Intracellular pH Measurements Using Perfluorocarbon Nanoemulsions

We
report the synthesis and formulation of unique perfluorocarbon
(PFC) nanoemulsions enabling intracellular pH measurements in living
cells via fluorescent microscopy and flow cytometry. These nanoemulsions
are formulated to readily enter cells upon coincubation and contain
two cyanine-based fluorescent reporters covalently bound to the PFC
molecules, specifically Cy3-PFC and CypHer5-PFC conjugates. The spectral
and pH-sensing properties of the nanoemulsions were characterized in vitro and showed the unaltered spectral behavior of dyes
after formulation. In rat 9L glioma cells loaded with nanoemulsion,
the local pH of nanoemulsions was longitudinally quantified using
optical microscopy and flow cytometry and displayed a steady decrease
in pH to a level of 5.5 over 3 h, indicating rapid uptake of nanoemulsion
to acidic compartments. Overall, these reagents enable real-time optical
detection of intracellular pH in living cells in response to pharmacological
manipulations. Moreover, recent approaches for in vivo cell tracking using magnetic resonance imaging (MRI) employ intracellular
PFC nanoemulsion probes to track cells using 19F MRI. However,
the intracellular fate of these imaging probes is poorly understood.
The pH-sensing nanoemulsions allow the study of the fate of the PFC
tracer inside the labeled cell, which is important for understanding
the PFC cell loading dynamics, nanoemulsion stability and cell viability
over time.