Motivation and Research Journey

It becomes evident that various mechanical cues act as key regulatory factors, affecting processes such as cell spreading, movement, differentiation, and proliferation. Thus, it is unsurprising that the external physical forces of cellular microenvironment were found to modulate the nanoparticle uptake kinetics and processing by living cells. Consequently, it is crucial to reveal how mechanics influence cell-nanoparticles interactions. Despite this, our knowledge of how localized physical forces impact the internalization and processing of nanoparticles by cells is still quite limited. Addressing such complex question requires combining efforts of different scientific fields.

The Laboratory of Biophysics of Institute of Physics of the Czech Academy of Sciences, led by Dr. Oleg Lunov, merges physical and biological research approaches to create interdisciplinary platform for studying nanomaterials-cell interactions and effects of physical forces on cellular behavior. The laboratory has established a solid research line in the field of cell-nanomaterial interactions. We uncovered hidden toxicological aspects of iron oxide nanoparticle-based contrast agents for magnetic resonance imaging. We revealed that particles passively accumulate in liver Kupffer cells, where they localize in lysosomal compartments^{1, 2}. Lysosomes degrade the sugar shell of the particles, and when iron oxide is exposed, it triggers sustained JNK activation via excessive accumulation of reactive oxygen species^{1, 2}. Further, we analyzed the potential mechanisms of toxicity caused by nanoparticle-induced lysosomal dysfunction^{3, 4}. Then, we summarized and critically evaluated the adverse effects of existing clinically approved nanomedicines. Importantly, we have shown that the physicochemical properties have a great influence on the interaction of nanoparticles with cells, the route and kinetics of excretion, and subsequently on toxicity. We hypothesized that adverse reactions could be associated with nanodrug-induced lysosomal dysfunction⁵.

Bearing knowledge of nanoparticle-cell interactions, we asked the further question of what is currently known about how extracellular mechanical cues may affect and/or change those interactions. Indeed, it turned out that our understanding of how cellular uptake and processing of nanoparticles are biased by mechanical cues is still rather limited. At this point, the laboratory welcomed a young, talented PhD student, Petra Elblová, who took over the project on nanoparticle-cell interactions.

Figure 1. Petra Elblová (PhD student at the Laboratory of Biophysics) and Oleg Lunov (Head of the Laboratory of Biophysics).

Key Findings

Petra's first task was to review and critically analyze evidence regarding the impact of mechanical cues on cell-nanoparticle interactions. The literature review revealed several mechanical factors influencing cell fate and, consequently, cell-nanoparticle interactions. These factors include hydrostatic pressure, fluid shear stress, tensile force, extracellular matrix (ECM) stiffness, and extracellular fluid viscosity.

Most research has focused on how ECM stiffness affects cell-nanoparticle interactions, likely due to the availability of commercial cell culture platforms that allow modulation of stiffness. However, as is common in relatively unexplored areas of scientific research, different studies sometime

s show conflicting effects of the same mechanical cues on nanoparticle-cell interactions. This inconsistency can be partially attributed to the variability in nanoparticle and cell models used in the studies. Despite this, some trends have emerged.

Several studies have shown a positive correlation between increased particle uptake and ECM stiffness. Other research suggests that substrate stiffness influences both cell adhesion and particle internalization, with softer substrates enhancing particle uptake. The cell type also predisposes the response to external stiffness. Regarding tensile forces, similar to ECM stiffness and shear stress, tensile forces can either promote or inhibit nanoparticle uptake by cells.

It is important to note that the effects of mechanical stimuli on nanoparticle uptake can be reflected by regulating the mechanical properties or stress state of the cell or by altering the endocytosis pathways (Fig. 2). We further analyzed the potential molecular cellular mechanisms responsible for the impact of mechanical stimuli on the total uptake level of nanoparticles.

Figure 2. Schematic representation of molecular mechanisms how different mechanical cues (tensile force, extracellular fluid viscosity, hydrostatic pressure, and shear stress) regulate nanoparticle uptake by cells. Red arrows indicate tensile forces, green arrows indicate shear stress, blue arrows indicate impact of extracellular fluid viscosity, and black arrows refer to hydrostatic pressure, yellow arrows indicate effect of ECM stiffness. ECM - extracellular matrix, ECF - extracellular fluid viscosity, YAP - yes-associated protein, ROS -reactive oxygen species. Created with BioRender.com. Reprinted from open-access article ref.⁶ under the terms of the Creative Commons CC BY license.

Mechanical stimuli, such as ECM stiffness and architecture, can significantly affect the intracellular mechanical state of cells, altering their mechanical properties and/or influencing the stress distribution within the cell. These changes may ultimately regulate nanoparticle uptake. However, studies on how mechanical stimuli-driven changes in the intracellular mechanical state affect nanoparticle uptake are currently very limited.

It has been found that ECM stiffness regulates cellular uptake of nanoparticles via actin cytoskeleton remodeling. In soft ECM, round cells lack stress fibers, with only discrete bright spots observed. Conversely, cells on stiff ECM display significantly more aligned stress fibers compared to those on intermediate ECM. Emerging evidence suggests that mechanical stimuli may regulate nanoparticle uptake by altering endocytosis pathways (Fig. 2). For example, it was found that stiffness may regulate nanoparticle uptake by modulating clathrin expression, thereby affecting the endocytosis rate.

Significance and Future Outlook

Overall, regulation of nanoparticle uptake by substrate stiffness through modulation of endocytosis via cytoskeletal and motor proteins appears to be a general mechanism observed across different cell lines and with various nanoparticles (Fig. 2).

Additionally, we highlighted the challenges and future perspectives in this research area. Physical factors exert diverse effects on the interaction between nanomaterials and cells, and their impact can vary depending on the specific cell type. The type of cell and its tissue origin introduce additional complexities, making it difficult to establish a universal rule for how nanomaterials will interact with cells.

Most studies addressing the influence of physical factors on nanomaterial-cell interactions use standard cell lines as cellular models. Only a limited number of studies utilize primary cell cultures to explore how mechanical cues bias nanoparticle-cell interactions. Another challenge is the relatively limited use of 3D cell culture models to study the effects of physical factors on nanoparticle uptake and processing by cells. While 2D systems provide more controllable and manageable environments for cell analysis, 3D culture systems offer a platform to study multiple physical factors simultaneously.

We hope this review serves as a catalyst for novel ideas and innovative approaches in designing safe and effective nanomedicines tailored for specific applications. A comprehensive understanding of how mechanical cues influence nanoparticle-cell interactions will provide valuable fundamental knowledge, ultimately enabling the formulation of a strategic roadmap for advancing the nanobio field toward successful clinical implementations.

References

1 O. Lunov, et al., Biomaterials, 2010, 31, 9015-9022.

2 O. Lunov, et al., Biomaterials, 2010, 31, 5063-5071.

3 K. Levada, et al., Nano Convergence, 2020, 7, 17.

4 A. Frtús, et al., Journal of Controlled Release, 2020, 328, 59-77.

5 M. Uzhytchak, et al., Advanced Drug Delivery Reviews, 2023, 197, 114828.

6 P. Elblova, et al., Discover Nano, 2024, 19, 106.