Annexin V-CLIO: A Nanoparticle for Detecting Apoptosis by MRI
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Rationale and Objectives
The aim of this project was to develop a superparamagnetic iron nanoparticle capable of separating apoptotic cells from non-apoptotic cells in vitro and capable of acting as a contrast agent for MRI in vivo. Annexin V, which selectively recognizes apoptotic cells, was conjugated to cross-linked monocrystalline iron oxide nanoparticles (CLIO's), which provides a label for changing MR signals.
Materials and Methods
Annexin V was purified from an Annexin V expressing E. coli clone obtained from J. Tait. To conjugate Annexin V to CLIO, sulfhydryl groups were introduced on Annexin V by treatment with SATA (N-Succinimidyl S-Acetylthioacetate). CLIO's were activated by SPDP (N-Succinimidyl 3-(2-pyridyldithio)propionate). After deprotection with hydroxylamine to generate free sulfhydryl groups, a disulfide bond formed, conjugating Annexin V to CLIO. The resulting nanoparticle had 2.7 Annexin V proteins per CLIO
Results
The functionality of the Annexin V on the Annexin V-CLIO nanoparticle was verified by its ability to magnetically separate apoptotic cells from healthy cells. Before separation a mixture contained 68.6% healthy and 31.4% apoptotic cells (FACS analysis, Fig. 1). After incubation with Annexin V-CLIO and application to magnetic columns, the apoptotic cells were almost completely removed (0.81%) (Fig. 1).
To test the ability of Annexin V-CLIO to detect apoptotic cells by MRI, a phantom MRI
Conclusion
A magnetic nanoparticle termed Annexin V-CLIO was synthesized and shown to be useful for separating apoptotic cells from healthy cells. By MRI Annexin V-CLIO allowed identification of cell suspensions containing a high percentage of apoptotic cells. Since CLIOs are successfully used for molecular imaging in vivo, we conclude that Annexin V-CLIO will be a useful MR contrast agent for imaging apoptosis in vivo.
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