Synthesis of an In vivo MRI-detectable Apoptosis Probe

Summary

Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

Abstract

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are evident. A means to detect apoptosis in its earliest, reversible stages would afford a pre-clinical 'window' during which preventive or therapeutic measures could be taken to protect the heart from permanent damage. We present herein a simple and robust method to conjugate human Annexin V (ANX), which avidly binds to cells in the earliest, reversible stages of apoptosis, to superparamagnetic iron oxide (SPIO) nanoparticles, which serve as an MRI-detectable contrast agent. The conjugation method begins with an oxidation of the SPIO nanoparticles, which oxidizes carboxyl groups on the polysaccharide shell of SPIO. Purified ANX protein is then added in the setting of a sodium borate solution to facilitate covalent interaction of ANX with SPIO in a reducing buffer. A final reduction step with sodium borohydride is performed to complete the reduction, and then the reaction is quenched. Unconjugated ANX is removed from the mix by microcentrifuge filtration. The size and purity of the ANX-SPIO product is verified by dynamic light scattering (DLS). This method does not require addition to, or modification of, the polysaccharide SPIO shell, as opposed to cross-linked iron oxide particle conjugation methods or biotin-labeled nanoparticles. As a result, this method represents a simple, robust approach that may be extended to conjugation of other proteins of interest.

Protocol

Adapted from prior study¹.

1. Conjugation of Annexin V to SPIO

1. Oxidize SPIO particles (Ocean Nanotech Inc., 5 mg/mL) for 1 hr at 20 °C in the dark in solution with 0.15 M sodium periodate (NaIO₄) (4:1 weight:weight ratio)².
2. Incubate the oxidized SPIO for 12 hr with purified ANX protein (1:1 ratio, weight:weight) in 0.15 M sodium borate (Na₂B₄O₇ 10H₂O) at 20 °C.
3. Reduce the mixture further for 1 hr with sodium borohydride (NaBH⁴)under the fume hood and then quench with 0.15 M Tris-HCl.
4. Separate the free ANX from ....

Discussion

The described conjugation method for linking SPIO to Annexin V exploits the side chain amine groups of Annexin and the carboxyl moieties of the SPIO nanoparticle. Through specific oxidation-reduction steps, covalent linkage of these compounds can be achieved, and the resulting functionalized nanoparticle can be isolated. This method may be generalized to other proteins and nanoparticles of interest.

The most critical steps are the oxidation and reduction conditions, carried out in the approp.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Superparamagnetic iron oxide	Ocean Nanotech, Inc.	ICK-40-005
Doxorubicin	Sigma	D1515-10MG
SuperMag Separator	Ocean Nanotech, Inc.	N/A
Zetasizer Nano DLS machine	Malvern, Inc	N/A