Antimikrobiyal Peptit ve Antibiyotik Kombinasyonu İçeren Lipozom Formülasyonlarının Geliştirilmesi

Abstract

In the treatment of infections caused by microorganisms, active substances are referred to as antimicrobial therapeutics. Due to the increasing resistance to antimicrobial therapeutics in recent years, AMPs have emerged as an alternative. AMPs interact with the cell membrane, causing rapid bacterial death. Liposomal formulations, due to their structural similarity to cell membranes, enhance dermal penetration. Therefore, AMP-loaded liposomal formulations aim to increase penetration and protect the active substance from environmental factors. Nisin is a peptide with antimicrobial activity against both Gram-positive and Gram-negative pathogens. It is particularly effective in inhibiting S. aureus, the most common pathogen intra dermal infections. When used alone, antimicrobial drugs often lead to resistance development and show limited efficacy during prolonged treatments. Combination approaches involving AMPs and drugs have been shown to reduce the likelihood of resistance development and significantly improve clinical outcomes. AZM, a broad-spectrum macrolide antibiotic, has low solubility and is one of the antibiotics with the highest resistance rates developed by S. aureus. Additionally, oral administration of AZM often results in undesirable side effects, making its topical application advantageous in infection treatment.

In the first stage of this thesis study, liposomal formulations containing nisin and AZM were prepared using different lipids (Egg PC, Soy PC, Lipoid S100, Phospholipon 90G, DPPC, and Lipoid S75). Characterization studies, including particle size (PS), polydispersity index (PDI), zeta potential (ZP), morphological properties, encapsulation efficiency, and stability, were performed. With the optimized formulation, studies on in vitro release, ex vivo permeation, and fluorescence microscopy imaging were conducted. It was determined that AZM-loaded liposomes prepared with Egg PC and Lipoid S75 showed homogeneous distribution (127.5 ± 10.22 nm and 88.13 ± 1.82 nm, respectively, with PDI ∼0.2) and remained stable (∼ -30 mV) for 1 month. IVRT studies revealed that approximately 200 µg/cm² AZM was released from Egg PC liposomes, and 250 µg/cm² from Lipoid S75 liposomes after 24 hours. In the second stage, the biocompatibility of optimized liposomal formulations was analyzed using dermal cell lines (HaCat and HDF). Results indicated that the high biocompatibility of the combined liposomes was attributed to the presence of nisin. In the third stage, the interaction of liposomal formulations with the skin was evaluated using QCM-D technology, with the stratum corneum (SC) isolated from human cadaver skin. Drug-loaded liposomes showed a significant mass increase (Δf) compared to empty formulations. Nisin-loaded liposomes demonstrated increased surface viscoelastic properties (ΔD) upon interaction with the SC. Finally, antimicrobial activity and biofilm eradication against S. aureus were evaluated. The combined liposomal formulation showed increased microbial inhibition zones compared to control solutions containing free nisin and AZM. Additionally, AZM-loaded liposomal formulations demonstrated enhanced antibiofilm effects compared to controls containing free active substances.

In conclusion, synergistic and innovative nisin- and AZM-loaded liposomal formulations were developed, validated by antimicrobial efficacy and safety studies. QCM-D analysis, introduced for the first time, offers insight into the interaction of topical formulations with human cadaver skin. This technique is a key tool for assessing nanoformulations' dermal interactions in preclinical ex vivo studies.