Preparation method and application of iron-containing lipid nanoparticles IO-LNPs with antibacterial function
IO-LNPs prepared by gradient solvent diffusion method solve the selectivity, drug resistance and safety issues of existing antibacterial treatment methods, achieve efficient bacterial killing and wound healing, and have broad application prospects.
Patent Information
- Application Number
- CN202510001889.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-02
- Publication Date
- 2025-10-10
- Estimated Expiration
- 2045-01-02
AI Technical Summary
Existing antibacterial treatments have problems with selectivity, drug resistance development, in vivo safety and biodistribution when facing complex infections, and have poor cost-effectiveness.
Antibacterial iron-containing lipid nanoparticles (IO-LNPs) were prepared using a gradient solvent diffusion method. Iron oleate was dissolved in a non-polar solvent and then injected into a polar solvent. The non-polar solvent was removed using a vacuum rotary evaporator to form stable LNPs, enhancing drug delivery efficiency and therapeutic efficacy.
The prepared IO-LNPs can induce bacterial ferroptosis, have good antibacterial activity, and are widely used in the fields of antibacterial and bactericidal effects. They are also simple to operate, highly stable, and have good biocompatibility, making them suitable for widespread use.
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Figure CN119868304B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of nanotechnology, and in particular 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 novel antimicrobial therapies has become a critical task for researchers worldwide. The widespread use of traditional antibiotics has led to a rapid increase in bacterial resistance, making the search for alternative therapies particularly urgent. Against this backdrop, nanotechnology, as an emerging approach, has gained increasing attention for its application in antimicrobial therapy. Lipid nanoparticles (LNPs) have become a research hotspot for drug delivery systems due to their excellent biocompatibility, ease of modification, and controllable release properties.
[0003] However, in the face of complex and changing infection situations, how to improve the effectiveness of antibacterial 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 iron-mediated lipid peroxidation reactions 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 is a key factor in the ferroptosis process. By using compounds or structures that can carry iron ions into bacterial cells, ferroptosis can be effectively promoted. For example, some iron chelators can bind to iron ions and be absorbed by bacteria, thereby inducing ferroptosis.
[0005] Inhibition of the antioxidant system: A key feature of ferroptosis is its reliance on lipid peroxidation, a process that can be enhanced by inhibiting the bacteria's own antioxidant defense system. For example, GPX4 (glutathione peroxidase 4) is a key antioxidant enzyme responsible for clearing intracellular peroxides. Inhibiting GPX4 activity can increase lipid peroxidation levels, thereby promoting ferroptosis.
[0006] Applications of nanomaterials: These materials can not only serve as effective iron ion carriers, but can also further promote the ferroptosis process through their unique physical and chemical properties, such as photothermal and magnetic effects. In addition, these nanoparticles often have good biocompatibility and controllable drug release properties, enabling 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 an urgent 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 form 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: 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 address the deficiencies in 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 into LNPs, enhances the design flexibility of antibacterial treatment plans, and improves the drug delivery efficiency and therapeutic effect, opening up a new path for antibacterial treatment and has important theoretical significance and application prospects.
[0013] The present invention adopts the 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 steps:
[0015] (1) Dissolve ferric oleate in n-hexane to obtain a solution A with a concentration of 10 mg / ml; dissolve soybean lecithin in anhydrous ethanol to obtain a solution B with a concentration of 0.3-2.7 mg / ml; dissolve DSPE-MPEG2000 in anhydrous ethanol to obtain a solution C with a concentration of 3-10 mg / ml; then, inject solution A and solution C into solution B, and rapidly stir at 1500 rpm for 3 minutes at room temperature to obtain an ferric oleate nanoemulsion;
[0016] The mass ratio of ferric oleate to soybean lecithin is 1:0.5-4; and 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 aqueous microporous filter membrane to obtain IO-LNPs with good dispersion.
[0018] The vacuum rotary evaporator was used at a speed of 150 rpm, for 6 min, at a temperature of 37°C.
[0019] The volume ratio of the iron oleate nanoemulsion after removing the n-hexane to the aqueous phase was 1:10; the stirring rate of the injected aqueous phase was 1500 rpm, and the stirring time was 3 min.
