Complex Formation of Amphotericin B in Sterol-Containing Membranes As Evidenced by Surface Plasmon Resonance

Abstract

Amphotericin B (AmB) is a membrane-active antibiotic that increases the permeability of fungal membranes. Thus, the dynamic process of its interaction with membranes poses intriguing questions, which prompted us to elaborate a quick and reliable method for real-time observation of the drug's binding to phospholipid liposomes. We focused on surface plasmon resonance (SPR) and devised a new modification method of sensor chips, which led to a significant reduction in the level of nonspecific binding of the drug in a control lane. With this method in hand, we examined the affinity of AmB for various membrane preparations. As expected, AmB exhibited much higher affinity for sterol-containing palmitoyloleoylphosphatidylcholine membranes than those without sterol. The sensorgrams recorded under various conditions partly fitted theoretical curves, which were based on three interaction models. Among those, a two-state reaction model reproduced well the sensorgram of AmB binding to an ergosterol-containing membrane; in this model, two states of membrane-bound complexes, AB and AB*, are assumed, which correspond to a simple binding to the surface of the membrane (AB) and formation of another assembly in the membrane (AB*) such as an ion channel complex. Kinetic analysis demonstrated that the association constant in ergosterol-containing POPC liposomes is larger by 1 order of magnitude than that in the cholesterol-containing counterpart. These findings support the previous notion that ergosterol stabilizes the membrane-bound assembly of AmB.

High-Throughput and Sensitive Assay for Amphotericin B Interaction with Lipid Membrane on the Model Membrane Systems by Surface Plasmon Resonance

“…Optimization of Immobilization of Model Lipid Membranes on the L1 Sensor Chip In previous studies, 20) the methodology using a modified CM5 sensor chip for the assay of the binding of AmB and the sterol-containing lipid membrane was reported using SPR, and the selectivity of AmB for sterol-containing lipid membrane was shown, indicating that AmB has a higher affinity for ergosterol membrane than cholesterol membrane. In this SPR study, two types of liposomes containing the sterol were used as model lipid membranes.…”

“…POPC-cholesterol (4 : 1, v/v) was used to mimic of mammalian membranes, while POPC-ergosterol (4 : 1, v/v) was used as a model fungal membrane. 20) We aimed to establish a better assay of AmB-lipid membrane interaction with our SPR system, considering the time-consuming process for sensor chip modification, the damage to the immobilized liposome on the chip due to repeated and consecutive AmB injection, the carryover between each binding assay, and the throughput and sensitivity. In other words, we tried to establish a high-throughput, sensitive, and reproducible binding assay of AmB-sterol containing lipid membrane using an l1 sensor chip, which is usually used for lipid-membrane systems.…”

“…[14][15][16][17][18][19] In the present study, we developed a high-throughput and sensitive assay of interactions of AmB with two model lipid membranes, which mimic mammal cell membrane and fungal membrane, using our established SPR technique, focusing on the analysis of the binding kinetics of lipid membranes. [14][15][16][17][18][19] Our ultimate target of the related study is to establish a valuable tool for predicting the selective binding and antimycotic activity of antifungals with the SPR system, including the toxicity properties of the target compounds.The sterol-containing lipid membrane of binding properties of AmB by SPR was previously reported by Mouri et al,20) and the method involved the kinetic data obtained from one AmB sensorgram, and the selectivity of AmB to a sterol-containing lipid membrane would be shown by SPR. There would be a few progressive points by our SPR technique, including the throughput and sensitivity and binding kinetic analysis with multiple sensorgrams.…”

“…The sterol-containing lipid membrane of binding properties of AmB by SPR was previously reported by Mouri et al,20) and the method involved the kinetic data obtained from one AmB sensorgram, and the selectivity of AmB to a sterol-containing lipid membrane would be shown by SPR. There would be a few progressive points by our SPR technique, including the throughput and sensitivity and binding kinetic analysis with multiple sensorgrams.…”

Lipid Membrane-Binding Properties of Amphotericin B Deoxycholate (Fungizone) Using Surface Plasmon Resonance

“…A POPC/cholesterol (4:1, v/v) mixture was used to mimic mammalian membranes, while a POPC/ ergosterol (4:1, v/v) mixture was used as a fungal membrane system. 18,26 The liposome mixture was prepared using PBS, isotonic dextrose solution, and water for injection in an effort to establish deal conditions to conduct steady liposome capture on the L1 chip. Also, these solutions were chosen because PBS is a typical buffer used for liposome preparation and other solutions are described as solutions for preparation of therapeutic medications in the Ethical Drug Package Insert of Fungizone.…”