Targeted Delivery of Linezolid Using Magnetic Nanoparticles to Enhance Osteomyelitis Treatment

Targeted Delivery of Linezolid Using Magnetic Nanoparticles to Enhance Osteomyelitis Treatment | 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 Targeted Delivery of Linezolid Using Magnetic Nanoparticles to Enhance Osteomyelitis Treatment mohammadjavad nasirpour, mohammadjavad raee, ahmad gholami This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6133116/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 Osteomyelitis, a challenging bone infection, is often complicated by formation of biofilms and antibiotic resistance. Traditional treatments typically involve prolonged high-dose antibiotic regimens, which can lead to various adverse effects. This research focuses on creating a targeted drug delivery system utilizing magnetic nanoparticles to improve the effectiveness of linezolid in the treatment of osteomyelitis. Magnetic nanoparticles were synthesized and functionalized with L-lysine. Subsequently, these nanoparticles were coated with chitosan and loaded with linezolid. The nanoparticles were characterized using TEM, FESEM, XRD, FTIR, and VSM. Antibacterial activity was assessed using the microdilution broth method against major osteomyelitis pathogens. The study successfully developed a novel drug delivery system based on magnetic nanoparticles. Compared to free linezolid The synthesized nanoparticles exhibited enhanced antibacterial activity. The nanoparticle structure facilitated deeper penetration of linezolid into bacterial cells, leading to a higher bactericidal effect and potentially lower required treatment doses. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 1. Introduction Osteomyelitis is a serious infectious disease that primarily affects bone marrow.(1) Treatment of osteomyelitis faces serious challenges due to the phenomenon of antibiotic resistance, biofilm formation, and intracellular infection by Staphylococcus aureus bacteria, which cause frequent recurrence of infection.(2) Due to the limited ability of antibiotics to penetrate bone tissue and the increased antibiotic resistance of bacteria within biofilms, treating osteomyelitis often requires lengthy courses of high-dose antibiotics, which can result in severe adverse effects.(3, 4) Conventional treatment for osteomyelitis typically involves a 4- to 6-week course of systemic antibiotics. However, the ideal duration of antibiotic therapy for chronic osteomyelitis remains unclear and the rate of re-infection is still high(5, 6) Prolonged linezolid therapy for osteomyelitis has been associated with several adverse events, including hematological toxicities such as thrombocytopenia (7)،anemia (8)and bone marrow suppression. Other reported side effects include fatigue, gastrointestinal intolerance and neuropathy.(9)There is a growing interest in exploring novel therapeutic strategies, particularly in the context of osteomyelitis treatment. Nanotechnology has emerged as a promising approach for targeted drug delivery and biofilm disruption. Specifically, superparamagnetic iron oxide nanoparticles (SPIONs) are being investigated for their ability to deliver antibiotics directly to infected tissues when guided by an external magnetic field. This method enhances antibiotic penetration and disrupts the biofilm matrix, potentially leading to more effective eradication of persistent infections.(10, 11) This study aimed to develop a new drug delivery system to improve the treatment of osteomyelitis By using magnetic nanoparticles coated with chitosan and loaded with the antibiotic linezolid, the goal was to increase the concentration of the drug at the infection site, reduce the overall drug dosage, and minimize side effects while combating antibiotic resistance. The antibacterial properties of this nanoantibiotic were assessed against four pathogenic bacterial strains, including Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa . 2. Material and methods Material All reagents were of analytical grade and used without further purification. FeCl 3 .6H 2 O, FeSO 4 .7H 2 O, NH 3, Acetic Acid and PBS were purchased from Merck. L-lysine, Linezolid and Chitosan (low molecular weight) were obtained from Sigma-aldrich. Method Preparation of IONPs L-lysine-functionalized IONPs synthesis was performed according to the previously reported co-precipitation method(12). Briefly FeCl3·6H2O and FeSO4·7H2O were dissolved in distilled water. An aqueous solution of iron salts was stirred at 70°C under a nitrogen atmosphere. After 30 min L-lysine were dissolved distilled water and added to the mixture. After another 30 min, NH3 33% was added until pH 11 was obtained and the reaction was followed for 1.5 h. At the end black nanoparticles were separated magnetically and washed 3 times with distilled water. Preparation of LZ-CS-IONPs : Initially, stock solutions were prepared by dissolving linezolid in distilled water at a concentration of 5 mg/mL and chitosan in 1% (w/v) acetic acid at a concentration of 0.2 mg/mL. Subsequently, iron oxide nanoparticles (IONPs), linezolid, and chitosan were combined in a 12:12:1 mass ratio and mixture was stirred at room temperature for 48 hours to facilitate drug encapsulation within the chitosan matrix. Finally, the resulting nanoparticles were magnetically separated and washed three 3 with distilled water. loading and release of linezolid from LZ-CS-IONP Linezolid loading onto LZ-CS-IONP was determined by quantifying unloaded linezolid in the supernatant following magnetic separation of the nanoparticles. The supernatant was analyzed using a UV spectrophotometer (PG Instruments Ltd.) at 251 nm. A standard curve was generated using serially diluted linezolid standards in distilled water. The amount of linezolid loaded onto the nanoparticles was calculated by subtracting the amount of unloaded linezolid (determined from the standard curve) from the initial amount of linezolid used in the preparation. Loading efficiency (LE%) and loading capacity (LC%) were then calculated using the following equations In vitro drug release