[0020] The antibacterial iron-containing lipid nanoparticles IO-LNPs prepared by the above method can induce bacteria to produce ferroptosis effect, and are 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 in this invention overcome the technical difficulty of preparing non-polar drugs into stable LNPs using traditional solvent diffusion methods. The prepared IO-LNPs exhibit excellent 7-day stability and are not susceptible to aggregation or degradation, making them suitable for long-term storage and transportation.
[0023] (2) The present invention has a simple process for preparing IO-LNPs, high reproducibility, a wide range of raw material sources, mild reaction conditions, easy synthesis, excellent biocompatibility, and suitability for widespread 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. The generated reactive oxygen species (ROS) and lipid peroxidation (LPO) can damage the microbial cell membrane, thereby effectively killing Gram-positive (S. aureus) and Gram-negative (E. coli) strains. They have excellent in vivo antibacterial properties. The materials used 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 (turbidimetric 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 treatment with different concentrations of IO-LNPs.
[0032] Figure 8 . Quantitative statistical results of the colonies of S. aureus prepared in Example 1 after being treated with IO-LNPs at different concentrations.
[0033] Figure 9 . Colony formation diagram of E. coli prepared in Example 1 after being treated with different concentrations of IO-LNPs.
[0034] Figure 10 . Quantitative statistical results of the E. coli colonies treated with different concentrations of IO-LNPs prepared in Example 1.
[0035] Figure 11 . Graph showing the biofilm inhibition effect of S. aureus after treatment with IO-LNPs prepared in Example 1.
[0036] Figure 12 . Graph showing the biofilm inhibition effect of E. coli after treatment with IO-LNPs prepared in Example 1.
[0037] Figure 13 . Graph showing the biofilm destruction effect of S. aureus after treatment with IO-LNPs prepared in Example 1.
[0038] Figure 14 . Graph showing the biofilm destruction effect of E. coli after treatment with IO-LNPs prepared in Example 1.
[0039] Figure 15 .The internal ROS level of S. aureus treated with IO-LNPs prepared in Example 1.
[0040] Figure 16 .The ROS level inside E. coli treated with IO-LNPs prepared in Example 1.
[0041] Figure 17 . MDA levels in S. aureus treated with IO-LNPs prepared in Example 1.
[0042] Figure 18 . MDA levels in E. coli treated with IO-LNPs prepared in Example 1.
[0043] Figure 19 .The growth curve of S. aureus after being treated with IO-LNPs prepared in Example 1 changes with time.
[0044] Figure 20 .The growth curve of E. coli after being treated with IO-LNPs prepared in Example 1 changes with time.
[0045] Figure 21 Live / dead staining of S. aureus after treatment with IO-LNPs prepared in Example 1.
[0046] Figure 22 . Live / dead staining of E. coli after treatment with IO-LNPs prepared in Example 1.
[0047] Figure 23 .SEM micromorphology of IO-LNPs prepared in Example 1 after treating S. aureus bacteria.
[0048] Figure 24 .SEM microscopic morphology of IO-LNPs prepared in Example 1 after treating E. coli.
[0049] Figure 25 .Cytotoxicity evaluation of IO-LNPs prepared in Example 1.
[0050] Figure 26 .Hemolysis results of IO-LNPs prepared in Example 1.
[0051] Figure 27 .Graphs showing the healing status of bacterial infection wounds in each group of mice prepared in Example 1.
[0052] Figure 28 .Images of wound healing traces of bacterial infection in each group of mice.
[0053] Figure 29 .Chart showing changes in the area rate of bacterially infected wounds in each group of mice.
[0054] Figure 30.The results of plating the wound skin tissue of each group of mice.
[0055] Figure 31 .The weight changes of mice in each group during the entire experimental period.
[0056] Figure 32 .H&E staining of wound tissues in mice infected with Staphylococcus aureus.
[0057] Figure 33 .Masson staining of wound tissue in mice infected with Staphylococcus aureus.
[0058] Figure 34 . Immunohistochemistry CD31 image of mouse wound healing.
[0059] Figure 35 H&E staining of organs from mice infected with S. aureus in each treatment group on day 13 of treatment. DETAILED DESCRIPTION
[0060] The present invention will be further described in detail below in conjunction with specific examples, but this 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 inventive method, steps or conditions are intended to fall within the scope of the present invention. Experimental methods and reagents for which specific conditions are not specified in the examples are based on conventional conditions in the art.
