Intracellular pH measurements using perfluorocarbon nanoemulsions

Abstract

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 (19)F 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.

Figure 1

Size distribution by intensity of nanoemulsions 20, 22 and 25.

Figure 2

Spectral properties of cyanine-PFPE conjugates in phosphate buffers. Inset (A) shows pH range for series. Absorbance spectra (A) of CypHer5-PFPE oil (14, 17 and 18) and (B) Cy5-PFPE oil (13, 16 and 18). Fluorescence emission spectra of CypHer5-PFPE (C) and Cy5-PFPE (D) nanoemulsions 22 and 21 respectively. Excitation wavelength was 630 nm.

Figure 3

Fluorescence spectra and pH response of ratiometric nanoemulsions 24 and 26 in phosphate buffers. Inset (A) shows pH range for series. Emission scans EX 630 nm (A) and EX 530 nm (B). pH-ratio calibration curves (C) normalized to maximum fluorescence ratio value (at pH 4.0), averaged from synchronous and fixed wavelength scanning methods. pK_a of both formulations is 6.8.

Figure 4

Synchronous scans (20 nm offset) of ratiometric nanoemulsion formulations 24-29 in phosphate buffers. Inset (24) shows pH range for series. Single component ratiometric control nanoemulsion 28 contains no Cy3-PFPE; 29 contains no CypHer5-PFPE. All spectra are normalized to EX 548 nm (Cy3).

Figure 5

Nanoemulsion uptake by ¹⁹F NMR. 9L cells labeled with ratiometric nanoemulsion 26. ¹⁹F NMR in 0.1% TFA of 9L cells labeled at 5 mg/ml. Cells were lysed for measurements.

Figure 6

Nanoemulsion uptake by ¹⁹F NMR and fluorescence methods. 9L cells labeled with ratiometric nanoemulsion 26. Dose curve by ¹⁹F NMR and, Cy3 and CypHer5 fluorescence. Cells were lysed for measurements.

Figure 7

Confocal microscopy images of ratiometric nanoemulsion 26 labeled 9L cells, showing channels: CypHer5 (A), Cy3 (B), DIC (C) and merged channels (D). Scale = 10 μm.

Figure 8

Flow cytometry of 9L cells labeled with ratiometric nanoemulsions. Representative FSC/SSC plot (A) and dot plot (B) for formulation 24; gated region marked by black line (A). Histograms are of live gated cells at (C) 575 nm for Cy3 and (D) 685 nm for CypHer5, and are overlaid control cells labeled with non-fluorescent nanoemulsion 23.

Figure 9

Comparison of ratiometric nanoemulsion formulations by flow cytometry. A plot of mean fluorescence ratio (685nm/575nm) against formulation ratio of nanoemulsions 24-27 using 9L cells labeled with ratiometric nanoemulsions. Data points are identified by formulation number.

Figure 10

(A) Comparison of pH-ratio curves of pH-clamped 9L cells labeled with ratiometric nanoemulsion 24, free nanoemulsion and autofluorescence correction. (B) Intracellular pH during nanoemulsion uptake showing raw and corrected data.

Scheme 1

Synthetic scheme of cyanine-NBoc conjugates.

Scheme 2

Synthesis of cyanine blended PFPE amides (CBPAs). Each CBPA is a mixture of PFPE derivatives where cyanine is Cy3, Cy5 or CypHer5 fluorogen and composition of each CBPA is: Cy3-PFPE-oil (12, 15 and 18), Cy5-PFPE-oil (13, 16 and 18) and CypHer5-PFPE-oil (14, 17 and 18).