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Identifying pathways of cellular nanoparticle uptake and early processing for novel nanomedicine applications

Periodic Reporting for period 3 - NanoPaths (Identifying pathways of cellular nanoparticle uptake and early processing for novel nanomedicine applications)

Reporting period: 2018-07-01 to 2019-12-31

Delivering drugs efficiently to the targeted disease still remains a central challenge for current therapies: even when (in many cases) we do have very good drugs to treat a disease, several other barriers need to be overcome to achieve good therapy and avoid side effects due to drug accumulation to undesired organs.
Nanomedicine promises to help to overcome these barriers by revolutionizing the ways drugs are delivered to their target. Nano-sized objects (with sizes between few and hundred nanometers) have in fact a unique capacity to enter cells and distribute within organisms. This capacity has made man-made nano-sized materials extremely attractive for their potential applications in nanomedicine to deliver drugs to their target and to reach places where drugs currently cannot arrive.
The first successes achieved so far clearly show nanomedicine potential. Despite this, relatively few nanomedicines so far have arrived to the clinic. Furthermore it has been showed that on average only 0,7% of the injected dose of currently available nanomedicines arrives to its target. Also, in many cases the processes by which cells interact with and process these nano-sized materials are not fully clear. Without this knowledge, developing efficient nanomedicines remains very challenging.

Within this context, this project is focused on understanding how cells internalize and process nano-sized objects. The knowledge gained will allow us to better understand how to design truly targeted drug carriers.
In particular, the Objectives of the project are:
- to characterise the mechanisms cells use to internalize nano-sized materials. This will be achieved by combining more classical transport and uptake studies in cells, such as those used so far in this field, to novel approaches from other disciplines, not yet applied to the study of uptake of nano-sized drug carriers.
- to develop novel methods to study how nano-sized drug carriers enter and are processed by cells, taking advantage of the unique properties of nanomaterials.
- to determine whether special sub-populations of cells (like for instance cells developed into cell barriers similar to those nanomedicines encounter in the body) internalize nano-sized materials in different ways.
By integrating the results obtained, the knowledge gained will allow to define guidelines for improving the design of successful nanomedicines.
In the first half of the project, great efforts have been focused in combining classical transport studies with recently developed methods not yet applied to the study of the mechanism drug carriers use to enter cells.
More in particular we are using RNA interference to shut down the expression of a panel of key proteins known to be involved in different uptake mechanisms. This allows us to determine their eventual role in the uptake of nano-sized drug carriers. In parallel, standard transport inhibitors have also been used. These compounds are known to be effective very quickly on cells, but often they lack specificity and can cause strong toxicity. The results obtained clearly show that careful controls are needed to ensure these inhibitors are used appropriately and to avoid generating artifacts.
An important result achieved combining these methods is that the layer of molecules that adsorb from the environment on the nano-carrier surface (the so called nanoparticle corona, for instance when nanomedicines are in contact with blood proteins after injection) can affect the mechanisms cells use to internalize these materials.

Furthermore, we have optimized protocols to grow endothelial cells into tight cell barriers, more similar to the cell barriers nanomedicines encounter in the body. Then we studied whether these barriers uptake and process nano-sized objects in different ways than the standard cell cultures used for laboratory testing. The results clearly show that when cells are differentiated into cell barriers, they process the same nanoparticles in different ways.

In parallel to this, in this first period major efforts have been dedicated in developing novel methods to use for the first time to the question of nanoparticle uptake for drug delivery.
More in particular, full genome screening approaches are being used to identify potential novel targets not yet associated with uptake processes in cells. Next to this we are developing new methods to isolate the cellular structures in which nanoparticles are internalised, by taking advantage of their unique properties. Then we are characterizing the structures isolated and in this way we will gain detailed information on how cells are processing these nano-sized materials.

Finally, we are using live cell imaging with a library of cells expressing fluorescently labeled variants of key proteins involved in uptake. This allows us to determine whether the selected transport proteins are involved in the uptake of nano-sized materials. Different methods for image analysis are being implemented and will be compared in order to extract quantitative information on colocalization between the nanoparticles and the different proteins.
Studying transport into cells is known to be extremely challenging. Several methods exist, but each presents some limits. For instance when artificially blocking one entry route to test if it is involved in nanoparticle uptake, cells can react by adapting and use a different route. Thus in most cases it is very hard to stop nanoparticles from entering cells and identify the mechanisms involved.
In order to tackle this problem, one unique feature of this project is that of combining several different classical methods currently available to study uptake into cells, with methods recently developed and never yet applied to the question of nanoparticle uptake into cells.
Similarly, by taking advantage of nanomaterial properties, novel methods are being developed to characterize how cells internalize nano-sized objects.
Each of these methods present well known limits and specific strengths, and thus alone they do not allow an accurate characterization of the mechanisms of uptake into cells. We believe that it is the combination of the results obtained with these very different approaches that will allow us to progress beyond the state of the art in the characterization of the mechanisms by which cells process nano-sized materials.
Next to this, the results obtained so far have highlighted two important aspects that require further attention in the field.
The first is the observation that cells, when developed into tight cell barriers process nanoparticles in a different way than individual cells in standard cell cultures typically used in the lab. Most of the barriers nanomedicines encounter in the body are such tight cell monolayers. Thus when studying nanomedicines in the lab, it is important to use similar cell models, more closely resembling the barriers nanomedicines encounter in the body.
The second interesting aspect is relative to the effect of the so called corona on nanoparticle uptake mechanisms in cells. Nano-sized drug carriers once in contact with biological fluids, such as for instance in the blood, are modified by the adsorption of molecules on their surface, forming a corona. It has been shown that this layer strongly affects the resulting interactions with cells. Interestingly our results show that the corona can also affect the mechanisms cells use to internalize the nanoparticles. This is another important aspect to take into consideration when testing nanomedicine efficacy in cells.