Intracellular pH measurements using perfluorocarbon nanoemulsions

Article abstract of DOI:10.1021/ja407573m

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.

Full text of DOI:10.1021/ja407573m

Article
pubs.acs.org/JACS
Intracellular pH Measurements Using Perfluorocarbon
Nanoemulsions
Michael J. Patrick,† Jelena M. Janjic,‡ Haibing Teng,† Meredith R. O’Hear,§,# Cortlyn W. Brown,∥,∇
Jesse A. Stokum,§ Brigitte F. Schmidt,† Eric T. Ahrens,*,§,⊥,¶ and Alan S. Waggoner†
†Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States
‡Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, Pennsylvania 15219, United States
§Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States
∥Department of Biological Sciences, University of Chicago, Chicago, Illinois 60637, United States
⊥Pittsburgh NMR Center for Biomedical Research, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States
S
* Supporting Information
ABSTRACT: We report the synthesis and formulation of
unique perfluorocarbon (PFC) nanoemulsions enabling intra-
cellular 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.
wavelengths (∼645/665 nm ex/em),3,4 compared with
1. INTRODUCTION
fluorescein (494/518 nm ex/em);12 thus, detection is less
Measurement of real-time intracellular pH is useful in the
likely to be confounded by cellular autofluorescence. Most
development of intracellular drug delivery systems and in vitro
recently, a CypHer5-based probe was developed by Grover et
pharmacology studies. A common uptake mechanism of protein
al. to measure pH variations in β2-adrenergic receptor
and nanoreagent delivery involves endocytosis,1,2 which results
in drug payload exposure to low pH in the lysosomal
compartments. The residence time of the vehicle and the
drug inside lysosomes can be measured, in principle, if a pH
trafficking and to study surface-to-endosome intracellular
transfer processes in dendritic cells.13
In this study, we have devised novel synthesis strategies to
incorporate pH-sensitive cyanine fluorochromes into perfluor-
ocarbon (PFC) nanoemulsion reagents. As described herein,
sensor is incorporated into the formulation. pH-sensitive
fluorochromes have been used to ascertain the pH of cellular
PFC nanoemulsions have received recent significant interest as
compartments.3−5 A protein or other macromolecule is labeled
19F MRI imaging agents for clinically relevant in vivo cell
with a pH-sensitive probe, and the fluorescence emission is
tracking14−18 and as theranostic vehicles.19,20 Here, we
conjugate CypHer5 to a linear perfluoropolyether (PFPE)
PFC molecule prior to nanoemulsion formulation. Additionally,
we have synthesized nanoemulsions containing blended PFPE
conjugates in the fluorocarbon phase that have a covalently
attached reference fluorophore with a different emission
monitored as it passes into and moves through the cell.3−5
Several fluorochromes are reported to have pH sensitivity,6
including BCECF, BCPCF, SNARF, cyanines, and fluores-
cein.5−8 Specifically, the pH-sensitive cyanine derivative,
CypHer5, developed by Mujumdar and Smith9−11 and
characterized by Briggs and Cooper,3,4 has been used as a pH
sensor of lysosomal compartments. The pKa varies among
CypHer5 analogs and ranges from 6.1 to 7.5. CypHer5 analogs
also have the advantage of longer excitation and emission
Received: July 23, 2013
Published: October 30, 2013
© 2013 American Chemical Society
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Scheme 1. Synthetic Scheme of Cyanine-NBoc Conjugates
Scheme 2. Synthesis of 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)
wavelength maximum (Cy3) that is insensitive to the pH
environment of the nanoemulsion. This feature allows
ratiometric measurement of absolute microenvironment pH
from a reference calibration curve. These new nanoemulsion
‘biosensors’ can be used to assay intracellular pH in proximity
to the nanoemulsion droplets in living cells using flow
cytometry and fluorescence microscopy. Development of a
fluorescent pH-sensing fluorocarbon nanoemulsion permits
tracking of the probe in the cell and reports on the pH of the
environment of the 19F probe. Additionally, our probes can
track intracellular localization and can potentially be used to
target site-specific organelles; importantly, they are durable,
self-delivering, and bright. Our method of design (pH sensor
conjugated to PFC) prevents separation of the pH sensor from
the fluorocarbon, which is essential to reporting the pH of the
fluorous phase location within the cell.
Formulation of stable and effective pH-sensing nano-
emulsions offers unique challenges. The fluorogenic portions
of the PFPE-biosensor conjugate must retain chemical and
photo stability during synthesis. Moreover, although CyDyes in
general have been useful as fluorescent labels for proteins and
antibodies, it was not clear that they would retain their spectral
properties once formulated into nanoemulsions by high-shear
microfluidization. Importantly, the final product must be
nontoxic to cells, readily taken-up by cells upon coincubation,
and retain sensor function within the intracellular milieu. We
briefly describe the process we have developed to optimize the
dye-PFPE conjugation, nanoemulsion formulation, cell labeling,
and fluorescence (pH) quantification. We demonstrate that the
CypHer5-PFPE labeled nanoemulsion retains its sensitivity to
pH following nanoemulsion formulation. Flow cytometry and
ratiometric fluorescence were used to generate a calibration
curve and show that the pH of the cellular compartment of 9L
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glioma cells that contain the internalized nanoemulsion changes
to approximately pH 5.5 over a 3 h period following labeling.
