ECM Mechano-Sensing Regulates Cytoskeleton Assembly and Receptor-Mediated Endocytosis of Nanoparticles.

It is possible to create sophisticated and target-specific devices for nanomedicine thanks to technological advances in the engineering of nanomaterials. When on target, these nanocarriers often have to be internalized by cells in order to accomplish their diagnostic or therapeutic function. Therefore, the control of such uptake mechanism by active targeting strategy has today become the new challenge in nanoparticle designing. It is also well-known that cells are able to sense and respond to the local physical environment and that the substrate stiffness, and not only the nanoparticle design, influences the cellular internalization mechanisms. In this frame, our work reports on the cyclic relationship among substrate stiffness, cell cytoskeleton assembly and internalization mechanism. Nanoparticles uptake has been investigated in terms of the mechanics of cell environment, the resulting cytoskeleton activity and the opportunity of activate molecular specific molecular pathways during the internalization process. To this aim, the surface of 100 nm polystyrene nanoparticles was decorated with a tripeptide (RGD and a scrambled version as a control), which was able to activate an internalization pathway directly correlated to the dynamics of the cell cytoskeleton, in turn, directly correlated to the elastic modulus of the substrates. We found that the substrate stiffness modulates the uptake of nanoparticles by regulating structural parameters of bEnd.3 cells as spreading, volume, focal adhesion, and mechanics. In fact, the nanoparticles were internalized in larger amounts both when decorated with RGD, which activated an internalization pathway directly correlated to the cell cytoskeleton, and when cells resided on stiffer material that, in turn, promoted the formation of a more structured cytoskeleton. This evidence indicates the directive role of the mechanical environment on cellular uptake of nanoparticles, contributing new insights to the rational design and development of novel nanocarrier systems.

Hemoglobin-Conjugated Gelatin Microsphere as a Smart Oxygen Releasing Biomaterial.

In this study, a novel micrometric biomaterial acting as a cyclic oxygen releasing system is designed. Human hemoglobin (Hb) is conjugated to the surface of gelatin microspheres (GM) to produce gelatin hemoglobin oxygen depot (G-HbOD). G-HbOD is obtained by means of two different conjugation strategies. The degree of conjugation of GM surfaces in terms of free amino groups by using HPLC is first evaluated. By following the strategy A (G-HbOD_A), Hb is conjugated to GM by means of the formation of a polyurethane linker. In the strategy B (G-HbOD_B) the conjugation occurs via amide bound formation. Physical and morphological differences between G-HbOD_A and G-HbOD_B are investigated by means of Fourier Transform Infrared Spectroscopy (FTIR), Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Differences in oxygen uptake/release kinetics are found depending on the conjugation strategy and it is proved that G-HbOD works under repeated cycles in microfluidic chip. Moreover, G-HbOD is also able to work as oxygen depot in the early stages of 3D cell cultures.

A supramolecular two-photon-active hydrogel platform for direct bioconjugation under near-infrared radiation.

A supramolecular hydrogel assembled from partially methacrylated polyethyleneimine (PEI) and with direct photopatterning capabilities at near-infrared (NIR) wavelengths is presented. The chemically modified branched PEI macromolecules were characterized by FTIR and NMR spectroscopy to quantify the degree of methacrylation. A highly hydrophilic polymer network with a water content up to 95% was prepared. The hydrogel microstructure in an aqueous solution was characterized using confocal laser scanning microscopy (CLSM), which revealed a porous network with large interconnected cavities. The photo-sensitive PEIMA hydrogel was activated by two-photon laser irradiation and micropatterns formed at its interface when probes with free carboxylic acid or hydroxyl groups were present in solution. Direct patterning of the hydrogel matrix with different biomolecules, and without additional photoinitiators, is demonstrated in two-photon microscopy experiments.

Design, synthesis and characterisation of a fluorescently labelled CyPLOS ionophore.

A novel fluorescently labelled synthetic ionophore, based on a cyclic phosphate-linked disaccharide (CyPLOS) backbone and decorated with four tetraethylene glycol tails carrying dansyl units, has been synthesised in 12 steps in 26% overall yield. The key intermediate in the synthetic strategy is a novel glucoside building block, serving through its 2- and 3-hydroxy groups as the anchor point for flexible tetraethylene glycol tentacles with reactive azido moieties at their ends. To test the versatility of this glucoside scaffold, it was preliminarily functionalised with a set of diverse probes--as fluorescent, redox-active or hydrophobic tags--either by reduction of the azides followed by condensation with activated carboxylic acid derivatives, or by a direct coupling with a terminal alkyne in a Cu(I)-promoted 1,3-dipolar cycloaddition. Tagging of the monomeric building block with dansyl residues allowed us to prepare a fluorescent, amphiphilic macrocycle, which was investigated for its propensity to self-aggregate in CDCl(3)--studied by means of concentration-dependent (31)P NMR spectroscopy experiments--and in aqueous solution, in which combined dynamic light scattering (DLS) and small-angle neutron scattering (SANS) measurements provided a detailed physico-chemical analysis of the self-assembled systems, mainly organised in the form of large vesicles. Its ion-transport properties through phospholipid bilayers, determined by HPTS fluorescence assays, showed this compound to be more active than the previously synthesised CyPLOS congeners. Solvent-dependent fluorescence changes for the labelled ionophore in liposome suspension established that the dansyl moieties are dispersed in environments with polarity intermediate between those of CH(2)Cl(2) and propan-2-ol, suggesting that the CyPLOS tentacles infiltrate the mid-polar region of the membranes.