Development of a combined In-Cell ELISA and flow cytometry method for quantification of uptake of PEGylated nanoparticles by Raw264.7 and HepG2 cells

Sammanfattning: Cancer is one of today’s most common causes of human mortality. For long, chemotherapeutics has been a conventional treatment of the disease, but due to its low precision and high frequency of side effects, this treatment has been highly debated. Nanosized particles, so called nanoparticles (NPs), have emerged as a promising tool for cancer treatment, due to their ability of selectively reaching tumor sites. Developing biocompatible NPs has turned out to be challenging, due to the particles’ tendency of interacting with proteins and immune cells present throughout the blood, interactions further leading to removal of the NPs from the bloodstream. This removal strongly reduces the circulation half-life of NPs in the blood, something that in turn reduces the therapeutic efficacy of the particles. Coating of NPs is often used as a tool for increasing the half-life of the particles in the blood. By attaching coating molecules to the surface of the NPs, protein interaction and subsequent removal of the particles from the bloodstream can be reduced. Polyethylene glycol (PEG) is one of the most common polymers used for coating of NPs. The main goal of this project was to study whether coating degree and PEG length of NPs had an impact on the uptake of these particles by Raw264.7 (macrophage cell line) and HepG2 (hepatocyte cell line) cells. To study the impact of coating on cellular uptake, organosilicophosphonate core NPs were coated with PEG of three different lengths, each at two different coating degrees. The coated NPs were characterized with respect to size, pH, charge, coating degree and tendency to aggregate in culture media. Analytical techniques such as gel permeation chromatography (GPC), dynamic light scattering (DLS), zeta potential measurements and inductively coupled plasma optical emission spectroscopy (ICP-OES) were used for characterization of the particles. Uptake of PEG coated as well as bare NPs by Raw264.7 and HepG2 cells was quantified by developing and using a combined method constituting In-Cell enzyme-linked immunosorbent assay (In-Cell ELISA), ELISA and flow cytometry. Finally, the intracellular distribution of NPs in Raw264.7 cells was studied using fluorescence microscopy. Indicatively, coating degree as well as PEG length had an impact on the cellular uptake of NPs. NPs coated with long-length PEG showed a lower uptake, compared to NPs coated with short-length PEG. In addition, NPs coated with a high amount of PEG showed a lower uptake than NPs coated with a low amount of PEG. Preliminary indications of differences in intracellular distribution of PEG coated NPs in Raw264.7 cells were seen.