Truszkowska Martyna, Ebert Melanie Lena, Afzal Khush Bakhat, Györgyi Váradi, Bernkop-Schnürch Andreas
Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.
Department of Medical Chemistry, Faculty of Medicine, University of Szeged, Dόm tér 8, H-6720 Szeged, Hungary.
Eur J Pharm Sci. 2025 Jul 18;212:107207. doi: 10.1016/j.ejps.2025.107207.
This study aimed to investigate the permeation behaviour and intracellular fate of hydrophobic ion pairs (HIP). HIP were formed by combining a daptomycin-derived model peptide (DD) with ethyl lauroyl arginate (ELA) and lipophilic fluorescent dye 4-(4-dihexadecylaminostyryl)-N-methylpyridinium iodide (DiA). A representative HIP (DD: ELA: DiA, molar ratio 1:4:0.5) was incorporated into self-emulsifying drug delivery systems (SEDDS) and characterized for size, zeta potential, stability, hemolytic activity, cytotoxicity, and cellular uptake. Permeability was assessed using the Parallel Artificial Membrane Permeability Assay (PAMPA) model and Caco-2 monolayers. SEDDS exhibited droplet sizes below 200 nm, a polydispersity index (PDI) < 0.4, positive surface charges, and high stability. Hemolysis studies indicated potential for endosomal escape, while dose-dependent toxicity became apparent after 4 and 24 h of incubation. Flow cytometry revealed enhanced cellular uptake: HIP and SEDDS increased internalization of DD by 12- and 32-fold, compared to free peptide. Permeation studies demonstrated marked improvements in DD transport. In the PAMPA assay, HIP and SEDDS increased passive diffusion by 2.8- and 6.5-fold. Similarly, in the Caco-2 model, HIP and SEDDS enhanced permeation by 17- and 57-fold, compared to free DD. DiA permeation remained minimal, suggesting that HIP disassociates intracellularly, allowing selective release of the peptide. These findings confirm that HIP enhances membrane permeation of DD and dissociates after uptake. The combination of HIP and SEDDS presents a robust strategy for improving the oral bioavailability of peptide therapeutics.
本研究旨在探究疏水性离子对（HIP）的渗透行为及细胞内命运。HIP由达托霉素衍生的模型肽（DD）与月桂酰精氨酸乙酯（ELA）及亲脂性荧光染料4-(4-二十六烷基氨基苯乙烯基)-N-甲基碘化吡啶（DiA）结合而成。将一种代表性的HIP（DD:ELA:DiA，摩尔比1:4:0.5）载入自乳化药物递送系统（SEDDS），并对其粒径、ζ电位、稳定性、溶血活性、细胞毒性及细胞摄取情况进行表征。使用平行人工膜渗透试验（PAMPA）模型和Caco-2单层细胞评估渗透性。SEDDS的液滴尺寸低于200 nm，多分散指数（PDI）<0.4，表面带正电荷且稳定性高。溶血研究表明其具有内体逃逸的潜力，而在孵育4小时和24小时后剂量依赖性毒性变得明显。流式细胞术显示细胞摄取增强：与游离肽相比，HIP和SEDDS使DD的内化增加了12倍和32倍。渗透研究表明DD转运有显著改善。在PAMPA试验中，HIP和SEDDS使被动扩散增加了2.8倍和6.5倍。同样，在Caco-2模型中，与游离DD相比，HIP和SEDDS使渗透率提高了17倍和57倍。DiA的渗透仍然最小，表明HIP在细胞内解离，允许肽选择性释放。这些发现证实HIP增强了DD的膜渗透性并在摄取后解离。HIP与SEDDS的组合为提高肽类治疗药物的口服生物利用度提供了一种有力策略。
