Preparation method and application of iron-containing lipid nanoparticles IO-LNPs with antibacterial function

Abstract

The invention belongs to the technical field of nanotechnology, and particularly discloses a preparation method and application of iron-containing lipid nanoparticles IO-LNPs with an antibacterial function. The iron-containing lipid nanoparticles (IO-LNPs) with an antibacterial function are synthesized by taking iron oleate, soya bean lecithin and DSPE-MPEG2000 as raw materials through a gradient solvent diffusion method, can induce a bacterial ferroptosis effect, has good antibacterial activity, can effectively kill gram positive (S.aureus) and negative (E.coli) bacterial strains, and has good biological membrane prevention and destruction capabilities on S.aureus and E.coli. The iron-containing lipid nanoparticles (IO-LNPs) have the advantages that the iron-containing lipid nanoparticles (IO-LNPs) with the antibacterial function can be used for preparing the iron-containing lipid nanoparticles (IO-LNPs), and the iron-containing lipid nanoparticles (IO-LNPs) can be used for preparing the iron-containing lipid nanoparticles (IO-LNPs); the iron-containing lipid nanoparticles IO-LNPs with the antibacterial function, prepared by the preparation method disclosed by the invention, show relatively high stability and biocompatibility under physiological conditions, have an excellent effect in antibacterial performance and are a safe and efficient medicament for resisting bacterial infection and promoting wound healing.

Description

Technical Field

[0001] The invention belongs to the field of nanotechnology, and specifically relates to a preparation method and application of iron-containing lipid nanoparticles IO-LNPs with antibacterial function. Background Art

[0002] In the field of antimicrobial therapy, with the increasing severity of antibiotic resistance, the development of new antimicrobial therapies has become an important task for researchers around the world. The widespread use of traditional antibiotics has led to a rapid increase in bacterial resistance, making it particularly urgent to find alternative therapies. In this context, nanotechnology, as an emerging method, has gradually attracted attention in the application of antimicrobial therapy. Among them, lipid nanoparticles (LNPs) have become one of the research hotspots of drug delivery systems due to their good biocompatibility, easy modification, and controllable release characteristics.

[0003] However, in the face of complex and changing infection situations, how to improve the effectiveness of antimicrobial treatment and reduce the development of drug resistance has become a key issue that researchers need to solve. In recent years, ferroptosis, as a new type of programmed cell death, has attracted the interest of researchers due to its unique mechanism of action on certain types of pathogens. Ferroptosis is a process that relies on lipid peroxidation reactions mediated by iron ions and leads to cell death. Existing methods for using ferroptosis to enhance antibacterial effects mainly focus on the following aspects:

[0004] Utilizing iron ion carriers: Iron ions are a key factor in the process of ferroptosis. By using compounds or structures that can carry iron ions into bacterial cells, ferroptosis can be effectively promoted. For example, some iron ion chelators can bind to iron ions and be absorbed by bacteria, thereby inducing ferroptosis.

[0005] Inhibition of the antioxidant system: An important feature of ferroptosis is its reliance on lipid peroxidation, and this process can be enhanced by inhibiting the bacteria's own antioxidant defense system. For example, GPX4 (glutathione peroxidase 4) is an important antioxidant enzyme responsible for clearing intracellular peroxides. Inhibiting GPX4 activity can increase lipid peroxidation levels, thereby promoting ferroptosis.

[0006] Application of nanomaterials: These materials can not only serve as effective iron ion carriers, but can also further promote the ferroptosis process through their special physical and chemical properties, such as photothermal effect, magnetic effect, etc. In addition, these nanoparticles often have good biocompatibility and controllable drug release properties, which can achieve more precise targeted therapy.

[0007] Although the above methods have shown good antibacterial potential under laboratory conditions, they still face the following challenges in practical applications:

[0008] The selectivity issue: How to ensure that these treatments only target pathogens and do not affect host cells, especially when the pathogens are located within the host, is a pressing issue that needs to be addressed.

[0009] Development of drug resistance: Although inducing ferroptosis provides a new antibacterial strategy, long-term use may prompt bacteria to evolve ways to counter this new mechanism and develop new drug resistance.

[0010] In vivo safety and biodistribution: For the use of nanomaterials, issues such as their long-term safety in the body, metabolic pathways, and possible side effects require further study.

[0011] Cost-effectiveness ratio: The research and development and clinical transformation of new technologies are often accompanied by high costs. Therefore, while ensuring efficacy, economic feasibility also needs to be considered. Summary of the invention

[0012] In order to solve the shortcomings of the prior art, the present invention proposes a method for preparing IO-LNPs based on a gradient solvent diffusion method. First, ferric oleate is dissolved in a non-polar solvent, and then the solution is injected into a polar solvent. The non-polar solvent is removed by using a vacuum rotary evaporator, and then an aqueous phase is added to the system to finally obtain stable LNPs. This process not only has simple preparation steps, but also overcomes the technical difficulties of preparing non-polar drugs into stable lipid nanoparticles by traditional solvent diffusion methods. The advantage of this method is that it allows a wider range of drug types to be encapsulated in LNPs, enhances the design flexibility of antibacterial treatment schemes, and improves the delivery efficiency and therapeutic effect of drugs, opening up a new path for antibacterial treatment, and has important theoretical significance and application prospects.

[0013] The present invention adopts a gradient solvent diffusion method to synthesize iron-containing lipid nanoparticles IO-LNPs with antibacterial function. The antibacterial iron-containing lipid nanoparticles prepared by the present invention induce bacterial ferroptosis effect, have good antibacterial activity, and have broad application prospects in the fields of antibacterial and bactericidal.