was performed by dispersing 600µg LZ-CS-IONP in PBS pH 7.4 and incubating them on a rotator at room temperature. At set time points, nanoparticles were magnetically separated, the supernatant collected, and replaced with fresh PBS. Released drug was quantified by UV spectrophotometry using generated standard curve. Characterization: S ynthesized nanoparticles were characterized by DLS(Dynamic light scattering, Microtrac, nanoflex), Zeta potential analysis(Zeta check, stabino, microtrack), FTIR spectroscopy with a KBr tablet(Bruker, Vertex 70, FTIR spectrometer), TEM (transmission electron microscopy, Zeiss, EM10C, 80 kV), FE-SEM(Field Emission Scanning Electron Microscope, Zeiss, sigma 300), VSM (Vibrating Sample Magnetometer Meghnatis Daghigh Kavir Co., Kashan, Iran), XRD(X-ray diffraction, Bruker, D8 Advanced, Cu-Kα) In Vitro Evaluation of Antibacterial Activity The antibacterial activity of various compounds against Enterococcus faecalis , Staphylococcus aureus , Escherichia coli , and Pseudomonas aeruginosa was evaluated using the broth microdilution method in 96-well plates. Bacterial suspensions, standardized to a 0.5 McFarland turbidity in Mueller-Hinton Broth (MHB), were dispensed into the wells. Ampicillin served as a positive control. Serial two-fold dilutions of each test compound were performed in triplicate to determine their antibacterial activity. Initial concentrations of pure linezolid and ampicillin were set at 32 µg/mL. For LZ-CS-IONP, the initial concentration was adjusted to deliver an equivalent amount of linezolid compared to the free drug. Plates were incubated at 37°C for 24 hours. After incubation, optical density (OD) was measured at 600 nm using a microplate reader. The minimum inhibitory concentration (MIC90), defined as the lowest concentration inhibiting 90% bacterial growth compared to a blank control (MHB only), was determined from the OD measurements. Antibacterial data were analyzed using IBM SPSS software. One-Way ANOVA followed by Tukey's post hoc test was performed to determine statistical significance. A p-value of ≤ 0.05 was considered statistically significant, and these results are denoted by an asterisk (*) in the corresponding figures. 3. Results and discussion DLS and Zeta potential The hydrodynamic diameter and zeta potential (surface charge) of iron oxide nanoparticles (IONPs) dispersed in distilled water were measured before and after chitosan coating and linezolid loading (Table 1). These modifications resulted in an increase in hydrodynamic diameter and a shift in surface charge from negative to positive. Table 1 Hydrodynamic Diameter and Zeta Potential of Nanoparticles Sample Hydrodynamic Diameter (nm) Zeta Potential (mV) PDI IONP 74.7 -50.3 0.08 LZ-CS-IONP 149.4 + 28 0.294 Figure 1 presents the particle size distribution of the nanoparticles obtained from dynamic light scattering (DLS) analysis. TEM and FESEM The morphology and actual size of the nanoparticles were investigated using TEM and FE-SEM. The resulting images (Figs. 2 and 3) revealed a roughly spherical morphology for both IONPs and LZ-CS-IONPs. The average particle sizes were determined to be 19.07 nm for IONPs and 23.08 nm for LZ-CS-IONPs. The particle size distribution, derived from TEM images, is presented in Fig. 4. FTIR The FTIR spectra of L-lysine and the L-lysine-functionalized magnetic nanoparticles (Lys-IONPs) are shown in Fig. 5. Two characteristic peaks at 450 cm⁻¹ and 578 cm⁻¹ confirm the presence of Fe-O bonds, indicating the formation of Fe₃O₄. The peaks at 1625 cm⁻¹ (bending vibration) and 3390 cm⁻¹ (stretching vibration) correspond to the OH groups on the nanoparticle surface. The peak at 1431 cm⁻¹ is attributed to C-O stretching vibrations, while the peak at 1625 cm⁻¹ (overlapping with the OH bending) is also associated with the C = O bond of the amino acid carboxyl group. C-H stretching vibrations were observed at 2910 cm⁻¹, suggesting the encapsulation of the nanoparticle surface with the long hydrocarbon chains of L-lysine. The peak at 3436 cm⁻¹ results from the overlapping stretching vibrations of the N-H bond. These findings indicate that the amino acids are anchored to the nanoparticles via their carboxyl groups and side chains.(12, 13) The FTIR spectrum of LZ-CS-IONPs compared to chitosan and linezolid is shown in Fig. 6. The Fe-O peak was observed at 574 cm⁻¹, with a slight shift, indicating that the chitosan-coated nanoparticle retained its magnetic phase. This shift suggests a change in the nanoparticle surface after coating and the formation of bonds between the nanoparticle and the hydroxyl groups of chitosan. New peaks, absent in the original IONP spectrum, were clearly observed in the LZ-CS-IONP spectrum. The C-O-C stretching vibration at 1014 cm⁻¹ is attributed to the chitosan monosaccharide ring. The peaks at 1088 cm⁻¹ (C-O stretching) and 1372 cm⁻¹ (C-N stretching) further confirm the presence of chitosan on the nanoparticle surface(14). The broad peak at 3430 cm⁻¹ is attributed to the overlapping N-H stretching vibrations of linezolid (3361 cm⁻¹) and chitosan (3444 cm⁻¹). A sharp peak at 1758 cm⁻¹ indicates the C = O bond of the carbonyl group in the oxazolidinone ring of linezolid. The C = C vibrations of the benzene ring in linezolid were observed at 1513 cm⁻¹. A broad peak at 3057 cm⁻¹ can be attributed to the C-H groups of the aromatic ring in linezolid(15, 16). Sharp peaks in the 700–800 cm⁻¹ region can be attributed bending vibrations of hydrogen atoms bonded to carbon atoms in the aromatic ring of linezolid. Figure 3 shows a comparison of the infrared spectra of IONP and LZ-CS-IONP. XRD Figure 8 shows the XRD patterns of IONPs) and LZ-CS-IONPs. Both samples exhibited characteristic diffraction peaks at approximately 30, 35.5, 43.3, 53.6, 57, and 62.8 degrees 2θ, corresponding to the (220), (311), (400), (422), (511), and (440) planes of Fe 3 O 4 . While the chitosan coating resulted in a decrease in peak intensities for LZ-CS-IONPs, no peak shifts were observed, confirming that the coating process did not alter the crystalline phase of the nanoparticles(17, 18). VSM Figure 9 confirms the superparamagnetic behavior of the synthesized nanoparticles due to the absence of hysteresis loops. The saturation magnetization decreased from 46.13 emu/g to 24.56 emu/g after chitosan coating, attributed to chitosan's diamagnetic nature.(14, 18) loading and release of linezolid from LZ-CS-IONP The maximum absorption wavelength (λmax) of linezolid was determined to be 251 nm using UV-Vis spectroscopy. A linezolid standard curve was then constructed by measuring the absorbance of a series of linezolid solutions with known concentrations at 251 nm. The standard curve equation was determined to be y = 0.0463x − 0.02 (Fig. 10). Linezolid loading onto IONP nanoparticles resulted in a loading efficiency (LE%) of 28.30 ± 0.667% and a loading capacity (LC%) of 20.57 ± 0.532%. The drug release profile from the LZ-CS-IONPs in PBS buffer is shown in Fig. 11.The release profile exhibits an initial rapid release within the first few hours, followed by a gradual decrease in the release rate after approximately 4 hours. Eventually, the release rate approaches zero, indicating minimal further drug release. In Vitro Evaluation of Antibacterial Activity The drug-loaded nanoparticles (LZ-CS-IONP) exhibited superior antibacterial activity compared to both IONP and CS-IONP against all four bacteria. For a more precise comparison of LZ-CS-IONP with free linezolid, the nanoparticle formulation's activity was normalized to its linezolid content and compared with free linezolid and ampicillin (standard control) (Fig. 12). LZ-CS-IONP's initial concentration was adjusted to match the linezolid concentration of the pure drug. Triplicate serial dilutions were tested against each bacterium, and viability graphs were generated. Comparing LZ-CS-IONP, pure linezolid, and ampicillin, LZ-CS-IONP showed enhanced antibacterial activity against Gram-positive bacteria, consistent with linezolid's known specificity. Based on equivalent linezolid concentrations, comparison of the antibacterial activity of LZ-CS-IONP with free linezolid revealed the following MIC 90 values: against Staphylococcus aureus , 2 µg/ml for free linezolid and 0.5 µg/ml for the nanoparticle-loaded formulation (equivalent to 2.43 µg/ml of LZ-CS-IONP); against Enterococcus faecalis , 4 µg/ml for free linezolid and 0.5 µg/ml for the nanoparticle-loaded formulation (equivalent to 2.43 µg/ml of LZ-CS-IONP); and against Escherichia coli , 16 µg/ml for free linezolid and 2 µg/ml for the nanoparticle-loaded formulation (equivalent to 9.75 µg/ml of LZ-CS-IONP). Notably, free linezolid did not achieve 90% growth inhibition of Pseudomonas aeruginosa at the highest tested concentration (32 µg/ml), indicating an MIC 90 > 32 µg/ml, whereas the nanoparticle-loaded formulation exhibited an MIC 90 of 8 µg/ml (equivalent to 39 µg/ml of LZ-CS-IONP). Loading drug within LZ-CS-IONP also improved linezolid's activity against Gram-negative bacteria, despite their low sensitivity to the pure drug. Based on the obtained viability results, the MIC90 values of the samples against the four tested bacteria were determined and are presented in the Table 2. Table2. MIC90 Values (µg/ml) Against Four Major Osteomyelitis Pathogens IONP CS-IONP LZ-CS-IONP LZ AMP s.aureus 156 156 2.43 2 4 E.faecalis > 156 156 2.43 4 4 E.coli 156 156 9.75 16 8 P.aeruginosa > 156 > 156 39 > 32 8 Conclusion The antibacterial assays revealed that LZ-CS-IONPs demonstrate significant antibacterial activity against the four principal pathogens implicated in bone infections: Staphylococcus aureus , Enterococcus faecalis , Escherichia coli , and Pseudomonas aeruginosa . Notably, the synthesized nanoparticles not only enhanced the antibacterial efficacy of linezolid against Gram-positive bacteria, as expected given linezolid's inherent activity profile, but also significantly improved its effectiveness against Gram-negative bacteria, extending its antibacterial spectrum. This enhanced activity against Gram-negative bacteria is likely attributable to the nanoparticle delivery system facilitating drug penetration through the outer membrane, overcoming a key resistance mechanism. Consequently, the designed nanostructure offers a promising strategy for linezolid delivery, leveraging synergistic effects between the nanoparticle components and improved drug penetration to enhance antibacterial efficacy and potentially reduce the required therapeutic dose, which could decrease the risk of adverse effects associated with high linezolid concentrations. This approach suggests a potential therapeutic strategy for challenging osteomyelitis infections, including those involving multidrug-resistant bacteria and biofilm formation. Declarations Author Contribution M.N wrote the main manuscript text and M.R and A.G References Jorge LS, Chueire AG, Rossit ARB. Osteomyelitis: a current challenge. The Brazilian Journal of Infectious Diseases. 2010;14(3):310-5. Zelmer AR, Nelson R, Richter K, Atkins GJ. Can intracellular Staphylococcus aureus in osteomyelitis be treated using current antibiotics? A systematic review and narrative synthesis. Bone research. 2022;10(1):53. 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Li G-y, Jiang Y-r, Huang K-l, Ding P, Chen J. Preparation and properties of magnetic Fe3O4–chitosan nanoparticles. Journal of alloys and compounds. 2008;466(1-2):451-6. Kafali M, Şahinoğlu OB, Tufan Y, Orsel ZC, Aygun E, Alyuz B, et al. Antibacterial properties and osteoblast interactions of microfluidically synthesized chitosan–SPION composite nanoparticles. Journal of Biomedical Materials Research Part A. 2023;111(11):1662-77. Additional Declarations No competing interests reported. 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. 