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 (supramolecular hacky sacks (SHS) particles). We aimed to determine whether this method could quantify protein encapsulation and track changes and if the particles could be tuned to bind to specific proteins. Our results showed that fluorescein isothiocyanate (FITC)-labeled proteins had apparent association constants in the micromolar range with hydrophobicity as the dominant factor enhancing the affinities. Confocal laser scanning microscopy (CLSM) imaging supported these results and provided additional information about the protein distribution within the particles. We also tested the feasibility of tuning the 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 noncovalent cross-linking caused by strong biotin/AVI interactions. CLSM images showed that the linker also impacted the 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 to use lower-affinity ligands would result in better strategies for modulating protein affinity for drug delivery applications.

Optimizing the Production of gp145, an HIV-1 Envelope Glycoprotein Vaccine Candidate and Its Encapsulation in Guanosine Microparticles.

We have developed a pipeline to express, purify, and characterize HIV envelope protein (Env) gp145 from Chinese hamster ovary cells, to accelerate the production of a promising vaccine candidate. First in shake flasks, then in bioreactors, we optimized the growth conditions. By adjusting the pH to 6.8, we increased expression levels to 101 mg/L in a 50 L bioreactor, nearly twice the previously reported titer value. A battery of analytical methods was developed in accordance with current good manufacturing practices to ensure a quality biopharmaceutical. Imaged capillary isoelectric focusing verified proper glycosylation of gp145; dynamic light scattering confirmed the trimeric arrangement; and bio-layer interferometry and circular dichroism analysis demonstrated native-like properties (i.e., antibody binding and secondary structure). MALDI-TOF mass spectrometry was used as a multi-attribute platform for accurate mass determination, glycans analysis, and protein identification. Our robust analysis demonstrates that our gp145 product is very similar to a reference standard and emphasizes the importance of accurate characterization of a highly heterogeneous immunogen for the development of an effective vaccine. Finally, we present a novel guanosine microparticle with gp145 encapsulated and displayed on its surface. The unique properties of our gp145 microparticle make it amenable to use in future preclinical and clinical trials.

Screening and Quantification of the Encapsulation of Dyes in Supramolecular Particles.

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 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.