Unravelling the mechanisms of recognition and internalization of nanoparticles by cells

Nanoparticles have great potential for drug delivery (nanomedicine), because of their size (few to hundreds of nanometers). They are bigger than molecular drugs, so they cannot diffuse into the cell. Instead they enter cells through specialized cell pathways, similar to those used by natural nano-sized objects, like proteins and viruses. This gives nanoparticles the potential to be used to deliver drugs only to specific cells, thus improving efficacy and reducing side-effects. Several nanomedicines have already reached the market, but the targeting to specific cells can still be improved and the mechanism by which nanoparticles are processed and internalized by cells often remains unclear. Most research studies well-known uptake pathways, however it might be that unconventional mechanisms are involved in the entry of nanoparticles into cells. In this thesis, several different methods have been used to study the role of known pathways in the uptake of nanoparticles. In particular, the impact of nanoparticle charge and mechanical properties on the mechanism of uptake has been investigated. Moreover, for the first time in this field, we have applied a method in which all cellular components can be screened for their involvement in nanoparticle uptake. In this way, we have been able to discover novel components and studied their role. Our results showed that nanoparticle uptake mechanisms depend on nanoparticle properties, but are also affected by the presence of biomolecules which adsorb on the nanoparticles, which can interact with multiple cell receptors. At a broader level, our results illustrate the complexity of nanoparticle-cell interactions

Interactions at the cell membrane and pathways of internalization of nano-sized materials for nanomedicine

Nano-sized materials have great potential as drug carriers for nanomedicine applications. Thanks to their size, they can exploit the cellular machinery to enter cells and be trafficked intracellularly, thus they can be used to overcome some of the cellular barriers to drug delivery. Nano-sized drug carriers of very different properties can be prepared, and their surface can be modified by the addition of targeting moieties to recognize specific cells. However, it is still difficult to understand how the material properties affect the subsequent interactions and outcomes at cellular level. As a consequence of this, designing targeted drugs remains a major challenge in drug delivery. Within this context, we discuss the current understanding of the initial steps in the interactions of nano-sized materials with cells in relation to nanomedicine applications. In particular, we focus on the difficult interplay between the initial adhesion of nano-sized materials to the cell surface, the potential recognition by cell receptors, and the subsequent mechanisms cells use to internalize them. The factors affecting these initial events are discussed. Then, we briefly describe the different pathways of endocytosis in cells and illustrate with some examples the challenges in understanding how nanomaterial properties, such as size, charge, and shape, affect the mechanisms cells use for their internalization. Technical difficulties in characterizing these mechanisms are presented. A better understanding of the first interactions of nano-sized materials with cells will help to design nanomedicines with improved targeting

Sources of Variability in Nanoparticle Uptake by Cells

Understanding how nano-sized objects are taken up by cells is important for applications within medicine (nanomedicine), as well as to avoid unforeseen hazard due to nanotechnology (nanosafety). Even within the same cell population, one typically observes a large cell-to-cell variability in nanoparticle uptake, raising the question of the underlying cause(s). Here we investigate cell-to-cell variability in polystyrene nanoparticle uptake by HeLa cells, with generalisations of the results to silica nanoparticles and liposomes, as well as to A549 and primary human umbilical vein endothelial cells. We show that uptake of nanoparticles is correlated with cell size within a cell population, thereby reproducing and generalising previous reports highlighting the role of cell size in nanoparticle uptake. By repeatedly isolating (using fluorescence-activated cell sorting) the cells that take up the most and least nanoparticles, respectively, and performing RNA sequencing on these cells separately, we examine the underlying gene expression that contributes to high and low polystyrene nanoparticle accumulation in HeLa cells. We can thereby show that cell size is not the sole driver of cell-to-cell variability, but that other cellular characteristics also play a role. In contrast to cell size, these characteristics are more specific to the object (nanoparticle or protein) being taken up, but are nevertheless highly heterogeneous, complicating their detailed identification. Overall, our results highlight the complexity underlying the cellular features that determine nanoparticle uptake propensity

