Delivery of Inorganic Polyphosphate into Cells Using Amphipathic Oligocarbonate Transporters

Abstract

Inorganic polyphosphate (polyP) is an often-overlooked biopolymer of phosphate residues present in living cells. PolyP is associated with many essential biological roles. Despite interest in polyP’s function, most studies have been limited to extracellular or isolated protein experiments, as polyanionic polyP does not traverse the nonpolar membrane of cells. To address this problem, we developed a robust, readily employed method for polyP delivery using guanidinium-rich oligocarbonate transporters that electrostatically complex polyPs of multiple lengths, forming discrete nanoparticles that are resistant to phosphatase degradation and that readily enter multiple cell types. Fluorescently labeled polyPs have been monitored over time for subcellular localization and release from the transporter, with control over release rates achieved by modulating the transporter identity and the charge ratio of the electrostatic complexes. This general approach to polyP delivery enables the study of intracellular polyP signaling in a variety of applications.

A Phosphoramidite Analogue of Cyclotriphosphate Enables Iterative Polyphosphorylations

“…In mammals, polyP is involved in the regulation of the blood‐clotting cascade, but the enzymes generating it remain elusive . Reports suggest polyphosphorylation as a new posttranslational modification of proteins, and cellular delivery of a polydisperse, labeled polyP has recently been achieved …”

“…[9] Reports suggest polyphosphorylation as an ew posttranslational modification of proteins, [10,11] and cellular delivery of ap olydisperse, labeled polyP has recently been achieved. [12] Therefore,a ccess to modified polyphosphates is highly desirable,y et the synthesis of such labile structures in monodisperse form is significantly understudied. [13] Approaches to analogues of polyP exist, but they rely on the biochemical synthesis of polydisperse polyP followed by modification resulting in P-amidate end-labeling.…”

A broad response to intracellular long-chain polyphosphate in human cells

“…While this remains an important caveat of our study, it is possible that untransfected cells contain localized pools of highly concentrated polyP that could impact pathways similar to those uncovered here. The Ppk1 system also provides a method to model the impact of high concentrations of intracellular polyP delivered via nanoparticles, which is being investigated as a method to deliver polyP intracellularly (Fernandes-Cunha et al, 2018). Long-chain polyP produced by Ppk1 may mimic the impact of bacterial long-chain polyP that could be released during infection and taken up by host cells.…”

Rapid stimulation of cellular Pi uptake by the inositol pyrophosphate InsP8induced by its photothermal release from lipid nanocarriers using a near infra-red light-emitting diode

“…An alternate approach has been to employ biocompatible nanoparticles as delivery vehicles. For example, guanidinium-rich oligocarbonate transporters 26 have proven to be successful. Liposomes have also been used to deliver a number of polyphosphorylated molecules into cells; appropriate lipid compositions have been described that facilitate endocytic liposomal entry into the cell, and then during the following 2–4 hours, fusion of the liposomal and endosomal membranes permits slow and gradual escape of the encapsulated cargo into the cytosol.…”