Efficacy of Liposome-Encapsulated Vancomycin Against Methicillin-Resistant Staphylococcus aureus

Efficacy of Liposome-Encapsulated Vancomycin Against Methicillin-Resistant Staphylococcus aureus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficacy of Liposome-Encapsulated Vancomycin Against Methicillin-Resistant Staphylococcus aureus Enkhtaivan Erdene, Odonchimeg Munkhjargal, Enkhjargal Dorjbal, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5084652/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bacterial infections significantly contribute to global morbidity and mortality, with antibiotic-resistant strains, such as Methicillin-resistant Staphylococcus aureus (MRSA), presenting severe treatment challenges. This study investigates the potential of liposome-encapsulated vancomycin as a novel treatment to combat antibiotic resistance. Phospholipids were extracted from egg yolk, and liposomes were prepared using the freeze-thaw method. The liposome-encapsulated vancomycin formulation was characterized using infrared spectroscopy, atomic force microscopy (AFM), and high-performance liquid chromatography (HPLC). Infrared spectroscopy confirmed the structural integrity and purity of the phospholipids, while AFM revealed a uniform liposome size, with an average diameter of 157 nm and a polydispersity index (PDI) of 0.0442, indicating high stability and suitability for drug delivery. The antibacterial efficacy of liposome-encapsulated vancomycin was tested against MRSA and Staphylococcus aureus , demonstrating that the encapsulated vancomycin inhibited bacterial growth at doses significantly lower than free vancomycin. Liposome-encapsulated vancomycin achieved 100% inhibition of MRSA and S. aureus at higher dilutions, while free vancomycin was only partially effective. These findings highlight the enhanced potential of liposome-based drug delivery in reducing bacterial load and overcoming antibiotic resistance. The study emphasizes the promise of nanotechnology in improving antibiotic efficacy, offering a potential solution to the global health crisis posed by antibiotic-resistant pathogens. Lecithin Liposome Vancomycin MRSA Nanocarriers Figures Figure 1 Figure 2 Introduction The natural environment allows humans to coexist with microorganisms, yet bacterial infections significantly contribute to the global rise in infectious diseases. Bacterial infections are a leading cause of morbidity and mortality worldwide with mortality from infectious diseases including community-acquired pneumonia (CAP) at 2.5 million annually, leptospirosis at 58,900, typhoid fever between 75,000 and 208,000, and diarrhea at 1,560 (O’Neill, 2016 ; Ventola, 2015 ; World Health Organization, 2014 ; Troeger, Blacker, et al., 2018 ; Rudd et al., 2020 ). High mortality rates from bacterial infections, particularly in premature infants and young children, are closely associated with the spread of bacteria resistant to multiple antibiotics. This resistance is linked to the functional and structural characteristics of bacteria, including mutations and the transfer of resistance genes (Costa et al., 2015 ; Crump, 2004 ; Lopman et al., 2016 ; Rudan, 2008 ; Troeger, Khalil, et al., 2018 ). According to the 2019 report "The Threat of Antibiotic Resistance in the United States," antibiotic-resistant pathogens cause over 2.8 million infections and more than 35,000 deaths annually. Europe reports 25,000 deaths per year due to antibiotic-resistant infections, while Asia (specifically India) reports 700,000 deaths annually. Projections indicate that by 2050, antibiotic-resistant bacterial infections will be among the leading causes of death globally (Cassini et al., 2019 ; Holmes et al., 2016 ; Laxminarayan et al., 2016 ; L. Liu et al., 2015 ; McAdam et al., 2012 ; Sands et al., 2021 ; World Health Organization, 2015 ). One of the leading causes of death from infectious diseases is Staphylococcus aureus ( S. aureus ). S. aureus not only persists on environmental surfaces but also colonizes the nasal cavity and skin of humans and animals, facilitating both direct and indirect transmission. Methicillin-resistant Staphylococcus aureus (MRSA), a common cause of skin infections, is resistant to most antibiotics, complicating treatment (Centers for Disease Control and Prevention (U.S.), 2019 ; Hiramatsu, 1997 ; Weiner et al., 2016 ). A 2023 World Health Organization (WHO) study reported S. aureus prevalence rates of 22.27% in the Americas, 16.57% in the Western Pacific, and 10.93% in Europe. Single studies in the Eastern Mediterranean and African regions showed prevalence rates of 8.55% and 9.04%, respectively. Countries with multiple surveys, such as the United States (23.78%), Great Britain (18.66%), and China (18.07%), exhibited the highest prevalence rates. Research indicates that approximately 33% of people carry S. aureus in their noses asymptomatically, with 2% carrying MRSA. While many carriers do not develop serious MRSA infections, significant progress has been made in reducing MRSA bloodstream infections, which decreased by 17.1% annually from 2005 to 2012. However, the decline slowed between 2013 and 2016, with no significant changes noted. Studies have reported a concurrent increase in MRSA CAP and a decrease in Streptococcus pneumoniae CAP strains following the implementation of pneumococcal vaccines, particularly in cases complicated by pleurisy. Consequently, staphylococcal infections are rising due to the increase in MRSA infections (Chambers & DeLeo, 2009 ; Laxminarayan et al., 2013 ; Mendelson et al., 2022 ; Tong et al., 2015 ). Beta-lactam antibiotics are commonly used to treat staphylococcal infections, but the emergence of beta-lactamase-resistant S. aureus strains poses significant challenges. Antibiotics such as vancomycin, linezolid, daptomycin, tigecycline, and telavancin are frequently used against MRSA in skin, soft tissue, and postoperative infections (Zetola et al., 2005 ). Monitoring and analyzing data on antibacterial drug use is crucial for developing and implementing policies and interventions to promote appropriate antibiotic use at the national level (Stryjewski & Corey, 2014 ). In Mongolia, amoxicillin, amoxicillin/beta-lactamase inhibitor, ampicillin, and doxycycline accounted for 75% of oral antibiotic consumption in 2018. A study in 13 Southeastern European countries found these drugs dominated antibiotic use. Antibiotics are categorized into three groups based on their mechanism of action: inhibition of protein synthesis, nucleic acid synthesis, and cell wall synthesis (Alkilani et al., 2015 ; C. Liu et al., 2011 ; Rabiei et al., 2020 ). Resistance to vancomycin is linked to the Van gene, which encodes various resistance phenotypes. Vancomycin resistance is classified into several gene clusters based on DNA sequences of homologs of the ligase Van gene, which encodes the key enzyme for synthesizing D-alanyl D-lactate or D-alanyl D-serine (D-Ala D-Ser). At least 11 Van gene clusters have been identified, corresponding to the vanA, vanB, vanD, vanF, vanI, vanM, vanC, vanE, vanG, vanL, and vanN phenotypes, conferring vancomycin resistance. Genes encoding D-alanyl D-lactate ligases, such as vanA, vanB, vanD, vanF, vanI, and vanM, typically confer high levels of vancomycin resistance, whereas genes encoding D-alanyl D-serine ligases, including vanC, vanE, vanG, vanL, and vanN, generally confer lower resistance (Allen & Cullis, 2013 ). Nanotechnology has revolutionized medicine, particularly in diagnosing diseases, improving methods for preventing and treating cancer, hereditary, and acquired diseases, producing new-generation drugs, and creating effective treatments. Since Aleika Benkham introduced this concept in 1965, advancements in liposome extraction and physicochemical methods have facilitated the modeling of biomembrane functions and liposome structures. Liposomes serve as model systems representing cell surface and biomembrane quality, playing a crucial role in experiments involving antibiotics, cancer drugs, and nucleic acids (Sercombe et al., 2015 ). Nanotechnology has transformed transdermal drug therapy by providing new strategies to overcome the skin's natural barrier (Sutradhar & Amin, 2013 ). Liposomes, developed based on nanotechnology, are drug delivery systems consisting of one or more lipid layers, spherical in shape, and surrounded by an aqueous core filled with hydrophilic compounds. The lipophilic layer, filled with hydrophobic compounds, is biocompatible and biodegradable. Nanosized drug carriers such as liposomes, niosomes, and micelles are designed to deliver drugs through the stratum corneum, the outermost layer of the skin, which poses a significant challenge for transdermal infections (Courvalin, 2006 ; Hiramatsu, 1998 ). Many liposomal formulations have been FDA-approved for clinical use due to their enhanced drug delivery and safety profiles. These include liposomal amphotericin B for fungal infections and lipoplatin preparations for breast cancer. Other formulations have shown consistent success in preclinical and clinical trials (Rybak, 2006 ). The necessity for active transport mechanisms for drugs to reach their biomolecules is emphasized in numerous studies, highlighting the importance