Pitfalls of Accurate Protein Determination inside PLGA Nanoparticles Using the Micro BCA Assay

Abstract

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 for the 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 the 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 PLGA nanoparticles using the micro BCA 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. We show differences between the methods, highlight the most common pitfalls, and show the importance of using correct standards in assessing EE.

Skin irritation and potential antioxidant, anti-collagenase, and anti-elastase activities of edible insect extracts

“…Although the Kjeldahl technique is regarded as the worldwide standard and therefore simple to compare findings with other laboratories, it does not measure real protein and can result in overestimation of protein owing to the use of the standard nitrogen correction factor 14 . On the other hand, the BCA technique, which is used in the present study, is based on two chemical reactions, including the biuret reaction, which reduces cupric ions (Cu 2+ ) to cuprous ions (Cu 1+ ) via peptide bonds and the chelation of one Cu 1+ molecule with two BCA molecules to form a bright purple complex which is spectrophotometrically detected 15 . Besides, BCA is simpler, ease of use, takes less time for the experiment, requires fewer instruments, high sensitivity, and tolerance of interfering species, e.g.…”

“…Besides, BCA is simpler, ease of use, takes less time for the experiment, requires fewer instruments, high sensitivity, and tolerance of interfering species, e.g. common surfactants 15 – 17 . The findings revealed that the protein contents analyzed by BCA in the current investigation were equivalent to those found in the prior study utilizing the Kjeldahl method.…”

Rational Design and Development of Polymeric Nanogels as Protein Carriers

“…[131,132] However, absorption by functional groups in the polymer/crosslinkers or scattering interferences due to NG concentration may lead to inaccurate results. [133][134][135][136] A frequently used strategy is based on labeled proteins that can be quantified by colorimetric or fluorometric methods. For example, Duan et al studied the loading of different FITC-labeled proteins into dual-responsive NGs to develop multifunctional carriers for combined cancer therapy.…”

“…[ 131,132 ] However, absorption by functional groups in the polymer/crosslinkers or scattering interferences due to NG concentration may lead to inaccurate results. [ 133–136 ] A frequently used strategy is based on labeled proteins that can be quantified by colorimetric or fluorometric methods. For example, Duan et al.…”

Avaliação da penetração/permeação cutânea da catalase livre, nanoencapsulada e peguilada visando um efeito antioxidante

“…Apolinário et al, 2019 descrevem que há uma superestimação da quantificação de proteínas quando a metodologia indireta é realizada, possivelmente, pela interação da cadeia polimérica, é possível aprisionamento de moléculas de enzima livre, que acabam sendo consideradas no cálculo da concentração de enzima encapsulada. Essa questão é abordada por outros autores, os quais levantam o questionamento sobre a possibilidade de falhas na estimativa da EE%, especialmente pela metodologia indireta (APOLINÁRIO et al, 2019;CLERICI et al, 2021).…”