[0014] The preparation method of iron-containing lipid nanoparticles IO-LNPs with antibacterial function comprises the following specific steps:

[0015] (1) dissolving ferric oleate in n-hexane to obtain a solution A with a concentration of 10 mg / ml; dissolving soybean lecithin in anhydrous ethanol to obtain a solution B with a concentration of 0.3-2.7 mg / ml; dissolving DSPE-MPEG2000 in anhydrous ethanol to obtain a solution C with a concentration of 3-10 mg / ml; then, injecting solution A and solution C into solution B, rapidly stirring at 1500 rpm for 3 min at room temperature, to obtain a ferric oleate nanoemulsion;

[0016] The mass ratio of ferric oleate to soybean lecithin is 1:0.5-4; the added amount of DSPE-MPEG2000 is 20% of the total mass of ferric oleate and soybean lecithin.

[0017] (2) Using a vacuum rotary apparatus to remove n-hexane from the iron oleate nanoemulsion, the obtained iron oleate nanoemulsion was injected into the aqueous phase, rapidly stirred, dialyzed with ultrapure water, and filtered through a 0.22 μm water-based microporous filter membrane to obtain IO-LNPs with good dispersibility.

[0018] The vacuum rotary evaporator was used with a rotation speed of 150 rpm, rotary evaporation for 6 min and a temperature of 37°C.

[0019] The volume ratio of the iron oleate nanoemulsion to the water phase after the n-hexane is removed is 1:10; the stirring rate of the injected water phase is 1500 rpm, and the stirring time is 3 min.

[0020] The antibacterial iron-containing lipid nanoparticles IO-LNPs prepared by the above method can induce ferroptosis in bacteria, and can be used to kill S. aureus and E. coli in vivo and in vitro, eliminate and destroy biofilms, and effectively inhibit the proliferation of S. aureus and promote wound healing in a skin infection model.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The IO-LNPs prepared by the gradient solvent diffusion method of the present invention can overcome the technical difficulty of preparing non-polar drugs into stable LNPs by the traditional solvent diffusion method. The prepared IO-LNPs have good 7-day stability, are not prone to aggregation or degradation, and are conducive to long-term storage and transportation.

[0023] (2) The present invention has a simple operation process for preparing IO-LNPs, high repeatability, a wide range of raw material sources, mild reaction conditions, easy synthesis, excellent biocompatibility, and is suitable for popularization and use. The prepared IO-LNPs have a small particle size, uniform dispersion, and high stability.

[0024] (3) The IO-LNPs prepared by the present invention can induce ferroptosis in bacteria, and the generated reactive oxygen species (ROS) and lipid peroxidation (LPO) can cause damage to the cell membrane of microorganisms, thereby effectively killing Gram-positive (S. aureus) and Gram-negative (E. coli) strains. It has excellent antibacterial properties in vivo, and the selected materials are all biodegradable materials with good biocompatibility and low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 .Potential distribution diagram of IO-LNPs prepared in Example 1.

[0026] Figure 2 .7-day particle size stability of IO-LNPs prepared in Example 1.

[0027] Figure 3 .Dilution factor stability of IO-LNPs prepared in Example 1.

[0028] Figure 4 .TEM image of IO-LNPs prepared in Example 1.

[0029] Figure 5 .Effect of IO-LNPs prepared in Example 1 on the survival rate of S. aureus (turbidity method).

[0030] Figure 6 .Effect of IO-LNPs prepared in Example 1 on the survival rate of E. coli (turbidity method).

[0031] Figure 7 . Colony formation diagram of S. aureus prepared in Example 1 after being treated with different concentrations of IO-LNPs.

[0032] Figure 8 .The corresponding quantitative statistical results of the colonies of S. aureus prepared in Example 1 after being treated with different concentrations of IO-LNPs.

[0033] Fig. 9 .The colony formation diagram of E.Coli prepared in Example 1 after being treated with different concentrations of IO-LNPs.

[0034] Fig.10 .The corresponding quantitative statistical results of the colonies of E. coli prepared in Example 1 after being treated with different concentrations of IO-LNPs.

[0035] Fig.11 . Graph showing the biofilm inhibition effect of S. aureus after treatment with IO-LNPs prepared in Example 1.

[0036] Fig.12 .Biofilm inhibition effect diagram of E.Coli after treatment with IO-LNPs prepared in Example 1.

[0037] Fig.13 . Graph showing the biofilm destruction effect of S. aureus after treatment with IO-LNPs prepared in Example 1.

[0038] Fig.14 .Biofilm destruction effect diagram of E.Coli after treatment with IO-LNPs prepared in Example 1.

[0039] Fig.15 .Internal ROS levels of S. aureus treated with IO-LNPs prepared in Example 1.

[0040] Fig.16 .The ROS level inside E. coli treated with IO-LNPs prepared in Example 1.

[0041] Fig.17 .The internal MDA level of S. aureus treated with IO-LNPs prepared in Example 1.

[0042] Fig.18 . MDA level inside E. coli treated with IO-LNPs prepared in Example 1.

[0043] Fig.19 .Growth curve of S. aureus treated with IO-LNPs prepared in Example 1 over time.

[0044] Fig. 20 .The growth curve of E. coli after being treated with IO-LNPs prepared in Example 1 over time.

[0045] Fig.21 Live / dead staining of S. aureus after treatment with IO-LNPs prepared in Example 1.

[0046] Fig. 22 . Live / dead staining of E. coli after treatment with IO-LNPs prepared in Example 1.

[0047] Fig.23 .SEM microscopic morphology of IO-LNPs prepared in Example 1 after treating S. aureus bacteria.