[0061] Example 1
[0062] Step 1: dissolve 10 mg of ferric oleate in 1 mL of n-hexane to obtain solution A; dissolve 5 mg of soybean lecithin in 15 mL of anhydrous ethanol to obtain solution B; dissolve 3 mg of DSPE-MPEG2000 in 1 mL of anhydrous ethanol to obtain solution C;
[0063] Step 2: inject solution A and solution C into solution B, and rapidly stir at 1500 rpm for 3 minutes at room temperature to obtain an iron oleate nanoemulsion.
[0064] Step 3: Use a vacuum rotator at a speed of 150 rpm for 6 minutes at 37°C to remove n-hexane from the iron oleate nanoemulsion. The obtained iron oleate nanoemulsion is injected into 10 mL of the aqueous phase, rapidly stirred at 1500 rpm for 3 minutes at room temperature, dialyzed with ultrapure water, and filtered through a 0.22 μm aqueous microporous filter membrane to obtain well-dispersed IO-LNPs.
[0065] The hydrated particle size and PDI of IO-LNPs were measured using a 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, 10 mg of iron oleate was dissolved in 1 mL of n-hexane to obtain solution A; 20 mg of soybean lecithin was dissolved in 15 mL of absolute ethanol to obtain solution B; 6 mg of DSPE-MPEG2000 was dissolved in 1 mL of absolute ethanol to obtain solution C;
[0068] Step 2, solution A and solution C were injected into solution B, and fast stirring at 1500 rpm was carried out at room temperature for 3 min to obtain iron oleate nanoemulsion.
[0069] Step 3, n-hexane in the iron oleate nanoemulsion was removed using a rotary evaporator at 150 rpm for 6 min at 37°C, and the obtained iron oleate nanoemulsion was injected into the aqueous phase, and fast stirring at 1500 rpm was carried out at room temperature for 3 min, and then dialysis was carried out using ultrapure water, and IO-LNPs with good dispersity were obtained by passing through a 0.22 μm water-based microporous filter membrane.
[0070] The hydrated particle size and PDI of the IO-LNPs were detected using a 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.
[0071] Example 3
[0072] Step 1, 10 mg of iron oleate was dissolved in 1 mL of n-hexane to obtain solution A; 20 mg of soybean lecithin was dissolved in 15 mL of absolute ethanol to obtain solution B; 6 mg of DSPE-MPEG2000 was dissolved in 1 mL of absolute ethanol to obtain solution C;
[0073] Step 2, solution A and solution C were injected into solution B, and fast stirring at 1500 rpm was carried out at room temperature for 3 min to obtain iron oleate nanoemulsion.
[0074] Step 3, n-hexane in the iron oleate nanoemulsion was removed using a rotary evaporator at 150 rpm for 6 min at 37°C, and the obtained iron oleate nanoemulsion was injected into the aqueous phase, and fast stirring at 1500 rpm was carried out at room temperature for 3 min, and then dialysis was carried out using ultrapure water, and IO-LNPs with good dispersity were obtained by passing through a 0.22 μm water-based microporous filter membrane.
[0075] The hydrated particle size and PDI of the IO-LNPs were detected using a 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: dissolve 10 mg of ferric oleate in 1 mL of n-hexane to obtain solution A; dissolve 40 mg of soybean lecithin in 15 mL of anhydrous ethanol to obtain solution B; dissolve 10 mg of DSPE-MPEG2000 in 1 mL of anhydrous ethanol to obtain solution C;
[0078] Step 2: inject solution A and solution C into solution B, and rapidly stir at 1500 rpm for 3 minutes at room temperature to obtain an iron oleate nanoemulsion.
[0079] Step 3: Use a vacuum rotator at a speed of 150 rpm for 6 minutes at 37°C to remove n-hexane from the iron oleate nanoemulsion, inject the obtained iron oleate nanoemulsion into the aqueous phase, rapidly stir at room temperature at 1500 rpm for 3 minutes, dialyze with ultrapure water, and filter through a 0.22 μm water-based microporous filter membrane to obtain IO-LNPs with good dispersion.