Complete details of the methods used are described in
Supporting Information.
absolute ethanol. Single component ratiometric controls (28−
29), containing only one dye, were made by blending PFPE-
amide (19) with either 12 or 14. The ratiometric contribution
of each dye was determined by fluorescence synchronous
excitation/emission (ex/em) scans in pH 5.8 phosphate
buffer3,4 using methods described in Supporting Information,
resulting in fluorescence ratios of the oils ranging from 1 to 10
times CypHer5 to Cy3 signal. Likewise, ratiometric PFPE-
conjugates were mixed with PFPE-oxide21 (at 4.9% v/v) with
the aid of an equal volume of absolute ethanol.
Blended PFPE conjugate oils behave as a stable and uniform
fluorous phase.21,24 The ratio of the CBPA to PFPE-oxide was
1:100; the fluorescent signals would be too saturated if CBPAs
were used at full concentration for the nanoemulsion and
would cause dye quenching and loss of signal.
2. RESULTS
2.1. Synthesis and Formulation of Nanoemulsions.
2.1.1. Synthesis of Cyanine-PFPEs. Linear PFPE was directly
conjugated to fluorescent probes by expanding the method-
ologies of Janjic et al.21,22 Starting from preparative high-
performance liquid chromatography (HPLC)-purified carbox-
ylic acids of fluorescent dyes,3,4,23 free acids were converted to
N-hydroxysuccinimidyl esters (NHS-esters) using N,N,N′,N′-
tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate
(TSTU) and then conjugated via amine groups to tert-butyl
carbamate (Boc)-protected diamine derivatives according to
Scheme 1. Dye-free acids (CypHer5, Cy3.29 or Cy5.29, 3, 1
and 2 respectively) were derivatized to make NBoc conjugates
(5, 6, 7) using ethylenediamine-NBoc in organic solvents and
purified by preparative HPLC. The Boc-protected dye was
purified by reversed phase HPLC, deprotected with trifluoro-
acetic acid (TFA), and conjugated to the highly reactive PFPE
ester as described elsewhere21 and as shown in Scheme 2.
When Cy3-PFPE and CypHer5-PFPE were both included in
the ratiometric nanoemulsion formulations, 3 M hydrochloric
acid was used instead of 1% TFA to improve Boc-group
deprotecting efficiency, which was >98% pure by analytical
HPLC. This was performed to ensure that during development
of pH calibration curves and cell pH measurements, fluorescent
signals from unconjugated precursors were eliminated.
PFPEs are highly hydrophobic and lipophobic, with limited
solubility in common organic solvents, but are soluble in
fluorinated solvents such as trifluorotoluene, trifluoroethanol,
and perfluorohexanes. The NBoc moiety was removed with
trifluoroacetic acid, and cyanine-amine conjugates (8, 9, or 10)
were mixed with ethanol and triethylamine, and reacted with
PFPE-methyl ester (11). More specifically, Cy3.29, Cy5.29, and
CypHer5 (8−10) were coupled at 1% mol ratio of PFPE ester,
yielding cyanine blended PFPE-amides (CBPAs). CBPAs are a
mixture of mono- and bis-conjugated oils (12−14 and 15−17,
respectively) or bis-amide PFPE (18) and were used without
further separation. Cy3.29-PFPE-oil is a mixture of products 12,
15, and 18; Cy5.29-PFPE-oil is compounds 13, 16, and 18, and
CypHer5-PFPE-oil is 14, 17, and 18 (Scheme 2).
It has been well documented that the longer the polymethine
bridge of a cyanine dye, the less stable the fluorophore.6
Because of the environmental sensitivities of the CypHer5
fluorophore, such as chemical reactivity or susceptibility to
decomposition, care was taken to preserve the compound
stability during synthesis and purification by HPLC (see
Supporting Information for details). For optimal stability while
in solution, CypHer5 derivatives were kept cold (4 °C) and
acidic; however, for long-term storage, cold (−20 °C), solid,
and dry conditions were used.
2.1.2. Blending of Cyanine-PFPEs. To make single-dye
nanoemulsions (20−22), CBPAs (12−15−18, 13−16−18, or
14−17−18) were mixed with PFPE-oxide21 (at 5% v/v) with
the aid of an equal volume of absolute ethanol. Likewise, PFPE-
amide (19) was used in place of CBPAs to make a
nonfluorescent nanoemulsion control (23). To make two-
dye, ratiometric nanoemulsions (24−27), CypHer5-PFPE
(nominally 14), and Cy3-PFPE oils (nominally 12) were
blended in molar stoichiometries using an equal volume of
Several formulations of CypHer5-PFPE to Cy3-PFPE were
blended in various stoichiometries (Table 1). CypHer5-PFPE
Table 1. Listing of nanoemulsion formulation products and
PFPE−conjugate composition. Ratio value (CypHer5:Cy3)
is post-formulation
nominal reagent
components (ratios)
product
nanoemulsion name
20
21
22
23
24
Cy3-PFPE
Cy5-PFPE
12
13
CypHer5-PFPE
PFPE-amide
14
19
ratiometric CypHer5-PFPE:Cy3-PFPE
(0.6:1)
14:12 (0.6:1)
25
26
27
28
29
ratiometric CypHer5-PFPE:Cy3-PFPE
(1:1)
14:12 (1:1)
14:12 (5:1)
14:12 (8:1)
14:19 (1:1)
19:12 (1:1)
ratiometric CypHer5-PFPE:Cy3-PFPE
(5:1)
ratiometric CypHer5-PFPE:Cy3-PFPE
(8:1)
single component ratiometric control
CypHer5-PFPE:PFPE-amide (1:1)
single component ratiometric control
PFPE-amide:Cy3-PFPE (1:1)
and Cy3-PFPE were blended in volume ratios of known
concentrations for ease of handling; however, the fluorescence
stoichiometry reported in the final formulation is by
synchronous fluorescence measurement in pH 5.8 phosphate
buffer, as described in Section 2.2.1. Initially, ultrasonication
was thought to be a good method to blend the CBPA oils prior
to emulsification, but CypHer5-PFPE oil (14, 17, and 18) did
not withstand the process. This was most likely due to the
localized heating that occurs during ultrasonication.25−27 For
this reason, high-pressure microfluidization was used to process
the nanoemulsions.
