In Vitro Assessment of Binding Affinity, Selectivity, Uptake, Intracellular Degradation, and Toxicity of Nanobody-Photosensitizer Conjugates

Photosensitizers have recently been conjugated to nanobodies for targeted photodynamic therapy (PDT) to selectively kill cancer cells. The success of this approach relies on nanobody-photosensitizer conjugates that bind specifically to their targets with very high affinities (k D in low nM range). Subsequently, upon illumination, these conjugates are very toxic and selective to cells overexpressing the target of interest (EC 50 in low nM range). In this chapter, protocols are described to determine the binding affinity of the nanobody-photosensitizer conjugates and assess the toxicity and selectivity of the conjugates when performing in vitro PDT studies. In addition, and because the efficacy of PDT also depends on the (subcellular) localization of the conjugates at the time of illumination, assays are described to investigate the uptake and the intracellular degradation of the nanobody-photosensitizer conjugates