[0080] The hydrated particle size and PDI of IO-LNPs were measured using a 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 Measurement 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 cell and the potential size of the IO-LNPs was measured using a Malvern particle size analyzer. The particle size of the IO-LNPs was monitored for 7 consecutive days. In addition, the IO-LNPs were diluted with different dilution factors and the hydrated particle size at different dilution factors was measured using a Malvern particle size analyzer. Figure 1 As shown in the attached figure, the Zeta potential of IO-LNPs is -12.3±1.73 mV. Figure 2 As shown in Figure 2, after IO-LNPs were placed in ultrapure water for 7 days, the hydrated particle size of 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, the particle size of IO-LNPs did not change significantly at different dilution factors, indicating 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. 20 μL of IO-LNPs were added dropwise to the carbon support film copper grid for negative staining. The sample was allowed to stand for 1 minute and then placed at room temperature overnight. After the copper grid was completely dry, the sample was observed using a transmission electron microscope. Figure 4 As shown in the figure, the IO-LNPs are uniformly spherical and well dispersed. The actual size of the IO-LNPs observed under TEM is approximately 40 nm. The above characterization results prove that the IO-LNPs have been successfully prepared.
[0085] Example 6
[0086] In vitro antibacterial activity detection of IO-LNPs prepared in Example 1
[0087] 1. In vitro antibacterial experiments of IO-LNPs
[0088] 1) In vitro antibacterial assay of IO-LNPs (culture medium turbidity method)
[0089] To investigate the effect of IO-LNPs on bacterial growth, overnight cultures of 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 measured using a microplate reader. 600 The bacterial survival rate was calculated based on the absorbance value. S. aureus continuously divides and proliferates in TSB liquid culture medium. After a period of culture, the TSB culture medium changes 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 600 nm to reflect the turbidity of the culture medium and indicate the growth of S. aureus. Figure 5 As shown, S. aureus medium 600 nm The absorbance value at 40 nm 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 at 600 nm of E. coli culture medium 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 test (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 The CFU / mL was incubated with IO-LNPs solution on a shaker (250 rpm, 37°C) for 1 h. The final concentrations of IO-LNPs were 1, 2, 4, and 8 mM. 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. Spread it evenly with a spreader 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 2 mM IO-LNPs was 44%, while that of S. aureus exposed to 8 mM IO-LNPs was only 12%. Therefore, the MIC50 of IO-LNPs against S. aureus is 2 mM, and the MIC90 is 8 mM.
[0093] To investigate the effect of IO-LNPs 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 (250 rpm, 37 ° C) and incubated for 1 h with IO-LNPs solution, where the final concentration of IO-LNPs was 2, 4, 8, and 16 mM. PBS was used as a negative control group. The post-treatment process was the same as above. Figure 9 and attached Figure 10 As shown in the figure, the number of colonies in the IO-LNPs group was significantly reduced compared to the control group. The survival rate of E. coli exposed to 4 mM IO-LNPs was 40%, while the survival rate of E. coli exposed to 16 mM IO-LNPs was 7%. Therefore, the MIC50 of IO-LNPs against E. coli is 4 mM and the MIC90 is 16 mM.
[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 to stain the biofilm. The crystal violet in the biofilm was then dissolved in 200 μL of 80% ethanol in a constant temperature shaker for 2 hours (250 rpm, 37°C), and the absorbance at 600 nm was measured using a microplate reader. Figure 11 As shown in Figure 2, only 25% of biofilm was formed when S. aureus was co-incubated with 8 mM IO-LNPs. Figure 12 As shown in Figure 3, only 32% of the 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, 96-well plates were first loaded with 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. The biofilm was then stained with 100 μL of crystal violet instead of the culture medium. 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 figure Figure 13 As shown in Figure 2, after incubation with 8 mM IO-LNPs for 1 hour, only 21% of the biofilm formed by S. aureus was retained. Figure 14 As shown, after 1 hour of incubation with 16 mM IO-LNPs, only 28% of the biofilm formed by E. coli remained. Therefore, IO-LNPs not only disrupt biofilm formation from the inside but also effectively dismantle established biofilms from the outside, thereby clearing the already formed biofilm. These results demonstrate that IO-LNPs have a potent biofilm-disrupting ability against both strains and suggest that IO-LNPs have broad-spectrum antimicrobial potential as a versatile material for wound management.
