contains 40% w/v dispersed perfluorocabon in phosphate
buffered saline (PBS) and retains more O2 concentrated in the
PFC droplets Therefore, it equilibrates to hypoxia at a slower rate
compared to diluted emulsions. Diluted emulsions equilibrate
close to hypoxic conditions in less than 10 min. This behavior
would be useful for in vivo applications to provide simultaneous
delivery and measurement of O2.
extracellular % O2, H1229 cells were incubated with 20 μM PTM
5 in PBS for 30 min. The supernatant was removed upon
differential centrifugation at 110 x g for 1 min, and found to
contain the entire PTM 5 EPR signal, without any loss in signal
intensity. The cells re-suspended in fresh PBS contained no
signal, indicating that there was no cellular uptake of the PTM
emulsion over this time.
For cellular respiration studies we opted to work with emulsions
containing 2 mM PTM 5 in PFC phase, suspended in PBS (0.5
mM final PTM 5 concentration). The stability of the emulsion
over time was measured by DLS analysis (Fig. 4B). Immediately
upon formulation (SD, Fig.16), and within 24 hours, the average
droplet size remains ~200 nm. The droplet size is well under 300
nm over the next 72 hours (Fig. 4B). PFC emulsions experience
a gradual increase in particle size according to the Ostwald
Ripening effect.36,37 However, the droplet size remains well under
500 nm for over two weeks without affecting oximetry
measurements. Considering the simplicity of the preparation
process, use of fresh formulations is recommended.
The EPR signal intensity remained stable during cellular
respiration studies and the concentration of the PTM radical
remained constant at 20 μM. This is an indication that PTM
esters formulated in PFC emulsions are protected from the
superoxide or other radicals generated during cellular respiration.
In conclusion, we have demonstrated that PTM radicals in PFC
emulsions can provide highly sensitive O2 measurements in
solutions and cellular systems. The high solubility of PTM
probes in these PFC emulsions along with their high O2 solubility
enables sensitive EPR based oximetry with excellent signal to
noise ratio. These formulations are well suited to measure O2
consumption in cells during cellular respiration. Many
pathologies, including ischemic vascular disease and cancer are
characterized by lack of O2 supply.38 In cancer, tumor hypoxia
limits the efficacy of radiation or other therapies in tumor
killing.39 Based on the efficacy of PFC emulsions as O2 carriers,
and the developments reported herein, PFC emulsions containing
PTM probes could be applied in the future for combined O2
delivery and O2 sensing to restore or enhance tissue oxygenation
Oximetry calibration curves were constructed for 0.5 mM PTM 5
stock solution in PBS, 5-fold dilutions to 100 μM, and 25-fold
dilutions to 20 μM. EPR measurements yielded virtually identical
oximetry calibration curves (Figs. 4D, 4E). With an EPR
measurement resolution of 5 mG, the slope of 16.8 mG/mmHg
provides a high O2 measurement sensitivity of 0.3 mmHg. This
remains unchanged between 20 μM-100 μM, which is a likely
concentration range to be used for in vivo delivery. The slope of
0.5 mM stock solution is 16.1 mG/mmHg (SD, Fig. 15). Time
dependent O2 depletion was followed via EPR spectroscopy (Fig.
4F). Similar to measurements in Fig. 3B, diluted solutions
equilibrate to hypoxic conditions within minutes, thus EPR
measurements can be used effectively to follow O2 consumption.
Acknowledgments
This work was supported by NIH grant EB016096.
Supplementary Material
We performed measurement of cellular O2 consumption in
human non-small cell lung carcinoma cells (NSCLC) cell line
H1229, which are shown in Fig. 4A. No significant cellular
toxicity was seen. Greater than 97 % cell viability was observed
even in the presence of high concentrations of the PFC emulsions
(Fig. 4C) for 1 hour.
Supplementary data associated with this work, includes
synthetic procedures, NMR, HRMS, Dynamic Light Scattering
analysis, and EPR spectra.
A low concentration of 20 μM PTM 5 in PBS emulsion was
sufficient to follow the O2 consumption in these cells where % O2
decreased to 0.71 % over 20 min and addition of KCN blocked
O2 consumption. To ensure that formulations report on
(5)
M. Joergensen, F. R., S. Andersson, T. Almen,
A. Aabye,; L. G. Wistrand, H. W., K. Golman, R. Servin, and P.
Michelsen, PCT Int. Appl. wo/9112024 1991.
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