![Author ORCID: We display the ORCID iD icon alongside authors names on our website to acknowledge that the ORCiD has been authenticated when entered by the user. To view the users ORCiD record click the icon. [opens in a new tab]](https://chemrxiv.org/engage/assets/public/chemrxiv/images/logos/orcid.png)
Abstract
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.
Supplementary materials
Title
Supporting Information File
Description
1. General experimental procedures
2. Dynamic Light Scattering and Zeta Potential measurements of SHS1 and SHS2 (Fig. S2, Table S1)
3. Density plots and histograms of SHS1 and SHS2 (Figs. S3-S20)
4. Binding isotherms of SHS1 and SHS2 (Figs. S21-S29; Tables S2, S3)
5. Correlation graphs of apparent protein affinities towards SHS particles (Figs. S23- S33)
6. Encapsulation efficiency (enrichment index) of SHS1 and SHS2 as determined by Confocal Scanning Laser Microscopy (Fig. S34, Table S4)
7. Synthesis and characterization of G-derivatives 3 and 4 (Figs. S35-S44)
8. Self-assembly NMR studies of G-derivatives 2, 3 and 4 (Fig. S45)
9. Dynamic Light Scattering and Zeta Potential measurements of biotinylated SHS particles (Fig. S46; Tables S5, S6)
10. Flow cytometry and encapsulation efficiency (enrichment index) studies of heteromeric assemblies (Figs. S47-S51)
11. Molecular Models (Fig. S52)
12. Supporting References
Actions