[0048] Fig.24 .SEM microscopic morphology of IO-LNPs prepared in Example 1 after treating E. coli.

[0049] Fig.25 .Cytotoxicity evaluation of IO-LNPs prepared in Example 1.

[0050] Fig.26 .Hemolysis results of IO-LNPs prepared in Example 1.

[0051] Fig. 27 .Graph showing the healing status of bacterially infected wounds in each group of mice prepared in Example 1.

[0052] Fig.28 .Images of wound healing traces of bacterial infection in each group of mice.

[0053] Fig.29 .Changes in the area rate of bacterial infection wounds in each group of mice.

[0054] Fig.30.The results of plating the wound skin tissue of each group of mice.

[0055] Fig.31 .The weight changes of mice in each group during the entire experimental period.

[0056] Fig.32 .H&E staining of wound tissues of mice infected with Staphylococcus aureus.

[0057] Fig.33 .Masson staining of wound tissue of mice infected with Staphylococcus aureus.

[0058] Fig.34 . Immunohistochemistry CD31 of mouse wound healing.

[0059] Fig.35 .H&E staining of organs from mice infected with S. aureus in each treatment group at day 13 of treatment. DETAILED DESCRIPTION

[0060] The present invention is further described in detail below in conjunction with specific examples, but it should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or replacements made to the method, steps or conditions of the present invention all belong to the scope of the present invention. The experimental methods and reagents without specifying the specific conditions in the examples are all in accordance with conventional conditions in the art.

[0061] Example 1

[0062] Step 1, dissolving 10 mg of ferric oleate in 1 mL of n-hexane to obtain solution A; dissolving 5 mg of soybean lecithin in 15 mL of anhydrous ethanol to obtain solution B; dissolving 3 mg of DSPE-MPEG2000 in 1 mL of anhydrous ethanol to obtain solution C;

[0063] Step 2, injecting solution A and solution C into solution B, rapidly stirring at 1500 rpm for 3 minutes at room temperature to obtain iron oleate nanoemulsion.

[0064] Step 3, using a vacuum rotator at a speed of 150 rpm for 6 min at 37°C to remove n-hexane from the iron oleate nanoemulsion, injecting the obtained iron oleate nanoemulsion into 10 mL of water phase, rapidly stirring at 1500 rpm for 3 min at room temperature, dialyzing with ultrapure water, and filtering through a 0.22 μm water-based microporous filter membrane to obtain IO-LNPs with good dispersibility.

[0065] The hydrated particle size and PDI of IO-LNPs were measured by Malvern particle size analyzer. The experimental results showed that the hydrated particle size was 130.06±8.48 and the PDI was 0.29±0.004.

[0066] Example 2

[0067] Step 1, dissolving 10 mg of ferric oleate in 1 mL of n-hexane to obtain solution A; dissolving 10 mg of soybean lecithin in 15 mL of anhydrous ethanol to obtain solution B; dissolving 4 mg of DSPE-MPEG2000 in 1 mL of anhydrous ethanol to obtain solution C;

[0068] Step 2, injecting solution A and solution C into solution B, rapidly stirring at 1500 rpm for 3 minutes at room temperature to obtain iron oleate nanoemulsion.

[0069] Step 3, using a vacuum rotator at a speed of 150 rpm for 6 minutes at 37°C to remove n-hexane from the iron oleate nanoemulsion, injecting the obtained iron oleate nanoemulsion into the aqueous phase, rapidly stirring at 1500 rpm for 3 minutes at room temperature, dialyzing with ultrapure water, and filtering through a 0.22 μm water-based microporous filter membrane to obtain IO-LNPs with good dispersibility.

[0070] The hydrated particle size and PDI of IO-LNPs were measured by Malvern particle size analyzer. The experimental results showed that the hydrated particle size was 183.73±26.46 and the PDI was 0.32±0.05.

[0071] Example 3

[0072] Step 1, dissolving 10 mg of ferric oleate in 1 mL of n-hexane to obtain solution A; dissolving 20 mg of soybean lecithin in 15 mL of anhydrous ethanol to obtain solution B; dissolving 6 mg of DSPE-MPEG2000 in 1 mL of anhydrous ethanol to obtain solution C;

[0073] Step 2, injecting solution A and solution C into solution B, rapidly stirring at 1500 rpm for 3 minutes at room temperature to obtain iron oleate nanoemulsion.

[0074] Step 3, using a vacuum rotator at a speed of 150 rpm for 6 minutes at 37°C to remove n-hexane from the iron oleate nanoemulsion, injecting the obtained iron oleate nanoemulsion into the aqueous phase, rapidly stirring at 1500 rpm for 3 minutes at room temperature, dialyzing with ultrapure water, and filtering through a 0.22 μm water-based microporous filter membrane to obtain IO-LNPs with good dispersibility.

[0075] The hydrated particle size and PDI of IO-LNPs were measured by Malvern particle size analyzer. The experimental results showed that the hydrated particle size was 137.13±28.08 and the PDI was 0.5±0.02.

[0076] Example 4

[0077] Step 1, dissolving 10 mg of ferric oleate in 1 mL of n-hexane to obtain solution A; dissolving 40 mg of soybean lecithin in 15 mL of anhydrous ethanol to obtain solution B; dissolving 10 mg of DSPE-MPEG2000 in 1 mL of anhydrous ethanol to obtain solution C;

[0078] Step 2, injecting solution A and solution C into solution B, rapidly stirring at 1500 rpm for 3 minutes at room temperature to obtain iron oleate nanoemulsion.

