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.

ABSTRACT: Inositol pyrophosphates (PP-InsPs), including diphospho-myo-inositol pentakisphosphate (5-InsP₇) and bis-diphospho-myo-inositol tetrakisphosphate (1,5-InsP₈), 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-InsP₇ analogue (i.e., 5-FAM-InsP₇) 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 10⁶ cultured cells. Finally, to interrogate the biological value of these procedures, we delivered 1,5-InsP₈ into HCT116 cells and showed it to dose-dependently stimulate the rate of [³³P]-Pi uptake; these observations reveal a rheostatic range of concentrations over which 1,5-InsP₈ is biologically functional in Pi homeostasis.

SUBMITTER: Wang Z 

PROVIDER: S-EPMC7891704 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature