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This study aimed to develop and characterize natamycin-loaded liposomal vesicles as a biocompatible delivery system to improve stability and achieve controlled release for potential topical application in the treatment of fungal infections. Methods: Formulations were prepared using two phospholipid mixtures (Lipoid S100 and Phospholipon 90H) via standard (thin-film) and proliposome methods. Evaluation included encapsulation efficiency (EE%), particle size, zeta potential, the polydispersity index (PDI), and rheological properties. In vitro release kinetics were compared to a natamycin solution. Antifungal efficacy was tested against four Candida strains to determine minimum inhibitory and fungicidal concentrations (MICs and MFCs, respectively) and biofilm inhibition, while biocompatibility was assessed via keratinocyte viability assays. Results: Formulations achieved high encapsulation (~90%). Natamycin incorporation improved homogeneity and reduced particle diameters, particularly in proliposome-derived vesicles, suggesting strong drug\u2013lipid interactions. Preparation method and lipid type significantly influenced properties; thin-film formulations showed a lower PDI and higher stability. Diffusion was twofold slower than the control, with Lipoid S100 proliposomes providing the most sustained release. The liposomes demonstrated robust antifungal activity (MICs: 0.00625\u20130.2 mg\/mL) and effective biofilm inhibition against C. krusei. While high concentrations moderately reduced keratinocyte viability, lower doses remained biocompatible and slightly stimulatory. Conclusions: Lipid composition and preparation methods have minimal impact on the physical properties and in vitro release profiles of natamycin liposomes. These vesicles provide a dose-dependent, biocompatible platform for the controlled delivery of antifungals, showing significant in vitro inhibitory activity against Candida growth and biofilm formation.<\/jats:p>","DOI":"10.3390\/ph19050710","type":"journal-article","created":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T13:32:21Z","timestamp":1777555941000},"page":"710","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development and Characterization of Natamycin-Loaded Liposomes for Potential Topical Application: Influence of Preparation Method and Phospholipid Composition"],"prefix":"10.3390","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2514-2542","authenticated-orcid":false,"given":"Natalija","family":"\u010cutovi\u0107","sequence":"first","affiliation":[{"name":"Institute for Medicinal Plants Research \u201cDr. Josif Pan\u010di\u0107\u201d, Tadeu\u0161a Ko\u0161\u0107u\u0161ka 1, 11000 Belgrade, Serbia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5647-2590","authenticated-orcid":false,"given":"Petar","family":"Batini\u0107","sequence":"additional","affiliation":[{"name":"Institute for Medicinal Plants Research \u201cDr. Josif Pan\u010di\u0107\u201d, Tadeu\u0161a Ko\u0161\u0107u\u0161ka 1, 11000 Belgrade, Serbia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1328-6223","authenticated-orcid":false,"given":"Tatjana","family":"Markovi\u0107","sequence":"additional","affiliation":[{"name":"Institute for Medicinal Plants Research \u201cDr. Josif Pan\u010di\u0107\u201d, Tadeu\u0161a Ko\u0161\u0107u\u0161ka 1, 11000 Belgrade, Serbia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1182-4895","authenticated-orcid":false,"given":"Andrea","family":"Pirkovi\u0107","sequence":"additional","affiliation":[{"name":"Institute for the Application of Nuclear Energy INEP, University of Belgrade, Banatska 31b, 11080 Belgrade, Serbia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2756-6517","authenticated-orcid":false,"given":"Ninoslav","family":"Miti\u0107","sequence":"additional","affiliation":[{"name":"Institute for the Application of Nuclear Energy INEP, University of Belgrade, Banatska 31b, 11080 Belgrade, Serbia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9016-9971","authenticated-orcid":false,"given":"Jovana","family":"Petrovi\u0107","sequence":"additional","affiliation":[{"name":"Department of Plant Physiology, Institute for Biological Research \u201cSini\u0161a Stankovi\u0107\u201d\u2014National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5394-0125","authenticated-orcid":false,"given":"Aleksandra A.","family":"Jovanovi\u0107","sequence":"additional","affiliation":[{"name":"Institute for the Application of Nuclear Energy INEP, University of Belgrade, Banatska 31b, 11080 Belgrade, Serbia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Lule, V.K., Garg, S., Gosewade, S.C., and Khedkar, C.D. 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