2.1.3. Formation of Nanoemulsions. Single dye nano-
emulsions containing Cy3-PFPE, Cy5-PFPE, or CypHer5-
PFPE and ratiometric nanoemulsions containing mixtures of
Cy3-PFPE and CypHer5-PFPE were prepared using the same
process. Blended PFPE oils (Section 2.1.2) were mixed with
surfactants comprising aqueous Pluronic F68 and polyethyle-
neimine solutions, while mixing with vortex (see Supporting
Information and Table S1 for formulation details), followed by
10−15 cycles through Microfluidizer (M110S, Microfluidics,
Inc., Newton, MA). The size of the final nanoemulsions was
150−175 nm, with a polydispersity index (PDI) of ∼0.10
(Figure 1), as measured by dynamic light scattering (DLS).
Nanoemulsions remained stable for over 12 months (Figure
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The emission signal with excitation at 630 nm increases at low
pH as expected (Figure 2C). As was demonstrated by Briggs
and Cooper,3,4 a plot of the emission signal versus pH allows
determination of the pKa for CypHer5 (Figure S17C). This
plot, normalized to the signal at pH 5.8, yields a pKa of 6.8 for
CypHer5 in the nanoemulsion.
The addition of pH-insensitive Cy3-PFPE to formulations
24−27 allowed quantification of nanoemulsion in labeled cells
and also ratiometric calibration of pH using both CypHer5 and
Cy3 signals (Figures 3 and S18). In these two-dye nano-
emulsions, the CypHer5 emission spectra were the same for
24−27 (Figure 3A) and matched the single-dye CypHer5-
PFPE nanoemulsion (22), as shown in Figure 2C. However,
when greater amounts of CypHer5 are added to the
nanoemulsion relative to Cy3, excitation of Cy3 at 530 nm
also excites CypHer5 on the blue side of its absorption band
(Figure 2A) to give fluorescence at 670 nm. This is more
noticeable for nanoemulsion 26, which has a higher content of
CypHer5 than 24 (Figure 3B). Because of this complication,
quantification of the relative Cy3 and CypHer5 compositions of
ratiometric nanoemulsions (24−27) was performed with
synchronous fluorescence scans. Simultaneously scanning
both excitation and detection wavelengths with a 20 nm
wavelength separation (the Stoke’s shift of both fluorophores)
enabled measurement of each signal without spectral spillover.
Using fixed wavelength excitation requires two emission scans
and results in two broad-band emission spectra, one
corresponding to each chromophore (as in Figure 3A,B); in
contrast, the variable excitation wavelengths of a synchronous
fluorescence scan produce a single spectrum that contains two
narrow-band peaks, corresponding to the maximum excitation
of each chromophore (as in Figure 4). Cy3 excites maximally at
Figure 1. Size distribution by intensity of nanoemulsions 20, 22, and
25.
S16). Further details and discussion of synthesis and
formulation are presented in Supporting Information.
2.2. Characterization of Nanoemulsions. 2.2.1. pH-
Sensitivity and Spectral Properties of Nanoemulsions. The
absorption and emission (em) spectra of a CypHer5-PFPE
nanoemulsion (22), without Cy3-PFPE, are shown in Figure 2,
along with spectrally similar and pH-insensitive Cy5-PFPE
nanoemulsion (21) for comparison. Absorbance spectra could
not be obtained from the conjugated dyes at low molar ratios in
the nanoemulsion due to interference from components of the
formulation; therefore, excitation (ex) spectra were obtained
instead (Supporting Information and Figure S17, panels E,F).
However, absorbance spectra could be directly obtained using
the PFPE-conjugates (12−14) solvated with ethanol (Figure
2A). The absorption spectrum of CypHer5-PFPE (14) changes
with pH, and the longer wavelength band dominates at low pH.
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.
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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 em 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. pKa of both formulations is 6.8.
544 nm, has an emission peak at 564 nm (Figure 3B), and
produces a pH-insensitive synchronous scan peak at 548 nm
(Figure 4). CypHer5, on the other hand, has a pH-sensitive
maximal absorption at 644 nm and emission at 664 nm (Figure
3A) with a pH-sensitive synchronous scan peak at 649 nm
(Figure 4). In the synchronous fluorescence spectra, the
intensity of each peak corresponds to the relative abundance of
each chromophore and thus allows formulation stoichiometries
to be compared. Nanoemulsions 28 and 29 contain only one
peak because these are single component ratiometric controls.
Ratiometric measurements using fixed wavelength and
synchronous scanning modes are discussed further in the
Supporting Information.
(24−27), regardless of ratiometric stoichiometry (Figure S20).
A pKa value of 6.8 is comparable to the values of the CypHer5
dye in aqueous buffers obtained by our lab and others.3,4 In
addition, similar plots for CypHer5-NBoc (7) and PFPE
nanoemulsions at different dye concentrations (nanoemulsions
24−27) in buffers yield the same pKa. This is interesting in
view of possible localization distributions the CypHer5
molecule might occupy within nanoemulsion droplets. The
result indicates that the CypHer5 fluorophores, which carry two
sulfonic acid groups, lie at the surface of the nanoparticles
exposed to the local polar environment. It is probable that the
CypHer5 fluorophores that are covalently bound to the PFPE
molecules are thus amphipathic (as in a large micelle) and
associate with nonpolar unlabeled PFPE molecules that form
the core of the nanoemulsion droplet.
