Quantifying and modulating protein encapsulation in guanosine-based supramolecular particles

The encapsulation of proteins is an effective way to preserve their structure and enhance their function. One exciting possibility is adjusting the protective agent to match the specific protein's characteristics to influence its properties. In a recent study, we developed a flow cytometry-based method to quantify the encapsulation of small molecule dyes in colloidal particles made from guanosine derivatives (SHS particles). We aimed to determine if this method could quantify protein encapsulation and track changes and if proteins could be tuned to bind to these particles. Our results showed that FITC-labeled proteins had apparent association constants in the micromolar range, with hydrophobicity as the dominant factor enhancing the affinities. Confocal laser scanning microscopy imaging supported these results and provided additional information about protein distribution within the particles. We also tested the feasibility of tuning avidin affinity (AVI) for SHS particles with a biotin ligand. We found that increasing the amount of biotin initially enhanced AVI binding, but then reached saturation, which we hypothesize results from non-covalent cross-linking caused by strong biotin/AVI interactions. CLSM images showed that the linker also impacted AVI distribution within the particles. Our strategy provides an advantage over other methods for quantifying protein encapsulation by being suitable for high-throughput analysis with high reproducibility. We anticipate that future efforts of using lower affinity ligands would result in better strategies for modulating protein affinity for drug-delivery applications.