand for 5 minutes. The remaining cream was washed off with warm water to ensure that the back skin was smooth and free of residual hair. Subsequently, a full-thickness skin defect was prepared next to the spine on the back of the mice using a 10 mm diameter circular punch. 10 μL of a 1.0 × 10⁻⁶ solution was then applied. 8 A CFU / mL suspension of methicillin-resistant Staphylococcus aureus was evenly applied to the wound surface, and a transparent waterproof membrane was then applied to fix the bacterial suspension and prevent it from spilling out.

[0061] Observe for 24 hours after modeling. If redness, swelling, and purulent exudate appear on the wound, the mouse infected wound model is considered to have been successfully established. The successfully modeled mice are randomly divided into groups and anesthetized again by inhalation of 2% isoflurane. The wounds are then photographed using a digital camera under the same lighting and distance to record the initial state.

[0062] Subsequently, 30 μL of 50 μM FACOD@DSPE formulation was evenly applied to the wounds of mice in each group, while the control group received PBS solution. After standing for 30 minutes, the wounds were irradiated with a 10W mercury lamp at an intensity of 100 mW / cm². 2 The light exposure lasted for 30 minutes. After the light exposure ended, the mice were anesthetized again, and the wound condition was photographed and recorded. Standardized photographs of the mouse wounds were then taken every 24 hours thereafter, and the observation and recording were continued for 14 days to dynamically monitor the wound healing process. After observation, the mice were euthanized, and wound tissue was collected for HE staining to observe tissue details.

[0063] from Figure 14It can be seen that the wound healing area of ​​the control group mice was relatively slow after 14 days, while the healing was faster in the FACOD@NPs treatment group. Furthermore, the FACOD@DSPE preparation applied to the wounds of mice in the FACOD@NPs treatment group did not cause any skin problems, and the mice's health and activity levels were no different from the control group. Therefore, applying FACOD@DSPE preparation to the skin surface of mice under light conditions does not affect the health and activity levels of the mice, and it does effectively promote wound healing (1.0 × 10¹²). 8 Wound healing of infection with CFU / mL methicillin-resistant Staphylococcus aureus suspension.

[0064] from Figure 15 It can be seen that when mice were treated with 1.0 × 10 8 The wound treated with a CFU / mL methicillin-resistant Staphylococcus aureus suspension showed obvious pathological changes including abnormal epidermal proliferation, dermal inflammatory infiltration, and local cell aggregation. These pathological changes are consistent with the histological characteristics of bacterial skin lesions, indicating that the mouse model of infected wounds was successfully established.

[0065] from Figure 16 It can be seen that when mice were treated with 1.0 × 10 8 The presence of CFU / mL methicillin-resistant Staphylococcus aureus suspension in the wound reduced abnormal epidermal proliferation, alleviated inflammatory response, and initiated repair of skin appendage structures. This suggests that applying 30μL of 50μMFACOD@DSPE formulation has a significant effect on improving skin lesions caused by pathogenic bacterial infection in mice.

[0066] This invention can be implemented in various ways and is not limited to the embodiments described. Those skilled in the art will understand that the invention can be implemented in other specific ways without changing the technical concept or essential features. Therefore, it should be understood that the embodiments described above are exemplary and not intended to limit the invention.

Claims

1. A method for preparing FACOD@DSPE nanoparticles, characterized in that, The preparation method includes: (1) Compound IV and compound COD were mixed and stirred overnight at room temperature, extracted using a separatory funnel, dried, filtered, separated and purified to obtain FACOD; (2) FACOD was encapsulated with distearate phosphatidylethanolamine-methoxy polyethylene glycol to construct FACOD@DSPE nanoparticles.

2. The method for preparing FACOD@DSPE nanoparticles as described in claim 1, characterized in that, The preparation method of the compound COD is as follows: A sodium hydroxide aqueous solution was added to compound III, and the mixture was stirred at room temperature for 30 minutes to obtain a first reaction mixture. Benzaldehyde was then added to the first reaction mixture, and the mixture was stirred at room temperature for 5 hours to obtain a second reaction mixture. The second reaction mixture was cooled to 0°C in an ice bath, K2O2 was added, and the mixture was stirred overnight while being heated to room temperature. The solution was acidified with hydrochloric acid, the precipitate was filtered, and the precipitate was washed with ethanol to obtain compound COD.

3. The method for preparing FACOD@DSPE nanoparticles as described in claim 2, characterized in that, The chemical formula of compound III is as follows: ; The chemical formula of the compound COD is as follows: The chemical name of the compound COD is 7-hydroxy-4-phenyl-3H-naphtho[2,3-c]furan-1-one.

4. The method for preparing FACOD@DSPE nanoparticles as described in claim 1, characterized in that, The preparation method of compound IV is as follows: (1) Compound I was added to anhydrous acetonitrile, mixed and cooled to 0°C, then dimethyl sulfide was added, nitrogen gas was introduced for protection, benzoyl peroxide was added and reacted at 0°C for 6 hours, pH was adjusted to 9, reaction was carried out overnight, extracted, dried, filtered and the filtrate was evaporated to dryness, purified and compound II was obtained. (2) Compound II was added to anhydrous dichloromethane, cooled to 0°C, and then sulfonyl chloride was slowly added. The reaction was continued at room temperature for 8 hours under nitrogen protection. The organic phase was extracted, dried, filtered, and the filtrate was evaporated to dryness. The mixture was then separated and purified to obtain compound IV.

5. The method for preparing FACOD@DSPE nanoparticles as described in claim 4, characterized in that, The chemical formula of compound I is as follows: The name of compound I is 2-nitrobenzyl alcohol; The chemical formula of compound II is as follows: , The chemical formula of compound IV is as follows: The chemical name of compound IV is 2-nitrobenzylchloromethyl.

6. The FACOD@DSPE nanoparticles prepared by the method described in any one of claims 1-5 are characterized in that, The structural formula of FACOD in the FACOD@DSPE nanoparticles is as follows: .

7. The use of the FACOD@DSPE nanoparticles as described in any one of claims 1-6 in the preparation of photoresponsive antibacterial and / or bactericidal formulations for animals.

8. The application of the FACOD@DSPE nanoparticles as described in claim 7 in the preparation of photoresponsive antibacterial and / or bactericidal formulations for animals, characterized in that, The antibacterial or bactericidal bacteria mentioned are drug-resistant Staphylococcus aureus.

9. The application of the FACOD@DSPE nanoparticles as described in claim 7 in the preparation of photoresponsive antibacterial and / or bactericidal formulations for animals, characterized in that, The antibacterial or bactericidal bacteria is Staphylococcus aureus.

10. A formulation containing FACOD@DSPE nanoparticles as described in any one of claims 1-6 at a concentration of 30-50 μM, characterized in that, The formulation is an animal photosensitizing antibacterial and / or bactericidal agent, and the bacteria killed or / and inhibited by the formulation are Escherichia coli and / or drug-resistant Staphylococcus aureus and / or Staphylococcus aureus.

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