ProxyPhos sensors for the detection of negatively charged membranes

Abstract

Membrane-embedded negatively charged phospholipids (MENCP) can be used as biomarkers for a range of biological processes, including early detection of apoptosis in animal cells, drug-induced phospholipidosis, and selective detection of bacterial over animal cells. Currently, several technologies for the detection of apoptosis and bacterial cells are based on the recognition of MENCPs, including the AnnexinV stain and PSVue™ probes. As probes, these technologies have limitations, the most significant of which is the need for washing the unbound probe away to achieve optimal signal. In contrast, a turn-on chemosensor selective for MENCP would address this shortcoming, and allow for a more rapid protocol for the detection of apoptosis, bacteria and for other relevant applications. In this work, the aim was to explore whether ProxyPhos chemosensors, previously reported by our group for the detection of proximally phosphorylated peptides and proteins, could be re-purposed for the detection of MENCPs. Six lead ProxyPhos sensors were screened against synthetic vesicles containing biologically relevant negatively charged phospholipids including phosphatidic acid (PA), phosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylserine (PS). Through these screens, ProxyPhos sensors exhibiting high selectivity for the detection of MENCPs over zwitterionic lipids were identified. Particular selectivity was observed for PA and CL. Sensitivity of the lead sensors for MENCPs was suitable for the detection of apoptosis: ProxyPhos detected vesicles containing as little as 2.5% PS and detected camptothecin-induced apoptosis in mammalian cells in flow cytometry experiments. The results suggest that ProxyPhos sensors can be used for the detection of MENCPs in synthetic vesicles and live mammalian cells.

Alginate Lyase Guided Silver Nanocomposites for Eradicating Pseudomonas aeruginosa from Lungs

“…This would greatly decrease the internalization of the nanoparticles by the mammalian cells because of their negatively charged cell membranes. 38 Furthermore, the FT-IR spectrum of Ag@ MON-AE showed the characteristic absorption bands of ceftazidime at 3416, 1756, 1619, 1537, 1365, 1186, and 1155 cm −1 , further demonstrating the successful loading of the drug in the silver nanoparticles (Figure 2c). 39 Briefly, the strong absorption bands at 3416, 1756, and 1619 cm −1 stand for the −OH group, stretching vibration of CO and CN, and vibration of the NH 2 and NH groups, respectively; the deformation band at 1612 cm −1 indicates the vibration of the The characteristic FT-IR absorption bands of the nanoparticles at different stages demonstrated that the silver nanocomposites were successfully loaded with alginate lyase and ceftazidime (Table S1).…”

“…Although the drug loading did not significantly change their diameter, the ceftazidime package greatly reduced the zeta potential of the nanoparticles from 30.06 to 7.09 mV (Figure a,b). This would greatly decrease the internalization of the nanoparticles by the mammalian cells because of their negatively charged cell membranes . Furthermore, the FT-IR spectrum of Ag@MON-AE showed the characteristic absorption bands of ceftazidime at 3416, 1756, 1619, 1537, 1365, 1186, and 1155 cm –1 , further demonstrating the successful loading of the drug in the silver nanoparticles (Figure c) .…”

Smart Bacterial Magnetic Nanoparticles for Tumor-Targeting Magnetic Resonance Imaging of HER2-Positive Breast Cancers

“…In addition, the ligation of negatively charged Cy5.5 on the proteins further lowered electric charges of the MamC-HAF-or MamC-THAFmodified magnetosomes (Figure 2c). Considering the negatively charged membranes of mammalian cells, 35 this could significantly reduce the nonspecific binding of bacterial nanoparticles to normal cells, 36 which is necessary for imaging agents in clinical applications. In addition, the presence of MamC-HAF or MamC-THAF on the magnetosomes was further confirmed by SDS-PAGE.…”

Time-lapse imaging of cell death in cell culture and whole living organisms using turn-on deep-red fluorescent probes

“…To date, several different fluorescence activation strategies have been explored that target intracellular or extracellular cell death biomarkers. 1718 In the specific case of “turn-on” fluorescent probes that target exposed PS, there are published cell studies using visible wavelength dyes, 19 or probes that exploit concentration dependent aggregation effects, 2021 but no demonstration of utility for in vivo imaging ( i.e. , imaging of the dead/dying cells in a living subject).…”