Screening and quantification of the encapsulation of dyes in supramolecular particles

04 August 2021, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


The encapsulation of therapeutic agents, like 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 the many publications 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 (FC) measurements 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 unveils insightful structure affinity relationships. In contrast to 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 high throughput 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.


flow cytometry
colloidal particles
high throughput screening

Supplementary materials

Supporting Information
General experimental procedures S11 Reagents S11 Instrumentations and abbreviations S11 1. Preparative protocols S11 1.1. Protocols for the preparation of SHS particles S11 1.2. Incubation with dyes S11 2. Additional data and figures referenced in main text (Figs. S1-S9; Tables S1-S2) S12 2.1. Dynamic light scattering and Z-potential studies S12 2.2. Flow cytometry measurements S13 2.3. Correlation plots S15 3. Additional flow cytometry data (Figs. S10-S38) S19 3.1. Histograms and density plots (Figs. S10-S38) S19 3.2. Saturation curves (Figs. S39-S52) S35 4. Confocal Laser Scanning Microscopy (CLSM) studies S42 5. Molecular modeling studies (Figs. S53-S66) S43 6. Supporting References S51


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