of ongoing research and development in this field. Materials and methods Methodology for the Extraction of Phospholipids : Gladkowski et al. (2012) developed a standardized protocol for isolating phospholipids from egg yolk, which served as the primary raw material for their research experiment. The procedure began with breaking the egg and weighing the yolk. The yolk was then mixed with acetone and continuously stirred using a magnetic stirrer for 10 minutes. The mixture was transferred to a glass flask and centrifuged at 3000 rpm for 10 minutes. After separation and drying of the precipitate, a mixture of chloroform and ethanol in a ratio of 2:1 was added, and the mixture was stirred thoroughly for three hours. The resulting solution was then filtered, and the organic solvent was separated to form a thin layer of lipids. Identification of phospholipids by infrared spectroscopy Infrared spectroscopy was employed to determine the absorption (vibration) spectrum of the phospholipids. The samples were measured in the wavelength range of 400-4000 cm -1 using a choline and tungsten light source with KBr pellets. Liposome preparation and drug encapsulation The freeze-thaw method was utilized for liposome preparation and drug encapsulation. A lipid film was prepared using egg-phosphatidylcholine (egg-PC) and cholesterol at a molar ratio of 4:1, with a total lipid content of 4 mg. The lipid films were rehydrated with 1.5 mL of phosphate-buffered saline (PBS) at pH 7.4 containing 0.01 g of vancomycin. A thin film was formed by connecting a round-bottom flask to a rotary evaporator and subjecting the solvent to three cycles of freezing at -20°C for 20 minutes and thawing at +45°C for 10 minutes. The heated vancomycin solution was added to the thin film, and the mixture was swirled in the round-bottom flask to form monolayer vesicles. After vortexing for 10 minutes, the round-bottom flask was removed from the rotary evaporator, and the contents were sonicated for 30 seconds. Immediately after sonication, the dispersion was extruded using a mini-extruder (Avanti Polar Lipids Inc., Alabaster, AL) to obtain a monodisperse liposome-encapsulated vancomycin formulation. The prepared liposome-encapsulated vancomycin was then flushed with nitrogen gas, and CMP solution was added at a molar ratio of 1:1 (CMP: DSPE-PEG-Mal). Thiol-maleimide click chemistry was allowed to proceed overnight to yield the CMP- liposome-encapsulated vancomycin formulation, followed by dialysis to remove excess vancomycin and CMP. Finally, the dialyzed CMP-liposome-encapsulated vancomycin was lyophilized in the presence of 20 mM sucrose. Particle diameter and polydispersity Particle diameter and polydispersity index (PDI) with atomic force microscopy (AFM). The final particle diameter was determined using the Stokes–Einstein equation, while the PDI was estimated using Nano DTS software (version 6.34). All measurements were performed in at least three sets of 10 runs at 25°C. The polydispersity index was calculated using the following formula: where std is the standard deviation of the diameter and avg is the average diameter. Bacteria culture methodology Staphylococcus aureus ATCC 25923 and MRSA ATCC 2758 were cultured in a normal medium and spread evenly on Baird-Parker (BP) agar (Biolab, Hungary), a selective medium. The cultures were incubated at 37°C for 24 hours. Colonies grown on BP agar were concluded to be Staphylococcus aureus . Two to three separate colonies were selected, and a bacterial suspension to 0.5 McFarland standard was prepared and diluted in 0.9% physiological solution. Liposome-encapsulated antibiotic solution To prepare the liposome-encapsulated antibiotic solution, 0.01 g of antibiotics was dissolved in 1 mL of distilled water. A working solution containing 100 μL of 0.8 μg antibiotic was prepared. For comparison, a pure antibiotic solution was prepared by dissolving 0.01 g of antibiotics in 1 mL of distilled water. A working solution containing 100 μL of 1 μg antibiotic was prepared. Microdilution method The microdilution method was performed using a 96-well reagent plate. Each well received 50-100 μL of antibiotic solution and an equal amount of bacterial suspension. For microdilution, six wells were used with Mueller-Hinton broth, with the antibiotic solution diluted to final concentrations of 1, 0.5, 0.25, 0.12, 0.06, and 0.03 μg/mL. Additionally, 10 μL of a 10 5 CFU/mL bacterial suspension ( S. aureus or MRSA) was added to each well. Negative controls included the antibiotic solution without bacteria, and positive controls included Mueller-Hinton broth with bacteria. The test wells contained Mueller-Hinton broth, MRSA, and the antibiotic solution. Evaluation of minimal inhibitory concentration (MIC) The minimal inhibitory concentration (MIC) of free vancomycin and liposome-encapsulated vancomycin was evaluated using the CLSI guidelines. The MIC was calculated as the lowest concentration of antibiotic that completely inhibited bacterial growth after 18-20 hours of incubation at 35-37°C. Analysis of high-performance liquid chromatography (HPLC) Analyses were performed on an Agilent 1260 Infinity HPLC system equipped with a C18 reversed-phase column. The mobile phase consisted of acetonitrile and water with 0.1% formic acid, with UV detection at 282 nm. The flow rate was set to 1 mL/min, and the injection volume was 10 µL. Data analysis included a standard vancomycin solution at 1000 ppm. Statistical analysis Statistical analysis was conducted using IBM SPSS Statistics software, version 22.0. Group differences were assessed through analysis of variance (ANOVA). Statistical significance was determined at a threshold of p ≤ 0.05. Results The yield of phospholipids extracted from egg yolk is 37.3%. Infrared spectroscopy was utilized to analyze the composition and purity of the isolated phospholipids. The spectrogram obtained, ranging from 400 nm to 4000 nm, confirms the distinct characteristics of the tissue-derived phospholipids. The spectrogram in Fig. 1 presents the infrared absorption characteristics of the phospholipids extracted from egg yolk, spanning wavenumbers from 4000 cm 1 to 500 cm 1 . Key absorption peaks and their corresponding functional groups are identified, providing insights into the composition and purity of the isolated phospholipids. Significant absorption bands are observed in the following regions:O–H Stretching (Decreased Intensity): The intensity of the absorption associated with the hydroxyl (-OH) group is reduced, indicating a lower presence of these groups in the sample. C–H Stretching: Peaks at approximately 2921 cm 1 and 2851 cm 1 correspond to the stretching vibrations of the C-H bonds in the aliphatic chains of the phospholipids. N(CH 3 ) 3 Group: A prominent absorption band at 1051 cm -1 is attributed to the stretching vibrations of the N(CH 3 ) 3 group, which is characteristic of phosphatidylcholine, a major component of egg yolk phospholipids. P = O Stretching: The region between 1226 cm 1 and 1051 cm 1 exhibits strong absorption due to the P = O stretching vibrations, indicative of the phosphate groups present in the phospholipids. P–O–C Stretching: An absorption peak at 819 cm 1 is assigned to the P–O–C stretching vibrations, confirming the presence of phosphate ester groups. The spectroscopic analysis demonstrates that the extracted phospholipids retain their structural integrity, as evidenced by the distinct absorption peaks corresponding to key functional groups. The purity of the phospholipids is inferred from the absence of extraneous peaks, indicating minimal contamination or degradation during the extraction process. In summary, the infrared spectrogram provides a detailed molecular fingerprint of the isolated phospholipids, validating the efficacy of the extraction method and confirming the presence of characteristic phospholipid functional groups. Analysis of Liposome Morphology Liposome size and uniformity are critical parameters in various applications, including drug delivery systems, nanotechnology, and materials science. This study employed Atomic Force Microscopy (AFM) to determine the height, diameter, and measurement error of liposomes. Figure 2 (a) displays the AFM image of liposome slices, where the positions of the slices are marked for further analysis. Figure 2 (b) shows the curves corresponding to these slices, and Fig. 2 (c) illustrates an example curve used to define the height and diameter of the liposomes. Height and Measurement Error: The average height of the liposomes was 43.2183 nm with a measurement error of 6.85 nm. AFM analysis confirmed their uniform size distribution and morphological uniformity. Analysis of Liposomes: High-Performance Liquid Chromatography (HPLC) was used to evaluate physicochemical properties. The retention time was 5.342 minutes, with a UV peak at 200 nm, confirming high purity (986 ppm). The mean diameter was 157.01385 nm with a standard deviation of 33.036 nm. A PDI of 0.0442 suggests a narrow size distribution, indicating uniformity and stability. Table 1 Height, measurement error, and physicochemical parameters Parameter Mean ± SD Error (nm) PDI Height (nm) 43.2183 6.85 - Particle size (nm) 157.01385 ± 33.036 - 0.0442 AFM and HPLC analyses