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Comparative internalization and recycling of different amphotericin B formulations by a macrophage-like cell line
P Legrand1, A Vertut-Doi, J Bolard
1Universite Pierre et Marie Curie, Paris, France.
Abstract:
The amount of amphotericin B (AmB) associated with cultured murine macrophage-like J774 cells, after incubation with various AmB lipid formulations, was determined by absorption spectroscopy. Large, negatively charged, AmB-containing, multilamellar vesicles and small cholesteryl sulphate-AmB complexes both enhanced the amount of AmB associated with J774 cells at 37 degrees C (up to 500-fold the extracellular concentration). In contrast, AmB-containing, small, negatively charged vesicles (AmBisome), positively charged, oligolamellar vesicles and mixed micelles showed a lower association of the antibiotic with cells, compared with AmB added from a solution in dimethylsulphoxide or Fungizone. Experiments performed at 40 degrees C showed a large reduction of AmB uptake for AmB preparations and AmB added from a solution in dimethysulphoxide or Fungizone, suggesting a high percentage of internalization of the antibiotic. Experiments in the presence of cytochalasin B resulted in a decrease of AmB uptake mainly for the preparations of large diameter, suggesting that these formulations were taken up by phagocytosis. A comparative study with Chinese hamster ovary cells, a model of non-phagocytic cells, showed a reduction in the take up of AmB. This reduction was always more marked when AmB was incorporated in lipid formulations. On the other hand, accumulation of the antibiotic in J774 cells was shown to be followed by its release from the cells in an unbound form, the extent of release depending on the type of vector used. The results suggest that in some cases macrophages can be considered as reservoirs of antibiotic, releasing free AmB in the medium.
Insights
Certain amphotericin B lipid formulations significantly increase drug association with macrophages, acting as reservoirs for the antifungal agent. This macrophage reservoir effect depends on the specific lipid formulation used.
Area of Science:
- Pharmacology
- Drug Delivery Systems
- Cell Biology
Background:
- Amphotericin B (AmB) is a critical antifungal agent, but its use is limited by toxicity.
- Lipid formulations of AmB aim to improve its therapeutic index.
- Understanding drug-cell interactions is crucial for optimizing AmB delivery.
Purpose of the Study:
- To investigate the association of various amphotericin B (AmB) lipid formulations with J774 macrophage cells.
- To determine the influence of lipid formulation characteristics on AmB uptake and release by macrophages.
- To explore the potential of macrophages as reservoirs for free AmB.
Main Methods:
- Incubation of cultured murine macrophage-like J774 cells with different AmB lipid formulations.
- Quantification of cell-associated AmB using absorption spectroscopy.
- Experiments conducted at varying temperatures (37°C and 40°C) and in the presence of cytochalasin B.
- Comparative studies using Chinese hamster ovary (CHO) cells.
Main Results:
- Large, negatively charged, multilamellar vesicles and small cholesteryl sulphate-AmB complexes significantly enhanced AmB association with J774 cells (up to 500-fold).
- AmBisome, positively charged vesicles, and mixed micelles showed lower cell association compared to AmB in DMSO or Fungizone.
- Reduced AmB uptake at 40°C suggested internalization, while cytochalasin B indicated phagocytosis for large-diameter formulations.
- Non-phagocytic CHO cells exhibited reduced AmB uptake, especially with lipid formulations.
- Accumulated AmB was released from J774 cells as unbound drug, dependent on the vector type.
Conclusions:
- Macrophage interaction with AmB varies significantly based on the lipid formulation's physicochemical properties.
- Certain AmB lipid formulations can lead to macrophages acting as reservoirs, releasing free AmB into the medium.
- These findings have implications for optimizing AmB delivery strategies and understanding its pharmacokinetics in vivo.