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Cytoplasmic fungal lipases release fungicides from ultra-deformable vesicular drug carriers.

Steinberg G - PLoS ONE (2012)

Bottom Line: With time, the lipid bodies were metabolized in an ATP-dependent fashion, suggesting that cytosolic lipases attack and degrade intruding TFVs.Indeed, the specific monoacylglycerol lipase inhibitor URB602 prevented Transfersome® degradation and neutralized the cytotoxic effect of Transfersome®-delivered terbinafine.As this mode of action of Transfersomes is independent of the drug cargo, these results demonstrate the potential of Transfersomes in the treatment of all fungal diseases.

View Article: PubMed Central - PubMed

Affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, United Kingdom. G.Steinberg@exeter.ac.uk

ABSTRACT
The Transfersome® is a lipid vesicle that contains membrane softeners, such as Tween 80, to make it ultra-deformable. This feature makes the Transfersome® an efficient carrier for delivery of therapeutic drugs across the skin barrier. It was reported that TDT 067 (a topical formulation of 15 mg/ml terbinafine in Transfersome® vesicles) has a much more potent antifungal activity in vitro compared with conventional terbinafine, which is a water-insoluble fungicide. Here we use ultra-structural studies and live imaging in a model fungus to describe the underlying mode of action. We show that terbinafine causes local collapse of the fungal endoplasmic reticulum, which was more efficient when terbinafine was delivered in Transfersome® vesicles (TFVs). When applied in liquid culture, fluorescently labeled TFVs rapidly entered the fungal cells (T(1/2)~2 min). Entry was F-actin- and ATP-independent, indicating that it is a passive process. Ultra-structural studies showed that passage through the cell wall involves significant deformation of the vesicles, and depends on a high concentration of the surfactant Tween 80 in their membrane. Surprisingly, the TFVs collapsed into lipid droplets after entry into the cell and the terbinafine was released from their interior. With time, the lipid bodies were metabolized in an ATP-dependent fashion, suggesting that cytosolic lipases attack and degrade intruding TFVs. Indeed, the specific monoacylglycerol lipase inhibitor URB602 prevented Transfersome® degradation and neutralized the cytotoxic effect of Transfersome®-delivered terbinafine. These data suggest that (a) Transfersomes deliver the lipophilic fungicide Terbinafine to the fungal cell wall, (b) the membrane softener Tween 80 allows the passage of the Transfersomes into the fungal cell, and (c) fungal lipases digest the invading Transfersome® vesicles thereby releasing their cytotoxic content. As this mode of action of Transfersomes is independent of the drug cargo, these results demonstrate the potential of Transfersomes in the treatment of all fungal diseases.

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Entry of terbinafine-loaded TFVs into the fungal cell.(A) Scanning electron micrograph of terbinafine-loaded Transfersome® vesicles. Bar represents micrometers. (B) Electron micrographs of an untreated hyphal cell (control) and after 30 min of incubation with fluorescent terbinafine-loaded TFVs (TBF in TFVs). The cell accumulates large droplets (asterisk). Bar represents micrometers. (C) Uptake of fluorescently labeled terbinafine-loaded Transfersome® vesicles. Prior to Transfersome® treatment, the cells show weak background fluorescence (0 min). Small and relatively faint fluorescent signals appear ∼1 min after adding fluorescent TFVs (1 min) and strong fluorescence is detected after ∼15 min of incubation. See also Movie S2. Bars represent micrometers. (D) Graph showing the kinetics of Transfersome® uptake. Each data point represents the mean ± standard error of the mean of 10–17 measurements. Note that uptake is rapid (K½ ∼2 min) and that uptake reaches a maximum (green dotted line). (E) Electron micrograph showing vesicles (arrowheads) attached to a hyphal cell treated with fluorescent terbinafine-loaded TFVs. Note that the attached vesicles are elongated (right lower corner). Bars represent micrometers. (F) Image series of electron micrographs of TFVs at different stages of entry. Note the deformation of the vesicles. Bar represents micrometers. (G) Bar chart showing the length and the width of vesicles attached to the outer fungal cell wall. Note that only those structures were measured that showed a lumen (e.g. the upper two vesicles in the image series given in F). Mean ± standard error of the mean of 16 measurements. Statistical significance difference at P<0.001 is indicated by double asterisks.
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pone-0038181-g002: Entry of terbinafine-loaded TFVs into the fungal cell.(A) Scanning electron micrograph of terbinafine-loaded Transfersome® vesicles. Bar represents micrometers. (B) Electron micrographs of an untreated hyphal cell (control) and after 30 min of incubation with fluorescent terbinafine-loaded TFVs (TBF in TFVs). The cell accumulates large droplets (asterisk). Bar represents micrometers. (C) Uptake of fluorescently labeled terbinafine-loaded Transfersome® vesicles. Prior to Transfersome® treatment, the cells show weak background fluorescence (0 min). Small and relatively faint fluorescent signals appear ∼1 min after adding fluorescent TFVs (1 min) and strong fluorescence is detected after ∼15 min of incubation. See also Movie S2. Bars represent micrometers. (D) Graph showing the kinetics of Transfersome® uptake. Each data point represents the mean ± standard error of the mean of 10–17 measurements. Note that uptake is rapid (K½ ∼2 min) and that uptake reaches a maximum (green dotted line). (E) Electron micrograph showing vesicles (arrowheads) attached to a hyphal cell treated with fluorescent terbinafine-loaded TFVs. Note that the attached vesicles are elongated (right lower corner). Bars represent micrometers. (F) Image series of electron micrographs of TFVs at different stages of entry. Note the deformation of the vesicles. Bar represents micrometers. (G) Bar chart showing the length and the width of vesicles attached to the outer fungal cell wall. Note that only those structures were measured that showed a lumen (e.g. the upper two vesicles in the image series given in F). Mean ± standard error of the mean of 16 measurements. Statistical significance difference at P<0.001 is indicated by double asterisks.