demonstrate that the liposomes have desirable size and stability characteristics, making them suitable for drug delivery and nanotechnology applications. Antibacterial Efficacy of Liposome-Encapsulated Vancomycin The study evaluated the antibacterial efficacy of liposome-encapsulated vancomycin compared to free vancomycin. It was determined that liposomal vancomycin inhibited bacterial growth at doses twice as low as those required for free vancomycin. The results are summarized in Table 2 . Table 2 Bacterial growth inhibitory activity of antibiotic-based liposomes Microdilutions Liposomal vancomycin Free vancomycin MRSA ATCC 2758 S. aureus ATCC 25923 MRSA ATCC 2758 S. aureus ATCC 25923 %(n) %(n) %(n) %(n) 10 1 100(10) 100(10) 50(5) 50(5) 10 2 100(10) 100(10) 0 0 10 3 70(7) 100(10) 0 0 10 4 10(1) 70(7) 0 0 10 5 0 30(3) 0 0 10 6 0 0 0 0 Liposomal vancomycin demonstrated superior efficacy in inhibiting the growth of both Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 2758 and Staphylococcus aureus ATCC 25923 strains. At dilutions of 10 1 and 10 2 , liposome-encapsulated vancomycin completely inhibited bacterial growth (100%), whereas free vancomycin achieved only 50% inhibition at 10 1 dilution and was ineffective at higher dilutions. For MRSA ATCC 2758, liposomal vancomycin maintained 70% growth inhibition at a 10 3 dilution, while free vancomycin showed no inhibitory effect at this dilution. At a 10 4 dilution, liposomal vancomycin exhibited 10% inhibition, whereas free vancomycin remained ineffective. For S. aureus ATCC 25923, liposomal vancomycin consistently inhibited 100% growth up to a 10 3 dilution and retained 70% inhibition at a 10 4 dilution. Free vancomycin showed no inhibitory effect beyond the 10 1 dilution. Overall, liposome-based vancomycin demonstrated significantly higher antibacterial activity compared to free vancomycin, achieving comparable or superior inhibition at lower doses. Statistical analysis confirmed the enhanced efficacy of liposome-encapsulated vancomycin with a p-value of 0.02, indicating a significant difference in performance. These results indicate that vancomycin-encapsulated liposomes have a distinct and more potent inhibitory effect on MRSA and S. aureus strains compared to free vancomycin. This suggests that liposomal formulations could enhance the therapeutic efficacy and potentially reduce the required dosage of vancomycin in clinical settings. Discussion The current study investigated the antibacterial efficacy of liposome-encapsulated vancomycin against Methicillin-resistant Staphylococcus aureus (MRSA), a significant global public health concern since its emergence in the 1960s. With a reported prevalence of 14.69% among 164,717 individuals across 29 countries, MRSA has remained a formidable pathogen due to its resistance to standard β-lactam antibiotics (A. Abdelkader et al., 2017 ; Surewaard et al., 2016 ). In the late 1990s, the advent of community-associated MRSA (CA-MRSA) infections further complicated the scenario, as these infections affected healthy individuals without traditional risk factors and displayed high virulence, primarily causing skin and soft tissue infections, necrotizing pneumonia, and necrotizing fasciitis. Given these challenges, our study focused on evaluating the potential of liposome-based vancomycin formulations. Liposomes, due to their lipid bilayer structure similar to cellular membranes, offer a unique advantage in antibiotic delivery. They enhance the concentration of antibiotics at the site of infection for extended durations, thereby improving antibacterial efficacy. This study's methodology involved extracting phospholipids from egg yolk using acetone, ethanol, and chloroform, resulting in a yield of 37.9%. This method was chosen based on previous research by Fahimeh Hajiahmadi et al. ( 2019 ), which demonstrated higher encapsulation efficiency of vancomycin using the freeze-thaw method over the ammonium sulfate gradient method (Hajiahmadi et al., 2019 ). Vancomycin was selected for encapsulation due to its widespread use in treating MRSA infections, particularly in Mongolia. Analytical techniques, including chromatography and UV spectroscopy, confirmed the integrity and purity of the encapsulated vancomycin, with consistent retention times and characteristic absorption peaks at 200 nm and 280 nm (Y. Liu et al., 2019 ). These results ensured the reliability of our encapsulation method and the stability of the antibiotic within the liposome formulation. Our microdilution studies demonstrated that liposome-encapsulated vancomycin exhibited significantly higher antibacterial activity against MRSA strains compared to free vancomycin [43]. Notably, liposomal vancomycin achieved complete growth inhibition at dilutions of 10^-1 and 10^-2, whereas free vancomycin only partially inhibited growth at 10^-1 and was ineffective at higher dilutions. Moreover, liposomal vancomycin maintained substantial inhibitory activity at 10^-3 and 10^-4 dilutions, underscoring its enhanced potency. These findings align with previous research by Abdelkader et al. (2012), Surewaard B. (2016), and Liu Y. (2019), which reported that liposome-encapsulated antibiotics are effective at lower doses than their free counterparts (H. Abdelkader & G. Alany, 2012 ; Y. Liu et al., 2019 ; Surewaard et al., 2016 ). The increased efficacy of liposomal vancomycin can be attributed to the sustained release and prolonged bioavailability of the antibiotic at the site of infection, ensuring higher concentrations over longer periods. From the above results it can be that the application of liposome-encapsulated vancomycin observed in this study has significant clinical implications. By reducing the required dosage and increasing the duration of effective drug concentration, liposomal formulations can potentially minimize the risk of resistance development and adverse side effects associated with high-dose antibiotic therapies. Additionally, the ability of liposomes to deliver antibiotics directly to the site of infection may improve treatment outcomes for severe MRSA infections, including those caused by CA-MRSA strains. Our study demonstrates the superior efficacy of liposome-encapsulated vancomycin over its free form, showing significant advantages in treating MRSA strains. The enhanced effectiveness is attributed to the improved bioavailability and sustained release of the antibiotic at the target sites, which facilitates higher concentrations at the infection site for extended periods. This results in a stronger therapeutic effect while reducing the required dosage, making it particularly beneficial for treating antibiotic-resistant pathogens like MRSA. Liposomal delivery systems can potentially minimize adverse side effects and decrease the likelihood of resistance development by allowing for lower dosages. The consistent efficacy of liposome-encapsulated vancomycin against various MRSA strains suggests it could be a versatile and reliable treatment option in clinical settings. Furthermore, the successful use of locally sourced phospholipids from egg yolk as a cost-effective alternative to commercially available lecithin highlights an innovative approach that could be especially beneficial in resource-limited settings. In summary, liposome-encapsulated antibiotics offer a promising advancement in the fight against antibiotic-resistant bacteria, providing improved bioavailability, sustained release, and reduced dosage requirements. Future research should aim to optimize these formulations further, evaluate their in vivo efficacy, and explore their potential in combination therapies to maximize clinical impact. Limitation of the study Financial limitations are detrimental to research. The most difficult aspect was using rare and advanced equipment. For example, such as Zeta (ζ) potential technique, and dynamic light scattering (DLS). It is important to determine the external characteristics of liposome formation by fatty acids when separating liposomes from raw materials. It is also necessary to check the dosage of the antibiotic after encapsulating it in liposomes. Declarations Conflicts of interest The authors declare that they have no conflicts of interest regarding the publication of this article. Ethics approval and consent to participate The IRB Committee of the Mongolian National University of Medical Sciences approved the study protocol (№2021/3 − 02, approved on 06/04/2022). Consent for publication All authors agree to be published. Author contribution Enkhtaivan Erdene conceived and designed the study, contributed to the acquisition, analysis, and interpretation of the data, and was responsible for drafting, editing, and submitting the manuscript. Odonchimeg Munkhjargal contributed to the acquisition and analysis of data, as well as the collection of samples. Baatarkhuu Oidov significantly influenced the study design and critically appraised the manuscript. Enkhjargal Dorjbal contributed to the study design. Ariunsanaa Byambaa contributed to the study design, data analysis, and interpretation, and reviewed the manuscript. All authors reviewed, discussed, and approved the final manuscript. Acknowledgments The authors would like to thank the Mongolian Academy of Science, Institute of Chemistry and Chemical Technology for providing the data of the experiment and for kind support. 