Role of Curvature-Sensing Proteins in the Uptake of Nanoparticles with Different Mechanical Properties

Nanoparticles of different properties, such as size, charge, and rigidity, are used for drug delivery. Upon interaction with the cell membrane, because of their curvature, nanoparticles can bend the lipid bilayer. Recent results show that cellular proteins capable of sensing membrane curvature are involved in nanoparticle uptake; however, no information is yet available on whether nanoparticle mechanical properties also affect their activity. Here liposomes and liposome-coated silica are used as a model system to compare uptake and cell behavior of two nanoparticles of similar size and charge, but different mechanical properties. High-sensitivity flow cytometry, cryo-TEM, and fluorescence correlation spectroscopy confirm lipid deposition on the silica. Atomic force microscopy is used to quantify the deformation of individual nanoparticles at increasing imaging forces, confirming that the two nanoparticles display distinct mechanical properties. Uptake studies in HeLa and A549 cells indicate that liposome uptake is higher than for the liposome-coated silica. RNA interference studies to silence their expression show that different curvature-sensing proteins are involved in the uptake of both nanoparticles in both cell types. These results confirm that curvature-sensing proteins have a role in nanoparticle uptake, which is not restricted to harder nanoparticles, but includes softer nanomaterials commonly used for nanomedicine applications.</p

Sources of Variability in Nanoparticle Uptake by Cells

Understanding how nano-sized objects are taken up by cells is important for applications within medicine (nanomedicine), as well as to avoid unforeseen hazard due to nanotechnology (nanosafety). Even within the same cell population, one typically observes a large cell-to-cell variability in nanoparticle uptake, raising the question of the underlying cause(s). Here we investigate cell-to-cell variability in polystyrene nanoparticle uptake by HeLa cells, with generalisations of the results to silica nanoparticles and liposomes, as well as to A549 and primary human umbilical vein endothelial cells. We show that uptake of nanoparticles is correlated with cell size within a cell population, thereby reproducing and generalising previous reports highlighting the role of cell size in nanoparticle uptake. By repeatedly isolating (using fluorescence-activated cell sorting) the cells that take up the most and least nanoparticles, respectively, and performing RNA sequencing on these cells separately, we examine the underlying gene expression that contributes to high and low polystyrene nanoparticle accumulation in HeLa cells. We can thereby show that cell size is not the sole driver of cell-to-cell variability, but that other cellular characteristics also play a role. In contrast to cell size, these characteristics are more specific to the object (nanoparticle or protein) being taken up, but are nevertheless highly heterogeneous, complicating their detailed identification. Overall, our results highlight the complexity underlying the cellular features that determine nanoparticle uptake propensity

Comparison of the uptake mechanisms of zwitterionic and negatively charged liposomes by HeLa cells

Zwitterionic molecules are used as an alternative to PEGylation to reduce protein adsorption on nanocarriers. Nonetheless, little is known on the effect of zwitterionic modifications on the mechanisms cells use for nanocarrier uptake. In this study, the uptake mechanism of liposomes containing zwitterionic or negatively charged lipids was characterized using pharmacological inhibitors and RNA interference on HeLa cells to block endocytosis. As expected, introducing zwitterionic lipids reduced protein adsorption in serum, as well as uptake efficiency. Blocking clathrin-mediated endocytosis strongly decreased the uptake of the negatively charged liposomes, but not the zwitterionic ones. Additionally, inhibition of macropinocytosis reduced uptake of both liposomes, but blocking actin polymerization had effects only on the negatively charged ones. Overall, the results clearly indicated that the two liposomes were internalized by HeLa cells using different pathways. Thus, introducing zwitterionic lipids affects not only protein adsorption and uptake efficiency, but also the mechanisms of liposome uptake by cells