AnxCLIO-Cy5.5 is an apoptosis-sensing magnetofluorescent nanoparticle with considerable translational potential. The sensitivity and diagnostic power of the agent has been demonstrated in several preclinical studies. In the current study we show that AnxCLIO-Cy5.5 is well tolerated and has no deleterious effects on the apoptotic cells it binds to. In fact, exposure to AnxCLIO-Cy5.5 seems to exert a membrane-stabilizing protective effect on apoptotic cells in vitro, and possibly in vivo. These results further underscore the translational potential of AnxCLIO-Cy5.5 as a diagnostic nanoparticle, as a therapeutic, and as a vehicle for the delivery of novel therapeutics.
The AnxCLIO-Cy5.5 nanoparticle consists of Annexin V (Anx), cross-linked iron oxide (CLIO), and the near infrared fluorochrome Cy5.5. Annexin V is a 35 kD protein that has been used as a radiotracer in humans and has an extensive safety record.5–7, 19
It is important to note that Annexin V is an endogenous protein produced by humans. Annexin levels in human plasma rise significantly in several disease states and a deficiency of annexin has been implicated in arterial thrombosis and autoimmune disease.20
A strong biological basis thus exists to suggest that the injection of annexin at the dose needed in diagnostic or theranostic nanoparticles would be well tolerated.
The exposure of apoptotic cells to the CLIO nanoparticle alone did not influence membrane integrity. Likewise, the use of unmodified annexin V did not produce a protective effect. This suggests that larger annexin constructs (dimers, oligomers and nanoparticle constructs) are needed to enhance membrane stability. Annexin dimers (diannexins) are currently being investigated as therapeutic agents in the settings of arterial thrombosis and organ transplantation.13
In addition to their size, these agents differ from unmodified annexin in that they exceed the renal threshold and have a long blood half-life. AnxCLIO-Cy5.5 likewise exceeds the renal threshold and has a blood half-life in mice of 2.7 hours.
Confocal microscopy in this study revealed that AnxCLIO-Cy5.5 remains on the surface of apoptotic cells for 3 to 4 hours, after which it is internalized. No changes in caspase-3 activity were seen in apoptotic cells exposed to AnxCLIO-Cy5.5. In addition, genomic analysis did not reveal changes in gene pathways likely to mediate cytoprotective effects. We thus hypothesize that the protective effect of AnxCLIO-Cy5.5 is mediated via direct mechanical stabilization of the apoptotic cell membrane in a critical time window soon after injury. This is consistent with prior studies showing that annexin forms a complex lattice upon binding to the apoptotic cell membrane.21, 22
The impact of this effect seems powerful enough to change the exponential rate constant of cell death and cause the kinetic (percent PI positive) curves of cells exposed and not exposed to AnxCLIO-Cy5.5 to continue to diverge.
The binding of unmodified Annexin V to phosphatidylserine on the surface of apoptotic cells has been well characterized.23
In solution, Annexin V exists as a monomer. When bound to phosphatidylserine, however, Annexin V forms trimers with a convex surface facing the cell membrane.21, 24
This leads to mechanical invagination of the membrane but conceivably could also buttress the cell membrane and attenuate outward bulging and rupture. Previous studies in lymphocytes have shown that Annexin V retards the development of cell death due to a membrane constraint effect.14
We hypothesize that AnxCLIO-Cy5.5 exerts a similar effect on apoptotic cells. However, the aim of the present study was to characterize the safety of AnxCLIO-Cy5.5, and further study will be needed to elucidate the detailed mechanism and extent of its protective effect.
Magnetic iron-oxide nanoparticles (MNP) have been used clinically for almost 2 decades. When injected intravenously, untargeted MNP are taken up by the cells of the reticulo-endothelial system and are degraded over a period of 7 to 10 days.25
The iron in the nanoparticle is metabolized much like endogenous iron, and is ultimately used to manufacture new red blood cells. Loading stems cells exogenously with very high concentrations of MNP has also been extensively studied and does not adversely affect their viability or metabolism.26
The metabolism of targeted MNP, such as AnxCLIO-Cy5.5, is less fully defined. These agents are taken up by cells that do not have the extensive degradative capacity of the cells in the reticulo-endothelial system.9, 25
Genome-wide gene expression analysis, however, did not reveal upregulation of gene sets related to inflammation, oxidative stress or apoptosis that would be consistent with AnxCLIO-Cy5.5 having a deleterious effect on the cell. These gene expression results are also consistent with recent data showing that labeling stem cells ex vivo with extremely high amounts of MNP did not produce any concerning changes in gene expression.27
The primary aim of this study was to determine whether AnxCLIO-Cy5.5 exerts a detrimental effect on apoptotic cells and accelerates their demise. This has been shown not to be the case. The suggestion in this study that AnxCLIO-Cy5.5 may actually be protective is an extremely interesting secondary result. The protective effect produced by AnxCLIO-Cy5.5 is smaller than reported with novel pharmaceuticals, such as caspase inhibitors. However, the production of even a small protective effect by a diagnostic imaging agent is noteworthy. It should also be noted that the translation of magnetic annexins into the clinical realm is highly feasible. Ferumoxytol (Advanced Magnetics, Lexington MA), a long circulating analogue of CLIO, was recently approved for clinical use and underscores the translational potential of AnxCLIO-Cy5.5. Annexin is a naturally occurring protein and has been extensively used in humans without safety concerns. Magnetofluorescent annexin constructs, analogous to AnxCLIO-Cy5.5, thus have the potential to become valuable diagnostic and theranostic nanoparticles in a broad range of clinical conditions.