Pitfalls of accurate protein determination inside PLGA nanoparticles using the microBCA assay

Cancer is one of the leading causes of death in the world and protein therapeutics play an important role in combating this disease. Novel nanocarriers are needed for optimal delivery, enhanced therapeutic effect, and protection of proteins. Poly Lactic-co-Glycolic Acid (PLGA) nanoparticles are commonly used, since they are non-toxic, biodegradable, and allow sustained release of the active pharmaceutical ingredient (API). Accurate quantification of the therapeutic inside these nanocarriers is essential for further development and precise in vivo experiments, especially for determining correct therapeutic dose. Bicinchoninic acid (BCA) assay is one of the most popular methods of protein quantification, known for its low sensitivity to common surfactants. However, large discrepancies between published results are often observed, with determined protein encapsulation efficiencies (EE) varying from 20 to 80%. We investigate the interference of excipients or the combination of excipients, on accurate EE determination of two different PLGA nanoparticle formulations using the microBCA assay. The EE was determined using multiple methods: by measuring the un-encapsulated protein (indirect approach) and directly by extracting the protein using sodium hydroxide and dimethyl sulfoxide extraction. We show significant differences between the methods, highlight the most common pitfalls, and show the importance of using correct standards in assessing EE.