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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-6133116","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":443801810,"identity":"8b75a39b-6f27-41b6-abd2-1248ca79efa7","order_by":0,"name":"mohammadjavad 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Patterns of IONP and LZ-CS-IONP\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6133116/v1/5e62ba1f2303cc81edcf4d1e.png"},{"id":81043687,"identity":"d4a45fd9-3de9-45d6-9be4-deeb9b6ee170","added_by":"auto","created_at":"2025-04-21 14:15:45","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":21074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVSM diagram of bare IONPs(a) and after coating with chitosan(b)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6133116/v1/e9b052275edeaeff337106f2.png"},{"id":81043685,"identity":"5888d84f-b0a9-45a5-beea-87bf5ffc8b2a","added_by":"auto","created_at":"2025-04-21 14:15:45","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":17205,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLinezolid UV Absorption Standard Curve\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6133116/v1/f9ec2484333b3fb833b621f2.png"},{"id":81043673,"identity":"417ded73-04c8-4063-8335-f4db324b880f","added_by":"auto","created_at":"2025-04-21 14:15:44","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":13646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCumulative Release of Linezolid from LZ-CS-IONPs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6133116/v1/235cdc2d444bdb9f8ab971ee.png"},{"id":81044676,"identity":"1a25b27e-89e8-4c69-951c-0009e6226abf","added_by":"auto","created_at":"2025-04-21 14:31:44","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":249465,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the Antibacterial Activity of LZ-CS-IONP and Free Linezolid\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-6133116/v1/af16638db5c7e6ccead724c5.png"},{"id":88217504,"identity":"da72f2c3-5fcf-4456-bad7-43c855719790","added_by":"auto","created_at":"2025-08-04 07:09:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3005716,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6133116/v1/c44c4652-bcbe-4385-b790-c4c98766eaf9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeted Delivery of Linezolid Using Magnetic Nanoparticles to Enhance Osteomyelitis Treatment","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOsteomyelitis is a serious infectious disease that primarily affects bone marrow.(1) Treatment of osteomyelitis faces serious challenges due to the phenomenon of antibiotic resistance, biofilm formation, and intracellular infection by Staphylococcus aureus bacteria, which cause frequent recurrence of infection.(2) Due to the limited ability of antibiotics to penetrate bone tissue and the increased antibiotic resistance of bacteria within biofilms, treating osteomyelitis often requires lengthy courses of high-dose antibiotics, which can result in severe adverse effects.(3, 4) Conventional treatment for osteomyelitis typically involves a 4- to 6-week course of systemic antibiotics. However, the ideal duration of antibiotic therapy for chronic osteomyelitis remains unclear and the rate of re-infection is still high(5, 6) Prolonged linezolid therapy for osteomyelitis has been associated with several adverse events, including hematological toxicities such as thrombocytopenia (7)،anemia (8)and bone marrow suppression. Other reported side effects include fatigue, gastrointestinal intolerance and neuropathy.(9)There is a growing interest in exploring novel therapeutic strategies, particularly in the context of osteomyelitis treatment. Nanotechnology has emerged as a promising approach for targeted drug delivery and biofilm disruption. Specifically, superparamagnetic iron oxide nanoparticles (SPIONs) are being investigated for their ability to deliver antibiotics directly to infected tissues when guided by an external magnetic field. This method enhances antibiotic penetration and disrupts the biofilm matrix, potentially leading to more effective eradication of persistent infections.(10, 11) This study aimed to develop a new drug delivery system to improve the treatment of osteomyelitis By using magnetic nanoparticles coated with chitosan and loaded with the antibiotic linezolid, the goal was to increase the concentration of the drug at the infection site, reduce the overall drug dosage, and minimize side effects while combating antibiotic resistance. The antibacterial properties of this nanoantibiotic were assessed against four pathogenic bacterial strains, including \u003cem\u003eEnterococcus faecalis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cp\u003e\u003cstrong\u003eMaterial\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll reagents were of analytical grade and used without further purification. FeCl\u003csub\u003e3\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO, FeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, NH\u003csub\u003e3,\u003c/sub\u003e Acetic Acid and PBS were purchased from Merck. L-lysine, Linezolid and Chitosan (low molecular weight) were obtained from Sigma-aldrich.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of IONPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL-lysine-functionalized IONPs synthesis was performed according to the previously reported co-precipitation method(12). Briefly FeCl3\u0026middot;6H2O and FeSO4\u0026middot;7H2O were dissolved in distilled water. An aqueous solution of iron salts was stirred at 70\u0026deg;C under a nitrogen atmosphere. After 30 min L-lysine were dissolved distilled water and added to the mixture. After another 30 min, NH3 33% was added until pH 11 was obtained and the reaction was followed for 1.5 h. At the end black nanoparticles were separated magnetically and washed 3 times with distilled water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of LZ-CS-IONPs\u003c/strong\u003e: Initially, stock solutions were prepared by dissolving linezolid in distilled water at a concentration of 5 mg/mL and chitosan in 1% (w/v) acetic acid at a concentration of 0.2 mg/mL. Subsequently, iron oxide nanoparticles (IONPs), linezolid, and chitosan were combined in a 12:12:1 mass ratio and mixture was stirred at room temperature for 48 hours to facilitate drug encapsulation within the chitosan matrix. Finally, the resulting nanoparticles were magnetically separated and washed three 3 with distilled