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The Vast and Varied Global Burden of Norovirus : Prospects for Prevention and Control. PLOS Medicine , 13 (4), e1001999. https://doi.org/10.1371/journal.pmed.1001999 McAdam, P. R., Templeton, K. E., Edwards, G. F., Holden, M. T. G., Feil, E. J., Aanensen, D. M., Bargawi, H. J. A., Spratt, B. G., Bentley, S. D., Parkhill, J., Enright, M. C., Holmes, A., Girvan, E. K., Godfrey, P. A., Feldgarden, M., Kearns, A. M., Rambaut, A., Robinson, D. A., & Fitzgerald, J. R. (2012). Molecular tracing of the emergence, adaptation, and transmission of hospital-associated methicillin-resistant Staphylococcus aureus . Proceedings of the National Academy of Sciences , 109 (23), 9107–9112. https://doi.org/10.1073/pnas.1202869109 Mendelson, M., Sharland, M., & Mpundu, M. (2022). Antibiotic resistance: Calling time on the ‘silent pandemic.’ JAC-Antimicrobial Resistance , 4 (2), dlac016. https://doi.org/10.1093/jacamr/dlac016 O’Neill, J. (2016). Tackling drug-resistant infections globally: Final report and recommendations. Rabiei, M., Kashanian, S., Samavati, S. S., Jamasb, S., & McInnes, S. J. P. (2020). Nanomaterial and advanced technologies in transdermal drug delivery. Journal of Drug Targeting , 28 (4), 356–367. https://doi.org/10.1080/1061186X.2019.1693579 Rudan, I. (2008). Epidemiology and etiology of childhood pneumonia . Bulletin of the World Health Organization , 86 (5), 408–416. https://doi.org/10.2471/BLT.07.048769 Rudd, K. E., Johnson, S. C., Agesa, K. M., Shackelford, K. A., Tsoi, D., Kievlan, D. R., Colombara, D. V., Ikuta, K. S., Kissoon, N., Finfer, S., Fleischmann-Struzek, C., Machado, F. R., Reinhart, K. K., Rowan, K., Seymour, C. W., Watson, R. S., West, T. E., Marinho, F., Hay, S. I., … Naghavi, M. (2020). Global, regional, and national sepsis incidence and mortality, 1990–2017: Analysis for the Global Burden of Disease Study. The Lancet , 395 (10219), 200–211. https://doi.org/10.1016/S0140-6736(19)32989-7 Rybak, M. J. (2006). The Pharmacokinetic and Pharmacodynamic Properties of Vancomycin. Clinical Infectious Diseases , 42 (Supplement_1), S35–S39. https://doi.org/10.1086/491712 Sands, K., Carvalho, M. J., Portal, E., Thomson, K., Dyer, C., Akpulu, C., Andrews, R., Ferreira, A., Gillespie, D., Hender, T., Hood, K., Mathias, J., Milton, R., Nieto, M., Taiyari, K., Chan, G. J., Bekele, D., Solomon, S., Basu, S., … Walsh, T. R. (2021). Characterization of antimicrobial-resistant Gram-negative bacteria that cause neonatal sepsis in seven low- and middle-income countries. Nature Microbiology , 6 (4), 512–523. https://doi.org/10.1038/s41564-021-00870-7 Sercombe, L., Veerati, T., Moheimani, F., Wu, S. Y., Sood, A. K., & Hua, S. (2015). Advances and Challenges of Liposome Assisted Drug Delivery. Frontiers in Pharmacology , 6 . https://doi.org/10.3389/fphar.2015.00286 Stryjewski, M. E., & Corey, G. R. (2014). Methicillin-Resistant Staphylococcus aureus : An Evolving Pathogen. Clinical Infectious Diseases , 58 (suppl 1), S10–S19. https://doi.org/10.1093/cid/cit613 Surewaard, B. G. J., Deniset, J. F., Zemp, F. J., Amrein, M., Otto, M., Conly, J., Omri, A., Yates, R. M., & Kubes, P. (2016). Identification and treatment of the Staphylococcus aureus reservoir in vivo. Journal of Experimental Medicine , 213 (7), 1141–1151. https://doi.org/10.1084/jem.20160334 Sutradhar, K. B., & Amin, Md. L. (2013). Nanoemulsions: Increasing possibilities in drug delivery. European Journal of Nanomedicine , 5 (2). https://doi.org/10.1515/ejnm-2013-0001 Tong, S. Y. C., Davis, J. S., Eichenberger, E., Holland, T. L., & Fowler, V. G. (2015). Staphylococcus aureus Infections: Epidemiology, Pathophysiology, Clinical Manifestations, and Management. Clinical Microbiology Reviews , 28 (3), 603–661. https://doi.org/10.1128/CMR.00134-14 Troeger, C., Blacker, B., Khalil, I. A., Rao, P. C., Cao, J., Zimsen, S. R. M., Albertson, S. B., Deshpande, A., Farag, T., Abebe, Z., Adetifa, I. M. O., Adhikari, T. B., Akibu, M., Al Lami, F. H., Al-Eyadhy, A., Alvis-Guzman, N., Amare, A. T., Amoako, Y. A., Antonio, C. A. T., … Reiner, R. C. (2018). Estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory infections in 195 countries, 1990–2016: A systematic analysis for the Global Burden of Disease Study 2016. The Lancet Infectious Diseases , 18 (11), 1191–1210. https://doi.org/10.1016/S1473-3099(18)30310-4 Troeger, C., Khalil, I. A., Rao, P. C., Cao, S., Blacker, B. F., Ahmed, T., Armah, G., Bines, J. E., Brewer, T. G., Colombara, D. V., Kang, G., Kirkpatrick, B. D., Kirkwood, C. D., Mwenda, J. M., Parashar, U. D., Petri, W. A., Riddle, M. S., Steele, A. D., Thompson, R. L., … Reiner, R. C. (2018). Rotavirus Vaccination and the Global Burden of Rotavirus Diarrhea Among Children Younger Than 5 Years. JAMA Pediatrics , 172 (10), 958. https://doi.org/10.1001/jamapediatrics.2018.1960 Ventola, C. L. (2015). The antibiotic resistance crisis: Part 1: Causes and threats. Pharmacy and Therapeutics , 40 (4), 277. Weiner, L. M., Webb, A. K., Limbago, B., Dudeck, M. A., Patel, J., Kallen, A. J., Edwards, J. R., & Sievert, D. M. (2016). Antimicrobial-Resistant Pathogens Associated with Healthcare-Associated Infections: Summary of Data Reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2011–2014. Infection Control & Hospital Epidemiology , 37 (11), 1288–1301. https://doi.org/10.1017/ice.2016.174 World Health Organization. (2014). Antimicrobial resistance: Global report on surveillance . World Health Organization. https://iris.who.int/handle/10665/112642 World Health Organization. (2015). Global action plan on antimicrobial resistance (Geneva, Switzerland: World Health Organization) . Zetola, N., Francis, J. S., Nuermberger, E. L., & Bishai, W. R. (2005). Community-acquired methicillin-resistant Staphylococcus aureus : An emerging threat. The Lancet Infectious Diseases , 5 (5), 275–286. https://doi.org/10.1016/S1473-3099(05)70112-2 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5084652","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":371992600,"identity":"17a92bbc-6d17-45c0-916c-e6df6e792d58","order_by":0,"name":"Enkhtaivan Erdene","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-6645-9354","institution":"Etugen Institute: Etugen University","correspondingAuthor":true,"prefix":"","firstName":"Enkhtaivan","middleName":"","lastName":"Erdene","suffix":""},{"id":371992601,"identity":"5766b97f-e2e6-489b-b976-59847434dab3","order_by":1,"name":"Odonchimeg Munkhjargal","email":"","orcid":"","institution":"Mongolian Academy of Sciences Institute of Chemistry and Chemical Technology","correspondingAuthor":false,"prefix":"","firstName":"Odonchimeg","middleName":"","lastName":"Munkhjargal","suffix":""},{"id":371992602,"identity":"54cfdced-7e8d-4c56-8a0b-f77ca91b1f22","order_by":2,"name":"Enkhjargal Dorjbal","email":"","orcid":"","institution":"Mongolian National University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Enkhjargal","middleName":"","lastName":"Dorjbal","suffix":""},{"id":371992603,"identity":"5bbcd5cc-7c2e-4919-ac69-0004c09bcd30","order_by":3,"name":"Baatarkhuu Oidov","email":"","orcid":"","institution":"Mongolian National University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Baatarkhuu","middleName":"","lastName":"Oidov","suffix":""},{"id":371992604,"identity":"a4a5398e-e564-4706-890a-17cc59411998","order_by":4,"name":"Ariunsanaa Byambaa","email":"","orcid":"https://orcid.org/0000-0001-8974-2892","institution":"Mongolian National University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ariunsanaa","middleName":"","lastName":"Byambaa","suffix":""}],"badges":[],"createdAt":"2024-09-13 14:52:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5084652/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5084652/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68711760,"identity":"6d9a4dbd-ee46-4936-abbf-15d46a73c23f","added_by":"auto","created_at":"2024-11-11 09:21:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":132729,"visible":true,"origin":"","legend":"\u003cp\u003eViolet-red spectrogram of phospholipids isolated from egg yolk\u003c/p\u003e","description":"","filename":"fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5084652/v1/253decf33aeed2d5c8e4095d.png"},{"id":68712940,"identity":"022b02e3-ecdf-49eb-86f3-197c12a5a4cd","added_by":"auto","created_at":"2024-11-11 09:29:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":154562,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAtomic Force Microscopy (AFM) Analysis of Liposome Morphology. