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Rapid stimulation of cellular Pi uptake by the inositol pyrophosphate InsP(8) induced by its photothermal release from lipid nanocarriers using a near infra-red light-emitting diode

Inositol pyrophosphates (PP-InsPs), including diphospho-myo-inositol pentakisphosphate (5-InsP(7)) and bis-diphospho-myo-inositol tetrakisphosphate (1,5-InsP(8)), are highly polar, membrane-impermeant signaling molecules that control many homeostatic responses to metabolic and bioenergetic imbalance...

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Detalles Bibliográficos
Autores principales: Wang, Zhenzhen, Jork, Nikolaus, Bittner, Tamara, Wang, Huanchen, Jessen, Henning J., Shears, Stephen B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Royal Society of Chemistry 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891704/
https://www.ncbi.nlm.nih.gov/pubmed/33659052
http://dx.doi.org/10.1039/d0sc02144j
Descripción
Sumario:Inositol pyrophosphates (PP-InsPs), including diphospho-myo-inositol pentakisphosphate (5-InsP(7)) and bis-diphospho-myo-inositol tetrakisphosphate (1,5-InsP(8)), are highly polar, membrane-impermeant signaling molecules that control many homeostatic responses to metabolic and bioenergetic imbalance. To delineate their molecular activities, there is an increasing need for a toolbox of methodologies for real-time modulation of PP-InsP levels inside large populations of cultured cells. Here, we describe procedures to package PP-InsPs into thermosensitive phospholipid nanocapsules that are impregnated with a near infra-red photothermal dye; these liposomes are readily accumulated into cultured cells. The PP-InsPs remain trapped inside the liposomes until the cultures are illuminated with a near infra-red light-emitting diode (LED) which permeabilizes the liposomes to promote PP-InsP release. Additionally, so as to optimize these procedures, a novel stably fluorescent 5-InsP(7) analogue (i.e., 5-FAM-InsP(7)) was synthesized with the assistance of click-chemistry; the delivery and deposition of the analogue inside cells was monitored by flow cytometry and by confocal microscopy. We describe quantitatively-controlled PP-InsP release inside cells within 5 min of LED irradiation, without measurable effect upon cell integrity, using a collimated 22 mm beam that can irradiate up to 10(6) cultured cells. Finally, to interrogate the biological value of these procedures, we delivered 1,5-InsP(8) into HCT116 cells and showed it to dose-dependently stimulate the rate of [(33)P]-Pi uptake; these observations reveal a rheostatic range of concentrations over which 1,5-InsP(8) is biologically functional in Pi homeostasis.