water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eloading and release of linezolid from LZ-CS-IONP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLinezolid loading onto LZ-CS-IONP was determined by quantifying unloaded linezolid in the supernatant following magnetic separation of the nanoparticles. The supernatant was analyzed using a UV spectrophotometer (PG Instruments Ltd.) at 251 nm. A standard curve was generated using serially diluted linezolid standards in distilled water. The amount of linezolid loaded onto the nanoparticles was calculated by subtracting the amount of unloaded linezolid (determined from the standard curve) from the initial amount of linezolid used in the preparation. Loading efficiency (LE%) and loading capacity (LC%) were then calculated using the following equations\u003c/p\u003e\n\u003cp\u003e\u003cimg 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BhsfPEZY8IHl1atX1wQgA4uPFF++fDkYxStzj1Hav//+ewi3YcOGEBcmTZqU5epeKE8++6/7vF8MX8iKZyYI7unTp4frvXv3Zu+//36ow1dffRXqgSBAmC1durT2QWZAQMVC4KWXXgoCgjKSztatW0tnaKdPn07OgMkLocWHbagPQotyAe3/+OOPZ2+//XbwAx5AAqFGfuT/5ptv1rWn4mKISzw+ZA3Kc9++fdmVK1eCYBzKzM9pkvwGOF0A61qsEbHmxrXsrOtwLfIBVbeexC8fJbZgJx5hLWXuIk4bsNs1QeI3sx6YCseanHU7cOBA+LByDPlbd8ptyxDHw641wRTUIVVn0rUfa6Z8lBsop213rskH4vxJw9bLxiUsa49q0zhP0im7H87w8Rlcl8BTnxnBuHHjggozYcKEYD927Fg2e/bsIlT/zCgfJLXZCjOOWP1iXezatWsDVK4yd0Ha+YCrpc3OHrMPq3pRnocffriwlUM4O2sirSNHjmSvvvpq4dI/M3vttdcK202YzeYCK1wze4xV3XPnztXVmQ9DT5kypbDVQ760l53JajeXdDWjpD3IV7NOZn1PPfVUuMaPe6JZMvmrfLBjx466uhKXGSXq6c6dO8NsTW1KnnPnzg3Xat+y2bQzfFzAdQEMOgmcsWPHBtULQYAbQgE11SL1jUF35syZoNZq0GptbcWKFeEXoQVl7inIF4G7fPnyLJ+JFK79UB4JhUYQDoEg4fD0009n+cylNtABVTilnp04cSL8UoavcvUVoUsbkY7AD6g3eZSpeajFEoaEQ51/6KGHgh1u3LgR3PmiPvmghgs2J8iHhwhtfunSpdpaJ3bibdu2LfwC1yKfuQX1dNmyZdmhQ4cK1354yCDccEe4US/Vxxlh+idyTidBTeFWSAUSqEy48ytQZ1BzUHtQdfr6+sL12bNna37EwUhNKnNPgXpFGNK0ahfoqEcVlEl5Ycg7rhtlwi8F9cIPtZNw5GnLjLqHG+1GvQlLuBjc1LbWKCz5kA5uykuoDLR9XAbytPkTlzoqvtRSpWtVftLAnbZV2cnLaQ3+4WenaZgtMWO0M6l2Q97MaBcsWFCzb968OSzWO05MR1RUu72eAjUAlYowGB1VwB31jd027ewJwktVcEYe2vbzzz/PnnnmmcKlM7DeheoI9AF2JFeuXBnsTmtBrWbMajnEwoOGsWnHq6DvaMynxm5LCfO4NtPoZLmm95q28ytVCZUANQAVyKos7Hxp98tpDdyvWGXtBPQHqZ30E7/v7YF2pu1RxeM2ZxxzP1C5tXxg1W7GKqo60Ie4f+2iIwIOIUZDxWitAwGWQmtRhLPXqbQcx0mj8cOvBaFkBVMK4sUCjvGq8QgIMQk0YEzrmA95Shi2g46oqGUny48ePRqmuRz0bITdVmf3qxkVhem1VN6USU27HacXYfy88sordceFUDHZvebfF4Pl+PHjdQex2dW/evVquEYdZcdZx3w0dtm9bgcdEXBlJ8v532Uu+QvbQM6fPx+207kZHDfgGsOxBXR7hCN+KWjgXKCXmlWrVhUhHaf3QZAxMUDIcbyFjZr169cXvoNDwixFuwRZKfngbjtkG0+PIXWcwML0mbjo8Oj6TIuZ+qLjE49pb6v0e/J142Y0mkbo2EpqPTxFSkVFHbXrs/hLZdVRGDvesdt/prSSts/gGp0szxt5wKFWC0+dvMxhJsd/+5jFMTNjFsexAQ5p5vp+Ebqe4aqo5OvGzWg0ZaDtMHb6+vqC5jTUUwj8iwStTHCQWQe6dXBaB8uVBwfa20HbBVyjk+W4b9myJVzjxxEAnRyPYQ3hrbfeKmw3setzFldRHecmWnPj73useUtdrRJyKf98thb+ucJkBX9e6rB48eLCNwvCU+Oaf4UQ3v6jpaXkg7ttME3VFr81mr4ynUVNlTvXKVUWVdVOk5lm48Y0u51b0I4zGmFMpY78xOPKgp8dm6igdieUnVR2SzFx2lpO0phu1w4qtFXAjQQSklbw0WBq3EZreMOF9HWDrdF6w0ijNceyTqfzhJj4QUBH0lplu6CceoDFA0BQTq37YFKoXrS3kNut+gBTuzqDw1usSRisLKYyQO05Pp5cZQIoRk+ywUD4RkIbf4xdtEUwpp6krQShRZtIoNJWmBjCyb1RW6hetu6k367F6VaQEs60Rdm5Twv1Vp9zmqcjx0RGIyyWsm7BGp89x/fggw9m48ePD9dV8PZW3tg7GNhQid/ma4nXMlgDafdrztnA4fVArL2oPLxFA7uFstGGzbzunHQWLlyY7d+/v3Dpf4OHfW3SaIJ65w+dwnYT3KvOfQJvMkmdHXUa4wJuCPCaHwkPBpw9HEmH5UweO7N20ZZrhABvyMWPcIA/fgrPxorisMPFoCjbOJF/p19zvmvXrvBapVjY2gHNAnSzrztXvR599NHQ1nLLZzDhWpCm2ppyKk3cORPJ+S7ameu47ITR/53t/yMJjxt21T0ua1k7puLiRphFixaFe2LTUxoW0lKdrB8bAvaBRR6pV6Lb/oShHLcyLuAGCR0oJTyAzknnTb0Cm9kKAzRXNYKfhCKdEcGQq77h1de8FUMCjRcwNnpqd8trzhnEejmksK8nh8G87lz10hEDyoEbrwAX1LXsleLs6PGBG07Yc2SB169zz4TisnNI3FxNzD788MOakMhV4yAcn3/++dBePJhEWTvauKRF3LVr14YXmLJDz/XGjRvr6p6r33X3l/7z8ssvhxcK0N5WwJGn7pfKn3olOv1tzJgxwf3s2bPhgXJLkzeEMwjs+lsM7qx/CdaQtD6Xd9i6RXMgLbsuQ1y7bqbd4TLwZ82LMFzLTp5cC/LmVuMH/MZ1wE48wlrK3IXSjv2pV7y2RDrNrAuqHkB4Gbv+ht3WkWu7Xoe/1vri+sZxKRf3SnCtvFQ/e13Vjql7hnu86UK+NmxZXPKx/cSWnzrTr2yZCNuo39xKuIAbJHQuTArb+RkMdDx1ajpivOgeD3gGlh2kdFSll0KDksFIXqQHuNsOjr8diClBQ3lxI5wVVmXugvJJAAgNujh8XK4yVC+g/WgH6wbY1VbkE+dHeSWkyDNuZ1sO7FYYW2FCOLVdVTuSf9wWoPLFkJa9v6m4QBmsQKa8hMXQp1RPoQ0xG+dWxVXUQYDKicomlTBF2SuweY8/dvzsvyYavfo67/whPdktqG0K38nXnKNe54O3VkY2HFCzUMNilbaZ153beoHaL0XZK8WJj0qqDQk2XfiOhF2PQnUFyo36G38nASgLSw58tUs0akdU1lz4hGsL7ca9B1sGyhjfX9WffNUOFy9eDN/oIK7ccuGONKx7JTplJx5txlqvVa1vWfrlnNMMPK1psrJm40nLkxp/nqB2RsFsA3dmB5rV8Ut4uRHHzpTw42lt0xHEIT074wM93fkVxCdd0qOMOpRJnvIjDkZP/TL3FMwgVB5+4zIBYcizilS9yFuzU0E9CKf2IW2Vkbi2/tw31Rf4VT7UUe5APqRDeAz5iEbtCOQTlxOYpaXSw27vL3FTbah8FBc/7ISlz2gWaO8D/vESwa2Iv7LcaQvMcjr9uvNmYNNn3bp1o/Y4ilOPq6hOy0Gt6obXnTcD6qoLt97BBZzTcjj/lqtSdecFuxHWv1hj07k2Z/TjKqrjOD2Lz+Acx+lZXMA5jtOzuIBzHKdncQHnOE6PkmX/B3JFOy2DwGYXAAAAAElFTkSuQmCC\"\u003e\u003c/p\u003e\n\u003cp\u003eIn vitro drug release was performed by dispersing 600\u0026micro;g LZ-CS-IONP in PBS pH 7.4 and incubating them on a rotator at room temperature. At set time points, nanoparticles were magnetically separated, the supernatant collected, and replaced with fresh PBS. Released drug was quantified by UV spectrophotometry using generated standard curve.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization: S\u003c/strong\u003eynthesized nanoparticles were characterized by DLS(Dynamic light scattering, Microtrac, nanoflex), Zeta potential analysis(Zeta check, stabino, microtrack), FTIR spectroscopy with a KBr tablet(Bruker, Vertex 70, FTIR spectrometer), TEM (transmission electron microscopy, Zeiss, EM10C, 80 kV), FE-SEM(Field Emission Scanning Electron Microscope, Zeiss, sigma 300), VSM (Vibrating Sample Magnetometer Meghnatis Daghigh Kavir\u003c/p\u003e\n\u003cp\u003eCo., Kashan, Iran), XRD(X-ray diffraction, Bruker, D8 Advanced, Cu-K\u0026alpha;)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn Vitro Evaluation of Antibacterial Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antibacterial activity of various compounds against \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e was evaluated using the broth microdilution method in 96-well plates. Bacterial suspensions, standardized to a 0.5 McFarland turbidity in Mueller-Hinton Broth (MHB), were dispensed into the wells. Ampicillin served as a positive control. Serial two-fold dilutions of each