Liposome comprising long chain fatty acids and antibacterial composition comprising same

Abstract

The present invention relates to a liposome comprising long-chain fatty acids and an antibacterial composition comprising same, and more specifically, to: a liposome comprising unsaturated long-chain fatty acids and additionally comprising phospholipids and cholesterol, and exhibiting an excellent antibacterial effect against bacteria such as MRSA; and a pharmaceutical composition.

Description

Liposomes containing long-chain fatty acids and antimicrobial compositions containing the same

[0001] The present invention relates to a liposome comprising long-chain fatty acids, cholesterol, and phospholipids, and an antimicrobial composition comprising the same, which exhibits an excellent antimicrobial effect against bacteria such as MRSA, and a pharmaceutical composition.

[0002] Bacterial infections have long posed a significant challenge to public health, exacerbated by the emergence and rapid spread of resistant strains. This increasing threat highlights the urgent need for innovative strategies to effectively combat bacterial infections. Among new approaches, fatty acids (FAs) are receiving considerable attention due to their broad efficacy against bacterial infections. Extensive research has demonstrated that FAs interact with bacterial membranes and possess antimicrobial properties. In particular, long-chain fatty acids (LCFAs) have garnered widespread attention due to their significant antimicrobial activity against Gram-positive bacterial strains. This interest has been driven by their intrinsic antimicrobial properties, broad solubility, and cost-effectiveness. However, the practical application of FAs is hampered by challenges such as limited solubility, micelle destabilization, and sensitivity to oxidation. To address these limitations, LCFAs must be encapsulated with supporting materials to enhance stability and functionality.

[0003] Various strategies for antimicrobial applications using FAs have been devised using emulsions, liposomes, and hydrogels. These strategies offer advantages such as high loading efficiency and controlled release of loaded FAs. Among these carrier systems, liposomes offer distinct advantages in facilitating the delivery of amphiphilic antimicrobial agents. This is achieved particularly through membrane fusion processes, which significantly enhance the efficacy of FA delivery. Furthermore, liposomes enable the integration of FAs into membrane structures, thereby altering their properties.

[0004] Membrane fluidity is a critical factor in bacterial metabolic processes, including the function of membrane-associated proteins, lipid bilayer permeability, and intracellular vesicle transport mechanisms. Membrane fluidity refers to the viscosity of the lipid bilayer within biological membranes, which influences the lateral mobility of components such as lipids and proteins. Insufficient membrane fluidity can disrupt essential cellular processes, such as cytokinesis. Another key concept, curvature stress, relates to mechanical tension within the membrane resulting from deviations from natural curvature. This stress affects the structural organization of the membrane and influences interactions with external substances, including fatty acids (FAs) and liposomes. Changes in membrane physicochemical properties can impair the structural integrity necessary to maintain biological activity. Previous studies have investigated the effects of liposomal fatty acids (LipoFAs) on bacteria. For example, liposomal oleic acid exhibits rapid fusion with the membrane of Staphylococcus aureus MW2, thereby inhibiting its growth. Similarly, liposomal linolenic acid displays fusion behavior even when Helicobacter pylori is in a dormant state. Furthermore, studies suggest that FAs can inhibit biofilm growth by regulating biofilm fluidity. However, the precise biophysical mechanism of biofilm disruption by FA-loaded liposomes remains unclear due to the complex nature of biological membranes. Despite these promising findings, there is a significant gap in our understanding of how FAs interact with biofilms at the molecular level when delivered via liposomes. Bridging this gap is crucial for optimizing therapeutic potential for bacterial infections.

[0005]

[0006] Against this backdrop, the inventors conducted extensive research to discover new substances with antibacterial efficacy. As a result, they discovered that liposomes utilizing long-chain fatty acids—more specifically, liposomal linolenic acid (LipoLNA), liposomal linoleic acid (LipoLLA), and liposomal oleic acid (LipoOA)—are excellent in combating bacterial infections and can be applied as antibacterial compositions, thereby completing the present invention.

[0007] Accordingly, the objective of the present invention is to provide a liposome containing a long-chain fatty acid that exhibits excellent antibacterial activity, and an antibacterial composition containing the same.

[0008] Another objective of the present invention is to provide a composition for reducing antibiotic resistance comprising liposomes containing long-chain fatty acids.

[0009] Another objective of the present invention is to provide a composition for preventing, improving, or treating bacterial infections comprising liposomes containing long-chain fatty acids.

[0010] Another objective of the present invention is to provide an antibacterial or antibiotic resistance-reducing use of liposomes containing long-chain fatty acids.

[0011]

[0012] The present disclosure is summarized as follows.

[0013]

[0014] 1. An antimicrobial composition containing liposomes comprising unsaturated long-chain fatty acids.

[0015] 2. The antibacterial composition according to claim 1, further comprising phospholipids and cholesterol, and comprising phospholipids : cholesterol : long-chain fatty acids in a weight ratio of 3 to 7 : 0.5 to 3 : 2 to 6.

[0016] 3. An antimicrobial liposome composition, wherein, in any one of the preceding paragraphs, the long-chain fatty acid is one or more selected from the group consisting of linolenic acid, linoleic acid, and oleic acid.

[0017] 4. In any one of the preceding claims, the phospholipid is egg phosphatidylcholine (EPC), phosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethane One or more selected from the group consisting of olamines (distearoylphosphatidylethanolamine, DSPE), phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), etc. Antibacterial composition.

[0018] 5. An antibacterial composition having a zeta potential of -200 to -20 mV or less, in any one of the preceding claims.

[0019] 6. An antibacterial composition having an average particle size of liposomes of 30-300 nm in any one of the preceding claims.

[0020] 7. An antibacterial composition having a polydispersity index (PDI) of 0.3 or less, in any one of the preceding claims.

[0021] 8. An antimicrobial composition that exhibits antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) in any one of the preceding claims.

[0022] 9. A composition for preventing, improving, or treating bacterial infections containing liposomes comprising unsaturated long-chain fatty acids.

[0023] 10. A composition for reducing antibiotic resistance containing liposomes containing unsaturated long-chain fatty acids.

[0024] The liposomes of the present invention exhibit excellent antibacterial activity, particularly against bacteria such as MRSA (methicillin-resistant Staphylococcus aureus), due to containing specific long-chain fatty acids, and can be usefully used as pharmaceutical and food compositions for the prevention, improvement, and treatment of bacterial infections such as MRSA.

