Interactions between biological cells and surrounding extracellular matrix (ECM) materials modulate many cell behaviors including adhesion and migration. One key example of this cellmatrix reciprocity is in the context of angiogenesis, the sprouting of new blood vessels from preexisting vasculature. Vascular endothelial cells (VECs) create and remodel the ECM during this process. In vivo, the surrounding fluid environment includes high concentrations of macromolecules, and is considered “crowded” in comparison to in vitro environments. Here, we quantified the amount and organization of collagen IV, a prominent ECM component of VECs, that was produced by these cells over four weeks in vitro in the presence or absence of macromolecular crowder (MMC) nanoparticles that approximated in vivo crowding. In the presence of MMCs, the amount and degree of alignment of collagen IV was greater. This ECM difference emerged within one week and was sustained for over four weeks. We explored the effect of initial cell density (cells/µm2) on this matrix production, to consider potential differences at a wound site versus an intact vessel. Moreover, we found the biophysical effect of MMCs to be unmodulated by secretions from an adjacent cell type in microvessels (pericytes). These results suggest that macromolecular crowding plays a direct role in remodeling the basement membrane, and that such crowding can be induced in vitro to more closely approximate the cell microenvironment.

Traditional methods of quantifying cell movement in response to a chemotactic factors provide either a binary count of cell migration in response to a known concentration of the factor of interest in solution, as in Boyden chamber assays, or a method of tracking cells to determine velocities across a solubilized protein gradient where exact concentrations vary over time and are difficult to define, as in the Ibidi chemotaxis gradient assay. Using a silane self-assembling monolayer (SAM)-based procedure pioneered by V Hlady and associates, we have developed an assay capable of covalently binding a wide variety of proteins to an optically transparent surface in a 2D pattern via amine linkages. The pattern was then verified by contact angle and Raman and X-ray photoelectron spectroscopy. This new assay provides greater control of protein concentration and gradient intensity than when using only solubilized proteins.

Nanoparticle-based vectors are fast becoming the main choice for nucleic acid delivery. Fluorescent nanoparticles have the added advantage of tracking the delivery process and also of tracking cells transfected with nucleic acids for cell therapy. Fluorescent upconversion nanoparticles (UCNs) are ideal candidates for tracking since they are excited by NIR light and hence have very low phototoxicity, high signal-to-noise ratio and enable imaging in deep tissues. UCNs coated with a layer of silica and adsorbed with siRNA or siRNA loaded into the mesoporous silica coating on the UCNs have been used for siRNA delivery. However the loading of siRNA is very poor since the silica coating is negatively charged and it repels the negatively charged siRNA limiting the amount of siRNA that can be adsorbed on the surface of nanoparticles. Here we report the use of a layer-by-layer approach to coat the UCNs with Poly-L-Lysine (PLL) and use it for delivery. Highly monodispersed UCNs were synthesized with an average size of 50 nm. They were then modified with PLL and STAT-3 siRNA was adsorbed on to the surface of the modified UCNs. The loading of the siRNA was found to be 60 % more efficient by this approach as compared to silica coated UCNs alone. The PLL-coated UCNs also were minimally cytotoxic as shown by MTS assay. The siRNA coated UCNs also efficiently transfected B16F0 cells and knocked down STAT3 significantly and also enabled cell imaging. Thus, This method shows good promise for siRNA delivery and tracking and this could also be extended to in-vivo transfection and tracking.