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e AFM Image of Liposome Slices, \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e) \u003cstrong\u003eCurves on Slices, \u003c/strong\u003e(\u003cstrong\u003eC\u003c/strong\u003e) \u003cstrong\u003eExample Curve Defining Height and Diameter on Slices\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5084652/v1/7fbd8d33ebbcb62574626657.png"},{"id":70902432,"identity":"dcf41f4a-7af5-4148-9321-ee446aaeef9f","added_by":"auto","created_at":"2024-12-09 06:05:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":889251,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5084652/v1/9ea82107-8d18-4cc4-ae8e-0a7cc3303aab.pdf"}],"financialInterests":"","formattedTitle":"Efficacy of Liposome-Encapsulated Vancomycin Against Methicillin-Resistant Staphylococcus aureus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe natural environment allows humans to coexist with microorganisms, yet bacterial infections significantly contribute to the global rise in infectious diseases. Bacterial infections are a leading cause of morbidity and mortality worldwide with mortality from infectious diseases including community-acquired pneumonia (CAP) at 2.5\u0026nbsp;million annually, leptospirosis at 58,900, typhoid fever between 75,000 and 208,000, and diarrhea at 1,560 (O\u0026rsquo;Neill, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ventola, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; World Health Organization, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Troeger, Blacker, et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rudd et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHigh mortality rates from bacterial infections, particularly in premature infants and young children, are closely associated with the spread of bacteria resistant to multiple antibiotics. This resistance is linked to the functional and structural characteristics of bacteria, including mutations and the transfer of resistance genes (Costa et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Crump, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Lopman et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Rudan, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Troeger, Khalil, et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). According to the 2019 report \"The Threat of Antibiotic Resistance in the United States,\" antibiotic-resistant pathogens cause over 2.8\u0026nbsp;million infections and more than 35,000 deaths annually. Europe reports 25,000 deaths per year due to antibiotic-resistant infections, while Asia (specifically India) reports 700,000 deaths annually. Projections indicate that by 2050, antibiotic-resistant bacterial infections will be among the leading causes of death globally (Cassini et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Holmes et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Laxminarayan et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; L. Liu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; McAdam et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sands et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; World Health Organization, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne of the leading causes of death from infectious diseases is \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e). \u003cem\u003eS. aureus\u003c/em\u003e not only persists on environmental surfaces but also colonizes the nasal cavity and skin of humans and animals, facilitating both direct and indirect transmission. Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA), a common cause of skin infections, is resistant to most antibiotics, complicating treatment (Centers for Disease Control and Prevention (U.S.), \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hiramatsu, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Weiner et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A 2023 World Health Organization (WHO) study reported \u003cem\u003eS. aureus\u003c/em\u003e prevalence rates of 22.27% in the Americas, 16.57% in the Western Pacific, and 10.93% in Europe. Single studies in the Eastern Mediterranean and African regions showed prevalence rates of 8.55% and 9.04%, respectively. Countries with multiple surveys, such as the United States (23.78%), Great Britain (18.66%), and China (18.07%), exhibited the highest prevalence rates.\u003c/p\u003e \u003cp\u003eResearch indicates that approximately 33% of people carry \u003cem\u003eS. aureus\u003c/em\u003e in their noses asymptomatically, with 2% carrying MRSA. While many carriers do not develop serious MRSA infections, significant progress has been made in reducing MRSA bloodstream infections, which decreased by 17.1% annually from 2005 to 2012. However, the decline slowed between 2013 and 2016, with no significant changes noted. Studies have reported a concurrent increase in MRSA CAP and a decrease in \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e CAP strains following the implementation of pneumococcal vaccines, particularly in cases complicated by pleurisy. Consequently, staphylococcal infections are rising due to the increase in MRSA infections (Chambers \u0026amp; DeLeo, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Laxminarayan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mendelson et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Tong et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBeta-lactam antibiotics are commonly used to treat staphylococcal infections, but the emergence of beta-lactamase-resistant \u003cem\u003eS. aureus\u003c/em\u003e strains poses significant challenges. Antibiotics such as vancomycin, linezolid, daptomycin, tigecycline, and telavancin are frequently used against MRSA in skin, soft tissue, and postoperative infections (Zetola et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Monitoring and analyzing data on antibacterial drug use is crucial for developing and implementing policies and interventions to promote appropriate antibiotic use at the national level (Stryjewski \u0026amp; Corey, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In Mongolia, amoxicillin, amoxicillin/beta-lactamase inhibitor, ampicillin, and doxycycline accounted for 75% of oral antibiotic consumption in 2018. A study in 13 Southeastern European countries found these drugs dominated antibiotic use. Antibiotics are categorized into three groups based on their mechanism of action: inhibition of protein synthesis, nucleic acid synthesis, and cell wall synthesis (Alkilani et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; C. Liu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Rabiei et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResistance to vancomycin is linked to the Van gene, which encodes various resistance phenotypes. Vancomycin resistance is classified into several gene clusters based on DNA sequences of homologs of the ligase Van gene, which encodes the key enzyme for synthesizing D-alanyl D-lactate or D-alanyl D-serine (D-Ala D-Ser). At least 11 Van gene clusters have been identified, corresponding to the vanA, vanB, vanD, vanF, vanI, vanM, vanC, vanE, vanG, vanL, and vanN phenotypes, conferring vancomycin resistance. Genes encoding D-alanyl D-lactate ligases, such as vanA, vanB, vanD, vanF, vanI, and vanM, typically confer high levels of vancomycin resistance, whereas genes encoding D-alanyl D-serine ligases, including vanC, vanE, vanG, vanL, and vanN, generally confer lower resistance (Allen \u0026amp; Cullis, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNanotechnology has revolutionized medicine, particularly in diagnosing diseases, improving methods for preventing and treating cancer, hereditary, and acquired diseases, producing new-generation drugs, and creating effective treatments. Since Aleika Benkham introduced this concept in 1965, advancements in liposome extraction and physicochemical methods have facilitated the modeling of biomembrane functions and liposome structures. Liposomes serve as model systems representing cell surface and biomembrane quality, playing a crucial role in experiments involving antibiotics, cancer drugs, and nucleic acids (Sercombe et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNanotechnology has transformed transdermal drug therapy by providing new strategies to overcome the skin's natural barrier (Sutradhar \u0026amp; Amin, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Liposomes, developed based on nanotechnology, are drug delivery systems consisting of one or more lipid layers, spherical in shape, and surrounded by an aqueous core filled with hydrophilic compounds. The lipophilic layer, filled with hydrophobic compounds, is biocompatible and biodegradable. Nanosized drug carriers such as liposomes, niosomes, and micelles are designed to deliver drugs through the stratum corneum, the outermost layer of the skin, which poses a significant challenge for transdermal infections (Courvalin, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Hiramatsu, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMany liposomal formulations have been FDA-approved for clinical use due to their enhanced drug delivery and safety profiles. These include liposomal amphotericin B for fungal infections and lipoplatin preparations for breast cancer. Other formulations have shown consistent success in preclinical and clinical trials (Rybak, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The necessity for active transport mechanisms for drugs to reach their biomolecules is emphasized in numerous studies, highlighting the importance of ongoing research and development in this field.