Background/Objectives: Here, we report the design, synthesis, and in vitro biological evaluation of a novel stimuli-sensitive nanotherapeutics based on cisplatin analog, cis-[PtCl2(NH3)(2-(3-oxobutyl)pyridine)] (Pt-OBP), covalently linked to a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via a pH-sensitive hydrazone bond. Methods: Two polymer-drug conjugates, P-Pt-A and P-Pt-B, were synthesized, differing in spacer length between the polymer chain and hydrazone bond, which in turn modulates their drug release kinetics. Results: The spacer based on hydrazone bond demonstrated satisfactory stability under blood-mimicking conditions while enabling selective release of the active drug intracellularly or even in the mildly acidic tumor microenvironment. Pt-OBP exhibits comparable or even superior cytostatic and cytotoxic activity to carboplatin across a panel of murine and human cancer cell lines, with the highest potency observed in FaDu cells representing human head and neck squamous cell carcinoma. Mechanistically, Pt-OBP induced significant phosphorylation of γ-H2AX and activation of caspase-3, indicating its ability to cause DNA damage with subsequent apoptosis induction. P-Pt-A retained moderate biological activity, whereas the slower-releasing P-Pt-B exhibited reduced potency in vitro, consistent with its drug release profile. Conclusions: Notably, free Pt-OBP induced rapid apoptotic cell death, surpassing carboplatin at early time points, and the polymeric conjugates achieved comparable pro-apoptotic activity after extended incubation, suggesting effective intracellular release of the active drug.

Institute of Macromolecular Chemistry of the Czech Academy of Sciences v v i Heyrovského sq 2 16206 Prague Czech Republic

Institute of Microbiology of the Czech Academy of Sciences v v i Vídeňská 1083 14220 Prague Czech Republic

NanoBioMedical Centre Adam Mickiewicz University Wszechnicy Piastowskiej 3 61 614 Poznan Poland

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