[0079] Step 3, using a vacuum rotator at a speed of 150 rpm for 6 minutes at 37°C to remove n-hexane from the iron oleate nanoemulsion, injecting the obtained iron oleate nanoemulsion into the aqueous phase, rapidly stirring at 1500 rpm for 3 minutes at room temperature, dialyzing with ultrapure water, and filtering through a 0.22 μm water-based microporous filter membrane to obtain IO-LNPs with good dispersibility.

[0080] The hydrated particle size and PDI of IO-LNPs were measured by Malvern particle size analyzer. The experimental results showed that the hydrated particle size was 148.70±22.06 and the PDI was 0.5±0.03.

[0081] Example 5

[0082] Hydration particle size stability, Zeta potential determination and TEM characterization of IO-LNPs prepared in Example 1:

[0083] After the IO-LNPs prepared in Example 1 were diluted 100 times with distilled water, 1 mL was taken into a sample pool and the potential size of the IO-LNPs was detected using a Malvern particle size analyzer, and the particle size of the IO-LNPs was monitored for 7 consecutive days. In addition, the IO-LNPs were diluted by different dilution factors, and the hydrated particle size under different dilution factors was detected using a Malvern particle size analyzer. Figure 1 As shown in Figure 2, the Zeta potential of IO-LNPs is -12.3 ± 1.73 mV. Figure 2 As shown in Figure 2, after the IO-LNPs were placed in ultrapure water for 7 days, the hydrated particle size of the IO-LNPs detected did not change significantly, indicating that the prepared IO-LNPs have high stability and are well dispersed in aqueous solution. Figure 3 As shown in the figure, under different dilution factors, the particle size of IO-LNPs did not change significantly, indicating that it has good stability. Such high stability provides a guarantee for subsequent in vitro and in vivo experiments.

[0084] In order to detect the successful preparation of IO-LNPs, the morphological characteristics of IO-LNPs were observed using a transmission electron microscope. Take 20 μL of IO-LNPs and drop it onto the carbon support film copper grid for negative staining. Let it stand for 1 minute and leave it at room temperature overnight. After the copper grid is completely dry, the sample is observed using a transmission electron microscope. Figure 4 As shown, IO-LNPs are uniform spherical structures with good dispersion, and the actual size of IO-LNPs observed under TEM is about 40nm. The above characterization results prove that IO-LNPs have been successfully prepared.

[0085] Example 6

[0086] In vitro antibacterial activity test of IO-LNPs prepared in Example 1

[0087] 1. In vitro antibacterial experiment of IO-LNPs

[0088] 1) In vitro antibacterial test of IO-LNPs (culture medium turbidity method)

[0089] In order to explore the effect of IO-LNPs on bacterial growth, overnight cultured S. aureus and E. coli were diluted and added to a 96-well plate and incubated with IO-LNPs. The final concentrations of IO-LNPs incubated with S. aureus were 1, 2, 4, and 8 mM, and the final concentrations of IO-LNPs incubated with E. coli were 2, 4, 8, and 16 mM. PBS was added as a control group. After 5 h, the turbidity of the culture medium changed, and the OD was detected by a microplate reader. 600 , the bacterial survival rate was calculated based on the absorbance value. S. aureus continued to divide and proliferate in TSB liquid culture medium. After a period of culture, the TSB culture medium changed from initially clear and transparent to relatively turbid. In order to study the in vitro antibacterial activity of IO-LNPs against S. aureus, the culture medium turbidity method was used to monitor the absorbance value at 600nm to reflect the turbidity of the culture medium to indicate the growth of S. aureus. As shown in the attached Figure 5 As shown, S. aureus medium 600 nm The absorbance value was negatively correlated with the concentration of IO-NLDs, indicating that IO-LNPs had a concentration-dependent inhibitory effect on S. aureus.

[0090] Similarly, E. coli continues to divide and proliferate in LB liquid culture medium. After a period of culture, the LB culture medium changes from initially clear and transparent to relatively turbid. In order to study the in vitro antibacterial activity of IO-LNPs against E. coli, the culture medium turbidity method was used to monitor the absorbance value at 600 nm to indicate the growth of E. coli. Figure 6As shown in the figure, the absorbance value of E. coli culture medium at 600 nm was negatively correlated with the concentration of IO-LNPs, indicating that IO-LNPs had a concentration-dependent inhibitory effect on E. coli.

[0091] 2) IO-LNPs antibacterial experiment (solid agar plate colony counting method)

[0092] In order to investigate the effect of IO-LNPs on the survival rate of S. aureus, the S. aureus bacterial solution in the logarithmic growth phase was diluted to 1×10 8 CFU / mL was placed in a shaker (250 rpm, 37°C) and incubated for 1 h with IO-LNPs solution, where the final concentration of IO-LNPs was 1, 2, 4, and 8 mM, and PBS was used as a negative control group. The volume of the bacterial solution and IO-LNPs solution was 100 μL. The sample after incubation was diluted to 2×10 4 Take 100 μL of the solution and drop it onto a solid agar plate. Use a spreader to spread it evenly and place it in a biochemical incubator for overnight culture. Take it out the next day, count and photograph the colonies on the agar plate, compare it with the PBS group, and calculate the bacterial survival rate. Set up 3 parallel samples for each group. Figure 7 and attached Figure 8 As shown, the number of colonies in the IO-LNPs group decreased in a concentration-dependent manner compared to the control group (PBS group). The survival rate of S. aureus exposed to 2mM IO-LNPs was 44%, and the survival rate of S. aureus exposed to 8mM IO-LNPs was only 12%. Therefore, the MIC50 of IO-LNPs to S. aureus is 2mM and the MIC90 is 8mM.

