Comparison of fluorescence-based methods to determine nanoparticle uptake by phagocytes and non-phagocytic cells in vitro

Abstract

At many portals of entry the relative uptake by phagocytes and non-phagocytic cells has a prominent effect on availability and biological action of nanoparticles (NPs). Cellular uptake can be determined for fluorescence-labeled NPs. The present study compares three methods (plate reader, flow cytometry and image analysis) in order to investigate the influence of particle size and functionalization and medium content on cellular uptake of fluorescence–labeled polystyrene particles and to study the respective method’s suitability for uptake studies. For comparison between the techniques, ratios of macrophage to alveolar epithelial cell uptakes were used. Presence of serum protein in the exposure solution decreased uptake of carboxyl-functionalized and non-functionalized particles; there was no clear effect for the amine-functionalized particles. The 200 nm non- or carboxyl-functionalized NPs were taken up preferentially by phagocytes while for amine-functionalized particles preference was lowest. The presence of the serum slightly increased the preference for these particles.In conclusion, due to the possibility of calibration, plate reader measurements might present a better option than the other techniques to (semi)quantify differences between phagocytes and non-phagocytic cells for particles with different fluorescence. In order to obtain unbiased data the fluorescent labeling has to fulfill certain requirements.

Quantification of Bioorthogonal Stability in Immune Phagocytes Using Flow Cytometry Reveals Rapid Degradation of Strained Alkynes

“… 58 Due to their size, the particles remain within the confines of the phago-lysosomal system. 59 The cells were then washed and chased for 3 or 24 h to determine to what extent acidification, thioreductase expression, 26 and proteolysis 60 , 61 in the matured phagosome contributed to bioorthogonal handle degradation. Cells were then fixed and permeabilized (allowing free entry of bioorthogonal reagents and neutralization of the phagosomal compartment), ligated with a complementary bioorthogonal fluorophore, before quantification of the two fluorescent signals by flow cytometry ( Figure 4 , Figures S8 and S9 ).…”

Nanotoxicologic Effects of PLGA Nanoparticles Formulated with a Cell-Penetrating Peptide: Searching for a Safe pDNA Delivery System for the Lungs

“…The surface charge of the NPs is also associated to potential toxic effects, with cationic NPs showing acute cytotoxicity that also trigger inflammation [17,20]. The increase in surface charge is a common strategy used to enhance NPs internalization [21], and showed to be essential for an efficient intracellular delivery (Table 2; Figure 3). In this study, adding CPP to the NPs, led to an increase in surface charge to a neutral zeta potential, with no further effect on both MTS and LDH assays, corroborating the results from Platel et al in which neutral and negatively charged NPs did not influence cell viability [20].…”

Design Considerations and Assays for Hemocompatibility of FDA-Approved Nanoparticles

“…The phagocytes can also endocytose cells and express certain antigens on their surface, thereby inducing secondary immune response. 1 The uptake of NPs by phagocytes can be visualized and quantified using different techniques such as electron microscopy, 97 fluorescence microscopy, 98 and flow cytometry. 99 Neutrophils phagocytosed TiO 2 , zinc oxide, and cerium dioxide NPs to different degrees, even though the NPs, were ingested by the same mechanism.…”