[0025]

[0026] Figure 1 is a schematic diagram of the experimental approach to the development of LipoFAs for antimicrobial applications. Liposomes were prepared via sonication and extrusion following the hydration of a lipid and cholesterol mixture. The membrane morphological changes induced in model bacterial membranes by LipoFAs were characterized in the following order: (A) bilayer formation, (B) fusion into the membrane, (C) budding, and (D) membrane disruption.

[0027] Figure 2 is a diagram showing the results of the characterization analysis of LipoFAs. Each panel of Figure 2 represents the following: (AC) Hydrodynamic size (diameter, nm) and PDI change of FFAs and LipoFAs of various compositions measured by DLS. (DF) Surface zeta potential (mV) change of FFAs and LipoFAs of various compositions measured by DLS over 24 hours (n = 3, mean ± standard deviation). (G) Representative cryo-electron microscopy images of (1) BareLipo, (2) LipoLNA, (3) LipoLLA, and (4) LipoOA (scale bar: 50 nm).

[0028] Figure 3 shows the results of the biophysical characterization analysis of membrane interactions and fusion with S. aureus MW2. It illustrates the viscoelastic fingerprints of the interactions between model Gram-positive bacterial SLBs and LipoFAs. Each panel in Figure 3 represents the following: (A) Frequency-dissipation (FD) curves for BareLipo, (B) 500 μg / mL LipoFAs, and (C) 15.625 μg / mL LipoFAs. The arrows in panel (A) indicate the interaction phase, and the downward arrows in panels (B and C) indicate the initiation of buffer washing. (D) Fluorescence image of fusion activity between RhB-labeled LipoFAs (red) and DAPI-stained bacteria (blue). (E) Analysis of fusion contact area quantified with RhB-conjugated liposomes. The value for the control group was 0. P values ​​< 0.05 were considered statistically significant (*p < 0.05, **p < 0.01, and ***p < 0.001). Control bacteria were cultured with PBS (scale bar: 5 μm).

[0029] Figure 4 is a diagram showing the results of a quantitative analysis of bright spot formation in Gram-positive model membranes. It presents the results of time-lapse microscopic observations of membrane morphological responses of SLBs induced by BareLipo, LipoOA, LipoLLA, and LipoLNA. Each panel of Figure 4 represents the following: (A) Sequential image snapshots depicting morphological changes in SLBs upon interaction with various concentrations of BareLipo and LipoFAs (scale bar: 20 μm). (B) Total number of high-intensity spots after sample treatment and (C) after washing (n = 5, mean ± standard deviation). The number was calculated using ImageJ.

[0030] Figure 5 illustrates the changes in membrane fluidity induced by LipoFAs in Gram-positive model membranes and the results of in vitro bacterial fusion analysis. Each panel of Figure 5 represents the following: (A) Summary of diffusion coefficients and migration fractions of Gram-positive model membranes before and after LipoFAs treatment. (B) Fluorescence micrographs at 0 and 2 minutes after photobleaching for model membranes treated with BareLipo, (C) LipoLNA, (D) LipoLLA, and (E) LipoOA (scale bar: 20 μm). (F) Membrane fluidity of S. aureus MW2 treated with LipoFAs and BareLipo for 1 hour. Laurdan GP index = (I 435 - I 490 ) / ( I 435 + I 490 ), where 435 and 490 are the emission intensities at 435 nm and 490 nm, respectively, when excited at 350 nm (n = 3, mean ± standard deviation). Statistical significance was determined as p < 0.05 (*p < 0.05, **p < 0.01, and ***p < 0.001).

[0031] Figure 6 illustrates the bactericidal effect of lipophilic liposomal fatty acids (LipoFAs) on Staphylococcus aureus MW2 and their influence on bacterial membrane integrity. Each panel of Figure 6 represents the following: (A) Exponential S. aureus MW2 cells were treated with LipoFAs for 4 hours, and bacterial viability was evaluated at 2-hour intervals. The detection limit was set to 2 × 10² CFU / mL, and error bars represent the standard deviation (SD) from three biological replicates. (B) Membrane permeability of actively growing S. aureus MW2 cells after LipoFA treatment was evaluated using SYTOX Green. Data represent the mean of three independent experiments. (C) ATP leakage from growing MRSA cells treated with LipoFAs for 10 minutes was evaluated using ATP emission spectrometry. Individual data points are indicated, and error bars represent the mean ± standard deviation (SD) (n=3). Statistical differences were analyzed using one-way ANOVA followed by Tukey's post-hoc test (* p < 0.1, **** p < 0.0001).

[0032] Figure 7 shows the results of an in vitro cell viability analysis for S. aureus MW2 after treatment with LCFAs and LipoFAs. Each panel of Figure 7 represents the following: (A) Cell viability analysis after treatment with LCFAs, BareLipos, and LipoFAs in mouse fibroblasts (L-929) and (B) human keratinocytes (HaCat) (n = 3, mean ± standard deviation).

[0033]

[0034] The present invention will be described in detail below.

[0035]

[0036] The term "one or more types" in the present invention means a "number" corresponding to one or more. In the present invention, if a certain composition is one or more types, it may preferably be one type, two or more types, three or more types, one to three types, or one to two types, but is not limited thereto. The term "one or more types" may be used interchangeably with the terms "one or more" or "one or more" in the present invention.

[0037]

[0038] In one aspect, the present invention relates to a liposome comprising a phospholipid, cholesterol, and a long-chain fatty acid, and an antimicrobial composition comprising the same. The antimicrobial composition may be a pharmaceutical composition or a food composition. More specifically, the liposome relates to a liposome comprising a phospholipid, cholesterol, and a long-chain fatty acid, and an antimicrobial composition comprising the same. The antimicrobial composition may be a pharmaceutical composition or a food composition.

[0039]

[0040] In one embodiment, the liposome may contain phospholipids, cholesterol, and long-chain fatty acids in a weight ratio of phospholipids:cholesterol:long-chain fatty acids of 3 to 7:0.5 to 3:2 to 6. More preferably, the weight ratio of phospholipids:cholesterol:long-chain fatty acids may be 5:4:1. According to the present invention, by having such a weight ratio, a liposome having physical stability and a particle size capable of having high stability, fusion ability, and penetration efficiency can be produced.

[0041] In one embodiment, the phospholipid is egg phosphatidylcholine (EPC), phosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), It may be one or more selected from the group consisting of phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), but is not limited thereto. Preferably, the phospholipid may be egg phosphatidylcholine.

