Membrane potential-dependent uptake of 18F-triphenylphosphonium--a new voltage sensor as an imaging agent for detecting burn-induced apoptosis

Abstract

Background: Mitochondrial dysfunction has been closely related to many pathologic processes, such as cellular apoptosis. Alterations in organelle membrane potential are associated with mitochondrial dysfunction. A fluorine-18 labeled phosphonium compound: (18)F-triphenylphosphonium ((18)F-TPP) was prepared to determine its potential use as a mitochondria-targeting radiopharmaceutical to evaluate cellular apoptosis.

Methods: Studies were conducted in both ex vivo cell lines and in vivo using a burned animal model. Uptake of (18)F-TPP was assessed in PC-3 cells by gamma counting under the following conditions: graded levels of extracellular potassium concentrations, incubation with carbonyl cyanide m-chlorophenylhydrazone and staurosporine. Apoptosis was studied in a burn animal model using terminal deoxynucleotidyl transferase dUTP nick end labeling staining and simultaneous assessment of (18)F-TPP uptake by biodistribution.

Results: We found that stepwise membrane depolarization by potassium (K) resulted in a linear decrease in (18)F-TPP uptake, with a slope of 0.62 ± 0.08 and a correlation coefficient of 0.936 ± 0.11. Gradually increased concentrations of m-chlorophenylhydrazone lead to decreased uptake of (18)F-TPP. Staurosporine significantly decreased the uptake of (18)F-TPP in PC-3 cells from 14.2 ± 3.8% to 5.6 ± 1.3% (P < 0.001). Burn-induced significant apoptosis (sham: 4.4 ± 1.8% versus burn: 24.6 ± 6.7 %; P < 0.005) and a reduced uptake of tracer in the spleens of burn-injured animals as compared with sham burn controls (burn: 1.13 ± 0.24% versus sham: 3.28 ± 0.67%; P < 0.005). Biodistribution studies demonstrated that burn-induced significant reduction in (18)F-TPP uptake in spleen, heart, lung, and liver, which were associated with significantly increased apoptosis.

Conclusions: (18)F-TPP is a promising new voltage sensor for detecting mitochondrial dysfunction and apoptosis in various tissues.

Keywords: (18)F-TPP; Apoptosis; Membrane potential; Mitochondria.

Fig. 1. Time course study and concentration study on ¹⁸F-TPP uptake by PC-3 cells

PC-3 cells were incubated with normal K concentration (5.4 μM). Time course study demonstrated time-dependent uptake kinetics of ¹⁸F-TPP by PC-3 cells. The cellular radioactivity increased gradually and reached the plateau at 60 minutes post incubation (Fig. 1 A: ANOVA, * vs. **: P<0.05; ** P>0.05). Cells incubated with ¹⁸F-TPP at different levels of extracellular concentration. The maximum uptake was shown at the concentration of 2 nM (Fig. 1 B: ANOVA, * vs. **: P<0.05; ** P>0.05).

Fig. 2. Effects of potassium and CCCP concentrations on ¹⁸F-TPP uptake by PC-3 cells

The uptake of ¹⁸F-TPP was decreased as the extracellular potassium concentration increased. At the highest potassium concentration of 172.8 μM, the uptake was the lowest, while at the lowest potassium concentration of 10.8 μM, the uptake was at the highest. An inverse linear correlation between the uptake and extracellular potassium concentration (slope: 0.62+/− 0.78; correlation coefficient: r=0.936+/− 0.11) was found (Fig. 2 A: ANOVA, * vs. **: P<0.05; ** P>0.05). Gradually increased concentrations of CCCP lead to decreased uptakes of ¹⁸F-TPP, which quickly reached a trough at CCCP concentration of 5μm (Fig. 2 B: ANOVA, * P>0.05).

Fig. 3. Effects of staurosporine and combined proapoptotic factors on ¹⁸F-TPP uptake

Staurosporine at 8μM lead to a significantly decreased uptake of ¹⁸F-TPP in PC-3 cells compared with the sham treated cells (Fig. 3 A: t test: P<0.001). Combination of proapoptotic factors clearly demonstrated similar effects on the uptake (Fig. 3 B: ANOVA: * vs. **p<0.05; **p>0.05). Note: CCCP: 50 μM/L; Val: 1 μg/ml; Low K= 2 μM/L; high K= 172.8 μM/L.

Fig. 4. Burn induced apoptosis in mouse spleen

TUNEL staining on the sections of the spleens from the sham mice demonstrated diffusely scattered apoptotic cells at a rate of 4.4 +/−1.8% in the white medulla (Fig. 4 A). By contrast, burn induced significant cellular apoptosis in the spleen presented an apoptotic index of as high as 24.6+/− 6.7 % (Fig. 4 B, C: sham vs. burn: t test, p<0.005). The majority of the apoptotic cells were present in the white medulla area.

Fig. 5. Effects of apoptosis on ¹⁸F-TPP uptake in spleen and ¹⁸F-TPP biodistribution

Time course study on the uptake of ¹⁸F-TPP by spleen in mice showed the maximum accumulation in 20 minutes after the injection of ¹⁸F-TPP via tail vein. This time point was chosen for biodistribution studies (Fig. 5 A: ANOVA, * vs. the rest, ** vs. #: P<0.05; ¤, ** vs. ¤, and ¤ vs. #: P>0.05). Tracer uptake in burned mouse spleens was reduces to 1.13±0.24% compared with 3.28 ±0.67% (t test: p<0.005) in spleens of sham treated mice (Fig. 5 B).

Fig. 6. ¹⁸F-TPP biodistribution

The biodistribution study showed significant reduction of ¹⁸F-TPP uptake in heart, lung, spleen, and liver in burned animals compared with sham treated ones(* burn vs. sham: t test, P< 0.05). No evident uptake differences were presented in other examined organs of burned animals by contrast with the sham treated ones (burn vs. sham: t test, P> 0.05).