Original ArticleNanobody–photosensitizer conjugates for targeted photodynamic therapy
Graphical Abstract
Conjugates consisting of nanobodies (NB) targeting the epidermal growth factor receptor (EGFR) and a traceable photosensitizer (PS) are here described and evaluated in vitro. EGFR-targeted NB–PS conjugates retain their binding specificity after PS conjugation and the biparatopic NB leads to a more effective intracellular delivery of PS. These NB–PS conjugates specifically induce cell death of EGFR overexpressing cells, in the low nanomolar concentration, while PS alone or the NB–PS conjugates in the absence of light induce no toxicity. These new NB–PS conjugates are promising in the field of targeted photodynamic therapy (PDT).
Section snippets
Nanobodies and PS conjugation
Nanobodies (NBs) 7D12, R2, and 7D12-9G8 were produced as described in the Supplementary Materials. The photosensitizer IRDye700DX (here named PS) was purchased from LI-COR (LI-COR Biosciences, Lincoln, Nebraska) as an N-hydroxysuccinimidine (NHS) ester. Conjugation of the PS to the NBs, purification and characterization of the NB–PS conjugates were performed as described in the Supplementary Materials.
Cell lines and culture conditions
The mouse fibroblast cell line NIH 3T3 2.2 (abbreviated 3T3 2.2) was described in Ref. 37; the
Production and characterization of NB–PS conjugates
The EGFR-specific NB 7D12 is employed for targeting the PS to EGFR expressing cells for PDT, and the non-relevant NB R2 is used as a negative control (Figure 1, A). In addition, the internalizing biparatopic NB 7D12-9G8 is used to investigate whether more effective internalization of PS would further increase the toxicity of these conjugates. Similarly to the previous study,28 NBs were conjugated to the PS (IRDye700DX) via random NHS-mediated coupling to lysine amino acids. After purification,
Discussion
Over the years, many efforts have been made in the field of photodynamic therapy (PDT) to target photosensitizers (PS) specifically to the site of interest (i.e. tumor).11, 12, 13, 14 Particularly in the context of photoimmunotherapy (PIT), monoclonal antibodies (mAbs) have been employed for this purpose. However, these conjugates have long half-lives and, consequently, antibody fragments have been used for more rapid and efficient clearance of PS from blood and normal tissues. In this study,
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Note: S. Oliveira is supported by the STW-NWO Veni grant number (11878). R. Heukers is supported by the Focus & Massa project of the Utrecht University and by a grant from arGEN-X, Gent, Belgium. The authors declare no conflict of interest.