Tuning Protein Nanoclusters With Molecular Crowding Agents (Depletants)

Stabilizing proteins at high concentration is of broad interest in drug delivery for treatment of cancer and many other diseases. Proteins have a tendency to undergo irreversible aggregation, gelation or precipitation at high concentrations due to unfolding caused by specific short ranged forces. We create highly concentrated antibody dispersions comprising dense equilibrium nanoclusters of therapeutic protein molecules, which upon dilution in vitro dissociate to individual protein molecules.  The key is to control nanocluster size and interparticle spacing within the clusters by manipulation of the particle colloidal forces.  The interaction potential, which depends upon the electrostatic, Van der Waals, steric and depletion forces, governs thermodynamic self-assembly and particle morphology. 

A hierarchy of intracluster and intercluster interactions has been controlled to provide colloidal stability, low viscosities, and stable protein for drug delivery. The clusters are formed by balancing depletion attraction between protein molecules against electrostatic repulsion. Our previous studies have focused primarily on trehalose as the depletant or crowding agent. In the current study we show substantial additional control over nanocluster morphology and the viscosity of the protein dispersions by varying the size and polarity of the crowding agent.  We also report new types of results for the effect of depletant on the nanocluster sizes as a function of the depletant properties.