These fluorescence scanning methods enabled the measure-
ment of pKa of 6.8 for the CypHer5 fluorophore in the
nanoemulsions (Figure 3C). The pKa curve profiles (normal-
ized to 1.0 at pH 4.0) were identical for two-dye nanoemulsions
The quantum efficiencies of the fluorescent nanoemulsions
could not be measured directly because light scatter from the
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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).
colloidal dispersion is very intense. However, the quantum
efficiencies of fluorophores Cy3 and CypHer5 have been
addressed previously by Mujumdar, Briggs, and Cooper.3,4,23
The values are likely to be unchanged with side-chain
modification. Since the spectra of the nanoemulsions are
comparable to those of the parent dyes, it is expected that the
quantum efficiencies of the nanoemulsions will likewise be
comparable to the parent dyes.
Optimization of the stoichiometry of CypHer5 relative to
Cy3 is essential in developing ratiometric reagents that are
suitable for platforms such as flow cytometry or fluorescence
microscopy; a ‘one size fits all approach’ may not be best. We
have observed that certain formulation stoichiometries are
better than others when used for spectral studies in cells and for
loading cells to be used during in vivo studies; it is essential to
have a strong Cy3 signal so that the autofluorescence of the
cells does not dominate quantification of the reference signal
(discussed further in Sections 2.3.2 and 2.3.3). The
synchronous scan method provides an easy way to determine
the amount of CypHer5-PFPE that should be added to the
formulation so that the CypHer5 to Cy3 signal ratios are in an
optimal range of 0.5−10, as displayed in Figure 4. However,
preformulation measurement of blended of CypHer5 and Cy3
PFPE-conjugate stoichiometries by synchronous scan did not
equal postformulation measurements, where CypHer5 content
was consistently 40% lower. We speculate that this apparent
dye loss is due to its decomposition of this sensitive
fluorophore during processing. Preformulation quantities of
CypHer5-PFPE were adjusted to anticipate dye loss during
processing (see Supporting Information).
2.2.2. Role of Energy Transfer. Forster resonance energy
̈
transfer (FRET) can occur when a pair of fluorophores has
overlapping excitation and emission spectra, and the molecules
are within close proximity. The emission of the shorter
wavelength fluorophore (donor) nonradiatively transfers its
excitation energy to the longer wavelength fluorophore
(acceptor), inducing fluorescence emission from the acceptor.28
Cy3 and Cy5 are a well-known energy transfer pair used for
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FRET measurements in biophysical studies. For this pair,
energy transfer can be observed when the donor and acceptor
are within 8 nm.29 Since CypHer5 and Cy5 are spectrally
similar when CypHer5 is protonated, there is potential for
FRET due to the Cy3-CypHer5 pairing. Efficient energy
transfer could potentially complicate pH calibrations and
estimation of the localized cellular pH of internalized
nanoemulsions. We observed no evidence of energy transfer
when Cy3 and CypHer5 were formulated in separate
nanoemulsions (29) and (28), and then mixed. This is not
surprising, as it is expected the average spacing of the two
fluorophores would not be close enough in mixed, but
separately formulated nanoemulsion droplets. However, if the
fluorophores are formulated within the same nanoemulsion
droplet, as in nanoemulsions 24−27, dye concentrations may
be sufficient to bring the fluorophores within FRET proximity.
Additionally, if FRET were to occur in our nanoemulsions, it
would be more probable when Cy3 and CypHer5 molecules
are present in equal quantities within the same nanoemulsion
droplet.
To investigate FRET, we used the nanoemulsion containing
the highest concentrations of Cy3 and CypHer5 (25). Because
of the spectral overlap of Cy3 and CypHer5, and hence their
energy transfer, depends on pH, nanoemulsion samples were
diluted into hydrochloric acid (50 mM) to ensure complete
protonation of CypHer5. The concentration of single-dye
control nanoemulsions (28 and 29) was adjusted to equal the
corresponding Cy3 or CypHer5 component of 1:1 ratiometric
nanoemulsion (25) using the emission maxima of Cy3 and
CypHer5 wavelengths accordingly (564 and 662 nm), with
excitation at 530 and 630 nm. The dyes were scanned
separately, and thus broad excitation or potential energy
transfer was excluded from concentration determinations.
Initially, the spectral properties of each dye were determined
individually using the single-dye controls. A nanoemulsion
containing only Cy3 (29) was excited at the Cy3 excitation
wavelength (530 nm), and the emission of Cy3 was measured
at the CypHer5 emission wavelength (662 nm). This
determined the amount of Cy3 emission spillover that could
mimic energy transfer. This spillover was 5% of the Cy3
emission maximum at 564 nm. Similarly, a nanoemulsion
containing only CypHer5 (28) was excited at the Cy3
excitation wavelength and the emission of CypHer5 was
quantified; the result was 18% of the CypHer5 emission
maximum at 662 nm, which indicates the efficiency of CypHer5
excitation at 530 nm. Finally, the same measurement done for
Cy3 alone (29) was repeated using a nanoemulsion containing
both Cy3 and CypHer5 (25), and the emission due to 530 nm
excitation comprised 33% of the CypHer5 emission maximum
at 662 nm. Additionally, for all nanoemulsions (25, 28, 29), the
direct emission intensities of Cy3 and CypHer5 (564 and 662
nm) were measured with excitation at 530 and 630 nm. The
instrument settings for all three nanoemulsions were identical.
