Nonprotecting Group Synthesis of a Phospholipase C Activatable Probe with an Azo-Free Quencher

Abstract

The near-infrared fluorescent activatable smart probe Pyro-phosphatidylethanolamine (PtdEtn)-QSY was synthesized and observed to selectively fluoresce in the presence of phosphatidylcholine-specific phospholipase C (PC-PLC). PC-PLC is an important biological target as it is known to be upregulated in a variety of cancers, including triple negative breast cancer. Pyro-PtdEtn-QSY features a QSY21 quenching moiety instead of the Black Hole Quencher-3 (BHQ-3) used previously because the latter contains an azo bond, which could lead to biological instability.

NIR Fluorescent Imaging and Photodynamic Therapy with a Novel Theranostic Phospholipid Probe for Triple-Negative Breast Cancer Cells

“…Pyro-PtdEtn-BHQ showed successful activation in DU145 human prostate cancer cells; − however, BHQ-3 has experienced stability problems in vivo due to the presence of an azo bond in the structure. ,− Complicated synthesis and short shelf life make its use impractical for clinical applications, and as a result, a more stable quencher is required. QSY21 has a similar quenching range (580–720 nm), but does not contain an azo bond and proves to be more stable. , …”

“…Pyro-PtdEtn-QSY and Pyro-PtdCho were synthesized following procedures reported previously (Schemes S1, S2). ,− In earlier studies, NIR Black Hole Quencher-3 (BHQ-3, absorbance range 620–730 nm) had been incorporated into the structure of the quenched probe. − The BHQ-3 quencher fully absorbed the fluorescence emitted by Pyro through Förster resonance energy transfer (FRET) . Pyro-PtdEtn-BHQ showed successful activation in DU145 human prostate cancer cells; − however, BHQ-3 has experienced stability problems in vivo due to the presence of an azo bond in the structure. ,− Complicated synthesis and short shelf life make its use impractical for clinical applications, and as a result, a more stable quencher is required.…”

“…The hydrophilic PEG prevents adsorption of proteins and cells, decreasing in vivo removal of the particles from circulation by the reticuloendothelial system . Due to their small size, LNPs should have low immunogenicity compared to large PEGylated liposomes. , For cell delivery, nanoparticles loaded with 13.5 mol % of either Pyro-PtdEtn-QSY or Pyro-PtdCho were prepared using an established protocol; the complete procedure is provided in the Experimental Section. Nanoparticle size distribution was determined by dynamic light scattering, using nanoparticles that did not carry the photosensitizer; these micelles had uniform size with diameter of 10.8 ± 2.9 nm (Figure S1), in agreement with our previous results…”

“…We have developed smart near-infrared (NIR) fluorescent phospholipid probes that fluoresce after selective cleavage by PC–PLC. ,, Smart probes that only fluoresce upon enzymatic activation specific to malignant cells are currently at the forefront of cancer imaging research, offering potential for improved selectivity. − PC–PLC shows a high degree of selectivity to phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) when a bulky substituent is attached to the sn -2 position of these phospholipids. − This principle is exploited in our probe, pyropheophorbide a- phosphatidylethanolamine-QSY21 (Pyro-PtdEtn-QSY, Figure a), which is selectively cleaved in the presence of PC–PLC …”

Detection and Differentiation of Breast Cancer Sub-Types using a cPLA2α Activatable Fluorophore

“…Efforts to improve signal to noise have been made through the targeting of fluorophores to cancer signatures with varying success 17–22 . An alternative solution is the use of enzyme targeted activatable fluorophores that remain non-fluorescent until selectively acted upon 23–31 . A single enzyme, cleaving multiple substrates, can result in rapid signal amplification 32 .…”