[0042] In one embodiment, the long-chain fatty acid may be an unsaturated long-chain fatty acid. Preferably, the unsaturated long-chain fatty acid may be a long-chain fatty acid having 16 to 20 carbon atoms. More preferably, the unsaturated long-chain fatty acid may be one or more selected from the group consisting of linolenic acid (3 double bonds), linoleic acid (2 double bonds), and oleic acid (1 double bond), having 18 carbon atoms, but is not limited thereto. In the present invention, the degree of unsaturation of the long-chain fatty acid plays an important role in the interaction mechanism, and a high degree of unsaturation can induce greater local curvature stress, increased membrane permeability, and significant ATP leakage, which can ultimately lead to enhanced bactericidal activity.

[0043]

[0044] In one embodiment, the liposome according to the present invention may have a particle size with a lower limit of 30 nm or more, 35 nm or more, 40 nm or more, 45 nm or more, or 50 nm or more, or an upper limit of 150 nm or less, 200 nm or less, 250 nm or less, 300 nm or less, 350 nm or less, 400 nm or less, 450 nm or less, or 500 nm or less, and may have a particle size having a range of combinations of the upper and lower limits. For example, although not limited thereto, the liposome according to the present invention may have a particle size of 30 to 300 nm, preferably 50 to 200 nm, more preferably 140 nm. According to the present invention, having such a particle size may result in high stability, fusion ability, and penetration efficiency.

[0045]

[0046] In one embodiment, a liposome according to the present invention may exhibit a zeta potential with a lower limit of -200 mV or higher, -150 mV or higher, -100 mV or higher, or -50 mV or higher, or an upper limit of -10 mV or lower, -15 mV or lower, -20 mV or lower, -25 mV or lower, or -30 mV or lower, and may have a zeta potential in a range of combinations of the upper and lower limits. For example, although not limited thereto, a liposome according to the present invention may preferably exhibit a zeta potential of -200 mV to -10 mV or -30 mV or lower, but is not limited thereto.

[0047]

[0048] In one embodiment, the polydispersity index (PDI) of the liposome according to the present invention may have an upper limit of 0.3 or less, 0.28 or less, 0.26 or less, and 0.24 or less, and a lower limit of 0.01 or more, 0.05 or more, and 0.1 or more, and may have a polydispersity index having a range of combinations of the upper and lower limits, but is not limited thereto.

[0049]

[0050] The liposome according to the present invention and the antimicrobial composition containing the same can be incorporated into a bacterial membrane and irregularly rearrange surrounding lipid molecules to significantly disrupt and damage the integrity of the membrane, thereby exhibiting excellent antimicrobial activity against various bacteria, preferably Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA).

[0051]

[0052] In an additional embodiment, the composition of the present invention may further comprise an antibiotic. Examples of the antibiotic may further comprise, but are not limited to, one or more antibiotics selected from the group consisting of oxacillin, tetracycline, erythromycin, ampicillin, and kanamycin.

[0053]

[0054] In one embodiment, the antimicrobial composition may be a pharmaceutical composition or a food composition. In the present invention, where the composition is a pharmaceutical composition, the pharmaceutical composition may be formulated and used in the form of a powder, granule, tablet, capsule, suspension, emulsion, syrup, oral formulation, aerosol, topical preparation, suppository, or sterile injectable solution according to conventional methods, and may additionally include one or more additives selected from the group consisting of a suitable antibiotic, carrier, excipient, and diluent commonly used in the manufacture of pharmaceutical compositions.

[0055] Specifically, the pharmaceutically acceptable carriers, excipients, or diluents included in the pharmaceutical composition of the present invention are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0056] When the pharmaceutical composition according to the present invention is used as a medicine, the preferred dosage may vary depending on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by a person skilled in the art.

[0057]

[0058] When the antimicrobial composition according to the present invention is a food composition, it may additionally include one or more additives selected from the group consisting of organic acids, phosphates, antioxidants, lactose, casein, dextrin, glucose, sugar, and sorbitol, although not limited thereto. The organic acid may be citric acid, fumaric acid, adipic acid, lactic acid, or malic acid, although not limited thereto; the phosphate may be sodium phosphate, potassium phosphate, acid pyrophosphate, or polyphosphate (polymerized phosphate), although not limited thereto; and the antioxidant may be a natural antioxidant such as polyphenol, catechin, alpha-tocopherol, rosemary extract, licorice extract, chitosan, tannic acid, or phytic acid, although not limited thereto.

[0059]

[0060] In addition, the present invention relates to a composition for reducing antibiotic resistance comprising the liposome as an active ingredient.

[0061] Since the composition for reducing antibiotic resistance of the present invention contains phospholipids, cholesterol, and long-chain fatty acids, which are the same active ingredients as the antimicrobial composition of another embodiment of the present invention, in specific weight ratios, redundant content is omitted to avoid excessive complexity in the description of this specification.

[0062]

[0063] The liposome according to the present invention and the antimicrobial composition containing the same are loaded with long-chain fatty acids exhibiting a high level of unsaturation and contain phospholipids, cholesterol, and long-chain fatty acids in specific ratios, resulting in increased membrane fluidity, permeability, and intracellular ATP leakage, and consequently exhibiting an enhanced bactericidal effect against bacteria including MRSA. Furthermore, by exhibiting reduced cytotoxicity, it highlights the potential as an effective antimicrobial agent with improved biocompatibility compared to free fatty acids.

[0064]

[0065] The present invention will be explained in more detail below through examples. These examples are merely for the purpose of explaining the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

[0066]

[0067] [Example]

[0068] The increasing prevalence of antibiotic-resistant bacteria has driven the need for alternative treatment strategies, and liposomal fatty acids (LipoFAs) are emerging as promising candidates due to their potent antimicrobial properties. Despite this growing interest, the detailed biophysical interactions between LipoFAs and bacterial membranes have not yet been sufficiently explored.

[0069] In this invention, the mechanistic interactions between liposomal linolenic acid (LipoLNA), linoleic acid (LipoLLA), oleic acid (LipoOA) and Gram-positive bacterial model membranes were systematically investigated using quartz crystal oscillator microbalance dissipation monitoring (QCM-D) and fluorescence microscopy. QCM-D analysis revealed that LipoOA exhibited the highest membrane fusion rate, followed by LipoLLA and LipoLNA. Fluorescence microscopy highlighted distinct morphological changes induced by each LipoFA: LipoLNA produced large membrane buds, LipoLLA formed smaller, high-density protrusions, and LipoOA induced rapid incorporation with uniform, high-density spots. Furthermore, fluorescence recovery after photobleaching (FRAP) demonstrated that LipoLNA significantly improved lipid mobility and membrane fluidity, which was confirmed by Laurdan generalized polarization measurements. The degree of unsaturation of lipoFAs was found to play a crucial role in the interaction mechanism; higher unsaturation induced greater local curvature stress, increased membrane permeability, and significant ATP leakage, ultimately resulting in enhanced bactericidal activity. Notably, liposomal formulations exhibited improved biocompatibility compared to free fatty acids. These findings provide valuable mechanistic insights into how lipoFAs disrupt bacterial membranes and support their potential application as alternative antimicrobial agents.

