Screening and Quantification of the Encapsulation of Dyes in Supramolecular Particles

Abstract

The encapsulation of therapeutic agents, such as drugs and vaccines, into colloidal particles offers an attractive strategy to enhance their efficacy. Previously, we reported the development of guanosine-based supramolecular colloidal particles suitable for encapsulating a broad array of guests ranging from small molecule drugs, like doxorubicin, to proteins, like GFP. Many biomedical applications of such particles require a precise determination of the amount of encapsulated therapeutic agents. Despite many studies describing the development of particle-based delivery systems, a general method for the precise and quick quantification of the encapsulated payload is still lacking. Here, we report a method based on flow cytometry measurements for complexes made from guanosine-based particles and a variety of commercially available fluorescent dyes. This method allows us to determine the apparent affinities of such dyes for two variants of these particles, which in turn provides insightful structure−affinity relationships. In contrast to the current methods, such as those that rely on fluorescence microscopy based on measurements of absorption/fluorescence of dissolved particles or on the supernatant of the solution, the reported method is suitable for highthroughput screening and more reproducible results. The protocol described here should be applicable to a wide variety of colloidal particles being developed around the world. Our group is currently expanding the scope to quantify the encapsulation of other molecules of biomedical interest, such as proteins and nucleic acids.

Quantifying and Modulating Protein Encapsulation in Guanosine-Based Supramolecular Particles

“…We recently reported the higher affinity of SHS particles for cationic dyes relative to their anionic counterparts . Based on this finding, we anticipated that a similar trend would hold for the proteins studied in this research.…”

“…In contrast with the behavior observed for small-molecule dyes, the EI values for the evaluated proteins only show a relatively weak correlation with the size of the SHS particle . For example, most EI values for small-molecule dyes toward SHS1 fall within the range of 4–20, while most EI values for the proteins tested range from 1 to 4 for the same SHS1 particle.…”

“…Figure presents confocal microscopy images and an enrichment index graph of heteromeric SHS particles. The enrichment index of these proteins, even after adding the biotin ligand, is lower compared to the encapsulation of small-molecule dyes, which suggests the potential for improvement using this strategy . The enrichment indexes of these particles demonstrate that incorporating the biotin ligand increases the affinity toward AVI .…”

“…The aim of this study is to measure the relative affinities of proteins for two kinds of SHS particles using a novel FC-based method recently developed by our group . We also wanted to examine if we can change the relative binding strength of a protein by adding an affinity ligand (Figure ).…”

“…(4) (B) After incubation, the Protein@SHS complex was further studied with various techniques like flow cytometry and confocal microscopy. (5) Adapted with permission from ref copyright 2021 American Chemical Society.…”