Stable Colloidal Drug Aggregates Catch and Release Active Enzymes

Abstract

Small molecule aggregates are considered nuisance compounds in drug discovery, but their unusual properties as colloids could be exploited to form stable vehicles to preserve protein activity. We investigated the co-aggregation of seven molecules chosen because they had been previously intensely studied as colloidal aggregators, co-formulating them with bis-azo dyes. The co-formulation reduced colloid sizes to <100 nm, and improved uniformity of the particle size distribution. The new colloid formulations are more stable than previous aggregator particles. Specifically, co-aggregation of Congo Red with sorafenib, tetraiodophenolphthalein (TIPT) or vemurafenib produced particles that are stable in solutions of high ionic strength and high protein concentrations. Like traditional, single compound colloidal aggregates, the stabilized colloids adsorbed and inhibited enzymes like β-lactamase, malate dehydrogenase and trypsin. Unlike traditional aggregates, the co-formulated colloid-protein particles could be centrifuged and re-suspended multiple times, and from re-suspended particles, active trypsin could be released up to 72 hours after adsorption. Unexpectedly, the stable colloidal formulations can sequester, stabilize, and isolate enzymes by spin-down, resuspension and release.

Internal Structure and Preferential Protein Binding of Colloidal Aggregates

“…Conversely, proteins are readily pelleted out of solution by the colloids, and they can be resuspended and released multiple times. 26 Indeed, when directly compared in competition with one another, the presence of even 20 μ g/mL DNA had no effect on the amount of AmpC that was bound, spun-down, resuspended, and then detergent-released from the colloids. These observations suggest that colloidal aggregates preferentially bind protein over either single-stranded or double-stranded DNA.…”

“…Indeed, enzymes bound to the colloids retained much more activity than those free in solution, suggesting that the colloids acted almost as chaperones. 26 …”

Three Essential Resources to Improve Differential Scanning Fluorimetry (DSF) Experiments

“…We suspected that dye binding to colloidal aggregates might underlie some of these cases. This hypothesis is based on work describing the formation of colloidal aggregates by small molecules as a recognized mechanism of pan-assay interference compounds (PAINS) [44][45][46] . To test this idea, we assembled a panel of eight compounds that have been reported to form colloidal aggregates, but that otherwise vary in chemical structure (Supplementary Figure 5) 47,48 .…”

Leveraging Colloidal Aggregation for Drug-Rich Nanoparticle Formulations

“…19,22 We and others have attempted to stabilize colloidal drug aggregates to further study their biological implications. 14,23 Previously, we demonstrated that coaggregation with azo-dyes can stabilize colloids, resulting in the maintenance of structural integrity in high ionic strength solutions and serum-containing media. 23 The incorporation of polymeric excipients, such as pluronics and polysorbates, remains an attractive method to stabilize colloidal aggregates due to the chemical diversity of polymers available and their ubiquity in pharmaceutical formulations.…”

“…14,23 Previously, we demonstrated that coaggregation with azo-dyes can stabilize colloids, resulting in the maintenance of structural integrity in high ionic strength solutions and serum-containing media. 23 The incorporation of polymeric excipients, such as pluronics and polysorbates, remains an attractive method to stabilize colloidal aggregates due to the chemical diversity of polymers available and their ubiquity in pharmaceutical formulations. Work by Taylor et al has shown that polymeric excipients can modulate the colloidal properties of drug aggregates; however, only modest improvements in stability have been achieved thus far.…”