[0070]

[0071] Experimental section

[0072] reagent

[0073] Egg L-α-phosphatidylcholine (Egg PC, cat# 840051), cholesterol (wool, >98%), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))] (Lysyl-PG), 1′,3′-bis[1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho]-glycerol (Cardiolipin), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (RhB-PE) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Linolenic acid (LNA), linoleic acid (LLA), oleic acid (OA), and 6-Dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan) were sourced from Sigma-Aldrich (St. Louis, MO). Tryptic soy broth (TSB), Mueller-Hinton (MH) agar, and media were purchased from Becton Dickinson (Sparks, MD). Phosphate-buffered saline (PBS) was purchased from Gibco (Carlsbad, CA). All solutions were prepared using deionized water treated with a Milli-Q system (>18 MΩ·cm) (Millipore, Billerica, MA).

[0074]

[0075] Preparation and Characterization of Antimicrobial Fatty Acids and Liposomes

[0076] LNA, LLA, and OA stock solutions were prepared by dissolving each FA in ethanol at a concentration of 50 mg / ml, and experimental concentrations were achieved by dilution with PBS. Liposomes were prepared using a vesicle extrusion technique by modifying a previously established protocol. LipoLNA, LipoLLA, LipoOA, and BareLipo (bare liposomes without added fatty acids) were synthesized by mixing Egg PC, cholesterol, and 15 mg of LNA, LLA, or OA in weight ratios of 5:1:4 and 9:1:0, respectively. These mixtures were combined with 1 mL of chloroform and dried using nitrogen at 50°C, then stored overnight in a desiccator to remove residual solvent. The dried lipid films were hydrated with 3 mL of sterile PBS buffer (pH 7.5). The generated lipid suspension was vortexed for 15 seconds and sonicated using a bath sonicator (RS PRO, Kuala Lumpur, Malaysia), followed by further sonication for 5 minutes using a 20 kHz sonication probe (QSonica, Newton, CT) with a 12 mm tip diameter at 20 kHz, 500 W, and 40% amplitude to generate small monolayer vesicles (SUVs). These final vesicles were extruded through a 400 nm polycarbonate (PC) membrane and then further extruded 21 times through 100 nm pores using a mini extruder (Avanti Polar Lipids Inc., Alabaster, AL). The hydrodynamic size and surface zeta potential of LipoLNA, LipoLLA, LipoOA, and BareLipo were analyzed using dynamic light scattering (DLS) and zeta PALS analyzers (Brookhaven Instruments, Holtsville, NY). BareLipo was used as a negative control, and all experimental characterization tests were performed three times at room temperature.

[0077]

[0078] Cryo-Electron Microscopy (Cryo-EM) Imaging

[0079] For cryo-EM imaging, 3 μl of the sample was prepared by placing it on a 400-mesh Tedpella lacey carbon copper grid coated with an ultrathin carbon film (Ted Pella, Inc.) and glow discharged in air for 60 seconds. Subsequently, the grid was blotted for 1 or 2 seconds (blot force 1) at 22°C and 100% humidity using an FEI Vitrobot Mark IV, followed by immersion in liquid ethane. Micrographs were acquired using a 300 kV Titan Krios cryo-transmission electron microscope equipped with a Selectris X imaging filter and a Falcon 4i direct electron detector. Images were acquired at a magnification of 53,000× within a pixel size of 2.4 Å / px.

[0080]

[0081] Geological preparation for supported lipid bilayer (SLB) formation

[0082] Gram-positive lipid membranes composed of the anionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), the cationic lipid 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))] (Lysyl PG), and the anionic lipid 1′,3′-bis[1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho]-glycerol (CL) were mixed with chloroform³ as reported in a previous study to achieve a total concentration of 0.3 mg / mL. The lipid mixture was dried under a nitrogen gas stream to obtain a dry lipid film, which was desiccated using nitrogen and vacuum-stored overnight to confirm the removal of organic solvents. To improve solubility, anionic and cationic lipids were dissolved in ethanol at 70°C for 1-2 minutes to achieve a concentration of 1 mg / mL. Before each experiment, this solution was further diluted in isopropanol.

[0083]

[0084] Crystal oscillator microbalance (QCM-D) experiment with dissipation

[0085] To investigate molecular-level interactions between liposomes and SLBs, QCM-D experiments were performed using a 4-channel Q-Sense E4 instrument (Q-Sense AB, Gothenburg, Sweden). QCM-D technology measures changes in frequency (Δf) and energy dissipation (ΔD) of a vibrating piezoelectric crystal oscillator sensor chip over time. These measurement signals provide mechanistic insights into changes in the mass and dissipation characteristics of the adsorbed film. A silicon dioxide-coated 5 MHz sensor chip (model number QSX 303, Biolin Scientific) was used. Prior to each experiment, the chip was washed with 1% SDS, DI water, and ethanol. Subsequently, the chip was dried with nitrogen gas and treated with oxygen plasma for 1 minute using an Expanded Plasma Cleaner (PDC-002, Harrick Plasma, Ithaca, NY). Bacterial model membrane SLBs were formed using the solvent-assisted lipid bilayer (SALB) technique. Each SLB was used only once per experiment. Initially, a baseline signal was recorded in an aqueous buffer solution (10 mM Tris, 150 mM NaCl, pH 7.5). The buffer solution was then replaced with an isopropanol solution, and 0.3 mg / mL of Gram-positive membrane lipids were added to the isopropanol solution. Subsequently, the solvent was exchanged with PBS to form the SLB. Once bilayer formation was complete, test liposome samples at concentrations of 500 μg / mL and 15.625 μg / mL were introduced into the PBS solution, followed by a final PBS rinse. All liquid samples were introduced into the measurement chamber using a peristaltic pump (Reglo Digital, Ismatec, Glattbrugg, Switzerland) at a flow rate of 50 μL / min. The temperature was maintained at room temperature (25.0 ± 0.5°C) throughout the experiment.Data collection was performed on the 3rd (n = 3), 5th (n = 5), 7th (n = 7), and 9th (n = 9) overtones using the Q-Soft software program (Biolin Scientific). The presented data was obtained specifically from the 5th overtone, and all data processing was completed using the Q-Tools (Biolin Scientific) and OriginPro (OriginLab, Northampton, MA) software programs.

