The study of poly(glutamate)/lysozyme complex-coacervation for sequestration, immobilization and release using a microfluidic dialysis setup.

Currently a challenge for the formulations of highly concentrated (>200g/L) solutions of protein-type drugs is preserving their stability. Stability refers to the protection against deleterious effects of various stresses that may occur during handling, storage and administration. Protecting a protein by immobilization/encapsulation into granules and releasing it in physiological conditions is by consequence essential. Complex coacervation with poly(amino acids) polyelectrolytes, holds great promises as a biodegradable non-toxic system. Coacervation with poly(glutamate) enabled for instance to prepare concentrated therapeutic antibodies with a decreased viscosity [1]. The complex pattern of ionic interactions between proteins and polyelectrolytes makes it however difficult to find the optimal association conditions. A priori, this requires for each protein to explore the phase diagram over a range of pHs and ionic strength. Herein, we illustrate with Lysozyme and poly(glutamate) how a microfluidic dialysis device improves the investigation of protein-poly(amino acid) coacervation, and sp. salt-triggered immobilization and release according to the polypeptide regioregularity vs racemic structure, and polymer peptide molecular weight.

We designed a chip with micro-dialysis chambers enabling in situ control and fast switch (minute time-scale) of the buffer’s conditions, while keeping unchanged the protein and polyelectrolyte concentration. The chip integrates a dialysis membrane that separates a flow channel from a microliter drop of sample. The device is compatible with fluorescence recovery after photobleaching (FRAP) to monitor the dynamics of labelled macromolecules in the coacervate.

By in situ equilibration with varying phosphate buffer concentrations, we determined the phase boundaries. We studied (i) the reversibility of the transition vs ionic strength at short time scale, but also the possibility to seek fibrillations on long time scales, (ii) regioregular vs racemic polymer structure having a straightforward impact on phase diagram by horizontally displacing the critical point, (iii) impact of the equilibrium ionic strength on the diffusion rate of poly(glutamate) (fluid to solid-like transition). (iv) Of note, the phase boundary was significantly broader when studied in the microchamber, as compared to mixtures prepared without in situ dialysis. At high concentrations (> 100 g/L protein), the release of co-ions upon coacervation contributes to the total ionic strength, resulting in the redissolution of the complexes. Owing to the drastic effects of equilibration with the buffer, the micro dialysis device offers accordingly a fast and reliable control that we believe shall be of interest in studies of various interpolymer assemblies and phase transitions.

[1] A. Lapenna et al., Mol. Pharm. 2024, 21(2), 982.