Biodynamers as prodrugs with controlled uptake and activity

Chemotherapy is one of the primary approaches for cancer treatment. However, its toxicity to healthy tissue often causes significant side effects, which is one of the main reasons why many potent drug candidates fail to gain therapeutic approval. Adenosine dialdehyde (ADOX), an adenosine analog used as an indirect methylation inhibitor that hinders S-adenosyl homocysteine hydrolase (SAHH), is one such promising candidate affected by this limitation. In this study, we developed a polymeric prodrug of ADOX to reduce its toxicity to non-cancerous cells under neutral conditions and to enhance its selectivity toward cancer cells in the acidic tumor microenvironment (TME). Specifically, we designed ADOX-biodynamers, alternative copolymers based on dynamic constitutional chemistry, that release ADOX from their backbone in response to the acidic pH of the TME. The ADOX-biodynamers were prepared by connecting two monomers, ADOX and amino acid hydrazides (Lys-Hz, His-Hz, Phe-Hz, and Glu-Hz), alternatively via pH-responsive dynamic covalent bonds, imines, and acylhydrazones. The dynamic covalent bonds allow degradation of ADOX-biodynamers, enabling the release of ADOX under acidic conditions. In particular, ADOX-Lys biodynamer (ALB) released ADOX under acidic conditions at a rate 8 times faster than in neutral conditions. In contrast, at pH 7.4, ALB maintained a stable polymeric structure, which suppressed ADOX activity and reduced toxicity up to 5 times compared to the free ADOX in non-cancerous cells, 16HBE14o-. However, when ALB was first exposed to acidic conditions, it regained its cytotoxicity toward three human cancer cell lines: HCT116, MCF-7, and SW480, recovering up to 60 % of the activity of free ADOX. In addition, ALB exhibited significantly higher cellular uptake under mildly acidic conditions (pH 6.4), with nearly twice the uptake compared to that at pH 7.4. Notably, in an in vitro tumor tissue model using HCT116 spheroids, ALB treatment resulted in a substantial reduction in spheroid size, achieving approximately a 40 % decrease after 7 days of treatment. As a result, the synthesized ADOX-biodynamers demonstrated improved safety toward non-cancerous cells in neutral pH, restored the activity of the free drug and increased cellular uptake in acidic conditions, and effectively penetrated into tumor tissue. These findings indicate that ADOX-biodynamers successfully address the limitations of ADOX and serve as an effective delivery system. This study highlights the potential of polymeric prodrugs utilizing dynamic covalent bonds as a promising strategy to simultaneously improve drug safety and therapeutic efficacy, supporting the development of potent therapeutic candidates.