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Engineered Trehalose Permeable to Mammalian Cells.

Abazari A, Meimetis LG, Budin G, Bale SS, Weissleder R, Toner M - PLoS ONE (2015)

Bottom Line: Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation.Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre).Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.

View Article: PubMed Central - PubMed

Affiliation: The Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, and Shriners Hospital for Children, Boston, Massachusetts, United States of America.

ABSTRACT
Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.

No MeSH data available.


Uptake of acetylated trehalose in hepatocytes.(A) Intracellular accumulation of trehalose, and (B) intracellular accumulation of (n)-O-Ac-Tre, after incubation with 30 mM concentration of trehalose, 2-, 4-, and 6-O-Ac-Tre. 8-O-Ac-Tre was not studied as it was not water soluble. Error bars represent SD (n = 3). (* denote p>0.05 and # denote p<0.05 between respective groups).
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pone.0130323.g003: Uptake of acetylated trehalose in hepatocytes.(A) Intracellular accumulation of trehalose, and (B) intracellular accumulation of (n)-O-Ac-Tre, after incubation with 30 mM concentration of trehalose, 2-, 4-, and 6-O-Ac-Tre. 8-O-Ac-Tre was not studied as it was not water soluble. Error bars represent SD (n = 3). (* denote p>0.05 and # denote p<0.05 between respective groups).

Mentions: We hypothesized that acetylation of trehalose would increase its membrane permeability, and that once in the cytoplasm, endogenous esterases would hydrolyze acetylated-trehalose to trehalose. To test this hypothesis, we measured the concentration of non-acetylated trehalose in rat hepatocytes after incubation with 30 mM solutions of trehalose and 2-, 4- and 6-O-Ac-Tre at regular intervals (Fig 3A). To reduce hydrolysis of acetylated trehalose by esterase activity after lysis, the cell lysate were immediately frozen or kept on ice until analyzed. The detection and time-dependent increase of trehalose in cell lysate suggests that both the penetration and the hydrolysis of acetylated trehalose occurred. However, a significant difference was observed in the amount of converted trehalose when comparing 6-O-Ac-Tre to 2- and 4-O-Ac-Tre. With 6-O-Ac-Tre, the trehalose concentration showed a time-dependent increase, suggesting continuous permeation and hydrolysis of 6-O-Ac-Tre. In the first hour of incubation, the concentration of fully deacetylated trehalose was below the detection limits of our trehalose assay (<1 mM), but increased linearly between 3–12 h of incubation with an approximate rate of 8.5 mM/h. After 12 h, the intracellularly-accumulated trehalose reached as high as 92 mM. This is more than 3 fold higher than the incubation concentration for 6-O-Ac-Tre. The amounts of trehalose accumulated in cells after incubation with 2- and 4-O-Ac-Tre were much smaller. Between 3–12 h, the trehalose concentration reached a plateau at ~8 mM in cells incubated with 2-O-Ac-Tre, while it gradually but slowly increased to ~17 mM in cells incubated with 4-O-Ac-Tre.


Engineered Trehalose Permeable to Mammalian Cells.

Abazari A, Meimetis LG, Budin G, Bale SS, Weissleder R, Toner M - PLoS ONE (2015)

Uptake of acetylated trehalose in hepatocytes.(A) Intracellular accumulation of trehalose, and (B) intracellular accumulation of (n)-O-Ac-Tre, after incubation with 30 mM concentration of trehalose, 2-, 4-, and 6-O-Ac-Tre. 8-O-Ac-Tre was not studied as it was not water soluble. Error bars represent SD (n = 3). (* denote p>0.05 and # denote p<0.05 between respective groups).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482662&req=5

pone.0130323.g003: Uptake of acetylated trehalose in hepatocytes.(A) Intracellular accumulation of trehalose, and (B) intracellular accumulation of (n)-O-Ac-Tre, after incubation with 30 mM concentration of trehalose, 2-, 4-, and 6-O-Ac-Tre. 8-O-Ac-Tre was not studied as it was not water soluble. Error bars represent SD (n = 3). (* denote p>0.05 and # denote p<0.05 between respective groups).
Mentions: We hypothesized that acetylation of trehalose would increase its membrane permeability, and that once in the cytoplasm, endogenous esterases would hydrolyze acetylated-trehalose to trehalose. To test this hypothesis, we measured the concentration of non-acetylated trehalose in rat hepatocytes after incubation with 30 mM solutions of trehalose and 2-, 4- and 6-O-Ac-Tre at regular intervals (Fig 3A). To reduce hydrolysis of acetylated trehalose by esterase activity after lysis, the cell lysate were immediately frozen or kept on ice until analyzed. The detection and time-dependent increase of trehalose in cell lysate suggests that both the penetration and the hydrolysis of acetylated trehalose occurred. However, a significant difference was observed in the amount of converted trehalose when comparing 6-O-Ac-Tre to 2- and 4-O-Ac-Tre. With 6-O-Ac-Tre, the trehalose concentration showed a time-dependent increase, suggesting continuous permeation and hydrolysis of 6-O-Ac-Tre. In the first hour of incubation, the concentration of fully deacetylated trehalose was below the detection limits of our trehalose assay (<1 mM), but increased linearly between 3–12 h of incubation with an approximate rate of 8.5 mM/h. After 12 h, the intracellularly-accumulated trehalose reached as high as 92 mM. This is more than 3 fold higher than the incubation concentration for 6-O-Ac-Tre. The amounts of trehalose accumulated in cells after incubation with 2- and 4-O-Ac-Tre were much smaller. Between 3–12 h, the trehalose concentration reached a plateau at ~8 mM in cells incubated with 2-O-Ac-Tre, while it gradually but slowly increased to ~17 mM in cells incubated with 4-O-Ac-Tre.

Bottom Line: Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation.Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre).Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.

View Article: PubMed Central - PubMed

Affiliation: The Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, and Shriners Hospital for Children, Boston, Massachusetts, United States of America.

ABSTRACT
Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.

No MeSH data available.