Visualization of cell death in mice with focal cerebral ischemia using fluorescent annexin A5, propidium iodide, and TUNEL staining.
Multimodal proteins, or proteins labeled with both fluorescent and magnetic reporter groups, can be used in a wide range of applications including FACS or fluorescence microscopy, MRI and or near-infrared based optical imaging, or to fractionate cells by magnetic cell sorting. A problem with multimodal proteins, however, is the need to maximize bioactivity, often achieved by minimizing the number of modification points of the protein, while attaching fluorescent and magnetic labels. Here we describe the synthesis of a magneto/optical form of annexin V, achieved by reacting the amino-CLIO nanoparticle with Cy5.5 and SPDP, to produce a fluorescent, sulfhydryl reactive nanoparticle. A single reactive sulfhydryl group was added to annexin V by reaction with SATA that preserved the protein's ability to bind apoptotic Jurkat T cells. Reacting SATAylated annexin V with an SPDP activated nanoparticle yielded Anx-CLIO-Cy5.5, a magneto/optical form of annexin V. The binding of Anx-CLIO-Cy5.5 was specific for apoptotic Jurkat T cells and had an EC(50) of 3.66 nM. This was comparable to the strength of the interaction of unmodified annexin V with apoptotic cells, measured as the displacement of FITC-annexin by annexin V (2.4 nM). Our conjugation strategy preserves the strength of the interaction between annexin V and apoptotic cells, while yielding a probe, Anx-CLIO-Cy5.5, that is readily detectable by standard MR imaging or NIRF optical methods.