[0093] To investigate the effect of IO-LNPs on E. coli, the E. coli culture solution in the logarithmic growth phase was diluted to 1×10 8 CFU / mL and IO-LNPs solution were placed in a shaker (250 rpm, 37°C) and incubated for 1 h, where the final concentration of IO-LNPs was 2, 4, 8, and 16 mM, and PBS was used as a negative control group. The post-treatment process was consistent with the above. Fig. 9 and attached Fig.10 As shown, the number of colonies in the IO-LNPs group was significantly reduced compared with the control group, and the survival rate of E. coli exposed to 4mM IO-LNPs was 40%, while the survival rate of E. coli exposed to 16mM IO-LNPs was 7%. Therefore, the MIC50 of IO-LNPs against E. coli is 4mM and the MIC90 is 16mM.

[0094] 2. Experiments on the inhibition and destruction of biofilm by IO-LNPs

[0095] 1) Inhibition experiment of IO-LNPs on biofilm

[0096] For biofilm inhibition studies, 100 μL of IO-LNPs (the final concentrations for incubation with S. aureus were 2 and 8 mM, respectively, and the final concentrations for incubation with E. coli were 4 and 16 mM, respectively) were added to a 96-well plate, and 100 μL of S. aureus and E. coli solutions (1×10 8 CFU / mL). The plate was placed in an incubator for 48 hours, and then 100 μL of crystal violet was added for biofilm staining. The crystal violet in the biofilm was then dissolved in 200 μL of 80% ethanol in a constant temperature oscillator for 2 hours (250 rpm, 37°C), and the absorbance at 600 nm was measured using a microplate reader. Fig.11 As shown in Figure 2, only 25% of the biofilm was formed when S. aureus was co-incubated with 8 mM IO-LNPs. Fig.12 As shown, only 32% biofilm was formed when E. coli was co-incubated with 16 mM IO-LNPs. These results indicate that IO-LNPs have a strong biofilm inhibition ability against both strains.

[0097] 2) Experiment on the destruction of biofilm by IO-LNPs

[0098] For biofilm disruption studies, 100 μL of the corresponding culture medium and 100 μL of S. aureus and E. coli solutions (1 × 10 8 CFU / mL). The plate was placed in an incubator for 48 hours, then the supernatant was discarded and 200 mL of IO-LNPs (the concentrations for incubation with S. aureus were 4 and 8 mM, and the concentrations for incubation with E. coli were 8 and 16 mM, respectively) were added and returned to the incubator for another hour. Then 100 μL of crystal violet was used to replace the culture medium for biofilm staining. The crystal violet in the biofilm was then dissolved in 200 μL of 80% ethanol in a constant temperature oscillator for 2 hours (250 rpm, 37°C), and the absorbance at 600 nm was measured using a microplate reader. As shown in the attached Fig.13 As shown in Figure 2, after incubation with 8 mM IO-LNPs for 1 h, only 21% of the biofilm formed by S. aureus was retained. Fig.14 As shown in the figure, after incubation with 16 mM IO-LNPs for 1 h, only 28% of the biofilm formed by E. coli remained. Therefore, IO-LNPs not only disrupted the formation of biofilm from the inside, but also effectively dismantled the formed biofilm from the outside, thereby clearing the already formed biofilm. These results show that IO-LNPs have a strong biofilm destruction ability against both strains, which also indicates that IO-LNPs have a broad-spectrum antibacterial potential and can be used as a universal material for wound management.

[0099] 3. Detection of ROS in bacteria by IO-LNPs

[0100] The bacteria were incubated with 10 μM DCFH-DA at 37 °C for 30 min and then washed three times with deionized water to remove unincorporated DCFH-DA. The bacteria were then incubated with the positive control (ROS), negative control (PBS), and IO-LNPs groups for 20 min. The changes in DCF fluorescence intensity were measured using a microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Fig.15 and attached Fig.16 As shown in the figure, after being treated with IO-LNPs, the ROS levels in S. aureus and E. coli increased significantly. This result suggests that IO-LNPs may trigger oxidative stress by increasing the ROS levels inside bacteria, leading to cell damage and even death.

[0101] 4. Detection of MDA in bacteria by IO-LNPs

[0102] In order to verify whether this oxidative stress is accompanied by lipid peroxidation, the malondialdehyde (MDA) content in bacterial cells was measured using a malondialdehyde (MDA) detection kit. 8 CFU / mL bacterial solution 100μL was added to 900μL PBS as the control group, and 1×10 8 100 μL of bacterial solution with CFU / mL and 900 μL of IO-LNPs were used as the experimental group. The supernatant was removed by centrifugation. After fully removing the liquid, the bacteria were flicked as much as possible, and 100 μL-200 μL of lysis buffer was added to mix them. Lysis was carried out on ice for 10 min. 200 μL of MDA detection working solution was added per well, heated at 100°C for 15 min, cooled in an ice bath, centrifuged at room temperature (5000 rpm, 10 min), 100 μL of supernatant was added to a 96-well plate, and OD was measured using an enzyme reader. 532 nm. As attached Fig.17 and attached Fig.18 As shown in the figure, the MDA detection kit detected that the MDA level in the IO-LNPs group was significantly higher than that in the PBS group in both S. aureus and E. coli, indicating that lipid peroxidation occurred inside them. This shows that IO-LNPs not only increased the ROS level inside the bacteria, but also triggered lipid peroxidation, which is a typical feature of ferroptosis.

