Optical calcium sensors: development of a generic method for their introduction to the cell using conjugated cell penetrating peptides

Abstract

Probes Encapsulated By Biologically Localised Embedding (PEBBLEs) are optical sensors with nanometer dimensions fabricated by microemulsion polymerisation. The most beneficial characteristic of these sensors is the protection offered by the sensor matrix which decreases interaction between the fluorophore and the cell. These sensors have been introduced to the cell by a number of methods; however this paper discusses the development of a generic method to facilitate inclusion of this type of sensor in the cell by a simple incubation step. This was achieved by covalent linkage of a synthetic Cell Penetrating Peptide (CPP) based on the Human Immuno-deficiency Virus (HIV) -1 Tat, to the external sensor matrix. Calcium sensors were used to demonstrate this approach to incorporate the sensors within the cell. Characterisation revealed the calcium sensors were approximately 30 +/- 7 nm in diameter with a slightly negative zeta potential. The sensors demonstrated a linear range of 0-50 microM with negligible interference from a range of cellular ions and protein. Leaching of entrapped dyes from the calcium sensors was determined as 3% in a 24 h period, while photobleaching of the entrapped dye was minimal over a 40 min period. The sensors ability to cross the cell membrane using the covalently attached synthetic Tat peptide is demonstrated. Cellular inclusion of the sensors occurred within a 30 min incubation period.

The delivery of PEBBLE nanosensors to measure the intracellular environment

“…CHO (Chinese-hamster ovary) cells [15] and pre-implantation embryos have had nanosensors successfully introduced by this method with good results, adequate loading and no discernible cellular changes, making this a viable method of introduction.…”

Electrochemical and optical sensing of reactive oxygen species: pathway to an integrated intracellular and extracellular measurement platform

“…Nanosensor fabrication was based on microemulsion polymerization techniques to obtain nanoparticles of an even size distribution (typically 50 nm in diameter based on AFFF (asymmetric field flow fractionation) characterization studies [28]). PEBBLE nanosensors for calcium [29], zinc [30], glucose [31] and pH [32] have all been reported. Numerous methods for introducing them into the intracellular environment have been reported including linking nanosensors to the cell penetrating TAT peptide [29], physical insertion via a gene gun [33] and utilizing innate cellular phagocytosis [34].…”

“…PEBBLE nanosensors for calcium [29], zinc [30], glucose [31] and pH [32] have all been reported. Numerous methods for introducing them into the intracellular environment have been reported including linking nanosensors to the cell penetrating TAT peptide [29], physical insertion via a gene gun [33] and utilizing innate cellular phagocytosis [34]. All of these methods have their limitations [33].…”

The development and in vitro characterisation of an intracellular nanosensor responsive to reactive oxygen species

“…The polyacrylamide PEBBLE nanosensors were synthesised using a modified version of the methods reported previously (Clark et al, 1999b;Coupland et al, 2008;Webster et al, 2005). In brief, a solution consisting of acrylamide and N,Nmethylenebisacrylamide (Sigma-Aldrich, UK) at a ratio of 1:3.37, respectively, was produced by dissolving in distilled water and sonicating for 5 min.…”

“…Membrane permeable 'free' dyes and particle-conjugated probes have been extensively reported in the literature (Chang et al, 2005a;Henderson and Chappell, 1993;Jiang et al, 1997;PalazzoloBallance et al, 2007). However, the use of fluorescent dyes encapsulated in the polyacrylamide matrix of PEBBLE nanosensors offers a number of advantages over previously reported probes (Clark et al, 1998;Webster et al, 2005). First, the bio-inert matrix of the PEBBLEs protects the intracellular environment from any potentially cytotoxic effects that long-term exposure to fluorescent probes may illicit.…”