test compound were performed in triplicate to determine their antibacterial activity. Initial concentrations of pure linezolid and ampicillin were set at 32 \u0026micro;g/mL. For LZ-CS-IONP, the initial concentration was adjusted to deliver an equivalent amount of linezolid compared to the free drug. Plates were incubated at 37\u0026deg;C for 24 hours. After incubation, optical density (OD) was measured at 600 nm using a microplate reader. The minimum inhibitory concentration (MIC90), defined as the lowest concentration inhibiting 90% bacterial growth compared to a blank control (MHB only), was determined from the OD measurements. Antibacterial data were analyzed using IBM SPSS software. One-Way ANOVA followed by Tukey\u0026apos;s post hoc test was performed to determine statistical significance. A p-value of \u0026le;\u0026thinsp;0.05 was considered statistically significant, and these results are denoted by an asterisk (*) in the corresponding figures.\u003c/p\u003e\n"},{"header":"3. Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eDLS and Zeta potential\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe hydrodynamic diameter and zeta potential (surface charge) of iron oxide nanoparticles (IONPs) dispersed in distilled water were measured before and after chitosan coating and linezolid loading (Table 1). These modifications resulted in an increase in hydrodynamic diameter and a shift in surface charge from negative to positive.\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eHydrodynamic Diameter and Zeta Potential of Nanoparticles\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHydrodynamic Diameter (nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZeta Potential (mV)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePDI\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIONP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e74.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-50.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLZ-CS-IONP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e149.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+ 28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.294\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eFigure 1 presents the particle size distribution of the nanoparticles obtained from dynamic light scattering (DLS) analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTEM and FESEM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphology and actual size of the nanoparticles were investigated using TEM and FE-SEM. The resulting images (Figs.\u0026nbsp;2 and 3) revealed a roughly spherical morphology for both IONPs and LZ-CS-IONPs. The average particle sizes were determined to be 19.07 nm for IONPs and 23.08 nm for LZ-CS-IONPs. The particle size distribution, derived from TEM images, is presented in Fig.\u0026nbsp;4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFTIR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe FTIR spectra of L-lysine and the L-lysine-functionalized magnetic nanoparticles (Lys-IONPs) are shown in Fig.\u0026nbsp;5. Two characteristic peaks at 450 cm⁻¹ and 578 cm⁻¹ confirm the presence of Fe-O bonds, indicating the formation of Fe₃O₄. The peaks at 1625 cm⁻¹ (bending vibration) and 3390 cm⁻¹ (stretching vibration) correspond to the OH groups on the nanoparticle surface. The peak at 1431 cm⁻¹ is attributed to C-O stretching vibrations, while the peak at 1625 cm⁻¹ (overlapping with the OH bending) is also associated with the C = O bond of the amino acid carboxyl group. C-H stretching vibrations were observed at 2910 cm⁻¹, suggesting the encapsulation of the nanoparticle surface with the long hydrocarbon chains of L-lysine. The peak at 3436 cm⁻¹ results from the overlapping stretching vibrations of the N-H bond. These findings indicate that the amino acids are anchored to the nanoparticles via their carboxyl groups and side chains.(12, 13)\u003c/p\u003e\n\u003cp\u003eThe FTIR spectrum of LZ-CS-IONPs compared to chitosan and linezolid is shown in Fig.\u0026nbsp;6. The Fe-O peak was observed at 574 cm⁻¹, with a slight shift, indicating that the chitosan-coated nanoparticle retained its magnetic phase. This shift suggests a change in the nanoparticle surface after coating and the formation of bonds between the nanoparticle and the hydroxyl groups of chitosan. New peaks, absent in the original IONP spectrum, were clearly observed in the LZ-CS-IONP spectrum. The C-O-C stretching vibration at 1014 cm⁻¹ is attributed to the chitosan monosaccharide ring. The peaks at 1088 cm⁻¹ (C-O stretching) and 1372 cm⁻¹ (C-N stretching) further confirm the presence of chitosan on the nanoparticle surface(14). The broad peak at 3430 cm⁻¹ is attributed to the overlapping N-H stretching vibrations of linezolid (3361 cm⁻¹) and chitosan (3444 cm⁻¹). A sharp peak at 1758 cm⁻¹ indicates the C = O bond of the carbonyl group in the oxazolidinone ring of linezolid. The C = C vibrations of the benzene ring in linezolid were observed at 1513 cm⁻¹. A broad peak at 3057 cm⁻¹ can be attributed to the C-H groups of the aromatic ring in linezolid(15, 16). Sharp peaks in the 700–800 cm⁻¹ region can be attributed bending vibrations of hydrogen atoms bonded to carbon atoms in the aromatic ring of linezolid. Figure\u0026nbsp;3 shows a comparison of the infrared spectra of IONP and LZ-CS-IONP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXRD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 8 shows the XRD patterns of IONPs) and LZ-CS-IONPs. Both samples exhibited characteristic diffraction peaks at approximately 30, 35.5, 43.3, 53.6, 57, and 62.8 degrees 2θ, corresponding to the (220), (311), (400), (422), (511), and (440) planes of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e. While the chitosan coating resulted in a decrease in peak intensities for LZ-CS-IONPs, no peak shifts were observed, confirming that the coating process did not alter the crystalline phase of the nanoparticles(17, 18).