[0086]

[0087] Time-course fluorescence microscope

[0088] Surface film morphological changes of SLBs on silicon dioxide substrates treated with LipoLNA, LipoLLA, LipoOA, and BareLipo were visually monitored using an epifluorescence microscope. These experiments were performed using an Eclipse TI-E inverted microscope (Nikon, Tokyo, Japan) equipped with a 60× magnification (NA = 1.49) oil immersion objective lens (Nikon). Micrographs were captured using an iXon EMCCD camera (Andor Technology, Belfast, Northern Ireland) featuring a 512 × 512 pixel resolution and a 0.267 × 0.267 μm² pixel size. Illumination was provided by a fiber-coupled mercury lamp (Intensilight C-HGFIE, Nikon) and a TRITC filter to excite fluorescently labeled bacterial phospholipids. SLBs were fabricated on glass coverslips as substrates contained within a flow chamber (sticky slide VI 0.4, Ibidi, Germany) using the SALB method. After SLB formation, the chamber was rinsed with PBS buffer solution, and the prepared LipoLNA, LipoLLA, LipoOA, and BareLipo were introduced at a flow rate of 50 μL / min. To investigate the time dependence of the effect of LipoFAs on SLBs, micrographs were taken every 5 seconds for 1 hour at room temperature. t = 0 seconds was set as the start time of test sample injection. The collected images were analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA).

[0089]

[0090] Fluorescence Recovery (FRAP) Measurement After Photobleaching

[0091] The lateral diffusivity of Rhodamine-PE lipid-labeled SLBs was evaluated before and after exposure to LipoFAs using the FRAP technique. The photobleaching process was performed by generating a circular dot with a diameter of 20 μm for 5 seconds using a 532 nm, 100 mW laser (Klastech Laser Technologies, Dortmund, Germany). Fluorescence micrographs were captured every 2 seconds for 120 seconds to track fluorescence recovery. Lateral diffusion coefficients were calculated from the FRAP data using the Hankel transform method implemented in Matlab (MathWorks, USA).

[0092]

[0093] Liquidity analysis

[0094] Dilute the overnight culture of S. aureus MW2 1:100 in 2 mL of TSB and OD 600 Incubated at 37°C until = 1.0. Then, the bacteria were co-incubated with 10 μM Laurdan in the dark for 10 minutes. After staining, the bacterial suspension was washed four times with PBS and concentrated twofold. This concentrated bacterial suspension was mixed with equal volumes of PBS, benzyl alcohol (BA), BareLipo, LipoLNA, LipoLLA, and LipoOA at twice the desired concentration. After incubating in the dark at room temperature for 1 hour, the fluorescence intensity of Laurdan was evaluated at emission wavelengths of 435 nm and 490 nm upon excitation at 350 nm using a spectrophotometer (Tecan Spark®, Tecan, Zurich, Switzerland). Membrane fluidity was GP = (I 435 - I 490 ) / ( I 435 + I 490 It was quantified using the Laurdan Generalized Polarization (GP) index, expressed as ). Benzyl alcohol at a concentration of 50 mM was used as a positive control.

[0095]

[0096] Liposome fusion with S. aureus MW2

[0097] The fusion between LipoLNA, LipoLLA, LipoOA, and S. aureus MW2 was investigated using fluorescence methods. Fluorescently labeled LipoLNA, LipoLLA, and LipoOA were prepared by mixing DMPE-RhB (0.5 mol%) with EggPC, cholesterol, and FFA. Subsequently, 1 mL of LipoFA suspension was mixed with 1.5 × 10⁶ CFU / mL S. aureus MW2. After 30 minutes of incubation, the bacteria were collected by centrifugation at 17,500 × g for 5 minutes and fixed in 2% (vol / vol) glutaraldehyde in PBS at room temperature for 20 minutes. The bacteria were washed and resuspended in 500 μL of DI water. For imaging purposes, 10 μL of the bacterial suspension was mixed with 10 μL of DAPI-containing mounting medium (Fluoroshield™ with DAPI, Sigma-Aldrich) and placed on a glass slide. Samples were imaged using the 63× oil immersion objective of a Zeiss Observer II. Unless otherwise specified, experiments were performed independently three times. Statistical analysis was performed using two-sided T-tests with Microsoft Excel (Microsoft, Redmond, WA, USA), and correlations with a p-value < 0.05 were considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001).

[0098]

[0099] Time-sterilization kinetics evaluation

[0100] The overnight culture of Staphylococcus aureus MW2 was diluted 1:10,000 in 25 mL of tryptic soy broth (TSB) and the OD 600Incubated at 37°C while shaking at 200 rpm until it reached 0.05. Then, the logarithmic phase cells were washed three times with PBS and the OD 600 The concentration was adjusted to 0.05. The culture medium was mixed with an equal volume of preheated TSB containing twice the target concentrations of the prepared LipoFAs and BareLipo, and then dispensed into a 96-well assay block (Bioneer cat# 90063, Daejeon, South Korea). The assay block was incubated at 37°C while shaking at 450 rpm. Samples were collected every hour, serially diluted 10-fold in PBS, and plated on cation-adjusted Mueller-Hinton (CaMH, BD cat# 212322) agar. After overnight incubation at 37°C, the number of colonies was determined to assess the number of viable cells. The experiment was repeated three times.

[0101]

[0102] Membrane integrity evaluation

[0103] Membrane permeability induced in MRSA by lipoFAs was evaluated using SYTOX Green dye (ThermoFisher cat# S7020), which binds to DNA but cannot pass through an intact cell membrane. S. aureus MW2 cells were washed three times with PBS, and OD 600... was adjusted to 0.4. SYTOX Green was added to the cell suspension to a final concentration of 5 μM, and the mixture was incubated in the dark at room temperature for 30 minutes. After incubation, 50 μL of the SYTOX Green-bacterial mixture was added to each well of a black clear-bottomed 96-well plate (Greiner Bio-One Cat no. 665090) containing LipoFA compounds at concentrations ranging from 16 μg / mL to 500 μg / mL. Fluorescence measurements were performed at room temperature for 1 hour using a BioTek Cytation 5 multimode reader (BioTek, USA), with the excitation and emission wavelengths set to 485 nm and 525 nm, respectively. The experiment was repeated three times.

