## 4 Discussion  
Good linearity for the dose dependency curve (Figure 2) indicates the absence of protein saturation with platinum under the experimental conditions. The slightly increased slope for the mass balance of the plasma may be attributed to the matrix effects of the residual organic carbon in the plasma samples [30; 31; 32; 33]. A good repeatability for parallel experiments (relative standard deviations, RSDs \(<\) 10%) was observed (Table 1). Significant retention of platinum for the control buffer after 4 h of dialysis is related to the macromolecular nature of the BP-Cx-1 ligand, reducing the pure drug's capability to cross the 8 kDa membrane. This was additionally validated by independent experiments using different dialysis durations (1-8 h, Supplementary Tables S1 and S2). The difference between 50% plasma at different dosage levels with a corresponding control PBS was evaluated, indicating a statistically significant difference (Table 1). Additionally, average binding to macromolecular species in plasma and the control buffer were calculated to be 76.5 \(\pm\) 6.0% and 57.6 \(\pm\) 5.0% (\(\pm\)standard deviation, SD), respectively. Taking into account the dilution of initial plasma 1:2 per volume with PBS before dialysis, the following fractions of 'free' (unbound to macromolecules) platinum were calculated: 42.4 \(\pm\) 5.0% and 11.8 \(\pm\) 3.0 % (\(\pm\)SD) for the control buffer and plasma, respectively. Considering the macromolecular nature of the BP-C1 lignin-derived ligand (average molecular weight of 1500-2000 Da [7]), the contribution of binding to plasma proteins may be considered to be ca. 60% (61 \(\pm\) 12%, \(\pm\)2 SD, \(n\) = 24), since ca. 15% of Pt is associated with polyphenic moieties, as identified by the control buffer experiments (see Supplementary Information for more details on calculations). Platinum-based drug plasma protein binding is known to be lower for drugs containing O-donor leaving ligands. For instance, carboplatin mostly releases active platinum moiety only intracellularly, showing ex vivo binding of 25-40% [34; 35]. Previous studies of platinum drug binding to plasma/serum proteins indicated that carboplatin is primarily bound to HSA (45-96%, depending on incubation times), followed by transferrin and immunoglobulin-G (both ca. 2%) [16; 36]. In vivo studies indicated the formation of reversible complexes of carboplatin with HSA [37]. Nevertheless, the intracellular distributions of cisplatin and carboplatin are similar, and their exhibited toxicity is not directly correlated with a total platinum concentration in target organs [38]. This is consistent with our previous studies for BP-C1 and other platinum drugs, indicating a high level of total platinum in kidneys and liver without the formation of toxic effects [23; 25]. Thus, BP-C1 demonstrates medium protein binding, comparable to carboplatin and considerably lower than that for cisplatin. A medium degree of binding of ca. 60% was demonstrated, indicating the maintenance of a high fraction of free drug in the bloodstream, facilitating its pharmacological activity. Further research on BP-C1 should focus on the effect of O-donor leaving ligands on its antitumor activity and the presence of potential cross-resistivity of certain tumours against platinum compounds. The following are available online at [[https://www.mdpi.com/article/10.3390/app112211008/s1](https://www.mdpi.com/article/10.3390/app112211008/s1)]([https://www.mdpi.com/article/10.3390/app112211008/s1](https://www.mdpi.com/article/10.3390/app112211008/s1)). Datasets for the protein-binding evaluations. The selection of optimal dialysis time is explained in Supplementary Table S1: Selection of optimal dialysis duration for cisplatin; 10 mL vertical dialysis tubes with 8-10 kDa cutoff in a Float-A-Lyzer G2 Dialysis Device (Repligen) were used for dialysis vs. 25 mL isotonic 0.9 % NaCl solution at room temperature. Pt was quantified by ICP-MS.