[0103] 5. Experiment on the effect of IO-LNPs on bacterial growth curve

[0104] Add 100 μL of bacterial solution (1×10 5CFU / mL) and 100 μL IO-LNPs (final concentration of 8 mM for incubation with S. aureus and 16 mM for incubation with E. coli), and PBS was used as the control group. The plate was placed in a constant temperature oscillator for 10 hours (250 rpm, 37°C) and the OD 600 Use a microplate reader to monitor each well every 1 hour. Fig.19 and attached Fig. 20 As shown in the figure, the bacterial growth is relatively slow in the first 1-2 hours of incubation, which is the retardation period of bacterial growth. After incubation for 4 hours, it can be found that the bacterial reproduction rate is accelerated, and it has entered the logarithmic phase. During the entire test period, the OD values ​​of S. aureus and E. coli in the control group (PBS) were 600 However, the OD of S. aureus and E. coli treated with IO-LNPs 600 The experimental results showed that IO-LNPs had a strong inhibitory effect on S. aureus and E. coli.

[0105] 6. Live / dead staining experiment of bacteria after IO-LNPs treatment

[0106] Take S. aureus and E. coli and enrich them in EP tubes, then add IO-LNPs (the final concentration of IO-LNPs incubated with S. aureus is 8mM, and the final concentration of IO-LNPs incubated with E. coli is 16mM) and incubate for 2h (250rpm, 37℃), then centrifuge with a refrigerated centrifuge (5000rpm, 4℃, 10min), collect the precipitate and remove the supernatant, add 20μL of Live / Dead reagent and vortex evenly, stain for 20min away from light, aspirate 10μL on a slide, cover with a coverslip, and observe with a laser scanning confocal microscope. As shown in the attached Fig.21 and attached Fig. 22 As shown in the figure, after co-incubation with IO-LNPs, they all showed strong red fluorescence, and the red fluorescence intensity of the MIC90 group was higher than that of the control group, indicating that the bacteria in the MIC90 group significantly lost their vitality. This effect may be related to the ROS generated by the Fenton reaction, because lipid peroxidation usually destroys the integrity of the membrane structure.

[0107] 7. SEM observation of IO-LNPs on bacterial micromorphology

[0108] Scanning electron microscopy (SEM) was used to observe the microscopic morphological changes of S. aureus and E. coli before and after the action of IO-LNPs. Take S.aureus and E.coli in EP tubes, centrifuge and enrich, and incubate with PBS and IO-LNPs (the final concentration of incubation with S.aureus is 8mM, and the final concentration of incubation with E.coli is 16mM) for 30min (250rpm, 37℃), centrifuge to get the precipitate, wash with PBS for several times, centrifuge, discard the supernatant, add 200μL 4% glutaraldehyde solution to the precipitate, keep it in the dark for 2h, centrifuge at 5000rpm, 4℃ for 5min, remove the supernatant, add 1mL 50% ethanol solution, blow evenly, dehydrate for 10min, centrifuge again, then dehydrate with 70%, 90%, and 100% ethanol solution in turn, wash with PBS, take 10μL and drop it on a clean silicon wafer, place it in a 6-well plate to air dry overnight, and observe with SEM. As shown in the attached Fig.23 As shown in Figure 2, for S. aureus, in the control group, typical spherical or elliptical cell structures can be observed. However, in the IO-LNPs-treated group, bacterial cells showed obvious deformation and aggregation. The cell surface became rough and irregular, which may indicate that the cell membrane was damaged or the internal substances of the cell were leaked. Similarly, as shown in Figure 2, Fig.24 As shown in the figure, the morphology of E. coli bacteria in the control group remained normal, presenting a long rod shape. However, after IO-LNPs treatment, the cell shape was severely distorted, the length was shortened, and obvious shrinkage appeared. This change may be due to the toxic effect of IO-LNPs on the cell wall or cell membrane, resulting in loss of cell integrity. These experimental results indicate that IO-LNPs can effectively cause morphological changes in these two representative bacteria, especially damage to the cell membrane and cell wall, by inducing ferroptosis mechanisms.

[0109] Example 7

[0110] Evaluation of biocompatibility of IO-LNPs prepared in Example 1

[0111] By counting, 200 μL of high-glucose DMEM medium containing HUVEC / L929 cells (8000 cells per well) was added to a 96-well plate. Incubate overnight until the cells adhere, discard the upper culture medium, add 200 μL of a series of IO-LNPs solutions of different concentrations, namely 0, 1, 2, 4, 8, 16, and 32 mM, and incubate for 24 hours. After the incubation, add 20 μL of MTT solution (5 mg / mL) and incubate in an incubator for 4 hours. Subsequently, remove the supernatant in the well, add 150 μL DMSO, shake on a shaker for 10 minutes, and use an enzyme reader to measure the absorbance of each well at 490 nm to calculate the cell survival rate. As shown in the attached Fig.25As shown, when the concentration was 0-16 mM, the survival rates of L929 and 3T3 cells were maintained above 80%, indicating that IO-LNPs had good biocompatibility at this concentration.

[0112] The good biocompatibility of IO-LNPs was further verified by hemolysis experiments. 200 μL of blood from ICR male mice was collected by orbital blood sampling, and impurities such as upper cell debris were removed by centrifugation. After repeated washing with PBS for 3 times, 100 μL of red blood cell sediment was resuspended with 500 μL PBS to prepare a 20% red blood cell suspension. A series of IO-LNPs solutions with different concentrations were prepared with PBS, namely 0, 1, 2, 4, 8, and 16 mM. Sterile water was used as the positive control group and PBS was used as the negative control group. 20 μL of red blood cell suspension was added to each group, and the mixture was incubated in the incubator for 2 hours before being taken out and centrifuged (4000 rpm, 10 min). The absorbance of the upper layer of each mixed solution after centrifugation was detected by an enzyme marker at 540 nm and the hemolysis rate was calculated. Photos were taken to record the state of each mixed solution after centrifugation and graphical analysis was performed. As shown in the attached figure Fig.26 As shown, IO-LNPs showed low hemolytic activity in all tested concentration ranges (1 mM to 16 mM). Specifically, the hemolysis rate measured at each concentration was less than 5%. This indicates that IO-LNPs have good biocompatibility with erythrocyte membranes even at relatively high concentrations.