Energy transfer was then estimated by deconstructing the total
emission intensity at 662 nm for nanoemulsion 25, using
information gained from these measurements. We observed
41% of the intensity of 25 was due to Cy3 emission signal that
spills over into the CypHer5 fluorescence channel (based on
percentage measured using 29), 54% of the signal intensity was
due to excitation of CypHer5 by 530 nm excitation (based on
percentage using 28), and the remaining 5% of the emission
intensity was attributed to energy transfer. With an estimated
5% efficiency, it is unlikely that energy transfer is a significant
interfering factor in our ratiometric nanoemulsion fluorescence
studies.
2.2.3. Fluorescence Stability of Bulk Ratiometric Nano-
emulsions. The fluorescence stability of bulk ratiometric
nanoemulsions (24−29) was characterized at 4 and 37 °C.
Nanoemulsion samples were taken every 1−2 weeks and
diluted (2% v/v) into pH 5.8 phosphate buffer. In fluorescent
synchronous scans, nanoemulsion fluorescent signals decreased
rapidly (100% of CypHer5 and 75% of Cy3 within 6 weeks) at
37 °C storage, while those kept at 4 °C were much more stable
(∼60% for CypHer5 and 50% for Cy3 over an extended 60
week period), further described in Supporting Information and
shown in Figure S21, top panel. Fluorescent ratios (CypHer5/
Cy3) were stable for several months at 4 °C, while at 37 °C,
ratios converged to 0 within 6 weeks due to decomposition of
CypHer5 (see Figure S21, bottom panel). For formulations
with a higher content of CypHer5 (27), fluorescence ratios
dropped 50% over 60 weeks, while those with a lower content
of CypHer5 (24), dropped only 25%. At 4 °C, both dyes
decompose at similar rates; however, the CypHer5 signal
dropped significantly relative to Cy3. For these reasons, bulk
ratiometric nanoemulsion products should be stored at 4 °C,
rather than at ambient or higher temperatures.
2.2.4. Stability of Fluorescence Signals under Cell Labeling
Conditions. Cell labeling experiments often subject nano-
emulsions to 37 °C, and media containing proteins or
biomolecules for many hours, thus the fluorescence stability
and pH sensitivity of the reagents in proteinaceous media were
evaluated. Ratiometric nanoemulsions 24−26 were diluted into
cell culture media [Dubelco’s modified Eagle medium,
(DMEM), containing 10% fetal bovine serum, 1% penicillin
streptomycin, 25 mM 4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid (HEPES) and free of Phenol Red indicator] or a
deionized water control at typical cell labeling concentration (1
mg/mL), and then incubated at 4 or 37 °C. Sample stability
and pH sensitivity of CypHer5 and Cy3 signals were measured
by fixed wavelength scans (ex/em 648/668 nm and ex/em 548/
568 nm), followed by subsequent dilution into high potassium
HEPES buffers5 to fix the sample pH during the measurement
(see Supporting Information for detail). Fluorescence signals of
CypHer5 and Cy3, and ratios of CypHer5/Cy3 normalized to
pH 5.5 value, were plotted; results are shown for nanoemulsion
26 (which contained the highest amount of CypHer5) in
Figure S22, panels A-B. When incubated in DMEM, Cy3
signals remained steady throughout 48 h, while CypHer5
decreased by 33% at both 4 and 37 °C. However, when
incubated in water at 37 °C, the CypHer5 signal decreased 45%
during the same time. The relative preservation of CypHer5
incubation in DMEM compared to water may be attributed to a
more shielded environment created by the media components
(such as serum proteins) that may make it less susceptible to
effects of degradation agents. Despite some loss of CypHer5
fluorescence signal, the pH sensitivity was retained in all
samples. Ratiometric pH function across pH 5.5−7.5 was
similar for each nanoemulsion/media combination when ratios
were normalized to pH 5.5 at the same time point (see Figure
S22C). Overall, through using normalized fluorescence ratios,
these results show that pH sensitivity of ratiometric nano-
emulsions is retained under typical cell labeling conditions.
2.3. Biological Evaluation of Ratiometric Nanoemul-
sions. Four of the ratiometric nanoemulsions prepared in this
study (24−27), were used to label 9L glioma cells, and these
cell preparations were then analyzed by three commonly used
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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.
fluorescence detection platforms, including a fluorescent plate
reader, fluorescence microscope, and flow cytometer. Optimal
CypHer5/Cy3 stoichiometries were determined for each
platform.
2.3.1. Nanoemulsion Cellular Uptake by 19F NMR and
Fluorescence Detection. Glioma cells (rat 9L) were co-
incubated with nanoemulsions 20−27 in media for 3 h at 37
°C, followed by a wash step with phosphate buffered saline
(PBS). The average nanoemulsion uptake during the labeling
period was determined using 19F NMR of cell pellets to
measure the 19F per cell, as described elsewhere.18
A
representative 19F NMR spectrum of a labeled cell pellet is
shown in Figure 5. At the optimum labeling doses, defined as
80% viability compared to unlabeled cells, the 19F loading
ranged from 0.1 to 1.0 × 1011 19F/cell (Figure S23).
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.