[0099] 3. Detection of ROS in bacteria by IO-LNPs
[0100] 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. Bacteria were then incubated with the positive control (ROS), negative control (PBS), and IO-LNPs group for 20 min. 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. Figure 15 and attached Figure 16 As shown in the results, after IO-LNPs treatment, ROS levels in S. aureus and E. coli increased significantly. This result suggests that IO-LNPs may trigger oxidative stress by increasing ROS levels in 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. 8 100 μL of bacterial solution was added to 900 μL of 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 experimental groups. The supernatant was removed by centrifugation. After fully removing the liquid, the bacteria were dispersed as much as possible. 100 μL-200 μL of lysis buffer was added to mix them. Lysis was carried out on ice for 10 minutes. 200 μL of MDA working solution was added to each well. The wells were heated at 100°C for 15 minutes, cooled in an ice bath, and centrifuged at room temperature (5000 rpm, 10 minutes). 100 μL of supernatant was added to a 96-well plate and the OD was measured using a microplate reader. 532 nm. As attached Figure 17 and attached Figure 18 As shown in the results, MDA detection kits were used to detect MDA levels in both S. aureus and E. coli, significantly higher in the IO-LNPs group than in the PBS group, indicating that lipid peroxidation occurred within the bacteria. This suggests that IO-LNPs not only increased ROS levels within the bacteria but also triggered lipid peroxidation, a typical characteristic 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 (8 mM final concentration for S. aureus and 16 mM final concentration for E. coli) and control group with PBS. The plates were placed in a constant temperature shaker for 10 hours (250 rpm, 37 °C) and the OD 600 The OD of each well was monitored every 1 hour using a microplate reader. As shown in FIGS. 1A and 1B, the OD of S. aureus and E. coli in the control group (PBS) continued to increase during the entire test period. However, the OD of S. aureus and E. coli treated with IO-LNPs did not increase. Figure 19 Figure 20 As shown in FIGS. 1A and 1B, the growth of bacteria was relatively slow during the first 1-2 hours of incubation, which was the lag phase of bacterial growth. After 4 hours of continuous incubation, the reproduction rate of bacteria increased, which entered the logarithmic phase. During the entire test period, the OD of S. aureus and E. coli in the control group (PBS) continued to increase. However, the OD of S. aureus and E. coli treated with IO-LNPs did not increase. The experimental results showed that IO-LNPs had a strong inhibitory effect on S. aureus and E. coli. 600 600
[0105] 6. Live / dead staining experiment of bacteria after IO-LNPs treatment
[0106] S. aureus and E. coli were enriched in EP tubes, and IO-LNPs (8 mM final concentration for S. aureus and 16 mM final concentration for E. coli) were added for co-incubation for 2 hours (250 rpm, 37 °C). Then, the precipitate was collected by centrifugation (5000 rpm, 4 °C, 10 min) and the supernatant was removed. 20 μL of Live / Dead reagent was added and vortexed uniformly. The staining was performed in the dark for 20 min, and 10 μL was taken onto a glass slide and covered with a cover glass. The laser scanning confocal microscope was used for observation. As shown in FIGS. 2A and 2B, after co-incubation with IO-LNPs, both S. aureus and E. coli 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 lost activity significantly. This effect may be related to the ROS generated by the Fenton reaction, as lipid peroxidation often destroys the integrity of the membrane structure. Figure 21 Figure 22
[0107] 7. SEM observation of the micro-morphology of bacteria treated with IO-LNPs
[0108] Scanning electron microscopy (SEM) was used to observe the micromorphological 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, add PBS to wash 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 solutions in sequence, 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 Figure 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, Figure 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 there was obvious shrinkage. 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, possibly by inducing ferroptosis.
[0109] Example 7
[0110] Biocompatibility evaluation 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 adhered, discard the upper culture medium, add 200 μL of a series of IO-LNPs solutions with 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 of 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 figure Figure 25As shown, the survival rates of L929 and 3T3 cells remained above 80% at concentrations of 0-16 mM, indicating that IO-LNPs have good biocompatibility at this concentration.