[0113] Example 8

[0114] In vivo antibacterial experiment of IO-LNPs prepared in Example 1

[0115] 1) Establishment and treatment of in vivo mouse wound model

[0116] In order to explore the effect of IO-LNPs on wound healing in mice infected with S. aureus, 9 ICR male mice were randomly divided into 3 groups, 3 mice in each group, as experimental subjects. First, an in vivo wound model was established. The hair on the back of the mouse was cleaned to make the surface bare and smooth. The surface of medical instruments (such as surgical scissors, tweezers, punchers, etc.) was cleaned and disinfected with 75% ethanol. An elliptical wound with a long axis of 2.2 cm and a short axis of 2.0 cm was made on the back of the mouse with a puncher. The excess skin at the edge of the wound was removed with surgical scissors and tweezers, and the blood oozing from the wound was wiped with a cotton swab. 50 μL of 1×10 8CFU / mL of S.aureus bacterial liquid was added to the wound. After the bacterial liquid on the wound was air-dried, the operation was repeated 3 times, and the mouse wound infection model was established for two consecutive days. After the wound model was established, the above three groups were divided into PBS, MIC50 and MIC90 groups for corresponding treatment. The control group was treated by dripping 100μL PBS on the wound; the MIC50 group was treated by dripping 100μL, 2mM IO-LNPs on the wound; and the MIC90 group was treated by dripping 100μL, 8mM IO-LNPs on the wound. The treatment was continued for 5 days, and photos were taken every day to record the changes in the mouse wounds. The wound area was recorded using Image J, and the wound healing rate was calculated using Origin. During the treatment period, the mice were weighed and recorded every day, and the pictures of the changes in the wound area of ​​the mice were sorted and calculated using ImagineJ software, and charts were drawn to analyze the data. As shown in the attached figure Fig. 27 , 28 As shown in Figure 29, the wounds in each group shrank to varying degrees over time, and the healing effect of MIC90 was better than that of MIC50 and the control group. Fig.31 As shown, the body weight of each group did not change significantly during the entire testing period, indicating that IO-LNPs have high biocompatibility.

[0117] 2) Bacterial plating of wound tissue

[0118] In order to explore the in vivo antibacterial effect of IO-LNPs, mice in each group were killed on the 13th day, and the wound skin tissue of each group of mice was removed using scissors and forceps disinfected with alcohol. Then, the removed skin of each group was placed in a labeled shaking tube containing 1 mL PBS and co-incubated (37°C, 250 rpm, 1 h). The co-incubated liquid was evenly spread on a solid plate culture medium and cultured in a biochemical incubator at 37°C for 12 h. Fig.30 As shown in the figure, the number of colonies in the MIC50 group and the MIC90 group was significantly less than that in the control group, and there was almost no colony in the MIC90 group. This result shows that IO-LNPs have a significant antibacterial effect.

[0119] 3) Mouse wound skin tissue staining

[0120] In order to explore the effect of IO-LNPs on wound healing of bacterial infection, mice in each group were killed on the 13th day, and the wound skin tissues of mice in each group were removed using scissors and tweezers disinfected with alcohol. The wound skin tissues were then immersed in the marked 4% paraformaldehyde fixative centrifuge tubes, and then sectioned and stained (H&E staining, Masson staining, CD31 immunohistochemical staining), and the section images were scanned and saved using a scanner. Fig.32As shown in Figure 2, both MIC90 and MIC50 treatments can effectively reduce wound inflammation, promote epithelial regeneration and granulation tissue maturation, and MIC90 has the best effect, which is consistent with the order of antibacterial effect and wound healing ability. Fig.33 As shown in the results, both MIC90 and MIC50 treatments can promote the production and maturation of collagen fibers and improve the structure and strength of healing tissues, and MIC90 treatment has the best effect, which indicates that IO-LNPs have a good ability to promote wound healing.

[0121] Angiogenesis plays an important role in wound healing. Capillaries are responsible for delivering nutrients and oxygen to the wound and removing excess debris. As a typical marker of endothelial cells, CD31 can be used for immunostaining to assess angiogenesis in the injured area. Fig.34 As shown in the figure, in the wound tissue treated with IO-LNPs, the expression of CD31 increased significantly, indicating that angiogenesis was activated. In particular, the MIC90 group had the highest blood vessel density, followed by the MIC50 group, which is consistent with the strength of the antibacterial effect. This may mean that the higher antibacterial efficacy promotes angiogenesis during wound healing. In the untreated control group, the expression of CD31 was low and the blood vessel density was significantly less than that of the treated group, suggesting that angiogenesis was slower under natural healing conditions.

[0122] 4) Mouse organ staining

[0123] In order to explore the in vivo biocompatibility of IO-LNPs, the mice in each group were killed on the 13th day, and the relevant organs (heart, liver, spleen, lung, and kidney) of the mice in each group were removed using scissors and tweezers disinfected with alcohol. The wound skin tissue was immersed in the marked 4% paraformaldehyde fixative centrifuge tubes, and then H&E staining was performed. The slices were scanned and saved using a scanner. Fig.35 As shown in the figure, the tissue structure of the visceral organs in the IO-LNPs-treated group was similar to that in the control group, and no signs of inflammation, necrosis or other pathological changes were observed. This indicates that IO-LNPs did not cause obvious tissue damage or toxic reactions to the main visceral organs at a given dose and had good biocompatibility.