To correlate 19F NMR signals with the fluorescent signals of
CypHer5 and Cy3 in the ratiometric nanoemulsions, 10% (by
volume) of cell lysate isolated during dose optimization was
read using a plate reader at fixed wavelengths (ex/em 530/564
nm for Cy3 and ex/em 630/664 nm for CypHer5). The
amount of nanoemulsion (ng/cell) taken up by the cells was
calculated from linear calibration curves of nanoemulsions 22−
27 in 0.1% TFA. The number of fluorine atoms per cell is
directly proportional to the mass of nanoemulsion per cell. The
concentration of bulk nanoemulsion (in mg/mL) is determined
before cell labeling by quantifying the number of fluorine atoms
using 19F NMR following methods described by Janjic.21
Additionally, the direct conjugation of the fluorescent probes to
the fluorocarbon ensured direct correlation of fluorescence
signals with 19F NMR signal. Excellent agreement between 19F
uptake per cell by NMR and CypHer5 and Cy3 uptake per cell
measured by fluorescent plate reader was observed when the
data were normalized to the highest labeling concentration and
unlabeled controls were subtracted as background (Figures 6
and S24). Normalized uptake data varied at the lowest labeling
concentration (0.125 mg/mL), where the signal of the
fluorescent dyes approached the noise level (these points
were not included on the plots). It should also be noted that
since the cells are lysed and the lysate contains 0.1% TFA,
CypHer5 is fixed in the protonated form and thus cannot
report the pH of the cellular material in this experiment; the
experiment only demonstrates correlation between both
fluorophore signals and 19F signals of the PFPE-conjugates.
These results indicate that all four ratiometric formulations are
well suited for fluorescent plate reader experiments, as the 19F
content of PFPE-loaded cells can be readily quantified from
Cy3 fluorescence. Importantly, simple and low-cost fluores-
cence plate reader measurements can thus be used to assay the
degree of cell labeling for in vivo MRI cell tracking
applications.14,18
2.3.2. Selection of Ratiometric Formulations for Fluo-
rescence Microscopy. Confocal microscopy was used to image
living 9L cells that were labeled at optimal doses with
ratiometric nanoemulsions 24−27 (see Supporting Informa-
tion). Using nonoverlapping emission filters and a spinning disk
confocal system, ratios of signals from the CypHer5 and Cy3
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channels were measured. Ratiometric nanoemulsion 26 (Figure
7) showed strong signals in the CypHer5 channel (Figure 7A,
tions similar to the corresponding single component control
nanoemulsions (28, 29), with Cy5-PFPE (21) being similar to
CypHer5-PFPE (22). When ratios of the mean fluorescence
histograms were calculated (mean 685 nm/mean 575 nm), the
ratios increased as the content of CypHer5 was increased in the
formulation stoichiometry (Figure 9).
2.4. Intracellular pH Measurement. 2.4.1. Generation of
Intracellular pH Calibration Curve. Ratiometric nanoemul-
sions were used to measure the intracellular pH in live cells
using flow cytometry. When a fluorophore is monitored at its
pH-sensitive wavelength, the magnitude of the signal depends
on the amount of probe present as well as the pH. Thus,
ratiometric detection methods are employed that use a (pH-
insensitive) reference signal at a separate wavelength to provide
a measure of the amount of probe present. CypHer5 alone can
also be used for pH measurements,3,4 but we found that this
approach is challenged by a significant amount of cellular
autofluorescence at shorter excitation wavelengths or a low
absorption coefficient at the isosbestic point. Thus, we
formulated nanoemulsions to include a separate pH-insensitive
probe in the sample (Cy3) that absorbs and emits at a separate
wavelength from the pH-sensitive probe. Also, the relative
proportions of Cy3 and CypHer5 in the nanoemulsion can be
tuned to provide an optimal detection system (see Section 2.3).
To calculate pH from fluorescence ratios in cells, a ratio-pH
calibration curve was initially generated, similar to Figure 3C.
Additionally, the cellular environment autofluorescence and
scatter impact the calibration and must be taken into account.
We employed methods described by Barriere et al.5 to obtain
the ratiometric calibration in cells. In brief, the calibration curve
is obtained by setting the pH of all intracellular compartments
to the chosen buffer pH by adding the hydrogen ionophores
nigericin and monensin to cells 5−30 min before the flow
cytometry measurements.
During nanoemulsion uptake in 9L cells using the above
incubation conditions, the Cy3 reference signal was used to
quantify the extent of CypHer5 (and 19F) incorporated during
cell labeling. Fluorescence ratios between reporter and
reference signals were calibrated intracellularly to external
high K+ phosphate buffers ranging from pH 5−8 using the pH
clamp reagents nigericin and monensin. Mean fluorescence
ratios 685/575 nm were calculated, normalized to the pH 5.0
value, and plotted against pH (Figure 10A). Data using the pH
clamp reagents showed small increases in side scatter as pH
became more acidic, which is indicative of cell swelling and
increased granularity. Minimal changes in viability and cell
swelling were observed at pH 5−8 during this treatment.
Cellular background autofluorescence was accounted for in
the calibration curves. Control cells labeled with nonfluorescent
nanoemulsion were treated in the same way using the pH
clamp methods. The mean fluorescence values of cells labeled
with nonfluorescent nanoemulsion 23 were subtracted from the
corresponding histograms, and the 685 nm/575 nm ratios were
recalculated and normalized to the maximum pH value (5.0)
and plotted against pH. These data were fit to Boltzmann
sigmoidal curves. Cellular autofluorescence was a significant
contributor to the Cy3 signal and altered the shape of
fluorescence ratio−pH calibration curves, as seen by the higher
ratio value at pH 8 relative to that of free nanoemulsion (Figure
10A). Background subtraction of nonfluorescent-labeled
controls shifted the ratio at pH 8 from 0.36 to 0.11, bringing
it closer to the ratio of free nanoemulsion (0.07), indicating
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.
red) and in the Cy3 channel (Figure 7B, green), which were
substantially above background noise. The differential interfer-
ence contrast (DIC) and fluorescence images confirmed that
the fluorescent labeling was confined to intracellular regions
consistent with an endocytosis uptake mechanism1,2 (Figure
7A−C). Also, the CypHer5 and Cy3 signals showed
colocalization as expected (Figure 7D, yellow). Formulations
with a higher content of CypHer5-PFPE (26, 27) showed
brighter fluorescence and thus are better candidates for
fluorescence microscopy. The low Cy3 signal in formulation
27 was barely detectable and thus nonoptimal compared to 26.