[0112] The IO-LNPs were further verified to have good biocompatibility through a hemolysis experiment. 200 μL of blood was taken from ICR male mice by the method of orbital blood sampling, and the upper cell debris and other impurities were removed by centrifugation. After being washed with PBS for 3 times, 100 μL of red blood cell precipitate was resuspended with 500 μL of PBS to prepare a 20% red blood cell suspension. A series of IO-LNPs solutions with different concentrations were prepared with PBS, i.e. 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 an incubator for 2 h. After that, the mixture was centrifuged (4000 rpm, 10 min). The absorbance of the supernatant of each group at 540 nm was detected by a microplate reader, and the hemolysis rate was calculated. The state of each group after centrifugation was recorded by taking a photo, and the results were analyzed by drawing a graph. As shown in FIG. 6, IO-LNPs showed low hemolytic activity at all tested concentrations (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 red blood cell membranes even at relatively high concentrations. Figure 26 As shown, the survival rates of L929 and 3T3 cells remained above 80% at concentrations of 0-16 mM, indicating that IO-LNPs have good biocompatibility at this concentration.
[0113] Example 8
[0114] In vivo antibacterial experiment of IO-LNPs prepared in Example 1
[0115] 1) Establishment and treatment of mouse wound in vivo model
[0116] In order to explore the effect of IO-LNPs on promoting wound healing of mice infected with S. aureus, 9 ICR male mice were selected and randomly divided into 3 groups, each group of 3 mice as experimental objects. First, an in vivo wound model was established. The hair on the back of the mouse was cleaned to make the surface smooth, the medical instruments (such as surgical scissors, forceps, puncher, etc.) were surface cleaned and disinfected with 75% ethanol, an oval wound with a long axis of 2.2 cm and a short axis of 2.0 cm was punched on the back of the mouse with a puncher, the excess skin around the wound was removed with surgical scissors and forceps, and the blood exuding from the wound was wiped with a cotton swab. 50 μL of 1 x 10 8CFU / mL of S. aureus bacterial liquid was added. 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 mouse wound area were sorted and calculated using ImagineJ software, and the data were drawn into charts for analysis. As shown in the attached figure Figure 27 、 28 As shown in Figures 29 and 30, 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. Figure 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) Wound tissue bacterial plating
[0118] In order to investigate 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. The removed skin of each group was then placed in a labeled shaking tube containing 1 mL of PBS and co-incubated (37°C, 250 rpm, 1 h). The co-incubated liquid was evenly spread on a solid plate culture medium and incubated in a biochemical incubator at 37°C for 12 h. Figure 30 As shown in the figure, the number of colonies in the MIC50 and MIC90 groups 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 investigate 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 respective labeled 4% paraformaldehyde fixative centrifuge tubes, and then the sections were stained (H&E staining, Masson staining, CD31 immunohistochemical staining), and the sections were scanned and saved using a scanner. As shown in the attached figure. Figure 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. Figure 33 As shown in the results, both MIC90 and MIC50 treatments could promote the production and maturation of collagen fibers and improve the structure and strength of healing tissue, and MIC90 treatment had the best effect, indicating 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. Figure 34 As shown, CD31 expression increased significantly in wound tissue treated with IO-LNPs, indicating activated angiogenesis. In particular, the MIC90 group had the highest blood vessel density, followed by the MIC50 group, consistent with the strength of the antimicrobial effect. This may mean that the higher antimicrobial efficacy promotes angiogenesis during wound healing. In the untreated control group, CD31 expression was low, and blood vessel density was significantly less than that in the treated group, suggesting that angiogenesis is slower under natural healing conditions.
[0122] 4) Mouse organ staining
[0123] In order to investigate the in vivo biocompatibility of IO-LNPs, mice in each group were sacrificed on the 13th day, and the relevant organs (heart, liver, spleen, lung, and kidney) of each group of mice were removed using scissors and tweezers disinfected with alcohol. The wound skin tissue was immersed in the respective labeled 4% paraformaldehyde fixative centrifuge tubes, and then H&E staining was performed. The slices were scanned and saved. Figure 35 As shown in the results, the tissue structure of the internal organs in the IO-LNPs-treated group was similar to that in the control group, with no signs of inflammation, necrosis, or other pathological changes observed. This indicates that IO-LNPs did not cause significant tissue damage or toxic reactions to major internal organs at a given dose and had good biocompatibility.