[0104]

[0105] Extracellular ATP leakage measurement

[0106] Extracellular ATP leakage from S. aureus MW2 cells was evaluated using the RealTime-Glo™ Extracellular ATP Assay (Promega, Madison, WI, USA). Logarithmic-phase S. aureus MW2 cells were washed three times with PBS and OD 600 The concentration was adjusted to 0.4. The ATP assay reagent mixture was prepared at a 4-fold concentration according to the manufacturer's instructions. The prepared LipoFAs and BareLipo were serially diluted in a black clear-bottom 96-well plate (Greiner Bio-One Cat no. 665090) to concentrations ranging from 16 μg / mL to 500 μg / mL. Then, 50 μL of the reagent mixture and 50 μL of the bacterial suspension were added to each well. The plates were incubated statically at 37°C for 20 minutes. After incubation, an additional 33.4 μL of the 4-fold reagent mixture was added to each well, and luminescence was measured using a BioTek Cytation 5 multimode reader. All experiments were repeated three times.

[0107]

[0108] Cell viability test

[0109] The effects of LipoLNA, LipoLLA, LipoOA, and BareLipo on cell viability were evaluated by measuring dehydrogenase activity in mouse fibroblast cells (L-929) (ATCC, USA) and human keratinocytes (HaCat) (CLS Cell Lines Service, Eppelheim, Germany). Cell viability was assessed using the cell counting kit-8 (CCK-8) (Dojindo Molecular Technologies, Rockville, MD). L-929 and HaCat cells were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Hyclone) in a humidified 5% CO₂ incubator at 37°C. L-929 cells were seeded into 96-well tissue culture plates at a density of 5 × 10³ cells per well, and HaCaT cells at a density of 1 × 10⁴ cells per well. After 24 hours of incubation, experimental samples ranging from 8 μg / mL to 128 μg / mL were added, and the cells were incubated for an additional 24 hours under the same conditions. After treatment, the cells were incubated for 2 hours in a 10% CCK-8 solution containing water-soluble tetrazolium salt (WST-8). Cell viability was determined by measuring sample absorbance at 450 nm using a microplate reader (Tecan Spark®, Tecan, Zurich, Switzerland). Each experiment was performed three times.

[0110]

[0111] Results and Discussion

[0112] Preparation and In Vitro Characterization of LipoFAs

[0113] The stability of the synthesized LipoFAs and FFA micelles was evaluated by monitoring changes in size and zeta potential over 24 hours using dynamic light scattering (DLS). Figures 2A–F show the size changes of FFAs and LipoFAs at a concentration of 5 mg / mL in PBS buffer, with samples diluted 10-fold for zeta potential measurements. All FFAs exhibited a significant increase in size over 24 hours. For example, the measured size of LNA increased from 626 ± 18 nm to 1016 ± 67 nm, LLA from 728 ± 35 nm to 1314 ± 45 nm, and OA from 885 ± 34 nm to 1523 ± 10 nm, indicating rapid aggregation of FFAs. Similarly, the size of LipoFAs loaded with 80% FAs (weight ratio 1:1:8) also increased significantly overnight; LipoLNA grew from 213 ± 4 nm to 813 ± 24 nm, LipoLLA from 236 ± 5 nm to 461 ± 11 nm, and LipoOA from 308.7 ± 9 nm to 404 ± 9 nm. This increase in final liposome size was attributed to structural changes in the FAs, with OA exhibiting denser packing with phospholipids than LNA. In contrast, LipoFAs with weight ratios of 8:1:1 and 5:1:4 showed better stability, maintaining a size within 150 nm for 24 hours. The polydispersity indices (PDIs) of FFAs and the corresponding LipoFAs with a weight ratio of 1:1:8 exceeded 0.3 over 24 hours, whereas the PDIs for LipoFAs with weight ratios of 8:1:1 and 5:1:4 remained below 0.24. This suggests that an excess of FAs leads to unstable liposomes. Additionally, the PDIs of the prepared LipoFAs increased slightly overnight, and higher PDIs were observed in the less stable group (Fig. 2A-C).

[0114] The zeta potentials of FFAs and LipoFAs were measured at 0, 6, 12, and 24 hours. It was observed that only liposomes with a weight ratio of 5:1:4 exhibited a consistent and stable trend consistent with zeta potential stability (Fig. 2D-F). Zeta potential, representing the effective charge on the liposome surface, is used to determine the stability and aggregation behavior of liposome formulations. This parameter serves as an indicator of the colloidal stability of liposomes, and higher absolute values ​​suggest enhanced particle stability. For LipoFAs with a weight ratio of 5:1:4, the surface zeta potentials were measured as -39 ± 1 mV for LipoLNA, -34 ± 2 mV for LipoLLA, -40 ± 2 mV for LipoOA, and -4 ± 0.4 mV for BareLipo. A significant decrease in surface zeta potential suggests the integration of FAs into the lipid layer, where carboxylic acid groups are deprotonated to COO at neutral pH. Liposomes with a diameter of less than 50 nm exhibit significant instability and a tendency to fuse due to high surface tension. Conversely, larger liposomes (>200 nm) tend to be stable but face penetration issues. Intermediate-sized liposomes (50–200 nm) offer relatively good stability, fusion ability, and penetration efficiency. Therefore, a weight ratio of 5:1:4 was selected due to the intermediate size (~140 nm) and physical stability. The size and morphology of the fabricated liposomes were further verified via cryo-electron microscopy (cryo-EM) (Fig. 2G), and both BareLipo and LipoFAs appeared spherical with distinct monolayers or bilayers, with a size of approximately 100 nm. The size difference between cryo-EM and DLS can be attributed to surface ions, which provide a larger dynamic diameter.

[0115]

[0116] Frequency and dissipation relationship of LipoFAs in Gram-positive model membranes (FD curve)

[0117] The frequency and dissipation relationships between the model membrane and LipoFAs were derived from QCM-D analysis (Fig. S1), which enabled real-time tracking of changes in the mass and viscoelastic properties of the membrane (Figs. 3A-C).

[0118] In Fig. 3A, the initial introduction of BareLipo to the surface of the model membrane resulted in adhesion, evidenced by a decrease in frequency and an increase in dissipation [-f, +D]. This stage represents the initial adsorption of liposomes to the membrane surface. In the second stage, a sustained increase in dissipation and a slight increase in frequency [+f, +D] were observed, suggesting the incorporation of lipid molecules into the model membrane. During the buffer wash in Step 3, both frequency and dissipation remained relatively unchanged, indicating that fusion between BareLipo and the membrane had occurred. Overall, higher dissipation values ​​demonstrated that the phospholipids altered the viscoelastic properties of the membrane.