Mentions: To investigate whether the TFVs can enter the fungal cell, TDT 067 was used that was labeled with 1,6-diphenyl-1,3,5-hexatriene, a dye that specifically labels lipid membranes [25]. Scanning electron microscopy of these fluorescent terbinafine-loaded TFVs indicated that the applied suspension contained vesicles of various sizes (Fig. 2A). This size variation was also found when non-fluorescent TFVs were visualized in scanning electron microscopy (not shown). However, using photon correlation spectroscopy TFVs were found to be of uniform size, with an average diameter of ∼90 nm and a size distribution width of 15 nm (Dr. U. Vierl, C.P.M. ContractPharma GmbH & Co. KG, Feldkirchen-Westerham, Germany, pers. communication), suggesting that the size variation observed in my experiments are most likely due to dehydration occurring during the preparation process.


Cytoplasmic fungal lipases release fungicides from ultra-deformable vesicular drug carriers.

Steinberg G - PLoS ONE (2012)

Entry of terbinafine-loaded TFVs into the fungal cell.(A) Scanning electron micrograph of terbinafine-loaded Transfersome® vesicles. Bar represents micrometers. (B) Electron micrographs of an untreated hyphal cell (control) and after 30 min of incubation with fluorescent terbinafine-loaded TFVs (TBF in TFVs). The cell accumulates large droplets (asterisk). Bar represents micrometers. (C) Uptake of fluorescently labeled terbinafine-loaded Transfersome® vesicles. Prior to Transfersome® treatment, the cells show weak background fluorescence (0 min). Small and relatively faint fluorescent signals appear ∼1 min after adding fluorescent TFVs (1 min) and strong fluorescence is detected after ∼15 min of incubation. See also Movie S2. Bars represent micrometers. (D) Graph showing the kinetics of Transfersome® uptake. Each data point represents the mean ± standard error of the mean of 10–17 measurements. Note that uptake is rapid (K½ ∼2 min) and that uptake reaches a maximum (green dotted line). (E) Electron micrograph showing vesicles (arrowheads) attached to a hyphal cell treated with fluorescent terbinafine-loaded TFVs. Note that the attached vesicles are elongated (right lower corner). Bars represent micrometers. (F) Image series of electron micrographs of TFVs at different stages of entry. Note the deformation of the vesicles. Bar represents micrometers. (G) Bar chart showing the length and the width of vesicles attached to the outer fungal cell wall. Note that only those structures were measured that showed a lumen (e.g. the upper two vesicles in the image series given in F). Mean ± standard error of the mean of 16 measurements. Statistical significance difference at P<0.001 is indicated by double asterisks.
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Related In: Results  -  Collection

