Polymer Masked–Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin–Colistin Conjugates

[Image: see text] Polymer masked–unmasked protein therapy (PUMPT) uses conjugation of a biodegradable polymer, such as dextrin, hyaluronic acid, or poly(l-glutamic acid), to mask a protein or peptide’s activity; subsequent locally triggered degradation of the polymer at the target site regenerates bioactivity in a controllable fashion. Although the concept of PUMPT is well established, the relationship between protein unmasking and reinstatement of bioactivity is unclear. Here, we used dextrin–colistin conjugates to study the relationship between the molecular structure (degree of unmasking) and biological activity. Size exclusion chromatography was employed to collect fractions of differentially degraded conjugates and ultraperformance liquid chromatography–mass spectrometry (UPLC–MS) employed to characterize the corresponding structures. Antimicrobial activity was studied using a minimum inhibitory concentration (MIC) assay and confocal laser scanning microscopy of LIVE/DEAD-stained biofilms with COMSTAT analysis. In vitro toxicity of the degraded conjugate was assessed using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. UPLC–MS revealed that the fully “unmasked” dextrin–colistin conjugate composed of colistin bound to at least one linker, whereas larger species were composed of colistin with varying lengths of glucose units attached. Increasing the degree of dextrin modification by succinoylation typically led to a greater number of linkers bound to colistin. Greater antimicrobial and antibiofilm activity were observed for the fully “unmasked” conjugate compared to the partially degraded species (MIC = 0.25 and 2–8 μg/mL, respectively), whereas dextrin conjugation reduced colistin’s in vitro toxicity toward kidney cells, even after complete unmasking. This study highlights the importance of defining the structure–antimicrobial activity relationship for novel antibiotic derivatives and demonstrates the suitability of LC–MS to aid the design of biodegradable polymer–antibiotic conjugates.