[0124] Comparative Example 1
[0125] In order to investigate 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 incubated with IO-LNPs and FAC solutions on a shaker (250 rpm, 37°C) for 1 h. The final concentrations of IO-LNPs and FAC were both 2 mM. 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 4 100 μL was added to the solid agar plate, spread evenly with a spreader, and placed in a biochemical incubator for overnight culture. The survival rate of S. aureus with 2 mM IO-LNPs was 44%, while the survival rate of S. aureus with 2 mM FAC was 80%. Similarly, 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 The CFU / mL was incubated with IO-LNPs and FAC solutions on a shaker (250 rpm, 37°C) for 1 hour. The final IO-LNP concentration was 4 mM, and PBS served as a negative control. The post-treatment process was identical to that described above. The E. coli survival rate was 40% with 4 mM IO-LNPs, while the survival rate was 50% with 4 mM FAC. These experimental results demonstrate that IO-LNPs are more effective than FAC against both S. aureus and E. coli.
[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 nano particle, and does not have Tyndall phenomenon.Yet, can form stable lipid nano particle, and have Tyndall effect by gradient solvent diffusion method.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 nano particle.
Claims
1. An iron-containing lipid nanoparticle IO-LNPs with antibacterial function, characterized in that: The iron-containing lipid nanoparticles IO-LNPs are synthesized 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; The preparation method of the IO-LNPs is as follows: (1) Dissolve ferric oleate in n-hexane to obtain solution A; dissolve soybean lecithin in anhydrous ethanol to obtain solution B; dissolve DSPE-MPEG2000 in anhydrous ethanol to obtain solution C; then, inject solution A and solution C into solution B and rapidly stir to obtain ferric oleate nanoemulsion; (2) The n-hexane in the iron oleate nanoemulsion was removed using a vacuum rotary evaporator, and the iron oleate nanoemulsion after the n-hexane was removed was injected into the aqueous phase, rapidly stirred, dialyzed with ultrapure water, and filtered through a 0.22 μm aqueous microporous filter membrane to obtain IO-LNPs with good dispersion.
2. A method for preparing iron-containing lipid nanoparticles 10-LNPs with antibacterial function according to claim 1, characterized in that: The preparation method of the IO-LNPs is as follows: (1) Dissolve ferric oleate in n-hexane to obtain solution A; dissolve soybean lecithin in anhydrous ethanol to obtain solution B; dissolve DSPE-MPEG2000 in anhydrous ethanol to obtain solution C; Subsequently, solution A and solution C were injected into solution B and rapidly stirred to obtain an iron oleate nanoemulsion; (2) The n-hexane in the iron oleate nanoemulsion was removed using a vacuum rotary evaporator, and the iron oleate nanoemulsion after the n-hexane was removed was injected into the aqueous phase, rapidly stirred, dialyzed with ultrapure water, and filtered through a 0.22 μm aqueous microporous filter membrane to obtain IO-LNPs with good dispersion.
3. The method for preparing iron-containing lipid nanoparticles 10-LNPs with antibacterial function according to claim 2, wherein The amount of DSPE-MPEG2000 added in step (1) is 20% of the total mass of iron oleate and soybean lecithin.
4. The method for preparing iron-containing lipid nanoparticles 10-LNPs with antibacterial function according to claim 2, wherein 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 iron-containing lipid nanoparticles 10-LNPs with antibacterial function according to claim 2, wherein The stirring in step (1) is carried out at 1500 rpm for 3 minutes at room temperature.
6. The method for preparing iron-containing lipid nanoparticles 10-LNPs with antibacterial function according to claim 2, wherein In step (2), the rotation speed of the vacuum rotary evaporator is 150 rpm, the rotary evaporation is performed for 6 minutes, and the temperature is 37°C.
7. The method for preparing iron-containing lipid nanoparticles 10-LNPs with antibacterial function according to claim 2, wherein The volume ratio of the iron oleate nanoemulsion after removing n-hexane in step (2) to the aqueous phase is 1:10; the stirring rate of the injected aqueous 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 to prepare antibacterial agents, and the bacteria are S. aureus and E. coli .
9. The use of the iron-containing lipid nanoparticles 10-LNPs with antibacterial function according to claim 8, characterized in that: The antibacterial agent is used for S. aureus and E. coli In vitro bactericidal, elimination and disruption of biofilms, and effective inhibition in skin infection models S. aureus Antibacterial agents that promote proliferation and wound healing.