In this project, we have used classical methods such as RNA interference and transport inhibitors to determine the role of known pathways in nanoparticle uptake. Next, we have used for the first time a genome-wide genetic screening and methods based on cell proteomics to identify novel targets involved in nanoparticle uptake by cells. By combining classical transport studies with advanced methods not yet applied to the study of the mechanism drug carriers use to enter cells, we have made important discoveries on how cells uptake and process nano-sized materials.
An important result achieved is that the layer of molecules that adsorb from the environment on the nano-carrier surface (the so-called nanoparticle corona, which forms –for example - when nanomedicines are in contact with blood proteins after injection) can affect the mechanisms cells use to internalize these materials. Additionally, we found that many complex interactions with multiple receptors occur at the cell surface and affect the mechanisms cells use for nanoparticle uptake. Importantly, even when specific receptors are involved, cells process nanoparticles in different ways in comparison to their endogenous ligands. These are all important aspects to take into account when designing targeted nanomedicines.
Next to this, thanks to the large genetic and proteomic screening performed, several new targets involved in nanoparticle uptake have been discovered.
In addition, we have developed a new method to determine where nanoparticles are located inside cells using flow cytometry. Flow cytometry is typically used to measure full cells, while we have used it to characterize the intracellular compartments in which nanoparticles are internalized and distributed. Thus, cells are exposed to nanoparticles and then lysed to recover all cell organelles. The organelles containing nanoparticles are quantified and characterized by flow cytometry and the kinetics of nanoparticle intracellular trafficking can be determined. Similarly, using fluorescence imaging to quantify nanoparticle location inside specific cell compartments over time, we have determined intracellular trafficking kinetics and showed how they change for nanoparticles of different size. Intracellular trafficking kinetics affect nanomedicine efficacy, thus it is important to determine how they can be tuned by changing nanomedicine design.
Furthermore, we have optimized protocols to grow endothelial cells into cell barriers, more similar to the barriers nanomedicines encounter in the body and we showed that these cell barriers process nanoparticles in different ways comparing to standard cell cultures used for laboratory testing.
Finally, we have studied how nanoparticle uptake varies in individual cells within a cell population and investigated some of the factors that lead to such variability.
Overall, the results generated have been published in 14 articles, and several other manuscripts are currently under review or in preparation for publication. The team has presented the results generated with 10 oral presentations and 13 posters in national and international conferences and 11 invited lectures (in national and international conferences and research visits to other Universities).
