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.

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.

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.

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.