The effects of cell culture conditions on premature hydrolysis of traceless ester-linked disulfide linkers

Abstract

Disulfide acids are important for traceless release mechanisms in prodrugs and drug delivery applications. Their ability to self-immolate and release cargo due to environmental stimulus is invaluable. However, complex reactivity patterns may be overlooked as assays increase in complexity or are conducted in media of increasing biological relevance. Conclusions drawn from preliminary characterization in simple phosphate buffers are often applied to in vitro studies in which more complex media are used (e.g. containing glucose, amino acids, FBS, and the cell surface). We developed a model disulfide incorporating a fluorogenic dye as a reporter group in order to explore the generality of the disulfide prodrug system, and used this to explore the stability of disulfide esters in various contexts of increasing complexity. We found that our reporter molecules prematurely released cargo in a series of cell-containing and cell-free assays. We systematically reverse-engineered the components of a complex cell medium and found that FBS was capable of interfering with disulfide-based prodrug linkers, triggering the release of conjugated 4-methylumbelliferone (4-MU) from representative reporter molecules. FBS consistently induced 4-MU release in complete media (i.e. DMEM and RPMI 1640), minimal essential media, and pure water. Signs of 4-MU release were mitigated when FBS was subjected to intense heat (>100 °C) or esterase-specific protease inhibitor cocktail (PIC), indicating that esterases from the serum were capable of triggering cargo release using a hydrolysis mechanism that is separate from the desired reductive cleavage pathway. These findings are important because they show that variance in models may hide unexpected results, which calls for more meticulous consideration of control experiments when developing stimulus-release agents for biological applications.