Internalization of annexin A5‐functionalized iron oxide particles by apoptotic Jurkat cells

Abstract

Apoptosis plays an important role in the etiology of various diseases. Several studies have reported on the use of annexin A5-functionalized iron oxide particles for the detection of apoptosis with MRI, both in vitro and in vivo. The protein annexin A5 binds with high affinity to the phospholipid phosphatidylserine, which is exposed in the outer leaflet of the apoptotic cell membrane. When co-exposed to apoptotic stimuli, this protein was shown to internalize into endocytic vesicles. Therefore in the present study we investigated the possible internalization of commercially available annexin A5-functionalized iron oxide particles (r1 = 34.0 +/- 2.1 and r2 = 205.0 +/- 10.4 mm(-1) s(-1) at 20 MHz), and the effects of their spatial distribution on relaxation rates R2*, R2 and R1. Two different incubation procedures were performed, where (1) Jurkat cells were either incubated with the contrast agent after induction of apoptosis or (2) Jurkat cells were simultaneously incubated with the apoptotic stimulus and the contrast agent. Transmission electron microscopy images and relaxation rates showed that the first incubation strategy mainly resulted in binding of the annexin A5-iron oxide particles to the cell membrane, whereas the second procedure allowed extensive membrane-association as well as a small amount of internalization. Owing to the small extent of internalization, only minor differences were observed between the DeltaR2*/DeltaR2 and DeltaR2/DeltaR1 ratios of cell pellets with membrane-associated or internalized annexin A5 particles. Only the increase in R1 (DeltaR1) appeared to be diminished by the internalization. Internalization of annexin A5-iron oxide particles is also expected to occur in vivo, where the apoptotic stimulus and the contrast agent are simultaneously present. Where the extent of internalization in vivo is similar to that observed in the present study, both T2- and T2*-weighted MR sequences are considered suitable for the detection of these particles in vivo.

Molecular MRI of Cardiomyocyte Apoptosis With Simultaneous Delayed-Enhancement MRI Distinguishes Apoptotic and Necrotic Myocytes In Vivo

“…Further study will thus be needed, under a range of conditions, to determine the optimal TE and dose of AnxCLIO-Cy5.5 to use. AnxCLIO-Cy5.5 accumulates principally on the outer surface of apoptotic CMs,30 with probe accumulation driven by a natural micro-environment. Decoupling the effects of iron-oxide and gadolinium will be more difficult when artificially high local concentrations of iron-oxide are encountered, such as those produced by intramyocardial injection of iron-oxide labeled stem cells.…”

Lactadherin functions as a probe for phosphatidylserine exposure and as an anticoagulant in the study of stored platelets

“…Annexin V also binds to other membrane lipids such as oxidized phospholipids and sulfatides [10,11]. In addition, annexin V can be internalized by stressed cells even when there is no PS exposure on the membrane [12,13], and Ca 2+ ‐dependent platelet activation makes annexin V an inapplicable probe for the detection of PS in stored platelets [14]. Thus, the extent to which annexin V fluorescence represents PS exposure on platelet membranes remains unknown.…”

A molecular MRI probe to detect treatment of cardiac apoptosis in vivo

“…However, application of this approach to the heart has been limited. Recently, other groups have conjugated ANX to cross-linked iron oxide, gadolinium chelates, and/or fluorochromes to image cardiac apoptosis in mouse models of ischemia-reperfusion, genetic injury, and atherosclerosis (5)(6)(7)(8). Here, we developed a different molecular MRI probe using two compounds approved individually by the FDA, human ANX and SPIO, and a clinical 3 T MRI scanner.…”