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pone-0038181-g002: Entry of terbinafine-loaded TFVs into the fungal cell.(A) Scanning electron micrograph of terbinafine-loaded Transfersome® vesicles. Bar represents micrometers. (B) Electron micrographs of an untreated hyphal cell (control) and after 30 min of incubation with fluorescent terbinafine-loaded TFVs (TBF in TFVs). The cell accumulates large droplets (asterisk). Bar represents micrometers. (C) Uptake of fluorescently labeled terbinafine-loaded Transfersome® vesicles. Prior to Transfersome® treatment, the cells show weak background fluorescence (0 min). Small and relatively faint fluorescent signals appear ∼1 min after adding fluorescent TFVs (1 min) and strong fluorescence is detected after ∼15 min of incubation. See also Movie S2. Bars represent micrometers. (D) Graph showing the kinetics of Transfersome® uptake. Each data point represents the mean ± standard error of the mean of 10–17 measurements. Note that uptake is rapid (K½ ∼2 min) and that uptake reaches a maximum (green dotted line). (E) Electron micrograph showing vesicles (arrowheads) attached to a hyphal cell treated with fluorescent terbinafine-loaded TFVs. Note that the attached vesicles are elongated (right lower corner). Bars represent micrometers. (F) Image series of electron micrographs of TFVs at different stages of entry. Note the deformation of the vesicles. Bar represents micrometers. (G) Bar chart showing the length and the width of vesicles attached to the outer fungal cell wall. Note that only those structures were measured that showed a lumen (e.g. the upper two vesicles in the image series given in F). Mean ± standard error of the mean of 16 measurements. Statistical significance difference at P<0.001 is indicated by double asterisks.
Mentions: To investigate whether the TFVs can enter the fungal cell, TDT 067 was used that was labeled with 1,6-diphenyl-1,3,5-hexatriene, a dye that specifically labels lipid membranes [25]. Scanning electron microscopy of these fluorescent terbinafine-loaded TFVs indicated that the applied suspension contained vesicles of various sizes (Fig. 2A). This size variation was also found when non-fluorescent TFVs were visualized in scanning electron microscopy (not shown). However, using photon correlation spectroscopy TFVs were found to be of uniform size, with an average diameter of ∼90 nm and a size distribution width of 15 nm (Dr. U. Vierl, C.P.M. ContractPharma GmbH & Co. KG, Feldkirchen-Westerham, Germany, pers. communication), suggesting that the size variation observed in my experiments are most likely due to dehydration occurring during the preparation process.

Bottom Line: With time, the lipid bodies were metabolized in an ATP-dependent fashion, suggesting that cytosolic lipases attack and degrade intruding TFVs.Indeed, the specific monoacylglycerol lipase inhibitor URB602 prevented Transfersome® degradation and neutralized the cytotoxic effect of Transfersome®-delivered terbinafine.As this mode of action of Transfersomes is independent of the drug cargo, these results demonstrate the potential of Transfersomes in the treatment of all fungal diseases.

View Article: PubMed Central - PubMed

Affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, United Kingdom. G.Steinberg@exeter.ac.uk

ABSTRACT
The Transfersome® is a lipid vesicle that contains membrane softeners, such as Tween 80, to make it ultra-deformable. This feature makes the Transfersome® an efficient carrier for delivery of therapeutic drugs across the skin barrier. It was reported that TDT 067 (a topical formulation of 15 mg/ml terbinafine in Transfersome® vesicles) has a much more potent antifungal activity in vitro compared with conventional terbinafine, which is a water-insoluble fungicide. Here we use ultra-structural studies and live imaging in a model fungus to describe the underlying mode of action. We show that terbinafine causes local collapse of the fungal endoplasmic reticulum, which was more efficient when terbinafine was delivered in Transfersome® vesicles (TFVs). When applied in liquid culture, fluorescently labeled TFVs rapidly entered the fungal cells (T(1/2)~2 min). Entry was F-actin- and ATP-independent, indicating that it is a passive process. Ultra-structural studies showed that passage through the cell wall involves significant deformation of the vesicles, and depends on a high concentration of the surfactant Tween 80 in their membrane. Surprisingly, the TFVs collapsed into lipid droplets after entry into the cell and the terbinafine was released from their interior. With time, the lipid bodies were metabolized in an ATP-dependent fashion, suggesting that cytosolic lipases attack and degrade intruding TFVs. Indeed, the specific monoacylglycerol lipase inhibitor URB602 prevented Transfersome® degradation and neutralized the cytotoxic effect of Transfersome®-delivered terbinafine. These data suggest that (a) Transfersomes deliver the lipophilic fungicide Terbinafine to the fungal cell wall, (b) the membrane softener Tween 80 allows the passage of the Transfersomes into the fungal cell, and (c) fungal lipases digest the invading Transfersome® vesicles thereby releasing their cytotoxic content. As this mode of action of Transfersomes is independent of the drug cargo, these results demonstrate the potential of Transfersomes in the treatment of all fungal diseases.

Show MeSH
Related in: MedlinePlus