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

Inositol pyrophosphates (PP-InsPs), including diphospho--inositol pentakisphosphate (5-InsP) and bis-diphospho--inositol tetrakisphosphate (1,5-InsP), 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 analogue (, 5-FAM-InsP) 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 cultured cells. Finally, to interrogate the biological value of these procedures, we delivered 1,5-InsP into HCT116 cells and showed it to dose-dependently stimulate the rate of [P]-Pi uptake; these observations reveal a rheostatic range of concentrations over which 1,5-InsP is biologically functional in Pi homeostasis.