Rapid stimulation of cellular Pi uptake by the inositol pyrophosphate InsP8 induced by its photothermal release from lipid nanocarriers using a near infra-red light-emitting diode

摘要

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.