Protein Compartments Modulate Fibrillar Self-Assembly.

A notable feature of complex cellular environments is protein-rich compartments that are formed via liquid-liquid phase separation. Recent studies have shown that these biomolecular condensates can play both promoting and inhibitory roles in fibrillar protein self-assembly, a process that is linked to Alzheimer's, Parkinson's, Huntington's, and various prion diseases. Yet, the exact regulatory role of these condensates in protein aggregation remains unknown. By employing microfluidics to create artificial protein compartments, the self-assembly behavior of the fibrillar protein lysozyme within them can be characterized. It is observed that the volumetric parameters of protein-rich compartments can change the kinetics of protein self-assembly. Depending on the change in compartment parameters, the lysozyme fibrillation process either accelerated or decelerated. Furthermore, the results confirm that the volumetric parameters govern not only the nucleation and growth phases of the fibrillar aggregates but also affect the crosstalk between the protein-rich and protein-poor phases. The appearance of phase-separated compartments in the vicinity of natively folded protein complexes triggers their abrupt percolation into the compartments' core and further accelerates protein aggregation. Overall, the results of the study shed more light on the complex behavior and functions of protein-rich phases and, importantly, on their interaction with the surrounding environment.

Assessing Differential Particle Deformability under Microfluidic Flow Conditions.

Assessing the mechanical behavior of nano- and micron-scale particles with complex shapes is fundamental in drug delivery. Although different techniques are available to quantify the bulk stiffness in static conditions, there is still uncertainty in assessing particle deformability in dynamic conditions. Here, a microfluidic chip is designed, engineered, and validated as a platform to assess the mechanical behavior of fluid-borne particles. Specifically, potassium hydroxide (KOH) wet etching was used to realize a channel incorporating a series of micropillars (filtering modules) with different geometries and openings, acting as microfilters in the direction of the flow. These filtering modules were designed with progressively decreasing openings, ranging in size from about 5 down to 1 µm. Discoidal polymeric nanoconstructs (DPNs), with a diameter of 5.5 µm and a height of 400 nm, were realized with different poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) ratios (PLGA/PEG), namely, 5:1 and 1:0, resulting in soft and rigid particles, respectively. Given the peculiar geometry of DPNs, the channel height was kept to 5 µm to limit particle tumbling or flipping along the flow. After thorough physicochemical and morphological characterization, DPNs were tested within the microfluidic chip to investigate their behavior under flow. As expected, most rigid DPNs were trapped in the first series of pillars, whereas soft DPNs were observed to cross multiple filtering modules and reach the micropillars with the smallest opening (1 µm). This experimental evidence was also supported by computational tools, where DPNs were modeled as a network of springs and beads immersed in a Newtonian fluid using the smoothed particle hydrodynamics (SPH) method. This preliminary study presents a combined experimental-computational framework to quantify, compare, and analyze the characteristics of particles having complex geometrical and mechanical attributes under flow conditions.

Leaf-Inspired Authentically Complex Microvascular Networks for Deciphering Biological Transport Process.

The vascular transport of molecules, cells, and nanoconstructs is a fundamental biophysical process impacting tissue regeneration, delivery of nutrients and therapeutic agents, and the response of the immune system to external pathogens. This process is often studied in single-channel microfluidic devices lacking the complex tridimensional organization of vascular networks. Here, soft lithography is employed to replicate the vein system of a Hedera elix leaf on a polydimethilsiloxane (PDMS) template. The replica is then sealed and connected to an external pumping system to realize an authentically complex microvascular network. This satisfies energy minimization criteria by Murray's law and comprises a network of channels ranging in size from capillaries (∼50 µm) to large arterioles and venules (∼400 µm). Micro-PIV (micro-particle image velocimetry) analysis is employed to characterize flow conditions in terms of streamlines, fluid velocity, and flow rates. To demonstrate the ability to reproduce physiologically relevant transport processes, two different applications are demonstrated: vascular deposition of tumor cells and lysis of blood clots. To this end, conditions are identified to culture cells within the microvasculature and realize a confluent endothelial monolayer. Then, the vascular deposition of circulating breast (MDA-MB 231) cancer cells is documented throughout the network under physiologically relevant flow conditions. Firm cell adhesion mostly occurs in channels with low mean blood velocity. As a second application, blood clots are formed within the chip by mixing whole blood with a thrombin solution. After demonstrating the blood clot stability, tissue plasminogen activator (tPA) and tPA-carrying nanoconstructs (tPA-DPNs) are employed as thrombolytics. In agreement with previous data, clot dissolution is equally induced by tPA and tPA-DPNs. The proposed leaf-inspired chip can be efficiently used to study a variety of vascular transport processes in complex microvascular networks, where geometry and flow conditions can be modulated and monitored throughout the experimental campaign.

Deciphering the relative contribution of vascular inflammation and blood rheology in metastatic spreading.

Vascular adhesion of circulating tumor cells (CTCs) is a key step in cancer spreading. If inflammation is recognized to favor the formation of vascular "metastatic niches," little is known about the contribution of blood rheology to CTC deposition. Herein, a microfluidic chip, covered by a confluent monolayer of endothelial cells, is used for analyzing the adhesion and rolling of colorectal (HCT-15) and breast (MDA-MB-231) cancer cells under different biophysical conditions. These include the analysis of cell transport in a physiological solution and whole blood over a healthy and a TNF-α inflamed endothelium with a flow rate of 50 and 100 nl/min. Upon stimulation of the endothelial monolayer with TNF-α (25 ng/ml), CTC adhesion increases from 2 to 4 times whilst cell rolling velocity only slightly reduces. Notably, whole blood also enhances cancer cell deposition from 2 to 3 times, but only on the unstimulated vasculature. For all tested conditions, no statistically significant difference is observed between the two cancer cell types. Finally, a computational model for CTC transport demonstrates that a rigid cell approximation reasonably predicts rolling velocities while cell deformability is needed to model adhesion. These results would suggest that, within microvascular networks, blood rheology and inflammation contribute similarly to CTC deposition, thereby facilitating the formation of metastatic niches along the entire network, including the healthy endothelium. In microfluidic-based assays, neglecting blood rheology would significantly underestimate the metastatic potential of cancer cells.