Synthesis and Characterization of an Epidermal Growth Factor Receptor-Selective RuII Polypyridyl-Nanobody Conjugate as a Photosensitizer for Photodynamic Therapy.

Johannes Karges,Kristof Zarschler,Marta Jakubaszek,Holger Stephan,Bruno Goud,Cristina Mari,Gilles Gasser

doi:10.1002/cbic.201900419

Abstract

There is a current surge of interest in the development of novel photosensitizers (PSs) for photodynamic therapy (PDT), as those currently approved are not completely ideal. Among the tested compounds, we have previously investigated the use of RuII polypyridyl complexes with a [Ru(bipy)2(dppz)]2+ and [Ru(phen)2(dppz)]2+ scaffold (bipy=2,2′‐bipyridine; dppz=dipyrido[3,2‐a:2′,3′‐c]phenazine; phen=1,10‐phenanthroline). These complexes selectively target DNA. However, because DNA is ubiquitous, it would be of great interest to increase the selectivity of our PDT PSs by linking them to a targeting vector in view of targeted PDT. Herein, we present the synthesis, characterization, and in‐depth photophysical evaluation of a nanobody‐containing RuII polypyridyl conjugate selective for the epidermal growth factor receptor (EGFR) in view of targeted PDT. Using ICP‐MS and confocal microscopy, we could demonstrate that our conjugate has high selectivity for the EGFR receptor, which is a crucial oncological target because it is overexpressed and/or deregulated in a variety of solid tumors. However, in contrast to expectations, this conjugate was found to not produce reactive oxygen species (ROS) in cancer cells and is therefore not phototoxic.

Highlights

The use of photodynamic therapy (PDT) has expanded the possible techniques in medicine to treat various types of cancer as well as bacterial, fungal or viral infections
The [Ru(phen)2(dppz-7-aminomethyl)](PF6)2 complex was synthesized as previously reported in nine synthetic steps.[4a]. The synthesis of the [Ru(phen)2(dppz-7-maleimidemethyl)](PF6)2 complex is already published but, in this study, a slightly different experimental procedure was employed.[4a]. The maleimide-containing Ru(II) complexes was prepared by reacting the [Ru(phen)2(dppz-7-aminomethyl)](PF6)2 complex with maleic anhydride. [Ru(phen)2(dppz-7maleimidemethyl)](PF6)2 was coupled to the poly-glycine chain via a thio-Michael addition reaction
As a benefit of the linkage to a 7C12 nanobody, the conjugate selectively accumulated at the epidermal growth factor receptor (EGFR)

Summary

Introduction

The use of photodynamic therapy (PDT) has expanded the possible techniques in medicine to treat various types of cancer (e.g., lung, bladder, oesophageal and brain cancer) as well as bacterial, fungal or viral infections. Due to the high reactivity of the latter, these can cause oxidative stress and damage in different cellular compartments (e.g., membrane, nucleus, endoplasmic reticulum, lysosome, mitochondria), leading to cell death.[1] Next to the already approved PDT PSs, which are based on a tetrapyrrolic scaffold (i.e. porphyrins, chlorins, phthalocyanines), the development of Ru(II) polypyridyl complexes as PDT PSs is receiving more attention due to their ideal photophysical and photochemical properties, which include, among others, high water solubility, high chemical stability and photostability, intense luminescence, large Stokes shifts, high 1O2 production.[1a-d, 2] These attractive features have allowed one of such complexes, namely TLD-1433, to enter into clinical trial as a PDT PS against bladder cancer.[3] Phase I has been recently completed.[2f] In this context, our group was able to demonstrate that Ru(II) complexes of the type [Ru(bipy)2(dppz)]2+ (bipy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2′,3′-c]-phenazine) and [Ru(phen)2(dppz)]2+ (phen = 1,10-phenanthroline) were effective PDT PSs (Figure 1).[1a, 2c, 4] As a highlight, we could demonstrate that some of these complexes were non-toxic in the dark and highly toxic upon light irradiation with IC50 values in the low micromolar range and a phototoxic index of up to >150.[2c] Based on the extended planar π-system of the dppz ligand, which is able to intercalate into the base pairs of the DNA, these compounds showed a preferable nuclear localization. Other frequently dividing cells in the organism (e.g. hair follicles, gastrointestinal tract, bone marrow) can be affected, leading to severe side-effects for the patients.[4a, 6] it is extremely important to increase the selectivity of PDT PS, for example, with the development of a suitable delivery system

Methods

Results

Conclusion