[0119] The frequency and dissipation relationships for LipoLNA, LipoLLA, and LipoOA are shown in Fig. 3B. Similar to the observations in Step 1 of BareLipo, the initial decrease in frequency indicates the adhesion of LipoFAs to the Gram-positive model membrane, characterized by [-f, +D]. The variation in the ∂D / ∂f ratio among LipoLNA, LipoLLA, and LipoOA was primarily attributed to differences in the rate of increase in dissipation. LipoOA exhibited the highest ∂D / ∂f values, suggesting a higher degree of incorporation into the model membrane as the membrane's viscoelasticity increased most rapidly (Fig. 3B). Furthermore, the consistent ∂D / ∂f ratio for LipoOA indicated that fusion begins upon adsorption to the model membrane. LipoLLA showed a slight increase in the ∂D / ∂f ratio, indicating minor impediments to liposome fusion and a slower fusion rate compared to LipoOA. Conversely, LipoLNA showed a noticeable change in the ∂D / ∂f ratio. The initial ∂D / ∂f ratio for LipoLNA (Fig. 3B) suggests slower incorporation into the model membrane, followed by a significant increase in the ∂D / ∂f ratio, which corresponds to a faster rise in dissipation for this sample. This may be attributed to the larger tail volume of LNA, which could hinder the incorporation rate. After rinsing with buffer at t = 100 min, the endpoints of the fD curves provided insight into changes in membrane properties. In Fig. 3C, the frequency changes of LipoFAs after buffer rinsing were -21.9 ± 0.1 Hz for LipoLNA, -8.6 ± 0.0 Hz for LipoLLA, and -3.4 ± 0.1 Hz for LipoOA, indicating a decreasing trend in mass loss and suggesting greater incorporation into the membrane components.

[0120] At a lower concentration of 15.625 μg / ml, membrane interactions were less pronounced (Fig. 3C), indicating a concentration-related effect of LCFAs. Due to the lower sample concentration, the adhesion process was less significant. Given the lack of a notable change in the surface zeta potential of LipoFAs, they should exhibit similar adsorption capacities resulting in similar frequency response. Interestingly, the frequency endpoints before buffer rinse were -49.7 ± 0.1 Hz for LipoLNA, -51.2 ± 0.0 Hz for LipoLLA, and -54.0 ± 0.2 Hz for LipoOA, respectively, demonstrating an increase in mass and indicating a stronger ability of OA to incorporate into the model membrane. Less change in mass was observed after buffer washing compared to higher LipoFA concentrations, and the final dissipation was highest for LipoOA, which is primarily a result of membrane instability caused by greater incorporation of OA into the model membrane.

[0121] To verify the fusion behavior of LipoFAs, the in vitro interaction between LipoFAs labeled with lipophilic RhB-PE fluorophores and S. aureus MW2 was investigated (Fig. 3D). The bacteria were stained with DAPI (blue), and only DAPI fluorescence was observed in PBS buffer solution. In contrast, when the bacteria were cultured with RhB-PE-labeled LipoFAs, a distinct RhB red fluorescence signal was observed around the bacteria, confirming fusion with the bacteria. Therefore, the fluorescence image is consistent with our QCM-D analysis.

[0122]

[0123] In vitro membrane fluidity changes induced by LipoLNA, LipoLLA, and LipoOA in Gram-positive bacterial biofilms

[0124] The incorporation of cis-unsaturated FAs into bacterial membranes can affect membrane fluidity by inducing the rearrangement of surrounding lipid molecules, thereby altering membrane properties. To evaluate these changes in membrane lipid behavior upon exposure to LipoFAs, FRAP measurements were performed to determine the diffusion coefficient and transport fraction of lipid molecules in a model membrane. As shown in Figure 5A, BareLipo increased the lateral lipid diffusion (diffusion coefficient) within the model membrane by 35.2%, and LipoLNA increased it by 18.4%, while LipoLLA and LipoOA decreased it by 37.8% and 91.8%, respectively. At the same time, the transport fraction of lipid molecules was 80.0 ± 3% for LipoLNA, 43.8 ± 0.8% for LipoLLA, and 25.7 ± 2.2% for LipoOA. The decrease in the diffusion coefficient suggests an increase in lipid molecule rearrangement to restore the membrane. Meanwhile, disturbance of membrane integrity will also reduce the diffusion coefficient and transport fraction. A higher degree of unsaturation results in a more irregular arrangement of lipid molecules, facilitating easier transport within the membrane. Conversely, the transport fraction of the exposed membrane was calculated to reflect the membrane's recovery rate. LipoOA exhibited the lowest transport fraction of 25.7 ± 2.2%, indicating that only one-quarter of the destabilized membranes were restored, suggesting less membrane integrity after exposure to LipoOA. In contrast, the presence of three unsaturated bonds in LipoLNA results in a more disordered arrangement of lipid molecules within the membrane.

[0125] To further verify the changes in membrane properties after treatment with LipoFAs, the fluidity of the bacterial membranes was evaluated using Laurdan Generalized Polarization (GP) intensity (Fig. 5F). The Laurdan GP values ​​for the membranes treated with LipoFAs were 0.457 ± 0.017 for LipoLNA, 0.471 ± 0.005 for LipoLLA, and 0.477 ± 0.006 for LipoOA. The results showed that S. aureus MW2 treated with LipoFAs exhibited increased membrane stiffness, which is consistent with the observations from the FRAP results and suggests that the incorporation of FAs into the membrane induces changes in fluidity.

[0126]

[0127] Evaluation of In Vitro Antimicrobial Activity, Membrane Permeability, and ATP Leakage of S. aureus MW2

[0128] To evaluate the antimicrobial activity of LipoFAs, time-bacterial kinetics analysis was performed on methicillin-resistant Staphylococcus aureus (MRSA) strain MW2 (Fig. 6A). LipoFAs showed a decrease in viability at 16 μg / mL. In particular, LipoLNA reduced viability by more than 2-log at 63 μg / mL and completely eradicated MRSA MW2 cells at 125 μg / mL. LipoLLA achieved a reduction of more than 2-log at 31 μg / mL and completely eliminated cells at 125 μg / mL. LipoOA showed a 1-log decrease in viability at 500 μg / mL.