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eMethodology for the Extraction of Phospholipids\u003cem\u003e:\u0026nbsp;\u003c/em\u003eGladkowski et al. (2012) developed a standardized protocol for isolating phospholipids from egg yolk, which served as the primary raw material for their research experiment. The procedure began with breaking the egg and weighing the yolk. The yolk was then mixed with acetone and continuously stirred using a magnetic stirrer for 10 minutes. The mixture was transferred to a glass flask and centrifuged at 3000 rpm for 10 minutes. After separation and drying of the precipitate, a mixture of chloroform and ethanol in a ratio of 2:1 was added, and the mixture was stirred thoroughly for three hours. The resulting solution was then filtered, and the organic solvent was separated to form a thin layer of lipids.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eIdentification of phospholipids by infrared spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInfrared spectroscopy was employed to determine the absorption (vibration) spectrum of the phospholipids. The samples were measured in the wavelength range of 400-4000 cm\u003csup\u003e-1\u003c/sup\u003e using a choline and tungsten light source with KBr pellets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLiposome preparation and drug encapsulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe freeze-thaw method was utilized for liposome preparation and drug encapsulation. A lipid film was prepared using egg-phosphatidylcholine (egg-PC) and cholesterol at a molar ratio of 4:1, with a total lipid content of 4 mg. The lipid films were rehydrated with 1.5 mL of phosphate-buffered saline (PBS) at pH 7.4 containing 0.01 g of vancomycin. A thin film was formed by connecting a round-bottom flask to a rotary evaporator and subjecting the solvent to three cycles of freezing at -20\u0026deg;C for 20 minutes and thawing at +45\u0026deg;C for 10 minutes. The heated vancomycin solution was added to the thin film, and the mixture was swirled in the round-bottom flask to form monolayer vesicles. After vortexing for 10 minutes, the round-bottom flask was removed from the rotary evaporator, and the contents were sonicated for 30 seconds. Immediately after sonication, the dispersion was extruded using a mini-extruder (Avanti Polar Lipids Inc., Alabaster, AL) to obtain a monodisperse\u0026nbsp;liposome-encapsulated vancomycin\u0026nbsp;formulation. The prepared\u0026nbsp;liposome-encapsulated vancomycin\u0026nbsp;was then flushed with nitrogen gas, and CMP solution was added at a molar ratio of 1:1 (CMP: DSPE-PEG-Mal). Thiol-maleimide click chemistry was allowed to proceed overnight to yield the CMP-\u0026nbsp;liposome-encapsulated vancomycin\u0026nbsp;formulation, followed by dialysis to remove excess vancomycin and CMP. Finally, the dialyzed CMP-liposome-encapsulated vancomycin\u0026nbsp;was lyophilized in the presence of 20 mM sucrose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParticle diameter and polydispersity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticle diameter and polydispersity index (PDI) with atomic force microscopy (AFM). The final particle diameter was determined using the Stokes\u0026ndash;Einstein equation, while the PDI was estimated using Nano DTS software (version 6.34). All measurements were performed in at least three sets of 10 runs at 25\u0026deg;C. The polydispersity index was calculated using the following formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" height=\"144\" width=\"729\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere std is the standard deviation of the diameter and avg is the average diameter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacteria culture methodology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923 and MRSA ATCC 2758 were cultured in a normal medium and spread evenly on Baird-Parker (BP) agar (Biolab, Hungary), a selective medium. The cultures were incubated at 37\u0026deg;C for 24 hours. Colonies grown on BP agar were concluded to be \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Two to three separate colonies were selected, and a bacterial suspension to 0.5 McFarland standard was prepared and diluted in 0.9% physiological solution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLiposome-encapsulated antibiotic solution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo prepare the liposome-encapsulated antibiotic solution, 0.01 g of antibiotics was dissolved in 1 mL of distilled water. A working solution containing 100 \u0026mu;L of 0.8 \u0026mu;g antibiotic was prepared. For comparison, a pure antibiotic solution was prepared by dissolving 0.01 g of antibiotics in 1 mL of distilled water. A working solution containing 100 \u0026mu;L of 1 \u0026mu;g antibiotic was prepared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrodilution method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe microdilution method was performed using a 96-well reagent plate. Each well received 50-100 \u0026mu;L of antibiotic solution and an equal amount of bacterial suspension. For microdilution, six wells were used with Mueller-Hinton broth, with the antibiotic solution diluted to final concentrations of 1, 0.5, 0.25, 0.12, 0.06, and 0.03 \u0026mu;g/mL. Additionally, 10 \u0026mu;L of a 10\u003csup\u003e5\u003c/sup\u003e CFU/mL bacterial suspension (\u003cem\u003eS. aureus\u003c/em\u003e or MRSA) was added to each well. Negative controls included the antibiotic solution without bacteria, and positive controls included Mueller-Hinton broth with bacteria. The test wells contained Mueller-Hinton broth, MRSA, and the antibiotic solution.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003eEvaluation of minimal inhibitory concentration (MIC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe minimal inhibitory concentration (MIC) of free vancomycin and liposome-encapsulated vancomycin was evaluated using the CLSI guidelines. The MIC was calculated as the lowest concentration of antibiotic that completely inhibited bacterial growth after 18-20 hours of incubation at 35-37\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003eAnalysis of\u003c/strong\u003e \u003cstrong\u003ehigh-performance liquid chromatography (HPLC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalyses were performed on an Agilent 1260 Infinity HPLC system equipped with a C18 reversed-phase column. The mobile phase consisted of acetonitrile and water with 0.1% formic acid, with UV detection at 282 nm. The flow rate was set to 1 mL/min, and the injection volume was 10 \u0026micro;L. Data analysis included a standard vancomycin solution at 1000 ppm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was conducted using IBM SPSS Statistics software, version 22.0. Group differences were assessed through analysis of variance (ANOVA). Statistical significance was determined at a threshold of p \u0026le; 0.05.\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe yield of phospholipids extracted from egg yolk is 37.3%. Infrared spectroscopy was utilized to analyze the composition and purity of the isolated phospholipids. The spectrogram obtained, ranging from 400 nm to 4000 nm, confirms the distinct characteristics of the tissue-derived phospholipids.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe spectrogram in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the infrared absorption characteristics of the phospholipids extracted from egg yolk, spanning wavenumbers from 4000 cm\u003csup\u003e1\u003c/sup\u003e to 500 cm\u003csup\u003e1\u003c/sup\u003e. Key absorption peaks and their corresponding functional groups are identified, providing insights into the composition and purity of the isolated phospholipids. Significant absorption bands are observed in the following regions:O\u0026ndash;H Stretching (Decreased Intensity): The intensity of the absorption associated with the hydroxyl (-OH) group is reduced, indicating a lower presence of these groups in the sample. C\u0026ndash;H Stretching: Peaks at approximately 2921 cm\u003csup\u003e1\u003c/sup\u003e and 2851 cm\u003csup\u003e1\u003c/sup\u003e correspond to the stretching vibrations of the C-H bonds in the aliphatic chains of the phospholipids. N(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e Group: A prominent absorption band at 1051 cm\u003csup\u003e-1\u003c/sup\u003e is attributed to the stretching vibrations of the N(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e group, which is characteristic of phosphatidylcholine, a major component of egg yolk phospholipids. P\u0026thinsp;=\u0026thinsp;O Stretching: The region between 1226 cm\u003csup\u003e1\u003c/sup\u003e and 1051 cm\u003csup\u003e1\u003c/sup\u003e exhibits strong absorption due to the P\u0026thinsp;=\u0026thinsp;O stretching vibrations, indicative of the phosphate groups present in the phospholipids. P\u0026ndash;O\u0026ndash;C Stretching: An absorption peak at 819 cm\u003csup\u003e1\u003c/sup\u003e is assigned to the P\u0026ndash;O\u0026ndash;C stretching vibrations, confirming the presence of phosphate ester groups.