## 3 Metal-Based Protein, Peptide and Antibody Drug Conjugates
As expected, all complexes interact with DNA, with a slight preference for TAT-9 derivatives, but unfortunately with minor differences with the non-bioconjugated metal derivatives. Furthermore, even the cellular uptake analysis has revealed the scarce impact of the use of CPPs, and no cytotoxicity towards HeLa and Hs-578T has been observed for either CPPs or the corresponding metal conjugated compounds (IC\({}_{50}\) > 100 uM) [88]. Figure 7: Pt(IV) complexes synthesized by [PERSON] and [PERSON]. In order to improve the panel of photosensitizers (PS) employed in photodynamic therapy (PDT), [PERSON] and [PERSON] have proposed the conjugation of [Ru(pphen)\({}_{2}\)(dmbipy)]+ complexes with Bombesin peptide bearing a \(N\)-terminal Cys. The latter is known have high affinity with human gastrin-releasing peptide receptor (GRPR), which is overexpressed in many solid tumours [89]. Complexes **42-43**, depicted in Figure 8B, were tested in PC-3, HT-29 and CT26 cancer cell lines in dark and light conditions, comparing the IC\({}_{50}\) values to the non bioconjugated metal complexes. Bioconjugation has permitted us to maintain the cytotoxicity only in dark conditions (IC\({}_{50}\) = 0.5-18 \(\upmu\)M) in both complexes. Moreover, the general decreasing in cellular uptake was observed in the CT26 line, showing the highest accumulation of drug in lysosomes [89]. In complex **44** (see Figure 8C), ruthenium has been conjugated to the N7C12 nanobody, a specific binder to epidermal growth factor receptor (EGFR)-expressing cells [90]. A  
Figure 8: (**A**) Ruthenium CPP conjugates; (**B**) Ruthenium Bombesin conjugates; (**C**) Ruthenium 7C12 conjugates; (**D**) Ruthenium Ab conjugates; (**E**) Ruthenium peptides. nanobody is a single chain domain, smaller than mAb, albeit with high stability, solubility, specificity, affinity and fast pharmacokinetics [82]. To encourage a specific site conjugation, a poly-glycine unit peptide was used as linker. The internalization of complex **44** into A431 cells is correlated to the capability of N7C12 to target EGFR in overexpressed cell lines after site-specific modification and is strongly dependent on the level of EGFR overexpression. The nanobody conjugation has not modified the photophysical properties of the metal complex, but no ROS production was detected, and consequently, no cytotoxicity in dark and in light conditions (IC\({}_{50}\) > 25 \(\upmu\)M due to complex precipitation) has been observed [90]. Compound **45** (Figure 8D) was reported in 2022 by [PERSON] and [PERSON]. It is an organometallic complex linked to an afflobody, a small robust protein engineered to bind a large number of target proteins or peptides with high affinity [91]. Compound **45** bears a Human Epidermal Growth Factor Receptor 2 (HER2) target which is able to activate gemcitabine prodrug in situ via bio-orthogonal reaction and release the drug in proximity of the cell membrane. Moreover, owing to its bifunctional properties, bioconjugated complex **45** induced DNA damage in positive breast cancer cells, thus blocking their proliferation. The result has been confirmed even in spheroids and zebrafish in vitro and in vivo models, respectively [91]. Among compounds **46**, depicted in Figure 8E, complex **46b**, prepared by [PERSON], [PERSON] and [PERSON], is the most active of the first family of metallocene peptides with inhibitor properties towards cell division cycle 25 (CDC25) [92]. Despite the promising ex vivo preliminary results, chemical modifications or caging are necessary to improve the anticancer activity and cell membrane permeability [92]. The immuno-positron emission technique (immune-PET) is a powerful tool to monitor the behaviour of radiolabelled antibodies in vivo in real time and it is therefore very useful to receive a rapid response in tumour diagnosis, immunotherapy and radioimmunotherapy. However, the structure of the bifunctional chelator (BFC) is crucial for the pharmacokinetics of the whole compound [93].