[0129] Subsequently, the effects of LipoFAs on the S. aureus MW2 membrane were explored based on SYTOX Green permeability and intracellular ATP leakage. S. aureus treated with LipoLNA exhibited a concentration-dependent increase in relative fluorescence units (RFU) over time, showing a sharp rise particularly at concentrations above 125 μg / mL (Fig. 6B), indicating a significant increase in membrane permeability. Similarly, LipoLLA treatment resulted in a substantial increase in permeability, with RFU rising noticeably at concentrations above 63 μg / mL. In contrast, LipoOA had a relatively low effect on permeability and showed less distinct concentration-dependent changes compared to LipoLNA and LipoLLA due to structural differences. LNA and LLA generate greater local stress on the lipid membrane compared to OA, which has a straighter tail. The bareLipo control group showed almost no change in permeability, indicating that the bacterial membrane remained stable in the absence of LCFAs.

[0130] LipoLNA also induced a concentration-dependent increase in ATP release, which was significant at concentrations above 250 μg / mL (Fig. 6C). The LipoLLA-treated group showed a similar trend and exhibited significant ATP leakage at higher concentrations. In contrast, LipoOA induced relatively low ATP leakage, with a significant difference observed at concentrations above 63 μg / mL. The BareLipo control group showed minimal ATP leakage, suggesting that there was no membrane disturbance in the absence of LCFAs. Treatment with LCFAs also resulted in ATP leakage in a concentration-dependent manner, but to a lesser extent than with LipoFAs. Overall, LipoFAs, particularly LipoLNA and LipoLLA, significantly disrupt membrane integrity. These results demonstrate that the unsaturated binding structure of these FAs significantly impairs biofilm integrity. These findings indicate that the unsaturated binding structure of LCFAs imposes high local stress on the membrane, thereby significantly compromising biofilm integrity.

[0131] In Vitro Cytotoxicity Evaluation of Manufactured LipoFAs

[0132] In addition to solubility issues associated with FAs, their inherent cytotoxicity represents a significant drawback that limits their application as antimicrobial agents. The cytotoxic effects of FAs have been well documented in numerous studies. Literature reports have linked FAs to cytotoxicity through various pathways, such as apoptosis, autophagy, and disruption of cellular pathways. These aspects hinder the utilization of FAs as antimicrobial agents and highlight the need to address this issue for the further development of effective therapeutic strategies. To investigate the in vitro cytotoxic effects of FFAs and LipoFAs, mouse fibroblasts (L-929) and human keratinocytes (HaCat) were used as experimental models.

[0133] Subsequent cell viability analyses were performed on mouse fibroblasts (L-929) and human keratinocytes (HaCat) (Figures 7A and 7B). FFAs began to exhibit cytotoxic effects in L-929 cells at a concentration of 125 μg / mL, reducing cell viability to approximately 80%. Conversely, LipoFAs maintained approximately 100% viability in both cell lines up to 125 μg / mL and over 90% viability at 250 μg / mL, exhibiting minimal toxicity. At 125 μg / mL, FFAs showed low viability in L-929 cells, with cell viability values ​​of 86.2% for free LNA, 75.7% for free LLA, and 85.4% for free OA. At higher concentrations, cytotoxicity increased, and in L-929 cells at 500 μg / mL, viability decreased to 28.4% for free LNA, 30.1% for free LLA, and 28.4% for free OA. In HaCat cells, viability decreased to 20.3% for free LNA, 21.7% for free LLA, and 20.9% for free OA at 500 μg / mL. In contrast, LipoFAs significantly improved cell viability, with approximately 70% of L-929 cells and over 40% of HaCat cells surviving at 500 μg / mL. These results suggest that LipoFAs exhibit low cytotoxicity at higher concentrations, supporting their potential as effective antimicrobial agents with improved biocompatibility compared to FFAs.

[0134]

[0135] conclusion

[0136] This study presents a detailed analysis of the interaction patterns between LipoFAs and Gram-positive model membranes using various biophysical and biological techniques. The study explores how LipoFAs fuse and integrate with phospholipid membrane components, resulting in changes to the membrane's mass and viscoelastic properties. LipoFA compositions formulated with optimal weight ratios exhibited a significant reduction in zeta potential of approximately -50 mV and an intermediate size of approximately 140 nm, indicating a tendency to fuse with Gram-positive model membranes. Biophysical experiments revealed that high levels of unsaturation in FA tails hinder integration and rearrangement within the Gram-positive model membrane, which is likely attributed to increased local curvature stress around the FAs. In contrast, FAs with lower unsaturation bonds are more easily incorporated and generate less curvature stress. Biological evaluations demonstrated that liposomes loaded with LCFAs exhibiting high levels of unsaturation result in increased membrane fluidity, permeability, and intracellular ATP leakage, consequently demonstrating enhanced bactericidal effects against MRSA. Furthermore, LipoFAs exhibited reduced cytotoxicity, highlighting their potential as effective antimicrobial agents with improved biocompatibility compared to FFAs. These findings provide significant insights into the biophysical mechanisms underlying FA-membrane interactions and offer a platform for the rational design of next-generation antimicrobial agents. By elucidating the relationships between FA structure, membrane interactions, and bactericidal efficacy, this invention lays the foundation for the development of customized lipid-based therapeutics that leverage specific structural characteristics to more effectively target bacterial membranes. Future research should focus on evaluating the in vivo efficacy of LipoFAs to validate their therapeutic potential in clinical settings. Investigating the ability of LipoFAs to disrupt biofilms clinically relevant to persistent infections represents another important direction for advancing these formulations.This research will play a crucial role in addressing the global challenge of antibiotic resistance and guiding the development of powerful next-generation antimicrobial therapies.

Claims

1. An antimicrobial composition containing liposomes containing unsaturated long-chain fatty acids.

2. In paragraph 1, further comprising phospholipids and cholesterol, An antibacterial composition comprising phospholipids : cholesterol : long-chain fatty acids in a weight ratio of 3 to 7 : 0.5 to 3 : 2 to 6.

3. An antimicrobial liposome composition according to claim 1, wherein the long-chain fatty acid is one or more selected from the group consisting of linolenic acid, linoleic acid, and oleic acid.

4. In claim 1, the phospholipid is egg phosphatidylcholine (EPC), phosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine, An antimicrobial composition comprising one or more selected from the group consisting of DSPE), phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS).

5. The antibacterial composition of claim 1, wherein the zeta potential is -200 to -20 mV or less 6. An antibacterial composition according to claim 1, wherein the average particle size of the liposomes is 30-300 nm.

7. An antibacterial composition according to claim 1, wherein the polydispersity index (PDI) is 0.3 or less.

8. An antimicrobial composition according to claim 1, which exhibits antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA).

9. A composition for preventing, improving, or treating bacterial infections containing liposomes containing unsaturated long-chain fatty acids.

10. A composition for reducing antibiotic resistance containing liposomes containing unsaturated long-chain fatty acids.

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