Preliminary pH calibration experiments in cells indicate that the
ratio change is readily observed when there is <3-fold difference
between the intensity of CypHer5 signals compared to Cy3
signals (data not shown).
2.3.3. Selection of Ratiometric Formulations for Flow
Cytometry. Flow cytometry was also used to detect 9L cells
labeled with ratiometric nanoemulsions 24−29 (see Section
2.3.1). After labeling, cells were washed, detached and
resuspended in PBS for flow cytometry analysis. Cy3 and
CypHer5 channel histograms of ratiometric nanoemulsions
24−29 were compared to nonfluorescent nanoemulsion 23 as a
control (Figures 8, S26−S27). CypHer5 and Cy3 signals were
readily differentiated from nonfluorescent nanoemulsion in
control nanoemulsions 28 and 29. However, Cy3 signals were
differentiable from nonfluorescent labeled cells only in
nanoemulsions that contained equal or greater amounts of
Cy3-PFPE compared to CypHer5-PFPE (such as 24 and 25);
while CypHer5 signal was differentiable from nonfluorescently
labeled cells in all four formulations (24−27) evaluated. For
this reason, nanoemulsion 24 was optimal for flow cytometry.
Single-dye nanoemulsions (20−22) were also evaluated using
flow cytometry and exhibited fluorescent population distribu-
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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 nonfluorescent nanoemulsion 23.
(575 nm) was used to quantify the extent of 19F (nano-
emulsion) incorporated during cell labeling, which increased
55% over the course of 0.5−3 h. Likewise, the 685 nm
CypHer5 signal increased 149% over the same period,
indicating both uptake and increased acidity (Figure S30).
Fluorescence ratios were calculated from histograms of gated
cells, and the pH was determined from the calibration curve
that was corrected for autofluorescence (see Supporting
Information for details). Results for both corrected and
uncorrected data are shown in Figure 10B. The intracellular
pH changed from ∼6.7 to ∼5.5 from 0.5 to 3 h; the same result
was obtained regardless of whether raw or corrected data was
used. The intracellular pH progressed to the terminal value of
∼5.5 at the end of the 3 h, suggesting nanoemulsion trafficking
from neutral cytosomes to acidic lysosomal compartments over
this time period. However, from the current data alone, it is
unknown if the ultimate nanoemulsion fate is the lysosome or if
it is recycled back to the cytosomes. Further study using our
ratiometric reagent to measure subcellular pH and with
confirmation using pharmacological agents is needed.
2.4.3. Experimental Parameters of pH Measurement. To
obtain reliable intracellular pH measurements, the same cell
preparation must be used to develop the calibration curve and
to do the cell measurement, and both should be performed
within a few hours of each other. Experimental data must be
normalized by the same value used to normalize the calibration
curve. Furthermore, it is important that cellular ratio and pH
calibration be performed with the same detection instrument
Figure 9. Comparison of ratiometric nanoemulsion formulations by
flow cytometry. A plot of mean fluorescence ratio (685 nm/575 nm)
against formulation ratio of nanoemulsions 24−27 using 9L cells
labeled with ratiometric nanoemulsions. Data points are identified by
formulation number.
that intracellular autofluorescence interference could be
corrected when subtracted from ratiometric measurements.
2.4.2. Calculation of pH during Nanoemulsion Uptake.
Using the calibration curve constructed in Section 2.4.1, we
measured the intracellular pH during uptake of nanoemulsion
by 9L cells. Measurements were made using ratiometric
nanoemulsion 24 at 30 min intervals for up to 3 h. The flow
cytometry measurements where made with cells suspended in
PBS without ionophores. The Cy3 fluorescence reference signal
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maintaining good discrimination of the Cy3 reference signal
over background. Using this reagent stoichiometry optimization
approach, further modifications can be made to the reporter or
reference fluorophore as additional application requirements
emerge.
3.2. Intracellular pH Measurement and Future
Applications. Ratiometric reagents were used to determine
the intracellular pH of 9L cells during nanoemulsion uptake
using flow cytometry. The Cy3 reference signal was used to
quantify the degree of nanoemulsion uptake during cell
labeling. Cellular autofluorescence was a contributor to the
Cy3 signal, and a simple autofluorescence subtraction method
was used to improve the accuracy of the calibration curve. The
pH changed from ∼6.7 to ∼5.5 throughout the progression of
the 3 h uptake experiment, indicating nanoemulsion migration
to a more acidic environment over the labeling time. PFC
nanoemulsion uptake has been studied extensively by Wickline2
using nonphagocytic 2F-2B cells. Janjic et al.21 suggested that
polyamine-driven nanoemulsion uptake is likely driving the
labeling in nonphagocytic cells. In the current study, using
nonphagocytic 9L cells, labeling studies suggest polyamine-
driven uptake mechanism, which was previously identified by
Godbey to be an endocytosis mechanism.1 Therefore, our PFC-
probe most likely reports the pH of endosomes.
