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

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.Nanoparticles are small particles that have emerged as a promising tool for cancer treatment. In order for these particles to efficiently treat cancer they have to avoid being eaten by cells from the immune system. This can be done by putting “invisibility jackets” on the particles. Cancer kills 8 million people every year and finding efficient cancer treatments has turned out to be hard. Many of the existing treatments kill cancer cells efficiently, but the problem is that healthy cells are affected as well. This causes unwanted side effects. Nanoparticles are small particles that have emerged as a novel tool for cancer treatment. Nanoparticles have the possibility to reach cancer cells without affecting healthy cells and in this way side effects can be reduced. However, treating cancer with nanoparticles is not completely without problems. One problem that may arise is that the nanoparticles may be removed from the body by cells called immune cells. Immune cells are supposed to protect the body from intruders. Even if the nanoparticles are used for a good reason (to treat the cancer) the immune cells will regard them as intruders. The immune cells will therefore remove the nanoparticles by eating them. This means that the particles will not reach the cancer cells, in turn meaning that they will not treat the cancer. The nanoparticles can however be prevented from being eaten if they become invisible to the immune cells. An “invisibility jacket”, made of molecules called PEG, can for example be put onto the particles. The PEG can be regarded as the textile fibers in the jacket. Like with all clothing, the quality of the invisibility jacket will vary depending on the material in it (i.e. the PEG). Just as with textile fibers PEG molecules can be very different. Some of them will generate good invisibility jackets, which will make the nanoparticles more invisible. Others will generate bad jackets. In an experiment the quality of different invisibility jackets was evaluated. Some of the jackets were made of long PEG, others of short or medium PEG. The number of PEGs in the jackets also varied; some jackets contained many PEG molecules, whereas others contained few. The quality of the different invisibility jackets was evaluated by putting them onto nanoparticles. The nanoparticles were then allowed to meet immune cells. If many nanoparticles were eaten by the immune cells, the invisibility jacket was regarded as bad (it did not make the particles invisible), whereas if few particles were eaten, the jacket was regarded as good (it made the particles more invisible). It was found that jackets made of long PEGs were better (they made the particles more invisible), compared to jackets made of short PEGs. It was also found that jackets containing many PEGs were better compared to jackets containing few PEGs