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVSM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 9 confirms the superparamagnetic behavior of the synthesized nanoparticles due to the absence of hysteresis loops. The saturation magnetization decreased from 46.13 emu/g to 24.56 emu/g after chitosan coating, attributed to chitosan's diamagnetic nature.(14, 18)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eloading and release of linezolid from LZ-CS-IONP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe maximum absorption wavelength (λmax) of linezolid was determined to be 251 nm using UV-Vis spectroscopy. A linezolid standard curve was then constructed by measuring the absorbance of a series of linezolid solutions with known concentrations at 251 nm. The standard curve equation was determined to be y = 0.0463x − 0.02 (Fig.\u0026nbsp;10). Linezolid loading onto IONP nanoparticles resulted in a loading efficiency (LE%) of 28.30 ± 0.667% and a loading capacity (LC%) of 20.57 ± 0.532%. The drug release profile from the LZ-CS-IONPs in PBS buffer is shown in Fig.\u0026nbsp;11.The release profile exhibits an initial rapid release within the first few hours, followed by a gradual decrease in the release rate after approximately 4 hours. Eventually, the release rate approaches zero, indicating minimal further drug release.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn Vitro Evaluation of Antibacterial Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe drug-loaded nanoparticles (LZ-CS-IONP) exhibited superior antibacterial activity compared to both IONP and CS-IONP against all four bacteria. For a more precise comparison of LZ-CS-IONP with free linezolid, the nanoparticle formulation's activity was normalized to its linezolid content and compared with free linezolid and ampicillin (standard control) (Fig.\u0026nbsp;12). LZ-CS-IONP's initial concentration was adjusted to match the linezolid concentration of the pure drug. Triplicate serial dilutions were tested against each bacterium, and viability graphs were generated. Comparing LZ-CS-IONP, pure linezolid, and ampicillin, LZ-CS-IONP showed enhanced antibacterial activity against Gram-positive bacteria, consistent with linezolid's known specificity. Based on equivalent linezolid concentrations, comparison of the antibacterial activity of LZ-CS-IONP with free linezolid revealed the following MIC\u003csub\u003e90\u003c/sub\u003e values: against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, 2 µg/ml for free linezolid and 0.5 µg/ml for the nanoparticle-loaded formulation (equivalent to 2.43 µg/ml of LZ-CS-IONP); against \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, 4 µg/ml for free linezolid and 0.5 µg/ml for the nanoparticle-loaded formulation (equivalent to 2.43 µg/ml of LZ-CS-IONP); and against \u003cem\u003eEscherichia coli\u003c/em\u003e, 16 µg/ml for free linezolid and 2 µg/ml for the nanoparticle-loaded formulation (equivalent to 9.75 µg/ml of LZ-CS-IONP). Notably, free linezolid did not achieve 90% growth inhibition of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e at the highest tested concentration (32 µg/ml), indicating an MIC\u003csub\u003e90\u003c/sub\u003e \u0026gt; 32 µg/ml, whereas the nanoparticle-loaded formulation exhibited an MIC\u003csub\u003e90\u003c/sub\u003e of 8 µg/ml (equivalent to 39 µg/ml of LZ-CS-IONP). Loading drug within LZ-CS-IONP also improved linezolid's activity against Gram-negative bacteria, despite their low sensitivity to the pure drug. Based on the obtained viability results, the MIC90 values of the samples against the four tested bacteria were determined and are presented in the Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable2. MIC90 Values (µg/ml) Against Four Major Osteomyelitis Pathogens\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIONP\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCS-IONP\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLZ-CS-IONP\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLZ\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAMP\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003es.aureus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE.faecalis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt; 156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE.coli\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eP.aeruginosa\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt; 156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt; 156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt; 32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n"},{"header":"Conclusion","content":"\u003cp\u003eThe antibacterial assays revealed that LZ-CS-IONPs demonstrate significant antibacterial activity against the four principal pathogens implicated in bone infections: \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. Notably, the synthesized nanoparticles not only enhanced the antibacterial efficacy of linezolid against Gram-positive bacteria, as expected given linezolid's inherent activity profile, but also significantly improved its effectiveness against Gram-negative bacteria, extending its antibacterial spectrum. This enhanced activity against Gram-negative bacteria is likely attributable to the nanoparticle delivery system facilitating drug penetration through the outer membrane, overcoming a key resistance mechanism. Consequently, the designed nanostructure offers a promising strategy for linezolid delivery, leveraging synergistic effects between the nanoparticle components and improved drug penetration to enhance antibacterial efficacy and potentially reduce the required therapeutic dose, which could decrease the risk of adverse effects associated with high linezolid concentrations. This approach suggests a potential therapeutic strategy for challenging osteomyelitis infections, including those involving multidrug-resistant bacteria and biofilm formation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.N wrote the main manuscript text and M.R and A.G\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJorge LS, Chueire AG, Rossit ARB. Osteomyelitis: a current challenge. The Brazilian Journal of Infectious Diseases. 