Intracellular pH measurements have utility in the develop-
ment of intracellular drug delivery systems and in vitro
pharmacology studies. PFC nanoemulsions are increasingly
being used for in vivo cell imaging studies14−16,18,30 and as
theranostic vehicles.19,20,31−33 A particularly compelling appli-
cation of the pH-sensing technology described herein is to
accelerate the development of emerging MRI-based cell
tracking technologies,14,17,18,21,34−40 one such technology of
clinical interest is called ‘in vivo cytometry’. In this platform
technology, cell populations of interest (e.g., therapeutic cells
such as stem cells destined for a patient) are labeled, tracked,
and quantified in vivo.14 Cells are initially labeled in culture
using PFC nanoemulsions.14,21 Following transfer to the
subject, cells are tracked in vivo using 19F MRI. The fluorine
inside the cells yields cell-specific images, with no background
signal. Images are readily quantified to measure apparent cell
numbers at sites of accumulation. In these MRI applications,
outstanding questions remain about the nature of the cellular
labeling dynamics and the nanoemulsion fate over the course of
labeled cell lifetime. A fluorescent, pH-sensing nanoemulsion
could potentially be helpful in characterizing intracellular
nanoemulsion behavior in vitro. More importantly, this same
technology could be used for routine quantification of the
degree of cell labeling of a patient’s cells in vitro, a parameter
that is crucial for in vivo cell quantification from the MRI data18
and for quality control purposes. For example, a second
(smaller) population of the patient’s cells could be labeled with
the ratiometric nanoemulsion and the uptake could be
measured using a multiwell plate reader. When in solution,
the background fluorescence of CypHer5 is low, but once the
nanoemulsion is consumed by cells it becomes significantly
higher, and cell labeling could be monitored in real-time,
without a wash step. An optical plate reader is rapid, low-cost
and commonplace in biomedical and clinical laboratories, and
thus is preferable over slow and more expensive 19F NMR
instruments, which is the current method of cell labeling
assessment. Once the validated or adjusted labeling dose of
fluorescent reagent is established on a small portion of the
patient’s cells, the remaining therapeutic cells would be labeled
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.
used for the cell measurements. For example, plate readers,
spectrofluorometers, flow cytometers, and imaging microscopes
usually have different excitation sources (wavelengths and
intensities), different emission monochrometers or interference
filters with different bandwidths, and different sensitivities for
detector sensitivity versus wavelength. For this reason, we used
an approach that is solely based on flow cytometry to obtain
cellular pH. With this approach, the flow cytometer settings
(filter sets, photomultiplier tube voltages, signal amplification)
are not changed between calibration and cellular measurements.
A calibration curve is needed for each fluorescence platform
used during pH measurements.
3. DISCUSSION AND CONCLUSIONS
3.1. Ratiometric Reagent Development. We describe a
novel design for PFC-based nanoemulsion reagents with
intracellular biosensing properties. A fluorescent pH sensor
was directly conjugated to PFPE molecules and formulated into
stable and nontoxic nanoemulsions. Four pH-sensitive
ratiometric formulations (24−27) of differing CypHer5:Cy3
stoichiometries were optimized for use with fluorescent plate
reader, flow cytometer, and fluorescence microscopy platforms.
All four ratiometric formulations were suitable for 19F NMR
and fluorescent plate reader applications. Quantification of 19F
NMR and fluorescence of both dye signals agreed in lysed cells.
Nanoemulsion 24 (0.6:1 CypHer5:Cy3) was best suited for
flow cytometry, while nanoemulsion 26 (5:1 CypHer5:Cy3)
was best suited for fluorescence microscopy. In both of these
platforms, the key requirement is to get strong CypHer5
reporter signals to monitor pH changes with as much
fluorescence response as possible over the pH range, while
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with nonfluorescent nanoemulsion and injected into the patient
for in vivo imaging.
Institutes of Health (P41-EB001977, P50-ES012359, R01-
CA139579, U54RR02224102). NMR instrumentation at CMU
was partially supported by NSF (CHE-0130903 and CHE-
1039870). Mass spectroscopy instrumentation at CMU was
funded by NSF (DBI-9729351).
Information about the fate of emulsions within the cells is
important to understanding the cell loading process and the
stability of the probe within cells over time. For example, if the
nanoemulsion enters the more acidic cellular environments,
such as the lysosomal compartments, the lower pH may lead to
break down of nanoemulsion components, including tracking
dyes and contrast agents, leading to loss of the labels within the
cells over hours or days in vivo. Additionally, the pH-sensing
reagent can report the health of cells that have engulfed the
emulsion, both in vitro and later in vivo following injection into
small animals. A common uptake mechanism of nanoreagent
delivery involves endocytosis,2 which results in drug payload
exposure to low pH in the lysosomes. The residence time of the
vehicle and the drug inside lysosomes can be measured with the
pH sensor incorporated into the formulation. This is important
for accurate determination of intracellular localization and the
nanoreagent fate. A number of pathways of internalization are
known, including clathrin-mediated endocytosis, pinocytosis,
phagocytosis, and micropinocytosis. Each mechanism differs in
the composition of the coat, size of the isolated vesicles, and the
fate of delivered substance.41 Endosomes may fuse with
lysosomes or may be recycled without significant acidification
to other compartments and the cell surface. Thus, development
of our pH-sensing nanoemulsion may make such studies central
to theranostic reagent development.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures, analytical data, and experimental
analyses, including formulation performance. Detailed synthesis
of fluorescent reagents, PFPE oils and nanoemulsions,
spectroscopy, DLS and NMR (1H, 19F) data, and additional
discussion; further experimental assessment of nanoemulsions
for cell labeling applications; biological evaluation through 19F
NMR and fluorescence, flow cytometry analysis, and confocal
microscopy; intracellular pH measurement by flow cytometry.
This information is available free of charge via the Internet at
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Corresponding Author
eta@ucsd.edu
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