Surface-Bound Gradient Deposition of Protein Nanoparticles for Cell Motility Studies

Abstract

A versatile evaporation-assisted methodology based on the coffee-drop effect is described to deposit nanoparticles on surfaces, obtaining for the first time patterned gradients of protein nanoparticles (pNPs) by using a simple custom-made device. Fully controllable patterns with specific periodicities consisting of stripes with different widths and distinct nanoparticle concentration as well as gradients can be produced over large areas (∼10 cm) in a fast (up to 10 mm/min), reproducible, and cost-effective manner using an operational protocol optimized by an evolutionary algorithm. The developed method opens the possibility to decorate surfaces "a-la-carte" with pNPs enabling different categories of high-throughput studies on cell motility.

Bacterial inclusion bodies are industrially exploitable amyloids

“…The fraction of functional polypeptides is not externally attached to the protein particles but it represents a structural part of the aggregates, being stable and tightly linked to IBs in aqueous solvents . The fact that an important fraction of the IB-embedded protein retains its biological activity and that solubility and functionality are not equivalent parameters (Garcia- have allowed to further explore IBs as immobilized enzymes for catalysis (Hrabarova, et al, 2015), as biocompatible materials for tissue engineering , Seras-Franzoso, et al, 2012, Tatkiewicz, et al, 2013, Tatkiewicz, et al, 2018 and as protein delivery agents for in vivo applications (Garcia- Fruitos, et al, 2012. Despite the exponentially growing number of examples supporting that IB functionality is a generic and transversal concept , it still remains unfamiliar to an important fraction of the scientific community that retains the obsolete concept linking protein solubility and functionality (Figure 1D).…”

“…For instance, IBs recovered from wild type cells are homogenous, while in DnaKand ClpAcells two IB populations with distinguishable stiffness and which remained segregated were produced . These mechanical properties are extremely important when using IBs as anchoring structures for the cultivation of the mammalian cells , Seras-Franzoso, et al, 2012, Tatkiewicz, et al, 2013, Tatkiewicz, et al, 2018, as stiffer IBs better stimulate cell attachment and/or proliferation (Novak, 2009).…”

High-Throughput Cell Motility Studies on Surface-Bound Protein Nanoparticles with Diverse Structural and Compositional Characteristics

“…These results are in agreement with our previous data, which showed that cells were freely moving inside corrals formed by IB-deposited surfaces surrounded by walls consisting of extremely high FGF-IB concentrations (500,000 particles/mm 2 ). 50…”

“…49 Surface-bound gradients of IBs consisting of the green fluorescence protein (GFP) have also been recently shown to influence cell motility. 50 Encouraged by the latter results, which were only based on physical cues (i.e., topography, geometry and roughness), we decided to study the effects of FGF-based IBs (FGF-IBs) that introduce a biochemical activity together with the physical cues to the substrate for promoting cell migration. Thus, cells not only could be stimulated by the topography of the substrate, but also by the biochemical activity of these inclusion body-like protein nanoparticles.…”

Stable anchoring of bacteria-based protein nanoparticles for surface enhanced cell guidance

“…These pNPs consisted of bacterial inclusion bodies (IBs) of green fluorescent proteins (GFPs) or fibroblast growth factors (FGFs). 27,[55][56][57][58] Such pNPs were produced through biological synthesis in recombinant bacteria obtaining highly pure aggregates, 24 which preserved the function of the protein of interest. 25 They were roughly spherical and showed diameters ranging from 50 to a few hundred nanometers, although their sizes and geometries can be tuned.…”