2010;14(3):310-5.\u003c/li\u003e\n\u003cli\u003eZelmer AR, Nelson R, Richter K, Atkins GJ. Can intracellular Staphylococcus aureus in osteomyelitis be treated using current antibiotics? A systematic review and narrative synthesis. Bone research. 2022;10(1):53.\u003c/li\u003e\n\u003cli\u003eGimza BD, Cassat JE. Mechanisms of antibiotic failure during Staphylococcus aureus osteomyelitis. Frontiers in immunology. 2021;12:638085.\u003c/li\u003e\n\u003cli\u003eKavanagh N, Ryan EJ, Widaa A, Sexton G, Fennell J, O\u0026apos;Rourke S, et al. Staphylococcal osteomyelitis: disease progression, treatment challenges, and future directions. Clinical microbiology reviews. 2018;31(2):10.1128/cmr. 00084-17.\u003c/li\u003e\n\u003cli\u003eSpellberg B, Lipsky BA. Systemic antibiotic therapy for chronic osteomyelitis in adults. Clinical infectious diseases. 2012;54(3):393-407.\u003c/li\u003e\n\u003cli\u003eHatzenbuehler J, Pulling TJ. Diagnosis and management of osteomyelitis. American family physician. 2011;84(9):1027-33.\u003c/li\u003e\n\u003cli\u003eCojutti PG, Merelli M, Bassetti M, Pea F. Proactive therapeutic drug monitoring (TDM) may be helpful in managing long-term treatment with linezolid safely: findings from a monocentric, prospective, open-label, interventional study. Journal of Antimicrobial Chemotherapy. 2019;74(12):3588-95.\u003c/li\u003e\n\u003cli\u003ePea F, Viale P, Cojutti P, Del Pin B, Zamparini E, Furlanut M. Therapeutic drug monitoring may improve safety outcomes of long-term treatment with linezolid in adult patients. Journal of antimicrobial chemotherapy. 2012;67(8):2034-42.\u003c/li\u003e\n\u003cli\u003eVeerman K, Goosen J, Spijkers K, Jager N, Heesterbeek P, Telgt D. Prolonged use of linezolid in bone and joint infections: a retrospective analysis of adverse effects. Journal of Antimicrobial Chemotherapy. 2023;78(11):2660-6.\u003c/li\u003e\n\u003cli\u003eZapata D, Higgs J, Wittholt H, Chittimalli K, Brooks AE, Mulinti P. Nanotechnology in the Diagnosis and Treatment of Osteomyelitis. Pharmaceutics. 2022;14(8):1563.\u003c/li\u003e\n\u003cli\u003eMok H, Zhang M. Superparamagnetic iron oxide nanoparticle-based delivery systems for biotherapeutics. Expert opinion on drug delivery. 2013;10(1):73-87.\u003c/li\u003e\n\u003cli\u003eRaee MJ, Ebrahiminezhad A, Gholami A, Ghoshoon MB, Ghasemi Y. Magnetic immobilization of recombinant E. coli producing extracellular asparaginase: an effective way to intensify downstream process. Separation Science and Technology. 2018;53(9):1397-404.\u003c/li\u003e\n\u003cli\u003eAntal I, Koneracka M, Kubovcikova M, Zavisova V, Khmara I, Lucanska D, et al. d, l-lysine functionalized Fe3O4 nanoparticles for detection of cancer cells. Colloids and Surfaces B: Biointerfaces. 2018;163:236-45.\u003c/li\u003e\n\u003cli\u003eKarimzadeh I, Aghazadeh M, Doroudi T, Ganjali MR, Kolivand PH. Electrochemical preparation and characterization of chitosan-coated superparamagnetic iron oxide (Fe3O4) nanoparticles. Materials Research Innovations. 2018;22(6):352-60.\u003c/li\u003e\n\u003cli\u003eGhataty DS, Amer RI, Wasfi R, Shamma RN. Novel linezolid loaded bio-composite films as dressings for effective wound healing: experimental design, development, optimization, and antimicrobial activity. Drug Delivery. 2022;29(1):3168-85.\u003c/li\u003e\n\u003cli\u003eWassif RK, Elkheshen SA, Shamma RN, Amer MS, Elhelw R, El-Kayal M. Injectable systems of chitosan in situ forming composite gel incorporating linezolid-loaded biodegradable nanoparticles for long-term treatment of bone infections. Drug Delivery and Translational Research. 2024;14(1):80-102.\u003c/li\u003e\n\u003cli\u003eLi G-y, Jiang Y-r, Huang K-l, Ding P, Chen J. Preparation and properties of magnetic Fe3O4\u0026ndash;chitosan nanoparticles. Journal of alloys and compounds. 2008;466(1-2):451-6.\u003c/li\u003e\n\u003cli\u003eKafali M, Şahinoğlu OB, Tufan Y, Orsel ZC, Aygun E, Alyuz B, et al. Antibacterial properties and osteoblast interactions of microfluidically synthesized chitosan\u0026ndash;SPION composite nanoparticles. Journal of Biomedical Materials Research Part A. 2023;111(11):1662-77.\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":"","lastPublishedDoi":"10.21203/rs.3.rs-6133116/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6133116/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOsteomyelitis, a challenging bone infection, is often complicated by formation of biofilms and antibiotic resistance. Traditional treatments typically involve prolonged high-dose antibiotic regimens, which can lead to various adverse effects. This research focuses on creating a targeted drug delivery system utilizing magnetic nanoparticles to improve the effectiveness of linezolid in the treatment of osteomyelitis. Magnetic nanoparticles were synthesized and functionalized with L-lysine. Subsequently, these nanoparticles were coated with chitosan and loaded with linezolid. The nanoparticles were characterized using TEM, FESEM, XRD, FTIR, and VSM. Antibacterial activity was assessed using the microdilution broth method against major osteomyelitis pathogens. The study successfully developed a novel drug delivery system based on magnetic nanoparticles. Compared to free linezolid The synthesized nanoparticles exhibited enhanced antibacterial activity. The nanoparticle structure facilitated deeper penetration of linezolid into bacterial cells, leading to a higher bactericidal effect and potentially lower required treatment doses.\u003c/p\u003e","manuscriptTitle":"Targeted Delivery of Linezolid Using Magnetic Nanoparticles to Enhance Osteomyelitis Treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 14:15:39","doi":"10.21203/rs.3.rs-6133116/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":"21b59371-92f5-4974-98ec-cd0ff3155ea7","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T07:09:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-21 14:15:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6133116","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6133116","identity":"rs-6133116","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}