An investigation of the effect of the protein corona on the cellular uptake of nanoliposomes under flow conditions using quartz crystal microgravimetry with dissipation

When nanoparticle delivery systems are immersed in biological fluids, a complex assembly of proteins forms on their surface, creating a protein corona. The protein corona alters the physicochemical properties, toxicity, biodistribution, cellular uptake, and immune response of the nanoparticles, and consequently, their therapeutic efficacy. Currently, there is a lack of <i>in vitro</i> methods to assess the effects of the protein corona on nanoparticle uptake under dynamic flow and assess their binding kinetics in real-time. Here, we introduce quartz crystal microbalance with dissipation (QCM-D) as an <i>in vitro</i> technique, capable of incorporating dynamic flow, to study the effect of the protein corona on the binding of nanoliposome (NLP) formulations to cell surfaces as a first step in their cellular uptake. The interactions of four NLP formulations (low PEGylated, high PEGylated, negatively charged and positively charged NLPs) with A375 melanoma and THP1 cell lines were assessed by QCM-D, before and after the formation of a protein corona. Through real-time recording of the frequency and dissipation shifts (Δ<i>f</i> and Δ<i>D</i>, respectively), the QCM-D results provided strong evidence of the role of the protein corona in the cellular interaction of these NLP formulations, with a variation in their adsorption kinetics depending on their initial composition. NLP's attachment to the cell surface was the lowest for PEGylated NLPs (<5%), while the positively charged NLPs showed the highest cellular attachment (≈100%), regardless of the presence of the protein corona or cell type. The effect of the protein corona was more pronounced for the negatively charged NLPs, where a significant reduction in the NLP attachment was observed. To complement the QCM-D data on the NLP attachment and to determine whether the NLP attachment leads to cellular uptake, confocal microscopy and flow cytometry were used to confirm NLP uptake by A375 and THP1 cells. Proteomic analysis revealed a differential composition of the protein corona on the various NLPs with possible implications for their sequestration and cellular uptake. Collectively, the findings suggest that QCM-D can be an important tool to study the binding of NLP formulations or other nanoparticles with cell membranes under dynamic flow, which very often differs from nanoparticle uptake under static conditions.