[0124] Comparative Example 1

[0125] In order to explore the effects of IO-LNPs and ferric ammonium citrate (FAC) on S. aureus, the S. aureus bacterial solution in the logarithmic growth phase was diluted to 1×10 8CFU / mL were placed in a shaker (250 rpm, 37°C) and incubated with IO-LNPs and FAC solutions for 1 h, where the final concentrations of IO-LNPs and FAC were both 2 mM, and PBS was used as a negative control group. The volume of the bacterial solution and IO-LNPs solution was 100 μL. The samples after incubation were diluted to 2×10 4 times, take 100 μL and drop it onto the solid agar plate, spread it evenly with a spreader, and place it in a biochemical incubator for overnight culture. The survival rate of S. aureus with 2mM IO-LNPs was 44%, while the survival rate of S. aureus with 2mM FAC was 80%. In order to explore the effects of IO-LNPs and FAC on E. coli, the E. coli culture in the logarithmic growth phase was diluted to 1×10 8 CFU / mL was placed in a shaker (250rpm, 37℃) with IO-LNPs and FAC solutions for 1h, where the final concentration of IO-LNPs was 4mM, and PBS was used as a negative control group. The post-treatment process was consistent with the above. The survival rate of E.coli with 4mM IO-LNPs was 40%, and the survival rate of E.coli with 4mM FAC was 50%. The above experimental results show that the antibacterial effect of IO-LNPs on S.aureus and E.coli is better than that of FAC.

[0126] Comparative Example 2

[0127] Ferric oleate is directly mixed with soybean lecithin and DSPE-MPEG2000 (concrete consumption is with embodiment 1), experimental result shows, direct mixing method can not form stable lipid nanoparticle, and does not have Tyndall phenomenon.Yet, stable lipid nanoparticle can be formed by gradient solvent diffusion method, and there is Tyndall effect.In addition, ferric oleate is a non-polar solvent, uses ethanol to dissolve, therefore selects normal hexane to dissolve.Inject ethanol by normal hexane, then inject water, this is gradient solvent diffusion method, and this method can form stable lipid nanoparticle.

Claims

1. An iron-containing lipid nanoparticle IO-LNPs with antibacterial function, characterized in that: The iron-containing lipid nanoparticles IO-LNPs are synthesized by using iron oleate, soybean lecithin and DSPE-MPEG2000 as raw materials through a gradient solvent diffusion method, wherein the mass ratio of iron oleate to soybean lecithin is 1:0.5-4.

2. A method for preparing the iron-containing lipid nanoparticles IO-LNPs with antibacterial function according to claim 1, characterized in that: The preparation method of the IO-LNPs is as follows: (1) dissolving ferric oleate in n-hexane to obtain solution A; dissolving soybean lecithin in anhydrous ethanol to obtain solution B; dissolving DSPE-MPEG2000 in anhydrous ethanol to obtain solution C; Then, solution A and solution C were injected into solution B and stirred rapidly to obtain iron oleate nanoemulsion; (2) Using a vacuum rotary apparatus to remove n-hexane from the iron oleate nanoemulsion, the iron oleate nanoemulsion from which n-hexane has been removed is injected into the aqueous phase, rapidly stirred, dialyzed with ultrapure water, and filtered through a 0.22 μm water-based microporous filter membrane to obtain IO-LNPs with good dispersibility.

3. The method for preparing the iron-containing lipid nanoparticles IO-LNPs with antibacterial function according to claim 2, characterized in that: The amount of DSPE-MPEG2000 added in step (1) is 20% of the total mass of ferric oleate and soybean lecithin.

4. The method for preparing the iron-containing lipid nanoparticles IO-LNPs with antibacterial function according to claim 2, characterized in that: The concentration of solution A in step (1) is 10 mg / mL; the concentration of solution B is 0.3-2.7 mg / mL; and the concentration of solution C is 3-10 mg / mL.

5. The method for preparing the iron-containing lipid nanoparticles IO-LNPs with antibacterial function according to claim 2, characterized in that, The stirring in step (1) is performed at 1500 rpm for 3 min at room temperature.

6. The method for preparing the iron-containing lipid nanoparticles IO-LNPs with antibacterial function according to claim 2, characterized in that: In step (2), the rotation speed of the vacuum rotary evaporator is 150 rpm, the rotary evaporation is 6 min, and the temperature is 37° C.

7. The method for preparing the iron-containing lipid nanoparticles IO-LNPs with antibacterial function according to claim 2, characterized in that: In step (2), the volume ratio of the iron oleate nanoemulsion after removing the n-hexane to the water phase is 1:10; the stirring rate of the injected water phase is 1500 rpm, and the stirring time is 3 minutes.

8. A use of the iron-containing lipid nanoparticles IO-LNPs with antibacterial function according to claim 1, characterized in that: The iron-containing lipid nanoparticles with antibacterial function are used for preparing antibacterial agents.

9. The use of the iron-containing lipid nanoparticles IO-LNPs with antibacterial function according to claim 8, characterized in that: The antibacterial agent is an antibacterial agent used for in vitro sterilization of S. aureus and E. coli, elimination and destruction of biofilm, and effective inhibition of proliferation of S. aureus and promotion of wound healing in a skin infection model.

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