\u003c/p\u003e \u003cp\u003eThe spectroscopic analysis demonstrates that the extracted phospholipids retain their structural integrity, as evidenced by the distinct absorption peaks corresponding to key functional groups. The purity of the phospholipids is inferred from the absence of extraneous peaks, indicating minimal contamination or degradation during the extraction process. In summary, the infrared spectrogram provides a detailed molecular fingerprint of the isolated phospholipids, validating the efficacy of the extraction method and confirming the presence of characteristic phospholipid functional groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAnalysis of Liposome Morphology\u003c/strong\u003e \u003cp\u003eLiposome size and uniformity are critical parameters in various applications, including drug delivery systems, nanotechnology, and materials science. This study employed Atomic Force Microscopy (AFM) to determine the height, diameter, and measurement error of liposomes. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003e(a)\u003c/b\u003e displays the AFM image of liposome slices, where the positions of the slices are marked for further analysis. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003e(b)\u003c/b\u003e shows the curves corresponding to these slices, and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003e(c)\u003c/b\u003e illustrates an example curve used to define the height and diameter of the liposomes.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eHeight and Measurement Error: The average height of the liposomes was 43.2183 nm with a measurement error of 6.85 nm. AFM analysis confirmed their uniform size distribution and morphological uniformity.\u003c/p\u003e \u003cp\u003eAnalysis of Liposomes: High-Performance Liquid Chromatography (HPLC) was used to evaluate physicochemical properties. The retention time was 5.342 minutes, with a UV peak at 200 nm, confirming high purity (986 ppm). The mean diameter was 157.01385 nm with a standard deviation of 33.036 nm. A PDI of 0.0442 suggests a narrow size distribution, indicating uniformity and stability.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHeight, measurement error, and physicochemical parameters\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eError (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePDI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeight (nm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43.2183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParticle size (nm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e157.01385\u0026thinsp;\u0026plusmn;\u0026thinsp;33.036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0442\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAFM and HPLC analyses demonstrate that the liposomes have desirable size and stability characteristics, making them suitable for drug delivery and nanotechnology applications.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial Efficacy of Liposome-Encapsulated Vancomycin\u003c/h2\u003e \u003cp\u003eThe study evaluated the antibacterial efficacy of liposome-encapsulated vancomycin compared to free vancomycin. It was determined that liposomal vancomycin inhibited bacterial growth at doses twice as low as those required for free vancomycin. The results are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBacterial growth inhibitory activity of antibiotic-based liposomes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eMicrodilutions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eLiposomal vancomycin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eFree vancomycin\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMRSA\u003c/p\u003e \u003cp\u003eATCC 2758\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMRSA\u003c/p\u003e \u003cp\u003eATCC 2758\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e%(n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%(n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%(n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e%(n)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100(10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100(10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50(5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50(5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 \u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100(10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100(10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 \u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70(7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100(10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 \u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10(1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70(7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 \u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30(3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 \u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLiposomal vancomycin demonstrated superior efficacy in inhibiting the growth of both Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) ATCC 2758 and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923 strains. At dilutions of 10\u003csup\u003e1\u003c/sup\u003e and 10\u003csup\u003e2\u003c/sup\u003e, liposome-encapsulated vancomycin completely inhibited bacterial growth (100%), whereas free vancomycin achieved only 50% inhibition at 10\u003csup\u003e1\u003c/sup\u003e dilution and was ineffective at higher dilutions.\u003c/p\u003e \u003cp\u003eFor MRSA ATCC 2758, liposomal vancomycin maintained 70% growth inhibition at a 10\u003csup\u003e3\u003c/sup\u003e dilution, while free vancomycin showed no inhibitory effect at this dilution. At a 10\u003csup\u003e4\u003c/sup\u003e dilution, liposomal vancomycin exhibited 10% inhibition, whereas free vancomycin remained ineffective.\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923, liposomal vancomycin consistently inhibited 100% growth up to a 10\u003csup\u003e3\u003c/sup\u003e dilution and retained 70% inhibition at a 10\u003csup\u003e4\u003c/sup\u003e dilution. Free vancomycin showed no inhibitory effect beyond the 10\u003csup\u003e1\u003c/sup\u003e dilution.\u003c/p\u003e \u003cp\u003eOverall, liposome-based vancomycin demonstrated significantly higher antibacterial activity compared to free vancomycin, achieving comparable or superior inhibition at lower doses. Statistical analysis confirmed the enhanced efficacy of liposome-encapsulated vancomycin with a p-value of 0.02, indicating a significant difference in performance.\u003c/p\u003e \u003cp\u003eThese results indicate that vancomycin-encapsulated liposomes have a distinct and more potent inhibitory effect on MRSA and \u003cem\u003eS. aureus\u003c/em\u003e strains compared to free vancomycin. This suggests that liposomal formulations could enhance the therapeutic efficacy and potentially reduce the required dosage of vancomycin in clinical settings.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe current study investigated the antibacterial efficacy of liposome-encapsulated vancomycin against Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA), a significant global public health concern since its emergence in the 1960s. With a reported prevalence of 14.69% among 164,717 individuals across 29 countries, MRSA has remained a formidable pathogen due to its resistance to standard β-lactam antibiotics (A. Abdelkader et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Surewaard et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the late 1990s, the advent of community-associated MRSA (CA-MRSA) infections further complicated the scenario, as these infections affected healthy individuals without traditional risk factors and displayed high virulence, primarily causing skin and soft tissue infections, necrotizing pneumonia, and necrotizing fasciitis.\u003c/p\u003e \u003cp\u003eGiven these challenges, our study focused on evaluating the potential of liposome-based vancomycin formulations. Liposomes, due to their lipid bilayer structure similar to cellular membranes, offer a unique advantage in antibiotic delivery. They enhance the concentration of antibiotics at the site of infection for extended durations, thereby improving antibacterial efficacy. This study's methodology involved extracting phospholipids from egg yolk using acetone, ethanol, and chloroform, resulting in a yield of 37.9%. This method was chosen based on previous research by Fahimeh Hajiahmadi et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which demonstrated higher encapsulation efficiency of vancomycin using the freeze-thaw method over the ammonium sulfate gradient method (Hajiahmadi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVancomycin was selected for encapsulation due to its widespread use in treating MRSA infections, particularly in Mongolia. Analytical techniques, including chromatography and UV spectroscopy, confirmed the integrity and purity of the encapsulated vancomycin, with consistent retention times and characteristic absorption peaks at 200 nm and 280 nm (Y. Liu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These results ensured the reliability of our encapsulation method and the stability of the antibiotic within the liposome formulation.