Freezing-mediated formation of supraproteins using depletion forces

• Published in: Journal of colloid and interface science Vol. 665; pp. 622 - 633
• Main Authors: Song, Jiankang, Tas, Roderick P., Martens, Max (C. M.), Ritten, Manon V.M., Wu, Hanglong, Jones, Elizabeth R., Lebouille, Jérôme G.J.L., Vis, Mark, Voets, Ilja K., Tuinier, Remco
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.2024
• Subjects: Arrested phase separation; Depletion interaction; Drug delivery; Freeze-drying; Micronization; Proteins; Proteins; Micronization; Drug delivery; Arrested phase separation; Depletion interaction; Freeze-drying
• ISSN: 0021-9797; 1095-7103
• DOI: 10.1016/j.jcis.2024.03.088

Hypothesis Long-acting formulations such as microparticles, injectable depots and implantable devices can realize spatiotemporally controlled delivery of protein drugs to extend their therapeutic in vivo half-lives. To efficiently encapsulate the protein drugs into such drug delivery systems, (sub)micron-sized protein particles are needed. The formation of micronized supraproteins can be induced through the synergistic combination of attractive depletion forces and freezing. The size of the supraproteins can be fine-tuned from submicron to several microns by adjusting the ice crystallization rate through the freeze-quench depth, which is set by the target temperature. Methods Supraprotein micron structures were prepared from protein solutions under various conditions in the presence and absence of nonadsorbing polyethylene glycol. Scanning electron microscopy and dynamic light scattering were employed to determine the sizes of the supraproteins and real-time total internal reflection fluorescent microscopy was used to follow the supraprotein formation during freezing. The protein secondary structure was measured before and after micronization by circular dichroism. A phase diagram of a protein–polyethylene glycol mixture was theoretically predicted to investigate whether the depletion interaction can elucidate the phase behavior. Findings Micronized protein supraparticles could be prepared in a controlled manner by rapid freeze-drying of aqueous mixtures of bovine serum albumin, horseradish peroxidase and lysozyme mixed with polyethylene glycol. Upon freezing, the temperature quench initiates a phase separation process which is reminiscent of spinodal decomposition. This demixing is subsequently arrested during droplet phase separation to form protein-rich microstructures. The final size of the generated protein microparticles is determined by a competition between phase separation and cooling rate, which can be controlled by target temperature. The experimental phase diagram of the aqueous protein–polyethylene glycol dispersion aligns with predictions from depletion theory for charged colloids and nonadsorbing polymers. Supraproteins of varying sizes emerge through the synergistic interplay of attractive depletion forces and freezing. This is demonstrated through aqueous protein-polyethylene glycol dispersions across different target temperatures. •Supraproteins can be prepared via freezing protein–polyethylene glycol in water dispersions.•The size of these supraproteins can be tuned via the depletion concentration and cooling rate.•Initial composition of protein–polyethylene glycol mixtures affects supraprotein formation.•The phase diagram of charged proteins and polyethylene glycol is quantified using depletion theory.•Proteins preserve their secondary structure during supraprotein formation process.