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Encapsulation of Living Leishmania Promastigotes in Artificial Lipid Vacuoles.

Guedes CE, Lima JG, Helfer E, Veras PS, Viallat A - PLoS ONE (2015)

Bottom Line: L. amazonensis promastigotes encapsulated in liposomes filled with RPMI-Dextran solution at pH 7.5 or 6.5 survived up to 96 h at 24°C.At 37°C and pH 5.5, parasites survived 48h.This method will also facilitate the study of the temporal evolution of biophysical properties of the PV during its maturation.

View Article: PubMed Central - PubMed

Affiliation: Centro de Pesquisas Gonçalo Moniz, Fiocruz, Laboratório de Patologia e Biointervenção, Rua Waldemar Falcão, 121, Candeal, Salvador, Bahia, Brazil.

ABSTRACT
After phagocytosis by mammalian macrophages, promastigote forms of Leishmania parasites settle inside intracellular parasitophorous vacuoles (PVs) in which they transform into amastigote forms and replicate. Here, using a variant of the 'inverted emulsion' method, we succeeded in encapsulating living L. amazonensis parasites in giant artificial liposomes that serve as model PVs. We were able to control the size of liposomes, the pH and the composition of their internal volume, and the number of internalized parasites per liposome. L. amazonensis promastigotes encapsulated in liposomes filled with RPMI-Dextran solution at pH 7.5 or 6.5 survived up to 96 h at 24°C. At 37°C and pH 5.5, parasites survived 48h. This method paves the way to identifying certain effectors secreted by the parasite and to unraveling specific mechanisms of fusion between the PV and intracellular vesicles of the host cell. This method will also facilitate the study of the temporal evolution of biophysical properties of the PV during its maturation.

No MeSH data available.


Related in: MedlinePlus

Liposomes encapsulating L. amazonensis promastigotes.The liposome membrane is composed of a EPC-PEG lipid mixture. Five million of L. amazonensis promastigotes per ml were used for the encapsulation process. (A) Liposomes containing multiple parasites. (B) Liposome with a single parasite with its flagellum at the right part of the parasite. Bright field images.
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pone.0134925.g002: Liposomes encapsulating L. amazonensis promastigotes.The liposome membrane is composed of a EPC-PEG lipid mixture. Five million of L. amazonensis promastigotes per ml were used for the encapsulation process. (A) Liposomes containing multiple parasites. (B) Liposome with a single parasite with its flagellum at the right part of the parasite. Bright field images.

Mentions: We successfully encapsulated L. amazonensis promastigotes inside EPC-PE-PEG liposomes containing sucrose, RPMI or RPMI-Dextran solutions. The RPMI-Dextran solution yields the highest encapsulation efficiency because its density matched that of parasites (see Fig 2). We also encapsulated L. amazonensis promastigotes in charged DOPC-DOPE-DOPS liposomes containing RPMI-Dextran solution. In this case, the divalent ions (such as Ca2+) released in the glucose solution after breakage of some liposomes at the interface interacted with the negatively charged lipids of the interface and of the liposome membrane. They mediated an attractive lipid-lipid interaction resulting in aggregation and sequestration of the liposomes at the lipid-oil/glucose solution interface. Addition of 15mM EDTA to the glucose solution enabled the detachment of liposomes from the interface and subsequent sedimentation toward the bottom of the chamber.


Encapsulation of Living Leishmania Promastigotes in Artificial Lipid Vacuoles.

Guedes CE, Lima JG, Helfer E, Veras PS, Viallat A - PLoS ONE (2015)

Liposomes encapsulating L. amazonensis promastigotes.The liposome membrane is composed of a EPC-PEG lipid mixture. Five million of L. amazonensis promastigotes per ml were used for the encapsulation process. (A) Liposomes containing multiple parasites. (B) Liposome with a single parasite with its flagellum at the right part of the parasite. Bright field images.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4524717&req=5

pone.0134925.g002: Liposomes encapsulating L. amazonensis promastigotes.The liposome membrane is composed of a EPC-PEG lipid mixture. Five million of L. amazonensis promastigotes per ml were used for the encapsulation process. (A) Liposomes containing multiple parasites. (B) Liposome with a single parasite with its flagellum at the right part of the parasite. Bright field images.
Mentions: We successfully encapsulated L. amazonensis promastigotes inside EPC-PE-PEG liposomes containing sucrose, RPMI or RPMI-Dextran solutions. The RPMI-Dextran solution yields the highest encapsulation efficiency because its density matched that of parasites (see Fig 2). We also encapsulated L. amazonensis promastigotes in charged DOPC-DOPE-DOPS liposomes containing RPMI-Dextran solution. In this case, the divalent ions (such as Ca2+) released in the glucose solution after breakage of some liposomes at the interface interacted with the negatively charged lipids of the interface and of the liposome membrane. They mediated an attractive lipid-lipid interaction resulting in aggregation and sequestration of the liposomes at the lipid-oil/glucose solution interface. Addition of 15mM EDTA to the glucose solution enabled the detachment of liposomes from the interface and subsequent sedimentation toward the bottom of the chamber.

Bottom Line: L. amazonensis promastigotes encapsulated in liposomes filled with RPMI-Dextran solution at pH 7.5 or 6.5 survived up to 96 h at 24°C.At 37°C and pH 5.5, parasites survived 48h.This method will also facilitate the study of the temporal evolution of biophysical properties of the PV during its maturation.

View Article: PubMed Central - PubMed

Affiliation: Centro de Pesquisas Gonçalo Moniz, Fiocruz, Laboratório de Patologia e Biointervenção, Rua Waldemar Falcão, 121, Candeal, Salvador, Bahia, Brazil.

ABSTRACT
After phagocytosis by mammalian macrophages, promastigote forms of Leishmania parasites settle inside intracellular parasitophorous vacuoles (PVs) in which they transform into amastigote forms and replicate. Here, using a variant of the 'inverted emulsion' method, we succeeded in encapsulating living L. amazonensis parasites in giant artificial liposomes that serve as model PVs. We were able to control the size of liposomes, the pH and the composition of their internal volume, and the number of internalized parasites per liposome. L. amazonensis promastigotes encapsulated in liposomes filled with RPMI-Dextran solution at pH 7.5 or 6.5 survived up to 96 h at 24°C. At 37°C and pH 5.5, parasites survived 48h. This method paves the way to identifying certain effectors secreted by the parasite and to unraveling specific mechanisms of fusion between the PV and intracellular vesicles of the host cell. This method will also facilitate the study of the temporal evolution of biophysical properties of the PV during its maturation.

No MeSH data available.


Related in: MedlinePlus