\u003c/p\u003e \u003cp\u003eOur microdilution studies demonstrated that liposome-encapsulated vancomycin exhibited significantly higher antibacterial activity against MRSA strains compared to free vancomycin [43]. Notably, liposomal vancomycin achieved complete growth inhibition at dilutions of 10^-1 and 10^-2, whereas free vancomycin only partially inhibited growth at 10^-1 and was ineffective at higher dilutions. Moreover, liposomal vancomycin maintained substantial inhibitory activity at 10^-3 and 10^-4 dilutions, underscoring its enhanced potency.\u003c/p\u003e \u003cp\u003eThese findings align with previous research by Abdelkader et al. (2012), Surewaard B. (2016), and Liu Y. (2019), which reported that liposome-encapsulated antibiotics are effective at lower doses than their free counterparts (H. Abdelkader \u0026amp; G. Alany, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Y. Liu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Surewaard et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The increased efficacy of liposomal vancomycin can be attributed to the sustained release and prolonged bioavailability of the antibiotic at the site of infection, ensuring higher concentrations over longer periods.\u003c/p\u003e \u003cp\u003eFrom the above results it can be that the application of liposome-encapsulated vancomycin observed in this study has significant clinical implications. By reducing the required dosage and increasing the duration of effective drug concentration, liposomal formulations can potentially minimize the risk of resistance development and adverse side effects associated with high-dose antibiotic therapies. Additionally, the ability of liposomes to deliver antibiotics directly to the site of infection may improve treatment outcomes for severe MRSA infections, including those caused by CA-MRSA strains.\u003c/p\u003e \u003cp\u003eOur study demonstrates the superior efficacy of liposome-encapsulated vancomycin over its free form, showing significant advantages in treating MRSA strains. The enhanced effectiveness is attributed to the improved bioavailability and sustained release of the antibiotic at the target sites, which facilitates higher concentrations at the infection site for extended periods. This results in a stronger therapeutic effect while reducing the required dosage, making it particularly beneficial for treating antibiotic-resistant pathogens like MRSA. Liposomal delivery systems can potentially minimize adverse side effects and decrease the likelihood of resistance development by allowing for lower dosages. The consistent efficacy of liposome-encapsulated vancomycin against various MRSA strains suggests it could be a versatile and reliable treatment option in clinical settings. Furthermore, the successful use of locally sourced phospholipids from egg yolk as a cost-effective alternative to commercially available lecithin highlights an innovative approach that could be especially beneficial in resource-limited settings. In summary, liposome-encapsulated antibiotics offer a promising advancement in the fight against antibiotic-resistant bacteria, providing improved bioavailability, sustained release, and reduced dosage requirements. Future research should aim to optimize these formulations further, evaluate their \u003cem\u003ein vivo\u003c/em\u003e efficacy, and explore their potential in combination therapies to maximize clinical impact.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLimitation of the study\u003c/h2\u003e \u003cp\u003eFinancial limitations are detrimental to research. The most difficult aspect was using rare and advanced equipment. For example, such as Zeta (ζ) potential technique, and dynamic light scattering (DLS). It is important to determine the external characteristics of liposome formation by fatty acids when separating liposomes from raw materials. It is also necessary to check the dosage of the antibiotic after encapsulating it in liposomes.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest regarding the publication of this article.\u003c/p\u003e\n\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eThe IRB Committee of the Mongolian National University of Medical Sciences approved the study protocol (№2021/3\u0026thinsp;\u0026minus;\u0026thinsp;02, approved on 06/04/2022).\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eAll authors agree to be published.\u003c/p\u003e\n\u003ch2\u003eAuthor contribution\u003c/h2\u003e\n\u003cp\u003eEnkhtaivan Erdene conceived and designed the study, contributed to the acquisition, analysis, and interpretation of the data, and was responsible for drafting, editing, and submitting the manuscript. Odonchimeg Munkhjargal contributed to the acquisition and analysis of data, as well as the collection of samples. Baatarkhuu Oidov significantly influenced the study design and critically appraised the manuscript. Enkhjargal Dorjbal contributed to the study design. Ariunsanaa Byambaa contributed to the study design, data analysis, and interpretation, and reviewed the manuscript. All authors reviewed, discussed, and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThe authors would like to thank the Mongolian Academy of Science, Institute of Chemistry and Chemical Technology for providing the data of the experiment and for kind support. We would like to express our gratitude to the management of Etugen University for the staff of the Department of Microbiology, Infection Prevention and Control, School of Biomedicine, Mongolian National University of Medical Sciences, those who provided the lab facilities. We express our deep gratitude to those who participated in this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbdelkader, A., El-Mokhtar, M. A., Abdelkader, O., Hamad, M. A., Elsabahy, M., \u0026amp; El-Gazayerly, O. N. (2017). Ultrahigh antibacterial efficacy of meropenem-loaded chitosan nanoparticles in a septic animal model. \u003cem\u003eCarbohydrate Polymers\u003c/em\u003e, \u003cem\u003e174\u003c/em\u003e, 1041\u0026ndash;1050. https://doi.org/10.1016/j.carbpol.2017.07.030\u003c/li\u003e\n \u003cli\u003eAbdelkader, H., \u0026amp; G. Alany, R. (2012). Controlled and Continuous Release Ocular Drug Delivery Systems: Pros and Cons. \u003cem\u003eCurrent Drug Delivery\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(4), 421\u0026ndash;430. https://doi.org/10.2174/156720112801323125\u003c/li\u003e\n \u003cli\u003eAlkilani, A., McCrudden, M. T., \u0026amp; Donnelly, R. (2015). 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Community-acquired methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e: An emerging threat. \u003cem\u003eThe Lancet Infectious Diseases\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e(5), 275\u0026ndash;286. https://doi.org/10.1016/S1473-3099(05)70112-2\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lecithin, Liposome, Vancomycin, MRSA, Nanocarriers","lastPublishedDoi":"10.21203/rs.3.rs-5084652/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5084652/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacterial infections significantly contribute to global morbidity and mortality, with antibiotic-resistant strains, such as Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA), presenting severe treatment challenges. This study investigates the potential of liposome-encapsulated vancomycin as a novel treatment to combat antibiotic resistance. Phospholipids were extracted from egg yolk, and liposomes were prepared using the freeze-thaw method. The liposome-encapsulated vancomycin formulation was characterized using infrared spectroscopy, atomic force microscopy (AFM), and high-performance liquid chromatography (HPLC). Infrared spectroscopy confirmed the structural integrity and purity of the phospholipids, while AFM revealed a uniform liposome size, with an average diameter of 157 nm and a polydispersity index (PDI) of 0.0442, indicating high stability and suitability for drug delivery.\u003c/p\u003e \u003cp\u003eThe antibacterial efficacy of liposome-encapsulated vancomycin was tested against MRSA and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, demonstrating that the encapsulated vancomycin inhibited bacterial growth at doses significantly lower than free vancomycin. Liposome-encapsulated vancomycin achieved 100% inhibition of MRSA and \u003cem\u003eS. aureus\u003c/em\u003e at higher dilutions, while free vancomycin was only partially effective. These findings highlight the enhanced potential of liposome-based drug delivery in reducing bacterial load and overcoming antibiotic resistance. The study emphasizes the promise of nanotechnology in improving antibiotic efficacy, offering a potential solution to the global health crisis posed by antibiotic-resistant pathogens.\u003c/p\u003e","manuscriptTitle":"Efficacy of Liposome-Encapsulated Vancomycin Against Methicillin-Resistant Staphylococcus aureus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-11 09:21:50","doi":"10.21203/rs.3.rs-5084652/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"385d16e1-b6d6-4d3a-b537-2630e3d9b969","owner":[],"postedDate":"November 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-09T05:57:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-11 09:21:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5084652","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5084652","identity":"rs-5084652","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}