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

Periodic Reporting for period 4 - NanoPaths (Identifying pathways of cellular nanoparticle uptake and early processing for novel nanomedicine applications)

Reporting period: 2020-01-01 to 2021-04-30

Delivering drugs efficiently to the targeted disease still remains a central challenge for current therapies: even when (in many cases) we have very good drugs to treat a disease, several other barriers need to be overcome to achieve good therapy and limit side effects due to drug accumulation to undesired organs.
Nanomedicine promises to help to overcome these barriers by using nano-sized objects (with sizes between few and hundred nanometers) to improve the delivery of drugs to their target and to reach places where drugs currently cannot arrive. Nano-sized object have in fact a unique capacity to enter cells and distribute within organisms. The successes achieved so far clearly show nanomedicine potential, including in the last year the development of nanomedicine-based RNA and DNA vaccines against Covid-19. Despite this, targeting drugs remains an important challenge for the treatment of many diseases.
In order to further improve the success of nanomedicine, a better knowledge of how cells interact with and process these nano-sized materials is required.
Within this context, this project focused on understanding how cells internalize and process nano-sized objects. The objectives of the project were:
- to characterise the mechanisms cells use to internalize nano-sized materials by combining more classical transport 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 types 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 and to study how uptake varies within individual cells in a cell population.
The knowledge gained helps understanding how to improve the design of successful nanomedicines.
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).
Studying transport into cells is known to be extremely challenging. Several methods exist, but each presents advantages and limits. For instance when artificially blocking one entry route, cells can react by adapting and using a different route for nanoparticle uptake. Because of this, in most cases it is very hard to stop nanoparticles from entering cells and identify the mechanisms involved. Thus, one unique feature of this project was 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. Additionally, by taking advantage of nanomaterial properties, novel methods have been developed to characterize how cells internalize these nano-sized objects. The combination of these very different approaches allowed us to progress beyond the state of the art in the characterization of the mechanisms by which cells process nano-sized materials and gain important insights, in particular in the early interactions at the cell membrane, and early trafficking.
Moreover, the large genetic and proteomic screening performed allowed us to identify many novel targets not yet associated to nanoparticle uptake.
Finally, different methods have been optimized (such as for instance the use of transport inhibitors, methods to develop cells into cell barriers, and methods based on fluorescence imaging to determine intracellular trafficking kinetics) and new methods have been developed (such as with organelle flow cytometry) to characterize the mechanisms by which nanoparticles are processed by cells. These methods are important outcomes of this project and are now available to the field to proceed further in the characterization of how nano-sized materials are processed by cells and the design of targeted nanomedicines.
Scheme illustrating the main questions addressed in the NanoPaths project